FREQUENTLY ASKED QUESTIONS FOR DIAGNOSTICS
Polymerase chain reaction (PCR) is a DNA based method of testing used to selectively target and replicate a specific region of DNA. Conventional PCR uses a forward and reverse primer containing nucleotides specifically arranged to compliment the target region of interest. A reaction containing the forward and reverse primers, DNA, random nucleotides, and an enzyme called polymerase, is placed into a thermocycler (a machine that very accurately manipulates temperature). The PCR process is started by heating the reaction to a high temperature, allowing the double-stranded DNA to ‘unzip’ into a single-stranded form. The unzipping leaves the single-stranded DNA exposed and ready for the annealing process which will take place next. The temperature is dropped significantly to allow the forward and reverse primers to bond to the single-stranded DNA. After primer annealing, the polymerase moves down the DNA strand, starting at the ends where the primers are located, and acts like a glue to stick the random nucleotides to the DNA strand as it goes, elongating and essentially making the single-stranded DNA double-stranded again. This is one full cycle. The next step is to heat the reaction to allow for unzipping of the DNA strand again. After many cycles of unzipping, annealing, and elongation, the original target region of DNA has been replicated countless times and is now present at a measurable quantity.
Real-time PCR is much more sensitive, specific, and reliable than conventional PCR. The addition of a dually labeled probe between the two primers allows data to be collected in real-time. The nucleotide at one end of the probe is tagged with a fluorescent marker, while the opposite end is tagged with a quencher marker. When in close proximity (i.e. when both markers are still attached to the probe) the quencher marker absorbs the energy from the fluorescent marker, thus prohibiting it from emitting a fluorescent signal. When a reaction containing the primer-probe set, DNA, random nucleotides, and polymerase is placed in a thermocycler the primers and probe attach themselves to the target DNA segment, and the polymerase anneals the random nucleotides to the DNA strand. When the polymerase reaches the dually labeled probe, it ‘kicks off’ the two markers, thus creating distance between them and allowing the fluorescent marker to emit a signal without interruption from the quencher marker. The laser reads the fluorescence after every cycle and displays the data on a computer screen in real-time. Being able to view the data after every cycle (as opposed to just at the end of all of the cycles as in conventional PCR) allows technicians to dismiss false positives and negatives. The addition of the probe also acts as a ‘safety check’ by greatly diminishing the chances of random, non-specific binding by the primers.
Our facility only uses real-time PCR for pathogen detection as it is much more reliable than conventional PCR. The avian sex determination test, however, is performed using conventional PCR.
Not only is real-time PCR sensitive, specific, and reliable it is also extremely time efficient. Results can be relayed in a matter of hours instead of days. This is crucial when treatment is dependent on a diagnosis. Also, quickly identifying the disease causing pathogen means proper biosecurity measures can be implemented to prevent disease spread. Commonly, clinical signs overlap making diagnosis difficult. PCR allows for the screening of many pathogens from one sample type.
PCR and serology are different techniques used to detect two different things; PCR detects the DNA of the pathogen (confirming the presence of the pathogen), while serology detects antibodies, a measure of the body’s immune response to a pathogen. For serology, it is commonly recommended that clients submit at least two samples at varying time points. The first sample acts as a baseline, while the second sample (submitted a few days or weeks later) will indicate an increased or decreased antibody level relative to the initial baseline sample. Serology testing, however, does not differentiate active infection from exposure or vaccination. For PCR, it is crucial to submit a sample type where the pathogen might be present. If the pathogen, and therefore the DNA of the pathogen, is not present in the sample type, then the PCR test will be negative, even if the animal is truly infected. This is why we have a list of appropriate sample types. For example, submitting a blood sample for a patient in respiratory distress is not a good idea because pathogens causing respiratory disease do not circulate in the bloodstream frequently or at all. In this case, the disease causing agent would be shed through the respiratory tract. Therefore, a nasal swab or wash would be a much more diagnostically significant sample to submit for PCR.
In positive cases: A positive PCR result means the DNA of the specified pathogen was detected in the provided sample type. In most cases this means the pathogen is likely the cause of disease as well. However, PCR cannot differentiate between live and dead organisms, and a few pathogens are carried by hosts without necessarily causing disease-like signs (equine herpesvirus-2 and equine herpesvirus-5 are good examples). Based on clinical signs, it is at the discretion of the veterinarian to decide if the pathogen is the cause of disease. Please see question # 13 in regards to vaccine detection.
