Molecular and CellularProbes (1995) 9, 25-32 Improved microplate immunoenzymatic assay of PCR products for rapid detection of Mycoplasma pneumoniae Antoine Vekris, 1 Frederic Bauduer7 Sophie Maillet, z Christiane B~b~ar 1 and Jacques Bonnet 2. 1Laboratoire de Bact~riologie, Universit~ de Bordeaux II, 146, rue L~o Saignat, 33076 Bordeaux cedex, and 21BGC-CNRS, 1, rue Camille Saint SaEns, 33077 Bordeaux cedex, France (Received 4 May 1994, Accepted 20 September 1994) We developed a microtitre hybridization assay for the detection of polymerase chain reaction (PCR) amplified sequences. For this, cloned Mycoplasma pneurnoniae 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. KEYWORDS: Mycoplasma pneumoniae, detection, PCR, microplate, non-isotopic labelling. INTRODUCTION Polymerase chain reaction (PCR) is a powerful tool for the detection and identification of infectious agents. Generally, a two-step method is used: first, am- plification and second, identification of the amplified products. The most commonly used identification procedure is gel electrophoresis. However, when dealing with clinical specimens non-specific am- plification is a common occurrence, a further analysis involving a hybridization step must be done to assess the specificity of the amplified products. This can be achieved by Southern or dot-blot hybridization. However, these techniques are not convenient for clinical laboratories because they are time-consuming and difficult to apply to the processing of a large number of samples. As the PCR technology is cur- rently finding a wide application in routine medical diagnosis, procedures for mass screening must be developed. Several techniques involving microplates and taking advantage of the specificity of hy- bridization have been proposed. A first format is based on the immobilization .of a capture nucleic acid molecule: oligonucleotide I or DNA 2'3 on a solid support and the hybridization of the PCR product labelled during its synthesis. A second format consists in immobilizing the PCR product and hybridizing it with a specific probe. 4 We describe here a fast, sensitive and very simple hybridization assay. Plasmid DNA containing a spe- cific sequence, covalently bound to microplate wells is used to capture PCR products labelled during their synthesis by incorporation of Dig-dUMP. We used it for detecting Mycoplasma pneumoniae sequences in clinical samples. After completion of this work, Gibellini et aL s * Author to whom correspondenceshould be addressedat: Laboratoired'lmmunologie et de Parasitologie, Universit~ de Bordeaux II, Bat lb, 146, rue L~o Saignat, 33076 Bordeauxcedex, France. 0890-8508195/010025+ 07 $08.0010 25 © 1995 Academic Press Limited