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Related Experiment Video

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A method for correcting underestimation of enteric pathogen genome quantities in environmental samples.

Pengbo Liu1, Nuhu Amin2, Rana Miah2

  • 1Center for Global Safe Water, Sanitation, and Hygiene, Rollins School of Public Health, Emory University, Atlanta, GA, USA..

Journal of Microbiological Methods
|September 3, 2021
PubMed
Summary

Quantitative PCR accurately detects enteric pathogens in environmental samples. A novel adjustment method corrects for PCR inhibition, improving pathogen quantification in water quality assessments.

Keywords:
Enteric pathogenEnvironmentPCR efficiencyPCR inhibitionQuantificationTaqMan real-time PCR

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Area of Science:

  • Environmental microbiology
  • Molecular biology

Background:

  • Environmental exposure to enteric pathogens presents significant health risks.
  • Accurate detection and quantification are crucial for understanding pathogen behavior and risk assessment.

Purpose of the Study:

  • To quantitatively detect five human-specific enteric pathogens in diverse environmental samples using TaqMan real-time PCR.
  • To develop and validate a method for adjusting pathogen quantification data affected by PCR inhibition and non-optimum PCR efficiency.

Main Methods:

  • Application of TaqMan real-time PCR assays for detecting Shigella/EIEC, Salmonella Typhi, Vibrio cholera, Norovirus, and Giardia.
  • Collection of environmental samples (open drains, canals, floodwater, septic tanks, ABR) in Dhaka, Bangladesh.
  • Development of a two-step method to correct for PCR inhibition and non-optimum PCR efficiency.

Main Results:

  • Quantitative PCR successfully detected multiple enteric pathogens in various environmental matrices.
  • Ultrafiltration samples exhibited significantly higher PCR inhibition compared to grab and sediment samples.
  • The developed adjustment method effectively corrected underestimation of pathogen quantities, with concentration increases ranging from 1.0% to 182.5% in ultrafiltration samples.

Conclusions:

  • Quantitative PCR assays are effective for enumerating multiple enteric pathogens in environmental samples.
  • The developed adjustment method is valuable for improving the accuracy of pathogen quantification, especially in samples with partial PCR inhibition.