PCR: A clinician's perspective (Proceedings)


PCR: A clinician's perspective (Proceedings)

Apr 01, 2008

When Should I Use PCR?

PCR-based diagnosis is ideally suited to detection of organisms that are not easily found on cytologic preparations, are slow-growing or difficult to cultivate, when a rapid diagnosis is required, when alternative methods are expensive or hazardous to laboratory staff, or when inhibitory substances such as antimicrobials may be present. PCR can also be used to search for evidence of an organism when the organism may have been inactivated in a biopsy or necropsy specimen. PCR can be used to assess clonality in cases with suspected lymphoma. Finally, PCR can be used to identify antimicrobial resistance.

How Do I Collect Samples For PCR?

When collecting samples for PCR, it is important to be aware that contamination can occur outside the PCR laboratory as well as within the PCR laboratory. Collecting samples for PCR should be performed aseptically, and disposable instruments and gloves should be used (eg. disposable scalpel blades, punch biopsy instruments).

If possible, prior to collecting the sample, the laboratory should be contacted to determine the optimal method of collection. If the laboratory is not available (eg. after hours cases), then the following are general guidelines. If you are looking for a DNA pathogen (ie all bacteria, fungi, some viruses), then either fresh or frozen tissue should be submitted. Repeated freeze-thaws should be avoided. A small amount of saline can be added, but excessive dilution should be avoided as it reduces the sensitivity of the assay. For blood samples, EDTA is usually fine. Tissues stored in formalin, especially for prolonged periods, are suboptimal for PCR because the formalin cross-links the DNA in the specimen. Tissues fixed briefly in formalin, then paraffin-embedded are preferable to tissues stored in formalin, because the formalin is removed during the embedding process. For RNA pathogens, special media may be requested to prevent degradation of the RNA. If this is not readily available at the time of specimen collection, the sample should either i) be kept refrigerated until the media is available (best for swabs or body fluids), no longer than 2-3 days, or ii) be frozen within half an hour of collection, and shipped on dry ice without thawing it in the interim (best for tissues).

Where Do I Send Samples For PCR?

There are several real-time PCR laboratories now in the US, although the list of assays offered vary. These include laboratories at IDEXX, UC Davis, Auburn University, the University of Tennessee, and Zoologix, in Chatsworth, CA. Some laboratories perform only conventional PCR. Antech has recently released FastPanel, a panel of real-time assays for companion animal infectious diseases, although at the time of writing, the details regarding the methodology and validation of this assay are unavailable.

How Do I Send The Samples?

The laboratory should be contacted if possible. Usually overnight FedEx is required. For DNA testing, samples should be held at room temperature or on ice. RNA should be sent either at room temperature (in special media), on ice, or frozen.

How Long Will It Take?

Most commercial laboratories are reporting results in 1 to 3 days from the time of sample submission. With the new rapid thermocyclers, results may become available on the same day as sample submission.

How Much Will It Cost?

The cost of PCR varies from laboratory to laboratory, but for a single target organism the cost is usually in the range of $40-60. The cost of testing for RNA pathogens is greater because of the necessity for the RT step. Many laboratories are offering discounted assay panels that allow testing for multiple pathogens that might be present within a single clinical specimen. Some laboratories will also provide sequence analysis, usually at a cost of around $100/product.

How Do You Interpret A PCR Test Result?

Factors influencing interpretation of a PCR test result, include:

1. The assay. It is important to know whether a laboratory is running conventional versus real-time PCR, because conventional PCR is more prone to contamination and false positives due to non-specific PCR products. If the assay is a multiplex assay, the sensitivity may be reduced due to consumption of reagents during PCR. It is important to find out whether the laboratory is including controls for inhibition, and has validated their assay.

2. The disease. If you run a panel, you may end up with an unexpected positive result. As with the result of any laboratory test, the result needs to be interpreted in light of the animal's clinical signs and the pathogenesis of the disease you are testing for. An understanding of the pathogenesis of infectious diseases and organism shedding patterns is crucial for interpreting positive PCR results. If the pathogenesis of a disease is largely immune-mediated, the organism you are testing for may be in such low concentrations it may be undetectable using the assay. If subclinical carrier states are common, a positive PCR result may not be significant to the animal's disease. If the assay detects organisms that an animal has received a modified live vaccination for, a positive result may not be important if the vaccine was recently administered. It also needs to be remembered that PCR detects both viable and non-viable organisms, so can be positive after antimicrobials have been administered.

3. The sample. The sample type can be important for interpreting PCR results. The organism you are testing for may not be present in the sample you have collected (ie. other sample types may have been more appropriate). The sample may not have been collected properly. Shipping conditions can also affect the chance of receiving a positive result.