Optimization of DNA extraction from heat-treated beef broth for bovine identification using urea-phenol-chloroform and salting-out methods

Abstract Meat adulteration, including substitution or mislabelling of high-value meats, remains a major concern in global food markets, affecting public health, consumer trust, and religious dietary compliance. Reliable detection of bovine DNA in heat-treated broth remains challenging due to thermal degradation, matrix complexity, and PCR inhibitors, creating a critical need for an optimized extraction protocol. This study evaluated two methods (urea-phenol-chloroform and salting-out), three meat-to-water ratios (1:1, 1:3, 1:5), and three broth-to-lysis buffer ratios (7:3, 5:5, 3:7; total 1000 μL) to optimize DNA extraction. DNA yield, purity (A260/280, A260/230), and qPCR Ct values targeting a 93 bp COX2 fragment were assessed. Three-way ANOVA revealed significant effects of extraction method and meat dilution (p < 0.001; p < 0.01), with multiple interactions. A multi-criteria scoring system identified the urea-phenol-chloroform method with a 1:1 meat-to-water ratio and a 5:5 broth-to-buffer ratio as optimal, yielding 1273.8 ± 173.8 ng/μL DNA, purity 1.84 ± 0.06, and Ct 25.35 ± 0.34. This protocol is simple, effective, and suitable for recovering DNA from broth-based matrices, supporting food authentication, halal verification, and traceability applications.