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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters
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A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

Published on: March 9, 2016

Effect of platform, reference material, and quantification model on enumeration of Enterococcus by quantitative PCR

Yiping Cao1, Mano Sivaganesan, Julie Kinzelman

  • 1Southern California Coastal Water Research Project Authority, Costa Mesa, CA 92626, USA. yipingc@sccwrp.org

Water Research
|November 6, 2012
PubMed
Summary

Quantitative polymerase chain reaction (qPCR) for detecting fecal bacteria in water requires method standardization. Reference materials significantly impact results, while platforms have minimal effect, highlighting the need for certified standards.

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Area of Science:

  • Environmental microbiology
  • Molecular biology
  • Water quality assessment

Background:

  • Quantitative polymerase chain reaction (qPCR) is vital for detecting fecal indicator bacteria in recreational waters.
  • qPCR enables same-day health advisories, with potential US EPA adoption for water quality testing.
  • Standardization is crucial for transitioning qPCR from research to routine water quality monitoring.

Purpose of the Study:

  • To compare qPCR performance and enterococci enumeration across different platforms, reference materials, and quantification models.
  • To identify key method variations influencing quantitative detection of fecal indicator bacteria.

Main Methods:

  • Compared three qPCR platforms (Applied Biosystem StepOnePlus™, BioRad iQ™5, Cepheid SmartCycler® II).
  • Evaluated two reference materials: lyophilized cells and frozen cells on filters.
  • Assessed two comparative CT quantification models: ΔCT and ΔΔCT.
  • Analyzed spiked and environmental water samples for enterococci enumeration.

Main Results:

  • Reference materials had the largest impact, causing ~0.5 log(10) unit variation.
  • qPCR platforms showed minimal effect, <0.1 log(10) unit difference.
  • Quantification models caused small differences in uninhibited samples but up to 8-fold (0.9 log(10) unit) in inhibitory samples.

Conclusions:

  • Standardized, certified reference materials are essential for reliable qPCR water quality testing.
  • Further research is needed to validate quantification models for PCR-inhibitory environmental samples.
  • Method variations, particularly reference materials and quantification models, significantly affect bacterial enumeration.