鸭病毒性肝炎病毒、禽流感病毒和新城疫病毒三重PCR诊断方法的建立及应用

根据GenBank中已登录的鸭病毒性肝炎病毒（DHV）、禽流感病毒（AIV）和新城疫病毒（NDV）的基因序列,设计了3对特异性引物,建立了DHV、AIV和NDV的多重PCR鉴别诊断技术,并对体系进行了优化和特异性、敏感性试验。结果表明,该体系所扩增的3种病毒基因片段大小分别为174bp（DHV）、264bp（AIV）和362bp（NDV）,且特异性、敏感性良好,能够分别检出1pg AIV、10pg DHV和10pg NDV的病毒RNA。对鸭临床样品的检测结果表明,该方法与病毒分离鉴定符合率90%以上,可用于临床样品的快速检测。

Establishment and application of a multiplex PCR diagnostic technology for DHV,AIV and NDV

According to the gene sequences of duck viral hepatitis virus（DHV）, avian influenza virus（AIV） and New- castle disease virus（NDV） available in GenBank,three pairs of specific primers were designed and synthesized to de- velop a multiplex PCR for simultaneous detection of DHV, AIV and NDV. The annealing temperature and primer, enzyme and dNTP concentration in the multiplex PCR were optimized and the specificity and sensitivity of the multi- plex PCR were tested and some clinical samples were detected by the multiplex PCR. In result,the gene fragments of 174 bp（DHV） ,264 bp（AIV）, 362 hp（NDV） were amplified from the RNA mixture which contained even little as 1 pg RNA of AIV and 10 pg RNA of DHV and NDV. The clinical samples detection showed that the developed multiplex PCR could rapidly and simultaneously diagnose DHV,AIV and NDV in duck.