Cargando…

Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay

Genetic engineering offers an attractive alternative in sugarcane breeding for increasing cane and sugar yields as well as disease and insect resistance. Bar transgenic sugarcane employing the herbicide tolerance is a useful agronomical trait in weed control. In this study, a loop-mediated isotherma...

Descripción completa

Detalles Bibliográficos
Autores principales: Zhou, Dinggang, Wang, Chunfeng, Li, Zhu, Chen, Yun, Gao, Shiwu, Guo, Jinlong, Lu, Wenying, Su, Yachun, Xu, Liping, Que, Youxiong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4782128/
https://www.ncbi.nlm.nih.gov/pubmed/27014303
http://dx.doi.org/10.3389/fpls.2016.00279
_version_ 1782419900741976064
author Zhou, Dinggang
Wang, Chunfeng
Li, Zhu
Chen, Yun
Gao, Shiwu
Guo, Jinlong
Lu, Wenying
Su, Yachun
Xu, Liping
Que, Youxiong
author_facet Zhou, Dinggang
Wang, Chunfeng
Li, Zhu
Chen, Yun
Gao, Shiwu
Guo, Jinlong
Lu, Wenying
Su, Yachun
Xu, Liping
Que, Youxiong
author_sort Zhou, Dinggang
collection PubMed
description Genetic engineering offers an attractive alternative in sugarcane breeding for increasing cane and sugar yields as well as disease and insect resistance. Bar transgenic sugarcane employing the herbicide tolerance is a useful agronomical trait in weed control. In this study, a loop-mediated isothermal amplification (LAMP) assay for rapid detection of the bar gene in transgenic sugarcane has been developed and evaluated. A set of six primers was designed for LAMP-based amplification of the bar gene. The LAMP reaction conditions were optimized as follows: 5.25 mM of Mg(2+), 6:1 ratio of inner vs. outer primer, and 6.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. The detection limit of the recombinant plasmid 1Ac0229 was as low as 10 copies in the developed LAMP, which was 10-fold higher sensitive than that of conventional PCR. In 100 putative transgenic lines, the bar gene was detected in 100/100 cases (100%) by LAMP and 97/100 cases (97%) by conventional PCR, respectively. In conclusion, the developed LAMP assay is visual, rapid, sensitive, reliable, and cost-effective for detection of the bar specific transgenic sugarcane.
format Online
Article
Text
id pubmed-4782128
institution National Center for Biotechnology Information
language English
publishDate 2016
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-47821282016-03-24 Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay Zhou, Dinggang Wang, Chunfeng Li, Zhu Chen, Yun Gao, Shiwu Guo, Jinlong Lu, Wenying Su, Yachun Xu, Liping Que, Youxiong Front Plant Sci Plant Science Genetic engineering offers an attractive alternative in sugarcane breeding for increasing cane and sugar yields as well as disease and insect resistance. Bar transgenic sugarcane employing the herbicide tolerance is a useful agronomical trait in weed control. In this study, a loop-mediated isothermal amplification (LAMP) assay for rapid detection of the bar gene in transgenic sugarcane has been developed and evaluated. A set of six primers was designed for LAMP-based amplification of the bar gene. The LAMP reaction conditions were optimized as follows: 5.25 mM of Mg(2+), 6:1 ratio of inner vs. outer primer, and 6.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. The detection limit of the recombinant plasmid 1Ac0229 was as low as 10 copies in the developed LAMP, which was 10-fold higher sensitive than that of conventional PCR. In 100 putative transgenic lines, the bar gene was detected in 100/100 cases (100%) by LAMP and 97/100 cases (97%) by conventional PCR, respectively. In conclusion, the developed LAMP assay is visual, rapid, sensitive, reliable, and cost-effective for detection of the bar specific transgenic sugarcane. Frontiers Media S.A. 2016-03-08 /pmc/articles/PMC4782128/ /pubmed/27014303 http://dx.doi.org/10.3389/fpls.2016.00279 Text en Copyright © 2016 Zhou, Wang, Li, Chen, Gao, Guo, Lu, Su, Xu and Que. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Plant Science
Zhou, Dinggang
Wang, Chunfeng
Li, Zhu
Chen, Yun
Gao, Shiwu
Guo, Jinlong
Lu, Wenying
Su, Yachun
Xu, Liping
Que, Youxiong
Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay
title Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay
title_full Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay
title_fullStr Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay
title_full_unstemmed Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay
title_short Detection of Bar Transgenic Sugarcane with a Rapid and Visual Loop-Mediated Isothermal Amplification Assay
title_sort detection of bar transgenic sugarcane with a rapid and visual loop-mediated isothermal amplification assay
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4782128/
https://www.ncbi.nlm.nih.gov/pubmed/27014303
http://dx.doi.org/10.3389/fpls.2016.00279
work_keys_str_mv AT zhoudinggang detectionofbartransgenicsugarcanewitharapidandvisualloopmediatedisothermalamplificationassay
AT wangchunfeng detectionofbartransgenicsugarcanewitharapidandvisualloopmediatedisothermalamplificationassay
AT lizhu detectionofbartransgenicsugarcanewitharapidandvisualloopmediatedisothermalamplificationassay
AT chenyun detectionofbartransgenicsugarcanewitharapidandvisualloopmediatedisothermalamplificationassay
AT gaoshiwu detectionofbartransgenicsugarcanewitharapidandvisualloopmediatedisothermalamplificationassay
AT guojinlong detectionofbartransgenicsugarcanewitharapidandvisualloopmediatedisothermalamplificationassay
AT luwenying detectionofbartransgenicsugarcanewitharapidandvisualloopmediatedisothermalamplificationassay
AT suyachun detectionofbartransgenicsugarcanewitharapidandvisualloopmediatedisothermalamplificationassay
AT xuliping detectionofbartransgenicsugarcanewitharapidandvisualloopmediatedisothermalamplificationassay
AT queyouxiong detectionofbartransgenicsugarcanewitharapidandvisualloopmediatedisothermalamplificationassay