H9N2亚型禽流感病毒和新城疫病毒混合感染环介导间接PCR鉴别方法的建立与评估

Establishment and evaluation of loop-mediated indirect PCR assay for detecting mixed infection of H9N2 subtype avian influenza virus and Newcastle disease virus

根据GenBank中禽流感病毒(AIV)的HA基因和新城疫病毒(NDV)的F基因序列保守性,分别设计2条序列相邻的特异性探针,标记于不同大小的拟南芥TOC1基因片段两端,作为报告基因,建立了AIV和NDV双重环介导间接PCR鉴别方法,AIV和NDV扩增片段大小分别为200,270 bp。结果表明,所建立的方法对AIV和NDV核酸样品可扩增出特异性目的片段,而传染性法氏囊病病毒(IBDV)、传染性支气管炎病毒(IBV)、鸡痘病毒(FPV)和鸡马立克病病毒(MDV)的核酸样品未见明显扩增;对AIV和NDV核酸样品检测的最低极限质量浓度分别为25.0,12.5μg/L;该方法特异性好、灵敏度高,2种病原同时检测不影响检测特异性和灵敏性。97份临床样本检测结果表明,环介导间接PCR对AIV和NDV的检出率分别为15.5%和27.8%,分别高于常规PCR的12.4%和23.7%,接近于SybrGreen实时PCR的15.5%和28.9%;Kappa一致性检验显示,环介导间接PCR、SybrGreen实时PCR 2种方法对AIV和NDV检测的结果高度符合,Kappa值分别为κ=0.92和κ=0.87。本试验成功建立了AIV/NDV双重环介导间接PCR检测方法,适用于临床上2种病原的快速鉴别诊断。

According to conserved sequence regions of the AIV HA gene and NDV F gene in the GenBank database,the specific dual-probes with adjacent sequence were designed respectively,which were labeled to ends of Arabidopsis thaliana TOC1 gene fragments of different sizes as reporter genes.The duplex loop-mediated indirect PCR assay was successfully established for the differential diagnosis of AIV and NDV,and the specific PCR products were 200 bp for AIV,270 bp for NDV,respectively.The test results showed that the samples containing AIV or NDV nucleic acids could be amplified the specific fragments by loop-mediated indirect PCR,but the samples containing IBDV,IVB,FPV,and MDV nucleic acids had no obvious amplification,and the minimum limit concentrations(inμg/L)of the AIV and NDV were 25.0 and 12.5μg/L,respectively.The method has good specificity and high sensitivity.The duplex loop-mediated indirect PCR was applied to the detection of 97 clinical samples.The results showed that the positive detection rates of AIV,NDV were 15.5%,27.8%respectively,higher than that of conventional PCR(12.4%,23.7%)and close to that of SybrGreen real-time PCR(15.5%,28.9%).The kappa consistency test showed that the loop-mediated indirect PCR and SybrGreen real-time PCR for the detection results of AIV,NDV were highly consistent,with Kappa values ofκ=0.92,κ=0.87,respectively.This study successfully established a duplex loop-mediated indirect PCR assay for detecting AIV/NDV,which can be applied to the rapid differential diagnosis of two pathogens in clinic.