Zhang Jie, Shi Changgen, Zhang Lei, Zhang Yan, Lu Qing, Wang Rongfang
Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.
Shanghai R&D Center, DiaSys Diagnostic Systems (Shanghai) Co., Ltd., Shanghai, 201318, China.
Heliyon. 2021 Apr 27;7(4):e06858. doi: 10.1016/j.heliyon.2021.e06858. eCollection 2021 Apr.
The aim of the present study is to develop and validate a reliable and simple application for genotyping serum amyloid A1 ().
The specific nested PCR was performed to amplify a product of gene. Two quenching probes (QPs) were designed for detecting two single nucleotide polymorphism (SNP) sites, rs1136743(C/T) and rs1136747(C/T) respectively for genotypes. The specific nested PCR and QPs of genotying was introduced into a fully automated genotyping system (I-densy, ARKRAY, Inc.), which enables the genotyping of from whole blood.
Six genotypes of (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 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.
This fully automated system is a rapid and reliable strategy for the genotyping and for its future clinical application.
本研究的目的是开发并验证一种用于血清淀粉样蛋白A1（）基因分型的可靠且简单的应用方法。
进行特异性巢式PCR以扩增基因的一个产物。设计了两种淬灭探针（QPs）分别用于检测两种单核苷酸多态性（SNP）位点，即rs1136743（C/T）和rs1136747（C/T）的基因型。将基因型分型的特异性巢式PCR和QPs引入全自动基因分型系统（I-densy，ARKRAY公司），该系统能够对全血中的进行基因分型。
通过采用熔解温度（TM）分析监测两种QPs的荧光强度，可以确定的六种基因型（α、β、γ、αβ、αγ和βγ）。使用基于荧光淬灭探针的方法和全自动I-densy系统对总共121份临床样本进行了基因分型，并通过PCR直接测序方法进一步进行序列确认。
这种全自动系统是用于基因分型及其未来临床应用的一种快速且可靠的策略。
