【作者】 徐晔；

【导师】 姚火春； 王恒安；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2006， 硕士

【摘要】 根据GenBank已公布的禽流感病毒H9亚型血凝素蛋白编码基因，设计了一对引物（H1和H2），RT-PCR扩增禽流感病毒A/Duck/Shanghai／02／99（H9）的HA基因，扩增片段与载体pUCm-T的连接产物转化大肠杆菌感受态细胞，重组质粒pUCm-T-HA经限制性内切酶酶切和PCR鉴定后进行测序。BlastN分析结果显示该分离株血凝素编码基因与已发表的鸭源禽流感病毒A/Duck／Shantou／1605／01（H9N2）和A／Duck／Hong Kong/Y280／97（H9N2）血凝素编码基因的核苷酸序列同源性为98％，从而从分子水平确定了该分离株为H9亚型禽流感病毒。 根据禽流感病毒M基因设计并筛选出两对引物M1和M2、M3和M4，以H1和H2、M3和M4建立了禽流感病毒的双重RT-PCR检测方法。检测80份临床样品，检出阳性样品17份，阴性样品63份，与鸡胚分离方法检出率相同且符合率为100％。同时该方法快速、简便，并且能在通过检测M基因确定是否为禽流感病毒的同时鉴定是否为H9亚型，从而为临床及实验室提供了一种快速、灵敏的检测AIV并鉴定其亚型的方法。 根据引物M3和M4扩增片断序列设计了探针M5，建立了快速检测A型流感病毒的荧光RT-PCR检测技术。该方法可检测到0.1EID₅₀，鸡胚分离可检测到1EID₅₀，说明该方法的敏感性高于鸡胚分离方法。从人工感染H9亚型禽流感病毒的发病鸡采集咽喉拭子、泄殖腔拭子进行荧光RT-PCR检测，结果显示人工感染后1-6天的咽喉拭子和泄殖腔拭子中都未检出病毒；人工感染后7-8天的咽喉拭子中都能检出和分离出病毒，但部分泄殖腔拭子中有未检出病毒的，表明咽喉拭子中病毒含量比泄殖腔拭子高。该方法简单、快速，可适用于禽流感临床快速诊断、活禽市场的禽流感监测和进出境口岸的检疫。更多还原

【Abstract】 According to the submitted nucleotide sequences of HA gene of H9 subtype avian influenza virus （AIV）, a pair of primer （H1 and H2） was synthesized and used to amplify the HA gene of A/Duck/Shanghai/02/99（H9） by RT-PCR using genome RNA as template, the ligation product of the amplified fragment with the linear vector pUCm-T was then transformed into E. coli competent cells. After identified by restrictive endonucleonase and PCR, the recombinant plasmid pUCm-T-HA was sequenced. The BlastN result showed that the cloned HA gene of A/Duck/Shanghai/02/99（H9） has 98% homology to that of A/Duck/Shantou/1605/01（H9N2） and A/Duck/Hong Kong/Y280/97（H9N2）.A method of diplex RT-PCR was developed to detect the Avian Influenza Virus （AIV）using a pair of primers （M3 and M4） for matrix（M） gene and another pair of primers （H1 and H2） for hemagglutinin（HA） gene. 17 positive samples were detected by the diplex RT-PCR among 80 clinical samples, which was same as the chicken embryo isolation method.An oligo-nucleotides M5 was designed for probing the M3 and M4 amplified fragment in the real-time RT-PCR for AIV detection. The sensitivity of this assay was up to 0.1EID₅₀, which was higher than the chicken embryo isolation method(1EID₅₀). 80 shares of gorge and cloaca swabs collected from the chicken which were infected by H9 subtype AIV were tested by the real-time RT-PCR. The results showed that the virus can be isolated and detected from all the gorge swabs in the 7th-8th day, while it can not be detected from all the corresponding cloaca swabs. It suggests that the virus content was higher in the gorge than that in the cloaca. The assay was simple and rapid and it could be applied to the clinical detection, the market supervision and export & import quarantine.更多还原