Molecular Pathogens, 2025, Vol.16, No.4, 147-158 http://microbescipublisher.com/index.php/mp 152 Bst enzyme), it can produce a large number of nucleic acid products within 30~60 minutes at constant temperature (usually 60 ℃~65 °C). Its amplification product is a series of DNA with circular structures of different lengths, with extremely high product volume. Since there is no need for a thermal cycler, the reaction can be completed with only a constant temperature water bath or heating block, which is very suitable for on-site inspection at the base level. For potato RNA viruses, the LAMP reaction usually combines a reverse transcription step (RT-LAMP): that is, in the same reaction system, the RNA is first converted to cDNA by reverse transcriptase, and then LAMP amplification is performed by Bst polymerase (Halabi et al., 2021). The design of LAMP primers is relatively complex, and it is necessary to ensure that the primers are well coordinated and avoid secondary structural interference. To improve specificity, loop primers can be added to shorten the amplification time and reduce false positives. LAMP technology has been used in potato virus detection. Raigond et al. (2019) established an RT-LAMP method for potato leaf roll virus (PLRV), optimized 6 primers and reaction conditions, and PLRV was detected by amplifying at a constant temperature of 63 ℃ for 30 minutes, and the overall sensitivity was more than 10 times higher than that of conventional RT-PCR. The advantage of LAMP is that it is fast, easy and does not require expensive instruments, but there is also a risk of false positives from nonspecific amplification (Raigond et al., 2019). 5.2 The advantages of rapid detection of recombinase polymerase amplification (RPA) Recombinase polymerase amplification (RPA) is another isothermal amplification technology developed in recent years. It is characterized by efficient amplification of DNA at low and constant temperatures of 37 °C~39 °C. The amplification time usually takes only 20~30 minutes (Wang et al., 2020). The RPA system uses recombinase to attach primers to the template double-strand, and the single-strand binding protein maintains the template melting state, and then expands the primers by DNA polymerase with strand displacement activity, thereby achieving exponential amplification. For RNA viruses, a step of reverse transcription can be added to form cDNA, and then RPA (RT-RPA). RPA requires extremely low equipment, only body temperature or a simple constant temperature device is required, and it is a truly fast on-site detection method. Compared with LAMP that requires 4~6 primers, RPA only needs one pair of primers to amplify. The design is relatively simple and the amplified product fragment is short (generally 100~200 bp), making it very suitable for use under resource scarcity. RPA's sensitivity is comparable to PCR. It has been reported that one-step RT-RPA detection of potato Y virus (PVY) can obtain results within 30 minutes at sensitive levels comparable to conventional RT-PCR (Cassedy et al., 2021). Recombinase-mediated amplification specificity is also high, but primers need to be optimized to avoid nonspecific amplification. For detection of RPA products, lateral flow strips (LFDs) or fluorescent probes can be used. 5.3 Operationality analysis of technology in low-resource environments For field monitoring or grass-roots seed potato testing stations in remote areas, the lack of complex instruments and insufficient professional staff are often obstacles. Because of its extremely low equipment requirements, isothermal amplification technology has outstanding advantages in low resource environments. Both loop-mediated amplification (LAMP) and recombinase amplification (RPA) can be done under constant temperature conditions, without the need for a PCR thermal cycler, can be operated with a constant temperature water bath or a pocket heating block, and even body temperature or on-site heating boxes can provide the required temperature. These technologies are easy to operate and have low requirements for personnel training: for example, LAMP can be used to judge the results of the solution color or turbidity change, and RPA can be read directly with the test strip directly with the naked eye, which is very intuitive (Kumar et al., 2023)). In contrast, qPCR and other people require expensive equipment, power supply and skilled technicians, and are not easy to implement in the field. Therefore, LAMP and RPA show excellent adaptability in non-laboratory environments, such as field seed potato sampling at the original breeding base, quarantine site at the origin, and entry phytosanitary quarantine. There have been cases that agricultural technicians in some developing countries can use handheld constant temperature equipment and LAMP reagents to determine whether seed potatoes are poisonous within 5~10 minutes in the field, greatly improving monitoring efficiency.
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