Improved Glass Bead-Vortex Oscillation Method for DNA Extraction from Diatom.

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.

[Diatoms in drowned and postmortem immersed rabbits' lungs].

OBJECTIVE: To investigate the numbers, sizes and types distribution of diatoms in drowned and postmortem immersed rabbits' lungs. METHODS: Sixty-two rabbits were randomly divided into drowning group (n = 30), postmortem immersion group (n = 30) and land death group (n=2), and the diatoms in each lung lobe were analyzed quantitatively and qualitatively by microwave digestion and scanning electron microscopy. RESULTS: In the drowning group, the diatoms were detected in each lung lobe with Cyclotella and Melosira in the majority. In the postmortem immersion group, Cyclotella was in the majority. And the diatoms weren't detected in some lung lobes in postmortem immersion. There were significant differences in the detection rates of upper lobe of left lung, middle lobe and cardiac lobe of right lung in two groups (P < 0.05). CONCLUSION: Based on the microwave digestion and scanning electron microscopy, the numbers, sizes and types distribution of diatoms in drowned and postmortem immersed rabbits' lungs can be analyzed and used as references for testing theory.

The effect of enzyme digestion time on the detection of diatom species.

This study is aimed at detecting diatom in lung, liver and kidney tissues using PCR - DHPLC technology after different periods of enzyme digestion to assess the effect of enzyme digestion on the detection of diatom species. Twenty Randomly selected experimental rabbits were drowned at the same place. Their liver, kidney, and lung tissues were removed for sampling. After the extraction of DNA from the samples, amplification was conducted with specific primers of the SSU gene of diatom. Then, an analysis was performed with agarose gel electrophoresis and DHPLC. Within 2 h-8 h, the amount of the diatom species found in the lung gradually increased over time and was statistically significant <. After 8 h, with enzyme digestion, the amount of the diatom species found in lung showed no significant increase (>). However, as for the liver and kidney, within 2h-6h, the amount of the diatom species gradually increased over time and was statistically significant <. After 6h, the fig. did not present significant growth (>). The amount of the diatom species found in the organs after different periods of digestion time had significant differences, which provides a reference for the detection of diatoms and also, has a good application prospect in the forensic identification of drowning.

Microwave Digestion--Vacuum Filtration-Automated Scanning Electron Microscopy as a sensitive method for forensic diatom test.

Diagnosis of drowning is one of the most difficult issues in forensic practice. A number of methods have been developed over the years to determine whether a person was drowned. Microwave Digestion-Vacuum Filtration-Automated Scanning Electron Microscopy (MD-VF-Auto SEM) method we developed is a new qualitative and quantitative method of diatom test for diagnosis of drowning. The new method is based on microwave digestion technique, vacuum filtration, and automated SEM, which would achieve a maximal recovery of diatoms and identify diatoms easily by SEM with high resolution. This study was designed to evaluate the sensitivity of this method, the recovery of diatom, and loss ratio of centrifugation, which were compared using the MD-VF-Auto SEM method and the conventional acid digestion method. Two groups of samples were designed in the study. Groups A (n = 20) and B (n = 20) were performed by MD-VF-Auto SEM method and the conventional acid digestion method, respectively. In addition, another eight water samples were centrifuged, and the diatoms in the supernatant and precipitate were counted and measured, respectively, in order to find out how many diatoms were lost after centrifugation. The difference between the two groups was statistically highly significant, and about 34 % of diatoms were lost after centrifugation at 4,000 rpm for 15 min. The results showed that the MD-VF-Auto SEM method was more sensitive and specific.

[Progress in application of microbeam X-ray fluorescence spectroscopy in forensic science].

