猪衣原体实时荧光定量PCR检测方法的建立及应用  

Development and Application of TaqMan-MGB Real-time PCR for Detection of C.cuis

作　　者：袁曾壮 王昱[2,3,4] 聂福平[2,3,4] 杨俊[2,3] 李应国[2,3] 肖进文[2,3] 王国民[2,3] 李贤良[2,3] 刘力[1] 

机构地区：[1]西南大学动物科技学院,重庆400715 [2]重庆出入境检验检疫局,重庆400020 [3]重庆市进出口食品安全工程中心,重庆400020 [4]重庆大学生物工程学院,重庆400030

出　　处：《动物医学进展》2014年第6期21-25,共5页Progress In Veterinary Medicine

基　　金：质检公益性项目(201310093);国家质检总局科技项目(2012IK028);重庆市科委科技创新能力建设项目(CSTC;2009CB1012)

摘　　要：为了建立猪衣原体（Chlamydia suis ）实时荧光定量 PCR（real-time PCR）检测方法，根据猪衣原体（C．cuis ）的 MOMP 基因，选取高度特异保守的区域，设计实时荧光定量 PCR 引物和探针，并构建了重组质粒。结果显示，该方法建立的标准曲线方程为：Y＝-3．2147x＋41．218，线性相关系数为0．9991，扩增效率为104．6％，最低检测量为1．7＆#215;102 copies ！μL。该检测方法特异性较好，与鹦鹉热亲衣原体、流产亲衣原体、沙眼衣原体、肺炎嗜衣原体、牛羊亲衣原体、鼠衣原体无交叉反应。批内和批间重复试验的变异系数在0．458％～1．174％范围内，具有较好的重复性；与普通 PCR 方法相比较，该方法具有较高的检出率。建立对猪衣原体的监测和流行病学调查都具有非常重要的意义。In order to establish quantitative PCR for detecting Chlamydia suis ,based on the MOMP gene of C .cuis,this study selected specific primers and probe from highly conserved region,the PCR reaction was developed by building the recombinant plasmid.The results showed that the detection limit of the assay was 1.7×10^2 copies/μL,the calibration curve equation：Y=-3.214 7x＋41.218,a correlation coefficient of R2 = 0.999 1,expanding efficiency rate 104.6%.The method used to detect Chlamydophila psittaci , Chlamydophila abortus ,Chlamydia trachomatis ,Chlamydophila pneumoniae ,Chlamydophila pecorum , Chlamydia muridarum had no cross reaction.The real-time PCR was reproducible,the test of its intra-and inter-coefficients of variation from 0.495% to 1.752%.Compared with conventional PCR method,the detection rate of the method established in this study is higher.This method is important for detecting C . cuis and epidemiological investigation.

关 键 词：猪衣原体 实时荧光定量PCR TaqMan-MGB 

分 类 号：S852.67[农业科学—基础兽医学]