Читать книгу Infectious Disease Management in Animal Shelters - Группа авторов - Страница 115

The detection of the nucleic acid of an infectious agent can be accomplished through the use of molecular assays such as PCR, reverse‐transcriptase PCR (RT‐PCR), and real‐time PCR. The ability of such assays to detect minute quantities of nucleic acid makes them among the most sensitive diagnostic testing methods available (Tizard 2013). Another important benefit of molecular assays is their ability to detect organisms in animals that are subclinical carriers or latently infected (Evermann et al. 2012), allowing for quicker identification of animals that may present an infectious disease risk to the population. This benefit may be particularly useful during the response to a disease outbreak when quantitative results are available to distinguish between vaccinates and infectious animals. However, as with antibody titer analysis and other methods of antigen detection discussed above, molecular detection of an organism merely indicates its presence. It does not indicate whether the detected organism is alive or dead or whether it is the cause of active disease. For these reasons, interpretation of results should take into account clinical signs and the possibility of latent stages of infection (e.g. herpesviruses, feline retroviruses). The high sensitivity of these assays makes careful sample collection and handling, as well as submission to a reliable laboratory with high‐quality control standards, of the utmost priority as sample contaminants will readily be detected. Molecular methods can be used to detect pathogens in samples of blood and tissue or, increasingly, in culturettes or swabs containing trace amounts of respiratory secretions or feces.

Molecular assays rely on the PCR to detect trace amounts of pathogen DNA. For RNA viruses (e.g. feline calicivirus, canine distemper virus), RT‐PCR may be performed. In this assay, reverse transcriptase is used to convert the viral RNA to DNA prior to the PCR process. Real‐time PCR allows for the quantification of DNA through the use of fluorescent labeling of the DNA (or a DNA probe), which can be tracked and quantified as the PCR progresses, allowing for the determination of initial DNA quantity, less susceptibility to contamination, and greater sensitivity of detection. Standard PCR techniques analyze DNA bands at the completion of a variable number of amplification cycles and rely on size discrimination for identification, which can result in comparatively lower precision and sensitivity of detection (Applied Biosystems n.d.; Tizard 2013). Real‐time PCR can also be completed on RNA viruses through reverse transcriptase real‐time PCR.

For the detection of common causes of both respiratory and GI disease, diagnostic laboratories offer real‐time PCR analysis of a variety of pathogens common to either dogs or cats. Such services carry the benefit of screening for a variety of pathogens with a single sample (and single fee), fast turnaround time (one to three days), and ready identification of potential co‐infections. Pathogen detection may be inhibited in aged samples or those from patients receiving antimicrobial therapy and, in the case of respiratory PCR panels, recent modified live‐virus vaccination can interfere with the interpretation of results. Samples for respiratory PCR analysis should include a conjunctival and deep pharyngeal swab collected on a sterile, plastic‐stemmed swab and placed in a sterile tube. Samples obtained via trans‐tracheal wash or bronchoalveolar lavage may also be accepted and would be most appropriate to collect from animals with suspected lower airway disease. Samples for GI PCR analysis should include fresh feces (minimum 1 gram) placed in a sterile container. Both respiratory and GI samples can be refrigerated for up to 10 days (IDEXX Laboratories 2017).