法医学杂志

法医学杂志 ›› 2011, Vol. 27 ›› Issue (4): 265-270.DOI: 10.3969/j.issn.1004-5619.2011.04.007

• 论著 • 上一篇    下一篇

常见嗜尸性昆虫mtDNA提取方法的比较

陈瑶清1，郭亚东2，李茂枝3，熊  枫2，李剑波3，蔡继峰2   

  1. （1. 湖南警察学院，湖南 长沙 410138； 2. 中南大学 基础医学院法医学系，湖南 长沙 410013； 3. 长沙市公安局，湖南 长沙 410008）
  • 发布日期:2011-08-25 出版日期:2011-08-28
  • 通讯作者: 通信作者：蔡继峰，男，副教授，主要从事法医昆虫学研究；E-mail：cjf_jifeng@163.com
  • 作者简介:陈瑶清（1974—），女，湖南湘乡人，硕士研究生，主要从事法医病理学研究；E-mail：chenyaoqing1994@21cn.com
  • 基金资助:

    国家自然科学基金资助项目（30672354）；公安部应用创新计划项目（2011YYCXHNST074）；2007年度湖南省优秀青年骨干教师培养项目（湘教通[2007] 214）

Comparison of mtDNA Extracting Methods for Common Sarcosaphagous Insects

CHEN YAO-QING1, GUO YA-DONG2, LI MAO-ZHI3, XIONG FENG2, LI JIAN-BO3, CAI JI-FENG2   

  1. （1. Hunan Police College, Changsha 410138, China; 2. Department of Forensic Medicine, School of Basic Medical Science, Central South University, Changsha 410013, China; 3. Changsha Public Security Bureau, Changsha 410008, China）
  • Online:2011-08-25 Published:2011-08-28

摘要： 目的 比较十六烷基三甲基溴化铵（cetyl triethyl ammonium bromide，CTAB）法、十二烷基硫酸钠-醋酸钾（sodium dodecyl sulfate-potassium acetate，SDS-KAc）法和十二烷基硫酸钠-蛋白酶K（sodium dodecyl sulfate-protein K，SDS-PK）法对法医学常见嗜尸性昆虫mtDNA的提取效果。 方法 随机采集放置在长沙地区室外草地家兔尸体上的大头金蝇、双色葬甲、金龟科、墨胸胡蜂4种72只常见嗜尸性昆虫，分别采用CTAB、SDS-KAc、SDS-PK 3种方法提取总DNA。核酸蛋白测定仪检测DNA纯度及浓度，用mtDNA特异性引物进行PCR扩增，琼脂糖凝胶电泳检测PCR产物，对PCR产物进行序列测定，将测序结果上传到GenBank。 结果 3种方法均能成功提取4种嗜尸性昆虫的mtDNA，SDS-PK法提取效果最好，CTAB法对陈旧性样本提取效果优于另两种方法，SDS-KAc法对各类样本的提取效果相近。 结论 实验中应根据不同情况，选择最恰当的提取方法。制备高质量DNA推荐使用SDS-PK法，陈旧性样本推荐使用CTAB法，在各类预实验中可采取低成本的SDS-KAc法。

关键词: 法医学, 昆虫学, 嗜尸性昆虫, DNA, 线粒体

Abstract: Objective To compare effects of three different methods for mtDNA extraction from common sarcosaphagous insects including cetyl trimethyl ammonium bromide （CTAB） method, sodium dodecyl sulfate-potassium acetate （SDS-KAc） method and sodium dodecyl sulfate-proteinase K （SDS-PK） method. Methods Seventy-two insects from four species [Chrysomya megacephala （Fabricius, 1784）, Eusilpha bicolor （Fairmaire, 1896）, Paraeutrichopus pecoudi （Mateu, 1954）, Vespa velutina （Lepeletier, 1836）] were collected from the corpses of the rabbits in Changsha district. The total DNA of above samples was extracted by CTAB, SDS-Kac and SDS-PK methods. The purity and concentration of DNA were examined by protein-nucleic acid spectrophotometry, and mtDNA were amplified by specific primers and PCR products were detected by agarose gel electrophoresis. Then PCR products were sequenced and subsequently uploaded to GenBank. Results mtDNA was successfully extracted with three methods from most of the samples. The SDS-PK method was better in DNA purity compared to other methods and the CTAB method was superior in extracting DNA from old samples, while SDS-KAc method showed no significant difference for extraction effects of different samples. Conclusion The most appropriate method should be chosen depending on different situations. SDS-PK method is expected to obtain high-quality DNA, while CTAB method is preferred in extracting obsolete samples. SDS-KAc method is low cost and can be used in various kinds of preliminary experiments.

Key words: forensic medicine, entomology, sarcosaphagous insects, DNA, mitochondrial

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