Molecular Diagnostics

Definition:  A test designated as PCR is a polymerase-chain-reaction test to detect DNA and is composed of three basic parts:

1. Extraction of the DNA from the sample,

2. Addition of sample DNA, one set of DNA nucleotide primers and other reagents     to a PCR Cycler machine for amplification of target DNA, and

3. Detection of target DNA by gel-electrophoresis

A test designated as RT-PCR is a reverse-transcriptase PCR test to detect RNA and is composed of the same three basic parts as PCR with the additional step using reverse-transcriptase enzyme to synthesize complementary DNA from the target RNA.  The complementary DNA is then run in the PCR test.

Nested PCR is a modification that uses two sets of nucleotide primers and two complete cycles of amplification; the second cycle of amplification further amplifies a target fragment of RNA originating within an

already amplified larger target fragment of DNA.  Nested PCR results in higher sensitivity than simple PCR or RT-PCR and is used for diseases that have very little target nucleic acid in tissue samples.

In Real-time PCR, amplification and detection of target sequences are achieved simultaneously in a single tube.  Real-time PCR reaction mixtures contain a single-strand DNA probe that can specifically hybridize with the target sequences that are being amplified.  The probe is labeled with a fluorescent dye at one end and a quencher molecule at the other end.  Since the probe hybridized to its specific target sequences gives stronger fluorescent signal than the unhybridized probe, the intensity of fluorescent signal detected during PCR is directly proportional to the amplification of the specific DNA fragment.  The advantages of real-time PCR include higher assay specificity, quicker turnaround time, and the potential for quantitative results.

PCR Requests

PCR tests are run on request only.  Additional charges will be incurred for each PCR test that is run on samples mailed to ADDL and on tissues collected during necropsy examination at ADDL.  When PCR tests are desired for various agents, each desired agent must be clearly requested on the ADDL submission form.  Alternatively, and only for animals submitted for necropsy, permission may be granted by checking the box on the submission for "For necropsy cases, permission is granted to run PCR tests deemed necessary by an ADDL pathologist."

• ·Prior arrangements are required for submission of >10 samples and for swine semen samples.

 It is best to collect samples with disposable instruments (plastic tableware often works well) into  Sterile Whirl Pak bags.  Optimum sample size per test is approximately 10-15 grams or mls. Eliminate air, roll the tops down, and seal by folding tabs.  Avoid use of instruments that may be contaminated from previous use.

 Tissues sent for PCR must be packaged separately in Whirl Paks and clearly labeled as to tissues included and tests requested.  Samples should be shipped overnight on ice packs in an insulated  shipping container.  All tissue samples received in a single bag will be pooled, and a single PCR  test will be run for each requested agent.  If the same PCR test is desired on tissues from different animals, or from different tissues from the same animal, samples must be submitted in separate Whirl Pak Bags.