Microbe Detection via FISH Analysis with Magnetic Concentration

The patent applies preliminary action by performing probe penetration enhancement treatment before hybridization. The method involves treating cells with a penetration enhancement solution containing detergents and enzymes to pre-open cell membranes and walls, allowing probes to enter cells more efficiently. This preliminary preparation significantly reduces the hybridization time while maintaining detection accuracy, directly resolving the contradiction between long analysis time and detection accuracy in conventional FISH methods.

The patent applies universality by developing a universal penetration enhancement solution that works across different cell types. The solution contains a combination of detergents (Triton X-100, SDS), enzymes (lysozyme, proteinase K), and chaotropic agents (guanidine HCl) that collectively address the diverse structural barriers of different cell types. This universal treatment protocol enables consistent probe entry efficiency and high analysis efficiency across Gram-positive bacteria, Gram-negative bacteria, and other microorganisms, eliminating the variability in analysis efficiency observed in conventional methods.

The patent applies the taking out principle by extracting the detection function from complex molecular biology instruments to simple fluorescence microscopy. Instead of requiring PCR amplification and sequencing equipment, the method directly detects microbial DNA or RNA in situ using fluorescently labeled probes that hybridize to target sequences. This extraction of the essential detection capability from the complex PCR workflow enables accurate microbial identification using only a fluorescence microscope, eliminating the need for expensive precision instruments and specialized facilities.

The patent applies periodic action by implementing a systematic multi-probe hybridization protocol where different fluorescently labeled probes are introduced in sequence. Each probe targets specific microbial groups (e.g., Gram-positive bacteria, Gram-negative bacteria, yeast) and is hybridized under optimized conditions followed by washing and imaging. This periodic, structured approach to multi-probe application maintains high analysis accuracy for each detection while enabling comprehensive identification of multiple microorganism types, resolving the contradiction between multi-detection capability and analysis accuracy.