Traditional PCR methods for detecting species adulteration in buffalo milk are both highly sensitive and non-quantitative, and therefore cannot distinguish incidental trace-level contamination from intentional fraudulent adulteration at commercially relevant concentrations.
To address these limitations, researchers in this study (USD $24.95 purchase required) developed a point-of-use quantitative, and amplification-free detection platform that directly correlates signal intensity with the degree of adulteration. Their 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. Two single-stranded DNA probes were designed, a gold nanoparticle-conjugated reporter probe and a biotin-labeled capture probe, to specifically hybridize with the buffalo mtDNA D-loop region. Bst DNA polymerase facilitates this process by unwinding the double-stranded mtDNA via strand displacement. Crucially, by omitting dNTPs from the reaction, all amplification is prevented. They report that this design eliminates the risk of aerosol contamination from amplification. Following hybridization, the mixture is applied to a lateral flow strip. The complexes migrate by capillary action, and biotin-streptavidin binding at the test line captures them to form a visible red band.
They report that, when using a portable reader, 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).
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