THE LAST DECADE HAS USHERED IN new technologies in molecular science. Techniques that were recently only research tools are becoming more widely used. The trend is to apply nucleic acid based tests toward disease diagnostics. Unknown organisms can be identified; closely related organisms can be differentiated; highly sensitive assays can be developed to detect small numbers of pathogens in complex samples; very fast results can be obtained in tightly controlled assays. These tests exploit the chemistry of natural DNA replication in vitro, allowing investigators to identify very specific sequences of a pathogenic genome.
Advances in nucleic acid sequencing technology and the computer-assisted comparisons of sequence data are allowing molecular biologists to have a greater impact on equine disease diagnostics. Sequencing a single microbial gene was once a project that would involve months of effort in highly specialized labs. The amount of work necessary to sequence all the genes on a microbial chromosome (genome) was nearly impossible to imagine 10 years ago. As a reference of magnitude, the genome of the bacteria Escherichia coli is composed of 4,377 genes on a double-stranded DNA molecule with a very specific sequence of 4,639,221 base pairs. Today, genes can often be sequenced within a week, and even microbial genome sequencing is becoming more commonplace. Technological advances are occurring that may soon allow microbial genome sequencing within a few hours.
The classification of bacteria is based in part on sequence comparisons of certain genes. The most commonly used is the gene encoding the 16S rRNA molecule, a sequence of about 1,400 bases in length. Databases of these sequences are now available that allow researchers to identify both conserved and unique regions of the sequence. Taxonomists can use this information to group bacteria in hereditary units and develop phylogenic trees. If a bacteria is isolated and its identification is difficult, the 16S gene can be readily sequenced and compared to the sequences of known organisms. A good example applying this directly to disease diagnostics is the bacterium causing most cases of nocardioform placentitis. The bacterium isolated had less than 97% homology to all known bacteria. Nucleic acid based tests indicated it was in the genus Crossiella, and it was named Crossiella equi. As a general rule if the unknown sequence is 98% identical or higher to the sequence of a known organism, the unknown is probably within the same species as the known organism. As more genes and complete genomes are sequenced, more complete and detailed information will become available.
A sequence that is unique to a pathogenic organism or gene (like a toxin gene) often becomes the target of a diagnostic nucleic acid test. These sequences are targeted because they can allow specific detection of that particular sequence in mixtures containing various other sequences. The most commonly used diagnostic nucleic acid test is the polymerase chain reaction test (PCR). The basic test process flows from isolation to amplification and ends with detection. Nucleic acids from a sample thought to contain a specific organism are first isolated and placed in a tube with primers that target the unique sequence. The chemicals responsible for nucleic acid synthesis (DNA polymerase, nucleotides, salts) are added, and the sample is placed in a machine that cycles temperature (thermocycler) and enables a chain reaction of DNA synthesis to occur. This amplification step is quick and dramatic. In as little as an hour a single molecule can be amplified over a billion times. Detection is qualitative (positive/negative) and usually achieved by observation of a band of the amplified DNA on an agarose gel. Positive amplification infers the target (pathogenic organism or gene) was present in the sample. Most nucleic acid tests are variations on this theme. Enhanced sensitivity and specificity can be attempted using a re-amplification step called “nested PCR” or by using a labeled probe to detect the amplified DNA. More than one target can be detected in a single reaction using multiplex PCR. RNA targets can be detected using RT-PCR that includes an initial reverse transcription step added before PCR amplification. Real-time PCR combines the amplification and detection steps and can be used for faster and even quantitative results. The ability to detect specific pathogens will continue to be enhanced by the advances in nucleic acid testing.
Dr. Stephen Sells or Dr. Mike Donahue, (859) 253-0571, firstname.lastname@example.org, Livestock Disease Diagnostic Center, University of Kentucky.