猪链球菌1、7、9型多重PCR诊断方法的建立及初步应用

Establishment and Application of Multiplex PCR Assay for Detection of Streptococcus suis Serotypes 1,7 and 9

为了建立猪链球菌(Streptococcus suis,SS)1、7、9型(SS1、SS7、SS9)的快速鉴别诊断和分型方法,本研究根据Gen-Bank已登录的SS1型特异性的CPS1I基因、7型CPS7H基因、9型CPS9H基因设计引物,以标准SS1、SS7、SS9株基因组DNA为模板,建立了SS1、SS7和SS9的多重PCR检测方法,并进行了特异性、敏感性和重复性试验;利用所建立的多重PCR检测方法对11份猪链球菌的临床分离纯化菌株进行了的应用检测。结果表明,成功建立了SS1、SS7和SS9的多重PCR检测方法,该方法的检测灵敏度可达100CFU/mL,特异性和重复性好;利用多重PCR检测方法对11份猪链球菌临床分离纯化菌株检测结果表明,11份样品中有3份样品为SS9阳性、2份样品为SS7阳性、1份样品为SS1阳性。本研究为临床上SS1、SS7、SS9的快速鉴别诊断提供了一种新方法。

A multiplex PCR assay for detection Streptococcus suis （S. suis, SS） serotypes 1, 7, 9 （SS1, SS7, SS9） was developed using the primers designed basing on the gene coding for CPSII, CPS7H and CPS9H coding for the capsule （CPS） of SS1, SS7 and SS9, respectively. The DNA of standard SS1, SS7 and SS9 strains were used as the positive control to establish the multiplex PCR assay. The sensitivity, specificity and repetition assay of multiplex PCR were tested, and 11 purified S. suis strains of culture samples taken from clinic suspicious S. suis infected pigs and having been testified by routine bacterial isolating and identification were detected by the established multiplex PCR assay. The results indicated that the multiplex PCR assay was successfully established. The specificity and the sensitivity of the multiplex PCR assay revealed that the multiplex PCR threshold was 100 CFU/mL of S. suis, and no products were amplified from the genomic DNA of the standard SS2 strain and the other 6 kinds of pathogenic bacterial microorganism acting as the negative control. The repetition test indicated that the duplex PCR was repeatable. Total 3, 2, 1 of 11 purified S. suis strains of culture samples displied the cPSgH, CPS7H, CPSII positive, respectively. The results showed that the established multiplex PCR method was highly specific and sensitive, and was suited to clinic rapid diagnosing of S. suis serotypes1, 7 and 9. .