多功能悬浮点阵技术快速检测肠道致病菌的初步应用研究

目的建立以多功能悬浮点阵技术为基础的临床常见肠道致病菌的快速检测方法。方法以细菌16S rDNA基因保守区序列设计1对通用引物,采用不对称PCR扩增7种临床常见肠道致病菌标准菌株,多功能悬浮点阵技术对不同菌株的PCR产物进行检测以验证相应菌种探针的特异性,最后对48份粪便标本进行肠道致病菌高通量快速检测。结果 7种临床常见肠道致病标准菌株的不对称PCR得到了大量单链产物,其产物用多功能悬浮点阵技术的检测特异性为100%,48份粪便标本不对称PCR产物可与相应探针发生特异性结合,且在多功能悬浮点阵技术的相应检测信号大于阴性对照3倍以上,5种细菌的多功能悬浮点阵技术检测结果与培养鉴定结果符合率100%,48份标本PCR产物均与志贺菌属探针发生杂交反应(阳性率100%)。结论 16S rDNA可以作为细菌快速鉴定的靶序列,不对称PCR产物可以显著提高与悬浮芯片杂交检测的灵敏度,多功能悬浮点阵技术在鉴定细菌方面具有简单快速、高通量、高检出率等特点,可以作为细菌快速鉴定的一种新方法,但无法鉴别志贺菌属和大肠埃希菌属。

Rapid detection of common enteropathogens using multi-analyte suspensim arrays

Objective To establish a new method for rapid detection of common enteropathogens based on Multi-analyte Suspension Arrays（MASA）.Method Seven species of enteropathogens were detected by unsymmetrical PCR.A pair of general primers based on highly conserved of 16S rDNA gene was designed for the above PCR.The amplified products of the 7 species of enteropathogens were detected by MASA,and 48 fecal samples were detected by MASA.Result The unsymmetrical PCR of the 7 species of enteropathogens produced a great quantity of single-strand nucleotides.The specificity of the seven enteropathogens′ probe were 100%.In the 48 fecal samples,the coincidence rate of MASA identification for 5 enteropathogens and cultures were 100%,and the detection rate of Shigella was 100% by MARA.Conclusion 16S rDNA gene may be used as the target sequence of rapid identification for bacteria.The unsymmetrical PCR products can significantly enhance the hybridization sensibility.MARA is a simple,rapid,high-throughput and high-performance method for identification of bacteria except Shigella and Escherichia.