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 of Diatoms Quantitative Analysis in the Diagnosis of Drowning / 法医学杂志

OBJECTIVES@#To retrospectively analyze diatom test cases of corpses in water and discuss the value of quantitative analysis of diatoms in the diagnosis of drowning.@*METHODS@#A total of 490 cases of water-related death were collected. They were divided into drowning group and postmortem immersion group according to the cause of death. Diatoms in lung, liver, kidney tissue and water sample were analyzed quantitatively by microwave digestion-vacuum filtration-automated scanning electron microscopy (MD-VF-Auto SEM) method. The ratios of content of diatoms in lung tissue and water sample (CL/CD) were calculated.@*RESULTS@#The results of diatom test for three organs (lung, liver and kidney) were all positive in 400 cases (85.5%); the content of diatom in lung, liver, kidney tissues, and water samples of drowning group were (113 235.9±317 868.1), (26.7±75.6), (23.3±52.2) and (12 113.3±21 760.0) cells/10 g, respectively; the species of diatom were (7.5±2.8), (2.6±1.9), (2.9±2.1) and (8.9±3.0) types, respectively; the CL/CD of drowning group and postmortem immersion group were (100.6±830.7) and (0.3±0.4), respectively.@*CONCLUSIONS@#Quantitative analysis of diatoms can provide supportive evidence for the diagnosis of drowning, and the parameter CL/CD can be introduced into the analysis to make a more accurate diagnosis of drowning.

Effects of Digestive Temperature and Time on Diatom Test / 法医学杂志

OBJECTIVES@#To study the effects of temperature and time for diatoms digestion and find out suitable digestive temperature and time.@*METHODS@#Eighty pieces of liver tissues were collected, each piece of tissue was 2 g, and 2 mL Pearl River water was added to each piece of tissue. The digestion temperature was set at 100 ℃, 120 ℃, 140 ℃, 160 ℃, 180 ℃ and the digestion time was set at 40, 50, 60, 70, 80 min. The liver tissue and water mixture were divided into 8 portions in each group. All the samples were tested by microwave digestive - vacuum filtration - automated scanning electron microscopy method. The quantity of diatom recovered and the quality of residue on the membrane were recorded.@*RESULTS@#When the digestion time was set to 60 min, there were statistically significant differences in the number of diatoms recovered at different temperatures (P<0.05). The maximum number of diatoms recovered was (28 797.50±6 009.67) at 140 ℃, and the minimum residue was (0.60±0.28) mg at 180 ℃. When the digestion temperature was set at 140 ℃, there were statistically significant differences in the number of diatoms recovered at different digestion times (P<0.05). The number of diatoms recovered was the highest at 40 min, it was up to (20 650.88±1 950.29), and the residue quality of each group had no statistical significance among different digestion time groups(P>0.05).@*CONCLUSIONS@#The effect of diatom digestion is related to temperature and time. When the digestion temperature was 140 ℃ and the digestion time was 40, 50 and 60 min, it is favorable for diatom test.

Pathway of Diatoms Enter Experimental Rabbits through the Lymphatic System of the Digestive Tract / 法医学杂志

OBJECTIVES@#To study whether diatoms can enter the body through the lymphatic system of the digestive tract.@*METHODS@#Twenty experimental rabbits were divided into the test group and the control group randomly, and intragastric administration was performed with 20 mL water sample from the Pearl River and 20 mL ultrapure water, respectively. After 30 min, lymph, lungs, livers and kidneys were extracted for the diatom test. The concentration, size and type of diatoms were recorded.@*RESULTS@#The concentration of diatoms of the test group was higher than that of the control group (P<0.05). In the test group, Stephanodiscus, Coscinodiscus, Cyclotella, Melosira, Nitzschia, Synedra, Cymbella, and Navicula were detected; in the control group, Stephanodiscus, Coscinodiscus and Cyclotella were detected. The long diameter and the short diameter of diatoms of the test group were higher than those of the control group (P<0.05). In the test group, 1-2 diatoms were detected in 3 lung samples and 2 liver samples, which were Stephanodiscus or Cyclotella, and no diatoms were detected in the kidney samples; in the control group, 1-2 diatoms were detected in 2 lung samples and 3 liver samples, which were Stephanodiscus or Coscinodiscus, and no diatoms were detected in the kidney samples.@*CONCLUSIONS@#Diatoms can enter the body through the lymphatic fluid, which is one of the reasons for the presence of diatoms in tissues and organs of non-drowning cadavers.

