Principles of Molecular Microbiology Testing Method

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Principles of Molecular Microbiology Testing Methods
Article in Infectious Disease Clinics of North America · January 2002

DOI: 10.1016/S0891-5520(05)70190-2 · Source: PubMed
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THE ROLE OF THE CLINICAL MICROBIOLOGY
LABORATORY 0891-5520/01 $15.00 + .OO
PRINCIPLES OF MOLECULAR
MICROBIOLOGY TESTING
METHODS
Donna Wolk, PhD, MHA, MT; Shawn Mitchell, MMSc, MT;
and Robin Patel, MD, FRCP(C)
Recent advances in molecular testing methods are changing the

practice of clinical microbiology and infectious disease. In the past de-
cade, nucleic acid technologies have complemented conventional culture,
antigen-based, and antibody-based methods for the detection, identifica-
tion, and epidemiological analysis of infectious microorganisms.", 76, Io4,
lo6,lo7,'09, 118 Until recently, molecular approaches to infectious disease
diagnosis have been limited to the detection of slow-growing, difficult-
to-cultivate, or uncultivatable microorganisms;29~ 83, 85 however, advances
in testing formats, including automated nucleic acid extraction instru-
mentation and rapid polymerase chain reaction (PCR)/target detection
formats, now make molecular diagnostic testing adaptable for use in
many clinical laboratories and applicable for a variety of common patho-
gens. Some assays offer the capability of detection, quantification, and
mutation screening in the same testing format, and may be useful in
screening for antimicrobial resistance-associated mutations.18,73
The number of commercially available assays continues to expand,
and scores of "home-brewed" molecular assays continue to develop.
Examples of infections for which the clinical use of molecular diagnostics
is relevant include group A streptococcal pharyngitis,", 36, 79,lo* herpes
simplex virus (HSV) encephaliti~,4~< 58, lo5central nervous system (CNS)
toxoplasmosis, progressive multifocal leukoencephalopathy (PML), hep-

atitis C virus (HCV) infection? 22 pulmonary tuberculosis (TB)," 13, 16, 25, 86,
From the Division of Clinical Microbiology (DW, PSM, RP) and the Division of Infectious
Diseases (RP), Mayo Clinic, Rochester, Minnesota
INFECTIOUS DISEASE CLINICS OF NORTH AMERICA
VOLUME 15 * NUMBER 4 * DECEMBER 2001 1157

95, loo, 117and urogenital infections caused by Chlamydia trachomatis and
Neisseria gonorrkoeae.lo,33, 44,89 Molecular tools are also used for quantifica-
tion of pathogens, such as HIV-1, to monitor therapy, or disease outcome.
In addition, studies of the genomic differences and similarities among
microorganisms of the same type are used to determine the epidemiol-
ogy of disease in public health outbreaks and in nosocomial infec-
t i o n ~ .lo8
~~,
Nucleic acid amplification technologies like PCR are important in
the diagnosis of infectious disease agents, because, in contrast to human
genetic disease (e.g., chromosomal polymorphism) detection where the
amount of DNA is plentiful, the amount of microbial nucleic acid found
in human infections may be limited. PCR was one of the first nucleic
acid amplification techniques to be broadly applied to the molecular
detection of microorganisms, and it is becoming standard methodology
in clinical and research microbiology laboratories. Since the original
description of PCR6* and the first report of Tkermus aquaticus (Taq)
DNA polymeraseg1(the enzyme used for PCR), numerous technological
advances have facilitated the application of this methodology to routine
clinical molecular microbiology diagnostics. In addition, a number of
non-PCR amplification molecular diagnostic methodologies are also
used.
Molecular methods continue to be valuable research tools to identify
new infectious causes of disease and to establish infectious links to acute
or chronic disease states. Adaptations of research methods will continue
to impact testing in clinical laboratories. For example, in the future, gene
expression profile analysis (i.e., mRNA assays) of humans infected with
specific pathogens may be used to help determine the nature of the
infection, or predict response to these pathogens. While the potential
applications of molecular research to clinical microbiology are innumera-
ble, it is important to understand that molecular tests performed in a
research setting differ in both style and substance from their diagnostic
laboratory counterparts.
As technology advances, additional commercial, Food and Drug
Administration (FDA)-approved methods will become available and
eventually enable even the smallest laboratory to apply molecular tech-
nologies to the detection of microorganisms. In the meantime, efforts
should continue to increase understanding of the strengths and limita-
tions of these new methods. Molecular diagnostic tests may enhance
diagnostic capabilities, but they should be interpreted within clinical
context and on the basis of individual laboratory performance. Extensive
clinical research and strict adherence to guidelines for method validation
are necessary to compare new molecular diagnostic techniques with
existing methodologies, to validate new technology when comparable
conventional techniques are unavailable, and to determine a method's
clinical ~ti1ity.l~
Results derived from these studies are vital to the practi-
cal implementation of molecular tests in the clinical laboratory and to the
assessment of their clinical utility and cost-benefit for patient testing.52
Although molecular diagnostic tests have traditionally been more expen-
sive than conventional diagnostic techniques, the ease with which some
PRINCIPLES OF MOLECULAR MICROBIOLOGY TESTING METHODS 1159
molecular tests can now be performed and the rapid results generated
by these methods can provide timely diagnosis and translate into overall
savings. For example, a rapid automated molecular test method may
replace labor-intensive cell culture methods used to detect viruses. Cost
savings may be realized, because rapid diagnosis may prevent invasive
diagnostic procedures (e.g., brain biopsies in patients with HSV encepha-
litis), limit unnecessary or potentially toxic empiric antimicrobial ther-
apy, and shorten hospital stays in expensive isolation rooms (e.g., for
patients with suspected pulmonary TB). Earlier detection of microbes
may also limit the spread of nosocomial and community-acquired infec-
tions.
PROBE HYBRIDIZATION METHODS
Nucleic acid probes are segments of DNA or RNA that can be

labeled with enzymes, antigenic substrates, chemiluminescent moieties,
or radioisotopes, and can bind with high specificity to complementary
sequences of nucleic acid. For many microorganisms, probes alone do
not have optimal sensitivity for direct detection of microorganisms in
clinical specimens. Nucleic acid amplification technology can overcome
this lack of sensitivity.
Oligonucleotide probes (usually defined as probes with fewer than
50 bases) are integral parts of several of the in vitro amplification
techniques to be discussed later. Oligonucleotide probes can, under
stringent reaction conditions, detect the change of a single nucleotide
within a given nucleic acid sequence. These probes can also be used
(in some instances) for direct detection of microorganisms in clinical
specimens or for identification of microbes isolated by culture. Probe-
based commercial kits, such as the PACE2 from Gen-Probe (San Diego),
directly detect C. trachornatis and N.gonorrhoem bacteria present in clini-
cal samples, producing results that are generally equivalent to culture
techniques in much less time. Use of such kits is described e1sewhere.l'
In situ hybridization, common in pathology laboratories, is a tech-
nique that uses intact cells (e.g., in formalin-fixed, paraffin-embedded
tissue) containing specific DNA or RNA as targets for hybridization
with labeled nucleic acid probes. The technique allows for visualization
of infected cells within the structure of the tissue itself and allows for
association of hybridization results with other morphologic changes in
the tissue. The sensitivity of this method may be limited.
QUALITY ISSUES
Molecular techniques developed as research tools have been applied

as "home-brewed" or "in-house" developed assays in clinical microbiol-
ogy laboratories. No universal standards exist for these assays. In 1995,
the National Committee for Clinical Laboratory Standards (NCCLS)
published guidelines for molecular diagnostic methods" and empha-
1160 WOLK et a1
sized that the guidelines did not contain standards, because molecular
methods were quickly evolving. In 2001, guidelines, as opposed to
standards, still appear to be more appropriate. Strategies to improve the
quality and partial standardization of molecular testing include labora-
tory requirements for certifi~ation,'~, 20* 66 proficiency surveys forwarded
and graded by an objective third party, and FDA-approval of commercial
assays. Currently FDA-approved molecular microbiology assays are
listed in Table 1. As with any laboratory assay, not all FDA-approved
molecular assays have identical performance characteristics. Although a
review of the clinical performance of each assay is beyond the scope of
this article, certain universal parameters are applicable, and their defini-
tions are important to understanding and interpreting molecular test re-
sults.
For molecular microbiology testing, validation data should be avail-
able to detail both the analytical and clinical specificity and sensitivity
for every "home-brewed" and commercial assay. For molecular tests,
analytical specificity refers to the ability of the assay to detect only the
organism or analyte it purports to measure. Clinical specificity is the
proportion of specimens from patients who do not have a specified
clinical disorder and whose test results are negative. Analytical sensitivity,
also called lower limit of detection, refers to the lowest number of organ-
isms that can be reproducibly detected by the assay. Clinical sensitivity is
the proportion of specimens from patients who have a specified clinical
disorder and whose test results are positive. Importantly, a molecular
diagnostic assay with a high analytical sensitivity may not provide
adequate clinical sensitivity if false-negative results occur, because the
target nucleic acid copy number in the clinical specimen is low.
For quantitative molecular microbiologic diagnostic testing, several
other terms are relevant. Linear range refers to the quantitative span over
which the assay provides results detecting a direct relationship between
the input target concentration and the output signal. The upper and lower
limits of quantification reflect the upper and lower ends of the linear
range. Importantly, the lower limit of quantification of a quantitative
assay may be higher than the analytical sensitivity of a qualitative assay.
Result interpretation can be confusing. For example, the lower limit of
quantification of the ~ ~ u n t i t u t i vHCV
e RNA assay, the HCV RNA 3.0
bDNA assay (Bayer Corporation, Tarrytown, N.Y.), is 520 IU/mL,
whereas the lower limit of detection of the qualitative HCV RNA assay,
the COBAS AMPLICOR'" HCV Test, version 2.0 (Roche Diagnostics
Corporation, Indianapolis, Ind.), is 50 IU/mL.
In an effort to standardize quantitative testing, laboratory collabora-
tions with the World Health Organization (WHO) have established the
WHO International Standards, standard reference materials with concen-
tration expressed as IU/mL, which can be used to calibrate, validate,
and compare quantitative molecular assays. Three quantitative standards
exist: HCV, hepatitis B virus (HBV), and HIV-1.65,92-94 Additional informa-
tion can be found by contacting the National Institute for Biological
Standards and Controls (NIBSC) at hftp://www.nibsc.ac.uk.
Table 1. EXAMPLES OF COMMERCIAL AMPLIFICATION ASSAYS FOR MOLECULAR DIAGNOSIS OF MICROBES INFECTING HUMANS (UNITED
STATES, 2001)t
Test Manufacturer Application Detection

