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Sensitive and high throughput quantification of abscisic acid based on quantitative real time immuno-PCR
BACKGROUND: Abscisic acid (ABA) functions as a stress phytohormone in many growth and developmental processes in plants. The ultra-sensitive determination of ABA would help to better understand its vital roles and action mechanisms. RESULTS: We report a new sensitive and high throughput quantitative...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6260876/ https://www.ncbi.nlm.nih.gov/pubmed/30534191 http://dx.doi.org/10.1186/s13007-018-0371-y |
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author | Su, Yi Li, Wei Huang, Zhigang Wang, Ruozhong Luo, Weigui Liu, Qing Tong, Jianhua Xiao, Langtao |
author_facet | Su, Yi Li, Wei Huang, Zhigang Wang, Ruozhong Luo, Weigui Liu, Qing Tong, Jianhua Xiao, Langtao |
author_sort | Su, Yi |
collection | PubMed |
description | BACKGROUND: Abscisic acid (ABA) functions as a stress phytohormone in many growth and developmental processes in plants. The ultra-sensitive determination of ABA would help to better understand its vital roles and action mechanisms. RESULTS: We report a new sensitive and high throughput quantitative real time immuno-PCR (qIPCR) method based on biotin–avidin linkage system for ABA determination in plants. ABA monoclonal antibody (McAb) coated on the inner surface of PCR well pretreated with glutaraldehyde. The pre-prepared probe complex, including biotinylated McAb, biotinylated DNA and streptavidin linker, was convenient for high throughput operations. Finally, probe DNA was quantified by real-time PCR. The detectable ranges were from 10 to 40 ng/L with a limit of detection (LOD) of 2.5 fg. ABA contents in plant sample were simultaneously analyzed using LC–MS/MS to validate the qIPCR method. The results showed that qIPCR method has good specificity and repeatability with a recovery rate of 96.9%. CONCLUSION: The qIPCR method is highly sensitive for ABA quantification for actual plant samples with an advantage of using crude extracts instead of intensively purified samples. |
format | Online Article Text |
id | pubmed-6260876 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-62608762018-12-10 Sensitive and high throughput quantification of abscisic acid based on quantitative real time immuno-PCR Su, Yi Li, Wei Huang, Zhigang Wang, Ruozhong Luo, Weigui Liu, Qing Tong, Jianhua Xiao, Langtao Plant Methods Methodology BACKGROUND: Abscisic acid (ABA) functions as a stress phytohormone in many growth and developmental processes in plants. The ultra-sensitive determination of ABA would help to better understand its vital roles and action mechanisms. RESULTS: We report a new sensitive and high throughput quantitative real time immuno-PCR (qIPCR) method based on biotin–avidin linkage system for ABA determination in plants. ABA monoclonal antibody (McAb) coated on the inner surface of PCR well pretreated with glutaraldehyde. The pre-prepared probe complex, including biotinylated McAb, biotinylated DNA and streptavidin linker, was convenient for high throughput operations. Finally, probe DNA was quantified by real-time PCR. The detectable ranges were from 10 to 40 ng/L with a limit of detection (LOD) of 2.5 fg. ABA contents in plant sample were simultaneously analyzed using LC–MS/MS to validate the qIPCR method. The results showed that qIPCR method has good specificity and repeatability with a recovery rate of 96.9%. CONCLUSION: The qIPCR method is highly sensitive for ABA quantification for actual plant samples with an advantage of using crude extracts instead of intensively purified samples. BioMed Central 2018-11-24 /pmc/articles/PMC6260876/ /pubmed/30534191 http://dx.doi.org/10.1186/s13007-018-0371-y Text en © The Author(s) 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology Su, Yi Li, Wei Huang, Zhigang Wang, Ruozhong Luo, Weigui Liu, Qing Tong, Jianhua Xiao, Langtao Sensitive and high throughput quantification of abscisic acid based on quantitative real time immuno-PCR |
title | Sensitive and high throughput quantification of abscisic acid based on quantitative real time immuno-PCR |
title_full | Sensitive and high throughput quantification of abscisic acid based on quantitative real time immuno-PCR |
title_fullStr | Sensitive and high throughput quantification of abscisic acid based on quantitative real time immuno-PCR |
title_full_unstemmed | Sensitive and high throughput quantification of abscisic acid based on quantitative real time immuno-PCR |
title_short | Sensitive and high throughput quantification of abscisic acid based on quantitative real time immuno-PCR |
title_sort | sensitive and high throughput quantification of abscisic acid based on quantitative real time immuno-pcr |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6260876/ https://www.ncbi.nlm.nih.gov/pubmed/30534191 http://dx.doi.org/10.1186/s13007-018-0371-y |
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