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| Development and Application of SYBR GreenⅡReverse Transcription Real-time Fluorescence Quantitative PCR Assay for Detection of Bamboo mosaic virus |
| ZHU Feng-Xiao1, 2, CHEN Jia-Lu1, ZHANG Zhi-Jun1, 2, * |
1 School of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou 311300, China;
2 State Key Laboratory of Subtropical forest cultivaion, Zhejiang A&F University, Hangzhou 311300, China |
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Abstract Bamboo mosaic virus (BaMV) is the only RNA virus that has been found to infect bamboo so far, seriously affects the economic value of bamboo.Therefore, development of a rapid and accurate detection method for the BaMV is of great significance for the prevention and control of BaMV. In this study, two pairs of specific primers according to the coat protein gene (CP) sequence of BaMV were designed . One sequence of BaMV CP gene were obtained from Phyllostachys bambusoides with mosaic symptoms by RT-qPCR.And the full-length sequence (GenBank accession number: KP256071) was 528 bp encoding 175 putative amino acid residues. The sequences shared more than 98% similarity at nucleotides level with those of Phyllostachys nigra BaMV strains.The recombinant plasmid was used as template for SYBR Green Ⅱ real-time PCR to generate standard and melting curves. The standard curve cycle threshold (Ct) had a good linear relationship with the logarithm of template concentration. Melting curve analysis indicated no primer-dimers and non-specific products in the assay. The amplification efficiency and correlation coefficient were 100% and 0.99, respectively. Repeat ability tests indicated that inter-assay variability of the Ct values was 1.5%. The purpose strips could be successfully amplified with the primers BaMV-F/BaMV-R only in the bamboo infected mosaic sample and the healthy young leaves without purpose strips, which indicated that the primers is highly specific. The sensitivity of RT-qPCR was 100 times higher than that of regular RT-PCR. The minimum detectable concentration of BaMV plasmid standard in RT-qPCR assay was 8.5×101 copies/uL.The presence of BaMV in 8 bamboo species were firstly detected by this method. The results show that the SYBR Green Ⅱ real-time fluorescent quantitative PCR method for detecting BaMV is sensitive, specific, and reproducible, and could be used for rapid detection of BaMV.
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Received: 29 October 2018
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Corresponding Authors:
zjzhang@zafu.edu.cn
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