摘要
目的利用荧光定量RT-PCR技术建立一种快速检测A、B型流感病毒的方法。方法根据A型流感M基因和B型流感HA基因的相对保守序列,分别设计一对引物及其相应的Taqman探针,建立、优化单一和双重反应体系后,利用10倍稀释法对已知滴度的流感病毒进行梯度稀释,检验方法的灵敏度并建立相对定量标准曲线。结果A、B型流感病毒双重反应的灵敏度为分别为2·56×10-5和5·0×10-5TCID50,标准曲线相关系数分别为0·997和0·998,扩增效率分别为114·1%和107·8%,说明双重反应中A、B型的引物、探针、模板之间无相互干扰,具有很好的稳定性和重现性。结论研究建立的荧光定量RT-PCR技术可以准确同时检测A、B型流感病毒,不仅灵敏度高、稳定性好,而且可以对病毒滴度进行定量检测。
Objective To develop a method of rapid detection of influenza virus A and B by multiplex real - time quantitative RT - PCR. Methods By the M gene of influenza virus A and HA gene of influenza virus B, a set of primers and Taqman probe were designed, respectively. After the real- time assay system was established, the sensitivity and the quantitative curve of the multiplex assay were determined using tenfold serial dilution of TCID50. Results For the influenza virus A and B, the sensitivity of the assay were 2.56 ×10^-5 and 5.0 ×10^-5 TCID50 and the regression coefficient of the quantitative curve were 0.997 and 0.998, respectively. The results suggested there was no interference between the primers and probes of influenza virus A and B. Conclusion The detection system based on multiplex real - time RT- PCR was rapid, sensitive and steady, by which the viral load could be detected also.
出处
《中国热带医学》
CAS
2005年第9期1782-1785,共4页
China Tropical Medicine
