ART-LAMP Assay For Detection Of Apple Chlorotic Leaf Spot Virus And Construction Of Infectious Clones

Apple is an important economic crop.In apple's high yield and good quality production,virus disease has become an important factor restricting the development of apple industry.Apple chlorotic leaf spot virus?ACLSV?is the highest detection rate,inner world apple distribution scope of one of the dangers of the most popular apple virus.Based on the research status of ACLSV and high quality for apple and the urgent need of production safety.This study aims to complete the following experiments:to established a method which uses reverse transcription loop-mediated isothermal amplification technology to detect ACLSV simply,quickly and high sensitively;using RT-PCR and RACE techniques,the complete genome of ACLSV,isolated from apple cultivar‘Hanfu'in Xingcheng Liaoning,was amplified;cloning vector constructed ACLSV infection sex,for subsequent mutual ACLSV copy,move,and the host do pathogenesis research and reform for apple gene function research of gene silencing carrier lay the foundation.The specific research results are as follows:1.The specific RT-LAMP method to detect ACLSV was established and the optimal amplification was achieved by incubation of 6 mM Mg²⁺,1.2 mM dNTPs,1M Betaine,1.6?M FIP/BIP?0.2?M F3/B3 with template RNA at 59?for 60 min.In the specificity test,only ACLSV test result was positive,and the control groups were negative.Sensitivity test,this method detected at least 10-3 times RNA dilution solution.The positive rate of RT-PCR in 23 apple leaf samples was 52.2%,RT-LAMP was 65.2%,and the detection rate of RT-LAMP was higher than that of RT-RCR,indicating that RT-LAMP was more sensitive2.The genome of ACLSV XC-HF has 7557 nucleotides,encoding three overlapping open reading frames?ORFs?.The nucleotide identity of complete genome between ACLSV XC-HF and the 19ACLSV isolates published in Gen Bank ranged from 68.90%to 84.40%.Phylogenetic analysis indicated that the concerned ACLSV isolates could be grouped into 6 groups,ACLSV XC-HF was in group JB,clustered with Chinese pear isolates JB,KMS,YH and Chinese Hawthorn isolate SY03.The recombinant event was identified in 1-338nt of ACLSV XC-HF in the recombination analysis.3.The ACLSV XC-HF was mechanically inoculated to the Chenopodiumamaranticolorand Malus sylvestriscv.R12740-7A,two hosts both showed the typical symptoms of yellow spots and chlorosis.4.This experiment successfully constructed ACLSV cloning vector infection sex,Nicotiana occidentalis appeared after inoculation ACLSV symptoms of infection,leaves yellow chlorosis,the gel electrophoresis to detect ACLSV was positive.