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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 detection method of influenza A (H1N1) virus based on nucleic acid sequence analysis. Methods: By optimizing the concentration of ATP sulfurylase and luciferase in the coke-sequencing reaction system, a sensitive pyrosequencing reaction system was established. The system was applied to a portable bioluminescence analyzer with low cost and miniaturization. The viral M, NP, HA gene fragment nucleic acid sequence. Results: The optimized pyrosequencing system can detect DNA samples down to 10 fmol, and the sensitivity of detection is more than 10 times higher than that of traditional pyrosequencing. Two samples were tested. According to the sequences of M, NP and HA genes, it was confirmed that they were all H1N1 infection. In addition, the resistance to M2 protein blockers (S31N mutation ) Results showed that there is S31N mutation in the virus, which is resistant to M2 protein blockers. Conclusion: The high sensitive coke sequencing system combined with portable bioluminescence analyzer has successfully realized the rapid and accurate low-cost detection of influenza A (H1N1) virus.