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U.S. Department of Health and Human Services

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Volume IV - 2.9 Polymerase Chain Reaction (PCR)

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Orientation and Training

Food and Drug Administration

DOCUMENT NO.:

IV-02
VERSION NO.: 1.7

Section 2 - Microbiology

EFFECTIVE DATE:
10-01-03
REVISED:
08-25-14

A.  Objective

  1. To understand the basic principles and applications of the polymerase chain reaction. 
  1. To familiarize the trainee with the techniques, procedures, and equipment used in PCR analysis.
  1. To detect genes associated with food borne pathogens.

B.  Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and in theory related to sample analysis. This may be accomplished through a series of designated training samples. Once the trainer is confident that the trainee can successfully and independently perform the analysis, the trainee will be issued a series of training samples.

  1. Test a sample for a defined gene(s) using PCR analysis.

Confirm the presence of the amplicon (e.g. gel electrophoresis, probe hybridization, DNA sequencing, etc.)

  1. Practice using a micropipette to dispense small liquid quantities.
  1. Practice using proper molecular biology techniques while opening and closing small reaction tubes and dispensing reagents.
  1. Learn how to calculate correct reagent quantities needed for master mix preparation.
  1. Trainer will demonstrate how to use a thermal cycler.
  1. Trainer will discuss which organisms can be analyzed using PCR.

C.  Questions

  1. What are some of the benefits to using PCR to detect microbial pathogens in foods?  What are some of the potential problems associated with using PCR to detect microbial pathogens in foods?
  1. What are the three major steps (processes) involved in the PCR cycle?
  1. Why would using an enrichment procedure before PCR analysis be useful?
  1. What components are needed in the PCR reaction mixture and what function does each element serve?
  1. Why is selection of the primers so important to the success of the reaction?
  1. Why are there forward and reverse primers?
  1. Why does a microbiologist need an excess quantity of primers in the reaction mixture?
  1. What is real-time PCR?  How does it differ from conventional PCR?
  1. Why always run a reagent control?
  1. List three ways to prevent contamination when performing PCR.
  1. In what ways can PCR be inhibited?