高分辨熔解方法的建立及在真菌感染患者CYP2C19遗传多态性检测中的应用

目的 探讨高分辨熔解方法 (high resolution melting,HRM)检测真菌感染患者CYP2C19遗传多态性的可行性.方法 建立HRM方法 检测CYP2C19 * 2和CYP2C19 * 3两个多态性位点的基因型,并与传统方法 PCR-限制性片段长度多态性(PCR-RFLP)及序列分析结果 相比较,进一步应用于47例真菌感染患者基因型的检测与分析.结果 2种方法 检测CYP2C19 * 2和CYP2C19* 3基因型结果 完全一致,与DNA序列分析结果 也相吻合,而HRM方法 操作更为简便,耗时更短.47份临床标本检测结果 显示,CYP2C19的2个多态性位点存在不同基因型,CYP2C19 * 1/ * 1所占比例为48.9%(23/47),CYP2C19 * 1/ * 2为25.5%(12/47),CYP2C19 * 1/ * 3为6.4%(3/47),CYP2C19 * 2/ * 2为12.8%(6/47),CYP2C19 * 2/ * 3为6.4%(3/47).结论 HRM方法 能有效检测CYP2C19基因多态性,且较PCR-RFLP方法 更为简便、快速.此外,CYP2C19基因在真菌感染患者中存在明显的遗传多态性.

Identification of CYP2C19 genetic polymorphism by high resolution melting technique in patients with invasive fungal infections

Objective To develop an assay based on high resolution melting (HRM) technique for detecting two major CYP2C19 alleles, CYP2CI9 * 2 and CYP2C19 * 3. Methods HRM curves were analyzed and genotypes of CYP2C19 * 2 and CYP2C19 * 3 were determined. The HRM results were compared with those obtained from PCR-restriction fragment length polymorphism (RFLP) assay or sequencing. HRM was further used in the genotyping in 47 patients with invasive fungal infections. Results There was excellent consistency in results obtained by HRM and PCR-RFLP. Genotypes were confirmed by DNA sequencing. CYP2C19 genotypes can be readily and rapidly detected with HRM assay. Various genotypes were detected in 47 clinical samples, 48.9% ( 23/47 ) were CYP2C19 * 1/* 1, 25.5% ( 12/47 ) were CYP2CI9 * 1/* 2, 6. 4% (3/47) were CYP2C19 * 1/* 3, 12. 8% (6/47) were C~P2C19 * 2/* 2, and 6. 4% (3/47) were CYP2C19 * 2/* 3. Conclusions HRM is a more rapid and sensitive method for detection of CYP2C19 genotypes compared with RFLP assay. CYP2C19 exhibits significant genetic polymorphism in patients with invasive fungal infection.