Bioscience Methods 2024, Vol.15, No.3, 91-101 http://bioscipublisher.com/index.php/bm 96 diagnostic tools are collectively driving a new era in molecular diagnostics for pet disease detection. These innovations are making diagnostics more accessible, efficient, and accurate, ultimately improving the health and well-being of pets. 5 Challenges and Limitations 5.1 Technical challenges Molecular diagnostics in veterinary medicine have seen significant advancements, particularly in techniques such as PCR, gene sequencing, and mass spectrometry. However, these technologies come with technical challenges. Quality control is paramount to avoid issues such as inhibitors, cross-contamination, and inadequate templates, which can lead to erroneous microbial identifications. Additionally, the integration of clinical, pathologic, and laboratory findings is essential for accurate diagnosis, as molecular testing alone is insufficient. The development and implementation of these technologies also require thorough validation to ensure their reliability and accuracy in routine diagnostics (Cai et al., 2014). 5.2 Cost and accessibility The cost of molecular diagnostic tests remains a significant barrier to their widespread adoption in veterinary practice. The choice of technology and equipment, along with the need for specialized personnel training, contributes to the high costs associated with these tests. Furthermore, the reimbursement by third-party payers is often limited, making it challenging for veterinary clinics to justify the investment in these advanced diagnostic tools (Figure 3) (Fortina et al., 2002; Belák et al., 2013; Kostyusheva et al., 2020). The development of cost-effective alternatives and the potential for point-of-care testing through technologies like "lab-on-a-chip" could help mitigate these issues in the future. Figure 3 Schematics of CRISPR-Cas9-based CRISPR-diagnostic method CASLFA (Adopted from Kostyusheva et al., 2020) Image caption: (A) Structure of the lateral flow device. The lateral flow device consists of a sample pad where the isolate is applied, a conjugate pad with pre-assembled AuNP-DNA probes, a test line and a control line. At the test line, complexes of CRISPR-Cas with the target biotinylated DNA and AuNP-DNA probes, hybridized with the stem-loop region of sgRNA, interact with pre-coated streptavidin at the test pad to produce a visible signal. At the same time, AuNP-DNA probes move further and interact with streptavidin at the control line. AuNP-DNA probes contain three regions, namely (1) polyA-polyT (poly A used for labeling with Au and polyT as a linker); (2) purple area for hybridization with the embedded probe in the control line and (3) yellow area used for hybridization with the engineered stem-loop region in sgRNA. (B) Schematics of CASLFA procedure. Isolated DNA is amplified with biotinylated primers using RPA or PCR. Amplicons are mixed with CRISPR-Cas9 detection complex and DNA probes and, after short incubation, applied to the lateral flow device (Adopted from Kostyusheva et al., 2020)
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