Detection of viable but nonculturable Escherichia coli O157:H7 using propidium monoazide treatments and qPCR
2012
Xiao, Xing-long | Tian, Cong | Yu, Yi-gang | Wu, Hui
Escherichia coli O157:H7 can enter into a viable but nonculturable (VBNC) state under stress conditions. The aims of the present study were to examine the influences of environmental factors on the survivability and culturability of E.coli O157:H7 and to develop an approach for accurate detection of VBNC E.coli O157:H7. The E.coli O157:H7 strain ATCC 6589 was inoculated into 3 induction microcosm models: (i) Luria–Bertani broth, (ii) sterilized tap water, and (iii) sterilized physiological saline solution. Our results showed that low temperature and nutritional starvation significantly impacted on the survivability of E. coli O157:H7 cells and that the in-vitro-induced VBNC cells were capable of resuscitating under normal temperature and appropriate nutrients. We tested the effectiveness of an approach combining propidium monoazide (PMA) treatment with real-time polymerase chain reaction (PMA–qPCR) for accurate quantification of total, viable, dead, and VBNC cells under different induction microcosm models. Our results indicated different threshold cycle (Ct) values for PMA-treated cells and untreated cells (ΔCt = 4.97, 4.29, and 3.30 for Luria–Bertani broth, sterilized tap water, and sterilized physiological saline solution, respectively). We determined the quantification limit of this PMA–qPCR approach to be 1 × 10² cells·mL–¹, providing sufficient sensitivity for detection of VBNC E.coli O157:H7 cells to no less than 100 cells·mL–¹. This study clearly demonstrated the feasibility and effectiveness of using PMA–qPCR to accurately quantify E.coli O157:H7 in a VBNC state.
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