Detection of giblets in chicken meat products using microRNA markers and droplet digital PCR assay

Abstract

Use of edible by-products like chicken giblets in processed meat products without proper ingredient declaration amounts to food adulteration. DNA based methods are not able to detect this type of intra species adulteration. Hence, organ specific microRNAs are employed in this study to develop an assay using digital droplet PCR (ddPCR). Eight miRNA target sequences were tested using Locked Nucleic Acid (LNA) miRNA primer (Exiqon, Qiagen, USA) in a QX200 Automated digital droplet PCR (Bio-Rad, USA). Annealing temperature and primer input volume were optimized using 20 μL ddPCR reaction. Specificity of miRNA primers were confirmed through melt curve analysis as well as for impact of gDNA contamination using SYBR Green based qPCR. In sensitivity evaluation, LODabs and LODrel were reported to be 2.5 copies/μL and 0.94 copies/μL. Separate cut-off values were established for each miRNA targets in raw as well as cooked samples. Validated assay detected gizzard using gga-mir-148a -3p, gga-mir-126 -5p and gga-mir-490 -5p, heart using gga-mir-218 -5p and gga-mir-499 -3p and liver using gga-mir-122 -5p in raw as well as cooked samples at 25 g/100 g substitution level. The miRNA-ddPCR methodology established in this study can be a valuable tool for accurate detection of tissue origin of meat products