Phrommanich Seksan, Suanjit Sudarat, Upatham Suchart, Grams Suksiri Vichasri, Kruatrachue Maleeya, Pokethitiyook Prayad, Korge Günter, Hofmann Annemarie
Biological Science Graduate Program, Faculty of Science, Burapha University, Bangsaen, Chonburi, Thailand.
Microbiol Res. 2009;164(4):486-92. doi: 10.1016/j.micres.2007.03.002. Epub 2007 Apr 24.
Quantitative detection of the oil-degrading bacterium Acinetobacter sp. strain MUB1 was performed using the SoilMaster() DNA Extraction Kit (Epicentre, Madison, Wisconsin) and hybridization probe based real-time PCR. The detection target was the alkane hydroxylase gene (alkM). Standard curve construction showed a linear relation between log values of cell concentrations and real-time PCR threshold cycles over five orders of magnitude between 5.4+/-3.0x10(6) and 5.4+/-3.0x10(2)CFUml(-1) cell suspension. The detection limit was about 540CFUml(-1), which was ten times more sensitive than conventional PCR. The quantification of Acinetobacter sp. strain MUB1 cells in soil samples resulted in 46.67%, 82.41%, and 87.59% DNA recovery with a detection limit of 5.4+/-3.0x10(4)CFUg(-1) dry soil. In this study, a method was developed for the specific, sensitive, and rapid quantification of the Acinetobacter sp. strain MUB1 in soil samples.
使用SoilMaster() DNA提取试剂盒（Epicentre公司，威斯康星州麦迪逊市）和基于杂交探针的实时荧光定量PCR对石油降解菌不动杆菌属MUB1菌株进行定量检测。检测靶点为烷烃羟化酶基因（alkM）。标准曲线构建表明，在5.4±3.0x10(6)至5.4±3.0x10(2)CFUml(-1)细胞悬液之间的五个数量级范围内，细胞浓度的对数值与实时荧光定量PCR阈值循环数之间呈线性关系。检测限约为540CFUml(-1)，比传统PCR灵敏十倍。对土壤样品中不动杆菌属MUB1菌株细胞进行定量分析，DNA回收率分别为46.67%、82.41%和87.59%，检测限为5.4±3.0x10(4)CFUg(-1)干土。本研究建立了一种对土壤样品中不动杆菌属MUB1菌株进行特异性、灵敏且快速定量的方法。