In negative cases: A negative PCR result can mean a number of things: 1.) no infection, 2.) the sample provided is free of the pathogen(s) requested, 3.) the pathogen(s) may be present, but at a quantity below our assays’ detection limit (about 2-10 copies of a gene), 4.) the pathogen is a rare mutation that is not detected by our current PCR assays.
Depending on the pathogen of interest, additional testing may be needed. As with any technology, PCR does have limitations, and is not the gold standard for all diagnostic testing. Please call the lab for pathogen specific recommendations.
The preferred sample types are those suggested by our veterinarians to be the ones most likely to host the pathogen(s) of interest. You may submit other sample types that are not on the list. It is important to note, however, that the likelihood of detecting the pathogen in other sample types may not be as great.
No, it is not necessary to send all the sample types listed. It is up to you to send the sample type that you think is most suitable for diagnosis based on the patient’s clinical signs. If you do send multiple types, please indicate on the form if you would like them pooled (see question 10) or tested individually. If you wish to test them individually we will charge you per sample type as outlined in question 10.
We pool multiple samples by mixing an aliquot of DNA from each sample type and testing this combined mixture of DNA for the pathogen(s) of interest. This is a great option if you are unsure where the pathogen is located. It is important to note, however that pooling a large number of samples may diminish pathogen detection by diluting out a weak positive. We recommend pooling no more than three sample types. For billing, if you pool samples you will only be charged for one test, instead of one test per sample type. The example below outlines these two options; pooling multiple sample types vs. testing each sample type individually.
|Option A: Pool samples|
|Patient Name:||Sample type(s):||Test requested:||Price:|
|“Harley”||POOLED whole blood, nasal swab||Canine Herpes Virus||$42.00|
|Option B: Samples tested individually|
|Patient Name:||Sample type(s):||Test requested:||Price:|
|“Harley”||whole blood||Canine Herpes Virus||$42.00|
|“Harley”||nasal swab||Canine Herpes Virus||$28.00*|
*$28.00 for the same test on a different sample type for the same patient.
Each assay is 99-100% specific to the available sequence information (National Center for Biotechnology, NCBI), sensitive enough to detect as few as 2-10 copies of the gene, and 95-100% efficient. Our primer/probe sequences are checked annually to make sure we use the most up to date sequence information. All our diagnostic assays undergo a strict validation process.
Serum is a component of blood from which blood cells and coagulation factors are removed. Because some pathogens invade the blood cells, sending a serum sample would eliminate detection by PCR. To ensure that our tests yield accurate results, the DNA contained within the blood cells must be tested. This is why whole blood (with EDTA additive, LTT) is preferred over serum.
We recommend freezing the sample if you need to hold it over the weekend or for longer periods of time (up to a week). If freezing is not an option, refrigeration can keep a sample fresh for up to five days. EXCEPTION: Refrigerate, DO NOT freeze, all fecal/environmental samples that will be submitted for Salmonella spp PCR. Our lab tests the sample directly for Salmonella and once again after a 20-hour selenite enrichment culture. The bacteria will be killed and will not grow in culture if freezing has occurred.
It is up to the discretion of the veterinarian. While antibiotics or other various treatments will not inhibit the PCR process, there is a good chance treatment will diminish the presence of the pathogen(s) beyond the detection limit of PCR. If possible, a pre-treatment sample that has been kept cold (either in the freezer or refrigerator) would be preferable for diagnosis.
In most cases, if we receive the sample by 11am we are able to FAX results by 5pm PST the same day. There are a few exceptions. If the sample does not pass quality control (see Quality Control section) then an additional 24-hours will be needed for retesting. Also, paraffinized tissues (FFPE) take 24-48 hours due to intricate processing techniques. Finally, any sample submitted for Salmonella spp. testing (or panels containing Salmonella spp.) will undergo a 20-hour selenite enrichment. Results will be relayed on the unenriched sample the same day as receipt, while the enrichment results will be relayed by close of the next day.
If you are in need of results and will be out of the office, we will be more than happy to call or email the veterinarian if you let us know ahead of time or indicate so on the sample submission form.
