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沙門氏菌LAMP可視化檢測方法的建立

點擊數:18255  時間:2019-03-04 14:57:36  來源: 中國動物檢疫      作者: 高志強等

為實現沙門氏菌的快速現場檢測,根據沙門氏菌 invA 基因編碼區序列,設計合成 1 套引物,通過對反應物濃度和反應條件優化,建立了沙門氏菌屬特異性 LAMP 檢測方法;通過對 invA 基因 8 個區域的 6 條引物配對進行等溫擴增,并在反應體系中加入 HNB 來指示反應結果,實現了反應的快速閉管檢測。靈敏度試驗顯示,建立的方法可以檢出稀釋至 3.6×101 CFU/mL 菌液所提取的 DNA;特異性試驗顯示,建立的方法與大腸桿菌 DNA、志賀氏菌 DNA、布魯氏菌試管凝集抗原 DNA、炭疽沉淀抗原 DNA 以及豬鏈球菌 2 DNA 不發生交叉反應,但能檢出雞白痢、禽傷寒沙門氏菌凝集抗原以及馬流產試管凝集抗原提取的 DNA;重復性試驗顯示,3 個稀釋度菌液 DNA 3 次重復檢測均為陽性;應用試驗顯示,從經增菌培養的 200 份進境猴糞拭子樣品中檢出陽性 2 份,與細菌分離鑒定檢測結果一致。結果表明,所建立的方法敏感性和特異性較高,重復性滿足實際要求,可用于進境動物樣品的沙門氏菌檢測。


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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