ISSN 1003-8280 CN 10-1522/R 中国疾病预防控制中心 主办
收稿日期: 2014-02-25
网络出版日期: 2014-08-20
基金资助
“十二五”国家科技支撑计划课题(2012BAK11B05);国家质检总局项目(2012IK223);广东出入境检验检疫局课题(2011GDK10,2013GDK36,2013GDK39);中山市课题(20123A298)
Direct amplification of barcode DNA from microtissues of medical vectors
Received date: 2014-02-25
Online published: 2014-08-20
Supported by
Supported by the National Key Technology R&D Program(No. 2012BAK11B05), Administration of Quality Supervision,Inspection and Quarantine Science and Technology Project(No. 2012IK223), Guangdong Entry-Exit Inspection and Quarantine Bureau Science and Technology Project(No. 2011GDK10, 2013GDK36, 2013GDK39)and Zhongshan City Science and Technology Project(No. 20123A298)
目的 为保证DNA条形码序列模板DNA来源的单一性,和最大程度减少凭证标本的形态破坏,建立一种从医学媒介生物微量组织中免提取,直接扩增DNA条形码序列的方法。方法 以螨、蚤、蜱单个个体以及小至蝇类后足1/5跗节的组织块为材料,通过优化裂解液浓度、裂解液和缓冲液配比,优化反应体系和反应条件,用KOD FX DNA聚合酶进行DNA条形码序列的直接扩增,反应产物测序,与NCBI基因库中的序列进行比较以验证其是否被污染。结果 裂解液优化为NaOH 50 mmol/L;裂解液和缓冲液配比优化为9∶1;反应体积优化为每50μl反应体系中2×KOD FX DNA聚合酶buffer(含Mg2 +)25μl、2mmol/LdNTP10μl、KOD FX DNA聚合酶(1U/μl)1μl、正向引物LCO1490(20μmol/L)、反向引物HCO2198(20μmol/L)各1μl;反应条件优化为:95℃3 min;98℃10 s,50℃30 s,68℃1 min,35个循环;68℃7 min。测序结果显示无污染,均为有效序列。结论 该方法操作简单而且省去了DNA提取的步骤,节约了时间和费用,适合螨、蚤、蜱单个个体以及小至蝇类后足1/5跗节的组织块DNA条形码序列的直接扩增。
胡佳,岳巧云,邱德义,吴可量,刘德星,汪小东,魏晓雅,陈健,廖俊蕾 . 医学媒介生物微量组织直接扩增DNA条形码序列方法的研究[J]. 中国媒介生物学及控制杂志, 2014 , 25(4) : 297 -300 . DOI: 10.11853/j.issn.1003.4692.2014.04.003
Objective To ensure the purity of template in amplification of barcode DNA from the microtissues of medical vectors and to minimize the morphological damage to specimens, a method of direct PCR without DNA extraction was established.Methods Individuals of mites, fleas, and ticks, as well as the first tarsus of metapodium from flies, were used as samples in this study. The concentration of lysis buffer and the ratio of lysisvs. stop buffer were optimized to determine the reaction conditions.KOD FX DNA polymerase was used instead of Taq DNA polymerase to directly amplify barcode DNA. The PCR product was sequenced and aligned with GenBank sequences using Blast to test whether the sequences were contaminated. Results The optimized lysis buffer was 50 mmol/L NaOH. The optimized ratio of lysisvs. stopbufferwas180μl∶20μl. The optimized reaction system(50μl) was determined as follows: 2×KOD FX DNA polymerase buffer (containing Mg2+) 25μl, 2mmol/LdNTP 10μl , KOD FX DNA polymerase (1U/μl)1 μl, forward primerLCO1490(20μmol/L)1μl, andreverseprimer HCO2198(20μmol/L)1μl. The reaction conditions were optimized as follows: 95℃3 min for pre-heating; 98℃10 s, 50℃30 s, and 68℃1 min for35 cycles, followed by extension 7 min at 68℃. No contamination was found by Blast alignment of amplified sequences.Conclusion The method established in this study is easy to operate, and omission of DNA extraction will save time and expenses. This method is suitable for direct amplification of barcode DNA from mites, fleas, ticks, and even the first tarsus of fly metapodium.
Key words： Direct PCR; Medical vector; Microtissue; Barcode DNA
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