Identification Of China Rose Viruses And Establishment Of Three Rapid Detection Methods

Rosa chinensis Jacq.is an ornamental plant of the genus Rosa in the family Rosaceae.It has a large cultivated area and serious virus disease occurs,and the virus spread mainly through scion or rootstock grafting.Due to the unique advantages of high-throughput sequencing,which can identify both known and unknown viruses,it has been widely used in virus detection in recent years.In this study,the virus species that infect China rose in Henan province,Jiangxi province,Jiangsu province,Guangdong province,Hunan province,Zhejiang province and Shanghai city were analyzed by high-throughput sequencing technology,and the infection situation of rose virus was determined.Three rapid detection methods were established for viruses with high infection rates.The main results were as follows:1.Identification of China rose virus species based on high-throughput sequencing technology:A total of 104 Chinese rose leaf samples with suspected virus symptoms were collected from Henan,Jiangxi,Shanghai,Guangdong,Hunan,Zhejiang,and Jiangsu province.The sequence information of 9 known viruses and 3 unknown viruses were obtained by mixed high-throughput sequencing analysis.The contigs of 9 known viruses with a highest similarity of 89.33%-100%between Apple mosaic virus（Ap MV）,Prunus necrotic Ringspot virus（PNRSV）,Rose spring dwarf-associated virus（RSDa V）,Rose yellow vein virus（RYVV）,Apple stem grooving virus（ASGV）,Rose cryptic virus 1（RCV1）,Rose leaf rosette-associated virus（RLRa V）,Blackberry chlorotic ringspot virus（BCRV）and Rose partitivirus（Ro PV）.The highest similarity between three unknown virus contig sequences and known viruses was only 38.16%-53.69%,which suggested that they might be new viruses.These 12 viruses were detected in China rose samples by RT-PCR,and the detection rates of different viruses ranged from 1.92%to 79.81%.RYVV,RCV1,ASGV and BFDa V were the high detection rates.The distribution statistics of China rose virus in 7 collection areas showed that RYVV,ASGV and RCV1 were the most common and existed in 7 areas.Henan province had the highest number of viruses,with nine,followed by Jiangxi and Guangdong with seven.The mixed infection rate of 104 China rose samples was 82.69%,and most single samples were infected by 2-4 viruses.2.Establishment of multiple RT-PCR detection system for China rose virus:In this study,two pairs of specific RT-PCR primers were designed for five China rose viruses with high detection rates,namely RCV1,ASGV,Ap MV,PNRSV and RSDa V.The primers were screened,and the reaction system,annealing temperature,primer concentration and other factors were optimized to establish a multiple RT-PCR detection system for five rose viruses.Then the sensitivity and field application of the system were tested.The results showed that the minimum virus concentration detected by the system was 2.56×10⁷ copies/μL.Eighteen virus-infected rose samples were randomly selected from the field and tested by single PCR and multiple RT-PCR.The results showed that these two methods had the same detection results,and most of the China rose was infected by different viruses.The multi-RT-PCR method established in this study can simultaneously detect five rose viruses,which provides technical support for epidemic surveillance and diagnosis of the virus.3.Establishment of RT-LAMP detection technology system for China rose virus:In this study,specific primers were designed for the CP gene sequences of three rose viruses,namely RCV1,ASGV and PNRSV,and RT-LAMP detection method was established by optimizing Mg²⁺concentration,d NTP concentration,reaction time and temperature in the reaction system.The method can detect the specific visualizations of the target virus under the condition of constant temperature（61℃）.There is no cross-reaction between the target virus and other viruses infecting China rose,and its sensitivity is 100-1000 times that of RT-PCR.The RT-LAMP method was used to detect 19 samples randomly in the field,and the results were consistent with those of RT-PCR.However,the method could realize visualization by adding dye directly,which had the characteristics of high sensitivity,rapid,convenient and good specific,and was suitable for the rapid diagnosis and field monitoring of PNRSV,ASGV and RCV1 infected China rose.4.Establishment of RPA-Cas12a detection technology system for China rose virus:In this study,specific RPA primers were designed to amplify the target virus for three kinds of rose virus with wide distribution,namely RCV1,ASGV and PNRSV.The optimal RPA reaction conditions were determined by optimizing the reaction time and temperature,and then corresponding cr RNA was designed and synthesized for each virus.After purification,factors such as Cas12a/cr RNA ratio,Cas12a concentration and FQ-reporter concentration in RPA-Cas12a reaction system were optimized,and plant RNA was obtained by acupuncture method.The RPA-Cas12a detection system of three rose viruses RCV1,ASGV and PNRSV was established.In this method,2-3 sterile needles were directly inserted into the tender stem of China rose to obtain nucleic acid,without the need to extract plant RNA,and the reaction could be completed by incubating at constant temperature for about 1 h,without any dependence on expensive instruments（such as PCR instrument,gel imager,etc.）.After the reaction,the results could be determined by directly irradiating with a flashlight under 365nm purple light,which could realize rapid visual detection of rose samples in the field.