摘要
为建立一种特异、快速的媾疫锥虫检测方法,本研究通过PCR方法从马媾疫锥虫动基体基因组中扩增得到395 bp的特异的保守序列,将其克隆到pMD-18T载体中构建重组质粒标准品,以10倍倍比稀释的质粒标准品为模板,进行SYBR GreenⅠ荧光定量PCR扩增并制作标准曲线,建立马媾疫锥虫荧光定量PCR的检测方法。结果表明:该方法的检测灵敏度可达到1拷贝/μL,并且与马属动物其他传染病无交叉反应。其组间及组内变异系数分别小于3.183%和3.842%。该方法的建立为快速及特异性检测马媾疫锥虫提供了有效的方法。
To develop a SYBR Green Ⅰ real-time PCR assay （qPCR） to detect Trypanosoma equiperdum, a 395 bp specific and consensus DNA sequence was amplified from the kinetoplast of T. equiperdum and cloned into plasmid pMD-18T. Standard curve was generated by using serial dilutions of recombinant plasmid. Detection results indicated that the assay has high sensitivity with a detection limit of 1 copies per reaction and no cross-reaction with other equine pathogens. Those results indicated that a convenient, specific, high performance and sensitive qPCR assay was established successfully for the detection of T. equiperdum.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2012年第9期724-727,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
国家质检总局科技项目(2008IK015)
