13种链球菌超抗原基因的双重实时荧光PCR检测方法的建立

目的建立一新的快速的链球菌超抗原基因检测方法,能快速、灵敏及特异地分析样本中存在的各种超抗原基因。方法根据13种链球菌超抗原基因的保守序列设计特异性引物和探针,利用13种超抗原的阳性标本作为模板来优化了反应体系与扩增条件,并验证其特异度与灵敏度。结果利用该研究建立的双重荧光PCR方法体系,可以特异性鉴定出13种链球菌超抗原,并且与其他呼吸道病原菌也没有交叉反应。另外,该检测体系的检测灵敏度高于普通PCR至少1~2个数量级(10~100倍)及以上。结论该研究建立的双重实时荧光PCR法能更加准确、快速地对链球菌菌株中的超抗原基因进行检测分析。

Development of a duplex real-time PCR assay for detecting 13 streptococcal superantigens

Objective To establish a duplex real-time PCR assay for rapid ,sensitive ,specific detection of 13 streptococcal supe-rantigens .Methods Primer and probe sequences were selected based on the highly conserved region from an alignment of nucleotide sequences of the 13 streptococcal SAgs .The reaction conditions of the duplex real-time PCR were optimized and the specificity and sensitivity of the duplex assay was evaluated using SAg positive strains .Results 13 SAgs were able to differentiated using the du-plex assay and there was no cross-reaction with non-Streptococcus bacteria .On the other hand ,the limit of detection of the duplex assay was at least one or two log dilutions higher than that of the conventional PCR .Conclusion The study of duplex real-time PCR assay can identified 13 streptococcal superantigens accurately and fastly .