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 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.

Change Rule of Diatom in Zhangweixin River in Dezhou / 法医学杂志

OBJECTIVES@#To study the annual variation of diatoms in Zhangweixin River, to provide theoretical support by using diatom examination to estimate the time and place of the corpse entering water, and to establish a diatom database.@*METHODS@#Samples were taken from 4 sampling sites in Decheng section of Zhangweixin River for 12 consecutive months. Non-metric multi-dimensional scaling (NMDS) analysis was performed on the species and content of diatom samples. Based on the sampling site of Tianqu Road, Sprensen similarity coefficient analysis was conducted with the data of other 3 sites in Decheng section and Leling section of Zhangweixin River and Ningjin section in previous studies.@*RESULTS@#The number of diatom species was positively correlated with diatom content. The average diatom content in different months ranged from 1 054 to 13 041/10 mL, and the species composition ranged from 8 to 16, with differences in dominant species. The similarity coefficient of diatom species within 2 km were all higher than 0.956 52, which could not be distinguished effectively. The similarity coefficients of Leling section and Ningjin section were 0.736 84 and 0.588 24 respectively, which could be effectively distinguished.@*CONCLUSIONS@#The species and content of diatom vary in different months in Zhangweixin River, and the composition of diatom species is different in different basins, which can provide reference for estimating the time and place of the corpse entering water in the river.

Distribution of Diatoms in the Navigable Sections of Beijing-Hangzhou Grand Canal / 法医学杂志

OBJECTIVES@#To establish a diatom database by analyzing the quatity, species distribution and differences of diatom in water samples of the whole navigable sections of the Beijing-Hangzhou Grand Canal, to provide a reference for the inference of the drowning site.@*METHODS@#Water samples were collected at 22 sites in the navigable sections of the Beijing-Hangzhou Grand Canal (Jining section to Yangzhou Section), and the diatoms at each site were qualitatively and quantitatively analyzed by using graphite digestion-scanning electron microscopy.@*RESULTS@#Sampling site T (Laohuaijiang River Line, Gaoyou City, Yangzhou City, Jiangsu Province) had the highest number of diatoms, while sampling site O (Siyang County, Suqian City, Jiangsu Province) had the lowest number of diatoms, with a large gap of 68 times. At sampling site Q (Jiangpu District, Huaian city, Jiangsu Province), there were 19 species of diatoms. The sampling site O had the least diatoms, with 7 species. There were no significant differences in species evenness and species diversity at each sampling site (P>0.05). Some sampling sites have characterized diatoms, such as Caloneis at station A (Taibai Lake, Weishan County, Shandong Province), Rhoicosphenia at station B (Nanyang Town, Weishan County, Shandong Province), Amphora at station I (Taierzhuang District, Zaozhuang City, Shandong Province) and Epithemia at station J (Pizhou 310 national highway, Xuzhou City, Jiangsu Province).@*CONCLUSIONS@#The species richness of diatoms gradually increased from north to south. Diatom species richness and species diversity might be higher in areas with complex environments and large population flow. Climate type has a certain influence on the distribution of diatoms.

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.

Construction and Application of YOLOv3-Based Diatom Identification Model of Scanning Electron Microscope Images / 法医学杂志

