Identification of vaginal fluid, saliva, and feces using microbial signatures in a Han Chinese population.

In recent years, forensic scientists have focused on the discrimination of body fluids using microbial signatures. In this study, we performed PCR-based detection of microbial signatures of vaginal fluid, saliva, and feces in a Han Chinese population. We investigated the 16S rRNA genes of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus iners, and Atopobium vaginae in vaginal fluid, the 16S rRNA and the glucosyltransferase enzyme genes of Streptococcus salivarius and Streptococcus mutans in saliva, and the 16S rRNA genes of Enterococcus species, the RNA polymerase ß-subunit gene of Bacteroides uniformis and Bacteroides vulgatus, and the α-1-6 mannanase gene of Bacteroides thetaiotaomicron in feces. As a result, the detection proportions of L. crispatus, L. gasseri, L. jensenii, L. iners, and A. vaginae were 15/16, 5/16, 8/16, 14/16, and 3/16 in 16 vaginal fluid donors, respectively. L. crispatus and L. jensenii were specifically detected in vaginal fluid; L. gasseri, L. iners, and A. vaginae were also detected in non-vaginal fluid. S. salivarius and S. mutans were not specifically detected in saliva. The detection proportions of Enterococcus species, B. uniformis, B. vulgatus, and B. thetaiotaomicron in 16 feces samples were 16/16, 12/16, 15/16, and 11/16, respectively. B. uniformis and B. thetaiotaomicron were specifically detected in feces. In addition, DNA samples prepared for the identification of body fluid can also be used for individual identification by short tandem repeat typing. The mean detection sensitivities of L. crispatus and L. jensenii were 0.362 and 0.249 pg/uL, respectively. In conclusion, L. crispatus, L. jensenii, B. uniformis, and B. thetaiotaomicron can be used as effective markers for forensic identification of vaginal fluid and feces.

[Application of the Peak Area Ratio of STR Loci to Amelogenin Locus in the Estimation of DNA Degradation].

OBJECTIVE: To explore the change rules of peak area ratio of STR loci to Amelogenin (AMEL) locus (STR/AMEL), a sex-determining gene in DNA degradation, and to evaluate the application of STR/AMEL value in the estimation of DNA degradation degree. METHODS: DNA was extracted from iliopsoas, and the variations of STR/AMEL value (Penta E/AMEL, Penta D/AMEL, FGA/AMEL) were analyzed after the artificial degradation was made by DNase I, and the changes of these three ratios of the iliopsoas naturally degraded in an outdoor environment were also analyzed. The regression curves were analyzed using the periods of DNA degradation and outside the body as the independent variable (x) and the STR/AMEL value as the dependent variable (y) and three curve equations under two conditions were established. RESULTS: Both under the conditions of artificial and natural degradation, STR/AMEL value had a negative relationship with the degradation time. The relationship between STR/AMEL and degradation time can be well simulated by the cubic function. R2 was over 0.99 under controlled degradation condition and over 0.86 under natural degradation condition. CONCLUSION: The STR/AMEL value (Penta E/AMEL, Penta D/AMEL, FGA/AMEL) is negatively related with the DNA degradation degree, which follows mathematical regression models strictly, and it might be applied to evaluate the DNA degradation degree.

[Development of a Forensic Multiplex Amplification STR Kit for 15 Autosomal STR Loci and 10 Y-STR Loci].

OBJECTIVE: To establish a multiplex STR genotyping method for autosomal STR and Y-STR loci in forensic biological practice. METHODS: Widely used autosomal STR loci and Y-STR loci were selected. A set of PCR primers was designed, and a 5-dye fluorescent labeled STR multiplex PCR reagent kit was developed. RESULTS: A kit was developed which can simultaneously detect 15 autosomal STR loci, 10 Y-STR loci, and an Amelogenin. CONCLUSION: The 15 autosomal STR plus 10 Y-STR kit in combination with capillary electrophoresis method was used to STR genotyping with accurate and reliable results. The new one-step testing kit can potentially be widely used in forensic cases and DNA databank in the future.

[Progress in Association between Genetic Correlation and Human Violent Behavior].

Human violent behavior is a complex behavior which is influenced by genetic and environmental factors. There is a trend in investigating the mechanism of violent behavior by using the genetic methods. This article reviews several candidate genes and advances in epigenetics which are associated with violent behavior. The prospects and significance of violent behavior research from the view of gene polymorphism and epigenetics are also discussed.

[Application of Ion Torrent PGM™ System in Detection of Fetal DNA in Maternal Plasma].

