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

摘要/Abstract

摘要： 【目的】2013年3月，中国国家卫生和计划生育委员会宣布在上海、安徽地区发现了人感染H7N9亚型流感病毒事件，由于这种新型重组H7N9流感病毒未曾有过感染人或者动物的报道，因此出现了一系列亟待解决的问题，引起了全世界范围的广泛关注。根据H7N9亚型禽流感病毒 HA和NA核苷酸序列，设计并合成靶基因为HA和NA的2对引物，建立快速检测H7N9亚型禽流感病毒的一步法RT-PCR检测方法。【方法】根据测序结果，用DNAStar生物软件进行同源性分析比较，选出H7和N9基因中高度保守且特异的核苷酸区域，用oligo6.0软件设计针对H7和N9基因的引物。用Trizol LS提取RNA，采用一步法Access RT-PCR扩增反应液，建立了一步法检测H7N9亚型禽流感病毒RT-PCR 方法。以H7N9亚型流感病毒为阳性对照，其他亚型流感病毒以及新城疫、传染性支气管炎、传染性法氏囊等其他禽病病原作为阴性对照，按所建立的反应体系和反应程序进行RT-PCR反应，验证所建立方法的特异性。对病毒含量为 10^6.5 EID₅₀·mL^-1 的 H7N9 亚型禽流感病毒尿囊液依次进行 10 倍倍比稀释，提取RNA用RT-PCR 方法检测，评价其敏感性。另外，采取双盲试验用荧光定量RT-PCR对该方法进行了比对验证。【结果】用H7亚型特异性引物检测 H1—H15 亚型禽流感病毒和鸡新城疫病毒等其他禽病病原，除H7亚型流感病毒外，其他样品均为阴性；用N9亚型特异性引物检测N1—N9 亚型禽流感病毒和其他禽病病原，仅当前流行的H7N9 亚型 AIV 样品有特异性目的条带，与其他N1—N9 亚型禽流感病毒和鸡新城疫病毒等其他禽病病原均无交叉反应。通过对 H7N9亚型禽流感病毒尿囊液进行10倍倍比稀释检测，证实该方法最低检出量为 1.4×10^2.5 EID₅₀·mL^-1，并可以从阳性棉拭子浸出液中扩增出目的基因片段。【结论】该RT-PCR 方法具有特异性强和准确率高的特点，可以作为H7N9亚型AIV核酸检测的一种有效候选方法。

关键词: 禽流感病毒, H7N9亚型, RT-PCR

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

王云鹤，包红梅，孙佳善，李雁冰，徐晓龙，王子龙，施建忠，曾显营，王秀荣，陈化兰. H7N9亚型禽流感病毒RT-PCR检测方法建立[J]. 中国农业科学, 2015, 48(15): 3050-3055.

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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