OBJECTIVES@#To construct a YOLOv3-based model for diatom identification in scanning electron microscope images, explore the application performance in practical cases and discuss the advantages of this model.@*METHODS@#A total of 25 000 scanning electron microscopy images were collected at 1 500× as an initial image set, and input into the YOLOv3 network to train the identification model after experts' annotation and image processing. Diatom scanning electron microscopy images of lung, liver and kidney tissues taken from 8 drowning cases were identified by this model under the threshold of 0.4, 0.6 and 0.8 respectively, and were also identified by experts manually. The application performance of this model was evaluated through the recognition speed, recall rate and precision rate.@*RESULTS@#The mean average precision of the model in the validation set and test set was 94.8% and 94.3%, respectively, and the average recall rate was 81.2% and 81.5%, respectively. The recognition speed of the model is more than 9 times faster than that of manual recognition. Under the threshold of 0.4, the mean recall rate and precision rate of diatoms in lung tissues were 89.6% and 87.8%, respectively. The overall recall rate in liver and kidney tissues was 100% and the precision rate was less than 5%. As the threshold increased, the recall rate in all tissues decreased and the precision rate increased. The F1 score of the model in lung tissues decreased with the increase of threshold, while the F1 score in liver and kidney tissues with the increase of threshold.@*CONCLUSIONS@#The YOLOv3-based diatom electron microscope images automatic identification model works at a rapid speed and shows high recall rates in all tissues and high precision rates in lung tissues under an appropriate threshold. The identification model greatly reduces the workload of manual recognition, and has a good application prospect.

Evaluation of Inspection Efficiency of Diatom Artificial Intelligence Search System Based on Scanning Electron Microscope / 法医学杂志

OBJECTIVES@#To explore the application values of diatom artificial intelligence (AI) search system in the diagnosis of drowning.@*METHODS@#The liver and kidney tissues of 12 drowned corpses were taken and were performed with the diatom test, the view images were obtained by scanning electron microscopy (SEM). Diatom detection and forensic expert manual identification were carried out under the thresholds of 0.5, 0.7 and 0.9 of the diatom AI search system, respectively. Diatom recall rate, precision rate and image exclusion rate were used to detect and compare the efficiency of diatom AI search system.@*RESULTS@#There was no statistical difference between the number of diatoms detected in the target marked by the diatom AI search system and the number of diatoms identified manually (P>0.05); the recall rates of the diatom AI search system were statistically different under different thresholds (P<0.05); the precision rates of the diatom AI system were statistically different under different thresholds(P<0.05), and the highest precision rate was 53.15%; the image exclusion rates of the diatom AI search system were statistically different under different thresholds (P<0.05), and the highest image exclusion rate was 99.72%. For the same sample, the time taken by the diatom AI search system to identify diatoms was only 1/7 of that of manual identification.@*CONCLUSIONS@#Diatom AI search system has a good application prospect in drowning cases. Its automatic diatom search ability is equal to that of experienced forensic experts, and it can greatly reduce the workload of manual observation of images.

Comparison among Four Deep Learning Image Classification Algorithms in AI-based Diatom Test / 法医学杂志

OBJECTIVES@#To select four algorithms with relatively balanced complexity and accuracy among deep learning image classification algorithms for automatic diatom recognition, and to explore the most suitable classification algorithm for diatom recognition to provide data reference for automatic diatom testing research in forensic medicine.@*METHODS@#The "diatom" and "background" small sample size data set (20 000 images) of digestive fluid smear of corpse lung tissue in water were built to train, validate and test four convolutional neural network (CNN) models, including VGG16, ResNet50, InceptionV3 and Inception-ResNet-V2. The receiver operating characteristic curve (ROC) of subjects and confusion matrixes were drawn, recall rate, precision rate, specificity, accuracy rate and F1 score were calculated, and the performance of each model was systematically evaluated.@*RESULTS@#The InceptionV3 model achieved much better results than the other three models with a balanced recall rate of 89.80%, a precision rate of 92.58%. The VGG16 and Inception-ResNet-V2 had similar diatom recognition performance. Although the performance of diatom recall and precision detection could not be balanced, the recognition ability was acceptable. ResNet50 had the lowest diatom recognition performance, with a recall rate of 55.35%. In terms of feature extraction, the four models all extracted the features of diatom and background and mainly focused on diatom region as the main identification basis.@*CONCLUSIONS@#Including the Inception-dependent model, which has stronger directivity and targeting in feature extraction of diatom. The InceptionV3 achieved the best performance on diatom identification and feature extraction compared to the other three models. The InceptionV3 is more suitable for daily forensic diatom examination.

