Technical Method
Development and evaluation of real-time fluorescence recombinase aided amplification assay without extracting nucleic acid for detection of adenovirus type 3
Ruihua Wang, Yi Zhang, Xingyu Xiang, Zhifei Zhan, Xinna Li, Xinxin Shen, Zhen Zhu, Ruiqing Zhang, Xueding Bai, Qingxia Duan, Guohao Fan, Hong Zhang, Xuejun Ma
Published 2019-12-30
Cite as Chinese J Exp Clin Virol, 2019, 33(6): 653-657. DOI: 10.3760/cma.j.issn.1003-9279.2019.06.020
Abstract
ObjectiveTo establish a real-time fluorescence recombinase acid amplification (RAA) method for the detection of adenovirus type 3(HAdV-3)without extraction nucleic acid.
MethodsAccording to the conserved sequence of adenovirus type 3 gene, a pair of primers and a probe were designed, and a real-time fluorescence RAA without extracting nucleic acid was established and optimizing the condition of DNA-free extraction. The sensitivity of the method was analyzed by a series of dilution and the specificity of the method was evaluated by detecting the original samples of other respiratory viruses. The clinical samples of HAdV-3 were detected and compared with the traditional real-time fluorescence quantitative PCR method for nucleic acid extraction.
ResultsThe sensitivity of the real-time fluorescence RAA method was as high as that of qPCR in the detection of 10 series diluted HAdV-3 strains. The highest corresponding CT value of qPCR was 36.87. The sensitivity of the real-time fluorescence RAA method was similar to that of the real-time fluorescence quantitative PCR method . There was no cross-reaction to other common types of respiratory viruses. The two method were used to detect 56 clinical samples at the same time, and the result were completely consistent.
ConclusionsWe provide the first report of the real-time fluorescent RAA assays for the detection of HAdV-3 without extracting nucleic acid and it has high sensitivity and specificity. Is suitable for rapid detection of HAdV-3 in clinical laboratories and on-site unite.
Key words:
Human adenovirus-3; Non-extracting; Recombinase acid amplification; Detection
Contributor Information
Ruihua Wang
Hunan Provincial Center for Disease Control and Prevention, Changsha 410005, China
University of South China, College of Public Health, Hengyang 421001, China
Yi Zhang
NHC Key Laboratory of Medical Virology and Viral Diseases, National Insitute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Xingyu Xiang
Hunan Provincial Center for Disease Control and Prevention, Changsha 410005, China
Zhifei Zhan
University of South China, College of Public Health, Hengyang 421001, China
Xinna Li
NHC Key Laboratory of Medical Virology and Viral Diseases, National Insitute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Xinxin Shen
NHC Key Laboratory of Medical Virology and Viral Diseases, National Insitute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Zhen Zhu
NHC Key Laboratory of Medical Virology and Viral Diseases, National Insitute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Ruiqing Zhang
NHC Key Laboratory of Medical Virology and Viral Diseases, National Insitute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Xueding Bai
NHC Key Laboratory of Medical Virology and Viral Diseases, National Insitute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Qingxia Duan
NHC Key Laboratory of Medical Virology and Viral Diseases, National Insitute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Guohao Fan
NHC Key Laboratory of Medical Virology and Viral Diseases, National Insitute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Hong Zhang
Hunan Provincial Center for Disease Control and Prevention, Changsha 410005, China
Xuejun Ma
NHC Key Laboratory of Medical Virology and Viral Diseases, National Insitute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
