Use of Two Reporter Dyes without Interference in a Single-Tube Rapid-Cycle PCR: α1-Antitrypsin Genotyping by Multiplex Real-Time Fluorescence PCR with the LightCycler

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Abstract
Background: α1-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 ∼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 α1-antitrypsin deficiency alleles.

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