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Introdução: A análise do DNA salivar é uma dos métodos de identificação humana relacionados à Odontologia Legal. A coleta do material genético salivar consiste num processo simples e pouco invasivo, por possuir grande potencial discriminatório tem sido amplamente aplicado em investigações criminais. Objetivo: Por meio de uma revisão de escopo, objetivou-se mapear o tempo de viabilidade da saliva humana em meio externo para fins de extração e purificação de DNA. Material e Métodos: A revisão foi conduzida seguindo o protocolo JBI e registrada (doi: 10.17605/OSF.IO/PN9ET). A estratégia de busca foi adaptada para as bases: Pubmed, Web of Science, Scopus, LILACS, Cochrane e Google Scholar, sem restrições sobre período de publicação ou idioma. Foram excluídos estudos que exploraram apenas as metodologias e materiais relacionados a extração de DNA , também aqueles que estudaram DNA de outros componentes que não a saliva. Resultados: Identificou-se 283 estudos. Após triagem inicial, 15 referências foram lidas na íntegra, sendo 6 incluídas na revisão, em função da confirmação da elegibilidade. Bitucas de cigarro, próteses dentárias, pastilhas de compressão dentária, cavidade oral e cartões de FTA foram os substratos descritos como fonte de saliva coletada. A viabilidade do DNA foi verificada em tempos que variaram de 1 dia à 11 anos. Conclusão: O protocolo de coleta e armazenamento das amostras é um fator que pode influenciar a quantidade e qualidade do material examinado, todavia, observou-se DNA viável em análise realizada uma década após a coleta da saliva e esse foi o tempo máximo de acompanhamento relatado nos estudos
Introduction: Salivary DNA analysis is one of the human identification methods related to forensic dentistry. Collection of salivary genetic material consists of a simple and poorly invasive process because it has great discriminatory potential has been widely applied in criminal investigations. Objective: Through a scope review, the feasibility time of human saliva was mapped in the external environment for DNA extraction and purification purposes. Material and Methods: The review was conducted following the JBI protocol and registered (doi: 10.17605/OSF.IO/PN9ET). The search strategy was adapted for the databases: Pubmed, Web of Science, Scopus, LILACS, Cochrane and Google Scholar, with no restrictions on period of publication or language. Studies that explored only methodologies and materials related to DNA extraction were excluded, as well as those that studied DNA from components other than saliva. Results: 283 studies were identified. After initial screening, 15 references were read in full, 6 of which were included in the review. Cigarette butts, dentures, dental compression tablets, oral cavity and FTA cards were the substrates described as a source of collected saliva. DNA viability was verified in times ranging from 1 day to 11 years. Conclusion: The sample collection and storage protocol is a factor that can influence the quantity and quality of the material examined, however, viable DNA was observed in an analysis performed a decade after saliva collection and this was the maximum reported follow-up time in the studies
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OBJECTIVES@#To provide a guideline for genealogy inference and family lineage investigation through a study of the mismatch tolerance distribution of Y-STR loci in Chinese Han male lineage.@*METHODS@#Three Han lineages with clear genetic relationships were selected. YFiler Platinum PCR amplification Kit was used to obtain the typing data of 35 Y-STR loci in male samples. The variation of Y-STR haplotypes in generation inheritance and the mismatch tolerance at 1-7 kinship levels were statistically analyzed.@*RESULTS@#Mutations in Y-STR were family-specific with different mutation loci and numbers of mutation in different lineages. Among all the mutations, 66.03% were observed on rapidly and fast mutating loci. At 1-7 kinship levels, the number of mismatch tolerance ranged from 0 to 5 on all 35 Y-STR loci, with a maximum step size of 6. On medium and slow mutant loci, the number of mismatch tolerance ranged from 0 to 2, with a maximum step size of 3; on rapidly and fast mutant loci, the number of mismatch tolerance ranged from 0 to 3, with a maximum step size of 6.@*CONCLUSIONS@#Combined use of SNP genealogy inference and Y-STR lineage investigation, both 0 and multiple mismatch tolerance need to be considered. Family lineage with 0-3 mismatch tolerance on all 35 Y-STR loci and 0-1 mismatch tolerance on medium and slow loci can be prioritized for screening. When the number of mismatch tolerance is eligible, family lineages with long steps should be carefully excluded. Meanwhile, adding fast mutant loci should also be handled with caution.