AMPLICORm and Roche Diagnostic Systems Mycobacterium tuberculosis* Qualitative or quantitative (varies with target)
AMPLICOR http://www.roche.com/diagnostics Chlamydia trachomatis* polymerase chain reaction (PCR)
MONITORm Neisseria gonorrhoeae* Can be automated on the COBAS instrument
HN-1'
Human T cell lymphotrophic virus
types I and I1
Hepatitis B virus (HBV)
Hepatitis C virus (HCV)"
Cytomegalovirus (plasma) (CMV)
Nuclisensm bioMeneux (formerly Organon HIV-1 Qualitative or quantitative (varies with target)
Teknika) CMV 67 mRNA (whole blood)* nucleic acid sequence-based analysis
http://www.nuclisens.corn (NASBA)
AmplifiedTM Gen-Probe M . tuberculosis* Qualitative transcription mediated
http://www.gen-probe.com C. trachomatis* amplification (TMA)
TMAm Bayer HCV Qualitative TMA
http://ww.bayerdiag.com/products/nad/
qualitivehcvrna.htm1
BDProbe Tecm BD Biosciences C. trachomatis" Qualitative strand displacement amplification
http://www.bd.com/biosciences N . gonorrhoeae* (SDA)
Hybrid CapturemTM
I1 Digene Human papilloma virus* Qualitative or quantitative (varies with target)
http://www.digene.com CMV (peripheral white blood cells)* hybrid capture assay
HBV
HN-1
bDNA Bayer HIV-1 Quantitative branched DNA @DNA) assay
http://www.bayerdiag.com/index3.html HBV
HCV
Lcxm Abbott Laboratories C. tmchomatis* Qualitative ligase chain reaction (LCR)
http://www.abbottdiagnostics.com N. gonorrhoeae*
c1 *Culture confirmation assays not listed

tFood and Drug Administration approved as of July, 2001
5
1162 WOLK et a1
Other important components of quantitative molecular assays in-

clude precision, accuracy, and the tolerance limit. Precision refers to the
agreement between replicate measurements of the same material. Accu-
racy refers to the ability of a method to reliably determine the true value
level of the particular target. The tolerance limit is the difference between
two sequential samples that can be significantly different and is the sum
of the biological variation in quantitation and the intra-assay variation
of the assay. For example, for HIV-1 quantitative assays, when biological
and intra-assay variation are considered, increases or decreases in HIV
concentration of at least three-fold typically reflect biologically relevant
changes in the level of viral replication. Of no less importance is the
sensitivity of the assay to detect the target in spite of nucleic acid
sequence variation of the target, which may occur in different strains of
the organism. For example, HIV-1 RNA viral load may not be equally
quantified among all subtypes using certain assays.12, lo3 A thorough
understanding of molecular microbiology assay parameters is important
for interpretation of laboratory results and for comparison of results
generated with different assays.23
For each molecular diagnostic assay, criteria for the appropriate
specimen must be defined. These include the optimal specimen source,
specimen volume, collection method, transport and storage conditions,
and specimen longevity. The sensitivity and specificity of an assay may
vary considerably if any of these conditions are altered. Typically, molec-
ular microbiologic assays are clinically validated using one or more
specimen source(s) (eg., blood or cerebrospinal fluid [CSF]) that may
contain the target organism. The choice of specimen plays a key role in
the performance and interpretation of test results. Even after deciding
that the desired specimen for a given target is blood, there remains a
number of specimen fraction options including plasma, serum, whole
blood, and various leukocyte fractions. As quantitative molecular assays
are becoming more common, careful definition of the optimal specimen
is gaining importance. For example, the clinical significance of viral
loads will vary depending, among other factors, on the specific specimen
assayed. Plasma or serum is preferred over whole blood or leukocytes
when the target microorganism is predominantly extracellular (e.g., HBV
DNA, HCV RNA, and HIV-1 RNA). By contrast, the ideal blood fraction
for molecular assays for quantitation of cytomegalovirus (CMV) in the
blood of transplant recipients has not yet been determined, although
many different fractions have been used. Adding complexity to this
issue, the concentration of target DNA in various blood fractions may
also vary depending on the method of storage and extraction. The
volume of specimen from which nucleic acid is extracted also varies
considerably. Most molecular diagnostic tests use specimen volumes of
200 p.L or less; however, in some cases larger volumes may be required.
This issue is critical when the number of infectious organisms per test
volume is small. All things considered, lack of standardization in speci-
men processing is an important reason why results may be difficult to
compare from laboratory to laboratory.
Just as antibiotic treatment may render a conventional blood culture
falsely negative, inhibitory substances (e.g., bilirubin, hemoglobin, lipids,

heparin), may interfere with various steps in molecular diagnostic
assays. Inhibitors can be present in patient specimens or introduced
during specimen collection or processing. Their interaction with nucleic
acid or critical enzymes, especially DNA polymerases, can prevent am-
plification of the target. Likewise, inhibitors may remove reaction com-
ponents (e.g., metals), which affect enzymatic substrates, resulting in
false-negative results.
The presence of such inhibitors can be determined by several meth-
ods. One approach is to incorporate amplification controls into the PCR
assay design. Spiking the patient’s specimen with known target DNA or
RNA and assaying t h s ”spiked” specimen along with the native patient
specimen ensures that a negative result is truly negative and not the
result of assay inhibition. When spiked specimens are assayed, the
negative predictive value of the assay is enhanced. Another approach
involves the use of internal amplification controls in a single sample.
These controls amplify concurrent to the target nucleic acid and are used
to detect the presence of inhibitors and can be used to evaluate the
quality and quantity of nucleic acid. To assess the adequacy of specimen
collection, human housekeeping genes like human p-globin or interleu-
kin-2 can be included to assess the presence of human cellular DNA. As
an example, if a throat specimen contains human DNA, this may assure
that an adequate specimen was collected.
Specimen preparation techniques are an important facet of the over-
all usefulness of molecular technologies used in clinical microbiology
laboratories. Optimal specimen preparation will efficiently release the
nucleic acid from the microbe, while preserving the integrity of the
nucleic acid target, removing inhibitors, sterilizing the specimen of viable
organisms, concentrating the target nucleic acid into a small volume (if
appropriate), and placing the target into an aqueous environment suit-
able for amplification. Many specimen preparation methods exist, but
may require considerable technical skill, because they are labor-intensive,
non-standardized, and prone to manipulation steps where template
cross-contamination may occur. Specimens are commonly treated with
chaotropic agents, alkaline pH, freeze-thaw cycles, sonication, heat, de-
tergents, and/or proteolytic enzymes to disrupt the cell membranes and
release nucleic acid.’O* Nucleic acid is commonly extracted with organic
solvents, and/or adsorption to silica in the presence of chaotropic agents
such as guanidinium salts, and/or precipitated in the presence of salts
and cold isopropanol. Novel nucleic acid capture technologies include
automated silica-based membrane capture technologies and magnetic
bead capture of nucleic acids.
Automation of specimen preparation is expected to provide rapid,
cost-effective, and consistent results. Results for automated nucleic acid
extraction are promising. Like some manual nucleic acid extraction
methods, however, PCR inhibitors may coextract with nucleic acid and
reduce the clinical and analytical sensitivities of the molecular assays.*”
43, Several new automated extraction instruments are available (see
Table 4).The ability to interface with high throughput PCR systems is an
Table 2. EXAMPLES OF AUTOMATED CLOSED-SYSTEM QUANTITATIVE REAL-TIME PCWHYBRIDIZATION INSTRUMENTS THAT USE FRET*,
TAQMANTM,AND MOLECULAR BEACON PROBES
Reaction
Instrument Source Format Cycling Capacity Comments
GeneAmpa5700 and Applied Biosystems Dehydrated reaction Thermal cycler 96 Run Designed for fixed hybridization temperature
PRISM@ ht tp://www.appliedbiosystems.com components in Potential for at least three to four probe sets
7700 Sequence plastic microplate per reaction
Detection Systems Fiber optic detection by way of CCD
Endpoint analysis of real-time data
Passive internal reference and internal PCR
controls
iCycler iQ System Biorad Plastic microplate or Thermal cycler 9&384/ Hybridization temperature can vary
http://www.bio-rad.comlicycler PCR tubes Run Light intensifier technology and CCDt
Real-time monitoring capabilities
NISTS -traceable temperatures
Gradient heating option
Dual sample blocks for independent runs
Lightcyclerm Roche Molecular Biochemicals Glass reaction tubes Rapid cycling 32/Run Hybridization temperature can vary
http://www.roche.com/diagnostics with ambient Potential for one to two probe sets per
air cooling reaction
Light detected by luminometer
Real-time monitoring capabilities
MX4000TM Stratagene Microplate, eight strip Thermal cycler 96/Run Potential for up to four probes per reaction
Multiplex http://www.stratagene.com/qgcu tubes or PCR tubes Internal control kits available
Quantitative PCR Broad emission detection range
System Molecular beacons only
Rotor Gene Corbett Research Thin-walled plastic Rapid cycling 32-72/ Run Two independent LED§ light sources
http://www/corbet tresearch.com PCR tubes or strip with ambient
Phenix Research Products tubes air cooling
h t tp://www.phenixl.cam
Smartcycler@System Cepheid Proprietary plastic Ceramic heating 1&96 f Run LED light source with silicon photodetectors
ht tp://www.smartcycler.com reaction tubes plate 16 independent &COREm modules per
http:/wwzu.cepheid.com. sample block
Fisher Scientific Up to four targets per reaction
http://www2$shersci. comlmain.j s p
‘FRET = fluorescent resonance energy transfer

tCCD = charge coupled device
$NET = National Institute of Standards and Technology
SLED = Light emitting diode
important advance of many new automated extraction systems. For

example, the Roche MagNA Pure (Roche Molecular Biochemicals, Indi-
anapolis) and the ABI PRISM 6700 instrument (Applied Biosystems,
TM
Foster City, Calif.) integrate with the Lightcycler PCR instrument

