Update on molecular techniques for diagnostic testing of infectious disease

Abstract

The era of diagnostic molecular biology has arrived for small animal clinicians, and it is a near certainty that assays such as the PCR and RT-PCR will become more widely available for a wider array of infectious agents. Already there is an extensive list of infectious diseases of dogs and cats that have been investigated with molecular tools. A partial list is included in box 1. An understanding of the advantages and disadvantages of the molecular techniques and some of the questions these techniques can answer for clinicians can serve practitioners well in their approach to the diagnosis of infectious diseases in dogs and cats. It is likely that additional applications of these tools to small animal medicine will become apparent as investigators use and refine them for their research purposes, or as new uses emerge from human medical applications. Clinicians also are likely to reap the benefits of this knowledge. Because samples often are acquired easily from clinical patients in most practice settings, access to these tools puts all clinicians in the group of discoverers of new, or variations of, infectious diseases and their clinical manifestations.

Fig. 1

Schematic representation of the polymerase chain reaction (PCR). DNA (a) in the test sample is heated to separate the two strands (b). The reaction is cooled to allow primer (short bold lines) annealing to the target DNA (c), then heated again to allow addition of nucleotides to extend the nascent DNA molecule (d). At the end of the cycle, the target DNA has been duplicated (e). Each new DNA can then itself be a targeted molecule for the next cycle of amplification. The process is repeated for 25–40 cycles, and then products are visualized following gel electrophoresis (f). The left hand lane represents DNA markers of a known size against which the size of the amplicons is measured.

Fig. 2

Schematic representation of restriction fragment length polymorphism (RFLP), or DNA fingerprinting, analysis. DNA (A) is cut with a restriction enzyme to produce a number of DNA fragments of varying lengths (B). The DNA fragments are separated by gel electrophoresis to produce a pattern unique to the organism and the enzyme used to cut the DNA. In this representation, six different samples were analyzed, with one of the six (in the 4th lane) clearly exhibiting a pattern different from the other five.