Application Progress of High-Throughput Sequencing Technology in Forensic Diatom Detection.

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.

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.

[Diagnosis of amniotic fluid embolism with blood samples by liquid-based cytology technique].

OBJECTIVE: To establish the diagnosis of amniotic fluid embolism with blood samples by liquid-based cytology technique and to study the validity of method. METHODS: The blood samples were collected from patients who suffered from amniotic fluid embolism. The components of amniotic fluid in blood samples were examined with blood smear by two direct smear methods (supernatant smear, sediment smear) and two liquid-based cytology methods (automatic smear, manual smear). The positive detection rate of each method was calculated. RESULTS: The positive detection rates of two liquid-based cytology methods (84.6% and 92.3%, respectively) were much higher than those of two direct methods (53.8% and 61.5%, respectively). CONCLUSION: The liquid-based cytology technique could improve the positive detection rate of amniotic fluid embolism.

DNA Extraction and Comparison between Old and Fresh Necrophilic Fly Samples.

A total of five samples of Chrysomya megacephala samples - three fresh samples, one sample stored in alcohol for 2 years, and one sample stored in dry sealed storage for 2 years protected from light only - were selected to investigate whether a blood DNA extraction kit could extract DNA from necrophilous flies and to determine whether alcohol could prolong the preservation of necrophilous flies' DNA. First, the blood DNA extraction kit was used to extract DNA from their thorax tissues. Then, the DNA purity and concentration were examined using a microplate reader and a fluorometer. Finally, PCR amplification and electrophoresis of the extracted DNA were done with necrophilic fly-specific primers located in the mitochondrial CO I gene sequence. The results showed that the DNA purity of all samples was greater than 2.0. The DNA concentration was observed to be of the following order: fresh samples > alcohol-preserved old samples > untreated, old samples. All samples had specific electrophoretic bands after PCR amplification. In conclusion, a blood DNA extraction kit can be used to extract DNA from necrophilic flies successfully, and the DNA concentration of fresh fly samples is greater than that of old fly samples. The flies can be stored in alcohol for a long time.

Extraction of Diatom DNA from Water Samples and Tissues.

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. Diatom testing is also an important research content in the field of the environment and plankton. The diatom molecular biology testing technology, which focuses on diatom DNA as the primary research object, is a new method of diatom testing. Diatom DNA extraction is the basis of diatom molecular testing. At present, the kits commonly used for diatom DNA extraction are expensive, which increases the cost of carrying out related research. Our laboratory improved the general whole blood genomic DNA rapid extraction kit and obtained a satisfactory diatom DNA extraction effect, thus providing an alternative economical and affordable DNA extraction solution based on glass beads for related research. The diatom DNA extracted using this protocol could satisfy many downstream applications, such as PCR and sequencing.

[Degenerate oligonucleotide primed-PCR technology establishment and its sensitivity test analysis].

OBJECTIVE: To establish a whole genome amplification testing system based on degenerate oligonucleotide primed-PCR (DOP-PCR) and to explore its reliability and sensitivity. METHODS: DOP-PCR amplified production was detected by fluorescent labeled multiplex STR amplification and capillary electrophoresis detection system to determine reliability and sensitivity of DOP-PCR system. RESULTS: DOP-PCR system was successfully established and the detection sensitivity reached 5 cells (30 pg) by pretreatment of DOP-PCR and then detection of STR genotyping. CONCLUSION: The system established in this study is reliable and more testing sensitive for forensic trace evidence.

[Effect of application of cow manure and green manure on corn yield and soil physical-chemical properties in land restoration area]. / 施用牛粪和绿肥对土地整治区玉米产量和土壤理化性质的影响.

