新城疫病毒一步法RT-PCR检测方法的建立及初步应用

目的建立新城疫病毒(Newcastle disease virus,NDV)一步法RT-PCR检测方法,并进行验证及初步应用。方法根据NDV F基因序列,设计合成了1对特异性引物,建立一步法RT-PCR检测方法,并验证其特异性及敏感性;应用建立的方法对23份疑似新城疫(Newcastle disease,ND)病料进行检测,并与血凝及血凝抑制试验检测结果进行比对。结果建立的一步法RT-PCR同时检测NDV、传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)和禽流感病毒(Avian influenza virus,AIV)H9亚型,仅NDV为阳性,IBDV和AIV H9均为阴性;该方法最低可检出约4 pg的NDV RNA;23份疑似ND病料,一步法RT-PCR检出11份阳性,血凝及血凝抑制试验检出13份阳性,两种方法的阳性符合率为85%。结论建立了NDV一步法RT-PCR检测方法,该方法特异性良好,敏感度较高,用时较短,可从分子水平上对NDV进行早期快速诊断和流行病学调查。

Development and preliminary application of one step RT-PCR assay for Newcastle disease virus

Objective To develop a one-step RT-PCR assay for Newcastle disease virus(NDV).Methods A pair of specific primers was designed according to the sequences of F gene of NDV,based on which a one step RT-PCR assay was developed and verified for specificity and sensitivity.Twenty-three clinic suspected samples were detected by the developed RT-PCR method,of which the results were compared with those by hemagglutination(HA) and hemagglutination inhibition(HI) tests.Results By developed RT-PCR method,the detection result of NDV was positive,while those of infectious bursal disease virus(IBDV) and avian influenza virus(AIV) subtype H9 were negative.The minimum detection limit of the method was 4 pg NDV RNA.Of the 23 clinic suspected samples,11 were judged as positive by RT-PCR,and 13 by HA and HI tests,indicating a positive coincidence rate of 85%.Conclusion One-step RT-PCR assay for NDV was developed,which was specific,sensitive and time-saving,and might be used for early rapid diagnosis and epidemiological investigation of NDV at molecular level.