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A novel method for rapid and sensitive detection of viable Escherichia coli cells using UV-induced PMA-coupled quantitative PCR.

ABSTRACT: We report a specific and sensitive method to improve the coupling of propidium monoazide (PMA) with DNA derived from killed cells of Escherichia coli using UV light of 365 nm. UV light of three different intensities mainly 2.4?×?103, 4.8?×?103, and 7.2?×?103 ?J/cm2 was applied to E. coli cells each for 1, 3, and 5 min. PMA was found to be successfully cross-linked with the DNA from killed cells of E. coli at 4.8?×?103 ?J/cm2 in 3 min leading to the complete inhibition of PCR amplification of DNA derived from PMA-treated heat-killed cells. In spiked phosphate-buffered saline and potable water samples, the difference of the Cq values between PMA-treated viable cells and PMA-untreated viable cells ranged from -0.17 to 0.2, demonstrating that UV-induced PMA activation had a negligible effect on viable cells. In contrast, the difference of the Cq values between PMA-treated heat-killed cells and PMA-untreated heat-killed cells ranged from 8.9 to 9.99, indicating the ability of PMA to inhibit PCR amplification of DNA derived from killed cells to an equivalent as low as 100 CFU. In conclusion, this UV-coupled PMA-qPCR assay provided a rapid and sensitive methodology to selectively detect viable E. coli cells in spiked water samples within 4 h.

SUBMITTER: Deshmukh R

PROVIDER: S-EPMC7203400 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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A novel method for rapid and sensitive detection of viable Escherichia coli cells using UV-induced PMA-coupled quantitative PCR.

Deshmukh Rehan R Bhand Sunil S Roy Utpal U

Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] 20191026 2

We report a specific and sensitive method to improve the coupling of propidium monoazide (PMA) with DNA derived from killed cells of Escherichia coli using UV light of 365 nm. UV light of three different intensities mainly 2.4 × 103, 4.8 × 103, and 7.2 × 103 μJ/cm2 was applied to E. coli cells each for 1, 3, and 5 min. PMA was found to be successfully cross-linked with the DNA from killed cells of E. coli at 4.8 × 103 μJ/cm2 in 3 ...[more]

PMID: 31654340

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Project description:Escherichia coli O157:H7 and Salmonella enterica are leading causes of foodborne outbreaks linked to fresh produce. Both species can enter the "viable but nonculturable" (VBNC) state that precludes detection using conventional culture-based or molecular methods. In this study, we assessed propidium monoazide-quantitative PCR (PMA-qPCR) assays and novel methods combining PMA and loop-mediated isothermal amplification (LAMP) for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. The performance of PMA-LAMP assays targeting the wzy gene of E. coli O157:H7 and the agfA gene of S. enterica and the performance of PMA-qPCR assays were compared in pure culture and spiked tomato, lettuce, and spinach. No cross-reaction was observed in the specificity tests. The values representing the limit of detection (LOD) seen with PMA-LAMP were 9.0 CFU/reaction for E. coli O157:H7 and 4.6 CFU/reaction for S. enterica in pure culture and were 5.13 × 103 or 5.13 × 104 CFU/g for VBNC E. coli O157:H7 and 1.05 × 104 or 1.05 × 105 CFU/g for VBNC S. enterica in fresh produce, representing results comparable to those obtained by PMA-qPCR. Standard curves showed correlation coefficients ranging from 0.925 to 0.996, indicating a good quantitative capacity of PMA-LAMP for determining populations of both bacterial species in the VBNC state. The PMA-LAMP assay was completed with considerable economy of time (30 min versus 1 h) and achieved sensitivity and quantitative capacity comparable to those seen with a PMA-qPCR assay. PMA-LAMP is a rapid, sensitive, and robust method for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce.IMPORTANCE VBNC pathogenic bacteria pose a potential risk to the food industry because they do not multiply on routine microbiological media and thus can evade detection in conventional plating assays. Both E. coli O157:H7 and S. enterica have been reported to enter the VBNC state under a range of environmental stress conditions and to resuscitate under favorable conditions and are a potential cause of human infections. PMA-LAMP methods developed in this study provide a rapid, sensitive, and specific way to determine levels of VBNC E. coli O157:H7 and S. enterica in fresh produce, which potentially decreases the risks related to the consumption of fresh produce contaminated by enteric pathogens in this state. PMA-LAMP can be further applied in the field study to enhance our understanding of the fate of VBNC pathogens in the preharvest and postharvest stages of fresh produce.

Dataset Information

A novel method for rapid and sensitive detection of viable Escherichia coli cells using UV-induced PMA-coupled quantitative PCR.

Publications

A novel method for rapid and sensitive detection of viable Escherichia coli cells using UV-induced PMA-coupled quantitative PCR.

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