Subject(s)
Male , Humans , Haplotypes , Chromosomes, Human, Y/genetics , Microsatellite Repeats , Mutation , Asian People/genetics , China , Genetics, PopulationABSTRACT
OBJECTIVES@#To investigate the efficacy of different numbers of microhaplotype (MH) loci and the introduction of different reference samples on the identification of full sibling, half sibling and differentiation between full sibling and half sibling kinships, and to explore the effect of changing mutation rate on sibling testing.@*METHODS@#First, a family map involving three generations was established, and four full sibling identification models, five half sibling identification models and five models distinguishing full and half siblings were constructed for different reference samples introduced. Based on the results of the previous study, two sets of nonbinary SNP-MH containing 34 and 54 loci were selected. Based on the above MH loci, 100 000 pairs of full sibling vs. unrelated individuals, 100 000 pairs of half sibling vs. unrelated individuals and 100 000 pairs of full sibling vs. half sibling were simulated based on the corresponding sibling kinship testing models, and the efficacy of each sibling kinship testing model was analyzed by the likelihood ratio algorithm under different thresholds. The mutant rate of 54 MH loci was changed to analyze the effect of mutation rate on sibling identification.@*RESULTS@#In the same relationship testing model, the systematic efficacy of sibling testing was positively correlated with the number of MH loci detected. With the same number of MH loci, the efficacy of full sibling testing was better than that of uncle or grandfather when the reference sample introduced was a full sibling of A, but there was no significant difference in the identification efficacy of the four reference samples introduced for full sibling and half sibling differentiation testing. In addition, the mutation rate had a slight effect on the efficacy of sibling kinship testing.@*CONCLUSIONS@#Increasing the number of MH loci and introducing reference samples of known relatives can increase the efficacy of full sibling testing, half sibling testing, and differentiation between full and half sibling kinships. The level of mutation rate in sibling testing by likelihood ratio method has a slight but insignificant effect on the efficacy.
Subject(s)
Humans , Siblings , Polymorphism, Single Nucleotide , DNA Fingerprinting/methodsABSTRACT
OBJECTIVES@#To calculate the likelihood ratios of incest cases using identity by descent (IBD) patterns.@*METHODS@#The unique IBD pattern was formed by denoting the alleles from the members in a pedigree with a same digital. The probability of each IBD pattern was obtained by multiplying the prior probability by the frequency of non-IBD alleles. The pedigree likelihoods of incest cases under different hypotheses were obtained by summing all IBD pattern probabilities, and the likelihood ratio(LR) was calculated by comparing the likelihoods of different pedigrees.@*RESULTS@#The IBD patterns and the formulae of calculating LR for father-daughter incest and brother-sister incest were obtained.@*CONCLUSIONS@#The calculations of LR for incest cases were illustrated based on IBD patterns.
Subject(s)
Male , Humans , Incest , Siblings , ProbabilityABSTRACT
OBJECTIVES@#To derive general formulas for calculating commonly used kinship index (KI).@*METHODS@#By introducing the Kronecker symbol, the formulas used to calculate the same KI under different genotype combinations were summarized into a unified expression.@*RESULTS@#The general formulas were successfully derived for KI in various case situations, including the paternity index, full sibling index, half sibling index, avuncular index, grandpaternity index, first-cousin index, and second-cousin index between two individuals without or with the mother being involved; grandpaternity index between grandparents and a grandchild without or with the mother being involved; half sibling index between two children with two mothers being involved; full sibling index among three children; and half sibling index among three children with no, one, or two mothers being involved.@*CONCLUSIONS@#The general formulas given in this study simplify the calculation of KIs and facilitate fast and accurate calculation through programming.