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 Quantitative Analysis of Diatoms in Lung Tissue for the Diagnosis of Drowning of Experimental Animals / 法医学杂志

Objective To discuss the application value of diatom examination in lung tissue for the forensic diagnosis of drowning. Methods The experimental animals were divided randomly into drowning, postmortem submergence and dying on land group. Diatoms in lung tissue and drowning fluid were analyzed quantitatively by microwave digestion-vacuum filtration-automated scanning electron microscopy diatom examination method. The ratios of content of diatoms in lung tissue and drowning fluid （CL/CD ratio） were recorded. Results The CL/CD ratios of experimental rabbits in the drowning group （5.82±3.50） were much higher than that of postmortem submergence group （0.47±0.35）; the CL/CD ratios of different parts of the lung lobes of experimental pigs in the drowning group were higher than that of postmortem submergence group （P<0.05）; in seawater, brackish water, river fresh water and lake fresh water, the CL/CD ratios of experimental pigs in the drowning group were higher than that of postmortem submergence group （P<0.05）. In animal experiments, all the cases with CL/CD ratio >1.6 were from drowning group. Conclusion CL/CD ratio is an indicator with good application prospects in the diagnosis of drowning.

Research Progress on Determination of Drowning Site / 法医学杂志

The bodies found in water are one of the most common types in forensic practice. The dis-covery site of the body is often not the drowning site. However, the determination of drowning site is vital for the identification of victim. Inorganic particles and planktons, such as granular impurities, diatoms and bacteria, are valuable markers for the diagnosis of drowning. By comparing the granular impurities and planktons in tissues and suspicious drowning mediums, the drowning site can be concluded based on their similarity of types and distribution, which has practical applied value. In this paper, the research progress on determination of drowning site is summarized to provide reference for the peers.

Diagnosis of drowning by detecting gyrB and 16S rRNA genes of Aeromonas hydrophila using PCR-capillary electrophoresis / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a method for diagnosis of freshwater drowning by amplifying gyrB and 16S rRNA genes of Aeromonas hydrophila using PCR coupled with capillary electrophoresis (CE).</p><p><b>METHODS</b>DNA samples were extracted from human, 18 planktons (including Candida albicans, Aeromonas hydrophila, and 16 species of algae), and 30 cases of tissue samples (including the lung, liver, and kidney, all examined with microwave digestion-vacuum filtration-automated scanning electron microscopy) from human cadavers, including 28 freshwater drowning victims and 2 with natural death. The DNA samples were amplified with the primer AH (for gyrB gene) and primer Ah (for 16S rRNA gene), and the products were analyzed with CE.</p><p><b>RESULTS</b>PCR amplification followed by CE yielded negative results for DNA of human, Candida albicans and 16 species of algae, whereas a positive result was found for Aeromonas hydrophila DNA with PCR products of 195 bp (with primer AH) and 350 bp (with primer Ah). In the 28 drowning cases, the detection rates of Aeromonas hydrophila using primer AH were 96.4% in the lung tissue, 71.4% in the liver tissue, and 60.7% in the kidney, as compared with the rates of 75.0%, 42.9%, and 32.1% using primer Ah, respectively. The positive rates for Aeromonas hydrophila in the organs of the drowning victims were 82.1% and 53.6% with primer AH and primer Ah, respectively. The detection showed negative results in the 2 cases of natural deaths. The two primers produced significantly different detection rates of Aeromonas hydrophila (P<0.05).</p><p><b>CONCLUSION</b>PCR coupled with CE for detecting gyrB gene of Aeromonas hydrophila has a high sensitivity in assisting a diagnosis of freshwater drowning. Detection of both the gyrB gene and 16S rRNA gene of Aeromonas hydrophila can yield more convincing evidence of the diagnosis of freshwater drowning.</p>

Value of specific 16S rDNA fragment of algae in diagnosis of drowning: an experiment with rabbits / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a method for amplifying specific 16S rDNA fragment of algae related with drowning and test its value in drowning diagnosis.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

DNA typing of minimal oral epithelial cell samples / 法医学杂志

OBJECTIVE@#To seek simple and cost-effective extraction technique to improve multiplex STR amplification from minimal oral epithelial cell samples.@*METHODS@#One hundred DNA samples of oral epithelial cells extracted by mini system Chelex-100 method were quantitated by ABI 7500 Real Time System and were then typed with Identifiler system in ABI 3130 Genetic Analyzer.@*RESULTS@#The DNA contents of different categories of samples were as followings: suck pipes (0.72-116.78 ng), cup edges (2.15-142.5 ng), mouths of drink bottle (1-34.65 ng), chopsticks (3.35-26.6 ng), fruit cores (0.294-21.4 ng), and poultry bones (0.88-5.88 ng). The mean successful typing rate for gender and more than 9 STR loci was about 59.38%. Except the addition or no addition of proteinase K to the samples, all other factors-C users' variation, sample extraction methods, and qualities and properties of the samples had considerable effects on the contents of extracted DNA.@*CONCLUSION@#Successful STR typing can be achieved in about 60% minimal oral epithelial cell DNA samples extracted by mini Chelex system.