The effects of cow manure and green manure on maize yield, soil respiration and soil physical-chemical properties in land restoration area was evaluated through field experiments. The results indicated that the maize yield and thousand-grain mass with cow manure were increased by 7.2%-29.9% and 2.5%-18.2%, respectively compared with the application of chemical fertilizer (CF), while the soil active organic carbon and organic matter contents of cow manure were 5.3%-34.6% and 8.0%-17.6% higher than that obtained in CF. The maize yield and thousand-grain mass were increased by 10.8%-15.6% and 4.5%-8.4% with application of green manure, respectively compared with CF. The content of active organic carbon in green manure was 14.1%-48.6% higher than that detected in CF. In the second year, the content of organic matter in green manure treatment was 7.2% higher than that of CF. The soil respiration rates under cow manure and green manure treatments increased by 20.0%-69.3% compared with CF. CF and green manure could improve the soil bulk density and increase the aggregate ratios of ＜0.01 mm and 0.05-1 mm fractions, respectively. On the other hand, the cow manure and green manure could decrease the soil total porosity and the capillary porosity. In conclusion, the application of cow manure and green manure in land restoration region could increase maize yield during the two consecutive seasons, which showed a positive response to improvement of soil physical-chemical properties.

[Constructing of STR slippage model by optimizing some factors].

OBJECTIVE: To construct STR slippage model and study factors involved in this procedure. METHODS: DNA samples were amplified with the technology of Degenerate oligonucleotide- primed PCR, then their products were taken as later DNA template and their STR genotype were analyzed by optimizing several factors. RESULTS: STR slippage model was constructed. CONCLUSION: Several factors were involved in the produce of STR slippage, such as amount of modulate DNA, concentration of MgCl2, property of DNA polymerase, motif sequence of STR loci, sample, etc.

[Forensic application of 9 Y-STRs fluorescence-labeled multiplex amplification system].

OBJECTIVE: In order to increase significantly the discriminatory potential of Y-STR systems available to the forensic community, we have developed a system capable of simultaneously amplifying 9 Y-STR loci by fluorescence-labeled multiplex PCR technique. METHODS: Primers of STR loci DYS434, GATA-A10, DYS438 and DYS439 were labeled with 6-FAM, primers of STR loci DYS531, DYS557, DYS448 were labeled with HEX, and primers of STR loci DYS456, DYS444 were labeled with TAMRA, respectively. PCR products were analyzed using capillary electrophoresis and GeneScan Software on the ABI Prism310 DNA Analyzer. Series experiments were carried out to evaluate the useful value in forensic application such as the sensitivity, male specificity and genotyping DNA different tissues of the same individual. RESULTS: 9 Y-STR loci were exactly determined following optimization of the polymerase chain reaction. In a sample of 120 males, a total of 105 different haplotypes was identified, 97 of them being unique. Overall, haplotype diversity was 0.996 8. It was proved that genotyping of these 9 Y-STR loci in some sexual crime should be prior to that of automosomal STR. CONCLUSION: The results suggest that the newly constructed 9-plex will be very powerful for establishing Y-STR database, population genetic studies and mixture stains identification.

Genetic polymorphisms of four STR loci on chromosome X and their forensic applications in a Chinese Han population.

OBJECTIVE: To add DXS7133, GATA198A10, DXS9896 and DXS6797 to the panel of forensically validated X chromosome markers, and apply the multiplex amplification system to a population study and forensic analysis on the Hans of Chengdu. METHODS: The PCR products were detected by the polyacrylamide gel electrophoresis and silver staining method. Hardy-Weinberg equilibrium of females was tested and every forensically interested value was calculated. RESULTS: Sequencing revealed that their common sequence motifs were tetranucleotide repeats. Population genetic data were obtained by analyzing 120 unrelated females and 100 males from Chengdu Han ethnic group. In this population, DXS7133, GATA198A10, DXS9896 and DXS6797 exhibited 6, 6, 11, 8 distinguishable alleles respectively. Chi-square test demonstrated that genotype frequencies in females did not depart from Hardy-Weinberg equilibrium. Power of discrimination for female samples for the four loci were 0.7962, 0.8021, 0.9675, and 0.9444. The parentage testing in 32 cases revealed a typical X-linked inheritance and no mutations. CONCLUSION: DXS7133, GATA198A10, DXS9896 and DXS6797, which are highly polymorphic in Chengdu Han population, are appropriate for individual identification and paternity testing involving a female child.

[Constructing standard allelic ladders for four short tandem repeat loci and employing them in a population study on Han Nationality of Chengdu in China].