TM
(Roche Molecular Biochemicals, Indianapolis) and the ABI PRISM '' 5700
and 7700 (Applied Biosystems, Foster City, Calif.), respectively, and can
be used to automate or partially automate both extraction and PCR
setups (Table 2).
TARGET NUCLEIC ACID AMPLIFICATION METHODS
Polymerase Chain Reaction
PCR is the most widely used nucleic acid target amplification tech-
nology. For PCR, a DNA sequence (template) is amplified in a buffered
reaction solution containing oligonucleotide primers, thermostable DNA
polymerase, deoxynucleotides triphosphates (dNTP), and magnesium or
manganese ions. Other additives, used to enhance amplification, are
common but not essential for the reaction to occur.
The reaction solution is placed into a thermal cycler, which heats
and cools the reaction components, exposing them to consecutive cycles
of varying temperatures. In each temperature cycle, three steps occur:
denaturation (heating to high temperatures to separate DNA into single
strands), primer annealing (lowering the temperature to allow for prim-
ers, synthetic oligonucleotide strands designed with a sequence that is
complementary to the ends of the original target gene sequence, to
anneal to the single stranded DNA and create a partial double strand),
and primer extension (addition of dNTPs to the ends of the bound
primers by DNA polymerase, thereby creating a new synthetic piece of
double-stranded DNA [the amplimer or amplicon], which is complemen-
tary to the original template strand). The annealing and extension steps
can be combined into a single step for some reactions. With the exception
of the first cycle, the number of amplicons will theoretically double with
each cycle (Fig. l),resulting in an exponential increase in the quantity
of amplicon, until the reaction reaches a plateau phase caused by deple-
tion of reaction components. After a 36-cycle amplification, amplicon is
present in the reaction tube at a theoretical concentration of approxi-
mately 6.9 X 1O1O copies per each original template (Fig. 1). The exact
concentration of amplicon is lower when the reaction efficiency is less
than 100%.
Traditionally, amplicons have been detected and identified by a
variety of methods. Many of these methods are manual, require subse-
quent manipulation of the PCR amplicon, and add substantial risk of
contamination to subsequent PCR reactions. One of the first methods
developed, gel electrophoresis, uses electrical current in an agarose or
polyacrylamide matrix to separate DNA fragments by size. Visualization
of DNA fragments is possible by staining the gel fragments with DNA
binding dyes (e.g., ethidium bromide). Using this methodology, ampli-
1166 WOLK et a1
5’ 3’
Double-stranded DNA template (target)
3, 5(
1 Denaturation
Denatured DNA template
.-
-
~
-
1
a,
0,
0
Pnrner annealing
3*=5< -
1
5-= 3,
1
Recycle target
Pnmerextension
(polyrnenzation)
-I
IDenaturation and pnrner anneal1
D
-- 1 to vanable lengthsfrands
Key
DForward primer
Reverse primer
Ccm 1st Strands

r-- synthesized
1 Pnrner extension
(polyrnenzation)
xca 2nd Strands
synthesized
5 Additional strands
synthesized
I Denaturation pnrner a n n e r l
I
I
-a,
Ln
Exponentialarnpbhcabon of arnplicon
(repmduction of target)
6
-
m
- I
0
I
.o
U
Q
I I
Figure 1. Polymerase chain reaction (PCR). A DNA sequence (template) is amplified in

a buffered reaction solution containing oligonucleotide primers, thermostable Ta9 DNA
polymerase, deoxynucleotide triphosphates (dNTPs), and magnesium or manganese ions.
The reaction solution is placed into a thermal cycler, which heats and cools the reaction
components, exposing them to consecutive cycles of varying temperatures. In each temper-
ature cycle, three steps occur: denaturation, primer annealing, and primer extension (see
text). The theoretical amount of product that accumulates is as follows: N (total) = N (initial)
2 , where “X’ is the number of cycles. The actual amount of product that accumulates is
as follows: N (total) = N (initial)(l +Y)”, where “Y” is the efficiency per cycle. Amplicon
can be detected by a variety of methods (see text).
fied DNA fragments of the expected size are assumed to be amplified

target Iol While gel electrophoresis is used in many research-
based PCR assays, the authors believe that the use of gel electrophoresis
alone does not permit the sensitivity or specificity necessary for clinical
microbiology diagnostic testing.
Several methods were subsequently developed to provide more
specific and sensitive detection of PCR amplicon. For instance, restriction
of enzyme digestion of amplicon can improve the specificity of gel
electrophoresis results, because restriction enzymes (enzymes that bind
to DNA at specific sites and cleave it into fragments) can be chosen so
that a unique pattern of DNA fragments results when gel electrophoresis
is performed. PCR products can also be subjected to a Southern blot
procedure followed by probe hybridization. In this procedure, DNA
from a gel is transferred (blotted) to a membrane. Then labeled oligonu-
cleotide probes, which are complementary to an internal region of the
target sequence, are hybridized (annealed) to the DNA on the membrane
(Fig. 2). Probes are detected by a variety of methods, including autoradi-
ography and chemiluminescence.lO'Another option for detection is in-
corporation of various chemicals (e.g., digoxigenen-11-dUTP) into the
amplicon during PCR.96Products are blotted and detected on the mem-
Molecular
marker
4
+ -b -b -b
Electrophoresis Place the gel on Hybridize specific

separates polymerase the membrane labeled probe to
chain reaction Transfer DNA to designated target
products according membrane region Visualize
to size alone (Lane 2) or label on membrane
size of restriction
enzyme digest
fragments(Lane 3)
I Stained DNA fragments on agarose gel
mms DNA on membrane

Hybridized probe sfgnai
Figure 2. Southern blot. Nucleic acids can be transferred from an electrophoresis gel to a
membrane by a variety of transfer methods (capillary, vacuum, electrophoresis). In this
depiction of Southern blotting by downward transfer, nucleic acid is transferred from the
gel to create a replica of the gel pattern on the membrane. Nucleic acid is linked to the
membrane, usually with ultraviolet light. The membrane is placed in a series of liquid
solutions, during which time labeled oligonucleotide probes, specific to a predetermined
target region, bind to the target on the membrane. Targetprobe complexes are visualized
by a variety of mechanisms, usually enzymatic, autoradiographic, or chemiluminescent.
1168 WOLK et a1
brane by using a second chemical reaction (e.g., anti-digoxigenin antibody:

alkaline phosphatase conjugate and a chemiluminescent
Several other variations of Southern blot technology are useful in
certain circumstancesbut are not as specific as traditional Southern blots,
because they do not provide information about the size of the target
DNA. Slot, dot, spot, and reverse blots involve application of target
nucleic acid sequences (amplicons) to the membrane without an electrc-
phoresis step. Slot, dot, and spot blots apply target DNA to a defined
area on a membrane; labeled probes are then hybridized to membrane-
bound DNA. Reverse dot blot, in contrast, uses membrane-bound probes
to hybridize targets in solution.
Besides membrane-based amplicon detection methods, a variety of
other options are possible. Probes can be bound to microtiter plate wells
or beads to capture amplicon. A specific "signal" probe may be added
to react with the captured target nucleic acid. In another variation,
PCR-enzyme-linked immunosorbent assay (PCR ELISA [DIG Detection],
Roche Diagnostics Corporation, Indianapolis), amplicon is hybridized to
oligonucleotide capture probes labeled with biotin. Biotinylated DNA
hybrids are then bound to a streptavidin-coated microtiter plate. Anti-
dioxigenin-peroxidase conjugate binds to the bound DNA hybrids, and
a color change is produced upon addition of 2.2-azino-bis (3-ethyl-benz-
thiazoline-6-sulfonic acid).
Solution (liquid) phase hybridization is another option. One exam-
ple of a solution hybridization reaction is the hybridization protection
assay (HPA [Gen-Probe, San Diego]). In the HPA assay, chemilumines-
cent probes hybridize to original target nucleic acid or to amplicons,
which have been produced by a previous amplification method. Then,
after chemical destruction of the non-hybridized probes, the chemilumi-
nescent label, "protected" within the hybrid, reacts under specific condi-
tions to produce light, which can be measured by a luminometer.
Non-hybridization methods may also be used to separate, quantify
or identify PCR amplicon. Examples include high-performance liquid
chromatography, mass spectrometry, and DNA sequencing method^.^, 59,
Recently described real-time PCR methods incorporate probe detec.
tion methods to provide homogeneous assays that perform amplificatior
and detection within one closed system.* These PCR-probe detectior
methods avoid the postamplification manipulation inherent to tradi.
tional methods and are described in detail in the subsequent discussions
Engineering and Operational Controls for

Amplification Technologies
Despite significant advances in technology, many challenges mus

be addressed to facilitate the widespread use of in vitro nucleic acic
amplification techniques. One of the most difficult challenges for clinica
laboratories has been prevention of nucleic acid contamination of othei
*References: 1,27,40,41,46,55,67,71,77,81,113
molecular assays performed in the same laboratory.6,50, 57, 74, 98 Sources of

contamination include crossover contamination from specimens con-
taining large numbers of target molecules or, most importantly, crossover
from manipulation of amplified nucleic acid. A positive PCR reaction,
for example, may contain as many as a trillion copies of an amplified
target sequence, each of which may serve as a substrate for further
amplification. Amplified nucleic acid may contaminate reagents, labora-
tory surfaces, ventilation ducts, and the skin, hair, and clothing of labo-
ratory personnel.
Specially designed molecular microbiology laboratories provide
physical separation of, and specially designed ventilation systems for,
preamplification and postamplification areas. Each phase of the testing
process, including the movement of patient specimens and personnel,
proceeds from preamplification to postamplification areas.57The Mayo
Molecular Microbiology Laboratory (Rochester, Minn.), for example, has
separate rooms for reagent preparation, specimen processing, and ampli-
fication and detection. Each room is designed to isolate potentially
contaminating aerosols by using a combination of controlled airflow,
two-door vestibule-type entrances with magnetic interlocks (permitting
only one door of the vestibule to be opened at a time), and ultraviolet
light fixtures (operated nightly) in the overhead lighting. The reagent
preparation and specimen processing rooms consist of positive-pressure
high-efficiency particulate air (HEPA)-filtered inner working areas con-
nected to a positive pressure hallway by negative-pressure vestibule
anterooms, creating net air flow into the vestibules (and exhausted to
the outside air). This prevents potentially contaminated aerosols within
the laboratory from escaping into the hallway and prevents aerosols
from the hallway from entering the inner laboratory. The amplification
and detection rooms, by contrast, are under negative pressure and are
exhausted from the building without being recirculated, a system analo-
gous to that found in TB isolation rooms. Various other physical contami-
nation control measures can be applied and are reviewed elsewhere.74,99
Despite the investment of these specifically constructed laboratories,
contamination events have still occurred.
Contamination control measures vary and include chemical, enzy-
matic (e.g., uracil-N-glycosylase [UNG]),or photochemical (e.g., isopsor-
alen) methods of inactivating PCR am~licon.*~, 51, 74 For enzymatic inacti-
vation using UNG, dUTP is incorporated into the PCR amplicon, in lieu
of dTTP, so that amplified target will contain dUTP, and native target
will contain dTTP. Any dUTP-containing amplification products, present
as contaminants, are cleaved by UNG prior to amplification in subse-
quent reactions (preamplification control). For photochemical inactiva-
tion, amplicons are generated in the presence of isopsoralen. Inactivation
occurs as postamplification irradiation of the reaction vessel with ultravi-
olet light causes the isopsoralen to modify the amplified DNA (by
forming cyclobutane monoadducts with pyrimidine bases) prior to open-
ing the reaction vessel. Modified DNA is unable to serve as a template
for further amplification, but is still capable of hybridization for use in
1170 WOLK et a1
various detection formats.51,99 Closed-system PCR reactions can also be

used to prevent contamination.
In laboratories where contamination prevention methods are used,
the demands for physical separation of preamplification and postampli-
fication areas may be reduced, but good laboratory practices still apply.
Closed systems and amplicon inactivation methods reduce contami-
nation risks, but concentrated template in clinical samples remains a
potential source of contamination for most molecular diagnostic tech-
niques. It is imperative that all PCR testing be designed to include a
sufficient number of negative controls (up to 10% to 20% of all sam-
ples),2O, e ~ 65,
, ln9 which are processed along with patient specimens
and positive controls, to provide a measure of assurance that no PCR
Contamination is occurring in the clinical laboratory. For each assay, a
low copy number positive control and reagent control should also be
included.11,20, 65. 109
Modifications of PCR
Several modifications of conventional PCR exist and require defini-

