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Objective To study the effect of silica in soil on the extraction of biological evidence DNA at the crime scene using the silica bead method.Methods Mud suspension and diluted blood were mixed to prepare biological samples mixed with dust and soil,which is to simulate biological evidence at the crime scene.Cell lysis was performed using heating lysis and guanidine salt chemical lysis,respectively.DNA was extracted using the silica bead method,amplified by PCR using Identifiler Plus kit and detected by capillary electrophoresis.The electrophoresis results were compared.Using mud suspension instead of silica beads to extract diluted blood DNA to validate the effect of silica in soil on the extraction of biological evidence DNA at crime scene using silica beads method.Results The complete STR loci were obtained after the extraction and amplification of 4 μL,20 μL dilute blood mixed with mud and lysed with heating cracking,whoes average peak heights arel 969.7±376.9 RFU and 9 706.7±349.8 RFU.For the 4 μL dilute blood mixed with mud guanidine salt chemical lysis,it cannot obtain complete STR loci after extraction and amplification.20 μL dilute blood mixed with mud guanidine salt was chemically cleaved and amplified to obtain complete STR loci with an average peak height of 1 899.8±801.3 RFU.After extraction and amplification by mud suspension instead of silica beads to extract 20 μL diluted blood DNA,complete STR loci were obtained.Conclusion Silicon dioxide in soil can bind to DNA in the presence of guanidine salts,leading to a decrease in the efficiency of recovering on-site biological evidence DNA using the silicon bead method.
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ObjectiveAt present, the matching reagents of commercially available rapid DNA instruments based on microfluidics chip technology are autosome short tandem repeat (STR) individual identification reagents. The non-recombining part of the human Y chromosome is widely used in forensic DNA analysis, particularly in cases where standard autosomal DNA profile is uninformative. Y-STR loci are useful markers to identify males and male lineages in forensic practice. In order to achieve rapid and fully integrated detection ofY-STR loci, this study constructed the RTyper Y27 microfluidic chip rapid detection system and validated the performance of this system. MethodsThe system was verified and evaluated by sensitivity, success rate, typing accuracy, peak height balance, sizing precision and accuracy, mock case sample tests, mixture detection ability, and inhibition tolerance. ResultsComplete Y-STR profiles can be obtained when the template amount of DNA standard 9948 was ≥8 ng, the number of blood cards was ≥3 pieces, and the number of oral swab scrapings was≥7 times. The success rate of fully integrated detection was 91.52%, and the concordance rates was 99.74% for 165 testing samples. The success rate of 115 blood spots in these samples was 90.43%, with a typing accuracy of 99.65%, the success rate of 50 buccal swabs was 94%, with a typing accuracy of 99.92%. There was no significant difference in typing accuracy between blood spots and buccal swab samples. The peak height ratio between different fluorescence channels was 89.81%. The standard deviation of allelic ladder for 10 runs was within 0.5 bp. The size differences between allele and corresponding allele in allelic ladder was within 0.5 bp. The maximum precision CV values within and between batches were 0.48% and 0.68%, respectively, which were lower than 15%. These data indicate that the system has good accuracy and precision. The system was capable of accurately typing oral swabs, blood cards, saliva cards, cigarette butts, blood swabs and seminal stains. Complete Y-STR profiles can be obtained and distinguish at the 1∶3 ratio of minor and major contributors in artificial male DNA mixtures. Complete Y-STR genotyping can be obtained under the interference of inhibitors, such as different concentrations of humic acid (50-400 mg/L), indigotin (20-100 nmol/L) and hemoglobin (100-500 μmol/L). ConclusionIn this study, the RTyper Y27 microfluidic chip rapid amplification system is combined with the Quick TargSeq 1.0 integrated system, and the Y-STR profile can be obtained in approximately 2 h. Through a series of verification experiments, the results show that the system has good repeatability, accuracy and stability, can meet the on-site Y-STR detection requirements, and can be used in forensic practice.
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Objective To genotype mixed samples with next generation sequencing and evaluate its prospects in forensic DNA application. Methods Three mixed biological samples from rapes cases and their reference samples were collected. DNA was extracted using the MagAttract M48 DNA Manual Kit(200). The ForenSeqTMDNA Signature Prep Kit was used for library preparation, and next generation sequencing was performed on the MiSeq FGx system. The ForenSeqTMUniversal Analysis v1.2.1 software was used for data analysis. NGS-based STR results were compared with CE-based genotypes. Results A single length polymorphic STR allele in the mixed profile could be recognized as two sequence polymorphic STR alleles from different donors, which would assist mixed profile analysis. Such phenomenon was observed in D3S1358, D9S1122 and D13S317 in this work. Conclusion Our results suggested that precision STR genotyping of mixed samples based on NGS can provide more information and hints for mixed STR profile separation.
