Improved Glass Bead-Vortex Oscillation Method for DNA Extraction from Diatom / 法医学杂志

OBJECTIVES@#To examine the effect of improving diatom DNA extraction by glass bead - vortex oscillation method.@*METHODS@#The DNeasy PowerSoil Pro kit was used as control, two plant DNA extraction kits with different principles (New Plant genomic DNA extraction kit and Plant DNA Isolation kit) and one whole blood DNA extraction kit (whole blood genomic DNA extraction kit) were selected to extract diatom DNA from lung tissue and water sample of the same drowning case. The combination of mass ratio of glass beads with different sizes and vortex oscillation time was designed, and the optimal DNA extraction conditions were selected with the addition of glass beads oscillation. The extracted products of the conventional group and the modified group were directly electrophoretic and detected by diatom specific PCR. Finally, all the extracts were quantified by qPCR, and the Ct values of different groups were statistically analyzed.@*RESULTS@#When the frequency of vortex oscillation was 3 000 r/min, the optimal combination of DNA extraction was vortex oscillation for 4 min, and the mass ratio of large glass beads to small glass beads was 1∶1. The DNeasy PowerSoil Pro kit was used as a reference, and the Ct value of 10 mL water sample was greater than that of 0.5 g tissue. The Ct values of the other three kits used for plant DNA extraction decreased after the glass beads-vortex oscillation method was used, and the Ct values of the tissues before and after the improvement were statistically significant (P<0.05). The whole blood genomic DNA extraction kit used in this study could successfully extract diatom DNA, the extraction of water samples was close to DNeasy PowerSoil Pro kit, after the modified method was applied to tissue samples, the difference in Ct value was statistically significant (P<0.05). However, when the three kits were used to extract diatom DNA from water samples, Ct values before and after the improvement were only statistically significant in New Plant genomic DNA extraction kit group (P<0.05).@*CONCLUSIONS@#The improved glass bead-vortex oscillation method can improve the extraction efficiency of diatom DNA from forensic materials, especially from tissue samples, by plant and blood DNA extraction kits.

Comparison of Application of MD-VF-Auto SEM Method and Plankton Gene Multiplex PCR System in the Diagnosis of Drowning / 法医学杂志

OBJECTIVES@#To compare the application effect of microwave digestion - vacuum filtration - automated scanning electron microscopy (MD-VF-Auto SEM) method and plankton gene multiplex PCR system in the diagnosis of drowning.@*METHODS@#Lung, liver and kidney tissue of 10 non-drowning cases and 50 drowning cases were prepared for further MD-VF-Auto SEM method analysis and plankton gene multiplex PCR system analysis. The positive detection rate of the two methods in each tissue was calculated.@*RESULTS@#The positive rate of the MD-VF-Auto SEM method detecting diatoms in drowning cases was 100%, and few diatoms were detected in the liver and kidney tissues of 6 non-drowning cases. By using the plankton gene multiplex PCR system, the diatom positive rate of drowning cases was 84%, and all the non-drowning cases were negative. There were significant differences in the positive rate of the liver, kidney tissues between MD-VF-Auto SEM method and plankton gene multiplex PCR system (P<0.05), as well as the total positive rate of cases. However, no significant differences were found in the positive rates of lung tissues (P>0.05).@*CONCLUSIONS@#MD-VF-Auto SEM method is more sensitive than plankton gene multiplex PCR system in diatom test. But the plankton gene multiplex PCR system can also detect plankton other than diatoms. Combination of the two methods can provide a more reliable basis for the diagnosis of drowning.

Application Progress of High-Throughput Sequencing Technology in Forensic Diatom Detection / 法医学杂志

Diatom detection is an important method for identifying drowning and throwing corpses after death and inferring the drowning sites in forensic examination of corpses in water. In recent years,high-throughput sequencing technology has achieved rapid development and has been widely used in research related to diatom taxonomic investigations. This paper reviews the research status and prospects of high-throughput sequencing technology and its application in forensic diatom detection.

Application of Chlorophyte ChlB Gene and Cyanophyte NIES Gene in the Detection of Drowning-Related Plankton / 法医学杂志

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.

Application of Specific Fragment Length Polymorphism of Algae rDNA in Identification of Drowning Cases / 法医学杂志

OBJECTIVES@#To identify the drop-off location of victims in drowning cases, and confirm whether it is a fatal drowning or the victim is thrown into the water after death by detecting part of 5.8S sequence and second internal transcribed spacer （ITS2） （5.8S+ITS2） of diatom rDNA in water and organs.@*METHODS@#Two cases identified by diatom examination, which received by Nanjing Municipal Public Security Bureau Forensic Center, were taken as the research objects. The difference of the population structure of algae in water and human tissue was analysed by length polymorphism of 5.8S+ITS2 marker.@*RESULTS@#In case 1, similar species of diatom were detected from victim's lung and liver tissues and the water sample. Two kinds of DNA fragments with length of 330 bp and 376 bp were detected from victim's lung tissue and the water sample using 5.8S+ITS2 marker, which could confirm the victim was drowning before death. In case 2, there was no diatom found in victim's lung and liver tissues. Only one kind of DNA fragment with length of 331 bp and low relative fluorescence unit （RFU） was obtained from victim's lung tissue using 5.8S+ITS2 marker, thus the victim was thrown into the water after death.@*CONCLUSIONS@#The experimental results of the two cases in present study are consistent with the actual facts and the result of the diatom microscopic examination. The difference of population structure of specific microorganism in water and human tissue can be detected by 5.8S+ITS2 marker, which can help to identify the drop-off location of victims in drowning cases, and confirm whether it is a fatal drowning or the victim is thrown into the water after death.