高灵敏焦测序结合生物发光分析仪检测甲型H1N1流感病毒

Detection of Influenza A(H1N1) Virus Using Highly Sensitive Pyrosequencing Combining Bioluminescence Analyzer

目的:建立基于核酸序列分析的快速、准确、低成本的甲型H1N1流感病毒检测方法。方法:通过优化焦测序反应体系中ATP硫酸化酶和荧光素酶的浓度,建立高灵敏的焦测序反应体系;将该体系应用于低成本、小型化的便携式生物发光分析仪,焦测序分析流感病毒M、NP、HA基因片段的核酸序列。结果:优化后的焦测序反应体系可检测低至10 fmol的DNA样本,检测灵敏度较传统焦测序提高了10倍以上。对两例样本进行检测,根据所测得的M、NP、HA基因特异性片段序列,可以确认其均为甲型H1N1感染;另外,对M2蛋白阻断剂耐药性标志位点(S31N突变)的测定结果显示该病毒存在S31N突变,为M2蛋白阻断剂耐药型。结论:高灵敏焦测序体系结合便携式生物发光分析仪成功实现了对甲型H1N1流感病毒快速、准确的低成本检测。

Objective： To establish a rapid, accurate and low-cost influenza A（H1N1） virus detection method based on nucleic acid sequence analysis. Methods： A highly sensitive pyrosequencing system was establised by optimizing the concentrations of ATP sulfurylase and luciferase respectively; the nucleic acid sequences of M,NP,HA gene fragments of influenza virus were analysed with a low-cost and portable bioluminescence analyzer by coupling the highly sensitive pyrosequencing system. Results： Compared with conventional pyrosequencing system, the sensitivity of the proposed method increased more than 10 times, and as low as 10 fmol of DNA samples can be detected. Based on the pyrosequencing results of the M, NP, HA genes, we confirmed the infection of influenza A（H1N1） virus in the two samples. The sequencing results of the gene coding M2 protein inhibitor drug resistance marker（S31N mutation）proved the presence of S31 N mutation, indicating that the virus is M2 protein inhibitor-resistant. Conclusion： A rapid, accurate and low-cost detection method of influenza A（H1N1） virus was established by combining the highly sensitive pyrosequencing system with a portable bioluminescence analyzer.