Triplex-colored nucleic acid lateral flow strip and multiplex polymerase chain reaction coupled method for quantitative identification of beef, pork and chicken

Abstract

Meat adulteration is one of the worldwide challenges for food quality control. Now, we developed a multiplex PCR and nucleic acid lateral flow strip (mPCR-NALFS) integrated method that employs triplex-color latex microspheres (LMs) as visual labels for simultaneous identification of beef, pork and chicken DNA. First, three primer pairs for beef, pork and chicken DNA are tailed with specific oligonucleotide sequences (anti-recognition/anti-detection/anti-control probes) as swing tags. Then, double-stranded amplicons obtained after mPCR amplification were flanked by tags. Finally, these amplicons can accurately hybridize with the pre-immobilized recognition probes on the conjugate pad, detection probes on the test lines and control probes on the control line, respectively. The LMs and recognition probe modified LMs were characterized by scanning electron microscopy, ultraviolet-visible spectroscopy and Fourier Transform Infrared spectroscopy. This strip can quantitatively detect beef, pork and chicken DNA within 10 min without compromising on accuracy compared to the electrophoresis. Specificity analysis revealed no cross-reactivity with meat derived from beef, pork, chicken, duck, mutton, rabbit, goose, ostrich, camel and horse meat. As low as 0.1 % (wt%) of beef, pork and chicken in the simulated adulterated meats were co-detected. This assay was successfully employed for adulteration identification in 28 commercial beef and lamb products.