H7N9亚型禽流感病毒RT-PCR检测方法建立

doi:10.3864/j.issn.0578-1752.2015.15.015

Abstract

Abstract: 【Objective】On March 2013, the National Health and Family Planning Commission of China announced that human infections with H7N9 subtype AIV had occurred in Shanghai and Anhui province, China. Because this novel reassortant (H7N9) virus had not previously been seen in either animals or people, the situation raises many urgent questions and global public health concerns. In this study, two pairs of RT-PCR primers were designed to target the haemagglutinin (HA) and neuraminidase (NA) genes of H7N9 virus, and a reverse-transcription PCR assay to rapidly detect the novel H7N9 subtype avian influenza virus was developed and evaluated. 【Method】 H7 and N9 primers were chosen based on the conserved regions of sequences that were designed and analyzed using DNASTAR and oligo 6.0. one-step RT-PCR assay for the detection of H7N9 virus was established with RNA extracted by using one-step access RT-PCR kit. The specificity of the RT-PCR assay was evaluated with H7N9 influenza virus as positive control and Newcastle disease virus, infectious bronchitis virus, infectious bursal disease virus and other avian respiratory viral pathogens as negative control. After specificity test by using clear background influenza virus and other pathogens, the detection limits of the assay were assessed with serial 10-fold dilutions of H7N9 influenza virus (10^6.5 EID₅₀·mL^-1). The specificity assays were evaluated using influenza A viruses of various genetic backgrounds and other avian pathogen. The sensitivity assays were determined using viral RNA extracted from serially diluted AIV-infected allantoic fluid. In addition, a blinded experiment was carried out to validate the accuracy of this assay in comparison with the results of real-time fluorescence RT-PCR. 【Result】The H7 HA can be detected by this assay, while other H1-H6 and H8-H15 subtype HA, as well as other avian pathogens were detected negative in specificity assay. Similarly, only the N9 NA related to the novel H7N9 virus was detected, the other N1-N9 NA were detected negative. Results of 10-fold dilution series of allantoic fluid by one step RT-PCR assays showed that detection limit of the assay was approximately 1.4×10^2.5 EID₅₀ per reaction. Furthermore, the assays showed clinical specificity for identification cloacal swabs of H7N9 virus.【Conclusion】The RT-PCR assay established in this study can be used as a referee method for early diagnosis of the avian-origin influenza A (H7N9) virus infection.

Key words: avian influenza virus, H7N9 subtype, RT-PCR

WANG Yun-he, BAO Hong-mei, SUN Jia-shan, LI Yan-bing, XU Xiao-long, WANG Zi-long, SHI Jian-zhong, ZENG Xian-ying, WANG Xiu-rong, CHEN Hua-lan. Development of RT-PCR Technique for Detection of H7N9 Subtype Avian Influenza Virus[J].Scientia Agricultura Sinica, 2015, 48(15): 3050-3055.

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Development of RT-PCR Technique for Detection of H7N9 Subtype Avian Influenza Virus

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