Development Of A Novel Panel For Blood Identification Based On Blood-specific CpG Linked SNP Markers

Objective:The origin of blood in the mixed stains was inferred based on blood-specific Cp G markers and adjacent SNP polymorphic markers,while individual identification of blood donors was identified,thereby simplifying the interpretation of complex mixtures.Methods:In this study,we employed blood-specific Cp G linked SNP markers(Cp G-SNP)for blood-specific genotyping and the linking of blood and its donor.The tissue specific Cp G markers were screened from the literature and further verified by combining bisulfite conversion with amplification-refractory mutation system(ARMS)technology.Meanwhile,adjacent SNP markers with a minor allele frequency(MAF)greater than 0.1 were selected within 400 bp upstream and downstream of the Cp G markers by retrieving the Common db SNP(153)dataset on human assembly(GRCh37/hg19)of the UCSC Genome Browser.SNP genotyping was performed using SNa Pshot technology on a capillary electrophoresis(CE)platform.Finally,a multiplex panel,including 19 blood-specific Cp G linked to 23 SNP markers,as well as 1 semen-specific Cp G,1 vaginal secretion-specific Cp G,and 1saliva-specific Cp G marker,was constructed successfully.In addition,Developmental validation studies,such as sensitivity,species specificity,aged and degraded samples analysis,and mixtures analysis,had then been performed to evaluate the efficacy of the new tool for forensic application.Results:1.A multiplex panel was successfully constructed,including 19 blood-specific Cp G markers(linked 23 SNP marker),a semen-specific Cp G marker,a vaginal secretion-specific Cp G marker,a saliva-specific Cp G marker and an internal control ACTB.Based on the capillary electrophoresis platform,the sources of four body fluids,blood,saliva,semen and vaginal secretions,could be accurately identified,and blood donors could be successfully typed.2.The detection sensitivity of multiplex panel was 15 ng,that is,when the input of bisulfite conversion was above 15 ng,accurate blood identification and personal identification could be realized.3.Even if the blood stains were preserved at room temperature for up to 9 months,the source of blood could still be accurately identified and the complete SNP typing could be obtained.4.When the blood stains was moderately degraded(4 < DI < 10),the source of blood could be effectively identified,and the ability of individual identification is weakened.5.The statistical results based on a one-way ANOVA indicated that the multiplex panel was age-independent.6.When the proportion of blood in mixed stains was as low as 1%,the existence of blood could be accurately detected.In addition,it could simultaneously identify super mixed stains containing blood,saliva,semen and vaginal secretions.7.The blood samples of 114 Han Chinese in Shanxi province were employed for SNP genotyping,and the CDP value of this multiplex was 0.99998.Conclusion:In this study,a multiplex panel based on Cp G-linked SNP compound marker was developed,which could accurately identify four kinds of body fluids including blood,saliva,semen and vaginal secretions,and could also identify blood donors in mixed stains.The successful construction of the panel will play a vital role for the comprehensive analysis of blood-containing mixtures in forensic practice.