马流产沙门氏菌的分离鉴定及其微量凝集抗体检测方法的建立与应用  

Establishment and Preliminary Application of Microagglutination Detection Method for Salmonella Abortus Equi

作　　者：郭奎 王宁 王金慧 初晓雨 赵语婷 郭巍[1] 刘荻萩[1] 胡哲[1] 王晓钧[1] GUO Kui;WANG Ning;WANG JinHui;CHU XiaoYu;ZHAO YuTing;GUO Wei;LIU DiQiu;HU Zhe;WANG XiaoJun(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069)

机构地区：[1]中国农业科学院哈尔滨兽医研究所/兽医生物技术国家重点实验室,哈尔滨150069

出　　处：《中国农业科学》2020年第10期2112-2121,共10页Scientia Agricultura Sinica

基　　金：“十三五”国家重点研发计划(2018YFD0502205)。

摘　　要：[目的]对流产马驹组织进行马流产沙门氏菌的分离和鉴定,以分离株制备凝集抗原,旨在建立一种敏感、特异、操作简便和高通量的微量凝集方法,实现对马流产沙门氏菌抗体进行快速检测.[方法]利用沙门氏菌显色培养基对流产的马驹脏器进行细菌分离,对紫色菌落进行革兰氏染色鉴定;提取细菌全基因组,利用16S rRNA进行PCR扩增和测序鉴定,并对菌株进行生化鉴定和血清型鉴定,对病因进行综合诊断.利用分离获得的阳性菌株进行灭活后作为凝集抗原,以马流产沙门氏菌标准阳性血清作为阳性对照,通过反应条件优化,建立微量凝集试验方法,并对该方法的特异性、敏感性、重复性进行了验证.与国家行业标准中的试管凝集试验方法进行对比,同时利用微量凝集对华北和西北各3个地方的共151份血清样品进行检测,并随机选取30份样品,利用微量凝集和试管凝集进行检测,并对两种方法检测的敏感性进行比较分析.[结果]各流产马驹组织在沙门氏菌显色培养基上划线结果,均可以获得大量紫色目标菌落;革兰氏染色结果表明,分离的菌株为革兰氏阴性短杆菌,16S rRNA测序证实分离株为沙门氏菌属,生化鉴定结果表明,分离的17株菌均符合马流产沙门氏菌的生化特性,但相比上世纪强毒株马流产沙门氏菌C77-1,均不能发酵鼠李糖;血清型鉴定证实分离株均为马流产沙门氏菌,将分离的17株马流产沙门氏菌分别命名为E.S-1-17.因此证实马匹流产均为马流产沙门氏菌感染所致.利用甲醛对E.S-1菌株进行了灭活处理,成功制备了马流产沙门氏菌凝集抗原,并建立了一种快速敏感的微量凝集检测方法,其中优化了最佳反应凝集抗原浓度为4亿/mL.其敏感性比试管凝集高1个滴度,其特异性与其他常见马传染病病原阳性血清无交叉反应,并且具有良好的重复性.利用微量凝集方法,对华北3个疫情区进行�【Objective】Salmonella abortus equi was isolated and identified from the tissue of aborted foal.The aim for the study was to establish a sensitive,specific,easy to operate and high throughput microagglutination method by using the isolated Salmonella abortus equi,so as to realize the rapid diagnosis of Salmonella abortus equi antibody.【Method】Selective medium for Salmonella was used for bacteria reproduction from aborted foal organs,and the purple colonies were selected and identified by Gram staining.The whole genome of bacteria was extracted,and 16S rRNA was used for PCR amplification and sequencing identification.Biochemical identification and serotype identification of the bacteria were carried out to make a comprehensive diagnosis of the etiology.The obtained positive bacteria was used as agglutinating antigen after inactivation,and the standard positive serum of Salmonella was used as positive control.The microagglutination assay was established by optimizing the reaction conditions,and then the specificity,sensitivity and repeatability of the method were verified.Meanwhile,a total of 151 serum samples from three different locations in north and northwest of China were detected with microagglutination.And 30 of these samples were randomly selected to be detected with both microagglutination and test tube agglutination,and the sensitivities of these two methods were compared and analyzed.【Result】A large number of purple target colonies could be obtained from the coloration culture medium of Salmonella.The results of gram staining showed that the isolated strains were gram-negative short bacillus,and 16S rRNA sequencing confirmed that the isolated strains were Salmonella.The biochemical identification results showed that all the 17 isolated strains were in line with the biochemical characteristics of Salmonella abortus equine,but could not ferment rhamnosus compared with C77-1,a strong strain of Salmonella abortus equi identified in China in the last century.Serotype identification confirmed that a

关 键 词：马流产沙门氏菌 分离鉴定 微量凝集 应用 马 

分 类 号：S852.61[农业科学—基础兽医学]