Su Yi, Li Wei, Huang Zhigang, Wang Ruozhong, Luo Weigui, Liu Qing, Tong Jianhua, Xiao Langtao
1Hunan Provincial Key Laboratory of Phytohormones and Growth Development, Hunan Agricultural University, Changsha, China.
2Southern Regional Collaborative Innovation Center for Grain and Oil Crops in China, Hunan Agricultural University, Changsha, China.
Plant Methods. 2018 Nov 24;14:104. doi: 10.1186/s13007-018-0371-y. eCollection 2018.
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.
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%.
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.
脱落酸（ABA）在植物许多生长和发育过程中作为一种应激植物激素发挥作用。对ABA进行超灵敏测定将有助于更好地理解其重要作用和作用机制。
我们报道了一种基于生物素-抗生物素蛋白连接系统的新型灵敏且高通量的定量实时免疫PCR（qIPCR）方法，用于植物中ABA的测定。将ABA单克隆抗体（McAb）包被在经戊二醛预处理的PCR孔内表面。预先制备的探针复合物，包括生物素化的McAb、生物素化的DNA和链霉亲和素连接体，便于进行高通量操作。最后，通过实时PCR对探针DNA进行定量。可检测范围为10至40 ng/L，检测限（LOD）为2.5 fg。使用LC-MS/MS同时分析植物样品中的ABA含量，以验证qIPCR方法。结果表明，qIPCR方法具有良好的特异性和重复性，回收率为96.9%。
qIPCR方法对实际植物样品中ABA的定量高度灵敏，具有使用粗提物而非经过深度纯化样品的优势。