Subject(s)
Female , Child , Humans , Paternity , Siblings , Genotype , Mothers , Models, GeneticABSTRACT
OBJECTIVES@#To derive the paternity index (PI) calculation formula of the alleged father (AF) when the AF is a relative (parent/child, siblings, grandparent/grandchild, uncle/nephew, first cousins) of the child's biological mother.@*METHODS@#For the case when the AF is related to the child's biological mother, the existence of the relationship in the numerator and denominator hypothesis of PI was considered. The genotype frequency of the AF was calculated by using the frequency formula in which the mother's genotype was considered, while the random male in the denominator was substituted as another relative of the mother's same rank. The PI calculation formula was derived to eliminate the effect of the relationship between AF and the child's biological mother.@*RESULTS@#When the AF and the biological mother have first, second and tertiary kinship, a more conservative PI was obtained from the PI calculation formula derived in this study compared with the PI calculation method which did not consider kinship.@*CONCLUSIONS@#The calculation method provided in this study can eliminate the effect of the relation of the AF and mother on the PI in incest cases, to obtain more accurate and conservative identification conclusions.
Subject(s)
Female , Humans , Male , Child , Paternity , Mothers , Genotype , FathersABSTRACT
OBJECTIVES@#To compare the application value of the likelihood ratio (LR) method and identity by state (IBS) method in the identification involving half sibling relationships, and to provide a reference for the setting of relevant standards for identification of half sibling relationship.@*METHODS@#(1) Based on the same genetic marker combinations, the reliability of computer simulation method was verified by comparing the distributions of cumulated identity by state score (CIBS) and combined full sibling index in actual cases with the distributions in simulated cases. (2) In different numbers of three genetic marker combinations, the simulation of full sibling, half sibling and unrelated individual pairs, each 1 million pairs, was obtained; the CIBS, as well as the corresponding types of cumulative LR parameters, were calculated. (3) The application value of LR method was compared with that of IBS method, by comparing the best system efficiency provided by LR method and IBS method when genetic markers in different amounts and of different types and accuracy were applied to distinguish the above three relational individual pairs. (4) According to the existing simulation data, the minimum number of genetic markers required to distinguish half siblings from the other two relationships using different types of genetic markers was estimated by curve fitting.@*RESULTS@#(1) After the rank sum test, under the premise that the real relationship and the genetic marker combination tested were the same, there was no significant difference between the simulation method and the results obtained in the actual case. (2) In most cases, under the same conditions, the system effectiveness obtained by LR method was greater than that by IBS method. (3) According to the existing data, the number of genetic markers required for full-half siblings and half sibling identification could be obtained by curve fitting when the system effectiveness reached 0.95 or 0.99.@*CONCLUSIONS@#When distinguishing half sibling from full sibling pairs or unrelated pairs, it is recommended to give preference to the LR method, and estimate the required number of markers according to the identification types and the population data, to ensure the identification effect.
Subject(s)
Humans , Siblings , Genetic Markers , Computer Simulation , Irritable Bowel Syndrome/genetics , Reproducibility of Results , GenotypeABSTRACT
OBJECTIVES@#To establish an analytical method for half sibling testing involving common three relatives' participation.@*METHODS@#Based on the half sibling testing scenarios with the known biological mother, grandfather or uncle, and two unidentified controversial half siblings participating, two opposing hypotheses were set. Lineage reconstruction according to Mendel's law of heredity was carried out, and the calculation formula of the half sibling kinship index was derived. Verification of actual cases was carried out and the results were compared with duo half sibling testing.@*RESULTS@#In the scenarios of the known biological mother, grandfather and uncle participating in half sibling testing, the kinship calculation formulae of 54, 91 and 99 genotype combinations for kinship index calculation were deduced respectively. The actual cases showed higher kinship indexes in trio half sibling testing compared with duo half sibling testing.@*CONCLUSIONS@#It is beneficial to obtain more genetic information for family reconstruction and improvement of the strength of genetic evidence for half sibling testing by adding known relatives.