OBJECTIVE: To explore the feasibility of detecting of Y-STR of fetal DNA in maternal plasma using Ion Torrent PGM™ System. METHODS: A total of 16 fetal DNA samples from maternal plasmas (8 cases from 38 weeks gestational age and 8 ones from 12 weeks) were prepared and a multiplex assay with 7 STR loci (DYS390, DYS391, DYS393, DYS438, DYS437, DYS456, DYS635) was designed for multiplex-PCR amplification. Using Ion Torrent PGM™ System, the results of Y-STR sequences and capillary electrophoresis were obtained and compared. RESULTS: Y-STR specific alleles were detected in the maternal plasma of all the pregnant women having male babies of second and third trimester, which were higher than that detected by capillary electrophoresis. Consistent Y-STR genotypes were observed between fetal DNA from maternal plasma and genomic DNA from the newborn babies. CONCLUSION: Based on Ion Torrent PGM™ System, the prenatal Y-STR detection method may provide a high-sensitive and high-throughput choice for prenatal STR detection in forensic testing.

[Allele-specific PCR and its application in forensic science].

Allele-specific polymerase chain reaction (AS-PCR) is a technique based on allele-specific primers, which can be used to analyze single nucleotide polymorphism (SNP) effectively including the transition, transversion and insertion/deletion polymorphism and has been exploited in the study of diseases research, molecular diagnosis, and forensic biological evidence. The article systematically reviews the principle, the detection methods, improvement of AS-PCR, and its research updates in the fields of autosome, Y chromosome and mitochondrial SNP, as well as its application in forensic science.

[AS-PCR assay for 20 mtDNA SNP typing and haplotype frequency].

OBJECTIVE: To develop a multiplex allele-specific PCR (AS-PCR) assay with three-color fluorescence labeling for mitochondrial DNA (mtDNA) SNP typing. METHODS: Based on the principle of AS-PCR, the primer sets were designed for 20 SNP located on the coding region of mtDNA and divided into 2 groups labeled with FAM and HEX fluorescence, respectively. A primer set included two forward (reverse) allelic specific primers with different sizes and a generic reverse (forward) primer. Blood samples from 200 unrelated individuals were analyzed by AS-PCR and capillary electrophoresis. Three random samples at least for each SNP site were examined and verified by direct sequencing. The haplotype frequency was investigated. RESULTS: Distinct electropherograms of 200 blood samples were obtained successfully. The typing results of direct sequencing were identical to those obtained from AS-PCR. The minimum detectable DNA concentration was 0.2 pg under the system of 10 microL. The sensitivity of the DNA concentrations ranged from 0.5 to 5 pg. The 200 individuals were assigned into 15 haplotype, and the haplotype diversity was 0.906 0. CONCLUSION: AS-PCR is a simple, rapid and efficient method for mtDNA SNP typing, and can be applied to forensic practice.

[Establishment of the multiplex genotyping system for 16 SNP loci on mtDNA].

OBJECTIVE: To establish a multiplex genotyping system of mtDNA SNP. METHODS: A multiplex analysis system of 16-plex mtDNA SNP loci was established with allele specific PCR and capillary electrophoresis genotyping technology. Fifty samples from unrelated Chinese Han individuals were typed with the multiplex system. The multiplex assay was validated by comparing with the direct sequencing method. RESULTS: The genotypes of all 50 samples were correctly determined by the multiplex system. The optimal genotypic graphs were obtained with an input DNA of 0.5-10 pg, and the typing results were completely consistent with those by direct sequencing method. CONCLUSION: The established multiplex system by allele specific PCR has high sensitivity, operational simplicity and high accuracy. It provides an effective and high output method for mtDNA SNP typing.

[Determination of the biological attribute of evidence at the scene using reverse transcription PCR and real-time fluorescent quantitative PCR].

OBJECTIVE: To explore the feasibility of biological method to identify the biological attribute of samples at crime scene. METHODS: Thirty samples of ten blood stains, ten saliva stains and ten semen stains were selected, and all the samples were processed by the routine method and biomolecular method, respectively. Both RNA and DNA were isolated using DNA-RNA co-extraction technology and the mRNA was converted into cDNA using reverse transcription PCR (RT-PCR). Three pairs of specific primers were designed for blood stain, saliva stain and semen stain based on the different target genes in three specific tissues and the primers were amplified using real-time fluorescent quantitative PCR. The differences in these biological samples were evaluated by melting temperature (Tm) values and the size of the amplification fragment. RESULTS: The Tm values of blood stain, saliva stain and semen stain were (84.5 +/- 0.2) degrees C, (76.9 +/- 0.3) degrees C and (88.5 +/- 0.2) degrees C, respectively. The length of PCR fragments of them was 177bp, 134bp and 294bp, respectively. CONCLUSION: Compared with the routine method, RT-PCR with real-time fluorescent quantitative PCR is highly specific, sensitive and reliable to identify the biological attribute of evidence, and can be potentially applied to determine evidence attribute in forensic practice.

