為建立一種檢測(cè)兔源肺炎克雷伯氏菌（Klebsiella pneumoniae）重組酶聚合酶擴(kuò)增方法（recombinasepolymerase amplification，RPA），根據(jù)肺炎克雷伯氏菌的phoE基因保守序列設(shè)計(jì)引物，擴(kuò)增片段大小為277 bp，并對(duì)反應(yīng)條件進(jìn)一步優(yōu)化，最終建立了適宜于快速準(zhǔn)確檢測(cè)兔源肺炎克雷伯氏菌的重組酶聚合酶等溫?cái)U(kuò)增方法。該方法可特異性檢測(cè)出兔源肺炎克雷伯氏菌，最低檢出細(xì)菌量為8.3×101 CFU/mL，靈敏度比傳統(tǒng)PCR高100倍。本研究建立的肺炎克雷伯氏菌RPA檢測(cè)方法特異性強(qiáng)、操作簡(jiǎn)單，從而為生產(chǎn)中的兔源肺炎克雷伯氏菌現(xiàn)場(chǎng)快速檢測(cè)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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