qpcr: rapid method tour · 2019. 6. 5. · • tv crime shows: ncis, csi-miami, etc. epa new...

EPA New England Regional Laboratory

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qPCR: Rapid Method Tour

Save The Harbor/Save The Bay

May 30, 2012

Scientific Excellence to Protect Public Health & the Environment


2 Scientific Excellence to Protect Public Health & the Environment

Overview PCR - Problem

PCR - Background & How it Works

PCR - Correlations

PCR - Present & Future


Scientific Excellence to Protect Public Health & the Environment 3

“Sweetheart, we won’t really know until tomorrow.”

“Mommy? When will we know if it’s safe to swim in the water?”

Problem: Bacterial culture methods yield

Results NEXT day.

Solution : Reliable rapid methods inform bathers of

Water Quality the SAME day.


Background  Polymerase Chain Reaction (PCR) is an

analytical tool applied from the branch of science called Genomics.

 Genomics is the study of organisms in terms of their DNA sequences and cellular activity in response to the environment.




Background


Dr. Kary Mullis received a Nobel Prize in chemistry in 1993 for his invention of the polymerase chain reaction (PCR).

Conceptualized in 1983 by Dr. Mullis, The process is hailed as one of the monumental scientific techniques of the 20th century.


Background -The Inventor



Genomics is the science of gene…

Identification

Structure, and

Function

How It Works



PCR is … •  AKA: Repetitive Molecular Photocopying

•  Utilizes Polymerase: natural enzyme catalyst DNA replication

•  Analytical method that mimics the process of cellular DNA duplication

•  Method Premise - Many copies are easier to identify than fewer copies

•  TV Crime Shows: NCIS, CSI-Miami, etc.


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to generate millions - billions of copies of target gene sequences through repetitive cycling of the 3 PCR steps

Polymerase Chain Reaction (PCR):

The goal of PCR is…

- uses the natural functions of the enzyme polymerase - to copy, genetic material (e.g., DNA or RNA)

- in a simple 3 step process:

1. Denaturation 2. Annealing 3. Extension

How It Works



WHY AMPLIFY DNA?

In traditional microbiology bacterial cells are grown or “amplified” to form colonies which can be easily identified and counted.

In PCR sections of DNA , unique target gene sequences, are copied or amplified instead of whole cells.

Target


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How It Works Target Gene

≈ 2n copies per cycle

1st cycle

2nd cycle

3rd cycle

4th cycle

From 1 original copy

35 - 45 PCR Cycles ≈ 40 - 60 minutes

235 = 34 Billion

Copies To Millions or Billions of Copies



Detection & Concentration



A Word About Correlations

•  Be Careful •  Know Why •  qPCR & cult. results corr. are…

•  Site specific •  Temporally specific

•  qPCR & epidemiology corr. are… •  More important

•  Show higher corr. than



1 10

100 1,000

10,000 100,000

1,000,000 10,000,000

Site

Ente

roco

ccus

/ 10

0mL

PCR CCE PCR CFU Enterolert MPN

Enterococci Concentration


qPCR & Epi Data



2012  Published new EPA qPCR Recreational Water

Method with DNA-based Rec Water Criteria  Source Tracking Apps & Research cont’d

  2012 eGST RARE   Human Vs. Nonhuman

  Fecal Indicator Organisms (FIOs)

  Tool Box


2013  qPCR Method Training


References & Links   EPA Genomics Task Force White Paper   Inside PCR

http://learn.genetics.utah.edu/code/cfpages/cite.cfm?title=PCR%20Virtual%20Lab

  Kary Mullis:

  http://www.karymullis.com/index.shtml



Questions




PCR Sample to Results


 PCR is a powerful analytical tool for supporting a broad spectrum of EPA Programs:

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Superfund: Risk Assessment

(e.g., Estrogen, Hg, etc.)

Bioremediation

NPDES: Toxicity Testing

TSCA:

Nanotechnology

TMDL: Microbial Source Tracking

Source Identification

CAFOs Drinking Water:

Rapid Screening

BEACHES:

Rapid Water Testing

Scientific Excellence to Protect Public Health & the Environment

How We Use It



Robustness



Specificity

qpcr: rapid method tour · 2019. 6. 5. · • tv crime shows: ncis, csi-miami, etc. epa new...

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