Whole blood is not a preferable sample type for PCR-based detection for the presence of Borrelia burgdorferi because spirochetes may be present for a limited time in blood. Therefore, a negative PCR result on blood does not rule-out Lyme disease. A punch biopsy within the area of the tick bite, lymph nodes in the region of the tick bite, muscle, fascia, or synovial (joint) membrane are ideal. Less frequently, the spirochetes end up in meninges.
The general rule is that any vaccine using DNA of the pathogen to illicit an immune response has the possibility of being detected by PCR for a short period of time post vaccination. This includes modified live vaccines which use an attenuated form of the virus/bacteria. The trace amount of DNA used in these vaccines will eventually be metabolized by the patient’s immune system and will no longer be detectable by PCR. The amount of time this takes varies depending on the vaccine and how it is administered (ex. intranasal versus intravenous). Unfortunately, not a lot of literature is available establishing concrete timelines. In general, however, most residual DNA will be shed/metabolized within two - four weeks post vaccination. If vaccination history is unknown, this should be taken into consideration. It is also important to remember to use PCR results in context with clinical signs. Please call the lab with questions regarding specific vaccines.
Yes. We have a program established with FedEx that allows you to save about 50% when shipping to our facility. You don’t pay anything upfront at the time of shipping, simply generate a shipping label online, print it, and send the package. We will bill you for shipping costs when we bill you for our testing services. Please see question #19 for enrollment instructions.
Enrollment is easy. Visit http://www.vetmed.ucdavis.edu/vme/taqmanservice/forms.html for an enrollment form. Fill out the form and FAX it back to the lab. Within one working day we will FAX you instructions with a username and password. Clients who enroll will receive ~50% off of standard FedEx shipping rates. More information can be found on the enrollment form.
Complete shipping instructions can be found in our sample submission packet or call the lab to have a copy faxed. All samples need to be shipped with an icepack in a Styrofoam container via overnight delivery. This preserves the integrity of the DNA/RNA during outside temperature fluctuations. If possible, please put the sample submission form in a separate plastic bag to protect it from condensation from the ice pack or leaky samples. All tubes should be wrapped in something padded (i.e. bubble wrap, vet wrap, paper) to prevent them from breaking during shipping. We receive deliveries from FedEx, IDEXX, DHL, and UPS. DO NOT ship samples through USPS. Many times the packages are retained at the main post office on central campus for several days before being distributed.
While IDEXX delivers to our facility every morning, we do not have an IDEXX account number. We recommend calling the IDEXX office in West Sacramento (916-373-9792) if you have questions in regards to shipping.
The DNA and cDNA (synthesized from the RNA) of every diagnostic sample is run with a standard housekeeping gene. The housekeeping gene varies depending on the sample type and species, but it is a gene that should be present in abundance. Therefore, the PCR should always be positive if the sample is of good quality, the nucleic acid extraction was successful, and if there is no inhibition during the amplification process. If the housekeeping gene is negative or very weak then we re-extract the nucleic acids from the back up sample, and run it through PCR a second time. If the housekeeping gene for the backup sample is again negative or weak, we consider the sample to “fail quality control” or in other words there are not enough cells present in the sample for accurate testing and/or the cells that were present have degraded beyond detection.
It is important to note that because all testing is done at a molecular level, determining the exact reason a sample failed quality control is not possible. We have listed the reasons samples fail quality control below and which sample types they are more likely to affect.
- Presence of dirt and/or plant material (feces or dirty swabs/washes). At the end of the DNA extraction process, we have DNA suspended in an aqueous solution. While not visible to the naked eye, particulate matter originating from the dirt can also find its way into this solution. Particulate matter can inhibit qPCR fluorescence (think of the dirt 'clouding' the qPCR reaction) which leads to a weaker signal (weaker signal = failing QC if the signal value is high enough).
Plant material, on the other hand, acts a bit differently. The chemicals found in plants can 'neutralize' the lysing of the cells in the first step of the DNA extraction process. If the cells aren't properly lysed, then the DNA can't be extracted efficiently.
2. Lack of DNA (acellular samples like CSF, urine, joint fluid). Simple enough, since PCR is a DNA-based technology, if there is a low concentration of DNA in the sample, you will get a weak signal for the housekeeping gene..
3. DNA degradation (any sample type, especially if older than four days, or if not kept cold after collection). With any sample type, as soon as a sample is collected, the DNA starts degrading. If the DNA degrades too much during transit, handling, or storage then the effect is the same as #2.
