Animal Inspection and Quarantine Laboratory, Technical Center, Guangdong Entry-Exit Inspection and Quarantine Bureau, No.66 Huacheng Dadao Ave., Zhujiangxincheng, Guangzhou, 510623, China,
Appl Microbiol Biotechnol. 2015 Jan;99(2):919-28. doi: 10.1007/s00253-014-6337-8. Epub 2015 Jan 6.
A novel multiplex detection array based on Luminex xMAP technology was developed and validated for simultaneous detection of five major viruses causing swine reproductive diseases. By combining one-step asymmetric multiplex reverse transcription polymerase chain reaction (RT-PCR) with xMAP bead-based hybridization and flow cytometry analysis, the resulting multiplex assay was capable of detecting single and mixed infections of PRRSV, PCV-2, PRV, CSFV, and PPV in a single reaction. The assay accurately detected and differentiated 23 viral strains used in this study. The low detection limit was determined as 2.2-22 copies/μL (corresponding to 0.5-6.8 fg/μL DNA template) on plasmid constructs containing viral fragments. The intra-assay and inter-assay variances (CV%) were low that ranged from 2.5 to 5.4 % and 4.1 to 7.6 %, respectively. The assay was applied to test field samples and detected single and mixed viral infections. The detection rate was higher than that of uniplex conventional PCR and RT-PCR methods. The detection of PRRSV by the bead-based multiplex assay was comparable with a commercially available real time RT-PCR kit. The test procedure on purified DNA or RNA samples could be completed within 2 h. In conclusion, the bead-based suspension array presented here proved to be a high-throughput practical tool that provided highly specific and sensitive identification of single and multiple infections of five major viruses in pigs and boar semen.
一种基于 Luminex xMAP 技术的新型多重检测微球芯片已被开发并验证，可用于同时检测导致猪繁殖障碍的五种主要病毒。通过将一步法不对称多重逆转录聚合酶链反应（RT-PCR）与 xMAP 微球杂交和流式细胞术分析相结合，该多重检测方法能够在单次反应中检测 PRRSV、PCV-2、PRV、CSFV 和 PPV 的单重和混合感染。该方法能够准确检测和区分本研究中使用的 23 种病毒株。在含有病毒片段的质粒构建体上，该方法的最低检测限为 2.2-22 拷贝/μL（相当于 0.5-6.8 fg/μL DNA 模板）。该方法的批内和批间变异系数（CV%）较低，范围分别为 2.5-5.4%和 4.1-7.6%。该方法应用于检测临床样本，可检测到单一和混合病毒感染。与单重常规 PCR 和 RT-PCR 方法相比，该检测方法的检测率更高。基于微球的多重检测方法检测 PRRSV 的结果与市售实时 RT-PCR 试剂盒相当。在纯化的 DNA 或 RNA 样本上进行检测的程序可在 2 小时内完成。总之，本文所介绍的基于微球的悬浮阵列被证明是一种高通量的实用工具，可高度特异性和敏感地鉴定猪和公猪精液中五种主要病毒的单一和多重感染。