OBJECTIVE: To solve the problems in the accuracy and standardization of short tandem repeats-polymerase chain reaction (STR-PCR) typing, the authors adopted the molecular clone technology in producing the standard allelic ladders of D1S1676, D2S2735, D11S1977 and D22S444 loci and applied them in a population study on the Hans in Chengdu, China. METHODS: PCR was used to produce several different allelic fragments of these loci. PCR products were eluted from the gel and re-amplified by PCR. The purified allelic fragments were then blunt-end subcloned individually into the pGEMR-T plasmid vectors and the recombinant were transfected into competent E.coli DH5alpha TM cells. The results of sequencing confirmed that the size and the construction of the inserts were correct. The recombinant plasmids DNA with the inserts were then used as template for re-amplification to generate the four loci standard ladders. RESULTS: The authors succeeded in producing large quantity of standard allelic ladder of these four loci, with which the genetic polymorphisms of these loci in Chengdu Han population of China were studied. CONCLUSION: This method is of high value for forensic DNA typing to construct standard ladders. D1S1676, D2S2735 loci are robust for forensic analysis in Chinese Han population, whereas the value of D11S1977 and D22S444 loci is limited.

[Study of Five STR Loci in A Chinese Han Population.].

To understanding the allele structure and genetic polymorphisms at five STR loci in Chinese Han population, and construct a preliminary database, EDTA-blood specimens were collected from unrelated individuals. DNA samples were extracted with Chelex method and were amplified by PCR technique. The PCR products were analyzed using both PAGE horizontal electrophoresis with discontinuous buffer system and automated fluorescence detection approach. As a result, three STRs consist of simple repeat motifs, while two STR contain a complex repeat structure. The STR polymorphisms at all of the five loci have been observed in Chinese Han population. In a word, the obtained data are beneficial to understanding the population genetics of the five STR loci in Chinese Han population. As a simple approach, the PAGE horizontal electrophoresis can be employed for typing the five STR markers.

[Forensic applications of the sequencing of mitochondrial DNA cytochrome oxidase subunit I and II gene for idenfication of Sarcosaphagous flies (diptera)].

OBJECTIVE: This study was based on a 278 and 635 bp region of the gene for cytochrome oxidase subunit I and I (CO I and CO II ) encoding region of mtDNA; the aim was to solve the problems in identifying Sarcosaphagous flies, particularly in the flies' larvae and eggs which could not be identified only by use of their morphologyical features. METHODS: Samples of sarcosaphagous flies and larvae were collected from those on the corps of rabbits on the grassland in the Huhhot district and of a pig on the sandy ground in the Dunhuang district. The mtDNA of flies and their larvae and eggs was extracted using the Chelex technique. Polymerase chain reactions were conducted on a Perkin-Elmer 9600 thermal cycler, followed by vertical non-denaturing polyacrylamide gelectrophoresis. PCR products were purified using the Nucleic Acid Purification Kit. Sequences of both strands were obtained by direct sequencing of the double-stranded PCR product using one of the PCR primers and the ABI PRISM Big Dye Terminator Cycle Sequencing Kit. Sequence reactions were electrophoresed on ABI Model 377 DNA Sequencers. A neighbour-joining tree using the Tamura and Nei model of nucleotide substitution was constructed using the MEGA2. 1 package. RESULTS: A 278 and 635 bp region of the gene for CO I and CO II encoding region of mtDNA of Sarcosaphagous flies and their larvae and eggs was noted to show the percentages for the sequence divergence within species (less than 1%) and the sequence divergence between species (above 3%). For species that diverged from all others by a relatively large percentage and had small within-species variation, the least percentages of sequence divergence were given which distinguished any individual within that species from any other species. CONCLUSION: A 278 and 635 bp region of the gene for CO I and CO II encoding region of mtDNA of Sarcosaphagous flies and their larvae and eggs has been effectively used for the molecular phylogeny and the identification of their species group. CO I and CO II, or CO II, is better than CO I.

[Practice value of whole genome amplification technology to be used in forensic science and analysis of its result].