tion. For multiplex PCR, two or more primer sets are used in the same
reaction mix. Multiplex PCR can be used to identify multiple microor-
, 72, 8o or multiple nucleic acid targets within one microorgan-
g a n i s m ~30,~32,
ism, in a single reaction mix. For nested PCR reactions to occur, one set
of primers is used initially. Then products of the first reaction are
amplified with fresh reaction components (in most cases) and a second
set of primers, internal to the first. Nested PCR can be likened to broth
enrichment techniques used to allow certain microorganisms to grow
prior to agar subculture and thereby increase the possibility of detection.
Although nested PCR may sometimes be more sensitive than non-
nested PCR, the nested method is prone to contamination by aerosolized
amplified DNA. Because amplicon is physically transferred to a fresh
reaction vessel and/or exposed to a second set of primers and fresh
reaction components prior to the second (nested) PCR step, amplicon
contamination of subsequent reactions can easily occur. The product of
the first reaction cannot be inactivated, because its activity as a template
must remain intact for the nested protocol to function. Nested PCR is
not recommended for use in clinical microbiology laboratories, unless
the reaction is modified for performance in a single vessel, which re-
mains closed throughout the amplification. A one-vessel nested ap-
proach can be accomplished by using physical separation of the two
reactions in the same vessel or by using different temperatures for
annealing the external and internal primer pairs. In sifu PCR is a general
term used to describe the amplification of DNA (or cDNA) inside an
intact cell (e.g., in a paraffin-embedded tissue section), and provides a
means of determining the disposition of a microbe within tissue. Reverse
transcriptuse-PCR (RT-PCR) is used to detect RNA, either because an
infectious agent’s nucleic acid is naturally present as RNA and not DNA
(e.g., HCV), or to detect transcribed microbial DNA as a marker of active

infection. In RT-PCR, an enzyme with reverse transcriptase activity is
used to create complementary DNA (cDNA) from an RNA target. The
cDNA is then amplified by PCR.
Various other modifications of PCR are used in clinical laboratories.
Broad-range PCR involves the use of PCR primers targeted to highly
conserved regions of DNA (e.g., 16s ribosomal DNA [rDNA]) so that,
depending on the primer sets used, the target can be amplified from
most bacteria. Broad-range PCR has been used in the research setting to
identify unknown pathogens (e.g., Tropheryma whippelii, the Whipple’s
disease-associated bacteria) and has applications in the clinical microbi-
ology laboratory.69,84 For example, the Applied Biosystems MicroSeqB
16s rDNA Bacterial Sequencing kit (Applied Biosystems, Foster City,
Calif.) is used to amplify 16s rDNA of bacteria isolated by culture.
Subsequent sequencing of the amplicon may be used for species-level
identification and is described in subsequent sections. Direct sequencing
of amplified 16s rDNA derived from patient specimens is also a possibil-
ity, although sensitivity and specificity issues need to be addressed prior
to application of this approach outside the research setting.
Quantitative PCR may be used to assess the amount of a microbe
present in a patient specimen70and can be performed in a number of
ways. Semi-quantitative or limiting dilution PCR, the most basic of
methods, involves titer determinations of the target template by end-
point specimen dilution prior to PCR. For more advanced, truly quanti-
tative molecular assays, an internal or external quantitative standard (or
calibrator) is incorporated. For quantitative competitive PCR, an internal
quantitative standard (or calibrator) is included in the amplification
reaction, along with the target sequences and competes with target
sequences in the PCR reaction; therefore, standard curves of the competi-
tor molecule correlate in inverse proportion to the concentration of
target. An example is the AMPLICOR HIV-1 MONITORTM Test (Roche
Diagnostics Corporation, Indianapolis), wherein the internal quantitative
standard is a constructed segment of nucleic acid that uses the same
primer binding sites as the target molecule, but has an internal sequence
that differs from that of the target sequence and therefore allows differ-
entiation from the target sequence. Current applications of quantitative
PCR testing include measuring of viral load for pathogens such as HIV-
1, CMV, HBV, HCV, and Epstein-Barr virus (EBV). Quantitative results
may be important for guiding therapy.
Rapid Real-Time Polymerase Chain Reaction
A significant advance in PCR technology is rapid PCR with simulta-

neous amplicon detection, rapid real-time PCR. In real-time PCR assays,
amplification and product detection are carried out in the same sealed
reaction vessel, tube, or well. A fluorescent signal is used for real-time
monitoring of PCR amplicon production.
1172 WOLK et a1
Closed PCR systems provide the single most important advance in

PCR technology of the last decade, because, by design, they substantially
lessen the risk of PCR amplicon contamination by avoiding postamplifi-
cation manipulation. These systems provide the opportunity for labora-
tories to perform PCR and amplicon detection in a simple and auto-
mated manner without the need for extensively engineered laboratory
space. Nevertheless, even with closed-system, real-time PCR procedures,
careful attention to unidirectional workflow, amplicon control, and am-
plicon inactivation protocols are favored, because template carryover
contamination can still occur, and liberation of amplicon through broken
reaction vessels can still lead to contamination events.
Another important advance is that many real-time PCR systems
have incorporated technology that enables a dramatic reduction in the
time required for assay performance. For example, real-time PCR per-
formed using the Lightcycler instrument can be completed in 30 to 40
TM
minutes (as opposed to several hours to days for conventional PCR and
postamplification amplicon detection).
Several fluorescent chemistry approaches can be used to detect PCR
amplicon in real-time. Real-time monitoring of non-specific fluorescent
dye incorporation (e.g., SYBR-Green I, Molecular Probes, Eugene, Ore.)
can be 38 Such nonspecific dyes bind to double-stranded DNA
generated during PCR and emit fluorescence, which can be monitored
by various detection systems. Because the dyes bind to all nonspecific
amplification products produced in the reaction, dye incorporation alone
does not ensure detection of a specific PCR product.
To improve specificity, melting curves can be performed. The ampli-
fied product will have a characteristic melting temperature, the se-
quence-dependent temperature at which half the strands have become
separated in solution, differentiating it from nonspecific amplification
products. To further ensure more specific hybridization and product
identification, protocols that incorporate specific internal probes are used
with or without melting temperature measurements. Several internal
probe formats exist, including hybridization probes63like fluorescence
resonance energy transfer (FRET) probes,14 hydrolysis which
use TaqMan (Applied Biosystems, Foster City, Calif .)
TM and
molecular beacon probes.110, 'I1
Hybridization probes use FRET technology, which is based on the
coupling of fluorescent dye pairs that have overlapping absorption and
emission spectra. Two probes designed to recognize single strands of the
amplicon are used, one labeled with a donor dye and the other with a
reporter dye. As DNA is amplified, probes hybridize to amplified DNA
during the annealing steps, and multicolor fluorescent dyes are brought
close to each other so that energy can be transferred between dyes. PCR
products are detected by monitoring the fluorescence produced when
energy transfers occur between these closely opposed fluorescent dyes
on stimulation from an external light source (Fig. 3).
Using FRET-based probe pairs, melting curve analysis can be per-
formed on amplicon by monitoring the melting temperature of the
probe from the amplicon. This permits specific identification of the
amplicon. After PCR is performed, the amplicon is heated to its dena-
Figure 3. Fluorescent resonance energy transfer (FRET). Two oligonucleotide probes (one
coupled to a fluorescent donor dye and the other to a fluorescent reporter dye) hybridize
close to one another (usually one to five nucleotides apart) on a target DNA strand at a
location internal to the PCR primer sites. An external light source stimulates the donor dye,
and energy, emitted from the fluorescent donor dye, excites the fluorescent reporter dye to
emit a specific wavelength of light.
turation temperature, and diminished fluorescent reporter signal is

observed. Because mismatches, such as point mutations or polymor-
phisms, combine with other amplicon characteristics, such as fragment
size and percentage of guanine and cystine (GC) content, to affect the
melting temperature of the probe:DNA hybrid, melting temperature
curves can be used to distinguish different PCR products with as few
as one base pair difference.78,87, 116
TaqManTMchemistry combines DNA polymerase with 5’ exo-
nuclease activity39,48 and a linear oligonucleotide hydrolysis probe car-
rying both a fluorescent reporter dye and a quencher molecule. The
hybridization probe binds to the denatured amplified target DNA
strands. Fluorescence of the reporter dye is prevented (quenched) by its
proximity to the quencher molecule. After each annealing cycle, DNA
polymerase, with 5’ exonuclease activity, will extend the specific target
strand and cleave the probe, which is specifically bound to the strand.
The exonuclease activity fragments the probe, and the quencher mole-
cule is separated from the reporter dye. The cleaved fluorescent reporter
dye will fluoresce when stimulated from an external light source. If
specific amplification of target DNA has occurred with cleavage of the
probe, reporter dye fluorescence will increase and accumulate with each
PCR cycle (Fig. 4).39,48
Molecular beacons are oligonucleotide probes produced in the shape
of a hairpin (rather than a traditional linear probe conformation). The
probes contain a fluorescent reporter dye and a quencher molecule. An
external light source stimulates the dyes, and fluorescence is measured
1174 WOLK et a1
Figure 4. TaqMann (Applied Biosystems, Foster City, Caif.) chemistry. Hybridization probes,
labeled with a fluorescent reporter dye and a quencher molecule, are bound to the dena-
tured target DNA strand. As PCR occurs and the opposing strand is generated, the 5'
exonuclease activity of DNA polymerase will hydrolyze the probe, removing the reporter
dye from its proximity to the quencher molecule and allowing measurable fluorescent signal
to accumulate on stimulation from an external light source.
in the annealing step when the molecular beacon is bound to the tar-
get.*1°While the probes are in a hairpin formation, the reporter dye and
quencher molecules are proximal and fluorescence is quenched. When
the probes bind to their targets in the PCR product, the hairpin structure
is linearized, and the reporter dye is separated from the quencher mole-
cule so that fluorescence is no longer quenched (Fig. 5).111
TaqMan chemistry, FRET, and molecular beacon technology can
TM
be used to monitor real-time endpoint (qualitative) PCR and real-time

quantitative PCR reactions. The amount of microbe-specific nucleic acid
in patient specimens is determined by comparing (using mathematical
algorithms) the exact cycle number at which target versus internal or
external quantitation standard amplification signal are detected above a
baseline or threshold value. Notably, real-time quantitative PCR assays
potentially offer wider linear ranges than conventional quantitative
PCR assays.
Several automated real-time PCR instruments that use the aforemen-
tioned chemistries are available. Real-time PCR assays are run on highly
specialized automated instruments that include optical systems to excite
the fluorescent dyes and detect fluorescent emissions at a variety of
wavelengths specific to the fluorescent dyes used. These systems have
Figure 5. Molecular beacons. Molecular beacons are single-stranded oligonucleotide

probes that are created using a specific probe, which is flanked by two inverted terminal
repeat sequences so that a stem-loop structure is formed. As the molecular beacon probe
opens and binds to a denatured DNA template, a quencher molecule (located on one arm)
is removed from the proximity of a fluorescent reporter dye (located on the opposing arm),
allowing the reporter dye to fluoresce on stimulation from an external light source.
1176 WOLK et a1
the potential to speed the results of PCR reactions and improve the
ability to multiplex PCR. Using different fluorescent dyes, multiple tar-
gets can be detected and distinguished in a single reaction. To date, no
commercial FDA-approved real-time test methods exist. The specifics of
available real-time PCR instruments are summarized in Table 2.
Non-PCR Target Nucleic Acid Amplification Methods
Several non-PCR target amplification methods exist, including tran-

