The sensitive detection of fluorescently labelled PCR products using an automated detection system

The polymerase chain reaction plays a central role in many detection assays and methods to improve the sensitivity and specificity of these detection systems are constantly being explored. In this study we investigated the use of an automated laser fluorescent system (ALF) in the context of DNA-based diagnostics for pathogenic bacteria. PCR products were generated using species-specific primer sets, one of which was labelled with a 5′ fluorescein. PCR products with a fluorescent label were detected on line with an ALF DNA sequencer and the sensitivity of detection was found to be comparable to that for DNA probe hybridization with a radioactive probe. The technology was successfully applied to the detection of Mycobacterium tuberculosis supplemented into sputum samples and to the detection of listeria in paraffin-embedded tissue samples.