| Plant viruses are widely distributed plant pathogens and and difficult to control,which are known as plant"cancers".The annual agricultural losses caused by plant viruses account for 10%of the total food crop production worldwide,and their prevalence and outbreaks pose a serious threat to food production and food safety.Therefore,establishment of accurate,sensitive and low-cost plant virus detection methods is important to prevent the spread of infectious plant diseases and ensure the safety of food production.In recent years,with the continuous development of agricultural modernization and globalization,plant viruses show characteristics of new occurrence,re-occurrence and mix infection,which bring new challenges to plant quarantine departments and agricultural production units in terms of rapid plant virus detection capability.Nanopore sequencing technology developed by Oxford Nanopore Technologies（Oxford,UK）is a novel sequencing platform with fast speed,long read length,device portability and real-time data processing,which proposes a new application model for rapid on-site detection of plant viruses.In order to explore the application of this technology in rapid identification of plant viruses,this project carried out research related to the rapid detection of plant viruses based on Nanopore sequencing technology,and established a multi-targeted sequencing technology solution for plant quarantine viruses,which can achieve rapid detection of 15 quarantine viruses in a single sequencing;established a non-targeted sequencing technology solution for unknown viruses,and successfully identified a novel plant virus.In addition,based on the genomic information of this virus,this project developed a detection technique combined loop-mediated thermostable amplified nucleic acid and lateral flow test strips technologies,which provides an important technology for the early detection of this novel virus.The main results are as follows:1.Multi-target sequencing technology solution for quarantine virusesTaking advantage of the characteristics that all plant viruses can produce RNA,a multi-target detection method for quarantine viruses was developed by combining specific multiple reverse transcription technology with nanopore c DNA sequencing technology.By obtaining the genomic information of 32 plant quarantine viruses in China,a specific reverse transcription primers pool was designed according to their structural characteristics;the RNA samples of 15 quarantine viruses were obtianed by in vitro transcription to generate the plant virus mix infection model.The model was used to evaluate the sensitivity and specificity of the multi-target detection method of known viruses based on nanopore sequencing technology.The results showed that in the samples with virus nucleic acid content as low as 0.05%,all the virus information to be tested could be obtained by sequencing for 1 hour,and the average sequencing coverage was 227x.Maize chlorotic mottle virus（MCMV）,potato mop top virus（PMTV）,southern bean mosaic virus（SBMV）and potato virus A（PVA）viruses were selected as the research objects for the detection and verification of actual samples.The results showed that using the established multi-target sequencing technology.All virus information to be tested can be obtained within 20 minutes of sequencing,and the average sequencing coverage is 155x.2.Non-targeted sequencing protocol for unknown virusesTo explore the application of nanopore sequencing technology to unknown viruses,c DNA amplification and library sequencing were performed on total RNA from suspected virus-infected bean,pepper and Chinaberry tree samples after a poly（A）-tailing reaction.6G data were obtained after 10h.The sequencing data were compared with virus database by Blast program,and it was found that bean was mainly infected by Peanut mottle virus（PMo V）with 85.6%gene coverage and99.4%similarity to the reference sequence.Pepper was mainly infected by Cucumber mosaic virus（CMV）with 99.4%,99.8%and 99.1%coverage of the three genomes and 97.5%,99.4%and 99.5%similarity,respectively.A new species of plant baculovirus with high similarity to Cacao swollen shoot virus（CSSV）was identified in Chinaberry tree samples（12.4%gene coverage,80.4%similarity）.The full-length genome sequence of the virus was obtained by PCR amplification,and evolutionary analysis showed that the virus had 70.8%sequence similarity to Cacao swollen shoot Ghana L.The virus was tentatively named Chinaberry badnavirus 1（Ch TBV1）.The discovery of this virus provides an application demonstration for the application of nanopore-based sequencing technology in the rapid detection of unknown viruses.3.Construction of rapid test strips for the detection of Ch TBV 1 based on LAMP-LFD technologyA LAMP-LFD visualization assay for the rapid identification of Ch TBV 1 was developed by combining the ring-mediated thermostatic amplification technique with the lateral flow nucleic acid test strip assay.Five LAMP primers and one isothiocyanate fluorescein-labeled probe were designed based on the ORF3 gene of Ch TBV1.The reaction conditions including reaction temperature,reaction time,polymerase concentration and Mg SO4concentration were also optimized.The results showed that the sensitivity of the technique was 100 times higher than that of the conventional PCR method,and the LAMP-LFD assay established in the study can be used as an effective means for early diagnosis and detection of Ch TBV1. |