4. Laboratory technician error. Certainly if a sample is mishandled, steps are missed during the DNA extraction or PCR process, or if reagents are mixed up or made incorrectly this can play a role in samples failing QC. Fortunately, this is the easiest error to 'catch' on our end. We have a strict Quality Assurance Protocol which includes running controls at every step of the process. Also, because all samples are batched each day and processed at the same time, if chemicals were mixed up or steps were missed it would affect the whole batch equally, most likely leading to all samples submitted that day failing QC.
Below is a very condensed version of our quality assurance procedures. These control measures include, but are not limited to the following*:
- All researchers are extensively trained in the areas of laboratory safety, sample preparation, reagent preparation, and data analysis. Training records are held onsite.
- The Real-time PCR Research and Diagnostics Core Facility has physically separate rooms for sample receiving/preparation, assay preparation, amplification and analysis. Separation of workflow greatly decreases the risk of cross contamination between samples and reagents.
- All samples are tested with a standard housekeeping gene. This ensures proper nucleic acid extraction and absence of inhibitors.
- Positive and negative controls are run for the diagnostic assays daily. This guarantees the assays are made correctly and no contaminants are present.
- Various areas within the lab are swabbed on a biweekly basis and tested for any pathogens that have tested positive within the previous two week interval. This ensures work areas are free from contamination.
*This is a small portion of the Quality Assurance program in place at the Real-time PCR Research and Diagnostics Core Facility. A complete guide can be found under our 'QC Guidelines/MIQE' tab.
Payment can be made via a credit card, personal or business check, or wire transfer (international clients). Please do not send cash. If paying by check, the invoice stub needs to be mailed with the check to the Cashier’s office in West Sacramento (address is provided on the invoice). All checks need to be made out to “The Regents of UC Davis” and please write the invoice # on the back of the check. The invoice # starts with 01-_______. Please call ahead of time if you plan on submitting a check with the sample so the check is for the correct amount.
If you would like to pay by credit card, please call the lab with your credit card information and the invoice number.
We no longer require FEIN/FTID#s for any client.
Billing is done at the end of every month. You can expect to receive your invoice by the second week of the month following your submission. If you need to know the exact cost of our services sooner than that, please feel free to call us!
Yes. If you would like to have multiple pathogens or panels added on to one sample type, then we do offer a discount. In general, we will charge full price for the most expensive test or panel, then $20 per additional pathogen or half price per additional panel. As always, please feel free to call the lab with specific pricing questions. Here are a couple of examples:
Example 1: Equine Vector-borne Panel ($80) and Potomac Horse Fever ($15, instead of $42) on one blood sample = $95 total.
Example 2: Feline Blood Donor Panel ($101) and Feline Vector-Borne Panel ($48, instead of $97) on one blood sample = $149.00 total.
Yes. Every sample submitted for EHV-1 testing is run with three different real-time PCR assays. The first is a universal EHV-1 assay located on the glycoprotein B gene. This test detects both the neuropathogenic (G2254) and the non-neuropathogenic (A2254) strains and is used for quantification in positive cases. The second detects only the neuropathogenic (G2254) strain, while the third detects only the non-neuropathogenic (A2254) strain. In positive cases, the report will include a viral load concentration (# of EHV-1 viral cells per million nasopharyngeal or blood cells), the strain type (neuropathogenic or non- neuropathogenic), and interpretation guidelines (see next question).
The disease stage can be determined using the individual results from each sample type. At different times during infection the nasal swab and whole blood can be positive for EHV-1. The following explanation is included in the report if the nasal swab and/or whole blood samples test positive:
PCR results for EHV-1 glycoprotein B gene are expressed qualitatively (positive or negative) and quantitatively in the case of a positive result. The viral load of a sample is calculated by the absolute number of EHV-1 (glycoprotein B gene) per million cells (either blood cells or nasopharyngeal cells). We are in the process of validating thresholds in order to better understand the viral kinetics of diseased and subclinical horses. Diseased horses (neurological or febrile) commonly have high (greater than 1*10^4) viral loads in nasal secretions. Subclinical horses commonly have low (less than 1*10^3) to moderate (1*10^3 - 1*10^4) viral loads only in nasal secretions. Viral load ranges are only suggestions and are not currently well-defined. The absolute number should be used judiciously and allow comparisons between samples taken at different time points in relation to treatment.
