Widada J, Nojiri H, Kasuga K, Yoshida T, Habe H, Omori T
Biotechnology Research Center, The University of Tokyo, Japan.
FEMS Microbiol Lett. 2001 Aug 7;202(1):51-7. doi: 10.1111/j.1574-6968.2001.tb10779.x.
The fluorogenic probe assay, competitive polymerase chain reaction (PCR) and co-extraction with internal standard cells were combined to develop a rapid, sensitive, and accurate quantification method for the copy number of a target carbazole 1,9a-dioxygenase gene (carAa) and the cell number of Pseudomonas sp. strain CA10. The internal standard DNA was modified by replacement of a 20-bp long region with one for binding a specific probe in fluorogenic PCR (TaqMan). The resultant DNA fragment was similar to the corresponding region of the intact carAa gene in terms of G+C content. When used as a competitor in the PCR reaction, the internal standard DNA was distinguishable from the target carAa gene by two specific fluorogenic probes with different fluorescence labels, and was automatically detected in a single tube using the ABI7700 sequence detection system. To minimize variations in the efficiency of cell lysis and DNA extraction between the samples, the co-extraction method was combined. A mini-transposon was used to introduce competitor DNA into the genome of other pseudomonads, and the resultant construct was used as the standard cell. After the addition of a fixed amount of the internal standard cells to soil samples, total DNA was extracted (co-extraction). Using this method, the copy number of the carAa gene and the cell number of strain CA10 in soil samples could be quantified rapidly.
将荧光探针分析、竞争性聚合酶链反应（PCR）以及与内标细胞共提取相结合，开发出一种快速、灵敏且准确的方法，用于定量目标咔唑1,9a - 双加氧酶基因（carAa）的拷贝数以及假单胞菌属CA10菌株的细胞数。通过用一段用于在荧光PCR（TaqMan）中结合特异性探针的序列替换20个碱基对长的区域，对内标DNA进行了修饰。所得DNA片段在G + C含量方面与完整carAa基因的相应区域相似。当用作PCR反应中的竞争物时，内标DNA可通过两种带有不同荧光标记的特异性荧光探针与目标carAa基因区分开来，并使用ABI7700序列检测系统在单个管中自动检测。为了最小化样品之间细胞裂解和DNA提取效率的差异，采用了共提取方法。使用一个微型转座子将竞争DNA引入其他假单胞菌的基因组中，所得构建体用作标准细胞。向土壤样品中加入固定量的内标细胞后，提取总DNA（共提取）。使用这种方法，可以快速定量土壤样品中carAa基因的拷贝数和CA10菌株的细胞数。
