One-step multiplex nested real-time RT-PCR assay for 2019-nCoV and Influenza A/B viruses detection
10.3760/cma.j.cn114452-20220301-00107
- VernacularTitle:一步法多重巢式荧光RT-PCR检测新型冠状病毒和甲、乙型流感病毒
- Author:
Kui ZHENG
1
;
Fangfang SUN
;
Cancan YAO
;
Jun DAI
;
Yongxia SHI
;
Xiaobo LI
;
Jicheng HUANG
Author Information
1. 广州海关技术中心卫生检疫研究所，广州 510403
- Keywords:
2019-nCoV;
Influenza virus;
Nucleic acid test;
Multiplex PCR
- From:
Chinese Journal of Laboratory Medicine
2022;45(11):1144-1149
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To develop a single-tube one-step multiplex nested real-time reverse transcription polymerase chain reaction (RT-PCR) assay for the simultaneous detection of 2019-nCoV, influenza A virus, influenza B virus and internal-control with human-derived gene.Methods:This study included 30 positive specimens for 2019-nCoV nucleic acid detection and 336 screening specimens collected from the arrivals at Guangzhou Baiyun Airport between February 2020 and February 2022. Sixty-four positive specimens of other respiratory pathogens were also collected from the arrivals at Guangzhou Baiyun Airport during the three-year period before the occurrence of COVID19 outbreak in 2020, and 7 positive viral strains of respiratory pathogens were provided by collaborative laboratories. In order to establish a set of multiplex nested real-time RT-PCR assay, a group of primers and probe combinations for a multiplex nested real-time RT-PCR was designed and screened according to a selection of nucleotide conserved regions of the ORF and N genes of 2019-nCoV and the M gene of influenza A and B viruses, while nested amplification primers and probe for the internal-control with human-derived gene were introduced. Then the prepared pseudovirus-positive quality control and sample discs were applied to evaluate the sensitivity and specificity. Clinical specimens were performed to validate the applicability of the method.Results:The results show that the established one-step multiplex nested real-time RT-PCR assay can specifically detect 2019-nCoV and influenza A and B viruses, with the limit-of-detection of about 125 copies/ml for 2019-nCoV and about 250 copies/ml for influenza A and B viruses. Totally 101 positive samples of various respiratory pathogens were detected, showing that the detection sensitivities of 2019-nCoV and influenza A and B viruses were 96.67%, 92.86% and 96.15%, respectively, with the specificity of 100%. No false-positive detection was found in the applied detection of more than 300 clinical samples.Conclusions:A one-step multiplex nested real-time RT-PCR assay for 2019-nCoV, influenza A and B viruses and human-derived gene internal-control was developed. The assay has good sensitivity and specificity and can be used for rapid screening of 2019-nCoV and influenza A and B viruses in high-volume samples.
