16S rDNA序列分析法在国际船舶压载舱沉积物病原微生物检测中的应用

〔目的〕探讨16S rDNA序列分析法在国境口岸监测外来病原微生物的可行性。〔方法〕按照0.5 mg/0.5 ml样品接种10 ml增菌液的比例将压载舱淤泥样品分别接种到营养肉汤和碱性蛋白胨水中,37℃过夜,震荡摇匀。增菌后划线接种在选择培养基上,过夜培养后挑取阳性菌落纯培养,按照试剂盒说明提取细菌基因组,扩增细菌基因组16S rDNA片段,将扩增产物纯化后进行序列分析。〔结果〕从48份样品中共检测12种细菌,其中检测到的霍乱弧菌有1株为B33株。这株霍乱弧菌最早在2004年分离自非洲东南部国家莫桑比克的贝拉港,是O1群霍乱弧菌的经典型与Eltor型杂交后产生的菌株,O139群霍乱弧菌尚未检测到。〔结论〕16S rDNA序列分析法可用作国境口岸外来微生物的监测和分析。

Application of 16S rDNA Sequence Analysis Method in Pathogens Detection from Ballast Sediments of International Vessels

Objective To discuss the feasibility of monitoring the invading microorganisms by 16S rDNA sequence analysis method.Method Sediments of ballast were added into nutrient broth and alkaline peptone water separately with the 0.5mg/0.5ml proportion at 37℃ overnight shaking condition.The Bacterial were crossed in the selective mediums after bacterial enrichment.Positive colonies were picked out after the overnight culture and 16S rDNA was amplified from genome DNA which is extracted from bacterial culture.PCR products were analyzed by direct sequencing, Result 15 kinds of bacteria were detected from 48 samples of ballast tank sediments which were collected from 45 international vessels and 12 kinds of them was pathogenic. In which, there was Vibrio cholera B33 which isolated from Beira, Mozambique in 2004. This strain was a hybrid between the classical and Ehor biotypes of Vibrio cholera O1.Vibrio cholerae O139 wasn＇t detected. Conclusion 16S rDNA sequence analysis method can be used as monitoring and analyzing of invading microorganisms at frontier port.