At different stages of the disease one or both samples may test positive for PHF. Generally, the bacteria can be found in the blood from Day 8 through Day 25 post infection (Day 0), and in feces from Day 13 through Day 28. Sending both sample types allows for a comprehensive diagnostic overview.
Positive PCR results for EHV-2 and EHV-5 should be interpreted carefully. A positive PCR result for either virus does not necessarily mean that it is the cause of clinical disease. It has been found that a subset of the healthy horse population can harbor EHV-2 or EHV-5, and even if treated will continue to host the pathogen(s).
We do have assays designed to detect the causing agents of EPM, Sarcocystis neurona and Neospora hughesi, however these pathogens are found in the blood or cerebral spinal fluid (CSF) of infected horses for a very short period of time. Therefore, PCR testing of neurologic horses using either blood or CSF is not recommended as it has the possibility of generating false negative results. PCR is best used for postmortem diagnostics on brain tissue. A concrete ante mortem diagnosis of EPM relies on the detection of antibodies against the two parasites in serum and/or CSF using the quantitative serological IFAT. The Immunology Lab at the Veterinary Medical Teaching Hospital at UC Davis offers such antibody testing (530)-752-7373.
You have one of two options for diagnosing S.equi subsp. equi:
a. Submit a nasal swab or guttural pouch wash for PCR testing. If negative, no infection/no detection of S.equi subsp. equi in the sample provided. If positive, retesting at a later time to determine carrier state is recommended.
We include a calculated bacterial load with the results if the horse is positive for S.equi subsp. equi. The bacterial load represents the number of S.equi subsp. equi cells per million nasopharyngeal cells. The bacterial load is meant to be compared during the course of infection as an indication of increased or decreased bacterial growth in relation to treatment.
b. Perform a single culture.
It is highly unlikely that an internal Streptococcus equi subsp. equi abscess will be detected by PCR in a whole blood sample. Once harbored at the abscess site, Streptococcus equi subsp. equi does not circulate in the blood stream frequently. The odds of collecting a blood sample containing the bacteria are very small.
Yes, we offer FIV PCR testing for clients in the United States and most other countries. We cannot perform FIV PCR for clients in Canada, however, as a patent prohibits us to do so until 2020. Currently, our facility tests four subtypes A, B, C, and F. There are also subtypes D and E, but they are not as common in the United States or are rare mutations.
Positive FIV PCR results:
- Almost always indicative of active FIV infection.
- Rarely, positive results may follow very recent FIV vaccination due to detection of vaccine virus.
Negative FIV PCR results:
- No infection.
- No viral DNA present in the sample submitted.
- Virus is present at quantities below our assay’s detection limit (5-10 copies of the gene).
- Virus has a rare mutation which is not detected by our assays (see question 36).
It is important when comparing PCR and serology results to keep in mind they are detecting two different things. PCR is a DNA test and therefore detects FIV proviral DNA. FIV serology testing detects antibodies against FIV. It is very possible, therefore, to have conflicting PCR and serology results. Below is a brief outline of interpretations and recommendations based on combined FIV PCR and serology results*:
Positive FIV PCR/positive FIV serology:
- Active FIV infection is most likely if vaccination was not performed recently and in the past.
Negative FIV PCR/negative FIV serology:
- No FIV infection
- Patient is in advanced stage of infection (feline AIDS) and cannot make antibodies. Virus cannot be detected by PCR due to mutation or insufficient quantity of viral DNA.
Positive FIV PCR/negative FIV serology:
- Active FIV infection. Patient has not seroconverted. Perform a follow up serology test at a later date. Alternatively, patient may be in advanced stages of infection (feline AIDS) and unable to make antibodies.
- No FIV infection. Patient was recently vaccinated, but has not seroconverted. PCR is detecting attenuated or residual DNA used in modified live vaccines.
Negative FIV PCR/positive FIV serology:
- No FIV infection. In kittens, serology testing can detect antibodies from the mother’s milk. Perform a follow up serology test at a later date.
- No infection. Patient was previously vaccinated and has developed antibodies.
- Active FIV infection. Patient is truly infected with FIV, but the subtype is not detected by our PCR assays, has a rare mutation (see question 36), or virus present is below the limit of diction of our assays.
*These are suggestions. Each case should be handled individually and diagnosis should be made at the discretion of the veterinarian based on all available testing options.
Yes, the assay will pick up the caviae strain of Chlamydophila that commonly infects guinea pigs/hamsters.