法医学杂志

法医学杂志 ›› 2021, Vol. 37 ›› Issue (1): 58-64.DOI: 10.12116/j.issn.1004-5619.2020.400219

• 技术与应用 • 上一篇    下一篇

绿藻ChlB基因和蓝藻NIES基因在溺死相关浮游生物检测中的应用

李欢1 ，徐曲毅2 ，刘超2 ，肖成3 ，赵建2 ，余仲昊3 ，杨幸怡2 ，李越2 ，万立华1   

  1. 1. 重庆医科大学基础医学院法医学教研室，重庆 400016；2. 广州市刑事科学技术研究所 法医病理学公安部重点实验室，广东 广州 510442；3. 南方医科大学法医学院，广东 广州 510515
  • 收稿日期:2020-02-23 发布日期:2021-02-25 出版日期:2021-02-28
  • 通讯作者: 万立华，男，教授，主要从事法医人类学教学及科研；E-mail：ccfw@cqmu.edu.cn
  • 作者简介:李欢（1994—），女，硕士研究生，主要从事溺死相关浮游生物DNA检测；E-mail：923664134@qq.com
  • 基金资助:
    民生科技攻关计划资助项目（2019030001，2019030012）

Application of Chlorophyte ChlB Gene and Cyanophyte NIES Gene in the Detection of Drowning-Related Plankton

LI Huan1 , XU Qu-yi2 , LIU Chao2 , XIAO Cheng3 , ZHAO Jian2 , YU Zhong-hao3 , YANG Xing-yi2 , LI Yue2 , WAN Li-hua1   

  1. 1. Department of Forensic Medicine, Faculty of Basic Medical Sciences, Chongqing Medical University, Chongqing 400016, China; 2. Key Laboratory of Forensic Pathology of Ministry of Public Security, Guangzhou Forensic Science Institute, Guangzhou 510442, China; 3. School of Forensic Science, Southern Medical University, Guangzhou 510515, China
  • Received:2020-02-23 Online:2021-02-25 Published:2021-02-28

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摘要： 目的 利用绿藻 ChlB 基因和蓝藻 NIES 基因，建立聚合酶链反应-毛细管电泳（polymerase chain reaction-capillary electrophoresis，PCR-CE）检测方法，验证该方法的特异性和灵敏度，并探讨其在溺死诊断 中的应用价值。 方法 针对GenBank中绿藻ChlB基因和蓝藻NIES基因的保守序列，设计特异性引物ChlB 和引物NIES，以此构建PCR-CE检测方法。扩增50种标准样本DNA。阳性标准样本梯度浓度检测，确定灵敏度。检测25例尸体肺组织样本（溺死20例，自然死亡5例），同时比较微波消解-真空抽滤-自动扫描电 镜法（microwave digestion-vacuum filtration-automated scanning electron microscopy，MD-VF-Auto SEM）的 同步检测结果。 结果 引物ChlB和引物NIES最低检测DNA含量分别为0.161、0.109 ng，可分别特异性扩 增水体广泛存在的绿藻（蛋白核小球藻）和蓝藻［铜绿微囊藻（产毒及不产毒）］，产物片段分别为156、182 bp，非溺死器官组织检测结果均为阴性。 结论 构建的方法灵敏度高、特异性好，能很好地应用于溺死相关浮游 生物检测，与MD-VF-Auto SEM法联用，可以增大溺死相关浮游生物的检测范围，提高溺死诊断的证据力。

关键词: 法医病理学, 法医遗传学, 溺水, 聚合酶链反应, 毛细管电泳, 蓝藻, 绿藻, ChlB基因, NIES基因

Abstract: Objective To construct a polymerase chain reaction-capillary electrophoresis （PCR-CE） detection method using ChlB gene and NIES gene, investigate the method’s specificity and sensitivity, and to evaluate its application value in drowning diagnosis. Methods The specific primers ChlB and NIES were designed for the conserved sequence of chlorophyte ChlB gene and cyanophyte NIES gene in GenBank to construct PCR-CE detection method; 50 species of standard DNA samples were amplified; the sensitivity was determined by gradient concentration detection of positive standard samples; 25 actual cadaver lung tissue samples （drowned：20, natural death：5） were detected, and the simultaneous detection results of microwave digestion-vacuum filtration-automated scanning electron microscopy （MD-VF-Auto SEM） were simultaneously compared. Results The minimum DNA detection concentration of primers ChlB and NIES was 0.161 ng and 0.109 ng, respectively, which could specifically amplify chlorophyte （Chlorella pyrenoidosa） and cyanophyte ［Microcystis aeruginosa （producing and not producing toxin）］ widespread in water. The product fragments were 156 bp and 182 bp, respectively. The results of non-drowning tissues were negative. Conclusion This method has high sensitivity and specificity. It can be applied to the detection of plankton related to drowning and combined with MD-VF-Auto SEM method, can increase the detection range of plankton related to drowning and improve the evidence power of drowning diagnosis.

Key words: forensic pathology, forensic genetics, drowning, polymerase chain reaction, capillary electrophoresis, cyanophyte, chlorophyte, ChlB gene, NIES gene

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