Xiangyi Pang, Nan Wang, Juan Zhang*, Wenyi Tian, Hongtao Lei, Huifu Yuan, Ailiang Chen, Yanger Liu*

Microchemical Journal；2026

Abstract:

Buffalo milk is a culturally significant dairy product in many regions, valued for its superior nutritional profile and traditional role in local diets and specialty food production. However, its high economic value and limited availability render it vulnerable to adulteration with lower-cost cow milk, which undermines food authenticity, consumer trust, and cultural heritage. Existing molecular methods like real-time PCR, while sensitive, are predominantly qualitative, labor-intensive, and susceptible to aerosol contamination. Critically, these methods cannot distinguish incidental trace-level contamination from intentional fraudulent adulteration at commercially relevant concentrations, nor can they provide a direct quantitative measure of adulteration. To address these limitations, we developed a rapid, quantitative, and amplification-free detection platform that directly correlates signal intensity with the degree of adulteration. Our approach utilizes the strand displacement activity of Bst DNA polymerase to enable target-probe hybridization without target amplification, in conjunction with a colloidal gold lateral flow strip (LFS) for visual readout and portable quantification. The colorimetric signal exhibits a strong linear correlation with the fraction of buffalo milk in adulterated samples, with a detection limit of 10% (v/v). The entire assay, from sample preparation to result readout, can be completed within 20 minutes without the requirement for thermocycling or specialized instrumentation. The present method offers a simple, portable solution for the on-site authentication of buffalo milk, enabling effective quantification of adulteration to support food integrity, regulatory compliance, and the preservation of culturally significant foods. Furthermore, this platform establishes a versatile framework for quantitative, species-specific nucleic acid analysis in complex food matrices.