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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
Author(s) -
Zhao Li,
Li Guixia,
Wang Ji,
Zhao Mengchuan,
Wang Le,
Feng Zhishan,
Ma Xuejun
Publication year - 2020
Publication title -
journal of medical virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 121
eISSN - 1096-9071
pISSN - 0146-6615
DOI - 10.1002/jmv.25686
Subject(s) - multiplex , real time polymerase chain reaction , virology , multiplex polymerase chain reaction , biology , polymerase chain reaction , reproducibility , respiratory system , microbiology and biotechnology , chemistry , chromatography , gene , bioinformatics , genetics , anatomy
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.