反向斑点杂交检测甲/乙型流感病毒及其基因分型的方法研究

Genotyping of Influenza Viruses by Reverse Dot Blot Method

目的建立一种特异、灵敏的检测甲/乙型流感病毒并对其进行基因分型的反向斑点杂交方法。方法应用生物软件针对甲/乙型流感病毒的膜蛋白(Matrix Protein)基因和血凝素(Hemagglutinin,HA)基因的保守区域设计引物及探针,通过优化PCR和杂交的反应条件,建立检测甲/乙型流感病毒并对其进行基因分型的反向斑点杂交方法,验证方法的特异性和敏感性,并与直接免疫荧光法分别对相同的临床咽拭子标本进行检测,比较两者的检测结果,以评价该方法的临床应用价值。结果该方法对甲/乙型流感病毒的检测具有特异性,对呼吸道合胞病毒、柯萨奇病毒A型、副流感病毒3型、鼻病毒和腺病毒3型均无交叉反应,检测灵敏度为102～103copies/μL。在111例发热门诊病人的咽试子标本中,免疫荧光检测出3例甲型流感病毒、0例乙型流感病毒,阳性率为2.7%;反向斑点杂交检测出3例甲型流感病毒,其中1例H1、2例H3、0例H5、0例H9,4例乙型流感病毒,阳性率为6.3%。结论初步应用结果表明,本研究建立的检测甲/乙型流感病毒并对其进行基因分型的反向斑点杂交方法具有特异、灵敏的特点,为该方法的后续临床应用奠定基础。

Objective To develop a rapid and specific genotyping method for the typing of influenza A and B viruses.Methods Primers and probes specific for the highly conserved regions of matrix protein（MP） gene and hemagglutinin（HA） gene of influenza A and B virus were designed.The assay conditions were optimized to improve the specificity and sensitivity.Throat swab samples were used for the testing of the sensitivity and specificity this reverse dot blot method and the results were compared with the direct immunofluorescence method. Results The developed assay method was highly specific and sensitive in the detection and genotyping of influenza A and B virus. None of the negative control samples showed positive reactions. Cross-reaction with rhinovirus and adenovirus was not observed. The detection limit of the assay was in the range of 102-103 copies/~L. Using the direct immunofluorescence method, 3 out of 111 samples（2.7%） were found to be positive for influenza A. Influenza B was not detected in these samples. Reverse dot blot method also detected influenza A virus in 3 samples, in which 2 samples were found to be H3 subtype. Hl, H5 and H9 subtype were not found. In addition, 4 samples（6.3%） were found to have influenza B virus. Conclusion The developed reverse dot blot method is sensitive and specific for the detection and genotyping of influenza A and B virus. This method may be useful for the detection of influenza virus in clinical samples.