Issues Facing Practitioners
Understanding the Older and the Newer Formats for Detection of Infection
Table 1 is a listing of the basic definitions of PCR methods. Several previous presentations at the ACVIM and at other veterinary
meetings have discussed PCR, and a recent paper indicates that a basic understanding of the different PCR test formats is
needed for continuing education of practitioners and diplomates1. While it is not necessary for the practitioner to be a molecular biologist, this table provides a short review of the definitions
of molecular testing.
Table1. Basic Description of Polymerase Chain Reaction Methods
Proper Procedures: Collection, Transport, Storage
The main advantage of genomic strategies for testing is that a live organism is not necessarily needed for diagnosis While
the fact that diagnosis based on molecular techniques does not require live organism, DNA and RNA are subject to the same
microbiological, biochemical and physical factors as live organism for degradation.6 Although in some situations, genomic DNA may be robust in its survival for forensic purposes, overgrowth and chemical contamination
of a small microbiological sample will result in false negative reactions for any of the aforementioned molecular techniques.
A sample collected using the correct media that is solely dedicated for PCR testing should be taken. For blood, EDTA or purple
top tubes are best. Use of EDTA as an anticoagulant for testing of virus in plasma or buffy coat is applicable for most genomic
techniques and viral culture techniques. However, if one wishes to culture plasma (such as that of a foal) for bacteria, EDTA
is bacteriostatic and a blood culture bottle must also be collected. Anticoagulants such as heparin can inhibit PCR reactions
especially when a kit extraction method is used which will likely the typical formats in most diagnostic laboratories.
For detection of nasal/respiratory pathogens, wooden swabs contain formalin which can inhibit PCR (fixed tissue methods are
performed on paraffin and formalin has been removed in the paraffin process). Thus, plastic, polypropylene sticks with Dacron
or rayon swabs, not those with calcium alginate, are essential for nasal swabbing.7 Furthermore, cotton allows bacteria and virus to become embedded in fibers, and frequently the extraction method calls for
direct extraction off the swab. This step will inactivate most viruses and bacteria because of the detergent used in the first
lysis step. Finally, use of a transport media is best for viruses because this will inhibit overgrowth of bacteria that may
inactivate and break down nucleic acids needed for the successful detection of viruses. The proteins in the viral media will
also assist in keep the virus in a biologically active state, so this is preferable for viral culture also.
Most testing has actually been validated on specimens collected under the best conditions without inhibitors and contamination
and these samples have been stored appropriately. Specimens requiring storage before shipping to a laboratory should not be
stored and the MOST optimal time for testing by PCR is less than 3 days in a sample stored consistently at 4°C. If one can
not ship a sample within 3 days we recommend that the sample be allowed to sit in a refrigerator for 20 minutes and then the
plasma drawn off and placed in a new tube without any anti-coagulants. For horses, do not centrifuge the sample! The white
blood cells of horses settle in this time and do not form a buffy coat that is adhered to red blood cells. This allows collection
of as few RBCs as possible.8 This is essential because as RBCs lyse in older samples, heavy protein contamination occurs and these particular proteins
(iron) are toxic to many PCR reactions. Many laboratories historically have indicated shipment at either 4°C or not to exceed
75°C. The latter is not appropriate for blood or nasal specimens due to the potential for hemolysis and bacterial overgrowth,
respectively. Ship all samples on ice packs (NO WET ICE) and then overnight for 24 hour arrival. Do not ship expecting successful
Saturday delivery and it is our experience that samples become lost more frequently with weekend deliveries.