Zammatteo N, Alexandre I, Ernest I, Le L, Brancart F, Remacle J
Laboratoire de Biochimie Cellulaire, Facultés Universitaires Notre-Dame de la Paix, Rue de Bruxelles 61, Namur, B-5000, Belgium.
Anal Biochem. 1997 Nov 15;253(2):180-9. doi: 10.1006/abio.1997.2352.
In this study, we compared the efficiency of capture DNA probes covalently bound onto magnetic beads or microplates for their hybridization with target human cytomegalovirus (HCMV) DNA amplicons. Polystyrene supports were first aminated by wet chemistry to allow covalent grafting of the capture probes. The level of amines grafted was three times higher on beads than on microwells. Increasingly higher sizes of capture probes were fixed on both supports and the best reaction yield ranged from 300 to 500 fmol. The sizes of the capture and detection probes were optimized in order to obtain high target DNA hybridization yield. Long capture probes were more accessible than short ones to the target, with faster kinetics of hybridization obtained on beads than on microplates. Sensitivity of the hybridization assay was then determined with a nonisotopic method and the detection limit found was 30 amol of HCMV amplicons on both supports. HCMV DNA extracted from clinical samples were amplified by PCR. The resulting amplicons were then analyzed using the optimized sandwich hybridization assay discussed here. The results perfectly fitted with the qualitative conclusions obtained after a nested PCR analyzed on agarose gel.
在本研究中，我们比较了共价结合在磁珠或微孔板上的捕获DNA探针与目标人巨细胞病毒（HCMV）DNA扩增子杂交的效率。首先通过湿化学方法对聚苯乙烯载体进行胺化处理，以实现捕获探针的共价接枝。磁珠上接枝的胺水平比微孔板上高两倍。在两种载体上固定了越来越大尺寸的捕获探针，最佳反应产量范围为300至500飞摩尔。对捕获探针和检测探针的尺寸进行了优化，以获得高目标DNA杂交产量。长捕获探针比短捕获探针对目标更易接近，在磁珠上获得的杂交动力学比在微孔板上更快。然后用非同位素方法测定杂交测定的灵敏度，在两种载体上发现的检测限均为30飞摩尔的HCMV扩增子。从临床样本中提取的HCMV DNA通过PCR进行扩增。然后使用本文讨论的优化夹心杂交测定法对所得扩增子进行分析。结果与在琼脂糖凝胶上分析的巢式PCR后获得的定性结论完全吻合。
