Phytopathology. 2003 Sep;93(9):1153-7. doi: 10.1094/PHYTO.2003.93.9.1153.
ABSTRACT Three polymerase chain reaction (PCR) techniques-standard PCR (Std-PCR), direct-binding PCR (DB-PCR), and immunocapture PCR (IC-PCR)-using degenerate primers were optimized and evaluated for the detection of begomoviruses. Tomato leaf samples were ground in three different extraction buffers and subjected to Std-PCR. The effect of the buffers on the detection limits of amplification of the virus (detection of the initial and end points of dilution) was determined. With the optimal extraction buffer determined by the first experiment, the antibody concentration and incubation conditions for IC-PCR were evaluated to determine the requirements for maximum capture of antigens during the capture phase of the technique. The incubation conditions of DB-PCR were also investigated to determine the most favorable conditions for adsorption of the viral template. The reproducibility of all assays was evaluated. With the results of the optimization experiments, the applicability of the three techniques to different plant species was assessed. Extracts of plant species belonging to three families were subjected to the optimized Std-, DB-, and IC-PCR protocols. Std- and IC-PCR both achieved reproducible detection of begomoviruses, but the detection limits and amplified band intensity for all plant species tested were superior for the latter. DB-PCR was an unreliable method of detection, because of poor reproducibility and low intensity of amplified bands. These results indicate that the optimized IC-PCR detection system using degenerate primers is the most effective for the detection of begomoviruses in clarified plant extracts.
摘要 使用简并引物的三种聚合酶链反应（PCR）技术——标准 PCR（Std-PCR）、直接结合 PCR（DB-PCR）和免疫捕获 PCR（IC-PCR）——经过优化和评估，可用于检测双生病毒。将番茄叶片样品在三种不同的提取缓冲液中研磨，并进行 Std-PCR。确定了缓冲液对病毒扩增检测限（检测稀释的初始和终点）的影响。通过第一个实验确定了最佳提取缓冲液后，评估了 IC-PCR 的抗体浓度和孵育条件，以确定在技术的捕获阶段最大程度捕获抗原的要求。还研究了 DB-PCR 的孵育条件，以确定吸附病毒模板的最有利条件。评估了所有检测的重现性。根据优化实验的结果，评估了三种技术在不同植物物种中的适用性。对属于三个科的植物物种的提取物进行了优化的 Std-PCR、DB-PCR 和 IC-PCR 方案。Std-PCR 和 IC-PCR 都可重复性地检测到双生病毒，但对于所有测试的植物物种，后者的检测限和扩增带强度都更优。DB-PCR 是一种不可靠的检测方法，因为重现性差且扩增带强度低。这些结果表明，使用简并引物优化的 IC-PCR 检测系统是澄清植物提取物中检测双生病毒最有效的方法。
