Two-color lateral flow nucleic acid assay combined with double-tailed recombinase polymerase amplification for simultaneous detection of chicken and duck adulteration in mutton.

We propose a visual strategy for simultaneous detection of chicken and duck ingredients in mutton by integration of a double tailed multiplex recombinase polymerase amplification (mRPA) assay with a multicolor nucleic acid hybridization lateral flow strip (mNAH-LFS) assay. Two colored gold nanoparticles (AuNPs), including gold nanospheres (red) and gold nanoflowers (blue), were synthesized and functionalized with the corresponding nucleic acid probe to target each analyte. The RPA product was analyzed by this two-colored mNAH-LFS assay which includes a red T 1 line for chicken adulteration and a blue T 2 line for duck adulteration. In this RPA reaction, special Spacer9 and tail nucleotide sequence-tagged primers (Tag+Spacer9+F-primer, Tag+Spacer9+R-primer) were used to obtain double-stranded amplicons with specific wobbled sequences that can directly bind with AuNPs-labeled probes (AuNSs-cT 1 hybridized with T 1 , AuNFs-cT 4 hybridized with T 4) and solid-phase probes (biotin-cT 2 hybridized with T 2 , biotin-cT 3 hybridized with T 3) on the mNAH-LFS producing a red or/and cyan color on the T lines. The detection limits of this mRPA-mNAH-LFS assay for chicken and duck were 0.01 % and 0.01 %, respectively. Finally, this mRPA-mNAH-LFS assay was successfully used to authenticate 59 commercial meat products. In conclusion, this mRPA-mNAH-LFS is a sensitive, accurate, and cost-effective approach for meat quality assurance and traceability. • A new double tailed mRPA with a mNAH-LFS was developed for mutton authentication. • Red AuNSs and blue AuNFs were synthesized as multicolor labels in the mNAH-LFS. • The specific RPA primers are uniquely designed with tail sequence and a spacer C9. • The sensitivity of this mRPA-mNAH-LFS assay for chicken and duck were 0.01 %. • This assay was successfully applied to the authentication of real mutton products. [ABSTRACT FROM AUTHOR]