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H9亚型禽流感病毒实时荧光RT-PCR检测方法的建立与应用

5716 2019-12-16 08:37:29 王楷宬,王素春,樊擎莹,等 中国动物检疫
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为建立一种快速准确检测H9亚型禽流感病毒基因的检测方法,根据GenBank中H9亚型禽流感病毒血凝素编码基因进行荧光检测引物和探针设计,建立了一种针对H9亚型禽流感病毒的荧光RT-PCR检测方法。利用该方法进行灵敏度和特异性检测,并用本方法和国家标准中的荧光RT-PCR检测方法,同时对临床样本进行检测。结果显示,仅H9亚型禽流感病毒出现了正常荧光检测曲线,而其他病毒及阴性对照均未出现;检测灵敏度为RNA终浓度10-4 ng/μL(1.30×104 copies/μL);临床样本检测结果与国标方法一致,符合率为100%。结果表明,该方法特异性强、灵敏度高,可用于H9亚型禽流感病毒检测。

Establishment and Application of a Real-time RT-PCR Assay for Detection

of H9 Subtype Avian Influenza Virus

In order to establish a method for detecting H9 subtype avian influenza virus(AIV-H9)rapidly and accurately,the fluorescence detection primers and probes were designed and synthesized according to hemagglutinin encoding gene of AIV-H9 registered in GenBank,and a fluorescence PCR assay was thereby established,and its specificity and sensitivity were tested. Then by the established assay and the RT-PCR stipulated in national standard,some clinical samples were simultaneously tested. The results showed that the normal fluorescence detection curve only appeared in AIV-H9 rather than other non-specific viruses and negative sample;its detection limit was 10-4 ng/μL(1.30×104 copies/μL)of RNA;the detection result of clinical samples by established assay was consistent with the national standard,and the coincidence rate was 100%. In conclusion,the assay could be used to detect AIV-H9 due to its strong specificity and high sensitivity.

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