Evaluation of a Novel 32 X-STR Loci Multiplex System in the Forensic Application

The AGCU X Plus STR system is a newly developed multiplex PCR kit that detects 32 X-chromosomal STR loci simultaneously.These are DXS6807,DXS9895,linkage group 1(DXS10148,DXS10135,DXS8378),DXS9902,DXS6795,DXS6810,DXS10159,DXS10162,DXS10164,DXS7132,linkage group 2(DXS10079,DXS10074,DXS10075),DXS981,DXS6800,DXS6803,DXS6809,DXS6789,DXS7424,DXS101,DXS7133,GATA172D05,GATA165B12,linkage group 3(DXS10103,HPRTB,DXS10101),GATA31E08 and linkage group 4(DXS8377,DXS10134,DXS7423).A major advantage of this kit is that it takes into account linkage between loci,in addition to detecting more X-STR loci.In order to evaluate the forensic application of 32 X-STR fl uorescence amplifi cation system,PCR settings,sensitivity,species specifi city,stability,DNA mixtures,concordance,stutter,sizing precision,and population genetics investigation were evaluated according to the Scientific Working Group on DNA Analysis Methods(SWGDAM)developmental validation guidelines.The study showed that the genotyping results of each locus were signifi cantly accurate when the DNA template was at least 62.5 pg.Complete profi les were obtained for the 1∶1 and 1∶3 combinations.A total of 209 unrelated individuals from Southern Chinese Han community,consisting of 84 females and 125 males,were selected for population studies,and 285 allele profi les were detected from 32 X-STR loci.The polymorphism information content(PIC)ranged from 0.2721 in DXS6800,to 0.9105 in DXS10135,with an average of 0.6798.DXS10135(PIC=0.9105)was the most polymorphic locus,with discrimination power(DP)of 0.9164 and 0.9871 for the male and female.The cumulative PD_(F),PD_(M),MEC_(trio) and MEC_(duo) valu es were all greater than 0.999999999.There were 78 different DXS10103-HPRTB-DXS10101 haplotypes among the 125 males,and the haplotype diversity was 0.9810.There was no signifi cant difference in the cumulative PD_(F),PD_(M),MEC_(trio) and MEC_(duo) values whether considering linkage or not.In summary,the new X-STR multiplex typing system is effective and reliable,which can be useful in human genetic analysis and kinship testing as a potent complement to autosomal STR typing.

Analysis of γ-Hydroxybutyrate in Human Urine by LC-MSIMS Method and Its Forensic Application

GHB （γ-hydroxybutyrate） is becoming popular recreational drugs. As a result of its strong sedative and amnesiac effects, GHB has been implicated in a number of DFSA cases. The natural presence of GHB in the human body and its rapid elimination after ingestion make it difficult to detect and to evaluate its roles in suspected GHB-facilitated assaults. The paper describes an analytical method for the determination of GHB in urine using LC-MS/MS. Samples were acidified by ammonium chloride solution and extracted with ethyl acetate, and then the extracts were analysed by LC-MS/MS. The limit of detection was 0.05 p.g/mL （S/N = 3）. The intra- and inter-day precision was within 10.0% at three concentrations. The methods were found to be sensitive, accurate, rapid and suitable for the forensic toxicology to test GHB in real cases.

Robust and efficient direct multiplex amplification method for large-scale DNA detection of blood samples on FTA cards

Deoxyribonucleic acid (DNA) damage arising from radiations widely occurred along with the development of nuclear weapons and clinically wide application of computed tomography (CT) scan and nuclear medicine. All ionizing radiations (X-rays, γ-rays, alpha particles, etc.) and ultraviolet (UV) radiation lead to the DNA damage. Polymerase chain reaction (PCR) is one of the most wildly used techniques for detecting DNA damage as the amplification stops at the site of the damage. Improvements to enhance the efficiency of PCR are always required and remain a great challenge. Here we establish a multiplex PCR assay system (MPAS) that is served as a robust and efficient method for direct detection of target DNA sequences in genomic DNA. The establishment of the system is performed by adding a combination of PCR enhancers to standard PCR buffer. The performance of MPAS was demonstrated by carrying out the direct PCR amplification on 1.2 mm human blood punch using commercially available primer sets which include multiple primer pairs. The optimized PCR system resulted in high quality genotyping results without any inhibitory effect indicated and led to a full-profile success rate of 98.13%. Our studies demonstrate that the MPAS provides an efficient and robust method for obtaining sensitive, reliable and reproducible PCR results from human blood samples.

