Integration of in-cassette lysis, purification, and lateral flow strips-based sensor for rapid and on-site detection of yak milk adulteration

Amplification-based nucleic acid testing has become a widely used method for food authenticity analysis. However, due to the need for specialized instrumentation, technical expertize, and trained operators, routine nucleic acid analysis can only be performed in a laboratory setting. For perishable products like yak milk, rapid and accurate detection of dairy components on-site is crucial for the automated management of the production process and improving the milk's quality at dairies. As a feasibility study, we developed an assay strategy for the simultaneous qualitative detection of yak and cow derived components in yak milk. We presented a handheld integrated cassette that combined rapid extraction and purification of dairy nucleic acids, loop-mediated isothermal amplification (LAMP), and visual detection of lateral flow strips (LFS). To enable visual detection of LAMP amplification products on LFS, we utilized a unique primer modification method that labeled the amplification terminator and single oligonucleotide sequence at the 5′ end of the loop primer. This modification avoids the use of antibodies and conventional fluorescein/biotin. The cassette allowed for DNA transfer, fluid drive, and liquid mixing by magnetic force and gravity alone, without the need for complex pumps and valves. The disposable cassette is easy to use, costs less than $1 to produce, has low instrument dependency, and is well suited to on-site detection. The platform can detect 0.1% of cow milk adulteration in yak milk within 50 min. This fully integrated "sample in-result out" detection procedure can also be extended to other detection areas.