H9亚型禽流感病毒间接免疫荧光检测方法的建立与比较

将禽流感病毒(AIV)接种犬肾上皮细胞(MDCK),24h后以鸡抗禽流感抗体和兔抗鸡荧光抗体进行间接免疫荧光试验(IFA),结果于细胞核、细胞质内观察到特异性的荧光。利用96孔细胞板培养MDCK细胞,以间接免疫荧光作为判定指标对禽流感病毒进行毒力测定,并与禽流感病毒通用荧光反转录-聚合酶链反应(荧光RT-PCR)检测方法进行比较。结果表明间接IFA、荧光RT-PCR对同一样品检测的最高稀释度分别为10-5.25TCID50和10-6,Ct<30,两种方法对禽流感病毒的检测敏感性均很高。但由于荧光RT-PCR仅对抽提的RNA进行检测,而间接IFA是对禽流感活病毒进行检测,因此在临床样品检测和动物产品检疫中间接IFA更具准确性和实用性。

Indirect immunity fluorescence assay on H9 subtype avian influenza virus and comparison with fluorescence RT- PCR method

Indirect immunity fluorescence assay(IFA) was developed and compared with reverse transference polymerase chain reaction(RT-PCR) method for the detection of H9 subtype avian influenza virus(AIV).AIV was first inoculated in Madin-Darby canine kidney cell(MDCK).IFA was performed using chicken antibody as the primary antibody and rabbit anti-chicken fluorescent antibody as the second antibody against AIV.Special fluorescents were observed in the nucleolus and cytoplast of the infected MDCK cells in 24h after inoculation.The pathogenicity of AIV in the MDCK cells cultured in 96-cell culture plate was determined by IFA and compared with that obtained by the fluorescence RT-PCR.The detectable limits of the IFA and the fluorescence RT-PCR on a same sample were 10-5.25 TCID50 and 10-6,Ct<30,respectively.The results indicated that both methods had high sensitivity for detection of the H9 subtype AIV.However,IFA was much more precise and practical in clinical examination and animal products quarantine than fluorescence RT-PCR.