穿刺巴斯德芽菌荧光定量PCR检测方法的建立与应用

Development and application of a real-time PCR system for detection of Pasteuria penetrans

以穿刺巴斯德芽菌16SrDNA片段为靶标,通过对荧光定量PCR反应条件的摸索,建立该菌的荧光定量PCR检测方法。所选靶点最适退火温度60℃,正、反向引物的最佳浓度搭配为900、300nmol/L;以Ct值和质粒拷贝浓度对数为坐标轴建立标准曲线,回归方程为y=-3.200×logx+34.43,R2值为0.998,PCR扩增效率为105.4%,对含穿刺巴斯德芽菌芽胞的土壤样品检测阈值为2×103个/g土壤。该方法敏感度高、特异性好,能够运用于穿刺巴斯德芽菌的定性、定量检测。

Based on the 16S rDNA sequence, a real-time PCR detection method for Pasteuria penetrans was developed. Its optimal annealing temperature was 60℃ and the most effective concentration of forward primer and reverse primer was 900 nmol/L and 300 nmol/L, respectively. There was a good linear relationship between logscaled copies of the plasmid and Ct values, and the regression equation was y =-3. 200 × logx ＋34.43, R2 value was 0. 998, and reaction efficiency was 105.4%. The threshold for P. penetrans detection from soil sample was 2 ×103 spores/g soil. This system is considered to be effective and specific to be used for qualitative and quantitative detection of P. penetrans.