scription-mediated amplification (TMA), nucleic acid sequence-based
amplification (NASBA), and strand displacement amplification
(SDA).8s,114 NASBA and TMA are isothermal (i.e., require no changes in
the temperature of incubation) amplification methods wherein RNA
target is reverse transcribed into cDNA, and then RNA copies are synthe-
sized using an RNA polymerase (Fig. 6). Modifications of TMA also
exist for its use with DNA targets. Gen-Probe Inc. (San Diego), currently
offers a TMA test for detection of Mycobacteriurn tubercuIosis, C. tvacho-
matis, and N. gonorrhoeae. A TMA assay for qualitative detection of HCV
is available from Bayer Corporation (Tarrytown, N.Y.). Bio Merieux
(Hazelwood, Mo.) offers NASBA testing for quantitative detection of
HIV-1 RNA and qualitative detection of CMV pp67 RNA.
SDA is an isothermal amplification technique that is based on the
ability of a restriction enzyme to generate a single-stranded site-specific
"nick in double-stranded DNA and the ability of a (5' to 3' exonuclease-
deficient) DNA polymerase to initiate replication at the nick and displace
the downstream non-template strand." Primers used in SDA contain
both a target-specific region and a recognition site for a restriction
enzyme. The restriction enzyme site is hemimodified, so that the restric-
tion enzyme does not produce a nick in one strand. Once the nick is
created by the restriction enzyme, the 3' end formed at the site of the
nick initiates DNA synthesis, thereby displacing the newly created single
strand. Each displaced strand is then available to anneal with amplifica-
tion primers. The process continues with repeated nicking, extension,
and displacement of new DNA strands, resulting in exponential ampli-
fication of the original DNA target (Fig. 7).
SIGNAL AMPLIFICATION METHODS
An alternative to enzymatic duplication of target nucleic acid is to

increase the signal generated from the probe molecule hybridized to the
target nucleic acid. This is referred to as signal amplification. Commonly
used signal amplification technologies include branched DNA @DNA)
and hybrid capture assays.
Quantitative bDNA assays are commercially available from Bayer
for HBV, HCV, and HIV-1. With bDNA, organisms are disrupted, and
Text continued on page 1181
Figure 6. Transcription-based amplification systems [nucleic acid sequence-based amplifi-

cation (NASBA) and transcription-mediated amplification (TMA)]. NASBA and TMA are
similar isothermal reactions that use an RNA target’ as the substrate for the enzyme
reverse transcriptase, which creates a complementary DNA strand (cDNA) from the RNA
template. RNA from the resulting RNA:DNA hybrid is degraded, reverse transcriptase
creates double-stranded DNA, and the DNA is used as a template for RNA polymerase,
so that multiple RNA copies of the DNA are produced. Results can be qualitative or
quantitative. NASBA uses avian myoblastosis virus reverse transcriptase, RNase H (an
enzyme that destroys RNA from RNADNA duplexes), and T, RNA polymerase. TMA
use a reverse transcriptase enzyme with endogenous RNase H (in addition to RNA
polymerase) activity.
*Modifications can be performed to allow the use of a DNA target.

CI
5 Table 3. EXAMPLES OF COMMERCIAL METHODS FOR DNA SEQUENCING
~ ~~~
Sequencer Manufacturer/Supplier Type Throughput Detection Comments

Gel-Based
ABI PRISM" 377 Applied Biosystems Gel-based sequencing 18-96 Lanes Argon ion laser Dye primer cycle
System http://wuw.appliedbios?lstems.com with fluorescent Analysis of DNA sequencing
detection molecules labeled Dye terminator cycle
with multiple sequencing
fluorescent dyes ABI PRISM" sequence
analysis
Software for base-calling
LI-COR-NEN" Li-COR Gel-based sequencing 61-96 Lanes Near infrared-producing Infrared dye primer
Global IR2 http://wuw.licor.
corn laser diodes cycle sequencing
Detection with scanning Infrared dye terminator
fluorescent microscope cycle sequencing
Long base sequence
capacity
Open-Genem Visible Genetics Gel-based sequencing 16 Lane Fluorescent detection of Dye primer chemistry
Sequencing http://www.visgen.com with fluorescent MicroCelm cassette tagged nucleic acids HIV protease inhibitor
System detection throughput is and reverse
SureFilP cartridge assay dependent transcriptase
containing database; proprietary
premixed interpretation codes
acrylamide provided
Capillary-Based
ABI PRISM@310 Applied Biosystems Single channel 1 Capillary Argon lasers Dye primer cycle
Genetic corn
http://www.appliedbiosystems. capillary Analysis of DNA sequencing
Analyzer POP* separation molecules labeled Dye terminator cycle
matrix with multiple sequencing
fluorescent dyes ABI PRISM@sequence
analysis
48-hour unattended
operation
Genescane analysis
software for fragment
sizing validation/
detection
ABI PRISM" Applied Biosystems 16 Capillary FOP" 16 Capillaries Argon lasers Dye primer cycle
3100 http:www.appliedbiosystems.com separation matrix Analysis of DNA sequencing
Genetic molecules labeled Dye terminator cycle
Analyzer with multiple sequencing
fluorescent dyes ABI PRISMa sequence
analysis
24-Hour unattended
operation
Genescan" analysis
sizing validation/
detection
ABI PRISM@ Applied Biosystems 96 Capillary POP 96 Capillaries Argon lasers Dye primer cycle
3700 DNA http:www.appJiedsystems.com separation matrix Analysis of DNA sequencing
Analyzer molecules labeled Dye termination cycle
with multiple sequencing
fluorescent dyes ABI PRISM" sequence
analysis
24Hour unattended
operation
Genescane analysis
sizing validation/
detection
MEGABASETM Arnersham Pharmacia Biotech l'olyacrylamide Confocal optics Dye primer cycle
100 http://www.apbiotech.corn matrix sequencing
Dye terminator cycle
sequencing patented
energy transfer (ET)
dye technology
CEQm 2000 Beckman Coulter Pre-mixed linear Eight capillaries in Diode laser Dye terminator cycle
http://www.beckman.com polyacrylamide gel cartridges WellRED dyes fluoresce sequencing
in the red spectral
region
Other
+ PSQm96 Pyrosequencing (Fig. 18) Sequencing by 96 Well plates Visible light Pyrosequencing
c1
J
. System http://www.pyrosequencing.com monitoring chemistry
\o synthesis Short sequences only
'Performance ophmized polymei

1180 WOLK et a1
Figure 7. Strand displacement amplification (SDA). SDA is an isothermal amplification

technique in which primers, containing both a target-specific region and a hemimodified
restriction enzyme site, combine with a DNA polymerase to create a synthetic double-
stranded DNA that contains the restriction enzyme site. A restriction enzyme generates a
site-specific “nick” in one of the two strands of double-stranded DNA. Once the strand is
nicked, DNA polymerase initiates synthesis at the 3‘ end of the nick site to create a new
DNA strand. Because the DNA polymerase used is deficient in 5’ to 3’ exonuclease activity,
it does not destroy the existing strand as it polymerizes the new strand. It simply displaces
the downstream non-template strand. Each displaced strand is then available to anneal
with more primers, and the process continues with repeated nicking, extension, and dis-
placement of newly formed DNA strands. The entire process results in exponential amplifi-
cation of the original DNA target.
nucleic acid is released and captured to a solid surface by multiple

capture probes.1o5Target probes hybridize to both the microbial nucleic
acid and signal amplification multimer (branched DNA or bDNA). Fi-
nally, enzyme-labeled probes hybridize with bDNA structures. Detection
occurs by way of a chemiluminescent process (Fig. 8). The amount of
signal generated is directly related to the amount of target nucleic acid
present in the specimen.
In the hybrid capture assay, an RNA or DNA probe hybridizes to a
DNA or RNA target, respectively. Hybrids are captured on a solid phase
and then coated with capture antibodies specific for RNA:DNA hybrids
(duplexes). Captured hybrids are detected using chemiluminescence
(Fig. 9). DiGene Corporation (Silver Spring, MD.) is a commercial sup-
plier for the hybrid capture assay. Assays using this method are available
for human papilloma virus (HPV), HIV-1, CMV, C. trackomatis, N. gonor-
rkoeae, and HBV.
Capture
probes
(microwell)
lrwTTn
Solid surface
(microwell)
I I
Capture
probes
'11
Capture
probes
ff
Target
probes
I
Signal
amplification
cc
cc
cc
EE
Enzyme-
labeled
+ Denatured target
DNA or RNA
(soiution) multimer probes
(branched DNA)
I Hybridtzatron of components
argel DNA or RNA
1 Substrate
Chemrlumrnescence
Figure 8. Branched DNA (bDNA) signal amplification. DNA or RNA is released from the
target organism, and hybridization of multiple probes occurs. Target nucleic acid is captured
by multiple capture probes to a solid surface. Target probes hybridize to both target nucleic
acid and the signal amplification multimer (bDNA). Enzyme-labeled probes are bound to
the branched DNA, and detection occurs by way of a chemiluminescent process in which
light is emitted in proportion to the original amount of target present.
1182 WOLK et a1
Figure 9. Hybrid capture. Single-stranded RNA or DNA probes are added to denatured
target DNA or RNA, respectively. DNA:RNA hybrids are formed and captured to a solid
support with antibody specific to the hybrids. Each hybrid can also bind multiple copies of
specific antibody, which is coupled to an enzyme. Captured hybrids are detected using
chemiluminescence.
PROBE AMPLIFICATION METHODS
Probe amplification technologies, wherein the end product of the

reaction is an amplified version of the original components used to
detect the target, include the Zigase chain reaction (LCR) (Abbott Labora-
tories, Abbott Park, Ill.), Q-beta replicase amplification (Gene-Trak System,
Framington, Mass.), Invader TM technology (ThirdWave Technologies, Inc.,
Madison, Wis.) and Cycling Probe Technologym (Velogene, ID Biomedical,
Vancouver, BC, Canada). The last three technologies will be described
later in the article.
In LCR, DNA template is denatured and subsequently hybridized
to probes that are located next to each other on the template.lo6Successful
ligation of the probes depends on the proximal positioning and perfect
base pairing of the 3’ end of one probe with the 5’ end of the other to
complete a complementary DNA strand (Fig. 10). Once the probes have
been ligated, the ligation product, which now mimics one strand of the
original target sequence, can serve as a template for ligation of more
probes. Accumulated ligation products are detected using labeled
probes. LCR-based assays are available for detection of C. trachomatis
and N . gonorrhoeae (Abbott Laboratories, Abbott Park, Ill.).
SEQUENCE-BASED ANALYSIS
D N A sequence analysis has several applications in the clinical micro-