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Objective Using circRNA detection technology to explore the feasibility of the application of circRNA molecules in the identification of body fluids. Methods Prepare three kinds of body fluid samples: semen, saliva and vaginal secretions. Total RNA was extracted from Qiagen RNeasy Micro kit and digested by RNase R to obtain circRNA. Reverse transcription PCR amplification and agarose gel electrophoresis analysis were performed to detect target products. Results CircRNA can be detected in all prepared samples. These results showed that the circRNA was widely present in common body fluids of forensic medicine, and had some application value. Conclusion The detection for circRNA can be compatible with the existing DNA detection technology, and its tissue specificity can be used as a new marker for identification of body fluid and has important research value.
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DNA mixture has been a problem to be conquered for a long time in the forensic study. The DNA mixtures can be mainly divided into two categories: one comes from the sex assaults, for which we have to detect the sperms among the large amount of vaginal epithelial cells; the other one is the combination of different common samples, such as blood mixtures, salvia-blood mixtures and so on. In recent years, deeper and deeper study on DNA mixtures has led a way to objectiveness and pluralism. Novel techniques, like fluorescence- or magnetic-activated cell sorting strategies,micromanipulation and acoustic different analysis can be utilized to separate sperms in the mixtures; as for the second sort, the application of massively parallel sequencing(MPS), microfluidic droplet, whole-genome amplification, emulsion PCR(ePCR) et al. rise up the chances to detect the minor contributor in the mixtures. Furthermore, the development of both traditional and novel biomarkers which includes STR, Y-STR, SNP, DIP, Microhaplotype, DIP-STR, SNP-STR,, mtDNA-SNP and so on, provide us more analyzing choices in mixture study. The last part of the assay focuses on the latest progresses of evaluating the number of contributors, the explanation theory and calculation software.
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In addition to obtaining DNA-STR typing of an evidentiary stain for individual identification and paternity tests, knowing the time since deposition (TSD) is also highly desired in forensics. To provide a reference for the research of predicting the TSD, this article reviews the reported optical, cell biological and molecular biological methods of determining the age of bloodstains domestic and overseas, and also introduces the application of microbial forensics, a new field of forensic science, to provide space-time clues of evidentiary stains.
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Objective This exploratory study aimed to assess effectiveness with ethylene oxide treatment for removing DNA contamination. Methods 98 different spiked samples such as saliva, dander, skin cell, hair, blood and cartilage were conducted with ethylene oxide treatment. After extraction of samples, the dna was amplified and then the STR analysis was performed with 3130xl or 3500xl. Results A 6h EO treatment results showed that two saliva stains of 44 samples STR profile were detected; Just one hair of 54 samples treated with ethylene oxide was detected contaminating DNA with EO treatment for 8 hours. Conclusion This work suggested that it was more successful to reduce DNA contamination by using ethylene oxide treatment.
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Objective To identify the half-sibling relationship by comprehensive using three different methods. Methods STR genotype was performed on A, A's mother, B and B's mother by using PowerPlex21 kit, AGCU Expressmarker 21+1 kit, Microreader 23sp-B kit and AGCU X-19 STR kit respectively. Based on the results of STR genotype and X-STR, we determined half-sibling relationship by ITO, discriminant function analysis and IBS. Results HIS was between 1.36×102and 2.09×105in ITO which indicated that A and B had the same father. The IBS and discriminant function analysis also had the same conclusion. Conclusion Comprehensive using multiple methods can obtain reliable result to identify the half-sibling relationship from the same father.
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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.
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Objective To analyze a large number of DNA mixture de-convolution data with stochastic simulation method. Methods Using the software in Identifiler, PP18D, AGCU EX20, PP21, AGCU EX20 & AGCU 21+1 system, 1 million groups of STR genotyping of mixture were analyzed. The average de-convoluted loci (L), the average de-convoluted combinatorial number (Π) were counted out. Results In identifiler, PP18D, AGCU EX20, PP21, AGCU EX20 & AGCU 21+1 system, L were 4.8, 5.8, 6.7, 7.0, 11.1, and Π were 3.71×104, 1.06×105, 3.34×105, 6.40×105, 2.48×1012. Conclusion The result in this paper has some guiding significance in DNA mixture de-convolution followed by DNA Database search.
