Use of two reporter dyes without interference in a single-tube rapid-cycle PCR: alpha(1)-antitrypsin genotyping by multiplex real-time fluorescence PCR with the LightCycler

Background: alpha(1)-Antitrypsin is the major plasma serine protease inhibitor. Its deficiency is mainly associated with the alleles PI S and PI Z and can lead to obstructive lung disease in adults and to liver cirrhosis during childhood. Methods: A multiplex PCR method has been established that uses two sets of primers to amplify the gene regions covering the PI S or PI Z mutations sites. Mutation detection was performed on the LightCycler by melting curve analysis of detection probes labeled with two different fluorescent dyes, LC-Red640 and LC-Red705. Results: Unequivocal genotyping results were obtained for all investigated samples in an assay time of similar to 30 min. The color compensation procedure greatly improved the readability of the resulting diagnostic melting curves. Conclusions: To our knowledge, this is the first report of simultaneous detection of two mutations in a single tube by PCR of genomic DNA and the use of two different reporter dyes with the LightCycler color compensation feature. This approach is a rapid, convenient, and economic alternative to other methods described to date for the detection of alpha(1)-antitrypsin deficiency alleles. (C) 2000 American Association for Clinical Chemistry.