Exploring The Application Of Insertion/Deletion Polymorphism In The Coding Region Of Body Fluid-Specific MRNA For Humoral Traceability

Objective: Body fluid identification has been one of the important research subjects in forensic genetics for a long time.In practical cases,determining the type of body fluid organization left at the crime scene and tracing the source of the individual can provide important clues and basis for the reconstruction of the crime scene and case investigation,and has significant practical application value.Traditional bodily fluid detection methods,such as chemical methods,immunological methods,protein catalytic activation tests,etc.,will consume a lot of time,waste precious materials,and it is difficult to ensure the accuracy of the results.In recent years,RNA analysis has gradually become the mainstream method for forensic body fluid identification.This study intends to construct a body fluid-specific mRNA analysis system containing coding region insertion/deletion(cInDels).There are 9humoral-specific cInDels,10 humoral-specific mRNA markers and 2 sex-specific mRNA markers.At the same time,the specificity,consistency,sensitivity,and mixed sample of the system were verified,aiming to provide new ideas and detection methods for forensic body fluid identification.Methods: For semen,saliva,vaginal fluid,menstrual blood and venous blood,9 body fluid specific cInDel markers were screened from the gnom AD database.At the same time,10 mRNA markers and 2 sex markers that had been verified in the literature were also included in the system;Primer Premier 5 software was used to design primers for each site,and fluorescent dyes were labeled in groups according to the length of amplified fragments for the construction of complex amplification system;RNA and genomic DNA of 50 body fluid samples were extracted according to the instructions of the kit,and RNA was reversely transcribed into c DNA.The c DNA was amplified by PCR using the constructed complex amplification system,followed by capillary electrophoresis typing;The specificity,consistency,sensitivity,mixture,species specificity,and repeatability of the complex amplification system were evaluated.Results: Based on the 3130 XL capillary electrophoresis platform,an mRNA fluorescence complex amplification system was successfully constructed for body fluid type identification and individual determination of body fluid source,including 19 mRNA markers:ODF1(rs752086061+rs757727788),TCP11(rs142993601+rs142118879),PRM1 and SEMG1 were semen-specific markers.KRT4,STATH and HTN3 were saliva-specific markers.GPAT3(rs35602893),CYP2B7P1,Lcris,FAM83D(rs534813259)were vaginal fluid-specific markers.COL6A3(rs35879189),STC1,LPAR3(rs58203951)and MMP11 were menstrual blood-specific markers.SLC4A1 and ALAS2 were venous blood-specific markers.XIST and RPS4Y1 were gender markers;The system was used to detect 50 samples(10 samples for each body fluid),and the tissue source of the above 5 body fluids was successfully detected;Consistency: The genotyping results of 9 cInDel markers observed in c DNA and g DNA samples were consistent,which further confirmed the accuracy of the detection results of this system;More than 2.5ng RNA templates could be successfully detected by this system;Using this system to detect mixed samples(semen and vaginal fluids,semen and saliva),successfully distinguish the source of body fluids in the mixed samples and match them to a specific donor;Species specificity: Venous blood and cheek swab samples from nine non-human samples(geese,donkeys,chickens,sheep,pigs,cattle,dogs,cats,and pigeons)were used to test the system,and no specific amplification peaks were observed.Conclusion: In this study,a fluorescence complex amplification system containing 9humoral-specific cInDels,10 humoral-specific mRNA markers and 2 sex-specific mRNA markers was innovatively constructed,which could detect the types of body fluids in semen,saliva,vaginal secretions,menstrual blood and venous blood,and provide cInDel typing information for the determination of the individual source of body fluids.It has been proved that the system has good specificity,consistency,sensitivity,species specificity,repeatability and the ability to detect mixtures.This study can provide a new idea for establishing a more perfect and reliable forensic body fluid source identification method.