基因芯片技术检测3种肠道病原微生物方法的建立

目的:建立一种运用多重PCR和基因芯片技术检测和鉴定伤寒沙门氏菌、痢疾杆菌和单核细胞增生利斯特菌的方法。方法:分别选取伤寒沙门氏菌染色体ViaB区域中编码调控Vi抗原表达的基因(vipR)、痢疾杆菌编码侵袭质粒抗原H基因(ipaH)和单核细胞增生利斯特菌溶血素基因(hlyA)设计引物和探针,探针3'端进行氨基修饰,下游引物标记荧光素Cy3。在优化的PCR和杂交反应条件下,进行三重PCR扩增,产物与包括3种致病菌特异性探针的基因芯片杂交。在评价基因芯片的特异性和灵敏度之后,对临床样本进行检测。结果:只有3种目的致病菌的PCR产物在相应探针位置出现特异性信号,其他阴性细菌均无信号出现;3种致病菌的检测灵敏度均可达到103CFU/mL;检测30例临床样本的结果与常规细菌学培养结果一致。结论:所建立的可同时检测伤寒沙门氏菌、痢疾杆菌和单核细胞增生利斯特菌的基因芯片方法快速、准确,特异性高,重复性好,为3种肠道致病菌的快速检测和鉴定提供了新方法和新思路。

Detection and Identification of Three Intestinal Pathogenic Microorganisms Using DNA Microarray

Objective:To develop a rapid,sensitive and specific assay of DNA microarray combined with multiplex PCR for simultaneous detection and identification of Salmonella typhimurium,Shigella dysenteriae and Listeria monocytogenes.Methods:vipR(ViaB region of Vi-positive S.typhimurium),ipaH(invasion-associated plasmid antigen H of S.dysenteriae)and hlyA(hemolysin A of L.monocytogenes)were as target genes for multiplex PCR respectively,and then three pairs of primers and captured oligonucleotide probes were designed and synthesized.Thus,all the olignucleotide probes were modified with 3' amino-group,and the reverse primers were labeled with fluorescein(Cy3).After multiplex PCR system and hybridization reaction were optimized,the multiplex PCR products were hybridized with DNA microarray,which contained specific probes of three pathogenic microorganisms.Then the reproducibility and specificity of DNA microarray assay were evaluated.Finally,this assay was applied to detected clinical specimens.Results:When PCR products from three target pathogens were hybridized with DNA microarray,the corresponding probes produced positive signals.Meanwhile,no signals appeared on the array for all non-target pathogens.The sensitivity of all three pathogens was 103 CFU/mL.The results from detecting 30 clinical specimens using DNA microarray were the same to that of the routine bacteriology method.Conclusion:It is proved that DNA microarray is a reliable and accurate assay for S.typhimurium,S.dysenteriae and L.monocytogenes simultaneously.Then this assay offers a new method and idea for detecting and identifying pathogenic microorganisms.