A high-quality reference genome of wild Cannabis sativa

Cannabis sativa is a well-known plant species that has great economic and ecological significance.An incomplete genome of cloned C.sativa was obtained by using SOAPdenovo software in 2011.To further explore the utilization of this plant resource,we generated an updated draft genome sequence for wild-type varieties of C.sativa in China using PacBio single-molecule sequencing and Hi-C technology.Our assembled genome is approximately 808 Mb,with scaffold and contig N50 sizes of 83.00 Mb and 513.57 kb,respectively.Repetitive elements account for 74.75%of the genome.A total of 38,828 protein-coding genes were annotated,98.20%of which were functionally annotated.We provide the first comprehensive de novo genome of wild-type varieties of C.sativa distributed in Tibet,China.Due to long-term growth in the wild environment,these varieties exhibit higher heterozygosity and contain more genetic information.This genetic resource is of great value for future investigations of cannabinoid metabolic pathways and will aid in promoting the commercial production of C.sativa and the effective utilization of cannabinoids.The assembled genome is also a valuable resource for intensively and effectively investigating the C.sativa genome further in the future.

Genome and transcriptome of Papaver somniferum Chinese landrace CHM indicates that massive genome expansion contributes to high benzylisoquinoline alkaloid biosynthesis

Opium poppy(Papaver somniferum)is a source of morphine,codeine,and semisynthetic derivatives,including oxycodone and naltrexone.Here,we report the de novo assembly and genomic analysis of P.somniferum traditional landrace‘Chinese Herbal Medicine’.Variations between the 2.62 Gb CHM genome and that of the previously sequenced high noscapine 1(HN1)variety were also explored.Among 79,668 protein-coding genes,we functionally annotated 88.9%,compared to 68.8%reported in the HN1 genome.Gene family and 4DTv comparative analyses with three other Papaveraceae species revealed that opium poppy underwent two whole-genome duplication(WGD)events.The first of these,in ancestral Ranunculales,expanded gene families related to characteristic secondary metabolite production and disease resistance.The more recent species-specific WGD mediated by transposable elements resulted in massive genome expansion.Genes carrying structural variations and large-effect variants associated with agronomically different phenotypes between CHM and HN1 that were identified through our transcriptomic comparison of multiple organs and developmental stages can enable the development of new varieties.These genomic and transcriptomic analyses will provide a valuable resource that informs future basic and agricultural studies of the opium poppy.

Simultaneous Determination of Heroin,Amphetamine and their Basic Impurities and Adulterants Using Microemulsion Electrokinetic Chromatography

Simultaneous separation of 17 species of heroin, amphetamine and their basic impurities and adulterants was conducted within 10 minutes by using capillary microemulsion electrokinetic chromatography. The influences of pH and 1-butanol cosurfactant on the separation were investigated, and 1-butanol was found to be a principal factor to improve separation efficiency.

The Application of X-STR： Two Case Reports

The usefulness of the X-chromosomal STRs （short tandem repeats） for forensic purposes seems to be restricted, but may be valuable in some paternity cases. Due to the particular mode of inheritance, X-chromosomal X-STR has the advantage in female traces identification against male contamination and in complex kinship cases, such as deficiency cases （mother-son or father-daughter）, grandparent-grandchild comparisons, half-sisters testing, etc. In this study, 4 blood samples from two paternity cases without father were examined with TYPER19 Amplification kit first, from the result, it is hard to judge the relationship between the mother-sons, then TYPERX19 kit was conducted to obtain the full profiles of the two pairs of the mother-son, from the genetypes of the samples, it can be inferred that neither of the two pairs of mother-son were in line with the kinship, which provides important evidence for the two paternity cases. All together, the X-chromosome marks included in the TYPERX19 kit can offer the possibility to solve complex kinship cases where autosomal STR markers cannot provide the information needed.