biology laboratory. For example, the technology can be used to deter-
mine HCV, HIV-1, HBV, and HPV genotypes; to determine the DNA
sequences of bacteria and fungi for microbial identification post-culture,
and to identify mutations associated with resistance to antimicrobial
agents.’15
A variety of DNA sequencing methodologies are available. Most
are based on a modification of Sanger dideoxynucleotide triphosphate
(ddNTP) chain termination chemistry in which synthetic ddNTP mole-
cules that lack 3’ hydroxyl groups normally present in the natural
molecules are added to a DNA synthesis reaction?’ Whenever a ddNTP
molecule is incorporated into the growing DNA strand in lieu of the
usual dNTPs, the synthesis reaction is terminated. For dye-terminator
cycle sequencing, four different fluorescent dyes are incorporated into
each of the four ddNTPs (ddATP, ddCTP, ddGTP, and ddTTP). The end
result is a collection of DNA fragments of different lengths, labeled with
different dyes depending on the terminal nucleotide (Fig. 11).Reaction
end products are separated by electrophoresis and excited and detected
by a laser. The resulting digital data are processed into electronic DNA
sequencing data. Sequence data are analyzed using special software and
displayed either as a linear string of one-letter nucleotide codes or as a
graphic presentation (electropherogram).
The generation of large numbers of DNA sequences has created
new bioinformatic needs for data archiving and updated, validated
1184 WOLK et a1
Figure 10. Ligase chain reaction (LCR). DNA template is denatured and subsequently
hybridized to probe sets that are designed to hybridize adjacent to each other on the target
strand. The hybridized probes are ligated (joined) by DNA ligase to form a ligation product,
which mimics one strand of original target sequence and can serve as a template for
ligation of more probes. In this figure, which reflects the commercially available LCR
method, a small gap, usually one to three nucleotides, remains after the probe sets
hybridize to target, preventing ligation in the absence of specific target. DNA polymerase
and deoxynucleotides are used to extend the probe and fill the gap region prior to ligation
with DNA ligase. Detection occurs as labeled strands are captured by antibody molecules
bound to microparticles. Enzyme-labeled antibodies are bound and catalyze an enzymatic
reaction to generate a signal.
Sequencing primer Sequence lo be determined

\ 1
/ -
ATTAGACGGA
Sequencing
CTA -TAATCTGCCTAGCTCC
Drirner bindinq site
I
r I I I
ddATPI* ddTTPO* ddGTPB* ddCTPeZa*
+ + +
+
dATP dTTP
+ +
dGTP dCTP
+
Fragments generated by
dye terminalor chemistry
{ A reaction
ATTAGA I
ATTAGACGGA I
T reaction
AT 0
ATT 0
G reaction
ATTAG
ATTAGACG
ATTAGACGG
C reaction
ATTAGAC eZa
Key
I Fluorescent dye # I labeling ddATP Sequence read from sequencing gel
0 Fluorescenl dye if2 labeling ddTTP
Fluorescent dye 113 labeling ddGTP
ezd Fluorescent dye 114 labeling ddCTP
* Fluorescent dye labeled ddNTPs ale

present ~nsmaller ~ o n ~ e n t i a l i ~ n ~
than dNTPS
gel
Electropherogram
A T T A G A C G 6 A
Figure 11. Dye-terminator cycle sequencing of DNA. PCR amplicon or cloned nucleic acid
sequences are purified and placed in a reaction mix with a primer designed to enable
synthesis of the DNA sequence of interest. The primer has one end that is fixed and cannot
support synthesis and another end onto which synthesis of a new strand will occur;
therefore, synthesis will occur in only one chosen direction. Four different fluorescent dyes
are incorporated into each of four dideoxynucleotide triphosphates (ddNTPs), which are
mixed in limited concentrations with larger amounts of unlabeled deoxynucleotide triphos-
phates (dNTPs) in a synthesis reaction. Synthesis terminates whenever a ddNTP is incorpo-
rated into a new strand instead of a dNTP. Strands of varying lengths are synthesized and
labeled as the terminal ddNTP is incorporated into the strand. Accumulated fragments are
separated by size using electrophoresis. During electrophoresis, labeled products are
visualized by fluorescence, with each of the four fluorescent dyes indicating which of the
terminal ddNTPs has been incorporated. Combining the terminal ddNTP information with
the fragment size allows for determination of sequence information in an electropherogram.
An example of an electropherogram is illustrated. Uni-directional sequencing is depicted,
but bi-directional sequencing (a forward and reverse sequence for each target strand) is
often used to obtain a more accurate consensus sequence.
1186 WOLK et a1
sequence information. Although the genomes of several microbes have

been sequenced, the vast majority of microbial genomes, even those of
clinically important microbes, have not yet been sequenced. Further-
more, although microbial genomic databases exist, there are limitations
to these databases. The Web-based genomic database GenBank is avail-
able from the National Center for Biotechnology Information (http:ll
www.ncbi.nlm.nih.gov) and was established in 1988 as a national resource
for molecular biology information. Related databases such as the ribo-
somal RNA WWW Server (http://www-rrna.uia.ac.be),the Ribosomal Da-
tabase Project I1 (http://www.cme.msu.edu/RDP/html/index.html), The Insti-
tute for Genomic Research (http://www.tigr.org), and the Ribosomal
Differentiation of Medical Microorganisms (hf tp://www.ridom.hygiene.uni-
wuerzburg.de) provide non-proprietary sources of genomic information.
While public databases contain a wealth of information, certain
caveats must be noted if the data are to be used by clinical microbiology
laboratories. The sequence and descriptive data included with each entry
may not undergo peer review, so any information contained in these
databases must be used with caution. Furthermore, although these are
large databases, one cannot be assured that they contain information for
every gene for every organism. For this reason, well-validated and
comprehensive databases are needed for use in clinical microbiology
laboratories. The MicroSeqB 16s rDNA Bacterial Sequencing kit com-
bines a PCR and cycle sequencing kit that can be used with a MicroSeqB
(Applied Biosystems, Foster City, Calif.) software and a MicroSeqB 16s
rDNA database for organism identification. Although this approach is
useful to aid in characterization of many organisms, its use is limited by
the relatively small size of the MicroSeqB database.
Perhaps the best use of sequence-based identification of microorgan-
isms is the combination of sequence analysis using a well-constructed
database with traditional phenotypic and morphologic organism charac-
teristics. This approach, referred to as polyphasic analysis, can be accom-
plished with a software program such as BioNumerics (BioSystematica,
Devon, United Kingdom). This software offers a relatively user-friendly
system designed to store and analyze both molecular sequence data and
phenotypic characteristics of an organism. Such software may improve
current organism identification schemes by enabling the routine use of
polyphasic algorithms for organism identification.
Several commercial systems have been designed for post-PCR prod-
uct sequencing and are described in Table 3. Of note, is the OpenGene'"
System (Visible Genetics, Toronto), which uses a modification of the
chemistry described above. Using this system and the TRUGENETM HIV-
1 Genotyping Kit for HIV-1, sequence data are obtained for protease and
reverse transcriptase genes and compared to a sequence database that
contains information about drug-resistant mutations. Sequence data are
regularly updated and interpreted using proprietary interpretation codes
and software.
The line probe assay (INNO-LiPA'", Innogenetics, N.V. Ghent, Bel-
gium and Alpharetta, Ga., and Bayer) is a modification of Southern blot
technology in which reverse hybridization takes place using multiple

specific sequences from a known target organism (e.g., HCV) arranged
on a strip (Fig. 12). PCR is performed using a biotinylated primer; the
amplicon is hybridized to the strip. Subsequently, a streptavidin-alkaline
phosphatase system is used to detect the hybridized biotinylated ampli-
con. The enzyme alkaline phosphatase acts on a chromogenic substrate
added to the system to enable color development at the locations on the
strip where specific DNA is bound. The result is a fingerprint of the
hybridization pattern from the PCR reaction. Patterns are compared to
standard genotypic patterns. Kits are available for HCV genotyping,
HIV protease and reverse transcriptase mutation analysis, and HBV
polymerase mutation analysis.
MOLECULAR EPIDEMIOLOGY
A variety of molecular techniques for epidemiologic strain typing

is available. Genomic DNA restriction site analysis involves comparison of
the number and sizes of fragments produced by digestion of DNA with
a restriction enzyme(s). Because of the specificity of restriction enzymes,
complete digestion of an organism's genomic DNA with a specific re-
Chromogen
n Color development
Alkaline phosphatase
4-
4 b
& Streptavidin
Probe (bound to strip)
Nitrocellulose
strip containing
probe sets
Figure 12. Line probe assay. The line probe assay is a strip-based detection system that
uses multiple probe combinations bound in a multi-line format to a nitrocellulose strip. The
assay is based on reverse hybridization, in which amplified PCR product is bound to the
probe combinations or probes already on the strip. Detection of product:probe complexes
is possible, because the PCR primer contains a biotin molecule, which is detected using a
streptavidin-alkaline phosphatase reporter system. The resulting pattern of hybridization is
interpreted.
1188 WOLK et a1
striction enzyme provides a reproducible array of fragments that can be

