In humans, peripheral blood cytokeratin 19 (CK19) mRNA-positive circulating tumor cells (CTCs) was utilized to identify early-stage breast cancer patients with micrometastatic disease who are at risk for disease progression and monitor treatment response in patients with advanced disease. To our knowledge, there has been little research regarding CK19 in canine mammary tumors (CMTs) using molecular methods. A droplet digital PCR (ddPCR) is proposed as a precise and sensitive quantification of nucleic acid targets. Hence, this study aimed to validate a newly designed assay for CK19 detection in canine blood and mammary tissue, along with the reference gene HPRT, by ddPCR. All primers and probes showed a precise match with the exon region of target genes. The assay exhibited PCR efficacy of 90.4% and 91.0% for CK19 and HPRT amplifications with linearity, respectively. The annealing temperature (Ta) for duplex ddPCR was 55 °C, providing the highest concentrations of both genes tested by the synthetic plasmid DNA. The limit of detection (LOD) of CK19 and HPRT were 2.16 ± 1.27 and 2.44 ± 1.31 copies/µL, respectively. Finally, the ddPCR assay was validated with canine peripheral blood, non-neoplastic mammary tissues and spiked samples. Our findings provide a new platform for CK19 studies in CMT diagnosis through blood and mammary tissues.
The alarming problem of meat adulteration emphasises the demand for accessible analytical approaches for food regulatory agencies to detect and, specially, to measure altered meat fractions. This study proposes a novel cross-species triplex droplet digital polymerase chain reaction (ddPCR) assay to simultaneously identify and quantify the ratios of pork/beef meat fractions from a total DNA content, including processed and autoclaved meat, without requiring a standard, achieving high sensitivity with a limit of quantification estimated at 0.1% (w/w) and a limit of detection down to 0.01% (w/w). A single copy nuclear gene, b-actin, was employed as a target, accompanied with myostatin gene as a cross-species target to quantify the meat background. The duplex assay provided a simultaneous quantification of pork and myostatin, whereas the triplex assay was able to detect pork, beef and myostatin with a decrease of technical error, cost and time.
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