[Development of Chinese forensic Y-STR DNA database].

Y chromosome is a male-specific paternal inherited chromosome. The STR markers on Y chromosome have been widely used in forensic practices. This article summarizes the characteristics of Y-STR and some factors are considered of selecting appropriate Y-STR markers for Chinese population. The prospects of existing and potential forensic applications of Y-STR profiles are discussed including familial excluding, familial searching, crowd source deducing, mixture sample testing, and kinship identifying. The research, development, verification of Y-STR kit, Y-STR mutation rate, and search software are explored and some suggestions are given.

[Genotyping and parental related methylation of SNRPN gene rs220030].

OBJECTIVE: To establish two methods by denaturing gradient gel electrophoresis (DGGE) and pyrosequencing for genotyping rs220030 (a SNP in the promoter region of small nuclear ribonucleoprotein polypeptide N, SNRPN). To establish an analytical technique for detecting CpG methylation status by pyrosequencing and to further investigate the feasibility of applying rs220030 to the determination of parental origin allele. METHODS: The rs220030 of 97 blood samples from individuals of Shanghai Han population were genotyped by DGGE, meanwhile the rs220030 of 25 blood samples of them were genotyped by pyrosequencing to compare the two methods in genotyping SNP. Pyrosequencing united bisulfite conversion method was applied to detect CpG methylation status of region upstream rs220030 of two random blood genealogical samples and investigate whether the methylation status was parental related. RESULTS: The rs220030 genotyping results of 97 blood samples detected by DGGE were 20 C homozygote, 29 T homozygote, and 48 C/T heterozygote. Twenty-five blood samples genotyped by pyrosequencing showed the same result with DGGE. The CpG methylation status of region upstream rs220030 of the child was similar to the mother. CONCLUSION: Compared with DGGE, pyrosequencing is more accurate, convenient, and suitable for large samples and high throughput SNP genotyping. Pyrosequencing united bisulfite conversion can be used to detect CpG methylation status precisely. It is feasible to apply rs220030 to parental origin allele determination.

[Genetic polymorphisms of the dinucleotide STR locus D6S261].

OBJECTIVE: To investigate the application of dinucleotide STR locus in paternity testing. METHODS: Dinucleotide STR locus D6S261 was selected and the paternity testing blood samples were amplified using 200 random blood samples, 16 family samples and 193 paternity test samples. Data of the PCR products were collected by 3130XL Genetic Analyzer and the genetic parameters of population were calculated by PowerStats v12. RESULTS: Fifteen alleles and 50 genotypes were found and H, DP, PE and PIC were 0.850, 0.953, 0.695, and 0.820, respectively. The typing results of both family samples and paternity test samples were accord with the law of inheritance, which no mutation was discovered. CONCLUSION: The genetic polymorphisms of D6S261 show good characteristics with low mutation rate and high stability. It can be an effective method to solve the indetermination caused by mutation in paternity testing if the stutter bands can be decreased.

[Forensic application of expressmarker 22 STR loci direct PCR amplification kit].

OBJECTIVE: To explore the application value of Expressmarker 22 STR loci direct PCR amplification kit. METHODS: One thousand nine hundred and forty-eight samples (including samples spotted on FTA cards, filter papers and case samples) were tested using Expressmarker 22 STR loci direct PCR amplification kit. At the same time, all were tested using Sinofiler kit, Identifiler kit and PowerPlex 16 kit respectively for comparison. The genotypes were compared at the same STR loci among these four kits to test the sensitivity and accuracy of Expressmarker 22 STR loci direct PCR amplification kit. RESULTS: 97.79% samples were successfully typed using Expressmarker 22 STR loci direct PCR amplification kit. The genotype profiles of the same samples using Expressmarker 22 STR loci direct PCR amplification kit were consistent with Sinofiler kit, Identifiler kit and PowerPlex 16 kit at the same STR loci. CONCLUSION: Expressmarker 22 STR loci direct PCR amplification kit can provide huge information and accurate results

[Rapid DNA identification using 6+1 STR kit and EX-Q20 electrophoresis].

