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Real Time RT-PCR

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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Related Experiment Video

Updated: Dec 10, 2025

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Published on: March 31, 2021

SARS-CoV-2 Detection using Real Time PCR by a Commercial Diagnostic Kit.

S Roy¹, S K Paul, T K Barman

¹Dr Sangjukta Roy, Lecturer, Department of Microbiology, Mymensingh Medical College (MMC), Mymensingh, Bangladesh;

Mymensingh Medical Journal : MMJ

|August 27, 2020

Summary

This summary is machine-generated.

A diagnostic kit effectively detected SARS-CoV-2 in Bangladesh laboratories. The real-time PCR test showed high sensitivity for identifying the virus, crucial for public health during the COVID-19 outbreak.

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Area of Science:

* Medical Microbiology
* Virology
* Public Health

Background:

* The emergence of SARS-CoV-2 caused a global public health crisis, leading to COVID-19.
* Limited virus isolates presented challenges for public laboratories in accurate SARS-CoV-2 detection.
* Early outbreak assessments indicated a wider spread than initially perceived.

Purpose of the Study:

* To evaluate the diagnostic performance of a specific real-time PCR kit for SARS-CoV-2 detection.
* To assess the kit's effectiveness in a clinical laboratory setting in Bangladesh.

Main Methods:

* A Novel Corona virus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing), a real-time reverse transcription polymerase chain reaction (rRT-PCR) test, was utilized.
* A total of 230 samples were analyzed from the microbiology department of Mymensingh Medical College.
* Detection of ORF 1ab, N gene, and IC gene was performed using specific fluorescent dyes (FAM, ROX, CY5).

Main Results:

* Out of 230 samples, 20 (8.69%) tested positive for SARS-CoV-2.
* Among positive samples, 55% amplified both ORF 1ab and N genes; 15% and 30% amplified only ORF 1ab or N gene, respectively.
* High amplification rates were observed for ORF 1ab (92.8%) and N gene (100%) with Ct values <38. Internal Control (IC) gene showed 70% positivity in positives and 100% in negatives.

Conclusions:

* The study kit demonstrated significant capacity for detecting SARS-CoV-2 in laboratory settings.
* The rRT-PCR diagnostic kit is a reliable tool for identifying SARS-CoV-2 in Bangladesh.
* The findings support the use of this kit for routine diagnostics during the COVID-19 pandemic.