Detection of four regulated grapevine viruses in a qualitative, single tube real-time PCR with melting curve analysis

被引：10
|
作者：
Aloisio, M. [1 ,2 ]
Morelli, M. [3 ]
Elicio, V. [4 ]
Saldarelli, P. [3 ]
Ura, B. [2 ]
Bortot, B. [2 ]
Severini, G. M. [2 ]
Minafra, A. [3 ]
机构：
[1] Univ Trieste, Dept Life Sci, Trieste, Italy
[2] IRCCS Burlo Garofolo, Inst Maternal & Child Hlth, Trieste, Italy
[3] Inst Sustainable Plant Protect, Bari, Italy
[4] Agritest Srl, Valenzano, Italy
来源：
JOURNAL OF VIROLOGICAL METHODS | 2018年 / 257卷
关键词：
Multiplex Real-time PCR; Melting curve; EvaGreen; Grapevine; Viruses; POLYMERASE CHAIN-REACTION; MULTIPLEX RT-PCR; SYBR GREEN; RAPID DETECTION; ASSAY; DIFFERENTIATION; VALIDATION; EVAGREEN; TYPE-2;
D O I：
10.1016/j.jviromet.2018.04.008
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
The detection of the four grapevine viruses (GLRaV-1, GLRaV-3, GFLV and ArMV) regulated in European Union plant material certification, requires sensitive and specific diagnostic tools. A strategy of simultaneous detection in a real-time single tube amplification was developed, based on the EvaGreen binding dye. The melting curve analysis (MCA) of the amplicons allows a qualitative detection of the four different virus targets in multiplex analysis. A plasmid dilution assay calculated an analytical sensitivity with an amplification threshold up to 100 copies of the target sequences. A small cohort of field grapevine samples, with a known status of infection by mixtures of the target viruses or free of them, respectively, was successfully tested for the evaluation of the amplicons Tm.
引用
收藏
页码：42 / 47
页数：6
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