Johnny Callahan, PhD - Senior Consultant, Thermo Fisher Scientific

Johnny Callahan, PhD

Senior Consultant, Thermo Fisher Scientific

I understand there are several methods for diagnostics testing, like ELISA and PCR. Does it matter which type of test is used on my samples?

The laboratory has many “tools” in its diagnostic toolbox, which include tests based on both classic and modern methods. The Enzyme Linked Immunosorbant Assay (ELISA) is an example of a classic laboratory method that can detect structural components of the organism, called “antigens,” in many different sample types, including tissues and fluids. The ELISA is also commonly used to detect antibodies in animals’ blood serum, which, when positive, indicates the animal has been exposed to a disease agent.

Many, but not all, infectious agents can be isolated in a culture, which is one of the oldest classic laboratory methods. If an organism is isolated by a culture method, that result is considered definitive. One of the most widely used modern methods is the real-time Polymerase Chain Reaction, often abbreviated as rPCR or qPCR. This test detects specific, unique genetic sequences found in the nucleic acid (DNA or RNA) of infectious organisms.

What are the advantages and limitations of these laboratory methods?

ELISA has several advantages compared to PCR. The technology is cheaper to perform on a cost-per-test basis; the required level of training usually is less, compared to PCR; and the equipment startup costs are much cheaper for ELISA. In addition, you cannot detect antibodies by PCR or culture. For this reason, ELISA is still widely used on a global basis, especially in countries with limited financial resources. ELISA limitations are that Antigen Capture ELISA (ACE) is less sensitive and less specific than PCR, and also is less specific when compared to culture for detection of the organism in veterinary samples.

Culture methods often are considered the reference method for detecting many disease agents. The advantage of culture methods is that isolated organisms can be used for further characterization of the disease agent and may be used to prepare vaccines. Limitations are that not all pathogens are easily cultured, or some organisms may be slow to grow and take weeks to issue a final lab report. Some organisms are dangerous to culture and pose an occupational risk to laboratory workers. Real-time PCR has the advantage in terms of sensitivity and specificity compared to the classic methods. Some diseases have a very low minimum infectious dose, so it is crucial that the screening method used to detect and control the disease has exquisite sensitivity. For many diseases, the ELISA test is not sensitive enough to meet this specification. As with any general statement, there are exceptions where ELISA sensitivity is adequate for routine use.

How does PCR work?

In PCR, the nucleic acid is extracted from a sample; added to a special mixture containing a buffered salt solution, enzyme(s), short fragments of oligonucleotide sequences that prime the reaction; and begin the test. In the PCR test, that special “master mix” mixture and the nucleic acid extracted from the test sample are cycled rapidly in a heating/cooling reaction that produces two new copies from one strand at the end of each cycle. Over a 40-cycle reaction, a few copies of DNA are exponentially copied/amplified until ultimately there are billions of copies of the target sequence. The results of the PCR reaction utilize specialized instruments that read fluorescent signals created during the reaction.

Is PCR better than ELISA?

No, one is not better than the other. Each just uses a different testing principle, and has advantages and limitations. The most efficient laboratory will use all of the tools in its diagnostic toolbox to maximize the advantages of each test method. For example, when testing for bovine viral diarrhea virus (BVDV) testing at Gold Standard Labs, the RNA extracted from 24 individual ear notch samples are pooled and run by PCR, since the incidence of the disease is low. Pooling allows the lab to maximize laboratory throughput with minimal risks of false negatives due to the high sensitivity of the PCR test. Retesting individual samples from a positive pool is prohibitively expensive if PCR is used exclusively. The optimal solution for retesting individual samples from pooled samples that are positive by PCR is to retest the individual samples that made up the pool by utilizing an ELISA test. This test algorithm of screening pools of 24 samples by PCR, then retesting the individual samples by ELISA, maximizes the benefits of the competing technologies while minimizing the overall testing cost.

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