Guidelines on PCR Technology
July 2006

BMH Guidelines On The Use Of Pcr Technology For The Detection Of Bacterial Foodborne Pathogens

Evaluation Division
Bureau of Microbial Hazards,
Food Directorate, Health Canada
Postal Locator: 2204A1
Ottawa, Ontario, K1A 0L2

E-mail: Don_Warburton@hc-sc.gc.ca

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1. Scope:

1.1. These guidelines apply to the use of PCR technology in 2 different situations:

1. during the "screening" for pathogens from enrichment broths or from purified colonies (Section 3.1), and
2. identification of foodborne pathogens that are purified on bacteriological agars (Sections 3.2 and 3.3).

Guidelines or position papers on the use of PCR technology for the detection of "viable but non-culturable" microorganisms, parasites and viruses may be forthcoming in another document.

1.2 Methods based on PCR technology to be used for compliance and/or regulatory purposes will be reviewed against this guideline on a case-by-base basis.

2. Definitions:

2.1 PCR Technology

PCR technology refers to nucleic acid amplification followed by diagnostic characterization (e.g., end-point PCR, real-time PCR, multiplex PCR, reverse-transcriptase PCR, NASBA, etc.).

2.2 Compendium Method

"Compendium method" refers to any validated and standard method published in the Compendium of Analytical Methods and appearing on the Food Directorate's (Health Canada's) website at http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php

MFLP or Laboratory Procedure in Volume 3.
A laboratory procedure is defined as a method not validated by an interlaboratory study or not meeting the attributes or specifications of an HPB Method.

MFHPB or HPB Method in Volume 2.
An MFHPB method is defined as a method that has been validated by an interlaboratory comparative or collaborative study.

See the document entitled "Procedure for the Development and Management of Food Microbiological Methods" in Volume 1 of this Compendium for further explanation of these methods.

3. Applications for PCR Technology:

Prerequisite: The PCR assay has been validated by performing evaluation studies for its purported application and must at minimum meet the status of a Laboratory Procedure (MFLP) in the Compendium.

3.1. Screening for a Target Microorganism:

PCR could be used as a screening test for a target microorganism:

1. in the pre-enrichment and/or selective enrichment broths prior to plating on selective or differential agars; or
2. on isolated colonies on selective media prior to completion of biochemical assays.

This approach could provide for the rapid screening of a large number of suspect colonies and broths in response to time constraints and health issues (such as in foodborne outbreaks and complex trade or compliance issues). All presumptive PCR positive samples must be confirmed using a validated confirmation method in the Compendium or equivalent1 as specified in the PCR method.

Note: When a broth is positive by PCR and viable target bacteria can NOT be isolated on selective agar, the analyst must follow recommended procedures as specified in the confirmation or PCR Method.

3.2. PCR Technology as an Alternate for "Rapid ID Kits".

When a method involving PCR technology is approved by the MMC (in consultation with HC and CFIA, as needed) for this purpose, then a minimal number of biochemical assays would be required for confirmation as specified in the method.

This will be dependent upon the number and specificity of the primers being used in the method, the circumstance (routine testing, compliance activity, or foodborne outbreak investigation), the target microorganism and the type of compliance activity being initiated.

The additional confirmatory tests are to be specified in each PCR method.

3.3. Confirmation of a Target Microorganism:

In the case where a Compendium method or equivalent¹ (enrichment steps and plating on selective agars) has been initiated and presumptive positive colonies are present on the selective agars, then PCR technology cannot be the only test for identification of the target microorganism unless approved otherwise (Section 3.4).

When a method involving PCR technology is approved by the MMC (in consultation with HC and CFIA , as needed), then a minimal number of biochemical assays must still be required to confirm the identification of the microorganism, as specified in the PCR method.

Factors determining the type and number of additional biochemical tests will be dependent upon the number and specificity of primers being used in the method, the circumstance (routine testing, compliance activity, or food borne outbreak investigation), the target microorganism and the type of compliance activity being contemplated. Additional confirmatory steps should be specified in each PCR method.

3.4. PCR Technology as the Sole Confirmation Step:

Prerequisite: The PCR assay has been validated by performing evaluation studies, including comparative and/or collaborative studies to obtain the status of a HPB Method (MFHPB) for its purported application in the Compendium.

3.4.1 Examples:

1. When a PCR method has met all criteria established by MMC (in consultation with HC and CFIA, as needed), then the PCR-based method may be used as the sole confirmation step for testing presumptive colonies from selective or non-selective agars.
2. A PCR method may be used provisionally when it is the only available confirmation step(s) for a targeted microorganism. Note: In this case, the method may have only obtained MFLP status. When illness(es) occur, and a new emerging pathogenic bacteria is suspected, even this status may not be necessary.

This will be dependent upon the number and specificity of primers being used in the method, the situation (routine testing, compliance activity, or food borne outbreak investigation), the target microorganism and the type of compliance activity being initiated and the amount of data supplied to MMC (in consultation with HC and CFIA as needed).

4. Further Information:

4.1. Internal PCR controls must be used in each amplification reaction to ensure that nothing is interfering with the amplification process.

4.2 Interpretation guidance will be given in each method. Expected results are to be included in the interpretation section of each method.

1. "Equivalent methods" are defined in the document on method development. The onus is on the laboratory to demonstrate that another method is equivalent to HC Laboratory Procedures or HPB Methods.