RESUMO
ABSTRACT: Objective To construct a polymerase chain reaction-capillary electrophoresis ï¼PCR-CEï¼ 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 ï¼drownedï¼ 20, natural deathï¼ 5ï¼ were detected, and the simultaneous detection results of microwave digestion-vacuum filtration-automated scanning electron microscopy ï¼MD-VF-Auto SEMï¼ 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 ï¼Chlorella pyrenoidosaï¼ and cyanophyte ï¼»Microcystis aeruginosa ï¼producing and not producing toxinï¼ï¼½ 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.
Assuntos
Chlorella , Diatomáceas , Afogamento , Diatomáceas/genética , Afogamento/diagnóstico , Humanos , Rim , Fígado , Pulmão , Plâncton/genéticaRESUMO
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 cyanophyte ［Microcystis 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.