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Objective To investigate the influences of drop components on the"coffee ring" test of DNA. Methods The DNA-EB drops containing SDS, NaCl, etc, were evaporated on glass slides. After evaporation, the fluorescent Deposition Pattern (DP) and Integrated Optical Density (IOD) was acquired with a gel imaging system. The dose-effect relationship was analyzed. Result When the non-DNA components concentration is low, the DP was still characteristiced by peripheral rings, subtle component-specific differences, such as tree-ring like structure and radial crack, existed. At high concentrations, DP was various, which may be ring + scattered dots (NaCl), central spot + small weak ring (SDS), concentric/tree-ring (TritonX-100) or ring + spot (H+), et al. Non-DNA components had little effect on IOD(0.5~2 times). Conclusion Non-DNA components in DNA-EB drops influences both the DP and IOD, but rough estimation of DNA concentrations is still effective. Moreover, analyzing the DP can provide more informations about sample purity and residue.
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High-resolution melting (HRM) analysis is a versatile method for variant scanning and genotyping. It involves amplification of the target in the presence of a saturation dye by the polymerase chain reaction (PCR). HRM analysis can be performed in one closed-tube, which does not require additional post-PCR separations and greatly reduces the possibility of contamination. HRM is faster, simpler, and less expensive than alternative approaches requiring labeled probes. Considering the many advantages of HRM analysis, many researchers have tried to apply this method to forensic research. This paper intends to summarize the principle, technical characteristics, limitations and application of HRM analysis in forensic science.
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Objective To analyze the 2496 biological samples collected from 1574 larceny cases and improve the application of DNA in the larceny cases. Methods Make a summary of the biological samples collection methods and DNA test results in different type of larceny cases. Results Touch samples have already become the most common type of biological samples in larceny cases, but their DNA test success rate are still low, more work should be done for the DNA mixed type to improve DNA hit. The DNA positive result rate had statistical difference in biological samples with different collection methods. For the Touch samples, flocked swab and original are the best collection methods. Conclusion More Valuable biological samples collected by the crime scene investigators are the Key factor in improvement of DNA detection capability, awareness of trace biological evidence and the touch sample collection technique are very important for the crime scene investigators.
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Objective From the perspective of making full use of database comparison function, giving certain guidelines to analyze mixed DNA profile,compare database,screen comparison results. Methods Using CPI to describe the identification of mixed DNA profile.Using CPBI to estimate reliability of individual samples being included. Results When CPI is less than 10-7, mixed DNA Profile is worth to be compared in database.When the number of alleles at one locus is more than 2, retain an additional allele will not reduce identification too much. According to the CPBI of the included samples,we can find the most reliable sample.
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Objective To investigate personal identification of mixed seminal stain of two individuals, we combined the detection of genotyping autosomal, Y and X STR and sequencing mtDNA hypervariable Ⅰ (HV Ⅰ ) region. Methods We analyzed autosomal, Y and X STR with commercial kit and separating and sequencing HVⅠfragments of mixed seminal stain from two males by SSCP electrophoresis. Results Four genetic markers of the high amount sample can be obtained when mixed ratio is more than 1:10. When the proportion of two samples is close, the suspect could be excluded or, to some extent, identified by comparing with our results. Conclusion The combined detection of four genetic marker systems can, to some degree, solve the personal identification from mixed seminal stain of two individuals.
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Objective This study aimed to assess the efficiency and purification of the Trace DNA extraction with a quantified method for the magnetic bead-based DNA extraction as performed on the Tecan Automated systems with TE-MAGS magnetic separator. Methods Serial dilutions of standard commercial DNA 9947A were used with the total DNA contents, 0.1ng, 0.2ng,0.3ng, 0.4ng,0.5ng, 0.6ng, 0.7ng, 0.8ng, 0.9ng,1ng,diluted progressively and a 1ng DNA (standard commercial DNA 9947A) admixed with 6 common DNA-PCR inhibitors were extracted on the Automated systems and then performed via Fluorogenic probe quantitative PCR and STR genotype for the quantification analysis of recovery and purification. Results The recovery rate of standard 9947A DNA diluted with 0.1~1ng was 38.92~60.01%, and 0.3ng and more DNA could observed the full STR profiles. For the different PCR inhibitors, above 94.5% of bile acid, collagen and urea were efficient removal during the purification process, and the hemoglobin, melanin and humic acid removal efficiency were about 97.5%, 97.85%, 82.14%, respectively.Conclusion Our results suggested that The TE-MAGS magnetic bead-based DNA extraction was suitable for the extraction of trace DNA with high recovery efficiency and purification ability.
