兔源性肺炎克雷伯氏菌PCR检测方法的建立

Establishment of PCR Method for Detection of Rabbit-derived Klebsiella Pneumoniae

试验拟通过建立兔源性肺炎克雷伯氏菌的PCR检测方法,为规模化兔场肺炎克雷伯氏菌的检测提供理论依据。以肺炎克雷伯氏菌的保守序列khe基因设计特异性引物,建立PCR反应体系,并对反应条件进行优化。选择不同菌株进行PCR扩增来检测该方法的特异性。接着对不同浓度的肺炎克雷伯氏菌DNA进行PCR扩增,以检测该方法的敏感性。对PCR反应体系和反应条件进行优化,结果显示当引物浓度为50μmol/L,退火温度为52℃时,特异性条带最亮。特异性试验结果显示只有肺炎克雷伯氏菌有特异性条带出现。敏感性试验结果显示建立的PCR检测方法能检测出的DNA最低浓度为1.83 ng/μL。研究所建立的PCR检测方法具有良好的敏感性和特异性,适合肺炎克雷伯氏菌的检测。

This experiment aims to provide theoretical basis for the detection of Klebsiella in large-scale rabbit farm by establishing a PCR detection method for Klebsiella pneumoniae in rabbits.Specific primers were designed with the khe gene of the conserved sequence of Klebsiella pneumoniae to establish PCR reaction system,and the reaction conditions were optimized.Different strains were selected for PCR amplification to test the specificity of the method.The sensitivity of the method was tested by PCR amplification of Klebsiella pneumoniae DNA at different concentrations.The PCR reaction system and reaction conditions were optimized.The results showed that the specific bands were brightest when the concentration of primer was 50μmol/L and the annealing temperature was52℃.The results of specific test showed that only Klebsiella pneumoniae had specific bands.Sensitivity test results showed that the minimum concentration of DNA detected by PCR method established in this study was 1.83ng/μL.The PCR method established in this study has good sensitivity and specificity,and is suitable for the detection of Klebsiella pneumoniae.