Sensitive and high throughput quantification of abscisic acid based on quantitative real time immuno-PCR
Abstract 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 quant...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2018-11-01
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| Series: | Plant Methods |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13007-018-0371-y |
| _version_ | 1819058577371824128 |
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| author | Yi Su Wei Li Zhigang Huang Ruozhong Wang Weigui Luo Qing Liu Jianhua Tong Langtao Xiao |
| author_facet | Yi Su Wei Li Zhigang Huang Ruozhong Wang Weigui Luo Qing Liu Jianhua Tong Langtao Xiao |
| author_sort | Yi Su |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-12-21T13:57:25Z |
| format | Article |
| id | doaj.art-3512758d8a7047818b2a2fc014bc333b |
| institution | Directory Open Access Journal |
| issn | 1746-4811 |
| language | English |
| last_indexed | 2024-12-21T13:57:25Z |
| publishDate | 2018-11-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj.art-3512758d8a7047818b2a2fc014bc333b2022-12-21T19:01:29ZengBMCPlant Methods1746-48112018-11-0114111010.1186/s13007-018-0371-ySensitive and high throughput quantification of abscisic acid based on quantitative real time immuno-PCRYi Su0Wei Li1Zhigang Huang2Ruozhong Wang3Weigui Luo4Qing Liu5Jianhua Tong6Langtao Xiao7Hunan Provincial Key Laboratory of Phytohormones and Growth Development, Hunan Agricultural UniversityHunan Provincial Key Laboratory of Phytohormones and Growth Development, Hunan Agricultural UniversityHunan Provincial Key Laboratory of Phytohormones and Growth Development, Hunan Agricultural UniversityHunan Provincial Key Laboratory of Phytohormones and Growth Development, Hunan Agricultural UniversityHunan Provincial Key Laboratory of Phytohormones and Growth Development, Hunan Agricultural UniversityHunan Provincial Key Laboratory of Phytohormones and Growth Development, Hunan Agricultural UniversityHunan Provincial Key Laboratory of Phytohormones and Growth Development, Hunan Agricultural UniversityHunan Provincial Key Laboratory of Phytohormones and Growth Development, Hunan Agricultural UniversityAbstract 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.http://link.springer.com/article/10.1186/s13007-018-0371-yqIPCRABABiotinAvidin |
| spellingShingle | Yi Su Wei Li Zhigang Huang Ruozhong Wang Weigui Luo Qing Liu Jianhua Tong Langtao Xiao Sensitive and high throughput quantification of abscisic acid based on quantitative real time immuno-PCR Plant Methods qIPCR ABA Biotin Avidin |
| 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 | qIPCR ABA Biotin Avidin |
| url | http://link.springer.com/article/10.1186/s13007-018-0371-y |
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