猪猝死症4种重要病原多重连接探针扩增鉴别检测技术的建立与应用

Simultaneous identification of pathogens causing porcine sudden death syndrome by using multiplex ligation-dependent probe amplification assay

为了快速检测引起猪猝死的病原,本研究针对临床引起猪猝死症的4种疾病病原(猪尼帕病毒、非洲猪瘟病毒、猪产气荚膜梭菌和猪胸膜肺炎放线杆菌)分别设计特异性探针,建立了同时检测这4种病原的多重连接探针扩增技术(MLPA)。对扩增产物进行毛细管电泳分析,结果显示该方法,探针之间没有交叉反应,特异性强。对不同病原核酸的最低检测限可以达到140拷贝μ/L～370拷贝μ/L,敏感性较高。应用该MLPA方法对129份临床样品进行检测,并与国家相关标准进行比较。结果显示,两种方法的符合率为100%,且该方法检测猪产气荚膜梭菌和猪胸膜肺炎放线杆菌的特异性和敏感性均达到100%,Kappa值均为1。该方法的检测结果与国家或行业标准PCR的检测结果一致性较高,且克服了后者无法同时进行多重检测的缺点,最多可以同时检测45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。该方法可推广到其它多重病原体的检测中,显示出广阔的应用前景。

In order to rapidly differentiate the pathogens that cause porcine sudden death, we developed a novel multiplex ligation-dependent probe amplification(MLPA) assay for the simultaneous detection of four clinically relevant pathogens of porcine sudden death syndrome, i.e., Nipah virus(NPV), African swine fever virus(ASFV), Clostridium perfringens and Actinobacillus pleuropneumoniae. The amplified MLPA products were analyzed by capillary gel electrophoresis. The results showed that the detection limit for each pathogen vary from 140 copies to 370 copies per MLPA assay. There were no cross-reactions among any of the probes. A total of 129 clinical samples were detected by the RT-MLPA assay and the results were confirmed by national reference standards. The MLPA assay showed specificities, sensitivities and kappa value of 100 %, 100 %and 1, respectively, for C.perfringens and A.pleuropneumoniae. In conclusion, the MLPA assay shows highly agreement with reference PCR assay, and overcome the shortcoming of mono-PCR assay, it allows analyses of up to 45 targets in a single reaction.The novel assay will provide an efficient and high-throughput tool for differential diagnosis. The MLPA method could be used in other multiplex pathogens detection and shows broad application prospect.