柑橘褪绿矮缩病毒dot-ELISA检测方法的建立及应用  

Establishment and application of dot-ELISA detection method for Citrus chlorotic dwarf-associated virus

作　　者：秦阳阳 陈香玲[2] 王甲军 叶肖 申世凯 周彦[1] QIN Yangyang;CHEN Xiangling;WANG Jiajun;YE Xiao;SHEN Shikai;ZHOU Yan(National Citrus Engineering and Technology Research Center,Citrus Research Institute,Southwest University,Chongqing 400712,China;Horticultural Research Institute,Guangxi Academy of Agricultural Sciences,Nanning 530007,Guangxi,China)

机构地区：[1]西南大学柑桔研究所国家柑桔工程技术研究中心,重庆400712 [2]广西农业科学院园艺研究所,南宁530007

出　　处：《果树学报》2022年第11期2181-2187,共7页Journal of Fruit Science

基　　金：国家重点研发计划(2019YFD1001802);财政部和农业农村部:国家现代农业产业技术体系(CARS-26-05B);重庆市自然科学基金项目(cstc2019jcyj-msxmX0557)。

摘　　要：【目的】柑橘褪绿矮缩病毒(Citrus chlorotic dwarf-associated virus,CCDaV)是一种新的柑橘病毒,为给我国柑橘脱毒苗木质量控制和田间监测提供技术支撑,建立了一种灵敏度高、特异性强的快速检测方法。【方法】通过序列分析和相似性比对,明确抗原靶标;构建CCDaV-CP的原核表达载体,诱导目的蛋白表达后作为抗原用于注射兔子,制备抗血清。运用Western blot检测效价。通过优化抗体浓度,建立CCDaV的dot-ELISA检测方法,明确其灵敏度,并用于田间疑似样品检测。【结果】以外壳蛋白(CP)的保守区域为靶标,成功构建了表达载体pET28a-CCDaV-CP,其在18℃,IPTG浓度0.5 mmol·L^(-1)时,诱导12 h,目的蛋白的表达量高。制备的特异性抗血清效价为1:3000。由此建立的dotELISA检测方法在提取液被稀释640倍时,仍能检测出CCDaV。应用该方法对42份田间疑似样品的检测结果与PCR法的符合率为94.73%。【结论】首次获得了CCDaV的抗血清,并以此建立了快捷、灵敏、准确的dot-ELISA检测方法,为CCDaV的快速检测和防控提供了技术支撑。【Objective】In 2015,a new citrus viral disease caused by Citrus chlorotic dwarf-associated virus(CCDaV)was discovered in Yunnan province of China,and since then it has spread in some important citrus-growing provinces of China,like Guangxi and Yunnan.At present,the main control strategy for prevention of CCDaV is based on using virus-free germplasm and propagation materials.To date,various techniques have been employed to detect CCDaV,including biological indexing,PCR and qPCR.However,these conventional detection techniques generally suffer from drawbacks.The purpose of this research was to establish a sensitive,accurate and high-throughput detection method for CCDaV,so as to provide technical support for the diagnosis and production of virus-free citrus plants in China.【Methods】The restriction enzymes Bam HⅠand XhoⅠwere added to the corresponding end of the CCDaV-CP gene sequence,respectively.Primers F(5’-GTGGACAGCAAATGGGTCGC■CCATGTAAAACACACACGGTGGATGTGAT-3’)and R(5’-CAGTGGTGGTGGTGGTGGTG■TTAATTT GATGTAGAATCATAAA AA TA CA-3’)were designed using SNAPGENE software based on a region(84-762 nt)of coat protein(CP)gene that was highly conserved across 15full CCDaV genome sequences available in GenBank.Total DNA was extracted from 0.1 g CCDaV-infected citrus sample and amplified by PCR with primers F/R.The reaction was conducted under conditions of initial 3 min denaturation at 98℃,34 cycles at 98℃for 10 s,60℃for 15 s,72℃for 40 s and extension for 5 min at 72℃.The PCR purified products and pET28a vector were digested by Bam HⅠand XhoⅠ,and after that they were ligated with T4 DNA ligase and transferred into Escherichia coli(E.coli)strain DH5αand finally plated onto Luria-Bertani(LB)agar containing Kanamycin(Kana).The expression strain Rosetta containing the recombinant plasmid was cultured at 37℃overnight,and transferred to a new medium at a 10%inoculum on tecond day,until OD600reached 0.4-0.6.Isopropyl-β-D-thiogalactoside(IPTG)was added to a final concentration of 0.1,0.3,0.5,0.7

关 键 词：柑橘褪绿矮缩病毒 外壳蛋白 抗血清 斑点酶联免疫吸附法 

分 类 号：S666[农业科学—果树学]