Lab 2a: Viral Nucleic Acid Tests

(NAT)

Reviewed by K. Francis, October 2021 1

 Nucleic acid tests (NAT)
• Nucleic Acid Tests (NAT) are
becoming increasingly
popular in the detection
and diagnosis of viral
infections.
• This technology has
reduced the use of some
other techniques such as
viral culture and serological
assays in the clinical
virology laboratory.
• Nucleic Acid Tests (NAT)
include techniques such as:
– polymerase chain reaction
(PCR)
– Reverse Transcription
polymerase chain reaction
(RT-PCR)
– real-time polymerase chain
reaction
– Reverse nucleic acid
sequencing
– genotyping
These methods have high
sensitivity and reproducibility.
 Polymerase Chain Reaction (PCR)
• PCR allows the synthesis of million of copies of a
targeted nucleic acid sequence.
• Now accepted as the gold standard for detecting
nucleic acids from a number of viruses.
• This chemical reaction occurs by means of the action of
a DNA polymerase that can copy a DNA strand.

• PCR is repeated cycling of three steps:

1. Denaturation
2. Annealing 1 cycle

3. Extension
• Each step happens at a different temperature
 1

2 4
 Polymerase Chain Reaction (PCR)
Detection
• After the PCR reaction, the product
of amplification can be detected
by:
– gel electrophoresis
– colorimetric detection -ELISA
Gel electrophoresis
• most commonly used
• results visualized by ethidium
bromide or non-toxic dyes such as
SYBR® green.
• The intensity of the band can be
used to estimate the amount of
product of given molecular weight
relative to a ladder.
• After electrophrasing, samples will
be separated to a number of bands
.
• Each band represents a fragment of 1. DNA ladder
DNA. 2. Positive Control
3. Negative Control
4. sample 1
5. Sample 2
 Variants of PCR
1. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
– RT-PCR is a PCR test that is designed to detect and measure RNA.
– Although initial PCR tests amplified DNA, many viruses and other
biological components (for example, influenza) utilize RNA as their
genetic material.
– RT-PCR differs from conventional PCR by first taking RNA and
converting the RNA strand into a DNA strand. (cDNA)
– This is done by essentially the same method for PCR described above
with the exception of using an enzyme termed reverse transcriptase
instead of the DNA polymerase.
– The reverse transcriptase allows a single strand of RNA to be
converted into a complementary strand of DNA (cDNA).
– Once that reaction occurs, the routine PCR method can then be used
to amplify the DNA.
– RT-PCR has been used to detect and study many RNA viruses.
 Variants of PCR
2. Real-time or Quantitative PCR (qPCR)
–Real-time or qPCR is used to quantify
the absolute or relative amounts of
target sequence in a sample.
–uses fluorescent dyes attached to
some of the small nucleotide strands
called probes.
–In this method, the target
amplification and detection steps
occur simultaneously
Real Time PCR

Reviewed by K. Francis, September 2016 9

 Interpreting real time PCR
• X-axis – PCR cycle number
• Y-axis – Fluorescence from the
amplification reaction

• Plot shows two phases:

1. exponential phase-the
experimental phase in which
the amount of PCR product
doubles with each cycle.

2. non-exponential phase –
consumption of one or more
component during the
reaction causes a plateau.
How to interpret results
• The Ct (cycle threshold) is
defined as the number of cycles
required for the fluorescent
signal to cross the threshold (ie
exceeds background level).
• Ct levels are inversely
proportional to the amount of
target nucleic acid in the sample
(ie the lower the Ct level the
greater the amount of target
nucleic acid in the sample).
– Cts < 29 are strong positive
reactions
– Cts of 30-37 are moderately
positive reactions
– Cts of 38-40 are weak reactions
RNA extraction
 Variants of PCR
2. Real-time or Quantitative PCR (qPCR)
–Real-time or qPCR is used to quantify
the absolute or relative amounts of
target sequence in a sample.
–uses fluorescent dyes attached to
some of the small nucleotide strands
called probes.
–In this method, the target
amplification and detection steps
occur simultaneously
Real Time PCR

Reviewed by K. Francis, September 2016 14

 Interpreting real time PCR
• X-axis – PCR cycle number
• Y-axis – Fluorescence from the
amplification reaction

• Plot shows two phases:

1. exponential phase-the
experimental phase in which
the amount of PCR product
doubles with each cycle.

2. non-exponential phase –
consumption of one or more
component during the
reaction causes a plateau.
How to interpret results
• The Ct (cycle threshold) is
defined as the number of cycles
required for the fluorescent
signal to cross the threshold (ie
exceeds background level).
• Ct levels are inversely
proportional to the amount of
target nucleic acid in the sample
(ie the lower the Ct level the
greater the amount of target
nucleic acid in the sample).
– Cts < 29 are strong positive
reactions
– Cts of 30-37 are moderately
positive reactions
– Cts of 38-40 are weak reactions
Record the Ct value for each sample
Interpret these Influenza A results
Read and interpret Adenovirus

Threshold

Pt
1
NC Pt. 2 PC
 Variants of PCR
3. Multiplex PCR
• In the same reaction mixture, two or more primer sets designed
for amplification of different targets are used.
• More than one target sequence in a clinical specimen can be co-
amplified in a single tube.
• However, the primers used must be carefully selected in order
that they have similar annealing temperatures and lack
complementarity.
• This kind of PCR is less sensitive than PCR with single primer set.
Multiplex PCR assays for viral respiratory pathogens and for
detection of viral infections of central nervous system have been
developed
 Samples for PCR
• Any material suspected of containing viral
genome.
• Sample must however be collected in acute state
of infection when viral load is highest.

Sample for Influenza PCR Sample for Zika PCR

• throat swab, • Serum
• nasopharyngeal swab, • Urine
• bronchial wash, • Salvia
• lung tissue,
• bronchial aspirate
 References
1. Leland,D., Ginocchio,C. 2007. Role of Cell
Culture for Virus Detection in the Age of
Technology. Clinical Microbiology Reviews,
20: 49–78

Reviewed by K. Francis, September 2016 24