摘要
目的建立特异敏感的荧光定量PCR (FQ-PCR)方法,用于新型H7N9流感病毒的快速检测。方法针对GenBank中新型H7N9流感病毒(2016年以后)的血凝素基因和神经氨酸酶基因序列的保守区域分别设计两对特异性引物和两条探针,经优化反应条件,建立一种基于TaqMan MGB探针技术的FQ-PCR方法,验证方法的敏感性、特异性和稳定性,对2017年采集的临床疑似感染样品进行检测,并与普通PCR方法进行比较分析。结果本研究成功建立的新型H7N9FQ-PCR检测方法在101～106拷贝/μl模板范围内有很好的线性关系,所得相关系数为0.999;对人工合成的H7和N9基因出现阳性扩增信号,但对鸡传染性法氏囊病病毒、鸡传染性支气管炎病毒、H1N2、H3N2、H6N2、H9N2流感病毒等病原对照未出现扩增;批内、批间试验变异系数在0.02%～0.05%;对H7和N9的最低检测模板浓度均为10拷贝/μl,比普通PCR方法的灵敏度高100倍;自30份疑似样品中检出4份阳性样品,与国家禽流感参考实验室复核结果一致。结论本研究成功建立了H7N9流感病毒双重FQ-PCR检测方法,为禽相关临床样本的新型H7N9早期检测提供了特异、敏感、快速的方法。
Objective To establish a sensitive real-time fluorescence quantitative RT-PCR method for the detection of novel H7N9 influenza virus. Methods Two sets of specific primers and probes were designed based on the sequences of HA and NA of novel H7N9 influenza virus(after 2016)available in GenBank.A duplex real-time RT-PCR assay based on TaqMan MGB probe technique was established.The sensitivity,specificity and stability of FQ-PCR were tested,and 30 clinically suspicious H7N9 infected samples were detected by the FQ-PCR assay and simultaneously compared with the conventional PCR method. Results The FQ-PCR assay showed high linear correlation at the template range of 101-106 copies/μl(linear correlation coefficient0.999);the amplicons of the FQ-PCR assay were on the H7 and N9genes while no other pathogens were amplified;the sensitivity of the assay was 10copies/μl which is 100 times higher than that of conventional PCR.Four positive samples were detected and the results were consistent with the national avian influenza reference laboratory(NAIRL). Conclusions A specific,sensitive and rapid duplex FQ-PCR assay was successfully established to detect novel H7N9 influenza virus.
作者
刘淑敏
王淑娟
班付国
赵明扬
王东方
赵雪丽
谢彩华
马震原
刘梅芬
闫若潜
LIU Shu-min;WANG Shu-juan;BAN Fu-guo;ZHAO Ming-yang;WANG Dong-fang;ZHAO Xue-li;XIE Cai-hua;MA Zhen-yuan;LIU Mei-fen;YAN Ruo-qian(Central Hospital of Zhengzhou City,Zhengzhou,Henan 450007,China)
出处
《中国病毒病杂志》
CAS
2019年第1期45-50,共6页
Chinese Journal of Viral Diseases
