NSF International, Microbiology Division, 789 Dixboro Road, Ann Arbor, MI 48105, USA.
J Ind Microbiol Biotechnol. 2011 Sep;38(9):1337-44. doi: 10.1007/s10295-010-0917-0. Epub 2010 Dec 4.
Raoultella terrigena ATCC 33257, a representative of the coliform group, is commonly used as a challenge organism in water purifier efficacy testing. In addition to being time consuming, traditional culturing techniques and metabolic identification systems (including automated systems) also fail to accurately differentiate this organism from its closely related neighbors belonging to the Enterobacteriaceae group. Molecular-based techniques, such as real-time quantitative polymerase chain reaction (qPCR) and enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting, are preferred methods of detection because of their accuracy, reproducibility, specificity, and sensitivity, along with shorter turnaround time. ERIC-PCR performed with the 1R primer set demonstrated stable unique banding patterns (~800, ~300 bp) for R. terrigena ATCC 33257 different from patterns observed for R. planticola and R. ornithinolytica. The primer pair developed from gyraseA (gyrA) sequence of R. terrigena for the SYBR Green qPCR assay using the AlleleID(®) 7.0 primer probe design software was highly specific and sensitive for the target organism. The sensitivity of the assay was 10(1) colony forming units (CFU)/ml for whole cells and 4.7 fg with genomic DNA. The primer pair was successful in determining the concentration (5.5 ± 0.3 × 10(6) CFU/ml) of R. terrigena from water samples spiked with equal concentration of Escherichia coli and R. terrigena. Based on these results from the ERIC-PCR and the SYBR Green qPCR assay, these molecular techniques can be efficiently used for rapid identification and quantification of R. terrigena during water purifier testing.
雷尔氏变形杆菌 ATCC33257 是大肠菌群的代表菌，常用于水质净化器功效检测的挑战菌。传统的培养技术和代谢鉴定系统（包括自动化系统）除了耗时之外，还不能准确地区分该菌与其密切相关的肠杆菌科菌属。基于分子的技术，如实时定量聚合酶链反应（qPCR）和肠杆菌重复基因间一致性（ERIC）-PCR 指纹图谱分析，因其准确性、重现性、特异性和敏感性以及较短的周转时间，是首选的检测方法。用 1R 引物对进行 ERIC-PCR 显示雷尔氏变形杆菌 ATCC33257 的稳定独特的带型（800、300bp），与植生雷尔氏菌和 ornithinolytica 的模式不同。从雷尔氏变形杆菌 gyraseA（gyrA）序列开发的引物对用于 SYBR Green qPCR 分析，使用 AlleleID(®)7.0 引物探针设计软件，对目标菌具有高度特异性和敏感性。该测定的灵敏度为 10(1)个细胞形成单位（CFU）/ml 全细胞和 4.7fg 基因组 DNA。该引物对成功地确定了用相同浓度的大肠埃希氏菌和雷尔氏变形杆菌污染的水样中雷尔氏变形杆菌的浓度（5.5±0.3×10(6)CFU/ml）。基于 ERIC-PCR 和 SYBR Green qPCR 分析的这些结果，这些分子技术可有效地用于水质净化器检测期间快速鉴定和定量雷尔氏变形杆菌。