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Objective To prepare a capillary electrophoresis sieving medium and apply it in GA118-16A genetic analyzer. Methods The white solid polyacrylamide (LPA) was prepared by polymerization and lyophilized. Through the swelling of the sol buffer, the sieving medium was obtained. The sieving medium was evaluated by 1) characterizing the parameters, including molecular weight, structure and viscosity, 2) applying in the GA118-16A genetic analyzer, including the spatial calibration, the spectral calibration and the STR analysis.. Results The prepared sieving medium Mw 1.8 x 105Da, Mn 1.2 x 105 Da, is of correct structure and high purity. The polydispersity was 1.5The spatial calibration and spectral calibration files can be established successfully in GA118-16A genetic analyzer, and the sieving medium can effectively separate the DNA fragments with 1bp difference. The STR profile is of sharp peaks, no impurity peaks, no tail, and no peak loss. Conclusion The sieving medium prepared by the method can be applied to domestic genetic analyzer such as GA118-16A.
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Objective Selecting Tibetan ancestry informative SNPs base on high-altitude adaptation genes of Tibetan. Methods We developed a multiplex assay constituted of 87 SNPs located on EPAS1 and EGLN1 genes based on Agena Mas-sARRAY? genotyping system. 183 samples from Tibet plateau were genotyped. The genotypes of 2504 samples from 26 populations were downloaded from the 1000 genomes. Genetic frequency and population structure were analyzed and compared between Tibetan population and worldwide populations. Results SNPs that have distinctive distribution between Tibetan and other populations could be used as Tibetan AISNPs (Ancestry informative SNPs). We selected 23 SNPs has a separated principal genetic component in STRUCTURE result. Conclusion These 23 AISNPs we selected could be combined into ancestry informative SNP panels to improve the resolution of ancestry inference in East Asian, and provide clues for cases of biological material ancestry inference.
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Objective The purpose of this study was to detect the degradation degree of long-term formalin fixed tissue and to compare the detection rate of STR with SNP. Methods DNA was extracted from 24 formalin-fixed tissues stored at -20 ℃ for five years, and the concentration and degradation index of DNA was quantified with Quantifiler? Trio DNA Kit. A 55-SNP multiplex SNaPshot assay and PowerPlex? 21 system were used to amplify SNP and STR loci, respectively. Results The results showed that the degradation indexes of 24 specimens were ranged from 1~8. The SNP genotypes of the 24 specimens were completely consistent with the non-degraded DNA from the same individuals and the successful genotyping rate was 100%. However, 33 allele dropouts were observed with STR genotyping in 8 samples, of which the degradation index was higher than 2.6, and the fragment size of the 75.8% allele was longer than 300bp. The likelihood ratio based on 16 typable STR loci in the sample was close to that onthe basis of 54 SNPs. However, likelihood ratio based on more than 17 STR loci was over that accord to 54 SNPs. There was a negative correlation between the fragment size of STR and the allele detection rate, and a negative correlation also observed between the degradation index of samples and the allele detection rate except for two samples with mild degradation. Conclusion This study validated that the long-term formalin-fixed tissues were susceptible to degradation, and the SNP was more suitable for detecting these tissues than STR typing system. However more SNP loci are needed to test in order to increase the discrimination power.
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Objective The magnetic beads direct adsorption method was used to extract the cell-free DNA (cfDNA) from three kinds of human humoral biological samples, including urine, saliva and blood, as to provide a reference for forensic cfDNA research and forensic inspection. Methods The cfDNA was isolated from humoral samples by centrifuging, and the cfDNA was extracted with the method of magnetic beads direct adsorption. Then the samples were sequentially amplified with Identifiler-Plus amplication kit, and the STR genotyping was detected by ABI 3500 Analyzer. Results The cfDNA was detected from all the three kinds of samples. The detection rate of cfDNA from the blood samples was 100%, the saliva was 90%, and the urine was 70%. Conclusion The results suggest that human humoral biological samples contain cfDNA. What's more, the magnetic beads direct adsorption method can be used to extract cfDNA efficiently and conveniently.