实时荧光定量聚合酶链反应检测血清2型猪链球菌的研究

Study on detection of Streptococcus suis serotype 2 by real-time PCR

目的基于TaqMan-MGB探针实时荧光定量聚合酶链反应(Real-time PCR)技术,建立针对血清2型猪链球菌的快速检测方法。方法针对血清2型猪链球菌的csp2 J基因序列,应用Beacon Designer 7.0,设计了引物和TaqMan-MGB探针,建立Real-time PCR检测方法;把目的片段克隆到pMD18-T载体制作标准曲线;以常见致病菌及条件致病菌验证引物探针的特异性;制备血液模拟标本评价本方法在临床标本中的应用。结果由标准曲线可知该方法可以检测到100拷贝/反应体系的目的基因,特异性检测显示只有血清2型猪链球菌有荧光信号,其他常见致病菌及条件致病菌均无荧光信号,血液模拟标本中3.9×102cfu/m l的细菌含量可被检出。结论以csp2 J为目的基因建立的血清2型猪链球菌Real-time PCR检测方法灵敏度高、特异性好、快速,可用于临床感染患者标本的初步筛查。

Objective To establish a rapid detection assay of Streptococcus suis serotype 2 with Taqman-MGB probe based on real-time PCR technology.Methods By using Beacon Designer 7.0,primers and Taqman-MGB probe were designed and the real-time PCR detection assay was established to detect the csp2J gene sequence of Streptococcus suis serotype 2.The target fragment was cloned into the pMD18-T vector to make standard curve.The specificity of the probe was verified with common and conditional pathogenic bacteria,blood simulating samples were prepared to evaluate the clinical application of this assay.Results The standard curve showed that the 100 copies csp2J gene per reaction could be detected by this assay,and specificity detection revealed that only the reaction of Streptococcus suis type 2 showed the fluorescence signal,while that of other common and conditional pathogenic bacteria showed no fluorescence signal,and the lowest testing limitation of blood simulating samples was 3.9×102CFU per ml.Conclusion The real-time PCR detection assay of Streptococcus suis serotype 2 with csp2J as target gene is sensitive,specific and rapid,which can be used in preliminary screening of samples of patients infected with Streptococcus suis type 2.