H1亚型猪流感病毒微滴数字RT-PCR定量检测方法的建立

Establishment of a Droplet Digital RT-PCR Method for Detection of H1 Subtype Swine Influenza Virus

本研究针对H1亚型猪流感病毒(swineinfluenzavirus,SIV)HA基因保守序列设计引物和探针,通过反应条件优化,建立了一种快速、准确检测H1亚型SIV的微滴数字RT-PCR定量方法。该方法特异性强,除H1亚型SIV外,H3、H5、H7亚型SIV以及猪瘟病毒、猪繁殖与呼吸综合征病毒等检测均为阴性;敏感性高,最低可检测4.7拷贝/μL;重复性好,对5个不同稀释度的H1亚型SIVRNA进行3次重复试验,每个稀释度的变异系数均小于5%。利用该方法对湖南省规模化养殖场收集的30份猪鼻拭子样品进行检测,发现与荧光RT-PCR检测结果一致。试验表明,该方法快速、准确、灵敏,可为H1亚型SIV的快速筛查及流行病学研究提供可靠的技术保障。

Based on the conservative sequence of HA gene of H1 subtype swine influenza virus(SIV),related primers and probes were designed,after optimization of the reaction conditions,a rapid and accurate droplet digital RT-PCR method was established for detection of H1 subtype SIV. The established method was with strong specificity, only H1 subtype SIV could be detected,while H1,H3,H5 and H7 subtype SIV,classical swine fever virus(CSFV) and porcine reproductive and respiratory syndrome virus(PRRSV)couldn't;the detection limit was 4.7 copies/μL, showing high sensibility;its reproducibility was also good,the variation coefficients of every dilution were all less than 5% when five different dilutions of SIV RNA were tested repeated for three times. Using the method,30 samples of pig nose swabs collected from scaled farms in Hunan Province were tested,and it was found that the result was consistent with that of real-time PCR. In short,it was concluded that reliable technical supports could be provided for rapid screening of H1 subtype SIV and relevant epidemiological study by the established method that was rapid,accurate and sensitive.