Sen Keya
Office of Water, Technical Support Center, US Environmental Protection Agency, 26W M.L. King Drive, Cincinnati, OH 43268, USA.
Can J Microbiol. 2005 Nov;51(11):957-66. doi: 10.1139/w05-089.
Existing biochemical methods cannot distinguish among some species of Aeromonads, while genetic methods are labor intensive. In this study, primers were developed to three genes of Aeromonas: lipase, elastase, and DNA gyraseB. In addition, six previously described primer sets, five corresponding to species-specific signature regions of the 16S rRNA gene from A. veronii, A. popoffii, A. caviae, A. jandaei, and A. schubertii, respectively, and one corresponding to A. hydrophila specific lipase (hydrolipase), were chosen. The primer sets were combined in a series of multiplex-PCR (mPCR) assays against 38 previously characterized strains. Following PCR, each species was distinguished by the production of a unique combination of amplicons. When the assays were tested using 63 drinking water isolates, there was complete agreement in the species identification (ID) for 59 isolates, with ID established by biochemical assays. Sequencing the gyrB and the 16S rRNA gene from the remaining four strains established that the ID obtained by mPCR was correct for three strains. For only one strain, no consensus ID could be obtained. A rapid and reliable method for identification of different Aeromonas species is proposed that does not require restriction enzyme digestions, thus simplifying and speeding up the process.
现有的生化方法无法区分某些气单胞菌属的物种，而基因方法又 labor intensive（劳动强度大）。在本研究中，针对气单胞菌的三个基因开发了引物：脂肪酶、弹性蛋白酶和DNA促旋酶B。此外，还选择了六个先前描述的引物组，其中五个分别对应维氏气单胞菌、波氏气单胞菌、豚鼠气单胞菌、詹氏气单胞菌和舒伯特气单胞菌16S rRNA基因的物种特异性特征区域，另一个对应嗜水气单胞菌特异性脂肪酶（水解脂肪酶）。这些引物组被组合用于一系列针对38株先前已鉴定菌株的多重PCR（mPCR）检测。PCR后，通过扩增子的独特组合来区分每个物种。当使用63株饮用水分离株对检测方法进行测试时，59株分离株的物种鉴定（ID）与生化检测确定的ID完全一致。对其余四株菌株的gyrB和16S rRNA基因进行测序表明，通过mPCR获得的ID对三株菌株是正确的。仅对一株菌株无法获得一致的ID。本文提出了一种快速可靠的鉴定不同气单胞菌物种的方法，该方法不需要限制性酶切，从而简化并加速了鉴定过程。
