Increasingly, molecular techniques are an important adjunct in diagnostic medicine. My focus here is diagnostic techniques for infectious diseases, although many techniques such as polymerase chain reaction (PCR) could be applied to genetic diseases, oncology, or other specialties as well.

The single most important new molecular technique for evaluating infectious diseases is PCR. This is a technique for finding pathogens directly and can be used typically with greater sensitivity, often comparably with culture, direct visualisation, special stains or antigen tests. PCR amplifies the deoxyribonucleic acid (DNA) of the pathogen typically to give a million times the amount of DNA that was present in the original sample, allowing us to visualise the DNA and make a specific and sensitive diagnosis.

PCR usually is very sensitive. A positive test indicates active infection either currently or in the past. This infection could be active or recent, since DNA may remain in the body for days after the pathogen has been killed. One potentially common problem with PCR is false-positive test results, which may occur with contamination during the set up. Also, PCR is only as good (specific) as the primers chosen for the test.

PCR Chemistry

Step 1 is to extract DNA from the sample. Common tissues to test are blood, skin, faeces and sputum. DNA may be fixed to some substrate, washed, separated from PCR inhibitors and extra protein, and then precipitated into water or a buffer. Commercial kits are available to do this. Another technique is to use a fine silica to hold the DNA, then digest protein and wash (this has worked well for us for faeces). For samples such as bacteria on a plate, simply boiling the bacteria in water may be adequate.

Step 2 is to add DNA to the PCR mix. PCR mix contains Taq DNA polymerase, dNTPs, a buffer, water, primers and magnesium. Taq comes from Thermus aquaticus, a thermostable bacterium that lives optimally at 72°C. As for other organisms, T. aquaticus has a DNA polymerase that allows it to copy DNA in order to replicate. Taq and its buffer are sold together. The deoxynucleotide triphosphates (dNTPs) consist of raw DNA bases (A, C, T and G) which will be used by the Taq to create new strands of DNA. Primers are critical for the reaction, because the cue for polymerisation is an area of DNA where there were two strands (e.g., the target plus an attached short primer) and then single-stranded DNA. Then the polymerase would jump in and start making more of the second strand.

Step 3 is recursive cycling. A sequence of denaturation, annealing and extension is repeated over and over, ultimately resulting in millions of copies of an initial target DNA. Double-stranded DNA denatures into single strands at 95°C. Then primers can attach to each strand. Next the reaction is brought down to a temperature where the primers can anneal to the single-stranded DNA, usually in the 50°C range though it depends on the particular primer. As DNA gets cooler, strands reattach. The higher the temperature, the more exact a match there needs to be between the two strands. Extension is the step where the Taq DNA polymerase copies the DNA, and occurs at 72°C. The 72°C is perfect because the polymerization step must be at a significantly higher temperature than the annealing step, or a total mix of double-stranded DNA mess would occur during annealing at low temperatures. After PCR is run, in a thermal cycler, the most common way to determine whether the test was positive is to run the samples out on agarose gels and then evaluate them under ultraviolet (UV) illumination. If results are positive, you will see fluorescing bands (due to the ethidium bromide) of specific lengths on your gel. If every lane contains the appropriate length bands, either every sample was positive or you have contamination. This is where the negative control is so important. If you have contamination, at minimum you need to re-run the PCR, again with negative control to ensure that you have eliminated the contamination, or at worst you need to re-extract the DNA.

Primers are short (approximately 20 bases long) fragments of DNA that are constructed to attach to a single-stranded target at specific locations, because you designed your primers to be a match to those locations. They should attach at only one site and flank a piece of target DNA from 50 to hundreds of base pairs long. However long the intervening sequence is, is the length of band you'll be looking for on your gel. It is up to the laboratory to design good primers in the first place. Designing PCR primers requires prior knowledge about the target. The primers must amplify a region that differentiates the target from other organisms. There is software to help find primers that will fit a given region, have a reasonable annealing temperature and so forth.

Sensitivity and Specificity of PCR

Optimally, the PCR is sensitive and specific. What determines specificity is the primer. Sensitivity is used in two ways for PCR. The laboratory sensitivity is the number of original target molecules in the sample. The more sensitive, the fewer copies would need to have been present for you to obtain a positive result. Sensitivities for some PCR tests run from 100 to 1000 organisms. This level of sensitivity can be improved with optimizing the amount of magnesium, doing a nested PCR (two sets of primers run sequentially), using quantitative real-time PCR and other techniques. Epidemiological sensitivity refers to how many true positives your test can detect. This is, of course, related to laboratory sensitivity: if there are few organisms, you may not get a positive test result. However, lack of sensitivity could also occur if there are different related strains of pathogen and the test only targets some of them, for example. Again, this is determined by primers.

DNA Probes

A DNA probe is a detector-conjugated length of DNA that will bind to a target specific to a disease you are testing for. The probe is often conjugated to a biotin molecule, so that when it attaches to the target, it can be visualized. A common use for DNA probes is to confirm PCR: since all you see with PCR is a band, you can't always be sure that your band is what you think it is. Probes may be used directly on tissue, although this application has lower sensitivity than PCR. There are few DNA probes in use at present in small animals. Examples are a Mycoplasma haemofelis probe used in blood samples and probes for avian chlamydophilosis (chlamydiosis).

Immunofluorescent Antibodies and Enzyme-Linked Immunosorbent Assay

There are some scenarios where you need to evaluate antibodies, not live agent or antigens. This could be because you think the patient has already recovered and you're testing for prior exposure or because you can't access the tissue where the pathogen would be. Common tests for antibodies include immunofluorescent antibodies (IFA) and enzyme-linked immunosorbent assay (ELISA).

The process for IFA is to fix antigen on a slide, react the slide with patient serum, react with a fluorescently labelled secondary antibody, and then read the slide under a fluorescent microscope. When you read the slide, you look for fluorescence in your slide that is in the right location. For example, herpesvirus inclusions can be in the cytoplasm or nucleus, while coronavirus inclusions are in the cytoplasm. This localisation increases specificity because you can rule out non-specific fluorescence if it doesn't localise where the antigen should.

IFA tests are easy to run and relatively inexpensive. However, IFA cannot be automated (in contrast to ELISA) and doesn't separate natural and vaccine reactions.

ELISA tests are performed in a 96-well plate format, typically with antigen layered in each well. As for IFA, patient serum is reacted and then a secondary antibody incubated as well. However, instead of reading fluorescence, the secondary antibody is conjugated with biotin or alkaline phosphatase, followed by enzymatic colour development. The ELISA can be read visually or in a plate reader. There are several advantages to ELISA. The test can be modified to a point-of-care test kit; ELISAs are inexpensive, can be automated, and have high sensitivity when used with the biotin-streptavidin reaction. The major cons are the possibility of false positives, and the difficulty evaluating results because there is no antigen visualisation.

Antigen ELISA is a modification in which antibody is applied to a well and then reacted with a patient sample to detect antigen. Examples are feline leukaemia virus (FeLV) and Cryptococcus neoformans, both of which work because of the large amount of antigen in natural infection.

Western Blot

In diseases such as Lyme disease and feline immunodeficiency virus (FIV), where IFA or ELISA tests have low specificity, western blots may be used for confirmation. Antibodies in the patient serum are reacted with pathogen proteins that have been separated out by size and charge on an electrophoretic gel. The pathogen may be cultured or purified, proteins are extracted and then separated on a gel. Then the proteins are reacted with patient serum. A secondary, conjugated antibody is applied.