A novel molecular quantitative method for rapid and sensitive detection of<i>Escherichia coli</i>from roof-harvested rainwater

Deshmukh, Rehan; Bhand, Sunil; Roy, Utpal

doi:10.1039/c9ay00587k

Cited by 8 publications

(14 citation statements)

References 32 publications

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“…These results also indicated that the samples under study contained high numbers of viable but non-culturable E. coli cells. The results obtained in our study are in agreement with the earlier reported data in which the application of qPCR yielded abundance of cell number when compared to standard plating method (5,17,22).…”

Section: Discussionsupporting

confidence: 93%

“…The analysis of the standard curve (Fig. 2) derived from the PMA-based RT-qPCR using DNA from E. coli strains (both E. coli O157:H7 and MTCC 3221) revealed that the LOD was approximately 7 femtogram, which is equivalent to approximately 1 cell of E. coli (5). However, the LOD of mSMAC was 1.8 CFU/100 mL.…”

Section: Sensitivity Of Bcig-smac and Pma-qpcrmentioning

confidence: 99%

“…The filter was then incubated on BCIG-SMAC agar at 37°C for determination of bacterial growth and colour. The membrane filtration method was used for both BCIG-SMAC agar and PMA-qPCR method (5). Sterility controls for membrane filters and bacteriological saline used for rinsing the filtration apparatus were also standardized.…”

Section: Membrane Filtration and Spiking Of Water Sam-mentioning

confidence: 99%

“…In order to effectively monitor future outbreaks, it is necessary to have availability of rapid yet specific methods for detection of E. coli cells. The quantitative PCR (qPCR) is one such method for detection of microbial pathogens mainly in terms of selectivity, specificity and rapidity (5,6). While the q-PCR alone cannot differentiate between the viable and non-viable used for microbiological monitoring of pathogens in a number of recent studies (11)(12)(13)(14)(15).…”

Section: Introductionmentioning

confidence: 99%

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BCIG-SMAC medium and PMA-qPCR for differential detection of viable Escherichia coli in potable water

Deshmukh

Bhand

Roy

2021

IJM

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Background and Objectives: Public health protection requires timely evaluation of pathogens in potable water to minimize outbreaks caused by microbial contaminations. The present study was aimed at assessing the microbiological quality of water obtained from Shantinagar (a rural area in the South Goa region of Goa, India) using 5-Bromo-4-Chloro-3-Indoxyl β-D-glu- curonide-Sorbitol MacConkey agar (BCIG-SMAC) medium and, propidium monoazide-quantitative polymerase chain reac- tion (PMA-qPCR) assay for differential detection and quantification of viable Escherichia coli cells in water samples. Materials and Methods: Membrane filtration method was used for both BCIG-SMAC medium and PMA-qPCR methods. To determine the efficiency of detection of viable cells, we first evaluated the PMA treatment protocol and established the standard calibration curves using previously reported primers. Results: PMA-qPCR detected as low as 7 femtograms of DNA of E. coli per qPCR reaction whereas the limit of detection (LOD) of BCIG-SMAC medium was 1.8 CFU/100mL. A total of 71 water samples spanning 2017-2018 have been analyzed using BCIG-SMAC medium and PMA-qPCR, of which 95.77% (68/71) and 7.04% (5/71) were found to be total E. coli and E. coli O157:H7, respectively. PMA-qPCR study showed the viable counts of total viable E. coli cells ranging from 3CFU/100mL to 8.2×102 CFU/100mL. The total E. coli CFU/100mL quantified by PMA-qPCR significantly exceeded (paired t-test; P<0.05) the number on BCIG-SMAC medium. Conclusion: The present study indicates that the microbiological quality of environmental water samples analyzed do not comply with the regulatory standard. Therefore, special attention is warranted to improve the overall portable quality of water in the perspective of public health.

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Section: Discussionsupporting

confidence: 93%

Section: Sensitivity Of Bcig-smac and Pma-qpcrmentioning

confidence: 99%

Section: Membrane Filtration and Spiking Of Water Sam-mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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BCIG-SMAC medium and PMA-qPCR for differential detection of viable Escherichia coli in potable water

Deshmukh

Bhand

Roy

2021

IJM

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“…In another work, Maheux et al [27] detected Escherichia fergusonii in a PCR targeting the tuf gene. Albeit, it has been extensively reported, neither β-D-glucuronidase activity nor uidA gene amplification is the full proof for the accurate molecular detection E. coli in the presence of this enzyme or gene has been reported in Flavobacteria and to a great extent in Shigella, Salmonella and Yersinia [38,40,41]. Contrarily, Fricker & Fricker [42] using uidA primer pair detected five non-E. coli coliforms in water samples.…”

Section: Polymerase Chain Reaction (Pcr) Methodsmentioning

confidence: 99%

Molecular Diagnostic Platforms for Specific Detection of Escherichia coli

Deshmukh¹,

Roy²

2023

Escherichia Coli - Old and New Insights

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Developing countries due to socio-economic conditions are more prone to frequent pathogenic outbreaks; inadequate sanitation and water quality monitoring are also responsible for such conditions. Therefore, it is of paramount importance to provide microbiologically safe food/water in order to protect public health. Several flaws in traditional culturing methods have sparked a surge in interest in molecular techniques as a means of improving the efficiency and sensitivity of microbiological food/water quality monitoring. Molecular identification of water contaminants, mainly Escherichia coli, has been extensively used. Several of the molecular-based techniques are based on amplification and detection of nucleic acids. The advantages offered by these PCR-based methods over culture-based techniques are a higher level of specificity, sensitivity, and rapidity. Of late, the development of a biosensor device that is easy to perform, highly sensitive, and selective has the potential to become indispensable in detecting low CFU of pathogenic E. coli in environmental samples. This review seeks to provide a vista of the progress made in the detection of E. coli using nucleic acid-based approaches as part of the microbiological food/water quality monitoring.

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

2019

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A novel molecular quantitative method for rapid and sensitive detection ofEscherichia colifrom roof-harvested rainwater

Abstract: Comparison of the sensitivity and specificity of CoDEX-qPCR with that of the U.S. EPA MI agar method 1604 and uidA, yaiO and tuf gene-based qPCR.

Cited by 8 publications

References 32 publications

BCIG-SMAC medium and PMA-qPCR for differential detection of viable Escherichia coli in potable water

BCIG-SMAC medium and PMA-qPCR for differential detection of viable Escherichia coli in potable water

Molecular Diagnostic Platforms for Specific Detection of Escherichia coli

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

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