A sensitive SYBR Green RT-qPCR method for grapevine virus E and its application for virus detection in different grapevine sample types
To develop a rapid and high-sensitivity method for detection of grapevine virus E (GVE), a SYBR Green based real-time fluorescence quantitative RT-PCR method (RT-qPCR) was established. This method could be used to detect GVE specifically, and the sensitivity was about 100 times greater than conventi...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2020-07-01
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| Series: | Journal of Integrative Agriculture |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S209531191962784X |
| _version_ | 1818438843944140800 |
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| author | Fang REN Zun-ping ZHANG Xu-dong FAN Guo-jun HU Meng-yan ZHANG Ya-feng DONG |
| author_facet | Fang REN Zun-ping ZHANG Xu-dong FAN Guo-jun HU Meng-yan ZHANG Ya-feng DONG |
| author_sort | Fang REN |
| collection | DOAJ |
| description | To develop a rapid and high-sensitivity method for detection of grapevine virus E (GVE), a SYBR Green based real-time fluorescence quantitative RT-PCR method (RT-qPCR) was established. This method could be used to detect GVE specifically, and the sensitivity was about 100 times greater than conventional RT-PCR. An excellent linear correlation (R2=0.997) and a high amplification efficiency (E=97.5%) were obtained from the standard curve of this method. Reproducibility tests revealed that the coefficients of variation in the intra- and inter-assay results were 0.31–1.03% and 0.82–2.62%, respectively, indicating a good reproducibility. The RT-qPCR method could be used to detect GVE in a wide range of grapevine sample types. The detection rates of RT-qPCR for nearly all sample types from different positions and seasons were higher than conventional RT-PCR. The detection rates in spring, summer, autumn and winter increased gradually. Samples in autumn and winter were best for detection, and the detection rates of most samples were 80–100%, which were 10 to 40% higher than conventional RT-PCR. In general, old petioles and branches were the best tissues for GVE detection. The detection rates of these samples in each season were all 100%, which were 20 to 40% higher than conventional RT-PCR. The second highest rates were in the old leaf, with detection rates for RT-qPCR of 80–100% in all seasons, which were 20 to 40% higher than conventional RT-PCR. GVE could be difficultly detected in young leaves by conventional RT-PCR, and the detection rates were only 0–50%, while by RT-qPCR the rates could increase to 0–80%. A total of 33 out of 363 samples (belonging to 68 cultivars) from 20 regions in China were detected to be positive by RT-qPCR (9.1%), which was more than twice the rate of the conventional RT-PCR (3.9%). |
| first_indexed | 2024-12-14T17:47:01Z |
| format | Article |
| id | doaj.art-d72febcfe6ae4da18cfe2258bd5648c7 |
| institution | Directory Open Access Journal |
| issn | 2095-3119 |
| language | English |
| last_indexed | 2024-12-14T17:47:01Z |
| publishDate | 2020-07-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Integrative Agriculture |
| spelling | doaj.art-d72febcfe6ae4da18cfe2258bd5648c72022-12-21T22:52:44ZengElsevierJournal of Integrative Agriculture2095-31192020-07-0119718341841A sensitive SYBR Green RT-qPCR method for grapevine virus E and its application for virus detection in different grapevine sample typesFang REN0Zun-ping ZHANG1Xu-dong FAN2Guo-jun HU3Meng-yan ZHANG4Ya-feng DONG5REN Fang, Tel: +86-429-3598137; National Center for Eliminating Viruses from Deciduous Fruit Trees, Research Institute of Pomology, Chinese Academy of Agricultural Sciences, Xingcheng 125100, P.R. ChinaNational Center for Eliminating Viruses from Deciduous Fruit Trees, Research Institute of Pomology, Chinese Academy of Agricultural Sciences, Xingcheng 125100, P.R. ChinaNational Center for Eliminating Viruses from Deciduous Fruit Trees, Research Institute of Pomology, Chinese Academy