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Molecular Pathogens 2025, Vol.16 http://microbescipublisher.com/index.php/mp © 2025 MicroSci Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher. Publisher MicroSci Publisher Edited by Editorial Team of Molecular Pathogens Email: edit@mp.microbescipublisher.com Website: http://microbescipublisher.com/index.php/mp Address: 11388 Stevenston Hwy, PO Box 96016, Richmond, V7A 5J5, British Columbia Canada Molecular Pathogens (ISSN 1925-1998) is an open access, peer reviewed journal published online by MicroSciPublisher. The journal is committed to publishing and disseminating all the latest and outstanding research articles, letters and reviews in all areas of molecular pathogens. The range of topics including isolation and identification of emerging pathogens viruses, pathogen-host interactions, genetics and evolution, genomics and gene regulation, proteomics and signal transduction, glycomics and signal recognition, virulence factors and vaccine design and other topical advisory subjects. All the articles published in Molecular Pathogens are Open Access, and are distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MicroSci Publisher uses CrossCheck service to identify academic plagiarism through the world’s leading plagiarism prevention tool, iParadigms, and to protect the original authors’ copyrights. MicroSci Publisher is an international Open Access publisher specializing in microbiology, bacteriology, mycology, molecular and cellular biology and virology registered at the publishing platform that is operated by Sophia Publishing Group (SPG), founded in British Columbia of Canada.
Molecular Pathogens (online), 2025, Vol. 16, No. 4 ISSN 1925-1998 http://microbescipublisher.com/index.php/mp © 2025 MicroSci Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher. Latest Content A Review of Molecular Diagnostic Techniques for Potato Virus Detection Jiamin Wang, Jiong Fu Molecular Pathogens, 2025, Vol. 16, No. 4, 147-158 Molecular Basis of Wheat Resistance to Fungal Diseases Wei Wang, Zhengqi Ma Molecular Pathogens, 2025, Vol. 16, No. 4, 159-170 Case Study: Successful Breeding of Rice Varieties Resistant to Bacterial Blight Dapeng Zhang, Zhonggang Li Molecular Pathogens, 2025, Vol. 16, No. 4, 171-181 Case Study of Downy Mildew Resistancein Grapevine: Molecular and Genetic Insights Ming Li, Chunyang Zhan Molecular Pathogens, 2025, Vol. 16, No. 4, 182-192 Hormonal and Genetic Control of Cucumber Responses to Pathogens Hongwei Liu, Shiying Yu Molecular Pathogens, 2025, Vol. 16, No. 4, 193-206
Molecular Pathogens, 2025, Vol.16, No.4, 147-158 http://microbescipublisher.com/index.php/mp 147 Review Article Open Access A Review of Molecular Diagnostic Techniques for Potato Virus Detection Jiamin Wang, Jiong Fu Hainan Provincial Key Laboratory of Crop Molecular Breeding, Sanya, 572025, Hainan, China Corresponding author: jiong.fu@hitar.org Molecular Pathogens, 2025, Vol.16, No.4 doi: 10.5376/mp.2025.16.0016 Received: 18 May, 2025 Accepted: 20 Jun., 2025 Published: 01 Jul., 2025 Copyright © 2025 Wang and Fu, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Wang J.M., and Fu J., 2025, A review of molecular diagnostic techniques for potato virus detection, Molecular Pathogens, 16(4): 147-158 (doi: 10.5376/mp.2025.16.0016) Abstract Potatoes are an important food and cash crop in the world, but viral diseases seriously affect their yield and quality. According to statistics, more than 40 viruses have been found in potatoes, which can lead to a yield loss of up to 50% in early or severe infection. Common potato viruses include Y virus (PVY), X virus (PVX), leaf roll virus (PLRV), A virus (PVA), M virus (PVM), S virus (PVS), and potato spindle tuber virus (PSTVd), etc. They occur widely in the main potato-producing areas of China. This study analyzed the molecular biological basis of potato viruses, as well as the principles and application progress of molecular diagnostic technologies such as polymerase chain reaction (PCR), real-time fluorescence quantitative PCR (qRT-PCR), isothermal amplification, high-throughput sequencing, emerging CRISPR/Cas and digital PCR, nanobiosensing, etc., and compared the sensitivity, specificity and applicable scenarios of each technology. The article also discusses the direction of multiple detection solutions and detection system optimization, and analyzes new achievements in potato virus detection in China and internationally, and looks forward to the future development trend of potato virus molecular diagnostic technology. Keywords Potato virus; Molecular diagnosis; Isothermal amplification; High-throughput sequencing; Rapid on-site detection 1 Introduction Potato (Solanum tuberosum) is the fourth largest food crop in the world and plays an important role in ensuring food security. However, various