Bioscience Methods 2024, Vol.15, No.3, 91-101 http://bioscipublisher.com/index.php/bm 93 targets are analyzed simultaneously, further enhances the efficiency and effectiveness of molecular diagnostics (Kurkela and Brown, 2009; Cai et al., 2014; Wright et al., 2021). Figure 1 Multiplexed CRISPR-based target detection (Adopted from Kaminski et al., 2021) Image caption: Left: pooled multiplexing of up to four targets, as implemented in SHERLOCKv2. Orthogonal CRISPR enzymes (PsmCas13b, LwaCas13a, CcaCas13b, AsCas12a), each cleaving preferentially different reporter molecules bearing different fluorophores (FAM, TEX615 (TEX), Cy5, hexachlorofluorescein (HEX)) with distinct absorbance and emission wavelengths. Right: in CARMEN, a distinct colour code is added to each PCR-amplified sample or Cas13-based detection mix. The Cas13-based detection mix contains Cas13a, a sequence-specific crRNA and a cleavage reporter. The colour-coded solutions are emulsified in fluorous oil, which creates nanolitre droplets. Droplets from all samples and detection mixes are pooled together and loaded into a microwell-array chip in one step to create all possible pairwise combinations. The droplet pair in each well is identified using fluorescence microscopy before they are merged by exposure to an electric field. Fluorescence microscopy is then used to monitor each Cas13-based detection reaction (Adopted from Kaminski et al., 2021) 3 Applications in Pet Disease Detection 3.1 Infectious disease detection Molecular diagnostics have revolutionized the detection of viral infections in pets. Techniques such as polymerase chain reaction (PCR) and droplet digital PCR (ddPCR) are highly sensitive and can detect viral DNA or RNA even in low quantities. These methods are particularly useful for identifying viral pathogens in both symptomatic and asymptomatic animals, aiding in early diagnosis and management (Lappin et al., 2009; Arora et al., 2023). The identification of bacterial infections in pets has also benefited from molecular diagnostics. PCR-based assays can detect bacterial DNA in various samples, including blood, tissue, and bodily fluids. This allows for rapid and accurate diagnosis, which is crucial for the timely treatment of bacterial infections. Molecular techniques are employed to detect parasitic infections in pets. These methods can identify the genetic material of parasites, providing a precise diagnosis that is essential for effective treatment. The use of molecular diagnostics in parasitology helps in detecting infections that might be missed by traditional methods. 3.2 Genetic disease screening Molecular diagnostics play a significant role in screening for inherited diseases in dogs. Techniques such as DNA sequencing and PCR enable the identification of genetic mutations associated with various hereditary conditions. This allows for early diagnosis and the implementation of preventive measures or treatments to manage these conditions. Similar to dogs, cats also benefit from molecular diagnostics for the screening of
RkJQdWJsaXNoZXIy MjQ4ODY0NQ==