入境微生物菌剂泛养副球菌检测方法的建立

Establishment of detection method for Paracoccus pantotrophus in entry microbe inoculums

目的建立入境微生物菌剂泛养副球菌的形态学、生化及分子生物学检测方法。方法用形态学和生化实验筛选最佳培养条件,确定以泛养副球菌ccdA基因为靶基因设计特异性引物和探针,建立泛养副球菌实时荧光PCR检测方法。通过平板计数典型菌落定量分析混合菌剂中的泛养副球菌。结果泛养副球菌的最佳培养条件为35℃±1℃48 h,最适合培养基为硫酸链霉素胰蛋白胨大豆琼脂。实时荧光PCR检测泛养副球菌的灵敏度为0.032 ng/反应,检测混合菌剂中泛养副球菌菌量的灵敏度可达260 CFU/ml。结论建立的泛养副球菌检测方法可以量化入境混合微生物菌剂菌种,有效识别评估混合菌剂中的活菌数。

Objective To establish a morphological,biochemical and molecular biological methods for the detection of Paracoccus pantotrophus from entry microbe inoculums. Methods The optimized culture condition and medium were screened by morphological and biochemical tests. Real-time PCR method was developed based on the specific primers and probes target to the ccdA gene. The number of typical colonies was counted to quantitatively analyze Paracoccus pantotrophus in the mixtured microbe inoculums. Results The optimized culture condition of Paracoccus pantotrophus was35℃±1℃ for 48 h,and the most suitable medium was streptomycin sulphate peptone soybean. The sensitivity of Real-time PCR for the detection of Paracoccus pantotrophus was 0.032 ng/reaction. The sensitivity of Paracoccus pantotrophus in microbe inoculums mixture was 260 CFU/ml. Conclusion The established method could be used to quantify Paracoccus pantotrophus in entry microbe inoculums,and effectively identify and evaluate the number of effective live bacteria.