Claudel Laure, Valeix Nicolas, Basmaciyan Louise, Pereira Bruno, Costa Damien, Vincent Anne, Valot Stéphane, Favennec Loic, Dalle Frederic
Laboratoire de Parasitologie-Mycologie, Plateforme de Biologie Hospitalo-Universitaire, 2 rue A. Ducoudray, BP 37013, CEDEX, 21070 Dijon, France.
UMR PAM Univ Bourgogne Franche-Comté-AgroSup Dijon-Equipe Vin, Aliment, Microbiologie, Stress, CEDEX, 21078 Dijon, France.
Microorganisms. 2021 Feb 2;9(2):297. doi: 10.3390/microorganisms9020297.
Nowadays, many commercial kits allow the polymerase chain reaction (PCR) detection of deoxyribonucleic acid (DNA) in stool samples, the efficiency of which relies on the extraction method used. Mechanical pretreatment of the stools using grinding beads has been reported to greatly improve this extraction step. However, optimization of this key step remains to be carried out. Indeed, many parameters could influence the pretreatment performances, among which the modulation of the speed and duration of the grinding step, in addition to the physicochemical features of the grinding beads, have never been evaluated to date. In this study, eleven commercial mechanical pretreatment matrixes (Lysis matrix tubes, MP Biomedical, Irvine, CA, USA) composed of beads with different sizes, shapes, and molecular compositions, were evaluated for their performances in improving oocyst DNA extraction before amplification by using our routinely used real-time PCR method. As expected, the eleven commercial mechanical pretreatment matrixes showed varying performances depending on the composition, size, and shape. All in all, the best performances were obtained when using the Lysing matrix, including ceramic beads with a median size (diameter of 1.4 mm).
如今，许多商业试剂盒可对粪便样本中的脱氧核糖核酸（DNA）进行聚合酶链反应（PCR）检测，其效率取决于所使用的提取方法。据报道，使用研磨珠对粪便进行机械预处理可大大改善这一提取步骤。然而，这一关键步骤仍有待优化。事实上，许多参数可能会影响预处理性能，其中研磨步骤的速度和持续时间的调节，以及研磨珠的物理化学特性，迄今为止从未进行过评估。在本研究中，我们使用常规的实时PCR方法，对11种由不同大小、形状和分子组成的珠子构成的商业机械预处理基质（裂解基质管，MP Biomedicals，美国加利福尼亚州欧文市）在扩增前改善卵囊DNA提取方面的性能进行了评估。正如预期的那样，这11种商业机械预处理基质根据其组成、大小和形状表现出不同的性能。总体而言，使用包含中值大小（直径为1.4毫米）陶瓷珠的裂解基质时获得了最佳性能。
