Molecular Pathogens, 2025, Vol.16, No.4, 147-158 http://microbescipublisher.com/index.php/mp 154 likely to be ignored. This case highlights the power of HTS in unknown or unexpected virus detection. Through HTS, all viruses present in the sample can be captured simultaneously without bias, greatly improving the chance of detecting unexpected pathogens. Figure 2 Transmission electron micrographs showing the particles in the saps of the infected leaves by negative preparation (Adopted from Dong et al., 2024) 7 Emerging Molecular Detection Technologies 7.1 Viral nucleic acid targeted detection mechanism of CRISPR/Cas system The CRISPR/Cas gene editing system that has emerged in recent years has been cleverly applied to the detection of pathogenic nucleic acids. The basic principle is to use Cas nuclease to specifically identify and cleave target sequences, thereby amplifying the signal and achieving high sensitivity detection. For potato RNA viruses, the most widely used are the CRISPR/Cas12 and Cas13 systems. Cas12a is a nuclease that targets double-stranded DNA. When complexed with a specific crRNA, it can be activated when the target DNA sequence is recognized and performs indiscriminate cleavage of any surrounding single-stranded DNA substrate (called "co-cleavage" activity) (Marqués et al., 2022). Accordingly, reporter probes (short ssDNA oligonucleotides) with fluorescence/quenching marks can be added to the reaction. The fluorescence of the probe is quenched in the initial state. Once Cas12a recognizes and cleaves the target virus DNA, the probe is cut off and releases the fluorescence signal to achieve real-time reports of the target existence. For RNA viruses, such as PVY, PVX, etc., the RNA is first reverse-transcribed and amplified into DNA by isothermal amplification (RPA or LAMP), and then Cas12a detects the amplification product. Another Cas13 protein can directly recognize RNA targets with
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