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目的建立一种快捷灵敏的禽流感病毒PCR-ELISA检测技术。方法针对禽流感病毒M基因(M)、H7基因(H7)、N9基因(N9)3个基因片段的保守序列,通过生物素与地高辛的特异性标记,最后通过酶联免疫吸附反应判断PCR扩增的有无。结果M、H7、N9 3个基因片段所能检测的最低拷贝数分别为1.43×103copy/μl、1.04×102copy/μl、8.80×103copy/μl,其敏感度比普通PCR扩增提高了10倍~100倍,且不同流感病毒亚型之间、不同种属病毒之间均不存在交叉反应现象。另外还检测了114份临床标本,结果与鸡胚培养匹配率达100.0%。结论本文通过特异性和敏感性实验显示此方法灵敏度高、特异性强,且操作简单,可以广泛地应用于流感病毒的实验室检测。
Objective To establish a rapid and sensitive PCR-ELISA for the detection of avian influenza virus. Methods The conserved sequences of three gene fragments of M gene, H7 gene and N9 gene (N9) of avian influenza virus were determined by enzyme-linked immunosorbent assay (ELISA) and biotin-digoxigenin PCR amplification with or without. Results The lowest detectable copy number of M, H7 and N9 gene fragments were 1.43 × 103 copies / μl, 1.04 × 102copy / μl, 8.80 × 103copy / μl, respectively. The sensitivity was 10 times ~ 100 times, and there is no cross reaction between different influenza virus subtypes and different species of viruses. In addition, 114 clinical samples were also tested and the result was 100.0% matching with chicken embryo culture. Conclusion In this paper, through specificity and sensitivity experiments show that this method is highly sensitive, specific, and easy to operate, can be widely used in laboratory testing of influenza virus.