Rapid identification of sheep species (Ovis aries) in meat samples using isothermal amplification method

Background and objectives
With the rising consumption of meat products, incidents of meat adulteration have become more prevalent and universal. One common form of adulteration is the substitution of lower-value red meats, such as goat or beef, for the more valuable species such as sheep meat. Consequently, there is a growing demand for rapid and accurate methods to identify meat species. While protein- and DNA-based methods, such as ELISA and PCR, are valuable tools for species identification, the short production and consumption chain of meat products necessitates rapid and accurate, point-of-care tests that can be conducted outside of well-equipped laboratories. In this study, we employed loop-mediated isothermal amplification (LAMP) for the specific identification of sheep meat. A set of four primers was designed targeting the mitochondrial cytochrome b (Cyt b) gene. The specificity and detection limit of these primers were evaluated and compared to the standard PCR method which is more common in food control laboratories.
Material and methods
In this study, a set of four LAMP primers targeting the Cyt b gene was designed using PrimerExplorer online software for the specific identification of sheep meat samples. Genomic DNA was extracted from raw tissue samples using the NaOH method (Alkaline lysis) and used in subsequent analyses. To assess primer specificity, LAMP assays were performed on a panel of target and non-target samples. Other than that, to determine the limit of detection of the designed primers and to compare the LAMP and PCR methods with each other, both assays were conducted on a serial dilution of sheep DNA. Finally, to optimize the diagnostic kit for field conditions, results were visualized using SYTO 24 dye.
Results
The results demonstrated that the designed LAMP primers were highly specific in identifying sheep meat from other non-target species. Moreover, the developed assay exhibited exceptional sensitivity, detecting as little as 17.1 pg/µL of sheep extracted DNA within 30 minutes of sampling, which represents a ten-fold increase in sensitivity compared to the conventional PCR method. Additionally, the use of SYTO 24 dye confirmed the feasibility of employing this method in field conditions, as the lower PCR product yield compared to LAMP renders SYTO 24 visualizations challenging in PCR assays.
Conclusion
Due to its simplicity and easiness of equipment, high sensitivity, specific function between close species and short amount of reaction time, this method holds significant potential for detecting meat adulteration, particularly in remote locations with limited laboratory access and educated personnel.