In order to establish a high-throughput identification technique that simultaneously detects six major pathogens including APP, HPS, PRRSV, Mhp, PCV-2 and CSFV, six pairs of primers and probes were designed based on the specific conservative sequences of the pathogens, a multiplex PCR system was developed, hybrid parameters were optimized, and evaluation of the technology was performed. The results showed that the present detection method had a sensitivity of 5.8 × 10²copies/μL for APP, 7.8 × 10³ copies/μL for HPS, 6.8 × 10³ copies/μL for Mhp, 6.3 × 10² copies/μL for PCV-2, 4.8 × 10³ copies/μL for PRRSV, and 5.5 × 10² copies/μL for CSFV, respectively; and it produced no cross reaction against the other nine pathogens like swine-origin pseudorabies virus, porcine parvovirus, Japanese B encephalitis virus, swine vesicular disease virus, vesicular stomatitis virus, foot-and-mouth disease virus, bluetongue virus, peste des petits ruminants virus and salmonella. Application of the multiplex oligonucleotide microarray established here to testing 285 clinical blood samples indicated a single infection rate of 18.2 % (52/285) and a mixed infection rate of 6.3 % (18/285) which were consistent with the results of the sequencing verification. This technique might serve as a rapid and high-throughput method of detection for epidemic investigation and clinical diagnosis of multiple pathogens.

为了建立可同时检测APP，HPS，PRRSV，Mhp，PCV-2和CSFV等六种主要病原体的高通量鉴定技术，根据病原体的特定保守序列设计了六对引物和探针。开发了多重PCR系统，优化了杂交参数，并对技术进行了评估。结果表明，本检测方法对APP的灵敏度为5.8×10 ²拷贝/μL，对HPS的灵敏度为7.8×10 ³拷贝/μL，对Mhp的灵敏度为6.8×10 ³拷贝/μL，对Mhp的灵敏度为6.3×10 ²拷贝/μL PCV-2，PRRSV为4.8×10 ³拷贝/μL,5.5×10 ²CSFV的拷贝数/μL；它与其他九种病原体没有交叉反应，如猪源伪狂犬病病毒，猪细小病毒，日本乙型脑炎病毒，猪水泡病病毒，水泡性口炎病毒，口蹄疫病毒，蓝舌病病毒，小反刍动物病毒病毒和沙门氏菌。此处建立的多重寡核苷酸微阵列在检测285种临床血液样本中的应用表明，单次感染率为18.2％（52/285），混合感染率为6.3％（18/285），与测序验证的结果一致。该技术可作为多种病原体的流行病调查和临床诊断的快速，高通量检测方法。