Rapid Detection of Live E. coli O157: H7 by PMA-qPCR Method

Abstract

Abstract: In this work, a method to detect live E. coli O157:H7 was established using propidium monoazide (PMA) coupled with quantitative polymerase chain reaction (qPCR). The minimum inhibitory concentration against DNA amplification from dead bacteria was 10 µg/mL, and PMA did not inhibit DNA application from live bacteria at concentrations equal to or lower than 20 µg/mL. Covalent cross-linking of PMA with DNA was induced by strong light illumination for 5 min and meanwhile, free PMA was completely inactivated, resulting in the avoidance of false negative results. When the live to dead bacteria ratio was more than 1%, PMA pretreatment allowed the elimination of DNA interference from thermally inactivated bacteria and therefore live bacteria could be quantified.

Key words: propidium monoazide (PMA), quantitative polymerase chain reaction (qPCR), live bacteria, detection

CLC Number:

References

[1]Riedy, M, Muirhead, K, Jensen, C, Stewart, C.Use of a photo-labeling technique to identify nonviable cells in fixed homologous or heterologous cell populations[J].Cytometry,1991,12(2):133-139
[2]Marouani Gadri, N.,et al , Potential of Escherichia coli O157:H7 to persist and from viable but non-culturable cells on a food-conduct surface subjected to cycles of soiling and chemical treatment[J].International Journal of Food Microbiology,2010,144(1):96-103
[3]蒋志国,杜琪珍.食品病原微生物快速检测技术研究进展[J].食品研究与开发,2008,29(3):165-170
[4]Nogva, H K.., et al, Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5’-nuclease PCR.[J].Biotechniques,2003,34(4):804-813
[5]Andreas Nocker, Katherine E Sossa, Anne K Camper.Molecular monitoring of disinfection efficacy using propidium monoazide in combination with quantitative PCR[J].Journal of Microbiological Methods,2007,70(2):252-260
[6]鲁玉侠,郭祀远,石磊,李琳.EMA-LAMP方法快速检测死/活的食源性沙门氏菌[J].食品科学,2009,30(22):324-327
[7]RawsthomenullH., C.N. Dock, & L.A. Jaykus.PCR-Based Method Using Propidium Monoazide to Distinguish Viable from Nonviable Bacillus subtilis Spores. Appl. Environ.[J].Microbiol,2009,:-
[8]Vesper nullS.,.,et al., Quantifying fungal viability in air and water samples using quantitative PCR after treatment with propidium monoazide (PMA) .[J].Journal of Microbiological Methods,2008,:-
[9]Mariana Fittipaldi, et al.Discrimination of infectious bacteriophage T4 virus by propidium monoazide real-time PCR[J].Journal of Virological Methods,2010,168:228-232
[10]Takashi SoejimaKen-ichiro Iida,Tian Qin,Hiroaki Taniai,Masanori Seki,and Shinichi Yoshida..Method To Detect Only Live Bacteria during PCR Amplification [J].Journal of Clinical Microbiology,2008,:-
[11]PM FratamiconullSK Sackitey, M Wiedmann and MY Deng. Detection of Escherichia coli O157:H7 by multiplex PCR[J].Journal of Clinical Microbiology,2008,46(7):2305-2313
[12]罗剑飞,林炜铁,郭勇.PMA与PCR结合的细菌活细胞检测方法[J].华南理工大学学报(自然科学版),2010,38(9):142-1146

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