viral diseases are widely distributed around the world and have become one of the main diseases that limit potato production. Viral infection will cause symptoms such as flower and leaves, shrinkage, and rolling leaves of potato plants, and the yield and quality of tubers will decrease. It is estimated that yield loss can reach 30%~50% or even higher in early or severe viral infections (Wu et al., 2013). China is a major potato producer, and the production cuts caused by virus disease cannot be ignored. In the survey of multiple potato-producing areas in China, the average detection rate of PVY is the highest, exceeding 60% in some areas, followed by PVS, PVX, etc. Because the virus can be transmitted through seed potatoes for a long time and leads to "degeneration", viral disease has become one of the important factors that restrict the continuous increase in potato crops. There are many types of potato viruses, and more than 60 types of potato viruses have been reported. PVX belongs to the genus Potexvirus. Infections alone are usually mild, but infection in combination with other viruses can lead to severe leaf rolls and necrosis (Rashid et al., 2021). PLRV belongs to the genus Polerovirus, is a cystos-limiting virus transmitted through aphids. It only invades the phloem, often causing potato rolls and premature aging. PVA, PVM, and PVS belong to the Potyvirus, Carlavirus and Carlavirus respectively, and are also popular in many places around the world. Since potatoes are asexual reproductive crops, these viruses are easily spread and accumulated through seed potatoes, resulting in variety degeneration. Therefore, the rapid and accurate detection of these major potato viruses is crucial to seed potato quality control and disease prevention and control. Traditional field symptom observation and indication plant biological identification has been used for the detection of potato viruses, but it often requires waiting for obvious symptoms to appear and take a long time, which is inefficient in large-scale seed potato testing. Although serological methods such as enzyme-linked immunosorbent assay (ELISA) were widely used in plant virus detection in the last century and had certain
Molecular Pathogens, 2025, Vol.16, No.4, 147-158 http://microbescipublisher.com/index.php/mp 148 sensitivity and automation potential, there is a problem of insufficient sensitivity for low concentrations of initial infection and shellless virus-like detection. With the development of molecular biology, nucleic acid-based molecular diagnostic technology has greatly improved the sensitivity and specificity of plant virus detection (Prinz et al., 2022). In addition, with the increase in international seed potato trade and transportation, quarantine departments of various countries are highly dependent on molecular testing methods to conduct virus testing of seed potatoes and tissue culture seedlings entering and leaving the country to prevent the spread of quarantine viruses. 2 The Molecular Biology Foundations of Potato virus 2.1 Viral genome structure and coding characteristics Most potato viruses are sense single-stranded RNA (+ssRNA) viruses, with different genome size and coding strategies. Taking potato Y virus (PVY) as an example, its genome is a single strand positive-strand RNA with a total length of about 9.7 kb and a long open reading frame. It translates to produce a polyprotein of about 350 kDa. It is then cut into 10 functional proteins by the virus-encoded protease into 10 functional proteins, including coat protein (CP), cofactor HC-Pro, nuclear sheath protein, etc. (Tam et al., 2012; Bartola et al., 2020). The genome of potato X virus (PVX) is about 6.4 kb, which is also +ssRNA, but the multi-open reading frame strategy is adopted to encode 5 proteins: the 5'-end ORF1 encodes RNA-dependent RNA polymerase (RdRp), the three ORF2-4 in the middle form a "tertiary gene cluster" that encodes three proteins related to movement (TGBp1-3), and the 3'-end ORF5 encodes the coat protein. The genome of potato leaf roll virus (PLRV) is smaller, about 5.9 kb, encodes a few proteins and needs to be reproduced by the host phloem cell environment. Potato M virus (PVM) and S virus (PVS) belong to the genus Carlavirus, with a linear +ssRNA, a length of about 8.5 kb, and contains 6 ORFs, presenting the typical genomic tissue structure of this genus virus. In addition, potato spindle tuber virus (PSTVd) is a circular single-stranded RNA with only about 360 nucleotides, which does not encode a protein but can interfere with host metabolism (Qian and Huang, 2025). 