摘要
目的建立一种运用多重PCR方法结合基因芯片技术快速、准确检测11种常见致病菌的方法。方法筛选志贺氏菌、肠炎沙门氏菌、伤寒沙门氏菌、大肠杆菌O157、副溶血性弧菌、普通变形杆菌、蜡样芽孢杆菌、金黄色葡萄球菌、单核细胞增生李斯特菌、产气荚膜梭菌、空肠弯曲菌的特异基因作为目的基因。设计相应的引物及探针,进行多重PCR扩增,制备寡核苷酸芯片。将多重PCR扩增产物与带有11种特异探针的基因芯片杂交。用扫描仪扫描,判定细菌种类。结果该基因芯片可特异性地检测11种致病菌,具有良好的特异性,基因组DNA检测灵敏度可达2×10-3ng。结论多重PCR结合基因芯片技术检测11种不同致病菌的方法特异性好,灵敏度高,具有较好的实用性。
Objective To establish a rapid,accurate detection method for 11 kinds of pathogenic bacteria using DNA microarray combined with multiplex PCR.Methods The special genes of these Bacterium including Shigella,Listeria monocytogenes,Escherichia coli O157,Staphylococcus aureus,Bacillus cereus,Campylobacter jejuni,Proteusbacillus vulgaris,C.perfringens,Vibiro parahaemolyticus,Salmonella typhi,Salmonella enteritidis were as target genes for multiplex PCR respectively,then primers and captured oligonucleotide probes were designed and synthesized.After multiplex PCR system and hybridization reaction were optimized,the multiplex PCR products were hybridizes with DNA microarray,which contained specific probes of eleven pathogenic bacterium.Scanner was used to determinant the types of bacterium.Results The DNA microarray assay can detect 11 kinds of pathogenic bacterium specially.The sensitivity of the DNA may reach 2×103 ng.Conclusion To detect 11 kinds of pathogenic bacteria using DNA microarray combined with multiplex PCR is specific,sensitive,practical.
出处
《中国实验诊断学》
北大核心
2011年第4期587-591,共5页
Chinese Journal of Laboratory Diagnosis
基金
国家质检总局课题项目(2009IK224)
