Smaller reaction volume of triplex taqman real‐time reverse transcription‐PCR assays for diagnosing coronavirus disease 2019

• Published in: Journal of Clinical Laboratory Analysis 2022-01, Vol.36 (1), p.e24137-n/a
• Main Authors: Dong, Wenxue, Yang, Xu, Li, Jing, Zhang, Zhiying, Liu, Lijun, Zhao, Zhipeng, Kang, Longli
• Format: Article
• Language: English
• Subjects: Biotechnology; Coronaviruses; COVID-19; COVID-19 Nucleic Acid Testing - methods; Disease transmission; DNA Primers; DNA probes; Gene banks; Genetic testing; Genomes; Humans; Influenza; Middle East respiratory syndrome; nucleic acid testing; Plasmids; Polymerase chain reaction; Polymers; Public health; Real-Time Polymerase Chain Reaction - methods; Regression analysis; Reproducibility; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction - methods; Reverse transcription; RNA, Viral; RT‐PCR; SARS-CoV-2 - genetics; SARS‐CoV‐2; Sensitivity and Specificity; Severe acute respiratory syndrome coronavirus 2; Thermal cycling; Viruses
• ISSN: 0887-8013; 1098-2825
• DOI: 10.1002/jcla.24137

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. In this work, 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. Surveillance of RNA‐based pseudovirus demonstrated that they were identified positive with respect to SARS‐CoV‐2, and the established PCR assays is achievable. The assays established provides a smaller reaction volume for diagnosing COVID‐19.