GROUP 5

Molecular
methods
ANDAYA AZADA BALANAY BANGIBANG BUQUEL BILAS
DANAO GABOT LADIA LOPEZ MATOTE PAGULAYAN
SIBALON SOTTO TUAZON VENTULA
TABLE OF CONTENTS

01 02
COMMON
APPLICATION OF
QUANTITATIVE
PCR
METHODS
 NAME OF THE PHARMACY

01
COMMON
QUANTITATIVE
METHODS
 NAME OF THE PHARMACY

POLYMERASE CHAIN REACTION

● common laboratory technique used to make many copies of a particular region of
DNA.
● GOAL: to make enough of the target DNA region that it can be analyzed or used in
some other way.
○ For instance, DNA amplified by PCR may be sent for sequencing, visualized
by gel electrophoresis, or cloned into a plasmid for further experiments.
PCR reagents
Template DNA
● Must be DNA only
● Plasmid DNA, bacterial DNA, cDNA, or gDNA can be
utilized as a template.
● The template DNA should be highly purified DNA
○ a purity of around ~1.80
○ quantity of up to 200ng
● works as a substrate for an enzyme when it is
denatured
● The good quality of extracted DNA can boost the
resulting efficiency of the polymerase chain
reaction.
dNTPs
● Deoxynucleotide triphosphates are artificially
synthesized nucleotides that bind to the growing
DNA strand.
● With the help of the Taq DNA polymerase, the
dATP, dGTP, dCTP and dTTP bind at their
complementary nucleotides on the growing DNA
strand.
PCR buffer
● help to boost reaction and amplification
efficiency
● maintains the constant pH of the reaction nearly
7.9 to 8.5 by keeping the constant chemical
environment for the PCR reaction.
● Mgcl2, DMSO, KCl, albumin, betaine, BSA,
glycerol, (NH4)2SO4, and formamide are some of
the chemicals commonly used in the PCR buffer.
 PRIMERS
-short sequence of nucleotides that
provides a starting point for DNA
synthesis
- given sequences that will make
them bind to opposite strands of
the template DNA

***Note :Taq polymerase can only make DNA if its

given primer
Primers bind to the template by
complementary base pairing
The PCR MACHINE
HOW DOES PCR WORK?
● PCR mimics what happens in cells
when DNA is copied (replicated)
prior to cell division, but it is
carried out in controlled
conditions in a laboratory.

● Test tubes containing the DNA

mixture of interest are put into
the machine, and the machine
changes the temperature to suit
each step of the process.
Taq Polymerase

● PCR requires a DNA polymerase

enzymes that make new strands of
DNA using existing strands as
templates
● Commonly used in PCR as DNA
polymerase
● Isolated from Thermus aquaticus
 COMMON QUANTITATIVE METHODS
PCR
Temperature Cycle Stages:
➔ Denaturation - heating to high temperatures to separate DNA into single strands,
typically at 95°C
➔ Annealing - lowering the temperature to allow the primers to anneal to a specific
region(s) of the single-stranded DNA and create a partial double strand
◆ primers (synthetic oligonucleotide strands that are complementary to the ends of the original target
gene sequence)
➔ Extension - (historically at 72°C), in which deoxynucleotide triphosphates are
added to the 3’ ends of the bound primers by a DNA polymerase, thereby creating
a new strand of synthetic DNA
 COMMON QUANTITATIVE METHODS
PCR
➔ The synthesized oligonucleotide sequence is complementary to
that of its original template strand.
➔ Amplicon, or PCR product - Double-stranded synthetic DNA
➔ Once the amplicon is created, it can then serve as a template and
be amplified further.
➔ This allows doubling of the number of amplicons with each cycle
and an exponential increase, a main feature of PCR.
 COMMON QUANTITATIVE METHODS
PCR
➔ In the PCR, a DNA sequence (template) is amplified in a buffered
reaction solution containing
◆ oligonucleotide primers
◆ thermostable Taq DNA polymerase
◆ deoxynucleotide triphosphates
◆ 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 temperature cycle three steps occur:
◆ Denaturation
◆ primer annealing
◆ primer extension
 COMMON QUANTITATIVE METHODS
PCR
➔ A mathematical description for the product accumulation within each
cycle is:
Yn = Yn - 1 * (1+Ev) with 0 ≤ Ev ≤ 1
➔ Ev - efficiency of the amplification
➔ •Yn - the number of molecules of the PCR product after cycle n
➔ •Yn – I - the number of molecules of the PCR product after cycle n – 1

