Real-time PCR Research and Diagnostics Core Facility

Diagnostic FAQ's

General

What is (conventional) PCR?
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.
What is Real-time PCR and how does it compare to conventional PCR?
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.
Does your facility use conventional or Real-time PCR?
Our facility only uses real-time PCR as it is much more reliable than conventional PCR
Why is Real-time PCR being used in veterinary diagnostics?
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 symptoms and signs overlap making diagnosis difficult. PCR allows for the screening of many pathogens from one sample type.
How does PCR differ from serology testing?
PCR and serology use different technologies 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.
How do I interpret PCR results?
In positive cases: A positive PCR result means the DNA of the specified pathogen was found within the provided sample type. In most cases this means the pathogen is likely the cause of disease as well. However, PCR can not differentiate between live and dead organisms, and a few pathogens are carried by hosts without necessarily causing disease like symptoms (Equine Herpesvirus-2 and EHV-5 are good examples). Based on clinical signs and symptoms, it is at the discretion of the veterinarian to decide if the pathogen is the cause of disease.
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.
Should I rely on PCR results alone?
Depending on the pathogen of interest, additional testing may be needed. As with any technology, PCR does have limits, and is not the gold standard for all diagnostic testing. Please call the lab for pathogen specific recommendations.
How do you choose the preferred sample types? Can I submit a sample type that is not on the list?
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.
There are multiple sample types per pathogen/panel listed on your submission form. Is it necessary to send all of the sample types?
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 (combined) or tested individually. If you wish to test them individually we will charge you per sample type as outlined in the next question.
What do you mean by "pool" samples? How does pooling affect the cost?

We pool multiple samples by either combining the samples and processing them as one for DNA extraction or by mixing an aliquot of extracted 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 total, instead of one test per sample type. The example below outlines these two options; pooling multiple sample types or 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
		Total:	$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*
		Total:	$70.00
			*$28.00 for the same test on a different sample type for the same patient.

What is the specificity/sensitivity/efficiency of your assays?
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 including testing with positive/negative controls to confirm proper assay design.
Why is serum not an acceptable sample type?
Serum is a component of blood from which blood cells and coagulation factors are removed from the plasma. 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.
How long can I hold a sample before shipping?
We recommend refrigerating the sample if you need to hold it over the weekend or for longer periods (up to five days).
Is it worthwhile to send a sample collected after the animal has started treatment?
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 with an antibiotic or antiviral may 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.
What is your turn-around time?
Results for samples received by 11 AM PST will be relayed within 24 hours (typically the morning after sample receipt). 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) samples take 24-48 hours due to intricate processing techniques.
Why is whole blood not recommended for the testing of Borrelia burgdorferi (Lyme disease)?
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.
Will your assays detect vaccine?
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 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.

Shipping

Do you offer a reduced rate shipping option?
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 below for enrollment instructions.
How do I enroll in the online FedEx reduced rate program?
Enrollment is easy. An enrollment form can be found on the Veterinary Diagnostics section of our website. Fill out the form and FAX it back to the lab (or call for an email address). Within one working day we will FAX/email you instructions with a userID and password. Clients who enroll will receive ~50% off of standard FedEx shipping rates. More information can be found on the enrollment form.
How do I ship a sample to you? How should the sample be packaged?
Complete shipping instructions can be found in our Sample Submission Packet or call the lab to have a copy emailed. 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.
Do you have an IDEXX account?
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.

Quality Control

What does it mean when a sample fails QC?
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.
What are the possible reasons for failure to pass QC?
Samples fail quality control for a variety of reasons, most of which depend on the sample type or how the sample is handled after collection. The longer a sample sits without being processed, the more likely the DNA/RNA will degrade. Also, some sample types are more susceptible to fail QC than others. Samples with typically low cell concentrations (mainly urine, CSF, and joint fluid) fail quality control simply because there is not enough genetic material present. PCR is a DNA test, if there is little or no DNA present in the sample then the testing is unreliable. While nasal swabs and feces rarely fail quality control, the most common reason is due to particulate contamination –excess soil/dirt/organic material (grass) which typically leads to inhibition of fluorescence during the PCR process.
What procedures are in place to ensure the accuracy and quality of your laboratory’s work/tests?
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 PCR 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.

Billing/Payment

What types of payments are acceptable?
Payment can be made via a credit card, personal or business check, or wire transfer (international clients). Please do not send cash. Your clinic will receive an invoice after testing.

If paying by check, the invoice stub needs to be mailed with the check to the Cashier’s office in Los Angeles (address is on the invoice). All checks need to be made out to “The Regents of UC Davis” and please write the invoice # on the check. Please call ahead of time if you plan on submitting a check with the sample so the check is for the correct amount.
What is an FEIN/FTID# and why do you require it for billing?
A Federal Tax Identification Number (FEIN or FTID#) is a nine digit number; two digits followed by a dash and seven more digits (ex: 12-3456789). Every business is required to have one by law as it is used to identify your company by the Internal Revenue Service (IRS). Please keep in mind that FEINs are public domain, and should not be regarded as the equivalent to your company’s social security number.
I submitted a sample a couple weeks ago and still have not received a bill. How long does it typically take to receive an invoice?
Because the University has so many checks and balances in regards to billing, receiving an invoice can take up to a month. If you need to know the exact cost of our services so you can bill your client, please feel free to call us!
Do you offer special pricing for additional/add-on tests?
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 $21 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 ($88) and Potomac Horse Fever ($21, instead of $46) on one blood sample = $109 total.

Example 2: Feline Blood Donor Panel ($111) and Feline Vector-Borne Panel ($53.50, instead of $107) on one blood sample = $164.50 total.

Large and Small Animal

Do you test for the neuropathogenic strain of Equine Herpesvirus 1?
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 absolute 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.
Why do you recommend submitting dual samples (whole blood and nasal swab) for EHV-1 testing?
The disease stage can be determined using the individual results for 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.
Why do you recommend submitting dual samples (whole blood and feces) for Neorickettsia risticii (Potomac Horse Fever) testing?
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 concrete diagnosis.
How should I interpret positive PCR results for EHV-2 or EHV-5?
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).
Do you test for EPM (Equine Protozoal Myeloencephalitis)?
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 530-752-8684.
What is the most accurate method for diagnosis of Streptococcus equi subsp equi (Strangles)?
You have one of two options for diagnosing S.equi subsp equi:
1.) 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.

2.) Perform a single culture.
I think my patient has an internal Streptococcus equi subsp equi abscess. Can I submit a whole blood sample for diagnosis?
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.
Do you test for Feline Immunodeficiency Virus (FIV)?
Yes, we offer FIV PCR testing. Currently, our facility tests for 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.
How should FIV PCR results be interpreted?
Positive FIV PCR results:
Almost always indicative of an 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.
How should FIV PCR results be interpreted in conjunction with FIV serology results?
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, 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.
Does the Chlamydophila felis/psittaci assay detect the isolate that infects guinea pigs/hamsters?
Yes, the assay will pick up the caviae strain of Chlamydophila that commonly infects guinea pigs/hamsters.