禽流感病毒H9N2亚型三重PCR检测方法的建立

›› 2014, Vol. 41 ›› Issue (11): 58-62.

禽流感病毒H9N2亚型三重PCR检测方法的建立

徐倩¹, 谢芝勋², 谢丽基², 罗思思², 黄莉², 黄娇玲², 曾婷婷², 谢志勤², 邓显文²

1. 广西大学动物科技学院, 广西南宁 530004;
2. 广西壮族自治区兽医研究所, 广西畜禽疫苗新技术重点实验室, 广西南宁 530001

收稿日期:2014-07-18 出版日期:2014-11-20 发布日期:2014-12-06
通讯作者: 谢芝勋 E-mail:xiezhixun@126.com
作者简介:徐倩(1987-),女,河北人,硕士生,研究方向: 兽医生物技术。
基金资助:
广西特聘专家专项经费(2011B020);广西自然科学基金(2013GXNSFDA019015);广西科技攻关重大专项(1222003-2-4);桂渔牧科(14-1)。

Development of a Triplex RT-PCR Assay for Detection of Avian Influenza Virus H9N2 Subtype

XU Qian¹, XIE Zhi-xun², XIE Li-ji², LUO Si-si², HUANG Li², HUANG Jiao-ling², ZENG Ting-ting², XIE Zhi-qin², DENG Xian-wen²

1. College of Animal Science and Technology, Guangxi University, Nanning 530004, China;
2. Guangxi Key Laboratory of Animal Vaccines and Diagnostics, Guangxi Veterinary Research Institute, Nanning 530001, China

Received:2014-07-18 Online:2014-11-20 Published:2014-12-06

摘要/Abstract

摘要： 为建立简便快速检测禽流感病毒(avian influenza virus,AIV)并同时区分出H9、N2亚型的方法,本试验根据基因库中H9亚型AIV的HA基因、N2亚型AIV的NA基因及AIV的M基因序列,分别设计了3对针对这3种基因保守序列的引物,建立了AIV H9N2亚型的三重PCR检测方法。应用该方法对H9N2亚型AIV模板进行PCR扩增,可得到3条与试验设计相符的目的条带,分别为313 bp (HA基因)、451 bp (NA基因)和667 bp(M基因);对非H9亚型的N2亚型AIV模板进行扩增,出现2条特异性扩增条带,即451 bp (NA基因)和667 bp(M基因);对非H9、N2亚型AIV模板进行扩增则只出现一条目的条带,即667 bp(M基因);对其他禽呼吸道病原体进行PCR扩增,结果均为阴性。敏感性试验结果显示此三重PCR方法最低检出限为10^-2 ng/μL。应用所建立的三重PCR方法对120份临床病料进行检测的结果与病毒分离鉴定结果一致。各项试验结果均表明,该方法对于禽流感病毒尤其是H9、N2亚型禽流感病毒的检测具有快捷、特异、灵敏的特点。

关键词: 禽流感病毒; H9亚型; N2亚型; M基因; 三重PCR

Abstract: A triplex reverse transcription-polymerase chain reaction (triplex RT-PCR) was developed to detect avian influenza viruses (AIVs) and H9 N2 subtype AIVs simultaneously. Three pairs of specific primers were designed according to the conserved regions on the sequences of H9 AIV HA gene, N2 AIV NA gene and AIV M gene in GenBank. It showed that all samples containing H9N2 subtype AIV could be amplified into three specific bands, 313 bp for HA gene, 451 bp for NA gene and 667 bp for M gene by this triplex PCR. All samples containing N2 subtype AIV with different HA genes (not H9) could be amplified into two specific bands, 451 bp for N2 subtype AIV and 667 bp for M gene. All samples containing other subtypes of AIVs (not H9 or N2) could be amplified into one specific band, 667 bp for M gene. No specific bands of the same sizes were amplified from genomic materials of other avian pathogens. The detection limit of triplex PCR was 10^-2 ng/μL. The results of triplex PCR for detection of clinical samples were coincident with that of the viral isolation completely. Our results demonstrated that the optimized triplex PCR assay was quick, specific and sensitive for detection of AIVs especially H9 and N2 subtype AIV.

Key words: avian influenza virus; H9 subtype; N2 subtype; M gene; triplex PCR

中图分类号:

S852.65

徐倩, 谢芝勋, 谢丽基, 罗思思, 黄莉, 黄娇玲, 曾婷婷, 谢志勤, 邓显文. 禽流感病毒H9N2亚型三重PCR检测方法的建立[J]. , 2014, 41(11): 58-62.

XU Qian, XIE Zhi-xun, XIE Li-ji, LUO Si-si, HUANG Li, HUANG Jiao-ling, ZENG Ting-ting, XIE Zhi-qin, DENG Xian-wen. Development of a Triplex RT-PCR Assay for Detection of Avian Influenza Virus H9N2 Subtype[J]. , 2014, 41(11): 58-62.

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