Historically, amplicon or product would be visualized using gel

electrophoresis, which has been replaced with real-time PCR methods that
combine target amplification and fluorescent detection of probe hybridization
within a single reaction vessel.
 COMMON QUANTITATIVE METHODS
Real-Time PCR
➔ This method utilize fluorescence tags whereby the number of fluorescent signals
increase until the reaction components are depleted, reaching a plateau phase
of amplification
➔ The amplification process is displayed as a plotted curve of the generated
fluorescent signals and is commonly called a response curve.
➔ cycle threshold (CT), crossover value, crossing threshold, or crossing point
- number of PCR cycles required to exceed the background fluorescence

➔ A direct and inverse relationship between the concentration of nucleic acid and
the CT values in the original specimen extract make it possible to quantify viral
loads.
 COMMON QUANTITATIVE METHODS
Real-Time PCR
➔ Since the fluorescent signal generated by the reaction is proportional to the
concentration of DNA in the reaction, real-time PCR methods can incorporate
standards that enable results to be reported in quantitative measures of nucleic
acid concentration
● viral copies/mL or International units IU/ml
➔ In general, measurements and readings that are to be used for quantitation
should be gathered during the exponential phase of PCR amplification, where
the amplification plot crosses the threshold
 COMMON QUANTITATIVE METHODS
Real-Time PCR
➔ Measurements taken during the lag phase or the plateau phase of amplicon
production will yield misleading results.
➔ Measurement of results during the exponential phases is referred to as
threshold analysis and contrasts to that of traditional gel electrophoreses,
referred to as endpoint analysis.
 COMMON QUANTITATIVE METHODS
Real-Time PCR
● Several fluorescent chemistries exist for use in real-time PCR assays to detect
PCR amplicon and fall into two main types:
○ intercalating dyes - nonspecific fluorescent dyes that intercalate with any
double-stranded DNA (e.g. SYBR Green)
○ sequence-specific DNA probes - consist of oligonucleotides labeled with a
fluorescent reporter which permits detection only after hybridization of the
probe with its complementary nucleic acid sequence has occurred
 COMMON QUANTITATIVE METHODS
Real-Time PCR
➔ In general, real-time PCR assays have the following commonalities
◆ A light source (e.g., LED, halogen lamp, or lasers with differing
wavelengths), housed within a specialized thermal cycler, stimulates the
reporter dye(s) to emit fluorescence, or in some cases, diminish
fluorescence, which can then be monitored by various detection systems.
◆ Target-specific hybridization can be assessed by incorporating additional
sequence-specific fluorescent probes into the assay; these bind to target
sites internal to the primer sites in the amplicon, and in some cases, to the
primers themselves, and serve as the detection method.
◆ The fluorescent signal generated as amplicon is produced, measured, and
converted by software to generate an amplification plot
 COMMON QUANTITATIVE METHODS
Real-Time PCR
➔ Several historical internal probe formats are still used today, including
◆ hybridization probes
◆ hydrolysis probes like those used in TaqMan chemistry
◆ molecular beacon probes
➔ Newly developed probes or primers include
◆ scorpion probes
◆ major groove binding (MGB) probes
◆ fluorescence-labeled locked nucleic acid (LNA) hydrolysis probes
◆ scalable target analysis routine (STAR) technology, measuring amplicon
accumulation through incorporation of labeled primers
 COMMON QUANTITATIVE METHODS
Real-Time PCR
➔ TaqMan chemistry, fluorescence resonance energy transfer, and molecular
beacon technology can be used to monitor real-time endpoint (qualitative)
PCRs as well as real-time quantitative PCRs.
➔ Typically, real-time quantitative PCR assays have wider linear ranges than
conventional quantitative PCR assays
 COMMON QUANTITATIVE METHODS
Nucleic Acid Sequence-based Amplification (NASBA)
➔ isothermal (41°C)
➔ transcription-based amplification method that sensitively detects RNA targets.
➔ Some NASBA techniques less efficiently amplify DNA, requiring that the target
DNA be in excess ( > 1,000-fold) over the RNA target or only in the absence of
the corresponding RNA target
➔ A major advantage of NASBA is attributed to the production of single-stranded
RNA amplicons.
➔ This ssRNA can then be directly used as a template for another round of
amplification, much like PCR, or it can be probed for detection without the need
for preliminary denaturation or strand separation
 COMMON QUANTITATIVE METHODS
Nucleic Acid Sequence-based Amplification (NASBA)
➔ Disadvantages of NASBA include:
◆ NASBA enzymes are not thermostable and thus can only be added after the
melting step
◆ primers are not incorporated in the amplicon and thus labeled primers
cannot be used for detection
◆ the length of the target sequence to be amplified efficiently is limited to
approximately 100 to 250 nucleotides.
 Specimen Collection: Blood
● Specimen: Whole Blood
● Container: Lavender/ EDTA
● Preferred Volume: 3.0 mL
● Minimum Volume: 1.4 mL
● Note: When drawing for multiple PCR tests (ex.
CMV PCR, EBV PCR, BKV PCR, ADV PCR Qnt), EACH
PCR test requires 1.4mL EDTA whole blood.
Specimen Collection: Blood
● Specimen: EDTA Plasma
● Container: Pearl white-top tube plasma
preparation tube (PPT)
● Preferred Volume: 1.0 mL
● Minimum Vol: 0.7 mL
● Note: When aliquotting for multiple PCR tests
(ex. CMV PCR, EBV PCR, BKV PCR, ADV PCR Qnt),
EACH PCR test requires 0.7mL EDTA plasma.
 Specimen Collection: Nasal Swab
During an anterior nares test, you will start by tilting
your head back. Then you or the provider will:

