Development and evaluation of a panel of multiplex one‐tube nested real time PCR assay for simultaneous detection of 14 respiratory viruses in five reactions. Issue 12 (14th February 2020)

• Record Type: Journal Article
• Authors: Zhao, Li; Li, Gui‐xia; Wang, Ji; Zhao, Meng‐chuan; Wang, Le; Feng, Zhi‐shan; Ma, Xue‐jun
• Is Part Of: Journal of medical virology. Volume 92:Issue 12(2020)
• Page Start: 3073
• Page End: 3080
• Publication Date: 2020-02-14
• Subjects: locked nucleic acid (LNA) -- multiplex one‐tube nested real‐time PCR (mOTNRT‐PCR) -- respiratory virus; Virology -- Periodicals; 616
• DOI: 10.1002/jmv.25686
• Languages: English
• ISSNs: 0146-6615

Abstract

Abstract: Multiplex real‐time quantitative polymerase chain reaction (mRT‐qPCR) assay is commonly used to detect respiratory viruses, however, the sensitivity is limited for most reports. A panel of locked nucleic acid based multiplex closed one‐tube nested real‐time PCR (mOTNRT‐PCR) assay consisting of five separate internally controlled RT‐qPCR assays was developed for detection of 14 respiratory viruses. The sensitivity and reproducibility of mOTNRT‐PCR panel were evaluated using plasmid standards and the specificity was evaluated using clinical samples. The clinical performance of mOTNRT‐PCR panel was further evaluated with 468 samples collected from patients with an acute respiratory infection and compared with individual real‐time PCR (RT‐qPCR) assay. The analytical sensitivities of mOTNRT‐PCR panel ranged from 2 to 20 copies/reaction, and no cross‐reaction with common respiratory viruses was observed. The coefficients of variation of intra‐assay and inter‐assay were between 0.35% and 8.29%. Totally 35 clinical samples detected by mOTNRT‐PCR assay panel were missed by RT‐qPCR and confirmed true positive by sequencing of nested PCR products. The mOTNRT‐PCR assay panel provides a more sensitive and high‐throughput method for the detection of 14 respiratory viruses. Research Highlights: A panel of mOTNRT‐PCR consisted of five separate internally controlled RT‐qPCR assay. Locked nucleic acid was used to develop the mOTNRT‐PCR assay panel. The sensitivity of mOTNRT‐PCR panel ranged from 2‐20copies/reaction using plasmid. mOTNRT‐PCR was more sensitive than reported individual RT‐qPCR assay.