双重实时荧光PCR快速检测猪链球菌2型方法的建立及应用

Establishmennt of dual real-time PCR for detection of type 2 Strepoccous suis and it's application

根据GenBank上公布的MRP与CPS2J的开放性阅读框保守序列,运用BEACON Desigener 2.0软件设计和合成了2对引物和其相应的荧光探针,优化各个反应物的浓度和实时荧光PCR循环程序,建立从组织中直接检测2型链球菌主要毒力因子MRP与CPS2J的实时荧光PCR方法。双重实时荧光PCR检测2型猪链球菌方法能够从病料中直接检测2型猪链球菌并可以检测出该菌的主要毒力基因MRP,运用该方法可以最低检测到24CFU/mL 2型猪链球菌,并且与其他血清型链球菌和其他病原菌无交叉反应,特异性良好。通过对8个样品在3个不同时间的检测,结果显示该方法的稳定性和重复性均很好。

On the basis of the obtained cors sequences of open reading frames of MRP and CPS2J,which were published in GenBank,we desigened two pairs of primers and it＇s fluorescent specific probe by the soft ware of BEACON Desigener 2.0. By optimized the concentrations of all actors and the loop program,we set up the method to detect the main virulence factors MRP,CPS2J in the type 2 Strepoccous suis from histological material. Dual real-time PCR for detection of type 2 Strepoccous suis can detect the type 2 Strepoccous suis from tissues enable to judgement of the type 2 Strepoccous suis＇virulence gene MRP, by the method we detected 24CFU/ml type 2 Strepoccous suis, and there was no consensual reaction between other serological type. We detected eight samples in three different periods. This method was proved to have a good stability and reproducibility.