Development of a Ladder-Shape Melting Temperature Isothermal Amplification Assay for Detection of Duck Adulteration in Beef

Abstract

Ladder-shape melting temperature isothermal amplification (LMTIA) is a newly developed technology, and the objective of this study was to establish its effectiveness for detection of duck adulteration in beef. LMTIA primers were designed with the prolactin receptor gene of Anas platyrhynchos as the target. The LMTIA reaction system was optimized, and its performance was compared with that of the loop-mediated isothermal amplification (LAMP) assay in terms of specificity, sensitivity, and limit of detection (LOD). Our results showed that the LMTIA assay was able to specifically detect 10 ng of genomic DNAs (gDNAs) of A. platyrhynchos, without detecting 10 ng of gDNAs of Bos taurus, Sus scrofa, Gallus gallus, Capra hircus, Felis catus, and Canis lupus familiaris. The sensitivity of the LMTIA assay was 1 ng of gDNAs of A. platyrhynchos; it was able to detect duck adulteration in beef with a 0.1% LOD. Although the LAMP assay could not clearly distinguish A. platyrhynchos from G. gallus, it had a sensitivity of 10 ng of gDNAs of A. platyrhynchos and a LOD of 1% duck adulteration in beef. This study may help facilitate the surveillance of commercial adulteration of beef with duck meat.