南方水稻黑条矮缩病毒dot-ELISA检测试剂盒的应用性能验证
|
摘要
以制备的抗南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)单抗2C2为核心,建立了检测水稻叶片和白背飞虱虫中SRBSDV的dot-ELISA试剂盒。试剂盒的灵敏度分析表明,当SRBSDV感染病叶稀释到1∶10 240倍(w/v,g/m L)、单头携毒白背飞虱稀释到1∶51 200倍(头/μL)时仍能检测到SRBSDV。建立的试剂盒检测感染SRBSDV的水稻和携毒白背飞虱呈阳性反应,而检测感染水稻黑条矮缩病毒、水稻矮缩病毒、水稻条纹病毒、水稻瘤矮病毒、水稻条纹花叶病毒、水稻锯齿矮缩病毒的病叶和健康水稻及非携毒白背飞虱呈阴性反应。试剂盒的田间样品检测结果与RT-PCR方法的检测结果的符合率达到100%,核酸测序和序列比对结果发现RT-PCR检测阳性的样品确实感染SRBSDV。试验结果表明,建立的检测试剂盒能准确、有效地检测田间白背飞虱及水稻样品中的SRBSDV,可为我国南方水稻黑条矮缩病的检测和诊断、预测预警及科学防控提供技术服务。
Based on monoclonal antibody(MAb) 2 C2 against southern rice black-streaked dwarf virus(SRBSDV), a dotELISA detection kit for detecting SRBSDV in rice plants and white-backed planthoppers was developed. Sensitivity analysis indicated that the detection kit could detect minimal viruses in infected rice leaf tissue extracts diluted at 1:10240(w/v, g/m L-1) and in one white-backed planthopper tissue extract diluted at 1: 6400(individual/μL). The specificity of the kit was confirmed by positive reactions of detection with SRBSDV-infected rice plants and viruliferous white-backed planthoppers,and negative reactions of detection with healthy, rice black-streaked dwarf virus-, rice stripe virus-, rice gall dwarf virus-,rice stripe mosaic virus-, or rice dwarf virus-infected rice plants, or non-viruliferous white-backed planthoppers. Field samples were detected using the developed dot-ELISA kit and RT-PCR respectively, and the detection results indicated that their coincidence rate was 100%. Nucleotide sequencing and sequence blast of the PCR-amplified product proved further that all positive samples detected by dot-ELISA kit were infected with SRBSDV. Those results indicated that the developed kit could accurately and effectively detect SRBSDV in field rice plants and white-backed planthopper samples. Therefore,the established dot-ELISA detection kit for SRBSDV can provide technique supports for the detection and diagnosis,forecasting and early warning and scientific control of southern rice black-streaked dwarf disease.
Based on monoclonal antibody(MAb) 2 C2 against southern rice black-streaked dwarf virus(SRBSDV), a dotELISA detection kit for detecting SRBSDV in rice plants and white-backed planthoppers was developed. Sensitivity analysis indicated that the detection kit could detect minimal viruses in infected rice leaf tissue extracts diluted at 1:10240(w/v, g/m L-1) and in one white-backed planthopper tissue extract diluted at 1: 6400(individual/μL). The specificity of the kit was confirmed by positive reactions of detection with SRBSDV-infected rice plants and viruliferous white-backed planthoppers,and negative reactions of detection with healthy, rice black-streaked dwarf virus-, rice stripe virus-, rice gall dwarf virus-,rice stripe mosaic virus-, or rice dwarf virus-infected rice plants, or non-viruliferous white-backed planthoppers. Field samples were detected using the developed dot-ELISA kit and RT-PCR respectively, and the detection results indicated that their coincidence rate was 100%. Nucleotide sequencing and sequence blast of the PCR-amplified product proved further that all positive samples detected by dot-ELISA kit were infected with SRBSDV. Those results indicated that the developed kit could accurately and effectively detect SRBSDV in field rice plants and white-backed planthopper samples. Therefore,the established dot-ELISA detection kit for SRBSDV can provide technique supports for the detection and diagnosis,forecasting and early warning and scientific control of southern rice black-streaked dwarf disease.
引文
[1]周国辉,温锦军,蔡德江,等.呼肠孤病毒科斐济病毒属一新种:南方水稻黑条矮缩病毒[J].科学通报,2008,53(23):3677-3685.
[2]刘万才,刘宇,郭荣.南方水稻黑条矮缩病发生现状及防控对策[J].中国植保导刊,2010,30(3):17-18.
[3]陈卓,李国君,范会涛,等.毒氟磷防治南方水稻黑条矮缩病药效研究[J].中国农学通报,2011,27(18):250-254.
[4]饶黎霞,王震成,吴建祥,等.2011—2012年三种水稻矮缩病在我国的分布及发生调查[J].植物保护,2014,40(3):151-156.
[5]周国辉,张曙光,邹寿发,等.水稻新病害南方水稻黑条矮缩病发生特点及危害趋势分析[J].植物保护,2010,36(2):144-146.
[6]王康,郑静君,张曙光,等.室内试验证实南方水稻黑条矮缩病毒不经水稻种子传播[J].广东农业科学,2010,371(7):95-96.
[7]吴建祥,饶黎霞,陈浙,等.检测南方水稻黑条矮缩病毒胶体金免疫试纸条的建立[J].植物保护学报,2017,44(6):1024-1032.
[8]刘欢,倪跃群,饶黎霞,等.南方水稻黑条矮缩病毒和水稻黑条矮缩病毒的单抗制备及其检测应用[J].植物病理学报,2013,43(1):27-34.
[9]吴凌,高维明,张洪友,等.孕酮ELISA试剂盒在牛奶乳汁检测中的初步应用[J].饲料加工与检测,2015,51(21):54-58.
[10]张蔚明,刘艳娟,周倩,等.南方水稻黑条矮缩病毒外壳蛋白p10的原核表达和抗血清制备及应用[J].湖南农业大学学报(自然科学版),2011,37(4):400-402.
[2]刘万才,刘宇,郭荣.南方水稻黑条矮缩病发生现状及防控对策[J].中国植保导刊,2010,30(3):17-18.
[3]陈卓,李国君,范会涛,等.毒氟磷防治南方水稻黑条矮缩病药效研究[J].中国农学通报,2011,27(18):250-254.
[4]饶黎霞,王震成,吴建祥,等.2011—2012年三种水稻矮缩病在我国的分布及发生调查[J].植物保护,2014,40(3):151-156.
[5]周国辉,张曙光,邹寿发,等.水稻新病害南方水稻黑条矮缩病发生特点及危害趋势分析[J].植物保护,2010,36(2):144-146.
[6]王康,郑静君,张曙光,等.室内试验证实南方水稻黑条矮缩病毒不经水稻种子传播[J].广东农业科学,2010,371(7):95-96.
[7]吴建祥,饶黎霞,陈浙,等.检测南方水稻黑条矮缩病毒胶体金免疫试纸条的建立[J].植物保护学报,2017,44(6):1024-1032.
[8]刘欢,倪跃群,饶黎霞,等.南方水稻黑条矮缩病毒和水稻黑条矮缩病毒的单抗制备及其检测应用[J].植物病理学报,2013,43(1):27-34.
[9]吴凌,高维明,张洪友,等.孕酮ELISA试剂盒在牛奶乳汁检测中的初步应用[J].饲料加工与检测,2015,51(21):54-58.
[10]张蔚明,刘艳娟,周倩,等.南方水稻黑条矮缩病毒外壳蛋白p10的原核表达和抗血清制备及应用[J].湖南农业大学学报(自然科学版),2011,37(4):400-402.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |