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 oxygenase,rbc L)基因的特异性引物,建立溺死尸体组织内硅藻rbc L基因的聚合酶链式反应-毛细管电泳(polymerase chain reactioncapillary 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rbc L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-rbc L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(20μL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(χ2=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-rbc L引物建立的PCR-CE法能快速检测硅藻rbc L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 drowning,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 comparing 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 outcomes 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 uL PCR system）. 35 cases of corpses （3 corpses land deaths, 2 waterborne corpse cases of non-drowning, 30 drowning 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 oositive rate was and 92.6% by PCR-CE. there was no statistical difference （x2=2.077 .P=0.g91 ）. 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.
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2018年第1期98-102,共5页
Journal of Chongqing Medical University
基金
公安部科技强警基础工作专项资助项目(编号:2017GABJC07)
公安部计划研究资助项目(编号:2015JSYJA03)
广东省省级科技计划资助项目(编号:2015A020217001)
大学生创新训练资助项目(编号:21712121302)
关键词
法医病理学
溺死
硅藻
聚合酶链式反应-毛细管电泳
forensic pathology
drowning
diatoms
polymerase chain reaction-capillary electrophoresis（PCR-CE）