GENETIC POLYMORPHISM OF SIX Y CHROMOSOMAL STR IN CHINESE HUI ETHNIC GROUP

Objective To study genetic polymorphism of 6 Y chromosomal STR in Hui ethnic group living in Ningxia Hui ethnic autonomous region, in order to evaluate their usefulness in forensic science and enrich the Chinese genetic information resources. Methods We investigated 101 unrelated, healthy, male individuals of Hui ethnic group and studied their allelic frequency distribution and haplotype diversity of 6 Y chromosomal STR. Primer for each loci was labeled with the fluorescent by FAM (blue) or TAMRA(yellow). The data of Hui ethnic group were generated co-amplification, GeneScan, genotype, and genetic distribution analysis. Results 31 alleles and 43 phenotype(DYS385) were detected, with the frequencies ranging from 0.0099-0.7129. Out of a total of 101 individuals, 96 showed different haplotypes; 91 were unique; 5 were found 2 times. The haplotype diversity for 6 Y-STR loci was 0.9990. Conclusion The date obtained can be valuable for individual identification, paternity testing in forensic fields and for population genetics because of 6 Y-STR loci high polymorphism.

HPLC-MS/MS Determination of Oleandrin and Adynerin in Blood with Solid Phase Supported Liquid-Liquid Extraction

[Objectives]To optimize the determination method of oleandrin and adynerin in blood. [Methods]High performance liquid chromatography-mass spectrometry( HPLC-MS/MS) was applied to determine oleandrin and adynerin in blood. The blood sample was dispersed and fixed on a solid phase supported liquid-liquid extraction column and eluted with ethyl acetate. The resulting eluent was used for chromatographic separation with Kinetex C_(18) column as the separation column and gradient elution was performed using 10 mmol/L ammonium formate solution containing 0. 1%( volume fraction) formic acid and acetonitrile as the mobile phase. In the tandem mass spectrometry analysis,the detection was carried out using the electrospray positive ion source multiple reaction monitoring mode. [Results] The mass concentration of oleandrin and adynerin showed linear relationship in the range of 2-100 μg/L. The limit of detection( 3 S/N) of the method was 0. 5 μg/L.A blank sample was used as the substrate for the spike recovery test. The recovery rate was in the range of 90. 0%-98. 0%,and the relative standard deviation( RSD) of the measured values( n = 6) was in the range of 2. 1%-7. 3%. [Conclusions]The method established in this experiment has the benefits of simple pretreatment,good recovery,high sensitivity and strong specificity,and is expected to provide an ideal method for the determination of such drugs in blood.

Detection of Three Common Organic Explosives Using Capillary Electrophoresis

In this article, a detection method for organic explosives by capillary electrophoresis (CE) is developed based on previous detection techniques. Firstly, a buffer solution consisting of 50 mmol·L-1 sodium dodecyl sulfate (SDS), 20 mmol·L-1 sodium tetraborate and 5% methanol was prepared and the UV detection in this buffer solution was conducted for three common organic explosives, including TNT, DNT and PETN. Then, the capillary UV detection method was investigated in terms of the transition time repeatability, the linear relationship between mass concentration and peak area and the limit of detection. The results revealed good reliability and stability of this method. In addition, these samples were characterized by photodiode array detector (PDA) to verify the qualitative results of UV detection.

Facing COVID-19:Forensic Doctors of Public Security Departments Should Improve Infected Cadaver Identification and Personal Protection Procedures

Because of the nature of the human identification system,forensic doctors working in public security departments are responsible for cadaver examination and conducting crime scene investigations.These processes contain inherent risks,which often include various injury,poisoning hazards,and probable exposure risks with virus such as COVID-19.This paper discusses the occupational protections used for forensic doctors,such as crime scene corpse identification,autopsy building construction,risk assessment,and protective measures.Finally,we suggest the introduction of relevant rules and regulations that could guarantee the stability and safety of crime scene investigations and cadaver examinations.These measures may be helpful for forensic institutes and doctors working in public security departments.

