多重荧光PCR检测Ⅱ型猪链球菌方法的建立及应用

建立了一种快速检测Ⅱ型猪链球菌（Streptococcus suis）的多重荧光PCR（multiplex real-time polymerase chain reaction）方法。首先，从GenBank中获得Ⅱ型猪链球菌荚膜抗原编码基因簇中的cps2I和溶菌素释放蛋白基因mrp，用PrimerExpress2．0设计引物和Taqman荧光探针，在ABl7500荧光PCR仪上进行荧光PCR检测。荧光曲线表明该多重荧光PCR可以特异性地检测Ⅱ型猪链球菌，而参考猪链球菌、大肠杆菌等细菌和空白对照均为阴性；检测方法灵敏度达10个细菌的最小检测量，整个过程仅需60min；稳定性试验表明，2次检测所得的ct值之间无统计学差异（P〉0．05）。本试验建立的多重荧光PCR检测Ⅱ型猪链球菌的方法快速、特异性强，灵敏度高，稳定性好，适合于大批量样品的检验，可广泛用于出入境检疫和动物防疫监督部门的疫情监测。

Establishment and application of multiplex real-time PCR to detect Streptococcus suis serotype 2

A rapid multiplex real-time polymerase chain reaction(PCR) for detection of Streptococcus suis serotype 2 was established in this study. Primers and taqman probes were designed according to cps2I (capsular polysaccharide 2I) and mrp (muramidase released protein) genes with primer express 2.0. Multiplex real-time PCR amplifying curve showed that this method could successfully amplify Streptococcus suis serotype 2, but reference Streptococcus suis strain, E.coli, Salmonella, Staphylococcus aureu, Shigella, Listeria monocytoge and blank control were all negative. 10-fold dilution of Streptococcus suis serotype 2 were used to measure the sensitivity of multiplex real-time PCR. 10 bacteria could be detected in one PCR reaction. To examine the stability of the multiplex real-time PCR, positive control was detected at two different times with 20 repeats of each. Results showed that Ct number of two times have no statistic difference (P>0.05), thus this method has a reliable stability. This reaction can be completed within 60 min. The newly-built multiplex real-time PCR has high sensitivity, good specificity, reliable stability, so it has potential to apply in entry-exit inspection and quarantine.