Abstract:
Mastitis is a prevalent disease affecting dairy cows, leading to significant economic losses in the dairy industry.
Conventional diagnostic methods such as microbiology and PCR are expensive and time-consuming, emphasizing
the need for alternative diagnostic approaches. The field of novel diagnostics is expanding rapidly due to the
application of a modern molecular detection methods based on the CRISPR/Cas system. This system functions by
targeting specific genetic sequences of the pathogen, including Escherichia coli, and detects the presence of the
pathogen by employing a CRISPR RNA that complements the pathogen’s genetic sequence and a Cas12a enzyme
that cleaves the particular DNA sequence. In this paper, we present a novel pathogen detection technology that
combines the loop-mediated isothermal amplification (LAMP) reaction and Cas12a collateral activity. We have
successfully developed a rapid and precise method for identifying E.coli genomic DNA using the LAMP-Cas12a
technology, which exhibits high analytical sensitivity within an hour, detecting as low as 1.3 × 101 copies of
target DNA. This technology also has the ability to differentiate E. coli from other prevalent mastitis pathogens
such as Staphylococcus aureus and Streptococcus agalactiae. Furthermore, we employed a novel MbCas12a
nuclease, which demonstrated excellent diagnostic performance in our study when identifying E.coli isolates
isolated from bovine mastitis milk samples. The development of such new methods has the potential to expand
agricultural tools for use in point-of-care (POC) diagnostics.