● Gently insert a swab inside your nostril

● Rotate the swab and leave it in place for 10 to 15
seconds
● Remove the swab and insert it into your second nostril
● Swab the second nostril using the same technique
● Remove the swab
 Specimen Collection: Nasal Swab
During an NMT swab, you will start by tilting your head
back. Then you or your provider will:

● Gently insert a swab onto the bottom of the nostril,

pushing it until you feel it stopping
● Rotate the swab for 15 seconds
● Remove the swab and insert it into your second
nostril
● Swab the second nostril using the same technique
● Remove the swab
 Specimen Collection: Nasal Swab
During a nasopharyngeal swab:

● You will tip your head back.

● Your health care provider will insert a swab into
your nostril until it reaches your nasopharynx (the
upper part of your throat).
● Your provider will rotate the swab and remove it.
 Specimen Collection and Parameters
● EDTA and sodium citrated plasma
➔ Preferred specimen for HCV PCR
● Serum
➔ Acceptable for some assays if it is
centrifuged immediately after clot
formation and frozen
● Refrigerated (4°C) short-term storage of serum
or plasma
➔ Also acceptable
● EDTA plasma
➔ preferred specimen for HIV-1 assays
 Inhibitors and Interfering Substances
● Bilirubin
● Hemoglobin
● Lipids
● Heparin
● Food by- products
 Specimen Adequacy
● Assessment of specimen adequacy
➔ Primer sets for human housekeeping genes
like Human B-globin, B-actin, or GAPDH are
designed and included in the assay.
➔ Useful only ife cellularity is an indicator
of adequacy.
● DNA concentration within patient sample
➔ Measured spectrophotometrically to ensure
samples contain sufficient human DNA
 Applications of PCR
● Amplification of gene fragments as
fast alternative of cloning
● Modification of DNA fragments
● Sensitive detection of pathogenic
microorganisms
- Can detect the presence of viral
DNA before it turns into a killer
● Detection of mutations relevant for
inherited diseases such as
thalassemia and cystic fibrosis,
malignant transformation or tissue
typing
 Applications of PCR
● Analysis of genetic markers for the
forensic applications, for
paternity testing and for the
mapping of hereditary traits
● DNA analysis of archaeological
specimens
● Species-specific amplification of
DNA segments between
interspersed-repeat elements
● Study of gene expression
Human Immunodeficiency Virus 1 (HIV-1)
· According to WHO guidelines (2013), Viral load testing is
recommended as the preferred monitoring method to diagnose and
confirm antiretroviral (ART) treatment failure.

· However, if viral load is not routinely available, CD4 count and clinical
monitoring should be used to diagnose treatment failure.

· The WHO guidelines do not recommend HIV load testing at the time
of HIV diagnosis, but recommend testing

o 6 months into ART treatment

o every 12 months thereafter to detect any treatment failure.

