The present study introduces a novel Point-of-Care(POC)polymerase spiral reaction(PSR)assay for rapid,on-site buffalo tissue identification,particularly in resource-constrained settings.Polymerase spiral reaction addr...The present study introduces a novel Point-of-Care(POC)polymerase spiral reaction(PSR)assay for rapid,on-site 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.展开更多
文摘The present study introduces a novel Point-of-Care(POC)polymerase spiral reaction(PSR)assay for rapid,on-site 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.
