溺死相关硅藻rbcL基因检测技术研究

A study on detection of rbcL gene for diatoms involved in drowning

摘要:

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。

Abstract:

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.