五种禽呼吸道病病原多重PCR检测方法的建立和应用

Establishment and application of a multiplex PCR assay for simultaneous detection of AIV,NDV,IBV,ILTV and MG

根据GenBank中禽流感病毒(AIV)、新城疫病毒(NDV)、鸡传染性支气管炎病毒(IBV)、鸡传染性喉气管炎病毒(ILTV)和鸡毒支原体(MG)的基因序列,设计了5对特异性引物,建立了分别针对鸡AIV、NDV、IBV、ILTV和MG的多重PCR鉴别诊断方法,并进行了退火温度、引物浓度和TaqDNA聚合酶浓度的优化,以及特异性、敏感性试验和临床应用。结果表明,设计的5对引物对同一样品中的RNA(AIV、NDV、IBV)和DNA(ILTV、MG)进行多重PCR扩增,均同时得到5条片段大小与设计相符的特异性片段,即268bp(AIV)、321bp(NDV)、457bp(IBV)、662bp(ILTV)和732bp(MG);建立的多重PCR方法具有较强的特异性和较高敏感性,能检测出1pgAIV、1pgNDV、10pgIBV的RNA和1pgILTV、10pgMG的DNA。对鸡临床样品的检测结果证明,该方法在临床诊断方面具有广阔的应用前景。

According to the gene sequences of avian influenza virus（AIV）,Newcastle disease virus（NDV）,infectious bronchitis virus（IBV）,infectious laryngotracheitis virus（ILTV） and Mycoplasma gallisepticum（MG） available in GenBank,five pairs of specific primers were designed and synthesized to develop a multiplex PCR for simultaneous detection of AIV,NDV,IBV,ILTV and MG.The annealing temperature,primer concentration and enzyme concentration in the multiplex PCR were optimized and the specificity and sensitivity of the multiplex PCR were tested and some clinical samples were detected by the multiplex PCR.In result,the gene fragments of approximate 268 bp（AIV）,312 bp（NDV）,457 bp（IBV）,662 bp（ILTV） and 732 bp（MG） were simultaneously amplified from the same sample containing the RNAs of AIV,NDV and IBV and the DNAs of ILTV and MG.The specificity and sensitivity tests showed that as little as 1 pg RNA of AIV,1 pg RNA of NDV,10 pg RNA of IBV,1 pg DNA of ILTV and 10 pg DNA of MG could be detected by this multiplex PCR.The clinical samples detections showed that the developed multiplex PCR could be widely used to diagnose simultaneously the five pathogens of AIV,NDV,IBV,ILTV and MG in Chicken.