Massart Sébastien, De Clercq Deborah, Salmon Michel, Dickburt Catherine, Jijakli M Haissam
Plant Pathology Unit, Faculté Universitaire des Sciences Agronomiques de Gembloux, Passage des Déportés, 2, 5030 Gembloux, Belgium.
J Microbiol Methods. 2005 Jan;60(1):73-82. doi: 10.1016/j.mimet.2004.08.012.
A real-time PCR assay using a 3'-Minor Groove Binding (MGB) probe was developed for specific detection and monitoring of Candida oleophila (strain O), a biocontrol agent against Botrytis cinerea and Penicillium expansum, on harvested apples. The application of the RAPD technique on C. oleophila strains followed by reproducible sequence characterized amplified region (SCAR) amplifications allowed the identification of a semi-specific fragment of 244 bp, observed in the profiles of strain O and three other C. oleophila strains. After sequencing, polymorphisms (3%) were observed between the strain O sequence and the three other sequences. A 3'-Minor Groove Binding probe was designed to specifically match a region of the strain O sequence and was able to discriminate a single base mutation or a two-base difference in the corresponding sequences of the non-target strains. This specific detection method was applied to monitor strain O population, recovered by a washing buffer, from harvested apples. Population densities were calculated using an external standard curve consisting in a serial dilution of strain O cells in the washing buffer from untreated apples. Linearity in the standard curve was kept between 1.64 x 10(2) and 1.64 x 10(5) cfu cm(-2) of apple surface. During a first practical experiment, the calculated population densities were similar to those obtained by plating on semi-selective media. This new real-time PCR method is a promising tool to monitor quickly and specifically strain O population on apple surface in middle- or large-scale experiments.
开发了一种使用3'-小沟结合（MGB）探针的实时PCR检测方法，用于特异性检测和监测采后苹果上的嗜油假丝酵母（菌株O），该菌是灰葡萄孢和扩展青霉的生物防治剂。对嗜油假丝酵母菌株应用随机扩增多态性DNA（RAPD）技术，随后进行可重复的序列特征扩增区域（SCAR）扩增，从而鉴定出一个244 bp的半特异性片段，该片段在菌株O和其他三株嗜油假丝酵母的图谱中均有观察到。测序后，在菌株O序列与其他三个序列之间观察到多态性（3%）。设计了一种3'-小沟结合探针，使其与菌株O序列的一个区域特异性匹配，并且能够区分非靶标菌株相应序列中的单个碱基突变或两个碱基差异。将这种特异性检测方法应用于监测通过洗涤缓冲液从采后苹果中回收的菌株O群体。使用由未处理苹果洗涤缓冲液中菌株O细胞的系列稀释液组成的外标曲线计算群体密度。标准曲线的线性范围保持在苹果表面1.64×10²至1.64×10⁵ cfu cm⁻²之间。在首次实际实验中，计算得到的群体密度与通过在半选择性培养基上平板计数获得的密度相似。这种新的实时PCR方法是在中大型实验中快速、特异性监测苹果表面菌株O群体的一种有前景的工具。