Research Progress of Automatic Diatom Test by Artificial Intelligence / 法医学杂志

Diatom test is the main laboratory test method in the diagnosis of drowning in forensic medicine. It plays an important role in differentiating the antemortem drowning from the postmortem drowning and inferring drowning site. Artificial intelligence (AI) automatic diatom test is a technological innovation in forensic drowning diagnosis which is based on morphological characteristics of diatom, the application of AI algorithm to automatic identification and classification of diatom in tissues and organs. This paper discusses the morphological diatom test methods and reviews the research progress of automatic diatom recognition and classification involving AI algorithms. AI deep learning algorithm can assist diatom testing to obtain objective, accurate, and efficient qualitative and quantitative analysis results, which is expected to become a new direction of diatom testing research in the drowning of forensic medicine in the future.

Growth Kinetics of Nanofrustulum Shiloi Under Different Mixing Conditions in Flat-plate Photobioreactor

Abstract Diatoms are the major group of microalgae which have been utilized by the potential applications as food industries, aquatic feeds, cosmetics, biofuels, and pharmaceuticals. In this study, current approaches were made in order to determine growth rate, biomass productivity, protein, carbohydrate, lipid and fatty acid composition for Nanofrustulum shiloi cultures using both aeration and mixing conditions in flat-plate photobioreactor (PBR). Physical (the intensity of aeration, mixing, light intensity etc.) and chemical (nutritional materials) factors are affecting the growth and bioproduct contents of a diatom. Biomass and lipid productivities of N. shiloi were measured as 31.29 and 36.9622±0.0598 mg L-1 day-1 in flat-plate PBR having the combination of aeration and stirring system, respectively. A slightly higher amount of saturated fatty acids was detected in PBR having only bubbling system while the increase of mono- and poly- unsaturated fatty acids were found in PBR having the combination of aeration and stirring system. Flat-plate PBR design was also investigated for improving not only biomass but also the lipid productivity of N. shiloi.

The biofilm removal effect of MnO₂-diatom microbubbler from the dental prosthetic surfaces: In vitro study / 대한치과보철학회지

PURPOSE: The aim of this study is to evaluate the effectiveness of MnO₂-diatom microbubbler (DM) on the surface of prosthetic materials as a mouthwash by comparing the biofilm removal effect with those previously used as a mouthwash in dental clinic.MATERIALS AND METHODS: DM was fabricated by doping manganese dioxide nanosheets to the diatom cylinder surface. Scanning electron microscopy (SEM) was used to observe the morphology of DM and to analyze the composition of doped MnO₂. Stereomicroscope was used to observe the reaction of DM in 3% hydrogen peroxide. Non-precious metal alloys, zirconia and resin specimens were prepared to evaluate the effect of biofilm removal on the surface of prosthetic materials. And then Streptococcus mutans and Porphyromonas gingivalis biofilms were formed on the specimens. When 3% hydrogen peroxide solution and DM were treated on the biofilms, the decontamination effect was compared with chlorhexidine gluconate and 3% hydrogen peroxide solution by crystal violet staining.RESULTS: Manganese dioxide was found on the surface of the diatom cylinder, and it was found to produce bubble of oxygen gas when added to 3% hydrogen peroxide. For all materials used in the experiments, biofilms of the DM-treated groups got effectively removed compared to the groups used with chlorhexidine gluconate or 3% hydrogen peroxide alone.CONCLUSION: MnO₂-diatom microbubbler can remove bacterial membranes on the surface of prosthetic materials more effectively than conventional mouthwashes.

