A novel method of detecting alpha-1 antitrypsin deficiency of Z mutant (GAG(342)AAG) in a single PCR reaction using base-quenched probe

Background: Alpha-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.

Methods: Primers 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 (GAG(342)AAG) was analyzed according to the melting curve. Finally, the accuracy was confirmed by direct sequencing.

Results: Z 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 10(3) copies of template DNA with a clear melting valley and a complete concordance occurred between this method and the direct DNA sequencing.

Conclusion: The 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.