Human Immunodeficiency Virus 1 (HIV-1)
· Indicative of Treatment Failure: Plasma viral load > 1,000 copies/ml
on two consecutive measurements at least 3 months apart, in the setting
of adherence counseling.
o the need to change to second-line ART.
· The rationale for the threshold of 1,000 copies/ml was based on two
main sources of evidence.
o First, viral blips or intermittent low-level viremia (50 to 1,000
copies/ml) can occur during effective treatment but have not
been associated with an increased risk of treatment failure
unless low-level viremia is sustained.
o Second, clinical and epidemiological studies show that the
risk of HIV transmission and disease progression is very low
when the viral load is lower than 1,000 copies/ml.
Human Immunodeficiency Virus 1 (HIV-1)
· Treatment should not be withheld if laboratory capabilities are not
available and both CD4 and viral load testing should be performed only if
resources permit
· According to WHO Interim Technical Update on Implementing HIV
Viral Load Testing indicates: Threshold of 1,000 copies/ml can be
effectively utilized for dried blood spots used on multiple laboratory
platforms. However, the sensitivity at this threshold may be reduced.
· Test that uses Whole Blood: unreliable at lower thresholds (1,000
copies/mL), therefore higher threshold (3,000 to 5,000 copies/ml) should
be adopted since the preferred specimen type for HIV-1 quantification is
plasma.
Human Immunodeficiency Virus 1 (HIV-1)

· For HIV viral loads, It is the responsibility of the laboratory to provide

clinicians with an awareness of an assay’s inherent variability, as
QRT-PCR HIV assays exhibit differences among laboratories.

· Current therapy guidelines for HIV-1 state that virologic failure

occurs when the viral load exceeds 200 copies/ml; therefore, a physician
would need to confirm this change in the viral load with a subsequent
viral load measurement.
Human Immunodeficiency Virus 1 (HIV-1)

· Precision is reduced at lower concentrations and increased

variability is typically exhibited when target concentration is less than 200
copies/ml, so an HIV patient with consecutive undetectable viral load
results may, at some point, have a viral load result of 250 IU/ml and still
be well within the normal variability of the quantitative RT-PCR.

· A rise in the viral load may simply indicate a “viral blip,” that is, a
temporary rise that will resolve itself or it may continue to rise and
indicate an actual change in the patient’s status (virologic failure).
Human Immunodeficiency Virus 1 (HIV-1)

· For these reasons, a > 3-fold difference in viral load is often required
before decisions that would affect therapy are made, HIV-1 quantitation
may also vary with genotype or subtypes. Most of the Group M genotypes
are now accurately quantitated, but not all assays will accurately
quantitate or even detect Group O genotypes.

· There are two recent reports that serve as examples of the way in
which HIV-1 viral load assay performance, specifically imprecision, can
impact patient management and therapy.
Human Immunodeficiency Virus 1 (HIV-1)
● Naeth et al. evaluated patient samples at densities of 40, 80,
and 90 copies/ml. The authors were able to predict how
likely one could identify a real change in the viral load with a
value between 40 to 200 cps/ml.

● Ruelle et al. provides another example of viral load

performance at the clinical cutoff. Authors were able to
ascertain the ability of an assay to identify a significant
change in the viral load from 25 to 400 cps/ml (a 1.2-log
copies/ml change). This change in the viral load represents
a larger shift than DHHS considers virologic failure
 Hepatitis C Virus (HCV)
HCV Viral load
- relatively stable in untreated patients with chronic HCV
>> Viral load and HCV genotype are not good predictors of disease severity or progression
- Provides information about treatment response to antiviral therapy

Treatment goal for chronic HCV infection is to prevent complications:

1. normalize ALT
2. achievement of SVR (sustained virologic response) - absence of detectable viremia at the end
of treatment and 6 months later
- viral load <25 IU/mL

Quantitative HCV assays

- provide baseline value
- assessment of kinetics of therapy
- genotype 1 virus -> longer therapeutic courses
- genotype 2 and 3 -> better SVR rates
Hepatitis C Virus (HCV)
In a study by Wiesmann et al. (171), HCV patients were monitored for a
2-log drop from baseline to ensure therapeutic response.
- evaluated HCV assay reproducibility at 25 IU/ml using two
commercial HCV assays
- demonstrated that precision ranged between 23% CV and 51% CV

The use of a highly precise Six Sigma method could benefit patients, as it
can document a more accurate assessment of sustained virologic
response and clearance of HCV near the LLOQ.
Hepatitis C Virus (HCV)

Therapeutic monitoring simplified by real-time quantitative PCR

applications due to greater sensitivity compared to qualitative TMA-based
assays or bDNA-based formats.

