Molecular Pathogens, 2025, Vol.16, No.4, 147-158 http://microbescipublisher.com/index.php/mp 151 specificity and amplification efficiency are required: the primer length is generally around 20 bp to avoid secondary structure and mismatch; the product length should be 80~150 bp to ensure that the amplification efficiency is close to 100%. If the probe is TaqMan method, the fluorescent reporter group and quenching group are labeled. The probe sequence is located between the two primers and specifically binds to the target template. It is necessary to ensure high specificity and avoid complementarity with the primer during design. Potato virus qPCR primers/probes usually select conserved regions of viral coat proteins or replicase genes, so that viruses of different strains can be detected (Jiang et al., 2024). In the qPCR system, parameters such as annealing temperature and primer probe concentration need to be optimized to obtain good linearity and amplification efficiency (90%~110%) on the standard curve. 4.2 Multiple detection strategies and efficiency improvement methods Similar to traditional PCR, qRT-PCR can also achieve synchronous detection of multiple viruses through multiple designs. Multiple qPCR is usually labeled with different fluorescence reporter dyes or probes, allowing the amplification signal of each virus to be read on different fluorescence channels. A triple qPCR method for simultaneously quantitative detection of PVY, PLRV and PVX was established, and three fluorescent probes were labeled using FAM, HEX, and Texas Red, respectively. Different viruses correspond to different fluorescent colors, achieving simultaneous quantification of three viruses in a sample (Prinz et al., 2023). The key to the design of multiplex qPCR is to avoid mutual interference between primer probes: the length and Tm values of each target should be as close as possible to share a cyclic program; significant complementarity between primer probe sequences (Zhang et al., 2016). At the same time, the amplification efficiency of each channel can be balanced by debugging the concentration of each primer probe. Some commercial reagents have added "passivating agents" to alleviate competition in different amplification reactions. In addition to multiple qPCR, another strategy to improve throughput is to use nucleic acid microarray chip technology. This technology immobilizes hundreds of oligonucleotide probes on the chip, allowing hybridization to detect multiple viral nucleic acids in the sample at the same time, and is a powerful tool for high-throughput parallel detection. In potato virus detection, biochips have been used to screen multiple viruses and virus-like one-time, greatly improving the throughput of the test sample (Ravinder et al., 2017). However, chip technology usually requires supporting scanners and analysis software, which is costly for grassroots laboratories. Multiple qPCR can be implemented under the conditions of ordinary real-time PCR instruments, so it is more practical when detecting several target viruses. 4.3 Comparative analysis with traditional RT-PCR Compared with traditional endpoint PCR, real-time quantitative PCR has the advantages of higher sensitivity, accurate quantitative and high automation. In terms of sensitivity, viruses with lower copy number can usually be detected due to qPCR using fluorescent probes and real-time cumulative monitoring. For potato viruses, qRT-PCR also showed higher sensitivity, and it has been reported that it can detect less than 10 copies of PVY nucleic acid in a single tube reaction (Zhou et al., 2019). In terms of specificity, qPCR introducing the TaqMan probe requires double matching of primers and probes, which has better specificity; and the use of probes avoids false positives caused by primer dimers. Compared with traditional PCR, the results of qPCR do not require electrophoresis, and the instrument can automatically generate Ct values and amplification curves, reducing artificial errors. However, qPCR instruments and fluorescent reagents are costly and are not suitable for resource-constrained grassroots. In current practical applications, the two types of PCR have their own strengths: in cases where quantification of viruses are required such as seed potato breeding, qRT-PCR plays a huge role; and in ordinary quarantine testing, if it is only necessary to determine whether it is poisonous, traditional RT-PCR combined with electrophoresis is still a cost-effective means. 5 Isothermal Amplification Technology 5.1 Reaction mechanism and optimization conditions of loop-mediated isothermal amplification (LAMP) Loop-mediated isothermal amplification (LAMP) is a technology that amplifies nucleic acids in constant temperature conditions. Experts report for the first time that LAMP uses a set of 4 to 6 primers to identify 6 specific regions of the target sequence. Relying on DNA polymerases with strand displacement activity (such as
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