禽呼吸道7种病毒多重连接探针扩增检测技术的建立与应用

Simultaneous identification of viruses causing avian respiratory tract infection by using multiplex ligation-dependent probe amplification assay

建立了一种多重连接探针扩增技术(MLPA),能快速区分临床上引起禽呼吸道疾病的7种病原,即禽流感病毒(AIV)H9亚型、AIV H5亚型、AIV H7亚型、新城疫病毒(NDV)、禽偏肺病毒(aMPV)、禽传染性支气管炎病毒(IBV)和禽传染性喉气管炎病毒(ILTV)。对扩增产物进行毛细管电泳分析,结果显示,该方法对不同病原核酸的最低检测极限可以达到5.3×10^0~1.40×10^2拷贝/反应,特异性好,探针之间无交叉反应。应用MLPA方法对122份临床样品进行检测,并与国家或行业标准进行比较,结果显示AIV H7亚型、NDV、aMPV、IBV、ILTV的特异性和敏感性均达到100%。结果与国家或行业标准PCR方法的检测结果一致性高,且克服了国家或行业标准PCR方法无法进行多重检测的缺点。该方法最多可同时检测40多种病原,对于复杂症候群的快速和高通量检测具有重要临床意义,可推广到其他多重病毒的检测中,显示出广阔的应用前景。

We developed a novel multiplex ligation-dependent probe amplification(MLPA)assay for the simultaneous and differential detection of 7 clinically relevant pathogens causing avian respiratory tract infections,i.e.,avian influenza virus(AVI)H9,H5,and H7 subtype,Newcastle disease virus(NDV),avian metapneumovirus(aMPV),avian infectious bronchitis virus(IBV),and avian infectious laryngotracheitis virus(ILTV).The amplified products of MLPA were analyzed by capillary gel electrophoresis.The results showed that the limits of detection for each virus varied from 5.3×10^0-1.40×10^2 copies per MLPA assay.There were no cross reactions among any of the probes.Clinical samples were tested by MLPA assay and the results were compared with the national reference standards.The results showed that this assay achieved 100%specificity and sensitivity for the detection of AIV H7 subtype,NDV,aMPV,IBV,and ILTV.In conclusion,the MLPA assay shows highly agreement with reference PCR assay,and it overcomes the shortcoming of mono-PCR assay,allowing analyses of up to 40 targets in a single reaction.This MLPA assay will provide an efficient and high-throughput tool for differential diagnosis,it can be adapted to detect other types of viruses and shows broad application prospect.