Microbeam X-ray fluorescence (micro-XRF) spectrometry has been raised as an analytical technique of microbeam during the recent years. With its advantages of high sensitivity, small sample requirement, high testing accuracy and non-destruction, the technique is widely utilized in forensic science. This review bases on recent researches at home and abroad, describes its applications including identification of gunshot residue, visualization of fingerprints, discrimination of drug source, production process, and other material evidences of analysis in crime scene. Thanks to the advances in technology, intelligent and portable micro-XRF equipment has appeared to be applied. It is believed that it may be more popular and frequent in administration of forensic science in the near future.

[Determination of acid-resistant silicic particles in lung by micro-XRF and its application in diagnosis of drowning].

OBJECTIVE: Using microbeam X-ray fluorescence (Micro-XRF) analyzer for determination of acid-resistant silicic particles in lung, and to explore its potential application in diagnosis of drowning. METHODS: Thirty two white rabbits were divided randomly into drowning group (n=12), post-mortem immersion group (n=10) and control group (n=10). Lungs and water sample were collected for determination of area concentration of acid-resistant silicic particles using Micro-XRF method. RESULTS: The area concentration of acid-resistant silicic particles for the drowning water sample was 4.4 mm2/mL. For the lungs of drowning group, the post-mortem immersion group and the control group, the determined average values were (25.30 +/- 10.95) mm2/g, (1.68 +/- 0.63) mm2/g and (1.65 +/- 0.85) mm2/g, respectively, with a statistically significant difference between the drowning group and the other two groups. CONCLUSION: The area concentration of acid-resistant silicic particles in lungs may be used as an indicator of drowning. The method is highly sensitive and rapid. It provides a potential application in drowning diagnosis.

[Deep learning network-based recognition and localization of diatom images against complex background].

ObjectiveWe propose a deep learning network-based method for recognizing and locating diatom targets with interference by complex background in autopsy.MethodThe system consisted of two modules: the preliminary positioning module and the accurate positioning module. In preliminary positioning, ZFNet convolution and pooling were utilized to extract the high-level features, and Regional Proposal Network (RPN) was applied to generate the regions where the diatoms may exist. In accurate positioning, Fast R-CNN was used to modify the position information and identify the types of the diatoms.ResultsWe compared the proposed method with conventional machine learning methods using a self-built database of images with interference by simple, moderate and complex backgrounds. The conventional methods showed a recognition rate of diatoms against partial background interference of about 60%, and failed to recognize or locate the diatom objects in the datasets with complex background interference. The deep learning network-based method effectively recognized and located the diatom targets against complex background interference with an average recognition rate reaching 85%.ConclusionThe proposed method can be applied for recognition and location of diatom targets against complex background interference in autopsy.

The Diagnostic Value of Quantitative Assessment of Diatom Test for Drowning: An Analysis of 128 Water-related Death Cases using Microwave Digestion-Vacuum Filtration-Automated Scanning Electron Microscopy.

The value of diatom test for the diagnosis of drowning remains controversial. The conventional forensic diatom test with low sensitivity is not a useful tool to provide accurate information about diatom in the tissues and organs. To improve the sensitivity of the diatom test, we developed a novel method called the Microwave Digestion-Vacuum Filtration-Automated Scanning Electron Microscopy (MD-VF-Auto SEM) method which resulted in a high recovery of diatoms. In this article, we analyzed 128 water-related death cases. Our results showed that the MD-VF-Auto SEM method could achieve a much higher positive rate (0.97) in drowning cases. Large amounts of diatoms are retained, even concentrated, in the lung tissues during the intense breathing movement in drowning process. This might be useful for the diagnosis of drowning. Our research indicates that the MD-VF-Auto SEM method would be a valuable methodology in the study of diatom test for the forensic community.

Detection of Diatoms by a Combination of Membrane Filtration and Transparentness.

