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Updated: Oct 11, 2025
Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses
Published on: November 10, 2023
Smaller reaction volume of triplex taqman real-time reverse transcription-PCR assays for diagnosing coronavirus
Wenxue Dong1, Xu Yang1, Jing Li1
1Key Laboratory for Molecular Genetic Mechanisms and Intervention Research on High Altitude Disease of Tibet Autonomous Region, School of Medicine, Xizang Minzu University, Xianyang, China.
Insights
New TaqMan real-time PCR assays can accurately detect SARS-CoV-2, the virus causing COVID-19. These highly sensitive assays offer a reliable method for diagnosing COVID-19, aiding in disease control efforts.
Area of Science:
- Molecular Biology
- Virology
- Diagnostic Assays
Background:
- COVID-19 presents diagnostic challenges due to symptom overlap with other respiratory viruses.
- Accurate and rapid detection of SARS-CoV-2 is crucial for controlling the pandemic.
- No specific therapies currently exist, making early diagnosis paramount.
Purpose of the Study:
- To develop and validate novel TaqMan real-time PCR assays for detecting SARS-CoV-2.
- To establish sensitive and specific molecular diagnostic tools for COVID-19 identification.
Main Methods:
- Development of triplex TaqMan real-time PCR assays targeting ORF1ab, N, and E genes of SARS-CoV-2.
- Assessment of assay sensitivity down to 100 copies per PCR.
- Validation using RNA-based pseudovirus surveillance.
Main Results:
- Established PCR assays demonstrated high sensitivity for ORF1ab, N, and E genes (as low as 100 copies/PCR).
- Excellent linear correlations (R² > 0.99) were observed between Ct values and target gene copy numbers.
- Assays successfully identified SARS-CoV-2 in pseudovirus surveillance, confirming their utility.
Conclusions:
- The developed TaqMan real-time PCR assays provide a sensitive and specific method for COVID-19 diagnosis.
- These assays offer a smaller reaction volume, potentially improving efficiency in diagnostic settings.
Background:
Coronavirus disease 2019 (COVID-19) has had a devastating impact on public health services worldwide. Currently, there are no standard remedies or therapies for COVID-19. it is important to identify and diagnose COVID-19 to control the spread. But clinical symptoms of COVID-19 are very similar to those of other respiratory viruses.
Results:
As a result, the diagnosis of COVID-19 relies heavily on detecting pathogens. We established a bunch of triplex new TaqMan real-time PCR assays. Three sets of primers and probes (targeting the ORF1ab, N, and E genes, respectively) are poorly consistent with other human coronaviruses and the human influenza virus. The sensitivity of established PCR assays notices as few as 100 copies per PCR of the ORF1ab, N, and E genes. Meanwhile, standard curves concluded from constant PCR reaction all showed glorious linear correlations between Ct values and the polymer loading copy variety (correlation coefficient (R2 ) of ORF1ab, N, and E genes is 0.996, 0.991, and 0.998, respectively). Surveillance of RNA-based pseudovirus demonstrated that they were identified to be positive with respect to SARS-CoV-2 and that established PCR assays are achievable.
Conclusion:
The assays established provide a smaller reaction volume for diagnosing COVID-19.
