Advances in the Standards & Applied Sciences

Advances in the Standards & Applied Sciences

Development and Validation of a PCR Based Method for Species Identification of Cow and Buffalo Milk

Document Type : Original Article

Authors
Marjan Heidarzadeh 1
Maryam Jalili 2
1 Food Toxicology Research Group, Food Technology and Agricultural Products Research Center, Standard Research Institute (SRI), P.O. Box 31745-139, Karaj, Iran
2 Food, Halal and Agricultural Products Research Group, Research Center of Food Technology and Agricultural Products, Standard Research Institute, Karaj, Iran
10.22034/asas.2026.581880.1104
Abstract
Background and Objective: Food fraud through the substitution of high value milk with lower cost cow milk represents a significant challenge in the dairy supply chain. Reliable analytical methods capable of accurately determining the species origin of milk are therefore essential for ensuring product authenticity and consumer protection. The aim of this study was to develop and validate a PCR based method for the species identification of cow and buffalo milk.
Materials and Methods: Raw milk samples from cow and buffalo were collected from private farms. Genomic DNA was successfully extracted using the CTAB method in combination with a commercial extraction kit. Species specific primers were designed using bioinformatic analyses, and PCR amplification conditions were optimized to ensure efficient amplification of the target fragments. The developed method was validated by evaluating key performance parameters including specificity, sensitivity, limit of detection, repeatability, and robustness under varying experimental conditions.
Results: The results demonstrated that the designed primers exhibited high species specificity for cow and buffalo DNA. Cross reactivity tests confirmed the absence of amplification with non target DNA, indicating strong discriminatory power of the assay. The method also showed satisfactory sensitivity, repeatability, and robustness across different reaction conditions.
Conclusion: Overall, the validated PCR based method provides a rapid, sensitive, and reliable approach for the identification of cow and buffalo milk species origin. This method can serve as a practical tool for authenticity verification and quality control within the dairy industry.
Keywords
PCR
Species identification
Cow milk
Buffalo milk
Dairy authentication
Subjects
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Advances in the Standards & Applied Sciences
Volume 4, Issue 1
Autumn 2025
Pages 42-52