Molecular Pathogens, 2025, Vol.16, No.4, 147-158 http://microbescipublisher.com/index.php/mp 156 more multiple detections are based on PCR improvements. The difficulty of multiple PCR is that dimers or competitive reactions may occur between primers, so it is necessary to strictly verify the compatibility and product differences of each primer. Multiple capabilities can be further improved by introducing improved methods such as fluorescent probes and multiple ligation amplification. Some studies have also explored the binding of microarrays and PCR, such as hybridizing the amplified products to the probe chip after PCR amplification, taking into account both sensitivity and multiple levels. 8.2 The joint strategy of molecular detection and immunologic methods Although molecular diagnostic technology is highly sensitive, in some cases, combining traditional immunology methods can learn from each other's strengths and make up for each other's weaknesses, further improving detection efficiency and accuracy. One typical strategy is immune capture-PCR (IC-PCR), which means that virus particles are first captured from the sample using antibodies, and then RT-PCR amplification is performed directly on the capture. This allows detection to be achieved without purifying nucleic acids, greatly simplifying the processing process. It is reported that IC-RT-PCR is about 250 times more sensitive than direct RT-PCR when used to detect PLRV, because the antibody captures and concentrates low levels of viruses and removes PCR inhibitory substances. At present, IC-PCR has been used for the detection of potato leaf roll virus, Y virus, etc., and has achieved good results in practice. Another joint strategy is printing PCR (Print-capture PCR) and direct blotting PCR, where plant juice is directly blotted on the membrane, and lysate is added to elute it as a PCR template. This method avoids the cumbersome nucleic acid extraction steps and can quickly process large numbers of samples in the field. For example, it is reported that direct blotting PCR is used to simultaneously detect PVY and PLRV, which enables rapid screening of field samples. 8.3 Case analysis: establishment of a multiviral mixed infection detection system for seed potatoes exported inEurope In order to ensure the health of potato seed potatoes and meet the quarantine requirements of imported countries, some major European potato producing countries have established a multiviral joint testing system. Taking the Netherlands' seed potato testing system as an example, its process includes dual checks on field quarantine and laboratory testing. In the laboratory stage, biochip technology is used to perform high-throughput initial screening of seed potato samples. A chip can simultaneously detect more than 10 common viruses (including viruses) such as potato Y virus, X virus, leaf roll virus, A virus, S virus, M virus, spindle tuber virus. For the chip-positive samples, specific qRT-PCR/RT-PCR are used for confirmation respectively to quantify the virus content and determine whether it exceeds the certification standards. Under this system, tens of thousands of seed potato samples are tested in the Netherlands every year, and more than 90% of the samples are virus-free or within the safety threshold. A few batches detected with poison are eliminated or processed in a timely manner. The establishment of this multiviral detection system relies on the optimized combination of various detection technologies: the chip provides high-throughput screening capabilities, while PCR ensures accurate quantities of results. On the other hand, some countries also incorporate serological methods. For example, in France, seed potato certification uses ELISA screening for PVY and other samples, and then uses RT-PCR verification for suspected samples (Medina Cárdenas et al., 2015). Others use instructed plant bioassays as auxiliary to prevent missed examinations. Recently, the UK and others have tried to incorporate digital PCR into seed potato virus detection to accurately quantify latent toxic levels before exporting, ensuring that the zero-tolerance requirements of importers are met. These cases show that a complete potato seed potato virus detection system often does not rely on a single method, but combines the advantages of multiple technologies. Through the multi-level process of "screening + confirmation + quantification", it realizes efficient detection and management of multi-virus mixed infections. Such a system provides technical support for ensuring the safety of international potato seed potato trade, and is worthy of China's reference and improvement of our potato seed potato quarantine testing standards. Acknowledgements We would like to thank all colleagues involved in this study for their collaboration and contributions with Cuixi Biotechnology Institute, and we would like to thank the peer review for their anonymous revisions.
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