绿藻ChlB基因和蓝藻NIES基因在溺死相关浮游生物检测中的应用

目的利用绿藻ChlB基因和蓝藻NIES基因,建立聚合酶链反应-毛细管电泳(polymerase chain reaction-capillary electrophoresis,PCR-CE)检测方法,验证该方法的特异性和灵敏度,并探讨其在溺死诊断中的应用价值。方法针对GenBank中绿藻ChlB基因和蓝藻NIES基因的保守序列,设计特异性引物ChlB和引物NIES,以此构建PCR-CE检测方法。扩增50种标准样本DNA。阳性标准样本梯度浓度检测,确定灵敏度。检测25例尸体肺组织样本(溺死20例,自然死亡5例),同时比较微波消解-真空抽滤-自动扫描电镜法(microwave digestion-vacuum filtration-automated scanning electron microscopy,MD-VF-Auto SEM)的同步检测结果。结果引物ChlB和引物NIES最低检测DNA含量分别为0.161、0.109ng,可分别特异性扩增水体广泛存在的绿藻(蛋白核小球藻)和蓝藻铜绿微囊藻(产毒及不产毒)],产物片段分别为156、182bp,非溺死器官组织检测结果均为阴性。结论构建的方法灵敏度高、特异性好,能很好地应用于溺死相关浮游生物检测,与MD-VF-Auto SEM法联用,可以增大溺死相关浮游生物的检测范围,提高溺死诊断的证据力。

Application of Chlorophyte ChlB Gene and Cyanophyte NIES Gene in the Detection of Drowning-Related Plankton

Objective To construct a polymerase chain reaction-capillary electrophoresis(PCR-CE)detection method using ChlB gene and NIES gene,investigate the method’s specificity and sensitivity,and to evaluate its application value in drowning diagnosis.Methods The specific primers ChlB and NIES were designed for the conserved sequence of chlorophyte ChlB gene and cyanophyte NIES gene in GenBank to construct PCR-CE detection method;50 species of standard DNA samples were amplified;the sensitivity was determined by gradient concentration detection of positive standard samples;25 actual cadaver lung tissue samples(drowned:20,natural death:5)were detected,and the simultaneous detection results of microwave digestion-vacuum filtration-automated scanning electron microscopy(MD-VF-Auto SEM)were simultaneously compared.Results The minimum DNA detection concentration of primers ChlB and NIES was 0.161 ng and 0.109 ng,respectively,which could specifically amplify chlorophyte(Chlorella pyrenoidosa)and cyanophyteMicrocystis aeruginosa(producing and not producing toxin)]widespread in water.The product fragments were 156 bp and 182 bp,respectively.The results of non-drowning tissues were negative.Conclusion This method has high sensitivity and specificity.It can be applied to the detection of plankton related to drowning and combined with MD-VF-Auto SEM method,can increase the detection range of plankton related to drowning and improve the evidence power of drowning diagnosis.