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Related Concept Videos

Real Time RT-PCR02:57

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...
Single Nucleotide Polymorphisms-SNPs01:05

Single Nucleotide Polymorphisms-SNPs

A single nucleotide polymorphism or SNP is a single nucleotide variation at a specific genomic position in a large population. It is the most prevalent type of sequence variation found in the human genome. Point mutations that occur in more than 1% of the population qualify as SNPs. These are present once every 1000 nucleotides on an average in the human genome. Replacement of a purine with another purine (A/G) or a pyrimidine with another pyrimidine (C/T) is known as a transition. In contrast,...
Viral Mutations00:36

Viral Mutations

A mutation is a change in the sequence of bases of DNA or RNA in a genome. Some mutations occur during replication of the genome due to errors made by the polymerase enzymes that replicate DNA or RNA. Unlike DNA polymerase, RNA polymerase is prone to errors because it is not capable of “proofreading” its work. Viruses with RNA-based genomes, like HIV, therefore accrue mutations faster than viruses with DNA-based genomes. Because mutation and recombination provide the raw material for adaptive...
Coronavirus01:29

Coronavirus

Coronaviruses, including the severe acute respiratory syndrome coronavirus (SARS-CoV), are enveloped viruses characterized by their single-stranded, positive-sense RNA genome and helical nucleocapsid structure. The hallmark of these viruses is their club-shaped spike (S) glycoproteins that protrude from the viral envelope, facilitating attachment to host cells. Typically, coronaviruses infect the upper respiratory tract, often causing mild or asymptomatic disease. However, certain strains like...

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Related Experiment Video

Updated: Jun 19, 2026

Detecting SARS-CoV-2 Virus by Reverse Transcription-Loop-Mediated Isothermal Amplification
05:34

Detecting SARS-CoV-2 Virus by Reverse Transcription-Loop-Mediated Isothermal Amplification

Published on: September 8, 2023

SARS-CoV-2 Evolution and Its Implications for RT-PCR Diagnostic Performance.

Shubhangi Gupta1, Abhishek Chaudhary1, Sonika Bhatnagar1

  • 1Department of Biological Sciences and Engineering, Computational and Structural Biology Laboratory, Netaji Subhas University of Technology, Dwarka, New Delhi, India.

Journal of Medical Virology
|June 18, 2026
PubMed
Summary
This summary is machine-generated.

SARS-CoV-2 mutations can cause false negatives in RT-PCR tests. This study analyzed primer/probe targets, finding some genes like Orf1b-nsp14 are mutation-resilient, recommending monitoring and redesign for diagnostic accuracy.

Keywords:
RT‐PCRSARS CoV‐2diagnostic impactmutation ratesprimer/probe targets

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Last Updated: Jun 19, 2026

Detecting SARS-CoV-2 Virus by Reverse Transcription-Loop-Mediated Isothermal Amplification
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Detecting SARS-CoV-2 Virus by Reverse Transcription-Loop-Mediated Isothermal Amplification

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07:54

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

Published on: March 31, 2021

Area of Science:

  • Virology
  • Molecular Biology
  • Genomics

Background:

  • Mutations in SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) primer and probe targets can reduce the sensitivity of Reverse Transcription Polymerase Chain Reaction (RT-PCR) assays.
  • This reduced sensitivity can lead to false-negative results, hindering the detection of viral transmission and effective public health responses.

Purpose of the Study:

  • To evaluate the mutational landscape of SARS-CoV-2 primer/probe targets across different genes, variants, and time periods.
  • To identify high-risk and moderate-risk mutations impacting RT-PCR diagnostic accuracy.
  • To propose strategies for enhancing the reliability of SARS-CoV-2 RT-PCR testing.

Main Methods:

  • Analysis of 135,852 high-quality SARS-CoV-2 genomes from ten countries, five major variants, and three time periods (pre-vaccination, post-vaccination, recent).
  • Evaluation of 20 primer/probe sets targeting E, N, Orf1b-nsp14, RdRp, and S genes.
  • Classification of mutations as high-risk or moderate-risk based on positional susceptibility and mismatch burden; calculation of mutation rate, impact risk, and population frequency.

Main Results:

  • Most primer/probe targets for E, N, RdRp, and S genes exhibited increasing and geographically variable mutation rates over time, particularly pronounced in Omicron variants.
  • High-risk, high-frequency mutations were identified in targets for N-CCDC-forward primer, N-NIH-reverse primer, RdRp-Charité-forward primer, S-Young-forward primer, and S-Sigma (S1/S2)-probes.
  • Orf1b-nsp14 and specific N-gene targets (N-Chan, N-NIID, N-UCDC (N2)) demonstrated mutation resilience, suggesting sustained diagnostic reliability.

Conclusions:

  • Mutation accumulation in SARS-CoV-2 RT-PCR targets varies significantly by gene, variant, geography, and time, impacting diagnostic sensitivity.
  • Certain primer/probe sets are more susceptible to mutations, while others, like Orf1b-nsp14, remain reliable.
  • A strategy involving regular mutation monitoring, primer/probe redesign when mutation frequency reaches 10%, and use of mutation-resilient targets is recommended to maintain SARS-CoV-2 diagnostic accuracy.