The Transfer and Change of Paints in a Hit-and-run Motor Vehicle Accident

This work investigated transfer and change of paint evidences in a case of hit-and-run.Two kinds of attachments were found on the clothes of the victim and they were initially considered paint fragments from the vehicle causing the accident.Infrared spectroscopy(IR),scanning electron microscope-energy dispersive spectrometer,and Microspectrophotometry were applied for examination of paints and clothing fibers.Polyester was detected in one of the attachments and in the clothing fibers of the victim by IR.A traffic accident simulation experiment was designed and conducted to research whether the polyester attachments come from suspected vehicle paints or victim’s clothing fibers.The results showed that a melt mixture of transferred paints and clothing fiber was formed after a violent collision.Because the amount of transferred paints was too low to be detectable in the mixture,the components detected by IR were mainly from clothing fibers.Thereby,we inferred that only one kind of attachment and paint fragments existed on the clothes of the victim,and the polyester attachments cannot be used to indicate the composition of suspected vehicle paint.Clothing fibers and paints are both common trace evidence in traffic accident cases,and more attention should be paid to the examination of transferred paints on clothing fibers.

Distal-scanning common path probe for optical coherence tomography

In this paper,we present a distal-scanning common path probe for optical coherence tomography(OCT)equipped with a hollow ultrasonic motor and a simple and specially designed beam-splitter.This novel probe proves to be able to effectively circumvent polarization and dispersion mismatch caused by fiber motion and is more robust to a variety of interfering factors during the imaging process,experimentally compared to a conventional noncommon path probe.Furthermore,our design counteracts the attenuation of backscattering with depth and the fall-off of the signal,resulting in a more balanced signal range and greater imaging depth.Spectral-domain OCT imaging of phantom and biological tissue is also demonstrated with a sensitivity of∼100dB and a lateral resolution of∼3μm.This low-cost probe offers simplified system configuration and excellent robustness,and is therefore particularly suitable for clinical diagnosis as one-off medical apparatus.

The Exploration on the Anti-inhibitor Ability of the DNATyper^(TM)21 Kit

The DNATyper^(TM)21 kit has good species specificity,typing accuracy,and locus amplification balance,which can be used for forensic genetic analysis such as individual identification and paternity testing.To improve the anti-inhibitor ability of this kit,the addition of bovine serum albumin(BSA),bovine thrombin(BT),FastStart Taq DNA Polymerase,TaKaRa Ex Taq Hot Start Version(Ex Taq HS),MyFiTM DNA Polymerase,and Klentaq DNA Polymerase(Klentaq)as PCR enhancers to the PCR reaction system was explored in the presence of different concentrations of inhibitors,such as indigo,humic acid(HA),hemoglobin,heme,and melanin.The results revealed that BSA exhibited high efficiency in improving the detection rate of STR loci;BT only helped to overcome the typing inhibition caused by indigo,melanin,HA,and heme;Four types of DNA polymerase had different anti-inhibitor effects on different inhibitors,among which Ex Taq HS was more effective than the other three DNA polymerases,but displayed comparatively lower resistance to hemoglobin than BSA and BT.This study provides basic data for further optimization of the kit through comprehensive analysis and facilitates its application in daily forensic investigations.

Analysis on Applicability Evaluation of Forensic Science Standards in China

Standard applicability is the ability of a standard to be suitable for the prescriptive purpose under specified conditions.There is no mature applicability.evaluation index system of standards worldwide at present.We aim to construct the index system for evaluating the applicability of forensic science standard.The index system of applicability evaluation is proposed using a statistical analysis on the applicability of forensic science standards in China.We summarize the major problems and reasons in developing and revising the national and industry standards in China.Based on these,we put forward measures to improve standards’applicability,provide policy support for rectifying the standards,and lay the theoretical foundation for the construction,adjustment,and optimization of the forensic science standard system.

Development of China's Forensic Science in Statistics:2005-2016

In today's scientific fact-finding,forensic science bears the responsibility of ascertaining authenticity and restoring the truth.With the acceleration of China's internationalization and judicial reforms,forensic science has begun to play a prominent role injudicial trials,where its function and value have received unprecedented attention.In this article,the authors have reviewed 10 years of development in China's forensic science between 2005 and 2016.

