Quantification of pork, chicken and beef by using a novel reference molecule.

Area of Science:

• Food Science
• Molecular Biology
• Genetics

Background:

• Accurate identification of meat species is crucial for food authenticity and regulatory compliance.
• Existing methods for meat species identification can be complex or lack universal applicability.
• The cytochrome b (cyt b) gene is a common target for species identification due to its presence in mitochondrial DNA.

Purpose of the Study:

• To develop a novel reference standard for real-time quantitative PCR (qPCR).
• To establish a universal method for quantifying DNA from beef, pork, chicken, mutton, and horseflesh in minced meat.
• To create a tool for verifying the accuracy of food product labels.

Main Methods:

• Construction of a standard plasmid containing a target domain of the cytochrome b (cyt b) gene and an artificial DNA sequence.
• Design of specific primers (CO-F, CO-R) and a probe (CO-P) to detect the artificial sequence via qPCR.
• Quantification analysis using standard curves with DNA concentrations ranging from 10³ to 10⁷ copies per reaction.

Main Results:

• The constructed plasmid served as an effective reference molecule in qPCR assays.
• Standard curves for quantification analysis showed high linearity, with R² values between 0.998 and 0.999 for the five tested species.
• The method demonstrated high precision and reliability for measuring cyt b DNA copy numbers.

Conclusions:

• The developed standard plasmid provides a universal and accurate method for meat species identification in minced meat.
• This qPCR-based approach is valuable for verifying food product labels and ensuring consumer protection.
• The novel reference molecule facilitates reliable quantification of mitochondrial DNA for species authentication.