高致病性禽流感通用和H5亚型荧光定量PCR方法的建立

Rapid diagnoses of avian influenza virus and avian influenza H5 virus by real-time PCRs

目的:建立高致病性禽流感H5亚型荧光定量PCR方法.方法:根据禽流感病毒(AIV)M基因和H5-HA基因分别设计AIV通用和H5亚型特异荧光定量PCR引物和探针,优化反应体系和反应条件,分析该试剂的敏感性、特异性、重复性等.结果:经优化反应体系和反应条件后,AIV通用试剂和AIV-H5试剂的敏感性达到1×101.25EID50水平;与细胞培养比较,AIV-H5试剂Kappa吻合系数κ为0.991(P<0.01),统计学显示两种检测结果的吻合度很强;批内和批间精密度CT值的CV值均远小于10%,可重复性良好.结论:本项目研制的AIV通用试剂和AIV-H5试剂的敏感性、特异性、重复性均好,适合在禽流感疫情和疑似H5禽流感病例中检测使用.

AIM：To detect avian influenza virus subtype H5 by the real-time fluorescent quantitative PCRs were established.METHODS：It were designed that the primers and probes in universal assay based on AIV M gene and those in subtype H5 assay based on AIV H5 gene,and was optimized in a reaction system and a condition to improve the sensitivity,specificity and reproducibility.RESULTS：After optimization in the reaction system and the condition,the sensitivities of the universal assay and the H5 assay reached to 1×101.25 EID50;the Kappa coefficient between the cell culture and the Real time PCR of AIV H5 is 0.991（P〈0.01）,which statistically showed that there was the strong coincidence between two methods;the CV values of CT in the intra and inter batches were far less than 10%,which statistically showed that there was the excellent reproducibility.CONCLUSION：There were the excellent qualities of both assays in this studies in the sensitivity,specificity and reproducibility,which might been suggested for use to detect in avian influenza epidemic or human avian influenza H5 case.