应用多重荧光Real-time PCR快速鉴定猪链球菌2型

Rapid identification of Streptococcus suis type 2 with Multi Real-time PCR

目的建立能同时鉴定链球菌2型(S.suis2)和确定主要毒力标志MRP的快速检测方法。方法根据S.suis2CPS2J与MRP的保守序列,设计并合成了2对引物及其相应探针。通过优化各反应物的浓度、配比和循环程序,建立能从组织中直接检测CPS2J和MRP的实时荧光PCR方法。结果能在3h内对送检组织样品检测确定S.suis2型和MRP毒力基因,最低检测菌体浓度24CFU/ml。与普通PCR相比,敏感性在检测CPS2J时比普通PCR高至少10倍以上,而在检测MRP时比其高100倍。与葡萄球菌、化脓链球菌、沙门氏菌、S.suis1型、9型等无交叉反应。同一人在试剂放置0d、20d和60d检测,以及不同人用同一方法检测,变异系数均小于5%,差异不显著。用于检测SSsc0501攻毒后的样品,结果在心、肝、脾、肺、肾、淋巴结、脑中均检测到S.suis2阳性,MRP阳性。而在肌肉中,S.suis2和MRP均为阴性。结论建立的多重荧光Real-timePCR敏感性高、特异性强,稳定性和重复性好,能用于可疑S.suis2型组织样品的快速定型和毒力鉴定。

Two sets of primers and the corresponding fluorescent probes were synthesized according to the Cps2j and Mrp conserved gene sequences of Streptococcus suis type 2（S.suis 2）to establish the identification of S.suis 2 and the main virulence marker Mrp.The multi real-time PCR which could detect the presence of cps2j and mrp genes directly from tissues,was set up after optimization of reaction concentration,matching and cycle programme.The experimental results revealed that this kind of multi real-time PCR could detect S.suis 2 and the mrp gene in samples within 3 hours and at a concentration of 24 CFU/ml.Compared to the traditional PCR,its sensitivity to detect cps2j gene was at least 10 times and that to detect mrp gene was 100 times higher than that of the traditional PCR.There was no cross reaction with Staphylococci,Streptococcus pyogenes,S.suis type 1 and 9,and salmonella species.The coefficient of variation（CV）was less than 5%,no matter detected by different or the same individuals after the reagents had layed up for 0 day,20 days or 60 days.This established multi real-time PCR assay had virtue of good sensitivity,specificity,stability and reproducibility.Therefore,it can be used for rapid identification of S.suis 2 and the main virulence mrp gene in the suspected clinical materials.