Detection of avian influenza A/H7N9/2013 virus by real-time reverse transcription-polymerase chain reaction

• Published in: Journal of virological methods 2014-09, Vol.206, p.140-143
• Main Authors: Kang, Xiaoping, Wu, Weili, Zhang, Chuntao, Liu, Licheng, Feng, Huahua, Xu, Lizhi, Zheng, Xin, Yang, Honglei, Jiang, Yongqiang, Xu, Bianli, Xu, Jin, Yang, Yinhui, Chen, Weijun
• Format: Article
• Language: English
• Subjects: Avian influenza A/H7N9 virus; China; Humans; Influenza A Virus, H7N9 Subtype - genetics; Influenza A Virus, H7N9 Subtype - isolation & purification; Influenza, Human - diagnosis; Influenza, Human - virology; Molecular Diagnostic Techniques - methods; Pharynx - virology; Real-Time Polymerase Chain Reaction - methods; Real-time reverse transcription polymerase chain reaction (rRT-PCR); Reverse Transcriptase Polymerase Chain Reaction - methods; Sensitivity and Specificity
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2014.01.026

•We have developed the sensitive and specific assay for detecting influenza A virus H7N9.•It could efficiently differentiate the H7N9 infection from other infection with Influenza A virus.•The assay can detect influenza A virus H7N9 loads below 3.2×10−4HAUs. The first case of avian influenza A/H7N9 infection was reported in Shanghai in mid-February, 2013; by May 1, 2013, it had infected 127 people and caused 26 deaths in 10 provinces in China. Therefore, it is important to obtain reliable epidemiological data on the spread of this new infectious agent, a need that may be best met by the development of novel molecular methods. Here, a new method was described for the detection of avian influenza A/H7N9 using real-time reverse transcription-polymerase chain reaction (rRT-PCR). Using serial dilutions of avian influenza A H7N9 cultures, the detection limit of the assay was determined to be approximately 3.2×10−4 HAUs (hemagglutination units) for the H7 gene and 6.4×10−4 HAUs for N9 gene. In tests of serial dilutions of in vitro-transcribed avian influenza A H7 and N9 gene RNA, positive results were obtained for target RNA containing at least three copies of the H7 gene and six copies of the N9 gene. Thirteen throat swabs from H7N9 patients were tested; all tested positive in the assay. Specificity was evaluated by testing 18 other subtypes of influenza viruses; all tested negative. A total of 180 throat swabs from patients infected with influenza virus, including 60 from patients infected with seasonal influenza A/H1N1 virus, 60 from patients infected with pandemic influenza A/H1N1/2009 virus, 30 from patients infected with seasonal influenza A/H3N2 virus and 30 from patients infected with influenza B virus, were also tested; all tested negative.