2.2 The relationship between viral mutation, recombination and pathogenicity Potato viruses have produced abundant genetic variations in long-term evolution, and the pathogenicity of different strains and variant types tends to vary significantly. Taking PVY as an example, it is traditionally divided into three types: ordinary strain O, tobacco ordinary strain N, and chlorosis strain C according to serology and gene sequence. However, in recent years, many studies have shown that PVY can derive new recombinant strain types through gene mutations or recombination between different strains. These recombinant lines tend to exhibit novel biological properties and pathogenicity (Samarskaya et al., 2024). Viral recombination and mutations increase the complexity of diagnosis and may also break through the resistance of host varieties. The study found that different PVY lines have amino acid mutations in viral-causing genes such as genome P1, HC-Pro, and NIa, which determine the pathogenicity of the virus and the host range of infectiousness. In addition to PVY, different lines of potato X virus (PVX) also have genetic mutations between mild and severe strains, and when infected with PVA or PVY in combination can lead to "curve leaf mosaic disease" or even plant death. In the fields, potato plants are often infected by multiple viruses. Some surveys have found that it is not uncommon for a single potato to be infected with 2~3 viruses at the same time, and even a very small number of plants can carry 4~5 viruses (Rashid et al., 2021). Multiple infections may lead to recombination and interaction between viruses, enhancing pathogenicity to plants. 2.3 Molecular mechanisms of virus interaction with host There is a complex molecular interaction between potato viruses and host plants. On the one hand, viruses need to replicate and move using the host's organelles and enzyme systems; on the other hand, plants evolve antiviral defense mechanisms, including resistance gene-mediated resistance responses and RNA silencing. Studies have shown that there are multiple antiviral genes (resistance R genes) in potatoes. These R genes usually encode NBS-LRR-like resistance proteins, which can recognize virus-specific pathogenic factors and trigger defense responses such as hypersensitivity reactions (HR), limiting the virus in the early stages of invasion (Yin et al., 2017; Chen et al., 2022). Potatoes also inhibit viruses through RNA silencing mechanisms: When viral RNA
Molecular Pathogens, 2025, Vol.16, No.4, 147-158 http://microbescipublisher.com/index.php/mp 149 enters plant cells, it can trigger the small interfering RNA (siRNA) pathway to degrade viral RNA. However, viruses have also evolved a countermeasure strategy, and many potato viruses encode RNA silencing inhibitory proteins (VSRs). For example, the HC-Pro protein of PVY and the P25 protein of PVX are both powerful RNA silencing inhibitors that can bind to the host's silencing signaling pathway factors, thereby hindering the antiviral silencing response (Figure 1). Recent studies have found that certain defense-related proteins of the plant themselves can interact with virus silencing inhibitors to enhance resistance. Studies have identified that a type I protease inhibitor (PI) in potatoes can bind to P25 protein of PVX, hindering the silencing and inhibiting function of P25, thereby improving resistance to PVX (Shen and Wang, 2025). This interaction mechanism suggests that plants can use their own proteins to directly interfere with the function of viral pathogenic factors. Figure 1 The viruses accumulate and symptoms develop in the RiStEXA1 and control plants (WT) inoculated with PVYO (a), PVX (b), and PVM (c). Relative virus accumulation was determined using qRT–PCR with total RNAs extracted from the non-inoculated upper leaves at 10 and 15 dpi. Symptoms on plants and systemic leaves were observed and photos were taken at ~25 dpi. Data are presented as means ± SD (n = 3) relative to WT plants, and EF1α was used as the normalizer. Three independent experiments were performed with similar results. Asterisks indicate statistically significant differences according to Student’s t test (**P < 0.01) (Adopted from Chen et al., 2022) 3 Application of PCR and RT-PCR Technology in Virus Detection 3.1 Technical principles and testing process Polymerase chain reaction (PCR) is a technology to amplify specific DNA sequences in vitro. Since most potato viruses are RNA viruses, it is usually necessary to reverse transcription RNA into cDNA by reverse transcription (RT) during detection, and then PCR amplification, that is, RT-PCR. The basic principle is to use a pair of oligonucleotide primers targeting conserved regions of the viral genome to perform multiple rounds of denaturation, annealing and extension cycles on the template cDNA under the action of a thermally stable DNA
