反向线性探针杂交在检测实验动物沙门菌中的研究  

Study on the Reverse Linear Probe Hybridization for Detecting Salmonella in Laboratory Animals

作　　者：魏立雯[1] 韦莉[1] 张文露[2] 潘永全[1] 谭毅[1] 赖国旗[1] 徐永柱 

机构地区：[1]重庆医科大学实验动物中心,重庆400016 [2]重庆医科大学感染性疾病分子生物学重点实验室,重庆400016 [3]重庆市卫生服务中心,重庆400020

出　　处：《四川动物》2013年第2期195-198,共4页Sichuan Journal of Zoology

基　　金：重庆市科委科技创新能力建设项目<重庆市啮齿类实验动物工程技术研究中心>CSTC;2010CB5013;科技部项目<重庆创新药物孵化基地实验动物服务平台>2010ZX09401-306-1-6

摘　　要：目的建立简便、快速、准确、灵敏、特异的沙门菌检测方法。方法根据沙门菌argT基因序列设计通用引物和3'、5'均加有polyC的特异性探针。上游引物5'标记生物素,将探针线性固定在硝酸纤维素膜上,使沙门菌PCR扩增产物与探针进行杂交,通过优化杂交条件,建立反向线性探针杂交检测方法。利用该方法对重庆地区74只实验动物进行检测,同时与传统分离培养方法比较。结果反向线性探针杂交方法灵敏度高,对沙门菌PCR扩增产物在3ng/μL以上可有效检测。从细菌分离培养及DNA提取到PCR扩增及反向杂交结束仅需27h。该检测方法特异性高,对6种非沙门菌的检测中,其特异性为100%。应用传统分离培养方法和反向线性探针杂交方法分别检测42只KM小鼠和32只SD大鼠,两种方法检测结果一致性为100%。结论反向线性探针杂交检测方法,具有快速、可靠、敏感和特异的特点,可用于沙门菌感染时的检测,适合应用于实验动物沙门菌的监测。Objective To establish a simple,rapid,accurate,sensitive and specific detection method of salmonella in laboratory animals.Methods Based on sequence of argT,universal primers and specific probes were designed.5＇ of upstream primer was labeled by biotin.Specific probes,3＇ and 5＇ of which were added poly C respectively,were fixed on the nitrocellulose membrane linearly,so as to make the amplification products of salmonella to hybridize with probes.By optimizing the hybridization conditions,a reverse linear probe hybridization method（RLPH） was established.74 laboratory animals＇ serum samples from Chongqing were detected by this method,compared with traditional culture method.Results More than 3 ng/μL of PCR products of salmonella was effectively detected by RLPH.The whole process,containing bacterium＇s culturing,DNA extraction,PCR and RLPH,was finished within 27 hours.The specificity is 100%（6/6） to test six other non-salmonella samples.The consistency is 100%.Conclusion The reverse linear probe hybridization method to salmonella is rapid,reliable,sensitive and specific.

关 键 词：沙门菌 实验动物 聚合酶链反应 反向线性探针杂交 

分 类 号：Q95-331[生物学—动物学]