Ⅱ型猪链球菌快速鉴定的分子生物学方法  

Rapid identification of Streptococcus suis serotype 2 by molecular biologic method

作　　者：郭桐生[1] 鲍春梅[1] 崔恩博[1] 李永利[1] 曲芬[1] 聂为民[2] 李伯安[1] 毛远丽[1] 

机构地区：[1]解放军302医院临检中心,北京100039 [2]解放军302医院感染1科,北京100039

出　　处：《解放军医学杂志》2010年第3期244-245,共2页Medical Journal of Chinese People's Liberation Army

基　　金：军队"十一五"科技攻关项目(06G143)

摘　　要：目的鉴定疑似Ⅱ型猪链球菌血液感染的病原。方法将采集的疑似Ⅱ型猪链球菌血液感染者血液标本注入血培养瓶培养,阳性报警后,转种血平板,5%CO2孵箱培养。同时涂片行革兰染色,API20 strep生化鉴定。16S RNA通用引物PCR扩增,产物经克隆、测序后行Blast比对。针对Ⅱ型猪链球菌型抗原(CPS)、溶菌酶释放蛋白(MRP)和细胞外蛋白因子(EF)设计引物,PCR扩增鉴定。结果镜下细菌为革兰阳性球菌,呈短链状或成对排列。API20 strep鉴定编码为4640453:Ⅱ型猪链球菌(ID值92.3%),16S RNA测序结果为猪链球菌,同源性99%以上,3个特异基因均在相应位置出现条带。结论以Ⅱ型猪链球菌三个特异基因为靶基因的PCR方法能够准确快速地鉴定Ⅱ型猪链球菌。Objective To identify the pathogens of patients suspected to be infected by Streptococcus suis serotype 2. Methods Samples of patients＇ blood were collected into blood culture bottles, and they were then cultured in an instrument of Bact/Alert. After the instrument gave a positive alarm, the samples of positive bottles were then inoculated on Columbia agar containing 5% defibrinated sheep blood, chocolate agar and china blue agar plates. The plates were then incubated for 18-24h at 35℃ with 5% CO2. At the same time the samples were smeared and gram-stained. The isolates of pure culture were smeared and identified with biochemical method of API Strep20 system. Universal primers were used to amplify the genes of 16S RNA, and the objective fragments were cloned. The sequence analysis was then performed, and then the sequences were BLASTed. On the other hand, primers were designed based on the sequences of the capsule polysaccharide （CPS）, muramidase-released protein （MRP）, and extracellular factor （EF） gene. Then PCR were performed to identify Streptococcus suis serotype 2. Results Catalase-negative and Gram-positive coccal genera, arranged in the form of chains or pairs, were isolated. The isolates were identified to be Streptococcus suis serotype 2 by biochemical method （code 4640453; ID value 92.3%）. Sequencing 16S rDNA of these strains revealed that the strains were closely related to Streptococcus suis （99% identity）. At the same time, the aimed bands were showed on electrophoresis pattern, by which the PCR results supported the identification of Streptococcus suis serotype 2. Conclusion Molecular biologic method may be used to verify epidemics of Streptococcus suis serotype 2.

关 键 词：链球菌 猪 分子生物学 抗原 细菌 

分 类 号：R446.5[医药卫生—诊断学]