H1亚型禽流感病毒RT-RPA-LFD快速检测方法的建立及应用

Development of Rapid Detection Method for H1 Subtype Avian Influenza Virus RT-RPA-LFD

以H1亚型AIV的HA基因为研究对象,采用重组聚合酶核酸等温扩增技术(RPA),通过RT-PCR方法优选出1套引物,进而优化体系的反应温度、最佳探针及引物浓度,使用优化后的最佳反应体系进行敏感性及特异性试验,并探索该方法能达到的最短反应时间,利用侧向流动免疫(LFD)试纸条观测反应后的结果,最终建立检测H1亚型AIV的RT-RPA-LFD快速检测方法。结果表明:建立的RT-RPA-LFD快速检测方法在探针工作浓度为0.14 mmol/L,37℃条件下反应20 min,可最低检测到1.5×10^(2)copies/反应的H1亚型AIV RNA,其他常见家禽病原体未见阳性反应。该方法与RT-PCR及鸡胚分离鉴定测序结果完全一致,可满足H1亚型AIV临床快速鉴别诊断的需求,为基层兽医现场快速检测提供技术支撑。

Taking the HA gene of H1 subtype AIV as the research object,recombinant polymerase nucleic acid isothermal amplification technology(RPA)was used to select a set of primers by RT-PCR method,and the temperature of the reaction system and the probe and primer concentration were optimized.The sensitivity and specificity of the method were tested using the optimized reaction system,and the optimized system was used to explore the shortest reaction time.Based on the results of lateral flow dipstick(LFD),the RT-RPA-LFD rapid detection method for H1 subtype AIV was established.The results showed that the rapid RT-RPA-LFD assay for detection of H1 subtype AIV RNA could detect 1.5×10^(2)copies/response of H1 subtype AIV RNA at the probe working concentration of 0.14 mmol/L and 37℃for 20 min,and no positive reaction was found for other common avian pathogens,which was consistent with the results of RT-PCR and chicken embryo isolation and sequencing.The rapid detection method can meet the need of rapid clinical differential diagnosis of H1 subtype AIV,and provide technical support for the rapid detection of grass-roots veterinary field.