法医学杂志

法医学杂志 ›› 2008, Vol. 0 ›› Issue (3): 185-188+.

• 论文 • 上一篇    下一篇

扩增12S rRNA基因鉴定生物检材种属

骆宏;陆惠玲;周新晨;章雅清;姚亚楠;   

  1. 中山大学中山医学院法医物证学教研室,中山大学中山医学院法医物证学教研室,嘉禾县公安局,中山大学中山医学院法医物证学教研室,中山大学中山医学院法医物证学教研室 广东广州510080,广州血液中心,广东广州510095,广东广州510080,湖南嘉禾424500,广东广州510080,广东广州510080
  • 发布日期:2008-06-25 出版日期:2008-06-28

Species Identification of Biomaterials by Amplifying 12S rRNA Gene

LUO HONG1,2, LU HUI-LING1, ZHOU XIN-CHEN3, ZHANG YA-QING1, YAO YA-NAN1 (1. DEPARTMENT OF FORENSIC BIOLOGY, ZHONGSHAN MEDICAL COLLEGE, SUN YAT-SEN UNIVERSITY, GUANGZHOU 510080, CHINA; 2.GUANGZHOU BLOOD CENTER, GUANGZHOU 510095, CHINA; 3.JIAHE PUBLIC SECURI   

  • Online:2008-06-25 Published:2008-06-28

摘要： 目的建立一种能够在同一反应条件下鉴定多个具体物种,又满足简单、快速、特异、灵敏、准确等实用要求的种属鉴定方法。方法从GenBank中获取人、鸡、鸭、鹅、猪、兔、鼠、绵羊、水牛、狗、山羊等11个物种的12SrRNA基因序列,设计一对针对上述11个物种的通用引物和分别针对人、鸡和鸭的特异引物,同时扩增各物种12SrRNA基因。以通用引物扩增片段为内部对照,以特异引物扩增片段用于人、鸡和鸭的种属鉴定,并分别对人、鸡、鸭单一检材以及人和鸡、人和鸭、鸡和鸭等二元混合DNA进行鉴定。结果通用引物扩增,各物种均有400bp左右的扩增片段;特异引物只对各自目标种属有扩增产物,片段大小:人163bp、鸡286bp、鸭374bp;测序结果与GenBank既有序列比对,Identities分值:人100%、鸡99%、鸭100%;通用引物扩增人、鸡和鸭检材的灵敏度为2.5pg;特异引物扩增灵敏度人为2.5pg,鸡、鸭均为200pg;混合DNA中任一种DNA的含量只要高于检测灵敏度即可被准确检出,不受另一种DNA量的干扰;盲测结果准确。结论本方法可以利用同一种PCR反应条件鉴定多个物种的种属。

关键词: 法医物证学, 种属鉴定, 12SrRNA基因, 生物检材

Abstract: Objective To establish an accurate, simple, quick, specific and sensitive method for species identification by amplifying 12S rRNA gene with the same reaction system. Methods Based on the downloaded 12S rRNA gene sequences of eleven species (human, chicken, duck, goose, pig, rabbit, rat, sheep, bull, dog and goat) from GenBank, a pair of universal primers to eleven species and three pairs of specific primers to human,chicken and duck were designed. The amplicons amplified with universal primers were used for internal controls, and the amplicons amplified with specific primers were used as identification of human, chicken and duck. DNA was extracted from various samples including blood stains, fresh or freezing muscles, heat-treated muscles and hairs. Both single DNA of human, chicken or duck and mixed DNA of any two kinds of them were amplified. Results The lengths of universal amplicons were about 400 bp. The lengths of specific amplicons were 163 bp for human, 286 bp for chicken, and 374 bp for duck, respectively. No cross amplification was observed, indicating a high specificity of the specific primers. The identification rate was 100% for human, 99% for chicken, and 100% for duck, respectively. The detection sensitivity ranged from 2.5 pg to 200 pg of DNA concentration depending on species, even in mixtures of different species DNA without interference. Conclusion The method established could identify different species under the same reaction system .

Key words: forensic biology, species identification, 12S rRNA gene, biomaterial