应用xMAP液念芯片多重快速检测四种病原微生物的研究

目的:建立一种多重、快速、特异性好、灵敏度高的病原微生物检测方法。方法:根据GenBank数据库中的小肠结肠炎耶尔森氏菌、单核细胞增生性李斯特菌、产气荚膜梭菌、鼠疫耶尔森氏菌基因序列,分别针对ail、hly、cpe、3a基因设计4对引物和4条探针。通过重叠PCR扩增各目的基因并构建重组质粒,以该重组质粒DNA为模板,通过多重PCR同时扩增上述4个基因,建立xMAP液态芯片检测技术,在此基础上对标准菌株基因组DNA进行检测并验证该方法的特异性和敏感性。结果:xMAP液态芯片对质粒DNA和标准菌株基因组DNA的检测结果与多重PCR结果一致。该方法能在3.5 h内同时完成对4种病原菌的检测,特异性好,且敏感性要高于PCR方法,灵敏度最高可达200CFU/ml。结论:xMAP液态芯片技术是病原微生物的多重快速检测的新方法,具有很好的应用价值和前景。

Application of Luminex xMAP Technology in the Rapid, High-throughput, Multiplexed Detection and Identification of Four Pathogens

Objective:To develop a new rapid,sensitive,multiplexed technology for pathogens detection and identification.Meth- otis:According to sequences from GenBank,four sets of specific primers and four probes were designed and synthesized for ail gene of Yersinia enterocolitica,hly gene of Listeria monocytogenes,cpe gene of Clostridium perfringens,and 3a gene of Yersinia Pestis.The four gene fragments were synthesized by overlapping PCR,and inserted into cloning vectors and recombinant plasmid.Using the constructed plasmid DNA as the template,the four gene fragments were simultaneously amplified by the multiplex PCR,and the PCR conditions were optimized.The multiplex PCR product was detected by xMAP technology in which the reaction condition was analyzed and opti- mized.The multiplex PCR product from the pathogens genomic DNA,and the specificity and sensitivity of this method were identified. Results:The multiplex PCR products amplified from the plasmid DNA and the bacterial genomic DNA were detected by the xMAP tech- nology,and the results were similar to the multiplex PCR.This new method which was more specific and sensitive than PCR was capable of simultaneous detection of four pathogens in 3.5h,and its sensitivity was up to 200CFU/mL.Conclusions:The xMAP technology pro- vides a more efficient and useful method for rapid,multiplex detection of pathogens.Further more,it indicates that the xMAP liquid chip method is valuable for exploitation and application.