摘要
目的建立藻类16Sr DNA特异性片段扩增方法,探讨其在溺死诊断中的应用价值。方法 35只实验兔随机分组:生前入水组(溺死组)15只,死后入水组(空气栓塞致死后入水)15只,对照组(空气栓塞死后不作处理)5只;微波消解-真空抽滤-电镜扫描法检测过的20例水中尸体肝脏样本20份(硅藻阳性14份,阴性6份)。7种已知藻(直链藻、菱形藻、针杆藻、舟形藻,铜绿微囊藻,小环藻,小球藻)作为对照。提取组织样本及藻类DNA,扩增产物银染显带。结果生前入水组肺、肝、肾检出率分别为100%,86%,86%;死后入水组肺、肝、肾检出率分别为13%,0%,0%。对照组肺、肝、肾藻类未检出。生前入水组与死后入水组各种脏器中藻类检出率差异显著(P<0.05)。20份经微波消解-真空抽滤-电镜扫描法检测的水中尸体肝脏样本中,使用本方法15份样本结果为阳性(包括1份硅藻阴性样本)。7种藻类DNA扩增结果为阳性。结论本文所建立的PCR法检测藻类16Sr DNA灵敏度高,可对多种溺死藻类同时检测,有较好的应用前景。
Objective To establish a method for amplifying specific 16S rDNA fragment of algae related with drowning and test its value in drowning diagnosis. Methods Thirty-five rabbits were randomly divided into 3 the drowning group （n=15）, postmortem water immersion group （n=15, subjected to air embolism before seawater immersion）, and control group（n=5, with air embolism only）. Twenty samples of the liver tissues from human corpses found in water were also used, including 14 diatom-positive and 6 diatom-negative samples identified by microwave digestion-vacuum filtration-automated scanning electron microscopy （MD-VF-Auto SEM）. Seven known species of algae served as the control algae （Melosira sp, Nitzschia sp, Synedra sp, Navicula sp, Microcystis sp, Cyclotella meneghiniana, and Chlorella sp）. The total DNA was extracted from the tissues and algae to amplify the specific fragment of algae followed by 8% polyacrylamide gelelectrophoresis and sliver-staining. Results In the drowning group, algae was detected in the lungs （100%）, liver （86%）, and kidney （86%）; algae was detected in the lungs in 2 rabbits in the postmortem group （13%） and none in the control group. The positivity rates of algae were significantly higher in the drowning group than in the postmortem group （P〈0.05）. Of the 20 tissue samples from human corps found in water, 15 were found positive for algae, including sample that had been identified as diatom-negative by MD-VF-Auto SEM. All the 7 control algae samples yielded positive results in PCR. Conclusion The PCR-based method has a high sensitivity in algae detection for drowning diagnosis and allows simultaneous detection of multiple algae species related with drowning.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2015年第8期1215-1218,共4页
Journal of Southern Medical University
基金
公安部科技强警基础工作专项项目(2014GABJC020)