Subject(s)
Female , Humans , Siblings , Genotype , Mothers , Microsatellite RepeatsABSTRACT
OBJECTIVES@#To study the detection efficiency of trio full sibling with another known full sibling reference added under different number of autosomal STR typing systems.@*METHODS@#Based on 43 detection systems consisting of 13 to 55 representative autosomal STR loci, 10 000 true families (full sibling group) and 10 000 false families (unrelated individual group) were randomly simulated. The full sibling index (FSI) was calculated based on the method of family reconstruction. The cumulative sibling relationship index (CFSI) of 0.000 1 and 10 000 were used as the evaluation thresholds, and the detection efficiency parameters were calculated and compared with the identification of the duo full sibling testing.@*RESULTS@#With the increasing number of STR loci, the error rate and inability of judgement rate gradually decreased; the sensitivity, specificity, correct rate of judgment and other parameters gradually increased, and the system efficiency gradually improved. Under the same detection system, trio full sibling testing showed higher sensitivity, specificity, system efficiency and lower inability of judgement rate compared with duo full sibling testing. When the system efficiency was higher than 0.85 and inability of judgement rate was less than 0.01%, at least 20 STRs should be detected for trio full sibling testing, which was less than 29 STRs required by duo full sibling testing.@*CONCLUSIONS@#The detection efficiency of trio full sibling testing is superior to that of duo full sibling testing with the same detection system, which is an effective identification scheme for laboratories with inadequate detection systems or for materials with limited conditions.
Subject(s)
Humans , Siblings , Microsatellite Repeats/genetics , DNA Fingerprinting , Gene FrequencyABSTRACT
Tri-allelic pattern in autosomal STR is a common abnormal typing phenomenon in forensic DNA analysis, which brings difficulties and uncertainties to the evaluation of the evidence weight in actual cases. This paper reviews the types, formation mechanism, occurrence frequency, genetic pattern and quantitative evaluation of evidence of the tri-allelic pattern in autosomal STR in forensic DNA analysis. This paper mainly explains the formation mechanism and genetic patterns based on different types of tri-allelic pattern. This paper also discusses the determination of tri-allelic pattern and the quantitative method of evidence evaluation in paternity testing and individual identification. This paper aims to provide references for scientific and standardized analysis of this abnormal typing phenomenon in forensic DNA analysis.
Subject(s)
Humans , Alleles , DNA/genetics , Forensic Medicine , Gene Frequency , Microsatellite RepeatsABSTRACT
Kinship testing is widely needed in forensic science practice. This paper reviews the definitions of common concepts, and summarizes the basic principles, advantages and disadvantages, and application scope of kinship analysis methods, including identity by state (IBS) method, likelihood ratio (LR) method, method of moment (MoM), and identity by descent (IBD) segment method. This paper also discusses the research hotspots of challenging kinship testing, complex kinship testing, forensic genetic genealogy analysis, and non-human biological samples.
Subject(s)
Humans , DNA Fingerprinting , Forensic Genetics/methods , Forensic Sciences , PedigreeABSTRACT
With the advance of molecular biology, DNA analysis technology has been widely applied in forensic science. Non-human DNA analysis can be used in some special cases and has unique forensic value to provide investigation clues and trial basis. Animal DNA typing plays a more prominent role in the detection of all kinds of non-human DNA related cases and is the main content of forensic non-human DNA analysis. This paper reviews the development history, present situation, advantages and disadvantages of animal DNA typing according to its technology, characteristic, challenges facing forensic science application scenarios, and also its future development.
Subject(s)
Animals , DNA Fingerprinting , Forensic Medicine , DNA/analysis , Forensic Sciences , Molecular Biology , Forensic GeneticsABSTRACT
With the improvement of DNA methylation detection techniques, studies on age-related methylation sites have found more age-specific ones across tissues, which improves the sensitivity and accuracy of age estimation. In addition, the establishment of various statistical models also provides a new direction for the age estimation of tissues from different sources. This review summarizes the related studies of age estimation based on DNA methylation from the aspects of detection technology, age-related cytosine phosphate guanine site and model selection in recent years.