Sensitive qualitative assays are typically used to evaluate the

end-of-treatment response (EOT), since low levels of residual RNA can be
found in a proportion of conventional PCR-negative EOT samples.

Highly sensitive real-time quantitative PCR assays can be used to

evaluate the early virologic response of infections to discontinue
treatment in nonresponders and modify the treatment strategy
Hepatitis C Virus (HCV)
Less sensative quantitative test methods, like conventional PCR, are
used, use of the qualitative test methods in combination is recommended
to assess the rapid virologic response, EOT, and SVR

Quantification and genotyping of HCV RNA continue to evolve as a key

component of the therapeutic strategy for chronic HCV treatment,
especially as new therapeutics receive FDA approval for routine clinical
use

Selection of the most accurate and precise method to detect changes in

viral load will more readily confirm sustained virologic response, assess
viral clearance, identify virologic failure, and limit overall testing costs.
HBV
➔ The risk of development of cirrhosis and hepatocellular carcinoma in chronic
hepatitis B infected patients is a priority in the management strategy for
treatment, with HBV viral load testing playing a vital role.
➔ Serial monitoring of HBV DNA levels is the best approach to determine the
need for treatment.

● Low levels of HBV DNA (3 log10 IU/ml to 5 log10 IU/ml) may warrant
treatment and be linked to liver disease in those who are HBeAg negative or
have developed cirrhosis.
● Viral load testing at 24 weeks of treatment to characterize the virologic
response as complete, partial, or inadequate.
HBV
● Decrease in serum HBV DNA to undetectable levels by PCR assays, and a loss
of HBeAg in patients who were initially HBeAg positive is a virologic response
to therapy.
● While fulfilling the criteria for the virologic response, loss of HBsAg, as well as
a biochemical response or decrease in serum ALT to within normal ranges is a
complete response.
● Measurement of HBV viral loads is critical to determining the efficacy of
therapy in chronic HBV patients whether it is assessing for a total reduction or
the kinetics of a decrease in HBV DNA levels.
Cytomegalovirus (CMV)
✓ Clinical utility of quantitative viral loads for CMV has most
extensively been demonstrated in solid organ transplant (SOT)
recipients for prognostication of CMV disease, to:

● guide preventative treatment

● assess efficacy of treatment
● guide treatment duration, and
● indicate risk of CMV clinical relapse or antiviral drug resistance

✓ Exposure to CMV via infected saliva and body secretions is the

primary route of infection in immunocompetent persons and presents as
an asymptomatic or self-limited illness.
Cytomegalovirus (CMV)
Those at highest risk for CMV infection are:

● lung
● intestinal and
● pancreas recipients

Moderately at risk are: heart and liver recipients

Lowest risk of CMV infection and disease is in kidney recipients

Cytomegalovirus (CMV)
✓ Primary infection

is defined as receipt of an allograft from a CMV-seropositive

donor (CMV D+/R-) by a CMV-seronegative recipient, acquiring CMV by
natural transmission or from a CMV-seropositive blood product.

✓ Risk of CMV disease is higher for CMV Donor seropositive/Recipient

seropositive (D+/R+) patients than for CMV D-/R+ SOT patients

✓ The onset of CMV infection is delayed when a prophylactic

prevention strategy is employed within the first 3 months after SOT,
deferring CMV infection occurrence until sometime during the first 3
months, after completion of CMV prophylaxis.
Cytomegalovirus (CMV)

Three major strategies exist for prevention of CMV

disease:

● antiviral prophylaxis
● monitoring viral reactivation and
● a hybrid approach
 Cytomegalovirus (CMV)
Antiviral prophylaxis

● is managed most commonly with valganciclovir administered for a defined duration,

generally 3 to 6 months to all patients at risk for CMV disease. In contrast to
SOT recipients, use of ganciclovir as a prophylactic

● Use of aciclovir or valganciclovir for prophylaxis has the following potential

benefits when using these drugs in selected patients:

-reducing the need for hospital admission and/or IV preemptive therapy

-reducing indirect effects of CMV reactivation on immune status post-transplant,

and

-delaying CMV reactivation until the patient no longer requires

immunosuppression and has recovered from transplant-associated toxicity

Cytomegalovirus (CMV)

Monitoring viral reactivation

● Monitoring SOT and HCT patients for CMV reactivation using highly
sensitive nucleic acid amplification tests is an alternative
approach.

