Molecular Pathogens, 2025, Vol.16, No.4, 147-158 http://microbescipublisher.com/index.php/mp 155 crRNA and cleave adjacent reporter RNA molecules, which can also be used for RNA virus detection. The advantages of CRISPR detection lies in its extremely high specificity and sensitivity: Cas enzymes have strict requirements on the target sequence, and single-base mismatch will significantly reduce activity; at the same time, the signals generated by their enzyme cleavage can accumulate and amplify in a short period of time (Zhan et al., 2023). For on-site detection of potato virus, researchers have combined CRISPR with RPA isothermal amplification to develop a series of rapid detection methods. 7.2 Application prospects of digital pcr in absolute quantitative detection of virus Digital PCR (dPCR) is a third-generation PCR quantitative technology that randomly allocates sample templates into thousands of independent microreactions for PCR amplification, and then calculates the absolute number of initial templates based on the proportion of positive reactions. Unlike traditional qPCR dependency standard curves, digital PCR does not require reference standards to achieve absolute quantification of viruses, thus having higher quantitative accuracy and anti-interference ability. Digital PCR shows great potential in potato virus detection. For some latent infected viruses with low titers, digital PCR can detect extremely low levels of nucleic acid copy number to achieve early warning. A study compared qPCR with titer digital PCR (ddPCR) detection and found that the detection sensitivity of ddPCR to pathogens is about 10 times higher than that of qPCR. The minimum detected concentration of qPCR is 2.4 fg/μL, while ddPCR can reach 0.24 fg/μL. In addition, digital PCR calculates copy numbers through statistical principles, avoiding the impact of PCR inhibitors on amplification efficiency, and can obtain reliable results even in complex plant extracts. This is particularly beneficial for samples rich in polyphenols and polysaccharides such as potato tubers. 7.3 Feasibility of combining nanobiosensing technology with instant detection (POCT) The fusion of nanotechnology and biosensing provides new ideas for real-time detection of plant viruses (POCT). Nanomaterial-based biosensors have high specific surface area and special optical/electrical properties and can be used to build sensitive and fast field detection devices. In potato virus detection, common nanobiosensors include gold nanoparticle immunochromatography test strips, nanometal enhanced electrochemical biosensors, etc. For example, preparing side-flow immunochromatography test strips with colloidal gold-labeled antibodies can detect viral antigens in potato tissue within minutes. The scientific research team in Chongqing has recently successfully developed a nano-microsphere immunochromatography rapid diagnosis test piece for four major viruses, potato Y, M, S, and A., which can detect 4 viruses simultaneously within 5 minutes, and have a sensitivity of 1,000 times dilution and good specificity. This technology combines traditional diabodyne sandwich with nanometer tracing and replaces enzymes with nanoparticles as signal reports, allowing the results to be visualized visually without the need for complex instruments. Similarly, there are studies that use gold nanoparticles and immunomagnetic beads to build an electrochemical sensing platform, achieving ultra-sensitive detection of PLRV, and the detection limit is one order of magnitude higher than ELISA. The advantages of nanobiosensitive sensors are fast detection and easy operation, and are very suitable for field applications such as fields and ports. In particular, immunochromatography test strips are cheap and easy to promote on a large scale. 8 Multiple Detection and Detection System Optimization 8.1 Multiviral parallel detection capability of nucleic acid probes and microarray chips Faced with the common multiviral complex infection situations in potato production, the detection of a single pathogen can no longer meet the actual needs. To this end, multiple parallel detection technology has received attention. Among them, a microarray chip based on nucleic acid probes is an effective tool for achieving simultaneous detection of multiple viruses. Microarray chips can detect whether there are complementary sequences of multiple viruses in the sample in a single hybridization experiment by immobilizing hundreds of oligonucleotide probes onto a solid carrier. Chinese scientific researchers have built potato virus and virus-like biochips, integrating probes such as PVY, PVX, PLRV, PVS, PVM, PVA and PSTVd into one chip to conduct high-throughput screening of field samples. The results show that the chip detection is consistent with the RT-PCR results and multiple infection viruses can be found at one time, improving the detection efficiency. Of course, chip detection requires a dedicated fluorescence scanner, which has high cost and professional requirements. Currently
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