Subject(s)
DNA Methylation , Forensic Genetics/methods , CpG Islands , Forensic MedicineABSTRACT
OBJECTIVES@#To compare the effects of cell lysis method and magnetic beads method in forensic DNA identification and to explore these two methods in forensic DNA identification.@*METHODS@#The genome DNA of THP-1 cells in different quantities was extracted by the cell lysis method and magnetic beads method, and the DNA content was quantified by real-time quantitative PCR. The cell lysis method and magnetic beads method were used to type the STR of human blood with different dilution ratios.@*RESULTS@#When the numbers of THP-1 cell were 100, 400 and 800, the DNA content extracted by cell lysis method were (1.219±0.334), (5.081±0.335), (9.332±0.318) ng, respectively; and the DNA content extracted by magnetic beads method were (1.020±0.281), (3.634±0.482), (7.896±0.759) ng, respectively. When the numbers of THP-1 cells were 400 and 800, the DNA content extracted by the cell lysis method was higher than that by the magnetic beads method. The sensitivity of cell lysis method and magnetic beads method was similar in STR typing of human blood at different dilution ratios. Complete STR typing could be obtained at 100, 300 and 500-fold dilutions of blood samples, but could not be detected at 700-fold dilution. STR typing of undiluted human blood could not be detected by cell lysis method.@*CONCLUSIONS@#The cell lysis method is easy to operate and can retain template DNA to the maximum extend. It is expected to be suitable for trace blood evidence tests.
Subject(s)
Humans , Forensic Medicine , DNA/genetics , Real-Time Polymerase Chain Reaction , Magnetic Phenomena , DNA Fingerprinting/methods , Microsatellite RepeatsABSTRACT
Este estudo teve como objetivo analisar as características das amostras questionadas de tecidos rígidos enviadas para análise de DNA forense, especificando o tipo e a região anatômica alvo de coleta, bem como a viabilidade da amostra após o processamento. Caracterizada por ser de corte transversal, com abordagem quantitativa e descritiva, utilizou dados secundários extraídos de planilhas do Instituto, com os quais construiu banco de dados, posteriormente analisado. Os resultados demonstraram que ossos e dentes são coletados como amostra para os testes de DNA, representando o percentual de 47,4% de todos os exames recebidos no ano de 2020, sendo 99,5% provenientes de crimes contra a vida. Das amostras obtidas de tecido rígido, 42% tiveram processamento concluído (39,1% para dentes e 42,6% para ossos), 10% não concluído (17,2% para dentes e 8,5% para ossos) e 48% constavam como não informado. Todas as amostras compostas por dente não especificaram a classificação dentária. Já as amostras compostas por ossos corresponderam a fragmento ósseo não especificado em 81% dos casos, 13,4% eram fragmento de fêmur e o percentual restante distribuído entre fragmentos de clavícula, crânio, escápula, fêmur, manúbrio, osso longo, tíbia, vértebra, osso esterno, mandíbula e ulna, variando entre 1-2% para cada. Pode-se concluir que o perfil dos exames de DNA extraído de amostras de tecidos duros do Instituto pesquisado é composto por ossos e dentes, sendo em sua maioria por fragmentos ósseos de vários tipos, principalmente fêmur, sendo necessária a melhor qualificação dos tipos de amostra utilizados para extração
This study aimed to analyze the characteristics of questioned hard tissue samples sent for forensic DNA analysis, specifying the type and anatomical region targeted for collection, as well as the viability of the sample after processing. Characterized by being cross-sectional, with a quantitative and descriptive approach, it used secondary data extracted from the Institute's spreadsheets, with which it built a database, later analyzed. The results showed that bones and teeth are collected as samples for DNA testing, representing the percentage of 47.4% of all exams received in the year 2020, with 99.5% coming from crimes against life. Among the samples obtained from hard tissue, 42% had its processing completed (39.1% for teeth and 42.6% for bones), 10% were not completed (17.2% for teeth and 8.5% for bones), and 48% were listed as not informed. All tooth-composite samples didn´t specify the tooth classification. Bone samples corresponded to unspecified bone fragments in 81% of the cases, 13.4% were femur fragments, and the remaining percentage was distributed among fragments of clavicle, skull, scapula, femur, manubrium, long bone, tibia, vertebra, sternum bone, mandible, and ulna, ranging from 1-2% for each. Therefore, it can be concluded that the profile of DNA tests extracted from hard tissue samples at the researched institute is composed of bones and teeth, being mostly bone fragments of various types, especially femur, and a better qualification of the sample types used for extraction is needed
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In forensic physical evidence identification, the accurate identification of the individual origin and their body fluid composition of the biological samples obtained from the crime scene play a critical role in determining the nature of a crime. In recent years, RNA profiling has become one of the fastest developing methods for body fluids identification. Due to the characteristics of tissue or body fluid specific expression, various types of RNA markers have been proven to be promising candidate markers for body fluids identification in previous studies. This review summarizes the research progress of RNA markers in body fluids identification, including the RNA markers that have been effectively verified in current research and their advantages and disadvantages. Meanwhile, this review prospects the application of RNA markers in forensic medicine.
