A rapid and field-ready snap-chill polymerase spiral reaction (PSR) colorimetric assay for identification of buffalo ( Bubalus bubalus ) tissue

The present study introduces a novel Point-of-Care (POC) polymerase spiral reaction (PSR) assay for rapid, onsite buffalo tissue identification, particularly in resource-constrained settings. Polymerase spiral reaction addresses the limitations of existing detection methods by utilizing chemical -free snap-chill DNA extraction, highly specific mitochondrial d -loop gene primers for amplification, and a unique chromogenic indicator triad of hydroxy naphthol blue, malachite green, and phenol red for visual detection. The effectiveness of the chromogenic indicators was validated against SYBR Green I and agarose gel electrophoresis methods. The assay exhibits high specificity with no cross -amplification observed in common meats (beef, mutton, chevon, and pork), sensitivity down to 10 fg of buffalo DNA, and the ability to detect adulteration as low as 0.1% buffalo meat content. Comprehensive validation through diverse, blind, and processed samples, along with established compatibility with the commercially available DNA isolation kit and comparison with standard buffalo-specific PCR, confirms its accuracy. To the best of our knowledge, this represents the first report of a PSR colorimetric assay capable of identifying buffalo tissue within 90 min in resource-constrained settings.