Development And Application Of STR Locus Based Kits For Individual Identification And Kinship Identification Of Cattle

Objective: DNA analysis techniques have been widely used in forensic identification cases involving cattle.In this study,a fluorescent complex amplification system containing 13 bovine STR locus was constructed,and the forensic validation and systematic efficacy evaluation of the constructed amplification system were carried out to further investigate the genetics of bovine populations in order to evaluate whether the constructed bovine STR locus complex amplification system can meet the needs of species identification,individual identification and kinship identification of cattle in forensic identification.Methods: The STR locus was screened using published reports and the STR locus recommended by the International Society for Animal Genetics(ISAG)for animal genetic diversity research as a database.We designed primers for the selected STR locus and established a fluorescent complex amplification system for the STR locus.According to the requirements of the Scientific Working Group for DNA Analysis Methods(SWGDAM),the constructed fluorescent complex amplification system was tested for forensic science related tests such as sensitivity,species specificity,identities and reproducibility,Inhibitor,and accuracy.Results:1.In this study,we screened and successfully constructed a fluorescent complex amplification system containing 13 cattle STR locus(TGLA126,BT66,BT165,ETH10,CSSM0113,INRA005,BT54,BT61,INRA023,BM2113,G18833,UMN0929,INRA063).2.Sensitivity study: When the DNA template amount was 2ng,1ng,500 pg,250pg,125 pg,the amplification system had 100% allele detection rate.When the template amount was62.5pg and 31.25 pg,the detection rate of the amplification system was 64% and 27.3%,respectively.3.Species specificity study: DNA samples from 11 common species such as pig,sheep,dog,donkey,cat,rabbit,chicken,duck,monkey,rat and snake,and samples contaminated with 3human DNA sample standards(9947A,9948,2800M)will not interfere with the correct typing of bovine genomic DNA.4.Identities and repeatability study: samples collected from different parts of the same cattle were tested for consistent STR genome typing.Consistent DNA typing for the same sample in different laboratories,operated by different laboratory personnel.5.Inhibitor study: The five common PCR inhibitors set in the study were heme,humic acid,urea,indigo,and melanin.The correct typing of sample DNA was only interfered when the concentration of heme was higher than 200 μM,humic acid was higher than 100 ng/μL,urea was higher than 24000 ng/μL,indigo was higher than 5000 ng/μL,and melanin was higher than 400 ng/μL.6.Accuracy study: Most of the allele fragment length offsets are less than 0.2 bp.The migration rate tends to increase as the product fragment length increases,but is still within the resolution of 0.5 bp of the 3130 XL Genetic Analyzer.7.Population genetics study: The cumulative discrimination power rate of the 13 STR locus was 0.999 999 999 97;the cumulative probability of exclusion was 0.999 926 668 46.Conclusions: In this study,a fluorescent complex amplification system containing 13 STR locus was screened and innovatively constructed.The complex amplification system was validated by forensic genetics,which showed that the characteristics of species specificity,sensitivity,inhibitor and accuracy were better than those of existing detection systems and could be applied to many types of case samples.It can be used for cattle species identification,individual identification and parentage identification,and can provide a reliable technical means for judicial cases involving cattle meat adulteration and theft of domestic cattle.