猪链球菌种与9型猪链球菌二重PCR检测方法的建立及应用

为了建立猪链球菌(Streptococcus suis,SS)种与9型猪链球菌(SS9)的快速诊断方法,本研究根据GenBank已登录的SS种特异性基因gdh和SS9型特异性基因CPS9H设计引物,以标准SS9株基因组DNA为模板,建立了SS种和SS9的二重PCR检测方法,并进行了特异性、敏感性和重复性试验;利用所建立的方法对检测疑似猪链球菌感染猪临床样品,并与常规细菌分离鉴定方法进行了比对。结果表明成功建立SS种和SS9型猪链球菌二重PCR检测方法,该方法的检测灵敏度可达100个CFU,特异性和重复性好;利用该方法对34份临床分离自疑似猪链球菌感染样品的细菌培养物进行了应用检测试验,其中有11份样品为gdh阳性,11份gdh阳性样品中有3份样品同时为SS9阳性。本研究成功建立了SS种与SS9型猪链球菌二重PCR检测方法,可用于猪链球菌种和SS9型猪链球菌的快速诊断。

Establishment and application of Duplex PCR Assay for Detection of Streptococcus suis species and Serotypes 9

A duplex PCR assay for detection of Streptococcus suis (SS) species and Serotypes 9 (SS9) was developed using the primers designed based on the gene encoding glutamate dehydrogenase (gdh) of S. suis and cps9H gene encoding the capsule (cps) of S. suis serotypes 9 (SS9) . The DNA of standard SS9 strain was used as the positive control to establish the duplex PCR assay. The sensitivity, specificity and repetition of the PCR assay were tested, and 34 purified culture samples taken from clinic suspicious S.suis infected pigs were detected by the established duplex PCR assay in contrast to the routine bacterial isolation and identifying method. The results indicated the duplex PCR assay were successfully established. The specificity and the sensitivity of the duplex PCR assay revealed that the duplex PCR threshold was 100 CFU of S. suis, and no products were amplified from the genomic DNA of the other 6 pathogenic bacterial acting as the negative control. The repetition test indicated that the duplex PCR was reproducible. Total 11 of 34 purified strains suspicious Streptococcus infected of culture samples demonstrated the gdh positive, which was consistent with the results of the routine bacterial isolation and identifying method, and 3 of 11 gdh positive samples displayed the cps9H positive, too. Our study suggested that the established duplex PCR method was highly specific and sensitive,and was suited to clinic rapid diagnosing of Streptococcus suis species and Serotypes 9.