● Monitoring of CMV DNA load in HCT recipients should be performed

at least weekly for the first 3 months post hematopoietic stem
cell transplant and should continue to 6 to 12 months if the
patient has chronic graft versus host disease (GvHD) or prolonged
immunodeficiency.
Cytomegalovirus (CMV)
Hybrid approach

● A hybrid approach to reduce the incidence of late-onset CMV

disease uses antiviral prophylaxis that is followed by the
preemptive approach of monitoring viral loads and initiating
treatment in those who develop CMV reactivation above a predefined
viral threshold.

● CMV treatment should be continued until the quantitative viral

load declines below the predefined threshold or to an undetectable
level
Cytomegalovirus (CMV)
CMV Assays

● RNA targeted assays, though less sensitive than DNA, have been
developed to serve as a better indicator of clinical disease;
however, RNA is readily degraded and proper transport and
processing is critical.
● CMV DNA is rather stable in specimens and is rarely affected by
delayed sample processing, yet is less specific for active CMV
infection partly due to the ability of such assays to detect
latent viral DNA
● Quantitative CMV viral loads may be used to assess treatment
response utilizing viral load decline and viral load suppression
as indicators. Higher viral loads at diagnosis have been
associated with longer treatment duration.
Cytomegalovirus (CMV)
● Current treatment guidelines recommend viral eradication from
blood prior to discontinuing antiviral therapy.

● Use of assays calibrated to the WHO international reference

standard is supported by studies indicating that the presence of
detectable virus in the blood is highly associated with disease
relapse.

● Therefore, viral load suppression to levels < 137 (2.14 log10)

IU/ml as the end of treatment is significant of clinical disease
resolution
Epstein-Barr Virus

● Quantitative detection
○ commonly performed in T-cell-depleted allogeneic stem cell
transplant patients and many other disease conditions
● Use of EBV viral loads
○ enabled development of models for preemptive anti-B-cell
immunotherapy for EBV reactivation, and for reducing not only
the incidence of EBV lymphoproliferative disease (EBV-LPD), but
also the virus-related mortality
Epstein-Barr Virus

● Quantitative analyses of circulating EBV DNA in nasopharyngeal

carcinomas
○ demonstrated a positive correlation with disease stage and a
strong relationship with clinical events, as well as being of
prognostic importance
● For EBV-associated lymphomas
○ quantitative EBV DNA analysis has also been found to correlate
closely with clinical progress.
○ High variability existed until recently, when EBV quantitative
standards became available
Other Herpesviruses

● Quantitative viral load assays useful for establishing active virus

replication of other ubiquitous herpesviruses, like human HHV-6,
HHV-7, and HHV-8

● High rate of reactivation and asymptomatic excretion of these

viruses is difficult to correlate with clinical disease without the use of
quantitative assays, as qualitative assays cannot differentiate
latent from active viral infection
Other Herpesviruses

● HHV-6 in plasma likely originates from the lysis of infected

circulating blood cells as opposed to circulating cell-free viral
particles, thus quantification of HHV-6 DNA in whole blood has a
higher sensitivity for diagnosis of active HHV-6 infection
originating in lymphoid tissues rather than measuring DNA load in
purified peripheral blood mononuclear cells or plasma.
● HHV-6 viral load assays are not standardized or interchangeable;
furthermore, thresholds to guide treatment have not yet been
established. The literature supports a viral load of > 10^3 targets
per 10^6 PBMCs in stem cell transplant patients associated with
clinical symptoms.
BK Virus

● Widespread use of PCR for quantitation has confirmed that BK viruria

and viremia are reliably identified before the development of BK virus
associated nephropathy (BKVAN)
● Screening for persistent BK virus shedding, presence of decoy cells
in urine, or urine viral loads of > 107 copies/ml, has been shown to
identify patients at risk for developing BKVAN
● Monitoring peripheral blood for evidence of BK viremia through
quantitative real-time PCR testing is an important management tool
that allows for interventions that prevent nephropathy in renal allograft
patients.
 NAME OF THE PHARMACY

references

https://www.khanacademy.org/scienc
e/ap-biology/gene-expression-and-r
egulation/biotechnology/a/polymera
se-chain-reaction-pcr

https://geneticeducation.co.in/pol
ymerase-chain-reaction-pcr/

Specimen Requirements/Containers | Department of

Pathology | School of Medicine | University of California,
Irvine (uci.edu)
 GROUP 5

Arigatōgozaimasu!