of Agricultural Sciences, Xingcheng 125100, P.R. ChinaNational Center for Eliminating Viruses from Deciduous Fruit Trees, Research Institute of Pomology, Chinese Academy of Agricultural Sciences, Xingcheng 125100, P.R. ChinaNational Center for Eliminating Viruses from Deciduous Fruit Trees, Research Institute of Pomology, Chinese Academy of Agricultural Sciences, Xingcheng 125100, P.R. ChinaCorrespondence DONG Ya-feng, Tel: +86-429-3598278; National Center for Eliminating Viruses from Deciduous Fruit Trees, Research Institute of Pomology, Chinese Academy of Agricultural Sciences, Xingcheng 125100, P.R. ChinaTo develop a rapid and high-sensitivity method for detection of grapevine virus E (GVE), a SYBR Green based real-time fluorescence quantitative RT-PCR method (RT-qPCR) was established. This method could be used to detect GVE specifically, and the sensitivity was about 100 times greater than conventional RT-PCR. An excellent linear correlation (R2=0.997) and a high amplification efficiency (E=97.5%) were obtained from the standard curve of this method. Reproducibility tests revealed that the coefficients of variation in the intra- and inter-assay results were 0.31–1.03% and 0.82–2.62%, respectively, indicating a good reproducibility. The RT-qPCR method could be used to detect GVE in a wide range of grapevine sample types. The detection rates of RT-qPCR for nearly all sample types from different positions and seasons were higher than conventional RT-PCR. The detection rates in spring, summer, autumn and winter increased gradually. Samples in autumn and winter were best for detection, and the detection rates of most samples were 80–100%, which were 10 to 40% higher than conventional RT-PCR. In general, old petioles and branches were the best tissues for GVE detection. The detection rates of these samples in each season were all 100%, which were 20 to 40% higher than conventional RT-PCR. The second highest rates were in the old leaf, with detection rates for RT-qPCR of 80–100% in all seasons, which were 20 to 40% higher than conventional RT-PCR. GVE could be difficultly detected in young leaves by conventional RT-PCR, and the detection rates were only 0–50%, while by RT-qPCR the rates could increase to 0–80%. A total of 33 out of 363 samples (belonging to 68 cultivars) from 20 regions in China were detected to be positive by RT-qPCR (9.1%), which was more than twice the rate of the conventional RT-PCR (3.9%).http://www.sciencedirect.com/science/article/pii/S209531191962784Xgrapevinegrapevine virus EdetectionRT-qPCRconventional RT-PCR |
| spellingShingle | Fang REN Zun-ping ZHANG Xu-dong FAN Guo-jun HU Meng-yan ZHANG Ya-feng DONG A sensitive SYBR Green RT-qPCR method for grapevine virus E and its application for virus detection in different grapevine sample types Journal of Integrative Agriculture grapevine grapevine virus E detection RT-qPCR conventional RT-PCR |
| title | A sensitive SYBR Green RT-qPCR method for grapevine virus E and its application for virus detection in different grapevine sample types |
| title_full | A sensitive SYBR Green RT-qPCR method for grapevine virus E and its application for virus detection in different grapevine sample types |
| title_fullStr | A sensitive SYBR Green RT-qPCR method for grapevine virus E and its application for virus detection in different grapevine sample types |
| title_full_unstemmed | A sensitive SYBR Green RT-qPCR method for grapevine virus E and its application for virus detection in different grapevine sample types |
| title_short | A sensitive SYBR Green RT-qPCR method for grapevine virus E and its application for virus detection in different grapevine sample types |
| title_sort | sensitive sybr green rt qpcr method for grapevine virus e and its application for virus detection in different grapevine sample types |
| topic | grapevine grapevine virus E detection RT-qPCR conventional RT-PCR |
| url | http://www.sciencedirect.com/science/article/pii/S209531191962784X |
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