2. Materials andmethods

2.1. Patients

Blood samples were collected and prepared at the Third Affiliated Hospital of Guangzhou Medical University. Approval of the Sun Yatsen University ethics committee was obtained, and informed consent was obtained from all adult participants and the parents of the children participating. Peripheral blood samples were collected in EDTA tubes and were divided into two groups including 33 G6PD deficient patients and 22 healthy subjects. Patients had been accurately diagnosed prior to the onset of the study.

2.2. Enzyme assay

G6PD activity was determined using the modified G6PD/6PGD ratio method [33] or the WHO standard method as recommended by the World Health Organization [34,35].

2.3. Genotyping

Genomic DNA was extracted from the peripheral whole blood of patients using a modified version of the ammonium acetate salting-out method [36]. PCR of the G6PD and HBB exons and the adjacent intron regions was completed with the primers and PCR conditions described previously [2,37,38]. Anαthalassemia genotyping kit (Yaneng Bioscience,
China)was used to detect common alpha thalassemiamutations in China. All PCR products were purified and sequenced in both the forward and reverse directions using the ABI PRISM 3730 DNA sequencer (Applied Biosystems) by BGI to identify mutations. Data was collected and analyzed using Chromas 2.33 and BLAST.

译文

2 材料和方法

2.1病人来源

在广州医科大学附属第三医院采集和制备血液样本。获得中山大学伦理委员会的批准，并获得所有成年参与者和参与儿童家长的知情同意。采用EDTA管采集外周血样本，分为两组，G6PD缺乏患者33例，健康者22例。患者在研究开始前已被准确诊断。

2.2 酶法测定

G6PD活性采用改良的G6PD/6PGD比值法[33]或世界卫生组织推荐的WHO标准方法[34,35]。

2.3 基因型分型

基因分型采用改良版的醋酸铵盐析法[36]从患者外周血中提取基因组DNA。G6PD和HBB外显子及其邻近内含子区域的PCR用前面描述的引物和PCR条件完成[2,37,38]。使用α地中海贫血基因分型试剂盒(亚能生物科学，中国)检测中国常见的α地中海贫血突变。所有PCR产物均使用华大基因的ABI PRISM 3730 DNA测序仪(Applied Biosystems)进行正向和反向测序，以鉴定突变。使用Chromas 2.33和BLAST收集和分析数据。