PCR-DGGE法检测浮游生物16S rDNA在溺死鉴定中的应用

A PCR-DGGE-based method for identifying plankton 16S rDNA for the diagnosis of drowning

目的建立并评估PCR-DGGE法检测浮游生物对溺死鉴定的应用价值。方法大白兔30只随机分为3组:溺死组(n=12),死后抛尸组(n=12)和对照组(n=6);溺死组和死后抛尸组又分为2个亚组:东湖水域组(n=6)和墨水湖水域组(n=6)。死后提取心血和肺、肝、肾、脑等组织,匀浆后,采用Percoll密度梯度离心法分离浮游生物并提取其DNA,PCR扩增浮游生物特异的16S rDNA片段后分别用琼脂糖凝胶电泳及DGGE检测分析。2个溺死案例检材同法检验。结果溺死组各组织器官中浮游生物检测多呈阳性:肺(100%)、肝(83%)、肾(75%)、心血(83%)、脑(42%);死后抛尸组仅2例肺组织(16.7%)检出阳性;对照组全部阴性。溺尸肺组织DGGE分型图谱与相应溺死点水样分型图谱相似,而与非溺死点水样分型结果差异显著。2实际案例均呈阳性。结论本方法不仅有助于定性诊断溺死,而且通过比较产物的多样性可以推断溺死地点,在法医学溺死鉴定中具有较大实用价值。

Objective To develop and evaluate a PCR-DGGE-based method for identifying plankton 16S rDNA for the diagnosis of drowning. Methods Thirty white rabbits were divided randomly into three -groups： drowning group （n = 12）, postmortem submersion group （n = 12） and control group （n =6）, and the drowning and postmortem submersion groups were redivided respectively into two subgroups： Donghu Lake （n=6） and Moshui Lake （n=6） subgroups. After sacrificed, the heart blood, lung, liver, kidney and brain tissues of rabbits were removed and homogenized, and then the plankton was separated using Percoll density gradient centrifugation technique. After DNA extracted, the 16S rDNA of plankton was amplified with specific primers followed by agarose gel electrophoresis or denaturing gradient gel electrophoresis（DGGE） analysis. In addition, two human drowning cases were also tested with the method described above. Results Plankton was detected from most kinds of the tissues from drowning group： lung （ 100% ）, liver （ 83% ）, kidney （ 75% ）, heart bloods （ 83% ）, brain （ 42% ）. For postmortem submersion group, however, plankton was detected only from two cases of lung （ 16.7% ）, and none from control group. After analyzed with DGGE, the special fingerprint of plankton from the lungs of drowning rabbits was very similar to that from the water sample of drowning site, but not to the other site. By this method, the plankton was also detected from two human drowning cases. Conclusion The new PCR-DGGE-based method developed by this study was helpful not only to diagnosis of drowning, but also to discriminate the site of drowning, and is a potentially useful tool for diagnosing drowning.