Diagnosis of drowning remains a difficult issue in current forensic sciences. A large number of diatoms were lost by removing the supernatant after centrifugation in the conventional forensic diatom test. We developed a novel membrane filtration method to enrich diatoms from samples. A new solution using different ratios of acetic acid and eugenol is prepared to make the membrane transparent. These processes allow the diatom-containing membrane to be visualized and identified easily by light microscopy. The tissues contaminated by water rich in diatoms were detected by the new method for the recovery of diatoms. Eleven drowning cases were analyzed by both the new method and the conventional method to compare the sensitivity of both methods. The recovery of the novel diatom test method was 54.2 ± 23.1%. The positive rate of the novel method has been proven to be superior to the conventional method in the diagnosis of drowning.

A quantitative comparison analysis of diatoms in the lung tissues and the drowning medium as an indicator of drowning.

The presence of diatoms in the lung tissues, internal organs and bone marrow is considered as the supportive evidence in the diagnosis of death by drowning. Generally, the diatoms detected in the lung tissues are regarded as insignificant since these diatoms can be detected in the lung tissues of the postmortem immersion bodies. In this study, we analyzed the relationships between the numbers of the diatoms in the lung tissues and the drowning medium. We made a comparison analysis between the diatoms in the lung tissues and the drowning medium using the ratio of diatom numbers in both samples (L/D ratio), utilizing Microwave Digestion - Vacuum Filtration - Automated Scanning Electron Microscopy method. Our data indicate that the L/D ratios in victims of the drowning group were higher than the postmortem immersion group. A higher L/D ratio provides valuable information about the cause of death in drowning victims. Quantitative diatom analysis in the lung tissues, especially combined with the diatom analysis of the drowning medium, provides supportive evidence in determining if a body recovered in water was due to drowning or not.

A simple digestion method with a Lefort aqua regia solution for diatom extraction.

Presence of diatoms in tissues has been considered as a significant sign of drowning. However, there are limitations in the present extraction methods. We developed a new digestion method using the Lefort aqua regia solution (3:1 nitric acid to hydrochloric acid) for diatom extraction and evaluated the digestive capability, diatom destruction, and diatoms' recovery of this new method. The kidney tissues from rabbit mixed with water rich in diatoms were treated by the Lefort aqua regia digestion method (n = 10) and the conventional acid digestion method (n = 10). The results showed that the digestive capability of Lefort aqua regia digestion method was superior to conventional acid digestion method (p < 0.01); the structure of diatom remained almost intact; and the recovery of diatom was comparable to the conventional acid digestion method (p > 0.05). The Lefort aqua regia reagent is an improvement over the conventional acid digestion for recovery of diatoms from tissue samples.

[Evaluation of three methods for forensic diatom test].

OBJECTIVE: To compare the efficacy of three methods for forensic diatom test, namely strong acid digestion-centrifuge enrichment-light microscopy (SD-CE-LM), microwave digestion-membrane filtration-automated scanning electron microscopy (MD-ME-SEM), and microwave digestion-membrane filtration-light microscopy (MD-MF-LM). METHODS: Sixty samples were randomly divided into 3 groups for diatom test using three methods, and the sample preparation time, degree of digestion and recovery rate of diatoms were compared. RESULTS: The sample preparation time was the shortest with MD-MF-LM and the longest with SD-CE-LM (P<0.05). MD-ME-SEM and MD-MF-LM allowed more thorough tissue digestion than SD-CE-LM. MD-ME-SEM resulted in the highest total recovery rate of diatom, followed by MD-MF-LM and then by SD-CE-LM (P<0.05); the recover rate of different diatom species was the highest with MD-ME-SEM, followed by MD-MF-LM and SD-CE-LM (P<0.05). CONCLUSION: SD-CE-LM has a low recovery rate of diatoms especially for those with lengths shorter than 40 µm or densities less than 1/5. With a high recovery rate and accuracy in diatom test, MD-ME-SEM is suitable for diagnosis of suspected drowning cases. MD-MF-LM is highly efficient, sensitive and convenient for forensic diatom test.

Application of the microwave digestion-vacuum filtration-automated scanning electron microscopy method for diatom detection in the diagnosis of drowning.