Molecular Pathogens, 2025, Vol.16, No.4, 147-158 http://microbescipublisher.com/index.php/mp 150 polymerase (such as Taq enzyme), so that the target sequence multiplies exponentially. The PCR reaction is highly specific, and amplification bands are generated only when there is a viral nucleic acid matching the primer in the sample, thereby achieving qualitative detection of the virus. This method does not require high experimental conditions, and a conventional PCR instrument can complete the amplification process. The results of RT-PCR are usually observed by agarose gel electrophoresis to see the size and presence of the amplified band, and can be compared with the positive control to determine whether the virus exists. Due to its advantages of high sensitivity and strong specificity, RT-PCR is widely considered to be one of the "gold standard" methods for plant virus detection (Zhang et al., 2016). 3.2 Sensitivity and specificity analysis RT-PCR has extremely high sensitivity due to its high amplification efficiency and can detect extremely low concentrations of viral nucleic acids in the sample. Studies show that the detection sensitivity of RT-PCR on potato viruses can reach the pg order, which is hundreds to thousands of times higher than traditional ELISA. RT-PCR is highly specific and depends on the base complementary pairing of primers and templates. If the primers are designed properly, nonspecific amplification will be difficult to produce even if the gene sequences are similar between different viruses (Zhang et al., 2016). On the one hand, since the concentration of PCR amplification products is very high, it is easy to cause aerosol contamination, it should be strictly divided and a negative control should be set up in each batch of reactions to monitor the contamination. On the other hand, plant extracts may contain substances that inhibit PCR, such as polyphenols and polysaccharides, which need to be removed during RNA extraction and purification, or add bovine serum albumin (BSA) and other factors to relieve inhibition in the PCR system. In addition, internal standard controls (such as plant endogenous genes) can be added to the detection system to verify whether the PCR reaction is running successfully. 3.3 Interpretation of test results and quality control measures The detection results of RT-PCR are usually presented in the form of electrophoretic bands and need to be explained in conjunction with appropriate controls. Positive controls generally use positive templates known to contain target viruses, and their target bands show that the system responds normally; negative controls are reactions without templates (or healthy plant extracts), and no amplified bands should appear. The sample had a band of the same size as the positive control and the fluorescence intensity was sufficient, so it was judged to be positive for the virus; if there was no band, it was negative. In terms of quality control, in order to prevent false positives, PCR steps must strictly follow the principles of sterile operation and partitioning: extraction, preparation, amplification and electrophoresis should be carried out in different areas, and disposable filters should be used to avoid aerosol contamination. Once contamination is suspected, primers or enzyme preparations can be replaced and DNA removal treatments (such as UV irradiation, etc.) of the laboratory environment (Lee and Rho, 2015). For high polysaccharide/polyphenol samples, adsorbed impurities such as PVPP can be added during extraction, or additives can be added to PCR to improve amplification. Positive plasmids with known concentration gradients should also be included in the experiment to evaluate amplification efficiency and sensitivity. When detecting multiple viruses, primers need to avoid interference and competition between each other, and parallel detection of the multiple PCR system can be used (Rashid et al., 2021). 4 Real-Time Fluorescence Quantitative PCR (qRT-PCR) 4.1 Quantitative detection principle and primer/probe design principle Real-time fluorescence quantitative PCR (qPCR or qRT-PCR) is the real-time monitoring of product accumulation through fluorescence signals during PCR amplification, thereby achieving quantitative analysis of the number of initial templates (Zhou et al., 2019). Unlike traditional PCR, qPCR uses fluorescent dyes (such as SYBR Green) or specific fluorescence probes to indicate the production of amplification products, measuring fluorescence intensity per cycle and drawing an amplification curve. When the fluorescence signal reaches the preset threshold, the number of cycles (Ct value) is negatively correlated with the initial template amount, and the copy number of viruses in the sample can be calculated based on the standard curve. The principle of qRT-PCR for RNA viruses is one-step or two-step RT-PCR combined with fluorescence detection. In primer and probe design, higher
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
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.
