A cost-effective method for the rapid detection of chicken adulteration in meat using recombinase polymerase amplification combined with nucleic acid hybridization lateral flow strip

Meat adulteration is becoming increasingly prominent in the global market. Therefore, a robust, sensitive, and affordable rapid detection strategy for identifying meat species is urgently needed. In this study, we developed a novel strategy using chicken-specific recombinase polymerase amplification (RPA) coupled with nucleic acid hybridization lateral flow strip (NAH-LFS) for the rapid detection of chicken adulteration. In an isothermal RPA reaction, chicken mitochondrial DNA (as target) was amplified at 39 degrees C for 15 min. Both the forward and reverse primers contained three parts, namely the specific binding region, a special abasic spacer 9 (iSp9), and the tag sequence (forward primer tag, F-tag, and reverse primer tag, R-tag). The specific binding region and tag sequences were linked by iSp9, which could also inhibit extension by DNA polymerase, producing a doublestranded amplicon with two tag sequences (F-tag and R-tag) at the two opposite ends. The F-tag and R-tag can complementarily hybridize with probes on AuNPs and the test line, respectively, showing red color on the test line. This visual detection strategy does not require expensive equipment, and the entire detection procedure can be performed within 20 min. Specificity tests with 10 other animal samples showed no cross-reactivity. The RPA-NAH-LFS strategy could detect 0.01% (w/w) chicken in the chicken/beef meat mixture. To verify its utility, this assay was used to authenticate 30 commercial beef samples; the results were 100% consistent with those obtained using the PCR- agarose gel electrophoresis (AGE). In conclusion, the RPA-NAH-LFS assay serves as an accurate, sensitive, and cost-effective method to evaluate chicken adulteration and traceability in meat.