实时荧光定量PCR检测硅藻UPA基因在溺死诊断中的研究

A real-time qPCR method to identify diatom UPA gene for the drowning diagnosis

目的建立硅藻UPA条形码基因的实时荧光定量PCR检测方法,探讨其在溺死诊断中的应用价值。方法对Gene Bank中登录的硅藻UPA基因序列进行比对获得同源序列,据其设计硅藻门通用引物。对2种人体常见共生菌(大肠杆菌、双歧杆菌)、3种浮游细菌、15种浮游藻类、32具尸体(其中生前溺水死亡28例,死后抛尸1例,陆地死亡3例)的组织样本(肺、肝、肾)及尸体发现地水样提取DNA,采用设计的引物进行特异性、灵敏度、重复性试验,分析检材中硅藻检出情况,统计硅藻阳性率,以上述组织样本的微波消解-真空抽滤-自动扫描电镜法检测结果对建立的q PCR检测方法进行比较评估,比较该方法与PCR-毛细管电泳检测法的灵敏度。结果引物UPA99仅对硅藻标准株(针杆藻、舟形藻、直链藻、小环藻、菱形藻)DNA具有扩增,扩增产物熔解曲线平稳,峰尖且窄,熔解温度为(87±1)℃。以p MD18-T重组质粒为标准品,检测区间为1.56×10~2-1.56×10^(-5)ng/μL,建立实时q PCR方法的灵敏度为1.56×10^(-5)ng/μL,而PCR-CE方法的灵敏度为1.56×10-3ng/μL。批间及批内变异系数均低于2%,具有较高的重复性和稳定性。对于生前溺水尸体肺、肝、肾的检出率依次为89.3%,71.4%,64.3%。结论基于硅藻UPA基因设计通用引物,建立的q PCR硅藻检验法特异性高、灵敏度高、重复性好,应用于溺死诊断具有较好的应用前景。

Objective To establish a Real-time quantitative PCR method （qPCR） for the detection of diatom UPA barcoding genes and evaluate its application in the drowning diagnosis. Methods The homologous sequences of diatoms UPA gene was obtained by Blast from GeneBank, based on which the universal primers for diatoms were designed. DNA were extracted from 2 common human symbiotic bacteria （Escherichia coli, Bifidobacterium longum）, 3 species of planktonic bacteria, 15 species of planktonic algae, tissue samples （lung, liver and kidney） from human cadavers （28 drowning victims, 1 victims by non-drowning in the water, 3 victims deaths on land） in 32 cases. The specificity, sensitivity and repeatability of the designed primers were tested. The positive rates of diatoms detection in the drowning cases were calculated. The results of the real-time quantitative method were evaluated comparatively by Microwave Digestion-Vacuum Filtration-Automated Scanning Electron Microscopy （MD-VF-Auto SEM） and PCR- Capillary Electrophoresis （PCR-CE）. Results The results showed that the primers UPA99 had strong specificity for the diatomaceae （Synedra radians, Navicula sp., Melosira varians, Cyclotella sp. and Nitzschia sp.） DNA. The melting curve of the amplified product was smooth; the peak was narrow; the melting temperature was （87±1）℃. The sensitivity of qPCR method was 1.56×10-3ng/μL with the detection range of 1.56×10-3ng/μL-1.56×10-3ng/μL, in contrast with the PCR-CE method （1.56×10-3ng/μL）. This real-time PCR method showed high repeatability andstability with the coefficient of variation less than 2%. The detection rate of lung, liver and kidney was 89.3%, 71.4% and 64.3% respectively. Conclusion The established qPCR method, based on the universal primers designed for diatom UPA gene, has high specificity, high sensitivity and good repeatability. With a promising prospect for application, qPCR is suitable for drowning diagnosis.