Molecular Diagnostics

Molecular DiagnosticsMolecular Diagnostics

Molecular diagnostic assays detect nucleic acid (DNA or RNA) of infectious disease agents within test specimens. Test specimens may be swabs, fluids, blood, or tissues. A test simply designated as “PCR” is a polymerase-chain-reaction test to detect DNA and is composed of 3 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
  3. Detection of target DNA by gel-electrophoresis

RT-PCR is a reverse-transcriptase PCR test to detect RNA and is composed of the same 3 basic parts as PCR and an 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 2 sets of nucleotide primers and 2 complete cycles of amplification; the second cycle of amplification further amplifies a target fragment of DNA 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.

Real Time PCR (also called quantitative PCR) is the same as PCR except that it simultaneously amplifies and quantitates the amount of DNA after each PCR cycle. It is based on the detection and quantitation of fluorescence of a reporter dye attached to DNA probe which is specific for the amplified DNA of interest (target) The test is very specific and about 10,000 more sensitive than conventional PCR in detecting DNA. 

General guidelines for sample submission

Preferred specimens for each test are listed in the table below. Under special circumstances, we may test other specimens; however, please contact the lab before submitting these. Submit all samples as quickly as possible and keep them chilled during transport to the lab for best results (refer to “WADDL Sample Submission”). Formalin can degrade DNA or RNA over time, so paraffin-embedding should be done as quickly as possible for best results from fixed tissues.

BVD-PI (Persistent Infection) Ear Notch Testing Program
Number of Cattle TestedPCR Pool SizePCR Charge per HeadAg-ELISA charge per Head
<12 samplesNo PoolingNA$6.00
12-35 samples12$4.50$2.00 *
36-99 samplesup to 36$3.50$2.00 *
100 or more samplesup to 36$2.50$2.00 *

* BVD Ag-ELISA testing on individual samples within a PCR positive pool
   Non-Washington samples will be assesed a 50% surcharge. (See General Policies #2, above)

Also see: Bovine Viral Diarrhea Virus Persistent Infection (BVD-PI) Ear Notch Testing Program for Cattle Herds


Test Specifics

All cases are charged a $10.00 accession fee.  Available procedures and prices can be accessed in the WADDL Web Test Search Tool.  Type in "PCR" and click on Search.

Test Specimen Turnaround
(days)
Comments
Ave. Max.
Anaplasma marginaleEDTA blood, fresh tissue (lung, spleen) fixed tissue block38 
Avian influenzaTracheal or cloacal swabs (up to 5 pooled samples) in 2 ml BHI. Fresh lung, spleen, or intestine (no pooling for fresh tissue). Whole bird.27NAHLN Assay
Contact Lab
Avian paramyxovirus (Newcastle's Disease)Tracheal or cloacal swabs (up to 5 pooled samples) in 2 ml BHI. Fresh lung, spleen, brain, or intestine (no pooling for fresh tissue). Whole bird.27NAHLN Assay
Contact Lab
Bluetongue virusEDTA blood, fresh tissue (lung, spleen) fixed tissue block37 
BVD (blood)EDTA blood 36Pooled testing of EDTA samples available; contact lab
BVD (ear notch)Ear notch skin 36See BVD-PI (Persistent Infection) Ear Notch Testing Program
BVD (milk or tissue)milk, fresh tissue (lung, spleen) fixed tissue block37 
Classical swine fever (CSF, Hog Cholera) 27NAHLN Assay
Contact Lab
Epizootic hemorrhagic disease virus (EHD) EDTA blood, fresh tissue (lung, spleen) fixed tissue block37 
Canine influenza virusTracheal wash, BAL, fresh tissue, nasal swab, oropharyngeal swab, fixed tissue block27Lung or bronchial lymph nodes are best tissues
Clostridium perfringensFeces, intestinal tract tissue, isolate37Genotyping is done from bacterial isolates. Detection of types A, B, C, D, E, Beta 2, and enterotoxin gene
E. coli virulenceFeces, intestinal tract tissue, isolate36Assay is performed from bacterial isolates. Routine assay detects F5(K99), F41, eaeA (intimin), stx1, and stx2 genes.
Equine Herpesvirus-1 (EHV-1) neuropathogenic and wild type EDTA blood, nasal swab, lung tissue 7Shipping:  Ice pack
Flavobacterium psychrophilumTissue, isolate on slant or plate27Assay is performed from bacterial isolates.
Francisella Tularensis (Tularemia)EDTA blood Culture isolate27Zoonotic Disease Surveillance Program
Contact Lab
Fungal identification by universal primers and sequencingFixed tissue block (affected organ), fresh tissue.  Cultured isolates are not accepted.720 
Helicobacter spp.  37 
Malignant Catarrhal Fever virus (MCF)EDTA blood, Fresh or fixed tissues. Lymphoid tissues such as spleen, lymph nodes, lung, or kidney are best. Liver and intestine may be tested, but sometimes contain PCR inhibitors. Brain may be tested, but false negatives may occur due to very low viral replication in the brain. 27Species being tested and clinical history is essential for the lab to perform the proper assay. Real time PCR specific for ovine herpesvirus-2 is used for clinical cases only. Nested PCR is used for non-clinical carrier species or for other MCF viruses.
Mycobacterium aviumFresh or fixed tissues, culture37 
M. avium ssp. paratuberculosis (Johnes)Culture slant37Assay is performed from bacterial isolate
M. avium ssp. paratuberculosis (Johnes)Feces or fresh intestine48Contact laboratory for information on pooled fecal testing.
Mycobacterium spp. identification by universal primers and sequencing 720 
Mycoplasma bovisCulture broth (preferred) or plate37Culture may be done in WADDL Bacteriology lab. Speciation of bacterial isolate by PCR.
Myxobolus cerebralis (Whirling Disease) 37 
Neospora caninumFixed tissue block (brain or affected organ)47 
Renibacterium salmoninarum (BKD)Tissue pool (processed at WADDL)47WADDL Aquaculture Section preprocessed samples for PCR. Samples which test negative may be further tested by nested PCR as required by OIE or Blue Book standards for pre-shipment, at extra charge.
Rhodococcus equiTracheal wash15 
Toxoplasma gondiiFixed tissue block (brain or affected organ)47 
Tritrichomonas foetus

Prepucial wash or vaginal scraping using Biomed Diagnostics TF-transit tube or InPouch transport/culture system (available direct from Biomed Diagnostics (800) 964-6466 or from all major veterinary supply distributors)


37

Shipping:  Must be sent to WADDL by FEDEX or other 24-48 hour shipping method.  Keep sample at room temperature or 37 degrees C before shipping and ship without ice unless using method 2 (see details for method 2 on Trichomonas accession form)

West Nile virusWhole bird. Brainstem from horse. Brain, tissue pool from others. Mosquito pool (25 or less in 2.0 ml microfuge tube)38 
Yersinia pestis(Plague)Culture isolate27Zoonotic Disease Surveillance Program Contact Lab
Yersinia ruckeriSlant of plate with isolate. Tissue.37PCR done on isolated colonies.
Direct PCR / SequencingFresh or fixed tissue. Isolate (fungi, bacteria, mycobacteria) on slant720 
Cloning / Sequencing 1540 

NAHLN - National Animal Health Laboratory Network


Contact Lab for large volume testing