新医学
新醫學
신의학
NEW CHINESE MEDICINE
2015年
8期
524-527
,共4页
黄烁丹%占伟%邹婕%庄宇嫦%熊蓉%朱莎莎%张华能%栾国彦
黃爍丹%佔偉%鄒婕%莊宇嫦%熊蓉%硃莎莎%張華能%欒國彥
황삭단%점위%추첩%장우항%웅용%주사사%장화능%란국언
干血斑%地中海贫血%DNA 提取%荧光 PCR 熔解曲线法
榦血斑%地中海貧血%DNA 提取%熒光 PCR 鎔解麯線法
간혈반%지중해빈혈%DNA 제취%형광 PCR 용해곡선법
Dried blood spot%Thalassemia%DNA extraction%Fluorescence PCR melting curve analysis
目的：研究滤纸干血斑（DBS）DNA 的自动化提取方法及其在地中海贫血（地贫）基因诊断中的应用。方法将276份经静脉血标本基因检测确诊为地贫的孕妇外周血标本按照研究需要分别制成240份合格、18份渗透不良和18份重复滴血滤纸干血斑标本，并模拟不同的运输和储存条件。使用 Lab-Aid 820核酸提取仪对滤纸干血斑进行 DNA 提取，考察不同的起始血斑用量检测的准确度，对提取的 DNA 进行浓度和纯度分析，并分别使用荧光 PCR 熔解曲线法和 PCR 联合膜杂交法对DNA 进行地贫基因检测。结果276份滤纸干血斑标本提取的 DNA 浓度9.85～29.56 ng／μl，纯度1.63～1.95，均能满足试剂盒检测要求，其中2种基因型检测方法的检测结果100％符合，2种方法均检出地贫基因突变 IVS-Ⅱ-654（C ＞T）、-28（A ＞G）、CD17（A ＞T）、CD26（G ＞A）、CD41／42（-TTCT）、--αSEA、-α3.7、--α4.2、αCSα，所有结果均与使用静脉血提取 DNA 作为标本的检测结果100％符合，覆盖了目前临床上常见的各种基因型。结论DBS 标本对运输、保存要求较低，结合自动化核酸提取仪提取 DNA，操作简单、提取结果稳定，可用于临床筛查地贫基因。
目的：研究濾紙榦血斑（DBS）DNA 的自動化提取方法及其在地中海貧血（地貧）基因診斷中的應用。方法將276份經靜脈血標本基因檢測確診為地貧的孕婦外週血標本按照研究需要分彆製成240份閤格、18份滲透不良和18份重複滴血濾紙榦血斑標本，併模擬不同的運輸和儲存條件。使用 Lab-Aid 820覈痠提取儀對濾紙榦血斑進行 DNA 提取，攷察不同的起始血斑用量檢測的準確度，對提取的 DNA 進行濃度和純度分析，併分彆使用熒光 PCR 鎔解麯線法和 PCR 聯閤膜雜交法對DNA 進行地貧基因檢測。結果276份濾紙榦血斑標本提取的 DNA 濃度9.85～29.56 ng／μl，純度1.63～1.95，均能滿足試劑盒檢測要求，其中2種基因型檢測方法的檢測結果100％符閤，2種方法均檢齣地貧基因突變 IVS-Ⅱ-654（C ＞T）、-28（A ＞G）、CD17（A ＞T）、CD26（G ＞A）、CD41／42（-TTCT）、--αSEA、-α3.7、--α4.2、αCSα，所有結果均與使用靜脈血提取 DNA 作為標本的檢測結果100％符閤，覆蓋瞭目前臨床上常見的各種基因型。結論DBS 標本對運輸、保存要求較低，結閤自動化覈痠提取儀提取 DNA，操作簡單、提取結果穩定，可用于臨床篩查地貧基因。
목적：연구려지간혈반（DBS）DNA 적자동화제취방법급기재지중해빈혈（지빈）기인진단중적응용。방법장276빈경정맥혈표본기인검측학진위지빈적잉부외주혈표본안조연구수요분별제성240빈합격、18빈삼투불량화18빈중복적혈려지간혈반표본，병모의불동적운수화저존조건。사용 Lab-Aid 820핵산제취의대려지간혈반진행 DNA 제취，고찰불동적기시혈반용량검측적준학도，대제취적 DNA 진행농도화순도분석，병분별사용형광 PCR 용해곡선법화 PCR 연합막잡교법대DNA 진행지빈기인검측。결과276빈려지간혈반표본제취적 DNA 농도9.85～29.56 ng／μl，순도1.63～1.95，균능만족시제합검측요구，기중2충기인형검측방법적검측결과100％부합，2충방법균검출지빈기인돌변 IVS-Ⅱ-654（C ＞T）、-28（A ＞G）、CD17（A ＞T）、CD26（G ＞A）、CD41／42（-TTCT）、--αSEA、-α3.7、--α4.2、αCSα，소유결과균여사용정맥혈제취 DNA 작위표본적검측결과100％부합，복개료목전림상상상견적각충기인형。결론DBS 표본대운수、보존요구교저，결합자동화핵산제취의제취 DNA，조작간단、제취결과은정，가용우림상사사지빈기인。
Objective To study the method of automatic DNA extraction from dried blood spots (DBS)on filter pater and its application in the molecular diagnosis of thalassemia.Methods Peripheral blood specimens were collected from 276 pregnant women who were diagnosed with thalassemia through molecular di-agnostic procedures using venous blood samples.The 276 peripheral blood samples were used to prepare 240 standard DBS specimens,1 8 DBS specimens with incomplete blood penetration,and 1 8 DBS specimens with repeated blood blotting,according to the study protocol.Different transportation and storage conditions were simulated.DNA was extracted from the DBS onto filter paper with the Lab-Aid 820 Nucleic Acid Extraction System.The concentration and purity of the extracted DNA were analyzed with consideration of the different ini-tial amounts of blood used for the preparation of DBS.Fluorescence PCR melting curve analysis and the PCR-based reverse dot-blot method were then used to detect the gene mutations responsible for thalassemia in the pa-tients.Results The concentrations of the DNA extracted from the 276 DBS samples ranged from 9.85 ng/μl to 29.56 ng/μl,with a purity of 1 .63 ~1 .95,and met the requirements of the diagnostic kits.The results from the two genotyping methods were 1 00% consistent,and both methods detected gene mutations present in the common genotypes that are clinically associated with thalassemia,including IVS-Ⅱ-654 (C >T),-28 (A>G),CD1 7 (A >T),CD26 (G >A),CD41 /42 (-TTCT),--αSEA ,-α3.7 ,--α4.2 ,and αCS α.The genoty-ping results of the 276 DBS samples were completely consistent with the results from the assay in which DNA was directly extracted from venous blood samples.Conclusions DBS specimens do not require stringent con-ditions for transportation and storage.Automatic nucleic acid extraction and purification from DBS samples pro-vides a quick,simple,and reliable method and is suitable for clinical screening of the gene mutations associat-ed with thalassemia.
