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

  • Environmental Microbiology - Short Communication
  • Published:
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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 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 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.

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Acknowledgments

We immensely thank the Indian Council of Medical Research (ICMR), Govt. of India, for awarding senior research fellowship (SRF-8/6/5/ITR-F/2018) to Mr. Rehan Deshmukh. Mr. Arun Kumar Prusty’s kind assistance in setting up and usage of CL-1000L UV Crosslinker is highly acknowledged.

Funding

This research was supported by a grant from Centre for Research Excellence in Waste Water and Energy Management (CORE WWEM) Project of Birla Institute of Technology and Science—Pilani, K. K. Birla Goa Campus.

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Authors and Affiliations

  1. Department of Biological Sciences, Birla Institute of Technology and Science, Pilani, Goa Campus, NH17B Bypass, Goa, 403726, India

    Rehan Deshmukh & Utpal Roy

  2. Department of Chemistry, Birla Institute of Technology and Science, Pilani, Goa Campus, NH17B Bypass, Goa, 403726, India

    Sunil Bhand

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  1. Rehan Deshmukh
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  2. Sunil Bhand
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  3. Utpal Roy
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Correspondence to Utpal Roy.

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Deshmukh, R., Bhand, S. & Roy, U. A novel method for rapid and sensitive detection of viable Escherichia coli cells using UV-induced PMA-coupled quantitative PCR. Braz J Microbiol 51, 773–778 (2020). https://doi.org/10.1007/s42770-019-00161-8

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