PCR-DHPLC法检测硅藻SSU基因在溺死鉴定中的应用

The value of detection of diatom SSU gene based in PCR-DHPLC on identification of drowning

目的采用PCR-DHPLC法检测硅藻SSU基因,评估其在溺死鉴定中的应用价值。方法 60只实验兔随机分为生前溺死(水中溺毙)、死后入水(空气栓塞致死后入水)、对照组(空气栓塞致死后不做处理);溺死人体脏器组织;取各组织检材提取硅藻DNA,PCR扩增硅藻特异的核糖体小亚基(SSU)片段,用琼脂糖凝胶电泳检测、DHPLC检测分析。结果 6份硝酸消化法检测阴性的溺死人体器官组织检材经PCR及琼脂糖凝胶电泳检出5例阳性。生前溺死组肺、肝、肾硅藻检出率分别为100%、90%、85%,死后入水组仅肺检出硅藻(15%),对照组各组织均为阴性;生前溺死组检出率明显高于死后入水组(P<0.05)。10份溺死人体器官组织检材采用DHPLC法检出硅藻种类明显多于微波消解-扫描电镜法(P<0.05)。脏器检出硅藻种类与溺死点水样一致。结论采用PCR-DHPLC法检测硅藻SSU基因,有助于溺死鉴定和溺死地点的推断,具有法医学应用价值。

Objective To develop and evaluate a method for identification of drowning death through detection of diatom SSU gene by PCR-DHPLC.Methods Sixty rabbits were divided randomly into three groups： the drowning death group,the submerging after lethal aeroembolism group and using only lethal aeroembolism as control group.Samples from experimental rabbits and drowning peoples have been analyzed.DNAs were extracted from experimental samples,and then amplified by specific primers of diatom SSU gene.PCR productions were detected by agarose gel electrophoresis and DHPLC.Results For drowning human sample,5 samples were positive for diatom detection by PCR and electrophoresis detection from 6 drowning human samples which were negative by using nitric acid digestion to detect the diatom.For experimental rabbits,the positive rate for diatom detection were 100%,90% and 85% respectively in lung,live and kidney tissue sample from drowning death group,while no specific amplification products were detected in all samples of the control group and only 15% lung sample were detected for diatom in submerging after lethal aeroembolism group.The positive rate for diatom detection in the drowning death group was significantly higher than the submerging after lethal aeroembolism group and there has statistical significance（ P 0.05）,the DHPLC method for diatom detection of ten human tissues was significantly better than the microwave digestion-scanning electron microscopy（ sem） method and has statistical significance（ P 0.05）.The diatom species of the tissues was consistent to the drowning point water.Conclusion The PCR-DHPLC based method of detecting diatom SSU gene is not only feasible for the identification of death by drowning but also helpful to infer drowning location,which both have good application prospect in the forensic identification of drowning.