Identification of pork in raw meat or cooked meatballs within 20 min using rapid PCR coupled with visual detection

Meat adulteration is seriously damaging the interests of consumers. It is important to develop a rapid, simple, cost-effective and sensitive method to identify meat species. In this study, a pair of suitable pork-specific primers was determined by the comparison of three pairs of pork-specific primers and the annealing/extension temperature optimized here was set at 64 °C. Rapid polymerase chain reaction (PCR) with glass capillary as reaction vessel, which could complete 45 cycles in 5 min by using two ordinary water baths, was established to detect pork contents in raw beef meat or cooked beef meatballs. A SYTO 9-based visual detection method was used to evaluate the amplification results. The fluorescence signal of negative samples could be removed at 72 °C according to the results of original melt curves of positive and negative samples. Strong green fluorescence would be produced in positive samples while the color of negative samples still remained black under the blue light (470 nm). A simple and portable device was designed to prevent detection results disturbed by ambient light and make operation easier. As low as 0.01% pork contents in binary mixtures could be detected and the whole detection process could be finished in 20 min from sampling to results. The developed method would have great potential for rapid on-site detection of pork meat and identification of meat species.