滑液囊支原体RAA快速检测方法的建立与应用  

Establishment and application of a RAA method for rapid detection of Mycoplasma synoviae

作　　者：吴颖臻 杨福剑 杜文珍 陈莹 张宗尧 磨美兰[1] 黄腾[1] 谢庆华 黄裕富 唐雪梅 韦天超[1] WU Yingzhen;YANG Fujian;DU Wenzhen;CHEN Ying;ZHANG Zongyao;MO Meilan;HUANG Teng;XIE Qinghua;HUANG Yufu;TANG Xuemei∗;WEI Tianchao(College of Animal Science and Technology,Guangxi University,Nanning 530005,China;Guangxi Shenhuang Group Ltd.,Yuling 537000,China)

机构地区：[1]广西大学动物科学技术学院,广西南宁530005 [2]广西参皇养殖集团有限公司,广西玉林537000

出　　处：《畜牧与兽医》2023年第11期88-93,共6页Animal Husbandry & Veterinary Medicine

基　　金：玉林市科学研究与开发技术(玉字科攻201833016);广西肉鸡产业创新团队建设项目(nycytxgxcxtd-19-05);广西重点研发计划(桂科AB18294038)。

摘　　要：为了建立一种快速、简便的基于重组酶介导等温扩增技术(RAA)检测滑液囊支原体(MS)的新型方法,本研究针对MS vlhA基因设计了2对特异性引物。经过优化引物和反应条件,建立了MS的RAA新型检测方法,并对其特异性、敏感性和重复性进行评价,还将其敏感性与常规PCR方法进行比较。应用建立的方法对疑似或确诊的临床样品共100份进行检测。结果表明:建立的基础RAA法可在29℃恒温操作10~15 min即可完成对MS核酸模板的检测;RAA法与大肠杆菌、肠炎沙门菌、鸡毒支原体、新城疫病毒、禽流感病毒、禽传染性喉气管炎病毒、禽传染性支气管炎病毒和禽呼肠孤病毒等其他病原均无交叉反应;对MS基因组DNA的最低检出限为6.53×10^(5)copies/μL,比PCR方法敏感100倍;应用该方法检测了100份临床样本,结果71份阳性,与PCR检测结果一致。研究表明,建立的MS RAA方法具有特异、敏感、快速、简便的特点,方便基层实验室应用。To establish a novel rapid and simple method for detection of Mycoplasma synoviae(MS)based on the recombinase-aided am⁃plification(RAA)assay,two pairs of specific primers were designed to target the VlhA gene of MS in this study.By optimizing the primers sets and the reaction conditions,a new method for MS RAA detection was developed.Then,the specificity,sensitivity,reproducibility of the assay,and comparison of its sensitivity with that of the conventional PCR methods were evaluated.Finally,the established method was ap⁃plied to detecting 100 clinical samples from suspected or confirmed MS infectious chickens.The results showed that the established basic RAA method was able to detect MS nucleic acid templates within 10-15 min,operated at a single constant temperature of 29℃.The RAA assay was specific and non-cross reactive against other chicken pathogens,such as avian Escherichia coli,avian Salmonella enterica,Myco⁃plasma gallisepticum,Newcastle disease virus,avian influenza virus,avian infectious laryngotracheitis virus,avian infectious bronchitis virus and avian reovirus.This method exhibited high sensitivity with a detection limit of 6.53×105 copies/μL for MS genomic DNA,which was 100 times more sensitive than that of the PCR method.71 of the 100 clinical samples were detected positive by the RAA assay,which was con⁃sistent with the findings by PCR.This study indicated that the RAA assay established for MS here had excellent specificity,sensitivity,ra⁃pidity and simplicity;and promised easy operation at low resource settings.

关 键 词：滑液囊支原体 重组酶介导等温扩增技术 快速检测 

分 类 号：S852[农业科学—基础兽医学]