实时荧光PCR检测Ⅱ型猪链球菌方法的建立  

Establishment of Real-time Fluorescence PCR to Detect Streptococcus suis Serotype 2

作　　者：罗宝正[1] 薄清如[1] 陈竞帆[2] 徐海聂[1] 杨素[1] 杨建允[1] 沙才华[1] 廖秀云[1] 

机构地区：[1]珠海出入境检验检疫局,珠海519015 [2]华南农业大学生命科学学院,广州510642

出　　处：《农业生物技术学报》2006年第5期783-787,共5页Journal of Agricultural Biotechnology

摘　　要：建立了一种检测Ⅱ型猪链球菌(Streptococcussuisserotype2)的实时荧光PCR(real-timefluorescencepolymerasechainreaction)方法。用PrimerExpress2.0和Oligo6.0设计针对Ⅱ型猪链球菌荚膜抗原基因簇中cps2I基因的引物和Taqman荧光探针。通过常规PCR方法扩增得到81bpDNA片段,克隆到pMD18-T载体,测序表明得到的序列为目的基因片段。在Lightcycler荧光PCR仪上对Ⅱ型猪链球菌的扩增曲线表明,该实时荧光PCR具有良好的特异性,可成功地扩增Ⅱ型猪链球菌,而参考猪链球菌(S.suis)、大肠杆菌(Escherichiacoli)、沙门氏菌(Salmonella)、金黄色葡萄球菌(Staphylococcusaureu)、志贺氏菌(Shigella)、单增李斯特氏菌(Listeriamonocytoge)和空白对照都是阴性;该检测方法可达到检测10个细菌的灵敏度,反应过程仅需30min完成;在两个不同时间检测同一浓度的菌液,每次做20个重复,2次检测所得的Ct(threshold,阈循环)值之间无统计学差异(P>0.05),方法稳定性好。该实时荧光PCR检测Ⅱ型猪链球菌的方法可用于出入境检疫和动物防疫监督部门的疫情监测。A real-time fluorescence polymerase chain reaction （PCR） method to detect Streptococcus suis serotype 2 was established. Primers and Taqman probe were designed according to cps2I （capsular polysaccharide 2I） gene with software Primer Express 2.0 and Oligo6.0. Eighty-one base pair DNA fragment was amplified from S. suis serotype 2 genome DNA, and the PCR product was cloned into pMD18-T vector. Sequencing result confirmed that DNA fragment was target segment. Real-time fluorescence PCR amplification curve on Lightcycler showed that this method could successfully amplify S. suis serotype 2, while reference S. suis strain, E. coli, Salmonella, Staphylococcus aureu, Shigella, Listeria monocytoge and blank control were all negative, so it had good specificity. Ten-fold dilution of S. suis serotype 2 was used to measure the sensitivity of real-time fluorescence PCR. Ten bacteria could be detected in one PCR reaction and the reaction could be completed within 30 min. To examine the stability of the real-time fluorescence PCR, positive control was detected at two different times with 20 repeats. Result showed that Ct value of two times had no statistic difference （P 〉 0.05）, thus this method has a reliable stability. The newly-built real-time fluorescence PCR has potential to apply in entry-exit inspection and quarantine.

关 键 词：实时荧光PCR Ⅱ型猪链球菌 

分 类 号：S188[农业科学—农业基础科学]