Molecular Pathogens, 2025, Vol.16, No.4, 147-158 http://microbescipublisher.com/index.php/mp 153 6 High-throughput Sequencing (HTS) and Metagenomics 6.1 The advantages of HTS in the identification of unknown viruses and data processing flow High-throughput sequencing (HTS, also known as next-generation sequencing) is a technology that can determine millions of nucleic acid sequences in parallel. Applying it to nucleic acid analysis of plant samples can detect all viral sequences present without prior knowledge of the virus species. This makes HTS a powerful tool for discovering new and unknown viruses. By meta-transcriptionome sequencing of total RNA from potato samples, abnormal sequences can be found in the data, thereby identifying previously unreported viruses (Kenzhebekova et al., 2024). Unlike traditional PCR or serum methods that can only detect known viruses, HTS is able to capture any viral sequence in a sample "unbiased". In recent years, a variety of new potato viruses and virus-like viruses have been identified at home and abroad using HTS technology. For example, researchers discovered a new type of potato Y virus from papaya samples in Jordan through HTS, and whole genome sequencing confirmed it to be a new virus. In Yunnan, China, some people also use HTS to test new varieties of potatoes for viruses. It was found that they are latently infected with two emerging viruses, namely tomato spotted wilt virus (TSWV) and tomato sporadic mottled virus (TZSV), which have not been reported on potatoes in the past (Pacheco-Dorantes et al., 2025). These findings show that HTS can break through the limitations of traditional detection and expand viral spectrum analysis to unknown areas. The general process for HTS identification of viruses includes: sample total or small RNA extraction, library sequencing, bioinformatics analysis and sequence alignment annotation. Among them, biological information analysis is a key link. By splicing the massive reads data obtained from sequencing into a longer contig sequence, and then comparing it with known virus databases to identify the sequence of virus origin. 6.2 Bioinformatics analysis methods and database construction The amount of data generated by high-throughput sequencing is extremely large and requires in-depth exploration with the help of bioinformatics tools. For potato metagenome sequencing data, commonly used analysis processes include: data quality control, host sequence removal, virus sequence assembly and annotation, etc. Low-quality and linker sequences in sequencing reads are removed by quality control software such as Trimmomatic (Lambert et al., 2018). Then the clean reads are aligned with the potato reference genome, and the reads derived from plants are filtered out, so that the retained non-host reads may contain viral sequences. Next, use de novo assembly software (such as SPAdes, Megahit) to splice these reads into pieces. The resulting contigs sequence is then compared with the virus database. BLASTn or BLASTx can be used to compare it with the NCBI virus genome library to find similar sequences (Zhang et al., 2025). Generally speaking, if contig has high homology (e.g. >90%) to a known viral genome and covers most of its sequence, it can be judged that the virus exists in the sample. If there is a contig without obvious homolog, it may be a new viral sequence. Analysis of new viruses can further predict its open code reading frame and conservative motifs to determine the classification status. In order to improve the sensitivity of virus identification, some special software has been developed in recent years, such as VirusDetect, VirFinder, etc., to use k-mer features and machine learning to identify virus sequences from complex data. 6.3 Case analysis: using hts to discover latent viruses in new potato varieties in Yunnan, China Yunnan is located on the plateau with a diverse ecological environment and is a region with rich potato germplasm resources. When a breeding unit was promoting a new high-yield potato variety, it found that its growth performance in the field was poor but no obvious symptoms were seen. In order to check whether there is any latent virus infection, researchers conducted high-throughput sequencing analysis on asymptomatic plant samples of this variety. Through total RNA sequencing and bioinformatic analysis, two important foreign virus sequences were identified in the samples: one belongs to the genus Tospovirus (Tomato spotted virus (TSWV), and the other is Tomato zonate spot virus (TZSV). These two viruses have mainly infected Solanaceae crops in the past. They are not common in potatoes in Yunnan, but because of their transmission through thrips, potatoes may be transmitted in a mixed environment (Figure 2) (Yang et al., 2023; Dong et al., 2024). Further qPCR tests confirmed that nucleic acids with TSWV and TZSV were found in multiple samples of the new variety, but the plants did not show typical symptoms and were latent infections. Without HTS, these hidden mixed infections are
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
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
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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