Subject(s)
Forensic Medicine/methods , Body Fluids/chemistry , RNA/analysis , Feces , Forensic Genetics , Semen/chemistry , Saliva/chemistryABSTRACT
OBJECTIVES@#To investigate the genetic information of 57 autosomal InDel loci (A-InDels) included in AGCU InDel 60 fluorescence detection kit in the Beichuan Qiang population of Sichuan Province and evaluate its application value in forensic medicine.@*METHODS@#A total of 200 unrelated healthy individuals from Beichuan Qiang population of Sichuan Province were typing detected by AGCU InDel 60 fluorescence detection kit. Allele frequencies and population genetic parameters of the 57 A-InDels were statistically analyzed and compared with the available data of 26 populations.@*RESULTS@#After Bonferroni correction, there was no linkage disequilibrium between the 57 A-InDels, and all loci were in Hardy-Weinberg equilibrium. Except for rs66595817 and rs72085595, the minor allele frequencies of 55 A-InDels were above 0.3. PIC ranged from 0.298 3 to 0.375 0, CDP was 1-2.974 8×10-24, CPEduo was 0.999 062 660, and CPEtrio was 0.999 999 999. The calculation of the genetic distance showed that Beichuan Qiang population had the closest genetic distances with Beijing Han and South China Han populations, but far away from African populations.@*CONCLUSIONS@#The 57 A-InDels in AGCU InDel 60 fluorescence detection kit have a good genetic polymorphism in Beichuan Qiang population of Sichuan Province, which can be used as effective supplemental for individual identification and paternity identification in forensic medicine.
Subject(s)
Humans , Genetics, Population , Asian People/genetics , Polymorphism, Genetic , Gene Frequency , INDEL Mutation , China , Microsatellite Repeats , Genetic LociABSTRACT
OBJECTIVES@#To investigate the genetic polymorphism of InDel loci in SifalnDel 45plex system in the Han population in Jiangsu Province and the Mongolian population in Inner Mongolia, and to evaluate the effectiveness of the system in forensic medicine.@*METHODS@#SifaInDel 45plex system was used for genotyping in blood samples of 398 unrelated individuals from the above two populations, and allele frequencies and population genetic parameters of the two populations were calculated respectively. Eight intercontinental populations in the gnomAD database were used as reference populations. The genetic distances between the two studied populations and eight reference populations were calculated based on the allele frequencies of 27 autosomal-InDels (A-InDels). The phylogenetic trees and multidimensional scaling (MDS) analysis diagrams were constructed accordingly.@*RESULTS@#Among two studied populations, the 27 A-InDels and 16 X-InDels showed no linkage disequilibrium between each other and the allele frequency distributions were in Hardy-Weinberg equilibrium. The CDP of the 27 A-InDels in two studied populations were all higher than 0.999 999 999 9, and the CPEtrio were all less than 0.999 9. The CDP of the 16 X-InDels in Han in Jiangsu and Mongolian in Inner Mongolia female and male samples were 0.999 997 962, 0.999 998 389, and 0.999 818 940, 0.999 856 063, respectively. The CMECtrio were all less than 0.999 9. The results of population genetics showed that the Jiangsu Han nationality, Inner Mongolia Mongolian nationality and East Asian population clustered into one branch, showing closer genetic relationship. The other 7 intercontinental populations clustered into another group. And the above 3 populations displayed distant genetic relationships with the other 7 intercontinental populations.@*CONCLUSIONS@#The InDels in the SifaInDel 45plex system have good genetic polymorphism in the two studied populations, which can be used for forensic individual identification or as an effective complement for paternity identification, and to distinguish different intercontinental populations.
