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沙门氏菌LAMP可视化检测方法的建立

点击数:4272  时间:2019-03-04 14:57:36  来源: 中国动物检疫      作者: 高志强等

为实现沙门氏菌的快速现场检测,根据沙门氏菌 invA 基因编码区序列,设计合成 1 套引物,通过对反应物浓度和反应条件优化,建立了沙门氏菌属特异性 LAMP 检测方法;通过对 invA 基因 8 个区域的 6 条引物配对进行等温扩增,并在反应体系中加入 HNB 来指示反应结果,实现了反应的快速闭管检测。灵敏度试验显示,建立的方法可以检出稀释至 3.6×101 CFU/mL 菌液所提取的 DNA;特异性试验显示,建立的方法与大肠杆菌 DNA、志贺氏菌 DNA、布鲁氏菌试管凝集抗原 DNA、炭疽沉淀抗原 DNA 以及猪链球菌 2 DNA 不发生交叉反应,但能检出鸡白痢、禽伤寒沙门氏菌凝集抗原以及马流产试管凝集抗原提取的 DNA;重复性试验显示,3 个稀释度菌液 DNA 3 次重复检测均为阳性;应用试验显示,从经增菌培养的 200 份进境猴粪拭子样品中检出阳性 2 份,与细菌分离鉴定检测结果一致。结果表明,所建立的方法敏感性和特异性较高,重复性满足实际要求,可用于进境动物样品的沙门氏菌检测。


Development of a Visual LAMPTest Method for Samonella

In searching of rapid field detection methods forsalmonellaa set of primers were designed andsynthesizedwhich were 3 pairs of primers thatidentifying 8 distinct regions of invA gene of Salmonella for isothermalamplification. With optimizing the reactant concentration and reactionconditionsand by adding HNB to the reaction as anindicatora closed-tube LAMP method for rapid fielddetection of Salmonella was developed. Sensitivity test showed that the DNAsextracted from 3.6×101 CFU/mL bacterial suspension could be detected. Specificity testconfirmed that no cross reaction was found between the method with the DNAs ofGolibacillusShigellaBrucella agglutinating antigenanthrax precipitation antigen and type 2Streptococcus suisbut it could detect DNAs from pullorosisSalmonella typhi agglutinating antigen andAbortus-equi agglutination antigen. Repetitive test verified that the resultsof 3 experiments for DNAs in 3 diluted bacteria solutions were all positive.Application test indicated that 2 of 200 swab samples from manures of importedmonkeys were found positivewhich was identical with the result of bacterial isolation test. Alltest results proved that the method is featured with higher degree ofsensitivity and specificityand its repeatability allows practical applicationhence it would be a better approach todetect Salmonella in animal import quarantine.

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