【摘要】 目的临床上对流感的有效控制与治疗在于实验室快速与准确的诊断,这对于指导用药和避免滥用抗生素具有重要意义。RT_PCR在流感病毒检测中特异性和灵敏度较强,nestedmultiplexRT_PCR法可以在此基础上提高灵敏度。实验的目的是应用分子生物学方法对流感病毒进行快速诊断。方法我们结合流感病毒M基因、NS基因、HA基因的结构和进化特性设计多元引物达到流感病毒的型和亚型准确与快速的鉴定。结果甲型流感病毒以M基因保守区域设计引物,PCR后电泳带为413bp;甲型流感病毒以HA基因分亚型,H1N1亚型PCR后电泳带为348bp,H3N2亚型PCR后电泳带为291bp,H5N1亚型PCR后电泳带为326bp。结论应用优化的套式核酸体外扩增方法进行流感病毒的型与亚型的诊断是比较快速的,且所需要的病毒量少(0.01～0.1TCID50),灵敏度较高。更多还原

【Abstract】 Objective The rapid and sensitive detection of influenza viruses is benefited to the control and the treatment of influenza viruses.It is important to the selection of remedy and may avoid using antibiotics mistakenly.The molecularbiological method described can offer a rapid and sensitive means for dctecting influenza viruses. It is rapid and sensitive for the detection of influenza virus type and subtypes by RT-PCR.The sensitivity of detection was improved in nested multiplex RT-PCR. Methods Influenza virus type and subtype specific primers were selected for M-gene,NS-gene and HA-gene. Results Influenza A virus type specific primers were selected for M-gene.A product of the expected size (413bp) was obtained.This was comparable to the sensitivity reported for detection of influenza A viruses,using primers specific for influenza A virus HA genes.A product of the expected size (348bp) was obtained when human H 1N 1 viruses were tested.A product of the expected size (291bp) was obtained when human H 3N 2 viruses were tested.A product of the expected size (326bp) was obtained when avian H 5N 1 viruses were tested. Conclusion From the results of our work,we consider the optimized nested multiplex RT-PCR method described to offer a rapid and sensitive means for influenza A viruses.In multiplex subtyping or multiplex typing and subtyping simultaneously,the sensitivities for types and subtypes specific bands were low(0.01～0.1TCID 50) in the tested volume.更多还原