摘要
目的:用多重PCR从临床标本中同时检测猪链2型特异性及5个毒力相关基因,建立SS2实验室快速诊断及应急检测的核酸鉴定方法。方法:根据已报告的引物序列设计并合成8对引物(含1对内对照引物),经过PCR反应条件的优化与组合,采用同一PCR反应条件与循环程序,直接从临床标本或培养物中同时一次性扩增属特异性(tuf)、种特异性(16S rRNA)、荚膜多糖(cps2 J)、溶菌酶释放蛋白(m rp)、细胞外蛋白因子(ef)、溶血素(sly)和甘油醛-3-磷酸脱氢酶(gapdh)等基因;并对方法的特异性和敏感性进行评估。结果:运用所建立的方法可直接从临床疑似猪链感染患者的脑脊液、血液、双相培养液和血培养物中一次性扩增出上述8对引物的目的基因,并经基因序列分析、细菌分离培养和生化鉴定进一步证实。其敏感性为78 cfu(克隆形成单位)。结论:所建立的方法具有敏感、特异、快速、简便的特点,可应用于SS2感染应急检测、流行病学监测和实验室快速诊断。
Objective：To develop a multiplex PCR assay for simultaneous detection and identification of serotype 2 and five virulence - associated phenotypes of Streptococcus suis in clinical specimens from suspicious cases and establish a rapid genetic diagnosis for the emergent detection. Methods：By optimizing the PCR condition and grouping the 8 primer pairs, the streptococcic genus spesific gene （tuf）, S. suis serotype 2 gene （16S rRNA）, the bacterial b globin gene acting as an internal control and five distinct virulence -associated DNA targets, including the S. suis capsular polysaccharide （cps） gene, the muramidase -released protein （mrp） gene, the suilysin （sly） gene, the extracellular protein factor ef gene encoding the EF protein and the glyceraldehyde -3 -phosphate dehydrogenase （gapdh） gene, were amplified by a multiplex PCR with the same reaction condition and uniform PCR cycle. The sensitivity and specificity of the multiplex PCR assay were evaluated. Results： Eight aimed DNA targets are amplified simultaneously from clinical specimens including cerebrospinal fluid （ CSF）, blood, and culturing materials by using this multiplex PCR assay, and are confirmed further by gene sequence analyzing, bacteria isolation and biochemistry identification. The PCR sensitivity is 78 clone forming unit （cfu）. Conclusion：A sensitive, specific, rapid and simple multiplex PCR assay was established for the application of SS2 emergent detection,epidemiological survey and lab diagnosis.
出处
《中国卫生检验杂志》
CAS
2006年第9期1029-1032,共4页
Chinese Journal of Health Laboratory Technology
基金
浙江省医药卫生科学研究基金项目计划资助课题
关键词
人感染猪链球菌2型
多重PCR
应急检测
Streptococcus suis serotype 2 in human
Multiplex PCR assay
Emergent detection