Genetic analysis from forensic microsamples is a urgent, difficult task in forensic science, because it is frequently limited by the amount of specimen available in forensic practice, much effort has been carried out to resolve this difficulty. Whole genome amplification (WGA) technology, which was developing quickly in these years, has been thought to be a powerful, reliable and efficient strategy in analysis of minute amount DNA on many fields. In this review, we discuss its application in forensic science.

[DNA samples preparation from single cell and its application in sensitivity test].

OBJECTIVE: To establish a reliable, exact and practical method to prepare DNA samples for sensitivity-test purposes. METHODS: The micromanipulation method was employed to prepare exact quantity DNA samples used to study the sensitivity of Profiler Plus Kit-ABI PRISM 310 system. RESULTS: We succeed in establishing a micromanipulation method to prepare groups of DNA samples, which contain 1-11 cells in turn, and also succeed in using them to study the sensitivity of Profiler Plus Kit-ABI310 system. CONCLUSION: The method we have established is proved to be a reliable, exact and practical way to prepare DNA samples for sensitivity-test purposes.

[Genetic polymorphism of 3 STR loci on chromosome X and its forensic application in Chinese population].

To investigate the genetic polymorphisms of three short tandem repeats loci of chromosome X in Chinese Han population in Chengdu area and its use in forensic science. Three X-chromosome linked short tandem repeat loci were analyzed by PCR followed by polyacrylamide gel electrophoresis. Hardy-Weinberg equilibrium was tested and forensic interested value was calculated . The power of exclution of DXS6804, DXS9896 and GATA144D04 is 0.5990, 0.6220, 0.4280, respectively. The result showed that all the three STR loci were polymorphic among 100 unrelated females and 120 unrelated males from Chinese Han population. Chi(2) tests demonstrated that genotype frequencies in females did not depart from Hardy-Weinberg equilibrium. Three X-chromosome linked short tandem repeat loci have high polymorphism, they can be applied to forensic medicine and population genetics.

[Genetic polymorphisms of three STR loci on chromosome X and their forensic application in a Chinese Han population].

OBJECTIVE: To understand the allele structure and genetic polymorphism at three STR loci on chromosome X in Chinese Han population and make an evalution of their forensic application. METHODS: EDTA-blood samples were collected from the unrelated individuals in Chengdu, China. After being extracted with Chelex method, the DNA samples were amplified by PCR technique. The PCR products were analyzed by PAG electrophoresis and the approach of automated fluorescence detection. Hardy-Weinberg equilibrium of females was tested and every forensic interested value was calculated. RESULTS: The polymorphisms of all 3 STR loci were obtained from 100 unrelated females and 120 unrelated males from Chinese Han ethnic group. Chi-square tests on the genotype frequencies in females did not reveal deviations from Hardy-Weinberg equilibrium. CONCLUSION: The obtained data are beneficial to understanding the population genetics of the three STR loci in Chinese Han population. For forensic genetics, the obtained data can be used to calculate the probabilities dealing with the paternity test and the individual identification.

[Nutrient spatial variability of tobacco soil restoration area and fertility suitability level evaluation].

By using geographic information system technology (GIS) and geostatistics methods, this paper studied the spatial variability of soil properties and available nutrients in the new regulation area units located in Qingjiangyuan modern tobacco agriculture science and technology park (Enshi, Hubei), suburb of Enshi City and the Baiyang base of Lichuan City, and further evaluation of the soil fertility suitability index (SFI) was carried out by use fuzzy mathematics. The results indicated that the effects of land restoration on the soil available phosphorus content variability and spatial distribution were very obvious, possibly due to the landform characteristics and restoration extent. The effect of land restoration on soil pH was small, however, serious soil acidification was detected in the soil sampled from Baiyang (pH < 5.5). Low SFI was found in 77.6%, 17.1% and 31.4% of the soils taken from the suburb, Baiyang and Qingjiangyuan, respectively. In conclusion, attentions should be paid on soil acidification in Baiyang, soil fertility and equalization in the suburb, and soil fertility in the region of Qingjiangyuan with low SFI.