Periphytic diatoms from an oligotrophic lentic system, Piraquara I reservoir, Paraná state, Brazil / Diatomáceas perifíticas de um sistema lêntico oligotrófico, reservatório do Piraquara I, estado do Paraná, Brasil

Abstract: Knowledge of biodiversity in oligotrophic aquatic ecosystems is fundamental to plan conservation strategies for protected areas. This study assessed the diatom diversity from an urban reservoir with oligotrophic conditions. The Piraquara I reservoir is located in an Environmental Protection Area and is responsible for the public supply of Curitiba city and the metropolitan region. Samples were collected seasonally between October 2007 and August 2008. Periphytic samples were obtained by removing the biofilm attached to Polygonum hydropiperoides stems and to glass slides. The taxonomic study resulted in the identification of 87 diatom taxa. The most representative genera regarding the species richness were Pinnularia (15 species) and Eunotia (14 species). Five species were registered for the first time in Brazil and seven in the State of Paraná. Taxonomic and ecological comments of the species registered are provided.

Resumo: O conhecimento da biodiversidade em ecossistemas aquáticos oligotróficos é fundamental para planejar estratégias de conservação de áreas protegidas. Este estudo teve como objetivo conhecer a diversidade de diatomáceas em um reservatório urbano com condições oligotróficas. O reservatório do Piraquara I está inserido em uma Área de Proteção Ambiental em região de manancial e é responsável pelo abastecimento público de Curitiba e região metropolitana. Foram realizadas coletas estacionais entre os meses de outubro de 2007 e agosto de 2008. As amostras perifíticas foram obtidas pela remoção do biofilme aderido a caules de Polygonum hydropiperoides e de lâminas de vidro. O estudo taxonômico resultou na identificação de 87 táxons. Os gêneros mais representativos, considerando a riqueza de espécies, foram Pinnularia (15 espécies) e Eunotia (14 espécies). Cinco espécies de diatomáceas foram registradas pela primeira vez no Brasil e sete no estado do Paraná. Comentários taxonômicos e ecológicos também são apresentados para as espécies encontradas.

Application of 18S rDNA Clone Library to Detect Diatom Population Diversity in Dianchi / 法医学杂志

Objective To detect the diatom population diversity in Dianchi by constructing a 18S rDNA clone library. Methods DNA from diatoms in 6 water samples of Dianchi was amplified with diatom 18S rDNA specific primer.The 18S rDNA clone library was constructed, and clones were randomly selected for sequence. Sequence alignment was performed by BLAST. The diatom population distribution in Dianchi was analyzed and the phylogenetic tree of diatom 18S rDNA in Dianchi waters was established with the MEGA v7.0.14 software. Results Two hundred and forty clones were sequenced, with 167 diatom sequences obtained, including 11 diatom species such as Stephanodiscus, Diatoma, and Melosira. There were certain differences in diatom population distribution among the 6 samples. Conclusion The population distribution of diatom species in Dianchi shows unique features and the sequence analysis of diatom 18S rDNA has a certain reference value to the inference of forensic drowning sites.

Diatom assemblages associated with turtle carapaces in the Neotropical region / Diatomeas epizoicas de los caparazones de tortugas de la región Neotropical

Abstract Few studies have explored the ecology and interrelationship with other organisms of the many endangered freshwater turtle species inhabiting the Neotropical region. The focus of the current study was to shed light on the relationship between Neotropical turtle carapaces and primary producers, insofar as the surface of former constitutes a suitable substrate for the colonization and establishment of the latter. The under-explored relationship between turtle carapaces and the diatom assemblages inhabiting them was investigated and characterized in terms of taxonomic and biological traits (bio-volume, life-form, and attachment). The carapaces of seven native turtle species were surveyed. Among these carapaces, a total of 45 diatom taxa were found, and diatom taxa varied among turtle species. Podocnemis vogli and Podocnemis expansa supported more diverse diatom assemblages than Podocnemis lewyana, Rhinoclemmys diademata, and Rhinoclemmys melanosterna, which were dominated by Navicula spp. Analysis, further showed that carapace size did not explain differences in diatom diversity. However, a trait-based analysis suggests that both carapace roughness and an assemblage's successional stage might explain the differences in assemblage composition. Because turtles can serve as dispersal vectors, characterizing their epibiont diatom assemblages may contribute to our understanding of diatom distribution on larger scales, as well as, give us some clues as to the auto-ecology of turtles that help us to effectively determine conservation areas for these endangered species.(AU)