separated by agarose gel electrophoresis and visualized by staining with
ethidium bromide. Variations in the array of fragments generated by a
specific restriction enzyme are called restriction fragment length poly-
morphisms (RFLPs). Changes to RFLP patterns can result from gene
sequence rearrangements, insertions or deletions of DNA, or base substi-
tutions within the restriction enzyme cleavage sites. The disadvantage
of this technique is that the genomic restriction fragments are usually
too numerous and too closely spaced for easy analysis.
To simplify genomic DNA RFLP analysis, several techniques are
available to limit the number of fragments to be compared. One ap-
proach is to probe the genomic DNA restriction fragments with labeled
probes targeting multicopy nucleic acid sequences such as insertion
sequences, bacteriophage DNA, or rDNA (ribotyping). One automated
ribotyping system, the RiboPrintern Microbial Characterization System
(DuPont Qualicon, Wilmington, Del.), can generate, analyze, compare,
and store genetic ”fingerprint” information.
Another approach is to generate large fragments of chromosomal
DNA by using ”rare cutter” restriction enzymes that recognize infre-
quent restriction sites and allow for separation using special large-
fragment electrophoretic procedures. Because of shearing, intact DNA
required for the generation of large DNA fragments cannot be isolated
using conventional methods. Furthermore, large DNA fragments cannot
be efficiently resolved by conventional agarose gel electrophoresis.
Pulsed-field gel electrophoresis (PFGE) solves both problems. PFGE involves
embedding organisms in agarose, lysing organisms in situ, and digesting
the chromosomal DNA with restriction enzymes that cleave infrequently,
thereby generating large restriction fragments. Slices of agarose con-
taining chromosomal DNA fragments are inserted into the wells of an
agarose gel. Restriction fragments are resolved into a pattern of discrete
bands on the gel by an apparatus that switches the direction of current
in a predetermined ”pulsed-field” pattern and results in the smaller
fragments migrating faster through the gel than the larger ones. The
DNA restriction patterns of the isolates are then compared with each
other to determine relatedness (Fig. 13).’08 PFGE analysis provides a
global chromosomal overview, scanning most of the chromosome, but has
only moderate sensitivity, because minor genetic changes may go m-
detected. Interpretative guidelines for PFGE have been published.107,
PCR-based strain typing methods include repetitive element sequence-
based PCR, randomly amplified polymorphic D N A (RAPD) analysis, and
amplified fragment length polymorphism (AFLPm) analysis, among others.
Repetitive element sequence-based PCR uses consensus PCR primers to
amplify DNA sequences located between successive repetitive elements.
RAPD analysis (arbitrarily primed PCR) involves randomly amplifying
segments of the target DNA by using arbitrary primers that do not have
any known homology to the target sequence. RAPD analysis surveys
relatively undefined regions representing a small percentage of the chro-
mosome. The PCR amplicons are visualized on a gel following electro-
Table 4. EXAMPLES OF AUTOMATED METHODS FOR NUCLEIC ACID EXTRACTION
Instrument Source ChemistrylMethod Comments
ABI PRISMN 6700 Applied Biosystems Proprietary chemistry and flow Robotic workstation integrates with real-time
automated nucleic http://www.appliedbiosysterns.corn membranes PCR instruments (GeneAmp@5700 Sequence
acid workstation Detection System and the ABI PRISM@7700
Sequence Detection System)
AutoGenprep 960 AutoGen Modified phenol extraction +/ - Walk-away robotics with built in centrifuge
and AutoGenprep http://wwzu.autogen.corn proteinase K High-efficiency particulate air (HEPA) filtered
2000 and 3000 cabinets available
Autopure LSTM Gentra Systems Inc. Proprietary liquid phase chemistry Sample volumes up to 10 mL
http://www.gentra.com
Biomek 2000@ Beckman Coulter Magnetic capture Polymerase chain reaction preparation methods
laboratory http://www.beckrnan.com Automation of Magnetic Particle available
automation Dynal Biotech Concentrator (Dynal DNA Sample volumes of 5 pL to 50 mL
workstation http:www.dynalbiotech.corn D I R E C F kit)
Biorobotm 9604 Qiagen Lysis in guanidinium buffer Robotic workstation with manual centrifugation
http://www.qiagen.corn QIAampm silica-gel membrane with steps integrates with various systems
selective binding technology Sample volumes up to 200 pL
COBAS AmpliPrepTM Roche Molecular Systems Lysis in guanidinium buffer Robotic workstation integrates with COBAS
http://www.roche.corn/diagnostics Hybrid capture with streptavidin- For specific organisms (e.g., hepatitis B virus,
coated magnetic beads and hepatitis C virus, HIV-1 cytomegalovirus)
biotinylated probes Sample volumes of 50 to 350 p,L
GenoP-48 Geno Vision GenoPrepTMmagnetic bead-based Robotic workstation can transfer extracts to
http://www.genovision.corn capture nucleic acids microtubes
MagNA Pure LC Roche Molecular Biochemicals Lysis in guanidinium buffer Robotic workstation integrates with Roche
instrument http://www.roche.com/diagnostics Glass (silica) magnetic particle LightCyclerTM
capture with selective binding Sample volumes of 20 to 200 pL
technology
NucliSensm extractor Bio Merieux Lysis in guanidinium buffer Closed system integrates with nucleic acid
http://www.nuclisens.corn Silicon dioxide particles with sequence based analysis (NASBA) technology
selective binding technology Sample volumes of 10 IJ.Lto 2 mL
1190 WOLK et a1
Figure 13. Molecular strain typing by pulsed-field gel electrophoresis (PFGE). For PFGE,
organisms embedded in agarose are lysed in situ, and the chromosomal DNA is subse-
quently digested with a restriction enzyme or enzymes. Because the enzymes used for
PFGE are rare cutters (i.e., cleave infrequently), large (macrorestriction) fragments are
generated. Pieces of the agarose containing chromosomal DNA fragments are inserted
into the wells of an agarose electrophoresis gel. Restriction fragments are separated by
electrophoresis using an apparatus that produces orthogonal electrical fields that switch
the direction of current in a predetermined pulsed-field pattern. The result is a DNA
restriction pattern of discrete bands on the gel with smaller fragments migrating faster
through the gel than larger ones. The patterns of different microbial isolates are compared
with each other to determine relatedness.
phoresis, and the different patterns are compared with each other (Fig.
14). AFLPB Microbial Fingerprinting (Applied Bioscience, Foster City,
Calif.) produces a distinctive DNA fingerprint by selective PCR amplifi-
cation of restriction fragments of the entire microbial genome (Fig. 15).
The AFLPB procedure includes the preparation of an AFLP template
where genomic DNA is digested with two restriction enzymes (”rare
cutter” and ”frequent cutter”), which produce cohesive fragment ends
and cut DNA with different frequencies. Following digestion, genomic
restriction fragments are modified by ligation (joining) of synthetic,
double-stranded oligonucleotide adapters with ends complimentary to
those of the restriction fragments. Thus, after the ligation step, genomic
restriction fragments have termini of known sequences. Such an AFLP
template is submitted to highly stringent PCR amplification with primers
complementary to their targets. Amplified fragments are separated by
electrophoresis and visualized either directly by gel electrophoresis or
by the laser detection system of an automated sequencing instrument.
Multilocus sequence typing (MLST) involves sequencing portions of
several microbial housekeeping genes and is used primarily for epidemi-
Figure 14. Molecular typing by randomly amplified polymorphic DNA (RAPD) analysis.
Random primers are used to amplify various fragments of the DNA sequence. Amplicons
are separated by agarose gel electrophoresis and stained with ethidium bromide to enable
visualization of the DNA fragments. The different patterns are compared with each other to
determine relatedness of strains.
Figure 15. Molecular strain typing by amplified fragment length polymorphism (AFLP)
analysis. DNA restriction fragments are ligated to adapter molecules. Amplification of the
restriction fragments occurs by using primers complementary to the adapterhestriction site
sequences. Electrophoresis of the amplified restriction fragments is performed to generate
specific patterns (fingerprints) that are visualized by analyzing fragment patterns on a gel.
The different patterns are compared with each other to determine relatedness of strains.
1192 WOLK et a1
ologic This technique can generate reproducible isolate pro-

files that can be run against standardized databases.
EMERGING TECHNOLOGIES
Many new molecular methods are under development. Not all are
within the scope of this article; however, several may have applications
to clinical microbiology laboratories in the future.
The Q-beta replicase amplification system is a signal amplifications
system based on the function of an enzyme called replicase, which is
derived from the RNA virus Q-beta.45,49 The Q-beta replicase system
(Gene-Trak Systems, Framington, Mass.) is based on mass production of
RNA sequence by an enzyme called replicase. The probe composite
consists of a hybrid RNA built of the target-specific probe region, a
cleavage site for RNAse 111, and a substrate region for the replicase
enzyme. Probe bound to target is not susceptible to cleavage by RNAse
111; unbound probe is cleaved by RNAse 111. Bound probe is then released
from the target and, because of its replicase substrate site, amplified by
the replicase enzyme (Fig. 16).
Invader@technology (Third Wave Technologies, Inc.) is an isother-
mal homogenous signal probe amplification assay that uses an Invader@
probe, a signal probe, and the enzyme Cleavase (Third Wave Technolo-
gies, Inc., Madison, Wis.) (Fig. 17).53Two probes bind to the target, so
that the Invader@probe binds upstream of the signal probe, with the
probes overlapping by at least one nucleotide. CleavaseB recognizes the
specific structure formed by the hybridization of the two probes and
cleaves the signal probe. The cleaved signal probes are then replaced, so
that many signal probes are cleaved for each copy of target nucleic acid.
The cleaved signal probe is then detected by hybridization to a hairpin-
shaped probe that is labeled with two dyes. When the cleavage product
binds to the hairpin-shaped probe, a fluorescent signal is detected (see
Fig. 17).
A new sequencing based methodology, Pyrosequencing (Pyro- TM
sequencing, Westborough, Mass., and Upsala, Sweden) is a process by

which short fragments of DNA sequence are determined. With this
technique, a sequencing primer is hybridized to a DNA template and
incubated with DNA polymerase, ATP sulfurylase, luciferase, apyrase,
and the substrates, adenosine 5’-phosphosulfate, and luciferin (Fig. 18).
As complementary dNTPs are added to the reaction mix and incorpo-
rated into the strand, pyrophosphate is released in a quantity equal to
that of the amount of original incorporated nucleotides. ATP sulfurylase
is generated and drives the luciferase reaction so that light is generated,
monitored by a charged-coupled device camera, and converted to
graphed data of peaks called a pyrogram. The enzyme apyrase modifies
residual dNTPs prior to incorporation of the other nucleotide bases in
subsequent reactions.
DNA microarray technology (i.e., biochip, DNA chip, DNA microar-
ray, gene chip, genome chip, and gene array) has the potential to assess
Replicase
substrate + Denatured
DNA target
(J Cleavage site
for RNAse I l l v
Target-specific
probe region
Probe-bound
RNAse 111 cleavage
site is resistant to
cleavage with
RNAse 111
IIIIIIIII
n
I RNAse 111 cleaves unbound probe
I Q-betareplicase added
Probe released and amplified by Q-beta replicase to yield a detectable RNA signal
Figure 16. Q-beta replicase. Probes are produced to contain three regions, the target-
specific probe region, a cleavage site for RNAse 111, and a substrate region for the replicase
enzyme. Once the probe binds to the target, RNAse 111 is not able to cleave the probe.
Unbound probes are cleaved when RNAse 111 is added to the reaction. Bound probes are
then released from the template and, because of the replicase substrate site in the probe,
amplified by the Q-beta replicase enzyme. Amplification yields detectable quantities of the
RNA product.
and visualize thousands of nucleic acid targets simultaneously. The

underlining principle of DNA microarray technology is nucleotide hy-
bridization, which takes place on a solid platform (e.g., glass, silica, or
nylon) containing genetic material (e.g., cDNA or oligonucleotides) ar-
rayed in a predetermined fashion. When a chip is probed with cDNAs
that are made from an unknown sample of RNA from a tissue or cell,
complementary sequences will hybridize with various sequences already
positioned on the chip (Fig. 19). By monitoring the hybridization pattern
of the unknown cDNA, the identity of the hybridized nucleic acids can
be determined. The patterns can indicate which RNAs were in the tissue
and which genes are being expressed. Microarray technology also can
1194 WOLK et a1
Figure 17. Invader assay. A primary probe and an Invader oligo hybridize to a target
sequence. The Cleavase enzyme recognizes the single base pair overlap configuration of
the bound probes. The enzyme cleaves 5' flap of the primaty probe at the position of the
overlap, releasing it. At the isothermal reaction temperature, cleaved and uncleaved primary
probes cycle on the target readily, and multiple copies are cleaved. The accumulated 5'
flaps generate signal when each binds to a synthetic FRET cassette-5' flap triplet; the
fluorescent reporter dye is separated from the quencher molecule, allowing measurable
fluorescent signal to accumulate when exposed to an external light source. (Adapted from
a graphic illustration created by Jodi Hoeser, courtesy of Third Wave Technologies, Inc.,
Madison, Wis.)
be used to identify gene sequences and mutations associated with HIV-

1 drug resistance (e.g., Genechip@HIV PRT Plus Probe Array Design
for analysis of subtype B protease and reverse transcriptase genes; Affy-
metrix, Santa Clara, Calif.), although there is only limited support for its
clinical use. Other limitations of microarray technology include the high
costs for instrumentation and disposable supplies. In addition, the inabil-
ity to easily analyze the enormous amount of information potentially
available through this technology presents challenges.
Another example of probe amplification methods is Cycling Probe TM
Technology (CPT, VelogeneTM,ID Biochemical, Vancouver, British Co-

lumbia), an isothermal process that can be used to detect target DNA.
CPT is used to detect a variety of infectious organisms, including M .
tube~culosis,~ methicillin-resistant Staphylococcus aweus (MRSA)? 9, 17, 28
and vancomycin-resistant enterococci (VRE).60 Briefly, the technology
d&TP
Sequencing primer
1 DNA polymerase
PCR amplified DNAtemplate G A A C T G T C C T A G C A

VIA
.t
Pyrophosphate
1
ATP
ATP sulphurylase
Adenosine 5'phosphosulfate
1 L uciferase
L uciferin
.-
3 the number of nucleotides incorporated
tn
S
a Pyrogram
-
c
S
i
Nucleotide added
Figure 18. Pyrosequencing (Pyrosequencing,Westborough, Mass., and Upsala, Sweden).

