Veterinary PCR Diagnostics

Compared with traditional PCR assays, diagnostic assays based upon real-time PCR technology have increased speed and dynamic range; in addition, they enable quantitative analysis of gene copies and have the potential for increased specificity when nucleic acid probes are used. Optimized real-time PCR assays can also be highly sensitive, detecting as few as 1-10 copies of a target gene in a nucleic acid sample. Adopting real-time PCR in a diagnostic laboratory requires an understanding of these assays, including both the benefits and drawbacks unique to this technology. An overview of real time PCR applications is presented here, with an emphasis on practical issues that might affect implementation of real-time PCR testing in a diagnostic laboratory. Increased cleanliness and process controls are required in the laboratory, to prevent contamination of sensitive real-time PCR. Nucleic acid extraction procedures, using one of the many available chemistries, should be carefully optimized for reproducible, efficient extraction of nucleic acids that are free of PCR inhibitors. Reverse transcription of RNA adds an additional variable that can affect quantitative data. For the assay itself, different options have been developed for the detection of products in real-time, including dye-based assays, hydrolysis probes, and hybridization probes. Different options and the benefits and drawbacks of each are discussed. Finally, specific applications for real-time quantitative PCR assays in diagnostic laboratories are highlighted.

With increased use of real-time polymerase chain reaction technology in molecular diagnostics, consistent procedures for design, optimization and validation of molecular diagnostic methods are needed. This chapter describes a practical guiding principle that can be used in different steps of the design and validation of in-house developed real-time PCR assays. The use of the described guidelines leads to more efficient and standardized optimization and validation. Ultimately, this results in a reliable and robust molecular diagnostic assay. A statistical follow-up of the performance of the assay is included and can be achieved by determination of target values and reproducibility of internal quality controls. Since this guiding principle is independent of environment, equipment and specific applications, it can be used in any laboratory.

In the last decade, antimicrobial resistance has been widespread in several bacterial species. This increase in resistance could be associated with an increase in the use of different antimicrobials to treat infections caused by pathogenic bacteria. While resistance to antimicrobials is often attributed to known mechanisms, other mechanisms are still under investigation for many bacterial species. Detection of antimicrobial resistance often involves conventional agar, broth or disk diffusion assays. However, these methods can be cumbersome and time consuming compared to molecular methods. Consequently, several polymerase chain reaction (PCR) techniques have been developed to expedite the detection of antimicrobial resistance in bacterial pathogens. PCR-based technologies are rapid, sensitive and specific for detecting antimicrobial resistance. Application of such technologies in diagnostic laboratories can provide insight into emerging mechanisms of antimicrobial resistance in veterinary pathogens. In this chapter, we describe molecular mechanisms of drug resistance in microbial pathogens and the potential advantages and disadvantages of PCR-based methods.

The ideal diagnostic method for veterinary purposes, particularly for the field practitioner, must be reliable, cost-effective and demonstrate good sensitivity and specificity. Although an ideal test with such characteristics does not yet exist, in a short horizon the most probable tests that could reach those goals are those based on molecular biology, particularly real-time PCR and its analogues. PCRbased methods, as a powerful tool for pathogen detection, have been frequently used in the identification of veterinary bacterial pathogens. This chapter focuses on the PCR method, some related important variations and their applications for the diagnosis of veterinary bacterial infections. The concepts such as restriction fragment length polymorphism analysis, multiplex PCR, nested PCR, allele-specific PCR, reverse transcription-PCR, real-time PCR and DNA sequencing are also discussed. This chapter particularly emphasizes the PCR-based diagnostic assays for Brucella sp., Leptospira sp., Mycobacterium bovis, Staphylococcus aureus and Mycoplasma sp. Real-time PCRs that could quantify the presence of the bacterial agents in a reliable way and identify the antimicrobial resistance and virulence factors genes have revolutionized veterinary medicine, making the diagnosis of infectious diseases rapid, reliable and cost-effective.

Since the invention of PCR by Michael Smith and Kary Mullis, the last two decades have seen an explosion of PCR application in various aspects of biological and medical sciences. Due to its high sensitivity, versatility and reproducibility, PCR has become one of the standard procedures in diagnosis of almost all viral diseases in veterinary medicine. Unlike serological methods, which rely on the presence of specific antibodies, and may lead to false positive or false negative results, PCR detects the presence or absence of the pathogen, thereby providing a better measure of the viruses. Furthermore, with the advent of real-time PCR, researchers now can quantify the amount of virus that is present at different sites in the animal, thereby gaining the ability to determine the stage of infection. Variations of PCRs also allow phenotypic characterization between different viral isolates and between wild-type viruses and vaccines, while allowing simultaneous diagnosis of multiple viruses. PCR has become one of the most commonly used methods in diagnosis of viral disease in livestock and companion animals, and with the development of automated technologies and multiplex PCR systems, which vastly elevate the throughput of PCR assays, increased use of PCR-based techniques is expected in the future.

Animals are routinely exposed to parasites from different taxonomic groups resulting in significant morbidity, mortality and economic losses. Accurate identification of the responsible parasites is central to the understanding and management of these infections and associated diseases. Comprehensive approaches to facilitate the diagnosis of parasites and parasitic diseases will yield better insight into their basic biology, epidemiology, pathogenicity, and the development of treatment strategies. Traditionally, the diagnosis of parasitic infections mainly relies on testing for the presence of parasites through direct fecal examination, blood smears, etc, but clinically, it is often difficult to elucidate the entire offending organism. Techniques for diagnosis of parasites such as counting parasites are often time-consuming, difficult, inaccurate, of limited sensitivity, and occasionally unpleasant. While the majority of parasites exhibit multiple stages during the course of their life cycles, the nucleic acids extracted from them during these different periods remain identical. PCR, providing exquisite sensitivity and specificity for detection of nucleic acid targets, has become one of the most important tools in parasite diagnostics. Real-time PCR has simplified and accelerated PCR procedures and has reduced complications associated with traditional PCR, such as cross-contamination. Molecular biology tools, such as PCR, are increasingly relevant to veterinary parasitology. This chapter focuses on the application of real-time PCR for parasite detection and differentiation, exemplified in protozoa, helminthes and arthropods, significant parasites in veterinary medicine and public health.

Recent advances in molecular biology are providing new opportunities for the diagnosis, prognosis and treatment of cancer. At two decades from its discovery, PCR with its hundreds of variants and improvements is still the keystone of molecular techniques which gave rise to such a revolution. Novel methods and techniques have been introduced in recent years which promise further breakthroughs. Unfortunately, in veterinary medicine, the unaffordable cost of new instrumentation has delayed their application and practical use in clinical settings. Nevertheless, thanks to PCR, the exciting era of molecular medicine has also begun in veterinary medicine. Due to the limited space available, this chapter cannot deal with all the potential applications of PCR in the cancer battlefield. Thus, the authors have focused their attention on those PCR applications concerned with for the diagnosis and prognosis of cancer in pets which are already currently available, albeit not diffusely, at both academic and private laboratories around the world. In some cases, such as in c-KIT somatic mutations, for the first time in veterinary medicine, a consensus panel of specialists has recommended the inclusion of a molecular assay in the staging work-up of a neoplastic disease (canine mast cell tumors). In addition to the role of the molecular biologist in developing, implementing and refining the molecular classification for routine clinical practice, it is necessary to discover and validate new targets able to provide accurate information regarding diagnosis, prognosis, treatment resistance, susceptibility or predisposition to toxicity, or the prediction of a therapeutic response.