Resumen De las especies de tortugas en estado crítico o vulnerable de conservación que habitan en la región Neotropical, son pocos los estudios que han ahondado en su ecología o relaciones con otros tipos de organismos. El presente estudio buscó caracterizar la asociación existente entre las diatomeas y el caparazón de especies de tortugas neotropicales, ya que este representa un sustrato adecuado para la colonización y establecimiento de este grupo de algas. Esta asociación fue estudiada considerando tanto la composición de especies como la de rasgos biológicos (biovolumen, forma de vida, y forma de adherencia) de la comunidad de diatomeas. En los caparazones de las 7 especies de tortugas muestreadas se encontraron un total de 45 táxones. La composición de táxones varió entre los caparazones de las especies. Se encontró una mayor diversidad en los caparazones de Podocnemis vogli y Podocnemis expansa, mientras que en Podocnemis lewyana, Rhinoclemmys diademata, y Rhinoclemmys melanosterna se encontró una comunidad de baja diversidad dominada principalmente por Navicula sp. Los resultados sugirieron que la rugosidad del caparazón junto al estado de la sucesión pueden explicar las diferencias observadas en la composición de táxones de los diferentes caparazones. Debido a que las tortugas pueden funcionar como vectores de dispersión, caracterizar las comunidades que pueden crecer sobre sus caparazones podría contribuir a entender la distribución de las diatomeas a escalas mayores. Además, esta caracterización podría darnos pistas clave para la delimitación de áreas de conservación efectivas para las especies más amenazadas.(AU)

Population Dynamics of Diatoms in a Spring-Neap Tidal Cycle in an Estuarine Creek of the Bonny River System

The present study focused on the aspects of diatom population changes through the spring-neap-spring tidal phases along the Elechi creek - an estuary of the Bonny River. Water samples at high and low tide were collected during a cycle of spring-neap-spring cycle between September and November 2015. Diatoms were counted at different magnifications of x250 and x400. Counting was in 100 fields of view along several transects. Total diatoms were calculated as a relationship between the area of grid (mm2), number of grids counted and area of counting chamber (1000 mm2). Diatoms density ranged from 1678 cells L-1 (high tide) to 3731 cells L-1 (low tide) with a mean value of 8.87 ± 6.64 cells L-1. During the lunar phases diatoms abundance ranged from 431 ± 0.2 cells L-1 (last quarter) to 396 ± 0.2 cells L-1 (new moon), to 430 ± 0.3 cells L-1 (full moon), to 421 ± 0.2 cells L-1 (first quarter). The result revealed evidence of changes in diatom abundance and variability. At low tide, the abundance and variability of diatom was higher than the observation at high tide. There was also a significant difference between mean counts of the diatoms during various moon phases; but there was no significant difference in the abundance between spring and neap tide. These findings are crucial for ecological inference during environmental assessment surveys. A consideration of these factors is suggested as necessary to avoid poor estimates that can drastically alter impact assessment conclusions.

The investigation of diatom species composition and database of diatom scanning electron microscope in the Pearl River of Guangdong province / 中国法医学杂志

Objective The investigation of diatom species composition and database of diatom scanning electron microscope in the Pearl River of Guangdong province. Methods Using the Lefort aqua regia digestion 19 different sampling sites (6 in the West River, 9 in the North River, and 4 in the East River) were sampled by us in June and September 2012.Water samples from each sampling site was digested and then observed by scanning electron microscopy. The diatom genera found in samples were recorded. Results 21 diatom genera, including Achnanthes, Cocconeis, Coscinodiscus, Cyclotella, Cymatopleura, Cymbella, Diatoma, Diploneis, Gomphonema, Gyrosigma, Hantzschia, Melosira, Navicula, Nitzschia, Pinnularia, Rhoicosphenia, Stauroneis, Stephanodisus, Surirella, Synedra, Tabellaria, were found in all the samples. Conclusion It is helpful to legal medical expert by using database of diatom scanning electron microscope in the Pearl River of Guangdong province. As hundreds of diatoms pictures were taken by SEM, it would be a valuable reference of diatoms identification for forensic experts as well as diagnosis of drowning place.

