Quantitative detection of Cymbidium mosaic virus by real time PCR

Abstract

The technique of SYBR Green-based quantitative real-time reverse transcription polymerase chain reaction (real-time RT-PCR) was applied to quantitative detect a 764 bp nucleotide sequence containing total coat protein (cp) gene of Cymbidium mosaic virus (CyMV). The plasmid containing the target sequence was constructed to prepare the standard curve and detect the sensitivity. The standard curve was drawn based on the linear relationship between the logarithm (base 10) of the quantity of target sequence and cycle threshold [C(T)]. While the concentration of plasmid DNA falling within the range of 2.6×10⁷ to 2.6×10² copies per tube established a regression equation, y=-0.3583x+10.32, and related coefficient: r²=0.995, The real-time RT-PCR assay for CyMV had a minimum detectable quantity of two copies per tube. The naturally infected samples of Phalaenopsis sp. and the artificially inoculated samples of Arachnis sp. with trace CyMV were quantitatively detected using this method. CyMV in the positive samples of Phalaenopsis sp. and Arachnis sp. was confirmed by DNA sequencing and cp gene homeology blast. The results showed that CyMV extracted from the leaves of orchid in Hangzhou, Zhejiang Province, China, could be derived from Kunming city (KM), Yunnan Province, China. This method characterized by high sensitivity, specificity, and precision is suitable for early diagnosis and quantitative detection of CyMV.

Keywords

Cymbidium mosaic virus (CyMV) / coat protein gene / quantitative detection / real-time reverse transcription polymerase chain reaction (real-time RT-PCR) / SYBR Green

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2014 Higher Education Press and Springer-Verlag Berlin Heidelberg