Multiplex polymerase chain reaction-restriction fragment length polymorphism assay discriminates of rabbit, rat and squirrel meat in frankfurter products

- First tetraplex PCR-RFLP to discriminate rabbit, rat and squirrel meat in food chain.
- Cross-specificity tested against 22 species practically and 45 species theoretically.
- Lower limit of detection tested until 0.1% meat under mixed matrices.
- Stability tested under extreme boiling and autoclaving treatments.
- Validated and cross-checked in commercial beef and chicken frankfurter matrices.

The demands for rabbit meat are rapidly growing and Rabbitry is becoming a mean of livelihood for many youths. Rats and squirrels are very close relatives of rabbits, could be hunted freely or raised in domestic farming and so could be substituted in expensive rabbit meat. This study, for the first time, developed and validated a tetraplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay to identify and discriminate rabbit, rat and squirrel meat under raw and processed foods. Four sets of primes amplified 123, 108, 243, and 141 bp fragments from rabbit, rat, squirrel and all eukaryotes, respectively. Specificity was confirmed through sequencing and RFLP analysis. When PCR products were digested with BtsIMutI and BtsCI enzymes, distinctive fingerprints (115 & 8 bp for rabbit; 64 & 44 bp for rat and 176 & 67 bp for squirrel) were obtained. The detection limit of the assay was 0.1% meat in frankfurter formulation.