Zoological Pathology Program, College of Veterinary Medicine, University of Illinois, Maywood, IL, USA
Molecular tools are exciting new diagnostic modalities used in a variety of areas of medicine including identification of infectious agents, evaluation of drug resistance, cancer detection, and prognosis. Molecular diagnostic techniques have become important supplements to conventional microbial identification and are particularly useful in non-domestic species as the assays target the pathogen negating the need for species-specific reagents. Most tests utilize polymerase chain reaction (PCR) based amplification of RNA or DNA targets as the basis of the assay. Commonly used techniques include PCR with sequencing of amplicons, real-time PCR, and more recently microarrays. Because of their sensitivity, specificity, and rapidity, the integration of molecular diagnostic techniques into routine diagnostics and pathogenesis research can enhance the diagnosis, treatment, and management of disease.
There are a variety of molecular techniques being utilized in a diagnostic setting. The most common include PCR and real-time PCR. Both techniques can be performed on RNA (in which case there is an initial reverse transcription step) or DNA extracted from a sample. With PCR, a positive result is identified either as a band of the appropriate size on an agarose gel or from sequencing the amplicon. False positives can occur if primers bind to non-target nucleotides (either non-specific binding or non-specific primers) resulting in a visible amplicon on a gel of an incorrect size, or possibly of the correct size. In the latter, sequencing of the PCR amplicon (product) provides an additional quality control measure to confirm the amplicon is the expected target. Real-time PCR utilizes fluorophores to quantify (either relative or absolute) the amount of target within a sample.3,4 Many real-time PCR assays couple the fluorophore to a specific probe increasing the specificity of the assay. Real-time PCR assays are particularly useful for identification of common pathogens or genes. With microarrays, a clinical sample can be screened for a variety of pathogens, gene expression, as well as for drug resistance.5 Pathogen microarrays typically contain sets of probes for strain, subspecies, species, genus, or higher taxonomic grouping. Microarray technology is more expensive and currently less available due to the requirement for specific equipment and arrays designed for the pathogens or genes of interest. Additionally, there have been concerns about sensitivity in comparison with real-time PCR; however, future advances are expected to improve the utility of this technique in a diagnostic setting. It is critical to remember that molecular tests only test for the presence of RNA or DNA and, in the case of pathogens, do not say anything about the viability of the organisms.
How to Get the Most from Your Sample
As with other diagnostic tests, submission of the correct sample is critical for obtaining the best result from a molecular test. Although obtaining samples can be challenging in non-domestic species, by considering the disease pathogenesis, judicious sample selection can optimize the concentration of your target and decrease the likelihood of a false negative result. Many tests utilize small volumes, typically less than 1 ml or 1 g and submission of large sample volumes is not necessary, but the submission should be representative. While the small sample size is a benefit of molecular tests, it is also a disadvantage if the sample is not obtained from the optimal site. For example, if testing a kidney for leptospirosis, it is important to sample an area of the kidney most likely to contain the organism. Blood can be tested for evidence of septicemia but again, the quantity utilized in nucleotide extractions is often small in comparison to the total blood volume of the animal and may not be representative of the process.
Once the appropriate sample is collected, it must be handled appropriately to ensure good quality RNA and DNA. RNA is more sensitive to degradation than DNA and caution should be taken with samples for RNA analysis (e.g., RNA viruses). Samples can be frozen immediately after collection or placed in preservative such as RNAlater (Ambion, Austin TX 78744-1832 USA). For blood samples, PAXGene tubes (PreAnalytiX, Franklin Lakes, NJ 07417 USA) are also useful for RNA preservation. Most samples for DNA can be shipped on ice packs; however, check with your individual laboratory for specific guidelines. If samples are collected for possible future testing, storage at -70°C is best. To inhibit buildup of frost, most -20°C freezers go through freeze-thaw cycles that can damage RNA and DNA. Molecular tests can also be used on archived formalin-fixed paraffin embedded tissues in some laboratories. However, formalin cross-links DNA causing it to break, restricting the size of the amplicon that can be obtained which can result in false negatives. Prompt processing of histologic samples into paraffin increases the chances of an accurate result.
With the proliferation of new molecular tests, it is important that assays be appropriately validated. The American Association of Laboratory Diagnosticians (AAVLD) recommends following the World Organization for Animal Health (The OIE) principles for development, validation, and quality control for molecular diagnostic assays (see http://oie.int/eng/normes/MMANUAL/A_summry.htm). These guidelines include information on optimization and standardization of reagents, assay repeatability, determination of analytic sensitivity and specificity, assay performance characteristics, determination of diagnostic sensitivity and specificity, and interpretation of results. Although some steps in assay validation are difficult given small population sizes (e.g., diagnostic sensitivity and specificity), every effort should be made to follow these guidelines in development of a new diagnostic test. The guidelines also include information on general laboratory quality control standards. Although assays targeting pathogens can be used across a variety of species, limited knowledge of host genomic DNA sequences can impact the utilization of assays in new species. Primers and probes should be checked for cross-reactivity with genomic DNA whenever new host species are tested and non-conforming results double checked for accuracy.
Generic Assays and Reference Libraries
When identifying unknown pathogens, generic or consensus primers are commonly used. These primers are designed by choosing areas of sequence similarity among closely related organisms (e.g., several isolates of a specific viral type). Identification of the targeted pathogen is then accomplished through sequencing of PCR amplicons and comparison of PCR products with reference libraries. One problem with these assays is that mixed infections can either result in unreadable sequence or one pathogen may obscure the presence of a second potentially more important pathogen. For example, a generic bacterial primer set may not work for identification of a sample from a non-sterile site or they may pick up a secondary colonizer rather than the real cause of a lesion. For these reasons, aseptic sample collection is as important in sampling for molecular as traditional culture-based tests when using generic primers. Another difficulty with generic primer sets is the lack of a reference library for sequence comparison. Many of the commercially available validated reference libraries are biased towards pathogens of importance in human medicine.1,2 Therefore, identification of some organisms requires utilization of other, less controlled, libraries that may include incorrectly identified sequences. Thus, results of sequence comparisons are only as good as the reference library, a major limiting factor when working with non-traditional species about which there is limited knowledge.
1. Fontana, C., M. Favaro, M. Pelliccioni, E.S. Pistoia, and C. Favalli. 2005. Use of the MicroSeq 500 16s rRNA gene-based sequencing for identification of bacterial isolates that commercial automated systems failed to identify correctly. J. Clin. Microbiol. 43(2): 615–9.
2. Hall, L., S. Wohlfiel, G.D. Roberts. 2004. Experience with the MicroSeq D2 large-subunit ribosomal DNA sequencing kit for identification of filamentous fungi encountered in the clinical laboratory. J. Clin. Microbiol. 42(2): 622–6.
3. Kaltenboeck, B., and C. Wang. 2005. Advances in real-time PCR: application to clinical laboratory diagnostics. Adv. Clin. Chem. 40: 219–59.
4. Mackay, I.M. 2004. Real-time PCR in the microbiology laboratory. Clin. Microbiol. Infect. 10(3): 190–212.
5. Miller, M.B., and Y.W. Tang. 2009. Basic concepts of microarrays and potential applications in clinical microbiology. Clin. Microbiol. Rev. 22(4): 611–633.