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兔源肺炎克雷伯氏菌重組酶聚合酶擴增檢測方法的建立

點擊數:2270  時間:2019-07-31 14:09:58  來源: 中國動物檢疫      

為建立一種檢測兔源肺炎克雷伯氏菌(Klebsiella pneumoniae)重組酶聚合酶擴增方法(recombinasepolymerase amplification,RPA),根據肺炎克雷伯氏菌的phoE基因保守序列設計引物,擴增片段大小為277 bp,并對反應條件進一步優化,最終建立了适宜于快速準确檢測兔源肺炎克雷伯氏菌的重組酶聚合酶等溫擴增方法。該方法可特異性檢測出兔源肺炎克雷伯氏菌,最低檢出細菌量為8.3×101 CFU/mL,靈敏度比傳統PCR高100倍。本研究建立的肺炎克雷伯氏菌RPA檢測方法特異性強、操作簡單,從而為生産中的兔源肺炎克雷伯氏菌現場快速檢測提供了一種新方法。

Establishmentof a RPA Assay for Detecting Klebsiella pneumoniae of Rabbit Origin

In order to establish a recombinasepolymerase amplification(RPA)assay for detecting Klebsiella pneumonia of rabbit origin,a pair of primers were designed based on the conservative sequenceof PhoE gene of Klebsiella pneumoniae,and the fragmentwith the length of 277 bp was amplified,then thereaction conditions were further optimized,finally,the RPA assay was established,by which,the Klebsiella pneumoniae of rabbit origin could be specificallydetected with the minimum bacteria of 8.3×101 CFU/mL,and its sensitivity was 100 times higher than that of traditionalPCR. Therefore,the method established in the study hadcharacteristics of strong specificity and simple operation,which would provide a new method for rapid detection of Klebsiellapneumoniae of rabbit origin.

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