L型滑液囊支原体实时荧光定量PCR方法的建立与应用  

Development and Application of a Quantitative Real-time PCR for L-type Mycoplasma synoviae

作　　者：刘子卿 任豆豆 孙丽红 王娟娟 孟晨晨 周守长 刘冠慧 许金朋 LIU Ziqing;REN Doudou;SUN Lihong;WANG Juanjuan;MENG Chenchen;ZHOU Shouchang;LIU Guanhui;XU Jinpeng(College of Life Science and Food Engineering,Hebei University of Engineering,Handan,Hebei 056038;Huayu Agricultural Science and Technology Co.,Ltd.,Handan,Hebei 056000;Hebei Laying Hen Industrial Technology Research Institute,Handan,Hebei 056000)

机构地区：[1]河北工程大学生命科学与食品工程学院,河北邯郸056038 [2]华裕农业科技有限公司,河北邯郸056000 [3]河北省蛋鸡产业技术研究院,河北邯郸056000

出　　处：《中国家禽》2024年第7期44-50,共7页China Poultry

基　　金：河北省教育厅科学研究项目(BJK2024031);河北省创新能力提升计划项目(225676150H);邯郸市科学技术研究与发展计划项目(22313014017)。

摘　　要：为建立一种快速、灵敏的检测我国优势基因型(L型)滑液囊支原体(MS)的方法,试验针对L型MS vlh A基因核苷酸序列保守区设计引物及Taq Man-MGB探针,将PCR扩增的vlh A基因克隆至p MD19-T载体,构建重组质粒vlh A-L作为阳性质粒标准品,建立实时荧光定量PCR(q PCR)方法,并检验该方法的特异性、灵敏性和重复性。结果显示:该方法特异性良好,与C型MS-H株及其他鸡常见病原不发生交叉反应;重组质粒vlh A-L最小检出浓度为1.94×10^(1) copies/μL;组内和组间重复性变异系数均小于1%;临床样品检测结果与PCR测序结果吻合。研究表明,建立的q PCR方法特异性强、灵敏性高、重复性好,适用于检测我国L型MS感染情况,还可满足监测免疫MS-H株疫苗鸡群L型MS感染的要求。In order to establish a real-time quantitative PCR for rapid and sensitive detection of the predominant genotype(L-type)of Mycoplasma synoviae(MS)in China,the primers and TaqMan-MGB probe were designed targeting the conserved region of vlhA gene nucleotide sequence.The vlhA gene amplified by PCR were inserted into the pMD19-T vector as the standard positive plasmid(vlhA-L)to establish the real-time PCR(qPCR).The specificity,sensitivity and repeatability of this method were assessed.The results showed that the method had good specificity and no cross-reactivity with C-type MS-H strain and other common chicken pathogens.The minimum detectable concentration of recombinant plasmid vlhA-L was 1.94×10^(1) copies/μL;The coefficients of variation within and between groups were both lower than 1%;The clinical sample detection results were consistent with the PCR sequencing results.These results indicated that the method had strong specificity,high sensitivity and good reproducibility,which could be suitable for the detection of L-type MS infection in China and meet the requirements of monitoring L-type MS infection in chicken flocks immunized MS-H vaccine.

关 键 词：滑液囊支原体 vlhA基因 L型 TAQMAN-MGB探针 实时荧光定量PCR 

分 类 号：S855.1[农业科学—临床兽医学]