Subject(s)
Humans , Phylogeny , Gene Frequency , Polymorphism, Genetic , Genetics, Population , Asian People/genetics , China , INDEL MutationABSTRACT
OBJECTIVES@#To establish a system for simultaneous detection of miR-888 and miR-891a by droplet digital PCR (ddPCR), and to evaluate its application value in semen identification.@*METHODS@#The hydrolysis probes with different fluorescence modified reporter groups were designed to realize the detection of miR-888 and miR-891a by duplex ddPCR. A total of 75 samples of 5 body fluids (including peripheral blood, menstrual blood, semen, saliva and vaginal secretion) were detected. The difference analysis was conducted by Mann-Whitney U test. The semen differentiation ability of miR-888 and miR-891a was evaluated by ROC curve analysis and the optimal cut-off value was obtained.@*RESULTS@#There was no significant difference between the dual-plex assay and the single assay in this system. The detection sensitivity was up to 0.1 ng total RNA, and the intra- and inter-batch coefficients of variation were less than 15%. The expression levels of miR-888 and miR-891a detected by duplex ddPCR in semen were both higher than those in other body fluids. ROC curve analysis showed that the AUC of miR-888 was 0.976, the optimal cut-off value was 2.250 copies/μL, and the discrimination accuracy was 97.33%; the AUC of miR-891a was 1.000, the optimal cut-off value was 1.100 copies/μL, and the discrimination accuracy was 100%.@*CONCLUSIONS@#In this study, a method for detection of miR-888 and miR-891a by duplex ddPCR was successfully established. The system has good stability and repeatability and can be used for semen identification. Both miR-888 and miR-891a have high ability to identify semen, and the discrimination accuracy of miR-891a is higher.
Subject(s)
Female , Humans , Male , Body Fluids/chemistry , MicroRNAs/analysis , Real-Time Polymerase Chain Reaction/methods , Saliva/chemistry , Semen/chemistryABSTRACT
OBJECTIVES@#To develop a rapid test for salivary bacterial community based on direct PCR (dPCR) and high resolution melting (HRM) curve analysis, to evaluate its application value in forensic medicine.@*METHODS@#The salivary bacteria were collected by centrifugation and then resuspended in Tris-EDTA (TE) buffer, and directly used as the template for amplification and HRM curve analysis (dPCR-HRM) of the 16S rDNA V4 region. The genotype confidence percentage (GCP) of the HRM profiles compared with the reference profile was calculated. The template DNA was extracted by traditional kit and then PCR-HRM (namely kPCR-HRM) was used as reference to validate the feasibility of dPCR-HRM. The gradient dilution templates, population samples and simulated salivary stains were analyzed by dPCR-HRM to evaluate its sensitivity, typing ability and adaptability.@*RESULTS@#Using dPCR-HRM method, the HRM profiles of salivary bacterial community were obtained within 90 minutes. The GCP between dPCR-HRM and kPCR-HRM was greater than 95.85%. For general individuals, the HRM type of bacterial community could be determined with 0.29 nL saliva by dPCR-HRM. The 61 saliva samples could be divided into 10 types. The typing of salivary stains deposited within 8 h was the same as those of fresh saliva (GCP>90.83%).@*CONCLUSIONS@#dPCR-HRM technology can be used for rapid typing of salivary bacterial community, and has the advantage of low cost and simple operation.