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

作者：Qin Li; Luo Guanghua^*; Zhang Jun; Xu Ning

来源：Clinica Chimica Acta, 2014, 427: 29-33.

DOI：10.1016/j.cca.2013.09.042

• Single nucleotide polymorphism
• Alphal-antitrypsin deficiency
• PIZ mutant

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 +/- 133 degrees C), while when the Z mutation was present, the TM was shifted to an obvious low TM (4138 +/- 0.9017 degrees 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.

• 出版日期2014-1-1
• 单位苏州大学