Highly sensitive nanozyme strip:An effective tool for forensic material evidence identification

During criminal case investigations,blood evidence tracing is critical for criminal investigation.However,the blood stains are often cleaned or covered up after the crime,resulting in trace residue and difficult tracking.Therefore,a highly sensitive and specific method for the rapid detection of human blood stains remains urgent.To solve this problem,we established a nanozyme-based strip for rapid detection of blood evidence with high sensitivity and specificity.To construct reliable nanozyme strips,we synthesized CoFe_(2)O_(4) nanozymes with high peroxidase-like activity by scaling up to gram level,which can be supplied for six million tests,and conjugated antibody as a detection probe in nanozyme strip.The developed CoFe_(2)O_(4) nanozyme strip can detect human hemoglobin(HGB)at a concentration as low as 1 ng/mL,which is 100 times lower than the commercially available colloidal gold strips(100 ng/mL).Moreover,this CoFe_(2)O_(4) nanozyme strip showed high generality on 12 substrates and high specificity to human HGB among 13 animal blood samples.Finally,we applied the developed CoFe_(2)O_(4) nanozyme strip to successfully detect blood stains in three real cases,where the current commercial colloidal gold strip failed to do.The results suggest that the CoFe_(2)O_(4) nanozyme strip can be used as an effective on-scene detection method for human blood stains,and can further be used as a long-term preserved material evidence for traceability inquiry.

Twenty-seven continental ancestry-informative SNP analysis of bone remains to resolve a forensic case

We employed our previously developed 27-plex ancestry-informative single nucleotide polymorphism(SNP)panel to infer the ancestral components of bone remains of a possible foreign pilot found in south-western China.For ancestry assignment of this unknown individual,we first obtained the 27-SNP genotype of the individual.Then,based on a reference database of 3081 individuals from 33 populations,we calculated the match probability and likelihood ratio using the self-developed software program Forensic Intelligence.Inferred ancestral components of this individual were calculated by structure at K=3.A complete profile was obtained for the individual using our multiplexed SNP assay.The European population was within one order of magnitude of the highest likelihood.The major ancestral component of this individual was 97.6%European.

Validation of the DNATyper^(TM)15 PCR Genotyping System for Forensic Application

We describe the optimization and validation of the DNATyper^(TM)15 multiplex polymerase chain reaction(PCR)genotyping system for autosomal short tandem repeat(STR)amplification at 14 autosomal loci(D6S1043,D21S11,D7S820,CSF1PO,D2S1338,D3S1358,D13S317,D8S1179,D16S539,Penta E,D5S818,vWA,D18S51,and FGA)and amelogenin,a sex‑determining locus.Several DNATyper^(TM)15 assay variables were optimized,including hot start Taq polymerase concentration,Taq polymerase activation time,magnesium concentration,primer concentration,annealing temperature,reaction volume,and cycle number.The performance of the assay was validated with respect to species specificity,sensitivity to template concentration,stability,accuracy,influence of the DNA extraction methods,and the ability to genotype the mixture samples.The performance of the DNATyper^(TM)15 system on casework samples was compared with that of two widely used STR amplification kits,Identifiler^(TM)(Applied Biosystems,Carlsbad,CA,USA)and PowerPlex 16®(Promega,Madison,WI,USA).The conditions for PCR‑based DNATyper^(TM)15 genotyping were optimized.Contamination from forensically relevant nonhuman DNA was not found to impact genotyping results,and full profiles were generated for all the reactions containing≥0.125 ng of DNA template.No significant difference in performance was observed even after the DNATyper^(TM)15 assay components were subjected to 20 freeze‑thaw cycles.The performances of DNATyper^(TM)15,Identifiler^(TM),and PowerPlex 16®were comparable in terms of sensitivity and the ability to genotype the mixed samples and case‑type samples,with the assays giving the same genotyping results for all the shared loci.The DNA extraction methods did not affect the performance of any of the systems.Our results demonstrate that the DNATyper^(TM)15 system is suitable for genotyping in both forensic DNA database work and case‑type samples.

Genetic distribution and forensic evaluation of multiplex autosomal short tandem repeats in the Chinese Xinjiang Mongolian group

To further enrich the genetic data of the Chinese Xinjiang Mongolian group,the genetic distribution and forensic parameters of 19 autosomal short tandem repeats(STRs)were investigated.Altogether,249 alleles were observed in these 19 STRs.The mean values of the polymorphism information content(PIC),match probability(MP),discrimination power(DP),and probability of exclusion(PE)for these 19 STRs were 0.7775,0.0699,0.9301,and 0.6085,respectively.Additionally,the cumulative DP and PE values obtained in the Mongolian group were 0.999 999 999 999 999 999 999 995 67 and 0.999 999 992 163,respectively.