A sequencing primer is hybridized to a DNA template. The first four dNTPs, in this example
dATP,* are added to the reaction. DNA polymerase catalyzes the incorporation of the dNTP
(dATP shown) into the DNA strand. The reaction is incubated with ATP sulfurylase, lucifer-
ase, apyrase, and the substrates, adenosine 5'-phosphosulfate and luciferin. As comple-
mentary dNTPs are added individually to the reaction mix, they are incorporated into short-
to medium-length DNA strands. As the reaction occurs, pyrophosphate is released in a
quantity equal to that of the amount of original incorporated nucleotides. As a result, ATP
is generated and drives an enzymatic luciferase reaction, so that light is generated.
Pyrograms (graphs of emitted light that occur during the reaction) are generated and can
be converted to nucleic acid sequence data. The enzyme apyrase will modify the residual
dNTPs before the addition of the next base incorporation. (Adapted from a graphic illustra-
tion, courtesy of Pyrosequencing,Westborough, Mass, and Upsala, Sweden.)
*Deoxyadenosine alfa-trio triphosphate is used as a substitute for dATP, since it is used

efficiently by DNA polymerase, but not recognized by the luciferase.
1196 WOLK et a1
Figure 19. DNA Microarray. (Hybridization experiment using the GeneChip Probe Array).
Various fluorescent signal intensities depicted by color-hybridization displays, are generated
on the probe array surface and digitized. A stronger signal is produced when there is an
exact match between the probe and the target; therefore, the resulting signal is a measure
of both sequence homology and the quantity of nucleic acid bound to the chip. Semi-
quantitative hybridization information is obtained. (From Affymetrix, Santa Clara, Calif.)
uses a synthetic probe, which is a chimera of DNA-RNA-DNA and is

labeled at the 5’ end with fluorescein dye and at the 3’ end with a biotin
molecule. The fluorescein/biotin-labeled probes are designed to anneal
with a specific DNA template. Once the probes are annealed, RNase H
is added to cleave the RNA portion of the bound probe. The shorter,
cleaved probe fragments will then dissociate from the target and release
the target for use in subsequent cycles. In the presence of a specific
Figure 20. Cycling Probe Technology. Cycling probe technology is a signal amplification
technology that uses target nucleic acid as a catalyst to convert intact probes to fragments.
A synthetic probe, which is a chimera of DNA-RNA-DNA that is labeled at the 5’ end with
fluorescein dye and at the 3’ end with a biotin molecule, anneals with a specific DNA
template. RNase H is added to cleave the RNA portion of the bound probe. Cleaved probe
fragments dissociate from the target and accumulate with each cycle. Target is released
from the probe for use in subsequent cycles. A colorimetric reaction will occur in the
presence of uncleaved probe; no color will occur in the presence of cleaved probes (specific
target). To detect the presence or absence of cleaved probes, an anti-fluorescein antibody
coupled with horseradish peroxidase is added, and the reaction is transferred to microtiter
wells coated with streptavidin. Only uncleaved probes will bind to the anti-fluorescein
antibody coupled with horseradish peroxidase.
Denatured DNA template

5'
1 Probe annealing
U
-
1
0
Probe degradation by RNAse H with
G subsequent melting of cut probe
I
3' 5'
4.
,?-
5 A*
I Transferred to streptavidin-coated solid surface

Washed to remove fluoremin-labeled fragment
Addition of antibody to fluorescein coupled

with horseradish peroxidase (HRP)
Key
Chimeric probe
@ Fluorescein dye
Add substrate For HRI? color developer; and
Streptavidin wash to remove unbound antibody
@ Biotin molecule
Antibody to fluorescein
Fr"-'.yy No binding
0 Horseradish peroxidase
, No color develops
Figure 20. See legend on opposite page

1198 WOLK et a1
target molecule, cleaved probes accumulate with each cycle. To detect

the presence or absence of cleaved probes, an anti-fluorescein antibody,
coupled with horseradish peroxidase (Ab/HRP), is added, and the reac-
tion is transferred to microtiter wells coated with streptavidin molecules.
(Alternatively, a nitrocellulose test strip has been manufactured for use
in the detection of uncleaved probes.) All probes will bind to streptavi-
din, but only the uncleaved probes will bind to the Ab/HRP. A substrate
for horseradish peroxidase is added and color development is measured
to determine the amount of uncleaved probe that is present in the well
(or the region of the strip). A colorimetric reaction will occur in the
presence of uncleaved probe. No color will occur in the presence of
cleaved probes (specific target) (Fig. 20).
SUMMARY
Molecular testing methods have the potential to replace many con-

ventional microbiology laboratory assays. Recent refinements in technol-
ogy have resulted in more user-friendly testing platforms. These plat-
forms are automated and have lowered risks for contamination,
decreased costs, and are faster than older platforms. The success of these
technologies depends on their successful application to patient care.
Quality issues include appropriate specimens for analysis, performance
characteristics of different analytical methods, optimal specimen proc-
essing, the effects of PCR inhibitors, and false-positive results caused by
contaminating nucleic acids. Quality control guidelines for molecular
microbiologic diagnostic assays are in their infancy and require further
development. Additionally, the problem of "too much" sensitivity
(brought on by the extreme sensitivity of these techniques coupled with
the potential presence of small numbers of pathogenic organisms in
asymptomatic individuals) should be considered. Potential problems
when monitoring therapy (because molecular detection techniques do
not generally have the ability to determine whether an organism is dead
or alive) can also occur.
Cost-effective test use, pathogen- or disease-targeted algorithms,
and standardized methods will be necessary for the true value of these
technologies to be realized. This is especially important, because, unlike
traditional culture methods, most molecular microbiology methods are
pathogen-specific. Clinicians familiar with the reasons why "pan-cul-
ture" (i.e., requesting all culture possibilities at once) is inadvisable
should not use the same irrational approach when requesting molecular
tests. The clinical usefulness of molecular testing will be maximized as
targeted algorithms are developed and an understanding of molecular
test ordering patterns is realized. Laboratory technicians and physicians
must continue to apply and combine theories of traditional microbiology,
clinical chemistry, and general medicine to the understanding and appli-
cation of molecular diagnostics.
ACKNOWLEDGMENTS
The authors acknowledge Jeffrey J. Germer, BS; Paul N. Rys, MS; and Nancy L.
Wengenack, PhD, for their insightful review of this article. They also acknowledge
Gretchen A. Thomason for her excellent secretarial assistance.
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Address reprint requests to

Robin Patel, MD, FRCP(C)
Mayo Clinic
200 First Street, S.W.
Rochester, MN 55905
e-mail: patel.robin@mayo.edu
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Principles of Molecular Microbiology Testing Methods

Article in Infectious Disease Clinics of North America · January 2002

Rapid Diagnostics: Bloodstream infections View project

Applications and Outcomes View project

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Recent advances in molecular testing methods are changing the

toxoplasmosis, progressive multifocal leukoencephalopathy (PML), hep-

INFECTIOUS DISEASE CLINICS OF NORTH AMERICA

VOLUME 15 * NUMBER 4 * DECEMBER 2001 1157

PROBE HYBRIDIZATION METHODS

Nucleic acid probes are segments of DNA or RNA that can be

Molecular techniques developed as research tools have been applied

Test Manufacturer Application Detection

c1 *Culture confirmation assays not listed

Other important components of quantitative molecular assays in-

falsely negative, inhibitory substances (e.g., bilirubin, hemoglobin, lipids,

‘FRET = fluorescent resonance energy transfer

important advance of many new automated extraction systems. For

Foster City, Calif.) integrate with the Lightcycler PCR instrument

TARGET NUCLEIC ACID AMPLIFICATION METHODS

Polymerase Chain Reaction

Denatured DNA template

Ccm 1st Strands

Figure 1. Polymerase chain reaction (PCR). A DNA sequence (template) is amplified in

fied DNA fragments of the expected size are assumed to be amplified

Electrophoresis Place the gel on Hybridize specific

I Stained DNA fragments on agarose gel

mms DNA on membrane

brane by using a second chemical reaction (e.g., anti-digoxigenin antibody:

Engineering and Operational Controls for

Despite significant advances in technology, many challenges mus

molecular assays performed in the same laboratory.6,50, 57, 74, 98 Sources of

various detection formats.51,99 Closed-system PCR reactions can also be

Several modifications of conventional PCR exist and require defini-

(e.g., HCV), or to detect transcribed microbial DNA as a marker of active

Rapid Real-Time Polymerase Chain Reaction

A significant advance in PCR technology is rapid PCR with simulta-

Closed PCR systems provide the single most important advance in

turation temperature, and diminished fluorescent reporter signal is

be used to monitor real-time endpoint (qualitative) PCR and real-time

Figure 5. Molecular beacons. Molecular beacons are single-stranded oligonucleotide

Non-PCR Target Nucleic Acid Amplification Methods

Several non-PCR target amplification methods exist, including tran-

SIGNAL AMPLIFICATION METHODS

An alternative to enzymatic duplication of target nucleic acid is to

Figure 6. Transcription-based amplification systems [nucleic acid sequence-based amplifi-

*Modifications can be performed to allow the use of a DNA target.

Sequencer Manufacturer/Supplier Type Throughput Detection Comments

'Performance ophmized polymei

Figure 7. Strand displacement amplification (SDA). SDA is an isothermal amplification

nucleic acid is released and captured to a solid surface by multiple

argel DNA or RNA

PROBE AMPLIFICATION METHODS

Probe amplification technologies, wherein the end product of the

D N A sequence analysis has several applications in the clinical micro-

Sequencing primer Sequence lo be determined

ezd Fluorescent dye 114 labeling ddCTP

* Fluorescent dye labeled ddNTPs ale

sequence information. Although the genomes of several microbes have

technology in which reverse hybridization takes place using multiple

A variety of molecular techniques for epidemiologic strain typing

Probe (bound to strip)

striction enzyme provides a reproducible array of fragments that can be