Fluorescent quenching probes based SAA 1 genotyping with a fully automated system

Abstract

Objective: The aim of the present study is to develop and validate a reliable and simple application for genotyping serum amyloid A1 (SAA1). Methods: The specific nested PCR was performed to amplify a product of SAA1 gene. Two quenching probes (QPs) were designed for detecting two single nucleotide polymorphism (SNP) sites, rs1136743(C/T) and rs1136747(C/T) respectively for SAA1 genotypes. The specific nested PCR and QPs of SAA1 genotying was introduced into a fully automated genotyping system (I-densy, ARKRAY, Inc.), which enables the genotyping of SAA1 from whole blood. Results: Six genotypes of SAA1 (α+/+, β+/+, γ+/+, αβ, αγ and βγ) could be determined by monitoring the fluorescence intensity of two QPs with melting temperature (TM) analysis. Total 121 clinical samples were SAA1 genotyped in the fluorescent quenching probes based method with a fully automated I-densy system and were further sequence confirmed with a PCR direct sequencing approach. Conclusion: This fully automated system is a rapid and reliable strategy for the SAA1 genotyping and for its future clinical application.

Keywords

• SAA1
• Genotyping
• Quenching probe
• I-densy
• Molecular diagnosis