OBJECTIVE: To establish a rapid STR genotyping method for individual identification. METHODS: Two hundred blood samples from FTA were collected. Equal amount of blood were collected by puncher and analyzed using two methods (6+1 STR kit in combination with EX-Q20 electrophoresis and Sinofiler kit in combination with POP4 electrophoresis). Consuming time and results of two methods were compared. RESULTS: 6+1 STR kit in combination with EX-Q20 electrophoresis method can obtain all genotyping results and be shorter time. CONCLUSION: 6+1 STR kit in combination with EX-Q20 electrophoresis method is used to STR genotyping with accurate, reliable results and this new method is potential value in mass personnel investigation and comparison in major criminal cases. It also can raise the work efficiency.

[Determination of bloodstain formation time by RNA analysis].

OBJECTIVE: To investigate the expression of 18S rRNA and beta-actin mRNA in bloodstain between 8 and 15 days after death and extrapolate the time of bloodstain formation. METHODS: RNA in dried bloodstain at different times was extracted, then quantified for 18S rRNA and beta-actin mRNA by real-time RT-PCR. The bloodstain formation time was deduced based on the changes of the ratio of 18S rRNA to beta-actin mRNA at different time points. RESULTS: The ratio of 18S rRNA to beta-actin mRNA increased gradually with time, indicating that rRNA and mRNA degraded in different rate with time. CONCLUSION; The ratio of 18S rRNA to beta-actin mRNA could be used for estimating the time of bloodstain formation in some period.

[Forensic analysis of LCN DNA using sample concentration methods followed by miniSTR genotyping].

OBJECTIVE: To optimize low copy number (LCN) DNA analysis methods for forensic STR genotyping. METHODS: Two groups of DNA sample, extracted using either Magnetic bead method or Chelex-100 methods, were previously amplified with a Identifiler PCR Amplification kit, but no genotype was detected. The DNA samples were concentrated using either a drying method or the Microcon-100 method, then amplified using an miniFiler PCR Amplification kit and genotyped. RESULTS: Among the 127 DNA samples, 47 samples, previously extracted using the Magnetic bead method, were genotyped with 36% success rate. Eighty samples, previously extracted using the Chelex-100 method, were genotyped with 30% success rate. CONCLUSION: The application of sample concentration methods and miniFiler kit can improve the success rate of LCN STR analysis.

[Validation of Differential Extraction Kit in forensic sexual assault cases].

OBJECTIVE: To evaluate the validity of Differential Extraction Kit in isolating spermatozoa and epithelial cell DNA from mixture samples. METHODS: Selective lysis of spermatid and epithelial cells combined with paramagnetic particle method were applied to extract the DNA from the mock samples under controlled conditions and forensic case samples, and template DNA were analyzed by STR genotype method. RESULTS: This Differential Extraction Kit is efficient to obtain high quality spermatid and epithelial cell DNA from the mixture samples with different proportion of sperm to epithelial cell. CONCLUSION: The Differential Extraction Kit can be applied in DNA extraction for mixed stain from forensic sexual assault samples.

[Application of MiniFiler kit in forensic DNA testing of low copy number template].

OBJECTIVE: To detect low copy number of DNA samples by using a newly launched commercial miniSTR detection kit (MiniFiler) in forensic practice. METHODS: Low concentration and/or challenged forensic DNA samples were analyzed according to protocols provided by the manufacturer (Applied Biosystems, Foster City, USA). RESULTS: DNA samples as low as 10 pg could be amplified by MiniFiler kit, and the optimal DNA quantity was 40 pg or above. CONCLUSION: MiniFiler kit can be used for analysis of low copy number STR.

[DNA genotyping of oral epithelial cells by laser capture microdissection].

OBJECTIVE: The STR genotypping of trace oral epithelial cells which are microdissected by laser capture microdissection system (LCM) is explored. METHODS: The oral epithelial cells are microdissected using a low-power infrared laser by VERITAS Microdissection Instrument. STR loci of Profiler Plus are detected by multiplex PCR procesures. RESULTS: DNA genotyping of 7-8 oral epithelial cells are succeeded, and DNA genotyping of 3-4 oral epithelial cells are failed. CONCLUSION: It is viable in genotyping of trace oral epithelial cells by Laser Capture Microdissection as a new technology of seperating single cell.

[Study on application of the whole genome amplification in LCN].

OBJECTIVE: To establish a method for whole genome amplification (WGA) based on (multiple displacement amplification MDA), and achieve DNA analysis of the human genome from low copy number (LCN). METHODS: DNA sample was used for WGA according to the REPLI-g kit protocol (QIAGEN, Germany). WGA product was used for DNA analysis according to the Applied Biosystems Profiler Plus kit protocol (ABI, USA). RESULTS: WGA product of DNA sample (10 pg) can be used for STR genotyping. CONCLUSION: WGA technology could be helpful for LCN DNA analysis.