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Sensitive and high throughput quantification of abscisic acid based on quantitative real time immuno-PCR
Yi Su1,2, Wei Li1,3, Zhigang Huang1,2
11Hunan Provincial Key Laboratory of Phytohormones and Growth Development, Hunan Agricultural University, Changsha, China.
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.
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