Vekris A, Bauduer F, Maillet S, Bébéar C, Bonnet J
Laboratoire de Bactériologie, Université de Bordeaux II, France.
Mol Cell Probes. 1995 Feb;9(1):25-31. doi: 10.1016/s0890-8508(95)90947-8.
We developed a microtitre hybridization assay for the detection of polymerase chain reaction (PCR) amplified sequences. For this, cloned Mycoplasma pneumoniae DNA containing a sequence complementary to the PCR products is first covalently bound to microtitre wells. These coated microplates can be stored for several months. Then, an aliquot of the PCR product, labelled with digoxigenin-dUTP during its synthesis is hybridized to the immobilized DNA. The use of a rapid hybridization buffer makes this step very short (5 min). Finally, the hybridization signal is detected by an anti-digoxigenin antibody conjugated with alkaline phosphatase. Compared to Southern or other microplate hybridization techniques, this method is cheaper, involved fewer steps and allows easy handling of a large number of samples. This method was used for detection of M. pneumoniae in a series of clinical specimens.
我们开发了一种用于检测聚合酶链反应（PCR）扩增序列的微量滴定杂交测定法。为此，首先将含有与PCR产物互补序列的克隆肺炎支原体DNA共价结合到微量滴定孔中。这些包被的微孔板可以储存数月。然后，在合成过程中用洋地黄毒苷-dUTP标记的PCR产物等分试样与固定化DNA杂交。使用快速杂交缓冲液使这一步骤非常短（5分钟）。最后，通过与碱性磷酸酶偶联的抗洋地黄毒苷抗体检测杂交信号。与Southern或其他微孔板杂交技术相比，该方法更便宜，步骤更少，并且便于大量样品的轻松处理。该方法用于一系列临床标本中肺炎支原体的检测。
