Future Perspectives

Point-of-care (POC) diagnostic tests are increas­ingly being integrated into the management of human diseases, but have yet to find a signifi­cant role in veterinary diseases (Manessis et al., 2019).

To date, the focus of POC in veterinary di­agnostics has been on lateral flow immunoas­says and microfluidic-based POC technologies (Busin et al., 2016). The uptake of veterinary POC applications may be suffering from two is­sues. First, previously developed POC tests for veterinary pathogens often rely on technologies that are invariably insensitive, and second, the cost-benefit of veterinary POCs, given their poor sensitivity, is often unfavourable and limits inter­est in commercial partners for their production and distribution.

However, the strengths of laboratory-based qPCR diagnostics (sensitivity, specificity and speed) have for some time now been considered to be integrated into POC, but field-based PCR technology is now a reality. For example, isother­mal assays such as recombinase polymerization amplification (RPA) offer a very simple design that is capable of detecting RNA as well and DNA, thus negating the requirement for a sepa­rate cDNA production step. This technique re­quires amplification at a single low temperature, after which the amplified product can simply be detected on a lateral flow device. Recent publica­tions have appeared reporting isothermal-based tests for bacterial pathogens of both terrestrial (Pasteurella multocida and Mycoplasma bovis) and aquatic animals (Francisella noatunensis subsp.

Various isothermal assays have been re­ported for the detection of MAP, including loop-mediated isothermal amplification (LAMP) (Enosawa et al., 2003; Trangoni et al., 2015, Trangoni etal., 2017; Sange et al., 2019; Yashiki et al., 2019) and RPA assays (Hansen et al., 2016; Zhao et al., 2018b). While LAMP assays offer some advantages, the complexities in the assay design can be prohibitive compared with the simpler RPA assays. Both studies using an RPA assay reported similar sensitivity to qPCR and equivalent specificity (Hansen et al., 2016; Zhao et al., 2018b). At present, the major draw­back to the integration of isothermal POC tests is the DNA extraction step. The issues and impli­cations of DNA extraction in conjunction with POC have been discussed (Ali et al., 2017), but once these have been overcome, isothermal as­says will find their place in POC diagnostics for paratuberculosis.

Alternative molecular diagnostic method­ologies may be the next major development in diagnostic and POC tests for paratuberculosis. Nanoparticles are already frequently used in sample preparation, bioassays and for enrich­ment, as they can be readily functionalized (Hahn et al., 2017; Gordillo-Marroquin et al., 2018; Markwalter et al., 2019). For detection of nucleic acids from a pathogen, however, signal amplification is often essential; nanoparticle- assisted signal amplification and detection methods are being developed to meet this need for diagnostic testing in tuberculosis (Teengam et al., 2017).

Pacheco, 2018). These involve engineered sen­sors that target DNA or RNA sequences specific to the pathogen and enable high sensitivity pathogen detection, with minimal processing of the clinical sample.

Detection of multiple RNA or miRNA bio­markers, rather than genetic elements of the organism itself, is an evolving field. Specifically, the identification of infection-associated gene expression biomarkers within cells found in milk or blood may be used to develop a rapid PCR- based diagnostic test capable of differentiating subclinical and clinical paratuberculosis (Purdie et al., 2012, 2019). This could be combined with concurrent detection of the pathogenic organ­ism to provide an overall picture of the biological status of an animal. Evidence has shown that in terms of biomarker-based detection assays, it is necessary to utilize multiple biomarkers to ad­equately evaluate disease status.

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