溺死相关浮游生物基因座的复合扩增体系

目的构建溺死相关浮游生物基因座的复合扩增体系,验证体系特异性并评估其在法医溺死诊断中的应用价值。方法针对溺死相关浮游生物同源基因序列,设计特异性引物,并用FAM、HEX、TAMRA、ROX、SIZ荧光染料分组标记,构建复合扩增体系;进行体系特异性验证;以16例实验猪样本评估体系检验效能;复合扩增体系、硅藻形态学MD-VF-Auto SEM检测法及硅藻rbc L基因PCR-CE检测法分别检测28例案件样本,比较分析应用效果。结果该复合扩增体系共包含14个基因座,可特异性扩增35种浮游藻类和3种浮游细菌,人、3种人体共生菌及8种浮游细菌均为阴性。以该体系检测溺死实验猪,阳性率为90%,非溺死实验猪均未检出;检测溺死案件样本,阳性率为96%,非溺死案件均未检出。复合扩增体系与MD-VF-Auto SEM法检测案件尸体肺、肝和肾的阳性率均不具有统计学差异(P>0.05),但其与硅藻rbcL基因PCR-CE法检测案件肝和肾的阳性率均具有统计学差异(P<0.05)。结论本文针对多种溺死相关浮游生物14个基因建立的复合扩增体系,具备多种靶物种的特异性遗传标记,且所需检材量小,提高了检测效能,在法医溺死鉴定中具有良好的应用前景。

Multiplex assay of drowning related plankton genes

Objective To establish a multiplex assay of drowning related plankton genes,verify its specificity and evaluate its application value in the forensic drowning diagnosis.Methods Based on homologous sequences of drowning related plankton,specific primers were designed in a 5-dye(FAM、HEX、TAMRA、ROX、SIZ)multiplex PCR system,and then the specificity was investigated.The preliminary positive rate of this system was investigated by 13 experimental pigs.The application value of this system was appraised by the analysis of 28 human cadavers,which were detected by this multiplex system,Microwave Digestion-Vacuum Filtration-Automated Scanning Electron Microscopy(MD-VFAuto SEM)and rbcL gene with PCR-Capillary Electrophoresis,respectively.Results This multiplex system included 14 loci,which could specifically amplify 35 species of planktonic algae,3 species of planktonic bacteria.Human being,3 species of common human symbiotic bacteria,8 species of planktonic bacteria could not be detected at any locus.For this multiplex system,the positive rate of drowning pigs was 90%,and all the non-drowning pigs were negative.There were no significant differences in the positive rate of human lung,liver and kidney between multiplex system and MD-VF-Auto SEM(P>0.05),but the positive rate of human liver and kidney detected by multiplex system was much higher than detected by rbcL gene with PCR-Capillary Electrophoresis(P<0.05).Conclusion The established multiplex system,targeted to 14 different genes of drowning related plankton,had less tissue consumption,higher positive rate.With a promising prospect for application in forensic drowning diagnosis,multiplex system is a better complementary to diatom tests.