A real-time qPCR method to identify diatom UPA gene for the drowning diagnosis / 中国法医学杂志

Objective To establish a Real-time quantitative PCR method (qPCR) for the detection of diatom UPA barcoding genes and evaluate its application in the drowning diagnosis. Methods The homologous sequences of diatoms UPA gene was obtained by Blast from GeneBank, based on which the universal primers for diatoms were designed. DNA were extracted from 2 common human symbiotic bacteria (Escherichia coli, Bifidobacterium longum), 3 species of planktonic bacteria, 15 species of planktonic algae, tissue samples (lung, liver and kidney) from human cadavers (28 drowning victims, 1 victims by non-drowning in the water, 3 victims deaths on land) in 32 cases. The specificity, sensitivity and repeatability of the designed primers were tested. The positive rates of diatoms detection in the drowning cases were calculated. The results of the real-time quantitative method were evaluated comparatively by Microwave Digestion-Vacuum Filtration-Automated Scanning Electron Microscopy (MD-VF-Auto SEM) and PCR-Capillary Electrophoresis (PCR-CE). Results The results showed that the primers UPA99 had strong specificity for the diatomaceae (Synedra radians, Navicula sp., Melosira varians, Cyclotella sp. and Nitzschia sp.) DNA. The melting curve of the amplified product was smooth; the peak was narrow; the melting temperature was (87±1)℃. The sensitivity of qPCR method was 1.56×10-5ng/μL with the detection range of 1.56×102ng/mL~1.56×10-5ng/μL, in contrast with the PCR-CE method (1.56×10-3ng/μL). This real-time PCR method showed high repeatability and stability with the coefficient of variation less than 2%. The detection rate of lung, liver and kidney was 89.3%, 71.4% and 64.3% respectively. Conclusion The established qPCR method, based on the universal primers designed for diatom UPA gene, has high specificity, high sensitivity and good repeatability. With a promising prospect for application, qPCR is suitable for drowning diagnosis.

The identification of the 16SrDNA of the diatoms in rabbits' internal organs in the summer and winter in Yongjiang River of Ningbo / 中国法医学杂志

Objective To evaluate the application value of identification on drown by detection 16SrDNA of the diatoms in rabbits' internal organs in summer month of July and winter month of December in YongJiang River of Ningbo. Methods 60 Rabbits were randomly and medially divided into three groups in summer and winter: drowning group, postmortem immersion group and using only lethal aeroembolism as control group. Specimen including heart, liver, lung and kidney from each rabbit were tested with diatom 16SrDNA PCR method. Results Compared with postmortem immersion group, detection rate of diatom 16SrDNA of heart, liver, lung, renal tissue in drowning group was significantly higher than that in summer month of July (P<0.05), In December, the 16SrDNA of the drowning group was detected in heart and lung tissues, There was no significant difference compared with postmortem immersion group (P>0.05) In summer month of July, detection rate of 16SrDNA of heart, liver, lung, renal tissues in drowning group was significantly higher than that in winter month of December (P<0.05). Diatom 16SrDNA of heart, liver, lung, kidney tissues in air embolism group were not detected In summer month of July and winter month of December. Conclusion With the higher detection rate of diatom 16SrDNA in drowning rabbit in summer, the diatom 16SrDNA PCR method can be used for the diagnosis of drowning in Yongjiang River of Ningbo; while in winter , it should be carefully apllied with the lower detection rate of diatom 16SrDNA.