Quantification of Viable but Nonculturable Escherichia coli O157:H7 by Targeting the rpoS mRNA
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- 作者：Yanming Liu ; Chuan Wang ; Christina Fung ; Xing-Fang Li
- 刊名：Analytical Chemistry
- 出版年：2010
- 出版时间：April 1, 2010
- 年：2010
- 卷：82
- 期：7
- 页码：2612-2615
- 全文大小：199K
- 年卷期：v.82,no.7(April 1, 2010)
- ISSN：1520-6882
文摘
Escherichia coli O157:H7 easily becomes viable but nonculturable (VBNC) under environmental stresses and escapes detection by current methods. Here, we report a unique method enabling the quantification of VBNC E. coli O157:H7 using a selective marker within the rpoS gene. A nucleotide at position +543 within the rpoS gene open reading frame was identified to be unique to E. coli O157:H7. Specifically designed primers and probe combinations were able to differentiate E. coli O157:H7 from closely related bacteria and other common bacteria. The application of this strategy correctly identified 36 clinical and bovine isolates of E. coli O157:H7. A one-step quantification method combining reverse transcription (RT) and real-time quantitative polymerase chain reaction (qPCR) was developed to provide a linear relationship (R2 > 0.99) of copies of RNA with threshold cycles (Ct) and the capability of detecting a single copy of rpoS RNA standards. This technique was used to determine the copies of the rpoS mRNA in culturable cells at different growth phases (mid-log, late-log, and stationary phase) to be 1.57, 0.56, and 0.41 copies/CFU, respectively. VBNC E. coli O157:H7 was determined to have one copy of the rpoS mRNA for every 10 cells, and no rpoS mRNA was detected in 106 dead cells and negative controls. This technique had a linear dynamic range over 6 orders of magnitude and >90% amplification efficiency for tap and river water samples. It was able to selectively quantify as few as 7 E. coli O157:H7 cells in pure culture, 9 culturable cells in tap water and river water, and 23 VBNC cells in river water, demonstrating the best quantification limits for culturable and VBNC E. coli O157:H7 in environmental water.