In this paper, a new approach to rapid pork detection was developed using an amplification-free and mix-to-read CRISPR-Cas12-based nucleic acid analytical strategy. An optimized guide RNA (gRNA) targeting the pork cytochrome b (Cyt b) gene was designed, which allowed specific identification of the target Cyt b gene in pork components. Activation of Cas12 protein to cleave single-stranded DNA probes with terminally labelled fluorophore and quencher groups then allowed confirmation of the presence of pork Cytb by reading the fluorescence signal. The assay allowed specific detection of pork in beef, mutton, and chicken products, The reliability of the method was tested on processed halal meat products - beef luncheon meat and spiced beef, as well as non-halal foods - sausage and dried pork slices.
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