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12th International Symposium on Bioluminescence & Chemiluminescence |
Symposium abstracts:
Roda, Aldo1, Mirasoli, Mara1, Venturoli, Simona2,
Baraldini, Mario3, Musiani, Monica2
1. Dept of Pharmaceutical Sciences, University of Bologna, Bologna, Italy
Email: roda@alma.unibo.it
2. Dept of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, Bologna, Italy
3. Inst. of Chemical Sciences, University of Bologna, Bologna, Italy
A new microtiter plate format was developed, which allows single-well multiple analyte detection. The format consists of a 15-well opaque polystyrene plate, each well divided in seven internal subwells. After immobilizing different probes in each internal subwell and applying the sample in all the volume of the main well, a multi-analyte detection in one sample can be performed by imaging a chemiluminescent signal with an ultrasensitive CCD camera. Detection and typing of six different human papilloma virus (HPV) genotypes in one well was performed by consensus PCR amplification of a sequence within the L1 open reading frame and ELISA identification of the specific HPV genotype. In particular, different HPV type-specific DNA probes were immobilized in each internal subwell. The target sequence, digoxigenin-labelled during amplification, was applied to the main well and incubated: the PCR product was able to specifically hybridise to the immobilized probe corresponding to its genotype. Subsequently, a peroxidase-labelled anti-digoxigenin antibody was added and the peroxidase activity was measured by means of a luminol-based chemiluminescent substrate and imaging with a CCD camera, allowing simultaneous single probe determination thanks to the spatial distribution of the chemiluminescent signal in the main well. The analytical performance of the method was evaluated.
This
is a preprint of an article accepted for publication in Luminescence: Copyright
2001 John
Wiley & Sons, Ltd (Wiley website)