A novel method of detecting alpha-1 antitrypsin deficiency of Z mutant (GAG342AAG) in a single PCR reaction using base-quenched probe

Abstract

BackgroundAlpha-1 antitrypsin (A1AT) is a protease inhibitor that protects the tissues from degradation by neutrophil elastase under certain pathological process. Alpha-1 antitrypsin deficiency (A1ATD) could associate with both lung and liver pathogenicities. Of all the deficiency alleles, Z mutant is the most common variant and causes severe complications. Here, we described a novel and quick method to detect Z mutant using the base-quenched probe technique in only one single PCR reaction. MethodsPrimers and probe were designed based on the base-quenched probe technique. Two vectors, representing the two genotypes, were constructed as amplification templates for validating the method. The Z mutant (GAG342AAG) was analyzed according to the melting curve. Finally, the accuracy was confirmed by direct sequencing. ResultsZ mutant could be accurately distinguished from the wild type. The wild type resulted in high melting temperature (TM) (48.64±1.33°C), while when the Z mutation was present, the TM was shifted to an obvious low TM (41.38±0.9017°C). The sensitivity reached a low of 103 copies of template DNA with a clear melting valley and a complete concordance occurred between this method and the direct DNA sequencing. ConclusionThe present described method is simple, quick and economic as well as suitable for large-scale genotyping studies and clinical testing of Z mutant in patients with emphysema and cirrhosis.