猪附红细胞体TaqMan荧光定量PCR检测方法的建立与应用  

Establishment and application of a TaqMan fluorescent quantitative PCR assay for detection of Mycoplasma suis

作　　者：高旭[1] 张守发[1] 许应天[1] 贾立军[1] 于龙政[1] 薛书江[1] 

机构地区：[1]延边大学农学院动物医学系,吉林延吉133002

出　　处：《中国兽医科学》2013年第5期505-509,共5页Chinese Veterinary Science

基　　金：国家自然科学基金资助项目(30860203);公益性行业(农业)科研专项(200903036-13)

摘　　要：为建立一种能定性与定量检测猪附红细胞体的方法,根据GenBank中猪附红细胞体的16SrRNA基因高度保守区设计了特异性引物和探针,经各反应条件的优化,建立了一种快速检测猪附红细胞体核酸载量的TaqMan荧光定量PCR方法。结果表明,该检测方法在107～101 copies/μL具有良好的线性关系(RSq=0.999)。能检测到模板的下限为30copies,灵敏度是普通PCR检测方法的100倍。该检测方法与其他猪常见病病原体无交叉反应,具有良好的重复性。对临诊疑似猪附红细胞体感染猪血液进行了检测,其检出率比常规PCR方法高10%。表明该方法可用于临床上猪附红细胞体的检测及定量分析。To establish a qualitative and quantitative method to detect Mycoplasma suis ,a pair of pri- mers and a probe were designed based on the high conserved region of M. suis 16 S rRNA gene retrieved from GenBank. A series of PCR and fluorescent quantitative PCR were carried out to optimize the reaction conditions and establish standard curves. The results showed that the method had a good linear relativity from 107 copies/μL to 101 copies/μL（RSq=0. 999）,and had a sensitivity of detecting 30 copies of hi. suis DNA,which was 100 times more sensitive than conventional PCR,and it was highly specific, good reproducibility and no cross reactivity. In clinical practice, a detection rate was 10% higher than conventional PCR. It could be applied for clinical diagnosis and quantification analysis of M. suis.

关 键 词：猪附红细胞体 TaqMan荧光探针 实时荧光定量PCR 临床检测 

分 类 号：S852.62[农业科学—基础兽医学]