快速诊断性DNA测序法鉴定致病性猪链球菌  

Rapid identification of pathogenic streptococcus suis by diagnostic DNA sequencing

作　　者：王洪敏[1,2] 柯昌文[2] 柯碧霞[2] 陈经雕[2] 邓小玲[2] 刘美真[2] 朱振宇[1] 杨杏芬[2] 

机构地区：[1]中山大学基础医学院生物化学与分子生物学教研室,广东广州510080 [2]广东省疾病预防控制中心

出　　处：《华南预防医学》2008年第1期13-17,共5页South China Journal of Preventive Medicine

摘　　要：目的尝试将诊断性DNA测序方法优化后用于猪链球菌的快速鉴定。方法5个来源于临床感染病例的可疑猪链球菌分离株,用诊断性PCR产物直接测序法分析其基因组上的16SrRNA,cps2J和mrp3个特征性靶基因的DNA序列;并对整个技术流程进行优化改进,缩短获得结果的时间。结果获得了5个可疑猪链球菌菌株的3个靶基因片段的DNA碱基序列;这5个菌株被扩增检测的基因区域,有相同的16SrRNA、cps2J和mrp基因片段,长度分别为336、437、520bp;将这3个靶基因片段序列上载到NCBI-BLAST服务器,与GenBank核酸序列数据库的已知序列进行比对分析,结果显示,该5个可疑菌株的被分析的3个靶基因片段与猪链球菌2型的同源性最高。优化后的方法在得到经过适当培养的纯菌后,在不超过7～8h内,就可以获得靶基因片段的DNA测序结果。结论该5个来源于临床感染病例的可疑菌株为血清2型猪链球菌;经优化改进的诊断性DNA测序方法可用于猪链球菌的快速鉴定。Objective To optimize genetic diagnostic method of DNA sequencing for fast identification of microbes. Methods 5 suspected S. suis isolates from clinical infected patients were subjected to PCR amplification of the 3 target genes （16S rRNA, cps2J, mrp） and subsequent DNA sequencing of these 3 amplified DNA fragments; Meanwhile, we optimized the total technical procedure of the diagnostic DNA sequencing to shorten the work time for the process. Results The 3 target gene fragments of the 5 suspected S. suis isolates were sequenced respectively and found identical gene fragment of 16S rRNA, cps2J and mrp in amplified region, with size of 336,437 and 520bp respectively. The sequenced 3 target gene fragments had the highest homology with counterpart genes of serotype 2 S. suis and short processing period of within 7-8h after optimizing. Conclusion 5 suspected S. suis isolates were identified as serotype 2 S. suis with the potential application of optimized diagnostic DNA sequencing method to rapidly identify microbes within 24 hours.

关 键 词：多聚酶链反应 DNA测序 猪链球菌 

分 类 号：R378.12[医药卫生—病原生物学]