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1. 广东工业大学轻工化工学院生物工程系,广州,510006
2. 广州市刑事科学技术研究所、公安部法医病理学重点实验室,广州,510000
3. 南方医科大学法医学院,广州,510515
纸质出版日期:2018,
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彭帆, 刘超, 徐曲毅, 等. 溺死相关硅藻rbcL基因检测技术研究[J]. 重庆医科大学学报, 2018,43(1):98.
Peng Fan, Liu Chao, Xu Quyi, et al. A study on detection of rbcL gene for diatoms involved in drowning[J]. Journal of Chongqing Medical University, 2018,43(1):98.
目的:筛选硅藻叶绿体核酮糖-1,5-二磷酸羧化酶大亚基(large subunit of ribulose-1,5-bisphosphate carboxylase oxyge-nase,rbcL)基因的特异性引物,建立溺死尸体组织内硅藻rbcL基因的聚合酶链式反应-毛细管电泳(polymerase chain reaction-capillary electrophoresis,PCR-CE)检测方法,探讨在溺死诊断中的应用价值。方法:采用荧光标记引物扩增硅藻rbcL基因,对21种标准藻株、5种浮游水生标准菌株、3种人体共生标准菌株和人类DNA扩增产物进行PCR-CE检测,验证引物特异性和灵敏度。检测溺死尸体组织样本中硅藻,并比对微波消解-真空抽滤-自动扫描电镜法(microwave digestion-vacuum filtration-automated scanning electron microscopy,MD-VF-Auto SEM)检测结果。结果:使用引物ND-rbcL对21种标准藻株、5种浮游水生标准菌株、3种人体共生标准菌株和人类DNA进行扩增,其中舟形藻、菱形藻、小环藻、变异直链藻、针杆藻和骨条藻6种硅藻的CE检测结果为阳性,DNA片段为197 bp,其他为阴性。6种硅藻DNA检测阳性的灵敏度分别为0.502、0.117、0.029、0.042、0.275和0.215 ng(20 μL扩增体系)。PCR-CE方法检测35例尸体(3例陆地正常死亡尸体,2例水中发现非溺死尸体,30例溺死尸体)的组织样本,肺脏、肝脏和肾脏硅藻检出率分别为100%、63.3%和53.3%,检测总阳性率为83.3%。当溺水死亡尸体脏器组织内硅藻含量>10个/10 g时,使用MD-VF-Auto SEM方法检测,其检测总阳性率为100%,PCR-CE方法检测,其检测总阳性率为92.6%,两者对检测溺死尸体脏器中硅藻阳性率无统计学差异( ?字2=2.077,P=0.491);当硅藻含量<10个/10 g时,PCR-CE方法检测为阴性,与MD-VF-Auto SEM方法检测溺死尸体各种脏器中硅藻阳性率具有统计学差异(P<0.05)。结论:基于ND-rbcL引物建立的PCR-CE法能快速检测硅藻rbcL基因,所需检材量小,易于推广,在溺死诊断中有较好地应用前景。
Objective:To establish the PCR method with capillary electrophoresis(PCR-CE) for the detection of rbcL gene of diatoms involved in drowning based on the specific primers for diatoms rbcL gene. Methods:With 29 microbes probably involved with drown-ing,a PCR-CE detection system with the primer for diatom rbcL gene labelled with fluorescent was established to verify the specificity and stability. The effectiveness of the PCR-CE method for the diatoms rbcL gene detection in corpses samples was evaluated by com-paring the results of MD-VF-Auto SEM. Results:Positive results(size=197 bp) were achieved with 6 diatoms species(Navicula sp.,Nitzschia sp.,Cyclotella sp.,Melosira varians,Synedra radians,Skeletonema) by the PCR-CE method,by contrast with negative out-comes for other species. The sensitivity of the above 6 species of diatoms DNA were 0.502,0.117,0.029,0.042,0.275,0.215 ng,respectively(20 μL PCR system). 35 cases of corpses(3 corpses land deaths,2 waterborne corpse cases of non-drowning,30 drown-ing cases) were detected,the detection rate of diatoms in the lungs,liver and kidney of the corpses was 100%,63.3% and 53.3%,respectively. The total positive rate was 83.3%. When the diatom content >10/10 g,the total positive rate was 100% by MD-VF-Auto SEM method. The total positive rate was and 92.6% by PCR-CE,there was no statistical difference( ?字2=2.077,P=0.491),otherwise,there was statistical difference(P<0.05). Conclusion:Because of less sample consumption,the PCR-CE method based on the ND-rbcL primer has a good prospect in forensic application for diatom detection,and leads to easy popularization in drowning diagnosis.
法医病理学溺死硅藻聚合酶链式反应-毛细管电泳
forensic pathologydrowningdiatomspolymerase chain reaction-capillary electrophoresis(PCR-CE)
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