从口腔黏膜拭子提取基因组DNA进行年龄相关性黄斑变性易感基因的检测  

Use of buccal swab as a source of genomic DNA for genetic screening in patients with age-related macular degeneration

作　　者：徐玉乐[1] 周海英[1] 徐军[1] 刘宁朴[1] 

机构地区：[1]首都医科大学附属北京同仁医院北京同仁眼科中心北京市眼科学与视觉科学重点实验室,100730

出　　处：《中华眼科杂志》2012年第2期114-118,共5页Chinese Journal of Ophthalmology

基　　金：国家重点基础研究发展（973）计划（2007CB512201）;北京市卫生系统高层次卫生技术人才培养计划（2009208）

摘　　要：目的 探讨应用口腔黏膜拭子提取基因组DNA进行渗出性年龄相关性黄斑变性（AMD）易感基因检测的可行性.方法 对照研究.选择已确诊的65例渗出性AMD患者.应用口腔拭子刮取患者不同部位（单侧颊黏膜与上下齿龈沟）的口腔黏膜,提取基因组DNA,应用分光光度计测定基因组DNA含量.抽取患者静脉血,应用PCR和限制性酶切分析法,对CFH、LOC387715及HTRA1位点进行基因型分析,比较口腔黏膜拭子取材与血液样本取材所获基因分型结果的一致性.口腔黏膜两种取材法提取的基因组DNA含量比较,采用Student t检验.结果 每位受试者分别获得3个口腔黏膜拭子,其中2个拭子分别从患者单侧颊黏膜取材,另1个拭子从患者上下齿龈沟处取材.于取材1周内检测基因组DNA含量.单侧颊黏膜取材患者基因组DNA含量为（3.17±1.46） μg,上下齿龈沟取材患者基因组DNA含量为（3.94±1.04） μg,口腔黏膜两种取材法间获取的基因组DNA含量差异有统计学意义（t=6.79,P＜0.05）.单侧颊黏膜拭子干燥保存（- 20℃）12个月后检测基因组DNA含量为（3.10±1.17） μg,与取材1周内的单颊黏膜拭子检测结果比较,差异无统计学意义（t =0.59,P＞0.05）.应用口腔黏膜拭子与血液样本提取的基因组DNA分型结果完全一致.结论 口腔黏膜拭子是一种简单、无创、可靠的获取基因组DNA的方法,可部分替代静脉血进行AMD的易感基因分析.口腔黏膜拭子干燥保存（- 20℃）12个月后基因组DNA含量未减少.Objective The collection of buccal cells with swabs provides a noninvasive method for obtaining genomic DNA for genetic screening.We aimed to study the feasibility of using buccal swabs for genetic screening in patients with exudative age-related macular degeneration （AMD）.Methods Blood and buccal swabs were collected for genetic analysis from 65 patients with exudative AMD.Genomic DNA was isolated from either blood or buccal swabs.The yield of genomic DNA from both sources was determined by spectrophotometer.Genotyping for CFH,LOC387715,and HTRA1 Polymorphisms was performed using a method of polymerase chain reaction （PCR） followed by restriction enzyme digestion.Results using genomic DNA from blood or buccal swabs were compared.Results Three swabs were obtained from each patient,2 from each side of buccal mucosa,and 1 from both upper and inferior gingival mucosa.From swabs with genomic DNA extracted within a week after sample collection,an average of （3.17 ± 1.46） μg genomic DNA was obtained from swab collected from the left or right side buccal mucosa,and （ 3.94 ± 1.04） μg from swab collected from the upper and inferior gingival mucosa,with relatively higher yield of genomic DNA from the upper and inferior gingival mucosa （ t =6.79,P 〈 0.05 ）.From swabs of the left or right side buccal mucosa after being stored at -20 ℃ for 12 months,an average of （3.10 ± 1.17） μg genomic DNA was obtained,which showed no statistically significant difference as compared to the yield of genomic DNA extracted from newly collected swabs （t =0.59,P 〉 0.05）.In all 65 patients,genomic DNA isolated from either buccal swabs or blood samples showed exactly the same results regarding the genotypes of CFH,LOC387715,and HTRA1 Polymorphisms. Conclusions Buccal swab is a simple,noninvasive,and reliable source for obtaining genomic DNA.Swabs stored for 12 months at - 20 ℃ provide similar amount of genomic DNA as the freshly collected swabs.

关 键 词：口腔黏膜 标本制备 基因组 DNA 黄斑变性 

分 类 号：R774.5[医药卫生—眼科]