禽流感病毒一步法RT-PCR检测方法的建立

为了能快速准确地对禽流感进行病原学诊断,根据流感病毒的M基因序列,在保守区内用Oligo4.0软件设计了1对引物,建立了一步法RT-PCR诊断方法,其目的片段大小为229 bp。通过对不同稀释倍数的H5亚型禽流感病毒尿囊液和棉拭子浸出液进行检测,结果显示,病毒尿囊液的最低检出稀释度为1∶104;阳性棉拭子的最低检出稀释度为1∶23。用病毒分离法和一步法RT-PCR同时检测人工感染鸡不同脏器、口咽及泄殖腔棉拭子样品,二者符合率为100%,但前者的检测灵敏度比后者高10~100倍。用该方法检测H1~H15亚型禽流感病毒和鸡新城疫病毒等其他14种禽病病原,所有禽流感病毒均有229 bp的目的条带出现,而其他14种禽病病原均无目的条带出现,表明该方法的特异性好。

Development of a one-step RT-PCR assay for detection of A subtype avian influenza virus

A one-step RT-PCR assay was developed to detect avian influenza virus(AIV).A pair of specific oligonucleotide primers was designed for the detection of AIV according to M gene sequences.Sensitivity for detection of allantoic fluid by the one-step RT-PCR reached 10 thousand dilutions and detection of swab samples reached 8 dilutions.The tissue and swab samples infected with AIV were simultaneity(detected) by the one-step RT-PCR and the virus isolation method.The sensitivity of the PCR assay was 10 to 100-fold lower than that of the virus isolation method but the PCR assay shared an excellent(100%) correlation with the conventional virus isolation method.H1-H15 subtypes of avian influenza virus and other avian diseases etiological agents were detected by the one-step RT-PCR assay.The results showed that the PCR assay was high specific,and displayed no cross reaction to other subtype or other avian diseases.Development of the one-step RT-PCR provides an effective technical support for the rapid diagnosis and surveillance of molecular epidemiology of avian influenza.