The cause of death for the decomposed corpses recovered from water is still a difficult issue in current forensic practice. In this article, we present two cases of bodies recovered from water with no positive findings to indicate the cause of their death. We apply both conventional acid digestion method as well as the microwave digestion-vacuum filtration-automated scanning electron microscopy method (MD-VF-Auto SEM) for diatom detection in different organs of both bodies. Our results indicate that MD-VF-Auto SEM method provide more accurate information and match further police investigation. This novel method would be a useful technique in assessing cause of death for body found in water.

[Value of specific 16S rDNA fragment of algae in diagnosis of drowning: an experiment with rabbits].

OBJECTIVE: To establish a method for amplifying specific 16S rDNA fragment of algae related with drowning and test its value in drowning diagnosis. METHODS: Thirty-five rabbits were randomly divided into 3 the drowning group (n=15), postmortem water immersion group (n=15, subjected to air embolism before seawater immersion), and control group(n=5, with air embolism only). Twenty samples of the liver tissues from human corpses found in water were also used, including 14 diatom-positive and 6 diatom-negative samples identified by microwave digestion-vacuum filtration-automated scanning electron microscopy (MD-VF-Auto SEM). Seven known species of algae served as the control algae (Melosira sp, Nitzschia sp, Synedra sp, Navicula sp, Microcystis sp, Cyclotella meneghiniana, and Chlorella sp). The total DNA was extracted from the tissues and algae to amplify the specific fragment of algae followed by 8% polyacrylamide gelelectrophoresis and sliver-staining. RESULTS: In the drowning group, algae was detected in the lungs (100%), liver (86%), and kidney (86%); algae was detected in the lungs in 2 rabbits in the postmortem group (13%) and none in the control group. The positivity rates of algae were significantly higher in the drowning group than in the postmortem group (P<0.05). Of the 20 tissue samples from human corps found in water, 15 were found positive for algae, including sample that had been identified as diatom-negative by MD-VF-Auto SEM. All the 7 control algae samples yielded positive results in PCR. CONCLUSIONS: The PCR-based method has a high sensitivity in algae detection for drowning diagnosis and allows simultaneous detection of multiple algae species related with drowning.

Detection of diatoms in water and tissues by combination of microwave digestion, vacuum filtration and scanning electron microscopy.

The detection of diatoms has been proposed to be useful in the diagnosis of drowning. In the presented paper, we describe a novel method for qualitative and quantitative analysis of diatoms in water and organs. Samples were treated by microwave digestion. The fluid obtained was vacuum-filtrated; its sediment on the membrane was coated and examined under the scanning electron microscope with the mode of automatic field scanning; and qualitative and quantitative diatom analysis was performed based on the images captured automatically and on manual identification of the suspected diatoms at a required higher magnification. The method is rapid, sensitive, labor-saving, and accurate for qualitative and quantitative diatom analysis. However, further scientific research in the field of automatic diatom identification using SEM images has to be done in order to automate the process of detection and identification of diatoms in water and tissues for the diagnosis of drowning.

Trajectory reconstruction through analysis of trace evidence in bullet-intermediate target interaction by SEM/EDX.

A young male was shot to death by a police officer with a Chinese Type 64 7.62 mm pistol when he was dealing with an aggravated assault arising from a traffic accident. By using scanning electron microscopy and energy dispersive X-ray spectroscopy (SEM/EDX), trace deposits on the discharged bullet and the intermediate target, i.e., a concrete telegraph pole at the scene, were identified to be from each other. The result demonstrated the bullet causing the death ricocheted from the concrete telegraph pole before striking the victim, thus indicating the incident was accidental. The case report illustrates the evidential value of trace materials derived, respectively, from discharged bullets and intermediate targets in bullet-intermediate target interaction for trajectory reconstruction. In addition, it indicates that the SEM/EDX method with its nondestructive nature, compared to other methods, may be more helpful in certain situations in determining the origins of trace evidentially valuable deposits on substrates.