禽肉产品中禽流感与新城疫病毒多重RT-PCR检测方法的建立

Detection and Differentiation of Avian Influenza and Newcastle Disease Virus by Multiplex RT-PCR

根据禽流感病毒NP基因和新城疫病毒F基因各设计1对引物,经优化单项RT-PCR(sRT-PCR)反应体系,建立的多重RT-PCR(mRT-PCR)同时检测禽流感、新城疫的极限分别为10-2.5 EID50/0.1 mL和10-3.75 EID50/0.1 mL,mRT-PCR检测禽流感的敏感性与sRT-PCR一致,mRT-PCR检测新城疫的敏感性较sRT-PCR敏感性下降2个数量级。特异性试验表明,mRT-PCR仅检测到不同亚型(H1、H3、H5、H6和H9)禽流感、不同毒力新城疫病毒,对其他9种禽病原体和SPF鸡尿囊液均未扩增出片断。mRT-PCR检测39株禽流感H1、H5和H9亚型病毒、41株不同宿主源新城疫病毒,结果与sRT-PCR、HI试验完全一致;mRT-PCR和病原分离法同时检测人工感染鸡的组织脏器,2种方法检测禽流感的符合率为93.8%(30/32),检测新城疫的符合率为95.2%(20/21);采用mRT-PCR和病原分离法同时对35份进口禽产品、32份鸡粪拭子检测,2种方法检测禽流感的符合率为100%(67/67),对新城疫的符合率为97.1%(65/67)。病原分离法的检出率虽高于mRT-PCR,经统计学分析,两者差异不显著。

A multiplex reverse transcription-polymerase chain reaction （mRT-PCR） assay for the detection of both Avian Influenza virus （AIV） and Newcastle disease virus（NDV） was developed and evaluated. The primers, specific for each virus, were designed from the nucleocapsid protein （NP） gene of AIV and the fusion protein （F） gene of NDV to amplify products of 502 and 421 nucleotides, respectively. Parameters （concentration of primer, Mg^2＋, dNTPs and Taq DNA Polymerase, PCR cycles, annealing temperature） of the single-virus RT-PCR（sRT-PCR）, which may have an essential influence on the detection limit, were optimized. Only AIV consisting of H1, H3, H5, H6, H9 subtypes or NDV composed of velogenic, mesogenic and lentogenic isolates were amplified and no amplification occurred with 9 other chicken pathogens and allantoic fluid of specific pathogen-free （SPF） chickens in the mRT-PCR. The PCR products of nine AIV and ten NDV isolates were sequenced to further confirm the specificity of mRT-PCR. The detection limit of AIV and NDV by mRT-PCR were respectively 10^25 EID50/0.1 mL and 10^375 EID50/0.1 mL, being the same sensitivity as sRT-PCR for AI and 100 fold decrease for ND compared to sRT-PCR. Eighty isolates with haemagglutinating activity consisting of H1, H5, H9 AIV and NDV of different origins （chicken, duck, goose, ostrich, pigeon and peacock） were compared by mRT-PCR and typing by haemagglutination inhibition （HI） test. They showed 100%（80/80） agreement. Olgan samples from the chickens experimentally infected with AIV or NDV were tested by mRT-PCR and Virus isolation（VI）, showing 93.8%（30/32 ） agreement for AIV and 95.2%（20/21 ） for NDV. Additionally, 35 samples from imported poultry products and 32 eloacal swab samples from Chinese domestic market were tested by mRT-PCR and VI, and showed 100% （67/67） agreement for AIV and 97.1%（65/67） agreement for NDV. These results showed that VI is more sensitive than mRT-PCR but that mRT-PCR proved to be a sensitive method for the simultaneous and rapid detection of AIV and NDV. This assay shows potential for poultry product testing and clinical diagnostic aoolications.