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园艺学报 ›› 2009, Vol. 36 ›› Issue (12): 1821-1826.

• 新技术与新方法 • 上一篇    下一篇

3种苹果潜隐病毒多重RT-PCR检测体系的建立

范旭东1 ;董雅凤1*;张尊平1 ;李丽丽2 ;裴光前1
  

  1. (1中国农业科学院果树研究所, 辽宁兴城125100; 2 沈阳农业大学园艺学院, 沈阳110161)
  • 收稿日期:2009-07-22 修回日期:2009-10-30 出版日期:2009-12-25 发布日期:2009-12-25
  • 通讯作者: 董雅凤

Multiplex RT-PCR Assay for Simultaneous Detection of Three Latent Apple Viruses

FAN Xu-dong1;DONG Ya-feng1*;ZHANG Zun-ping1 ;LI Li-li2 ; PEI Guang-qian
  

  1. (1Research Institute of Pomology, Chinese Academy of Agriculture Sciences, Xingcheng, Liaoning 125100, China; 2College of Horticulture, Shenyang Agricultural University, Shenyang 110161, China)
  • Received:2009-07-22 Revised:2009-10-30 Online:2009-12-25 Published:2009-12-25
  • Contact: DONG Ya-feng

摘要: 研究建立了能同时检测苹果茎沟病毒(Apple stem grooving virus, ASGV) 、苹果褪绿叶斑病毒(Apple chlorotic leaf spot virus, ACLSV) 和苹果茎痘病毒(Apple stem pitting virus, ASPV) 的多重RT-PCR方法。以复合感染3种病毒的苹果‘望山红’组培苗为试材, 对影响多重PCR的反应条件进行了一系列的调整和优化。多重RT-PCR体系灵敏性测验显示, 最低能从RNA总量187.5 ng的样品中检测3种病毒的存在。多重RT-PCR产物的序列与报道的病毒序列有较高的同源性, 12个田间苹果样品的多重RT-PCR检测结果与单一PCR的结果一致, 初步证明了多重RT-PCR检测的准确性。

关键词: 苹果, 多重RT-PCR, 检测, 苹果茎沟病毒, 苹果褪绿叶斑病毒, 苹果茎痘病毒中

Abstract: The multiplex RT-PCR assay was established to simultaneously detect Apple stem grooving virus (ASGV) , Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV). Using tissue culture apple seedling (Wangshanhong) infected by three latent apple viruses as sample, a series of factors affecting the multip lex RT-PCR were optimized. Sensibility test showed that it could simultaneously detect out ASGV, ACLSV and ASPV in 187.5 ng total RNA. The multiplex RT-PCR products were cloned and sent for sequencing, and the sequences show high homologies to the published virus sequences. Consistent results for virus detection of 12 apple samples in field were obtained by the multiplex RT-PCR and single RT-PCR system, suggesting the accuracy of the system in ASGV, ACLSV and ASPV detection.

Key words: apple, multiplex RT-PCR, detection, Apple stem grooving virus, Apple chlorotic leaf spotvirus, Apple stem pitting virus

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