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  News from the Maryland Veterinary Medical Association                                                    Winter 2012

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PCR for the Molecular Diagnosis of Infectious and Genetic Diseases: A Primer
by Celeste Clements, DVM, DACVIM

PCR or polymerase chain reaction technology has become an accessible and economical tool for diagnosing infections in veterinary patients and for identifying genetic mutations that are the basis for a variety of congenital and heritable diseases.  Detection of genetic fingerprints and paternity testing are additional applications.  PCR amplifies the target genetic material, which may be present in minute amounts, to produce many copies of DNA that reach a detectable level. 

Bacteria, viruses, rickettsiae, protozoa and fungi are examples of infectious organisms that may be recovered from whole blood, serum, tissue swabs, aspirates or fluids, urine or feces. Sample selection should be dictated by the tissue predilection or site of organism replication. Fresh or fresh- frozen samples are preferred, but DNA or RNA may be successfully extracted from formalin-fixed, paraffin embedded tissues in some cases, even after the results of histopathology are known.  The extracted genetic material is quite stable, but must be protected from contamination to maintain integrity.

With the use of real-time PCR a patient showing clinical signs of disease can be tested for multiple infectious agents in just a few hours.  The very high sensitivity of PCR also allows for the accurate identification of some infectious agents that previously have been difficult to diagnose, for example, erythrocyte pathogens such as Mycoplasma haemofelis aka Haemobartonella, or Babesia spp. PCR may permit early recognition of infections, even before seroconversion, which often takes several days to weeks.  PCR panels that test for

multiple infectious agents may be useful to screen blood donors or new pets that will be introduced into a household with an immunocompromised resident pet.  Large diagnostic laboratories are able to offer these combination tests for a very affordable price, with fast turn–around times.

 The sensitivity and specificity of PCR are generally high, but a positive result does not always predict clinical disease:  serologic conversion, with production of antibodies to a given pathogen, is often considered the gold standard for proof of disease.   Or, as in the case of FIP, demonstration of classical histopathologic findings confirms the diagnosis, not the demonstration of feline coronavirus antigen or antibody in circulation.  PCR is, then, a valuable complementary diagnostic tool, supporting the results of traditional serologic techniques for the detection of antibodies or antigen; histopathology; or other tests

A genetic mutation is responsible for multidrug sensitivity in some herding breeds, such as Collies and Australian shepherds, and other pure- and cross-breds:  dogs with the MDR1 mutation are at risk for serious, even fatal, intoxication with many drugs such as ivermectin, loperamide, or acepromazine.  PCR technology offers a simple way to prevent these adverse reactions to common medications by identifying at risk patients.  DNA can be recovered from a cheek swab collected by the owner, and mailed to the lab with relative ease and economy.  Genetic testing for polycystic kidneys, feline hypertrophic cardiomyopathy, Fanconi syndrome  and a host of other diseases are equally accessible dna_alltest.html.

Both conventional and real-time PCR processes begin with DNA or RNA extracted from patient samples. Heating opens the double helix and exposes small DNA or RNA fragments (100-600 bases in length), with their target genes, to a master mix of nucleotides, enzymes, and buffers to optimize enzyme function.  Specific primers, short sequences of DNA that span the sequence of DNA that one seeks to amplify, and a heat stable polymerase, often Taq polymerase, direct the assembly of multiple copies of DNA, or complementary DNA, as the mixture cools. The cycle of heating and cooling is repeated 40 times, amplifying the product DNA:  one strand of DNA is amplified to approximately1000 billion copies.  

In real-time PCR sequence-specific fluorescent markers are used to report the generation of product, and the resulting fluorescence is measured by a special sensor within the PCR machine.  At the end of each cycle the exponential increase in the DNA product is registered on a graph. Measurable cutoffs or particle counts can be established that suggest a pathogenic load of the infectious organism and distinguish it from vaccine strains.

Conventional PCR is a slower and more manual process with PCR reactions conducted in an individual tube and agar gel electrophoresis used to detect the final product at the end of the reaction. The result is qualitative rather than quantitative. The more automated real- time PCR uses stable premixed reagents in a closed reaction vessel that minimizes the risk of cross-contamination and efficiently produces genetic material that can be quantified during the course of the reaction. 

When choosing a laboratory veterinarians should look for quality assurances such as positive and negative controls; contamination protection in the facility, with separated workflows and physical and pressure gradients; prevention of reagent carry over; and contamination monitoring.

Consider asking these questions suggested by Dr. Katrina Mealy of WSU’s Veterinary Clinical Pharmacology Lab:

1.  How familiar are you with the laboratory and with the quality of other products/diagnostics they offer?

2.  Do personnel from the laboratory publish results of investigations involving the diagnostic test and/or the agent(s) tested?

3.  Does the laboratory have an expert that can answer questions about the disease and/or interpretation of the results or is it simply a molecular biology laboratory that has limited to no knowledge about the pathogens and their effects in dogs and/or cats?

4.  Does the laboratory have written standard procedures that are followed including quality control procedures?

5.  Does the diagnostic test sound too good to be true? It might be!

6.  Does the test make sense clinically? (Is it a blood test for a respiratory pathogen?)

Mealey, K (2007). Infectious Disease Diagnostics: Beware of the PCR Peddler. ACVIM Forum

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