RTPCR

1. G. Brindha
I MSc MICROBIOLOGY
Reverse transcription polymerase chain reaction

2. What is RT-PCR?
The most sensitive technique available for
detecting and quantifying mRNA is called
RT-PCR,
also called Reverse transcription
polymerase chain reaction and RNA PCR..

3. • The method uses an enzyme called reverse
transcriptase (proper name is RNA dependent
DNA polymerase) to synthesize a
complementary strand of DNA
(complementary DNA, or cDNA) from an RNA
template.
• The resulting cDNA is then used as template in
a PCR assay using DNA polymerase. Most
clinical microbiology laboratories use RT-PCR
assays to detect RNA viruses from clinical
specimens.

4. Principle of RT-PCR
• Reverse transcription and PCR amplification
can be performed as a two-step process in a
single tube or with two separate reactions. In
both cases, RNA is first reverse-transcribed
into cDNA, which is then used as the template
for PCR amplification.
• The primers used for cDNA synthesis can be
either non–sequence-specific primers (a
mixture of random hexamers or oligo-dT
primers) or sequence-specific primers.

7. Steps of RT-PCR
• The initial step of RT-PCR entails synthesizing a
cDNA complementary to RNA transcript with
reverse transcriptase.
• Many commercial reverse transcriptases are
blended enzymes that have different
properties, including enzymes that have
reverse transcriptase and DNA polymerase
features.

8. Steps of RT-PCR
• Thus, the same enzyme can be used in one-tube
RT-PCR. Many reverse transcriptases function
best at 42° C, so the first step of many RT-PCR
assays is incubation at 42° C for 30 minutes.
• Following this step, the temperature is raised to
95° C for 1 to 5 minutes to denature DNA and, for
several commercial enzymes, inactivate the
reverse transcriptase function of the enzyme.
• Raising the temperature also activates the DNA
polymerase function of blended enzymes.
• Standard PCR cycling is then performed and the
PCR products are analyzed.

9. One-step RT-PCR
• One-Step reagents are used to directly
amplify RNA samples on your real-time PCR
instrument. The reverse transcription (RT) and
qPCR steps are both conducted in the same
reaction well. ... One-step RT-PCR is most
commonly used to process many samples at a
time, for one or a few targets per sample.

10. What is two-step RT-PCR?
• With the two-step approach, the reverse
transcription of the RNA template is
performed first. Once completed, the
amplification of the cDNA is carried out in a
separate reaction.

11. Uses of RT-PCR
• RT-PCR is often used in clinical microbiology
laboratories to detect RNA viruses from
clinical specimens.
• RT-PCR can be used to quantify the amount of
viruses in clinical specimens
• Other applications of RT-PCR include
quantitative analysis of gene expression,
detection of human genes involved in
diseases, and detection of cancers from
human specimens.

12. What is a COVID-19 RT-PCR test?
• The polymerase chain reaction (PCR) test for COVID-
19 is a molecular test that analyzes your upper
respiratory specimen, looking for genetic material
(RNA) of SARS-CoV-2, the virus that causes COVID-
19.
• Scientists use the PCR technology to amplify small
amounts of RNA from specimens into DNA
• The PCR test has been the gold standard test for
diagnosing COVID-19 since authorized for use in
February 2020. It’s accurate and reliable.

13. Three steps to the COVID-19 PCR test:
Sample collection
The type of specimen collected when testing
Nasopharyngeal and Oropharyngeal
• Extraction
• PCR

14. RNA Extraction

15. PCR
..PCR..