Development Of The STR Multiplex PCR System Of Felis Catus And Its Application In Forensic

ObjectiveTo establish the STR fluorescent multiplex PCR system of Felis catus fluorescent multiplex PCR system was constructed and its technical performance was tested to evaluate the application value of this system in forensic science.Methods1.Developed Felis catus STR locus were collected based on literature reports and Gene Bank database.Primers were designed for the selected locus using software Primer Premier 5.0 and online tool Primer-BLAST.Agarose gel electrophoresis was performed after the amplification of each single locus to verify the amplification effect of primer design.2.The 16 autosomal STR loci and one sex identification loci were divided into four groups.The forward primers of each loci in each group were labeled with fluorescent dye（6 FAM HEX TAMRA and ROX）to explore and optimize the PCR reaction conditions of the combined amplification system,so that the amplification products of each loci basically met the requirement of balance and specificity3.According to the Scientific Working Group on DNA Analysis Methods,SWGDAM and International Society for Forensic Genetics（ISFG）required to conduct Forensic verification studies on the sensitivity,species specificity,accuracy and balance of the constructed cat fluorescence complex amplification detection system.A population genetic survey was conducted on 145 unrelated feline individuals.Results1.In this study,a multiplex amplification fluorescence detection system including 16autosomal STR locus（FCA733、FCA391、FCA740、F27、FCA453、FCA734、F85、F124、FCA742、FCA736、FCA730、FCA749、FCA559、FCA723、FCA441、FCA732）and 1 sex identification loci（SRY）was successfully constructed.2.In this study,forensic verification of the constructed compound amplification system showed that the sensitivity study showed that complete typing could still be obtained when the amount of DNA template was as low as 0.25ng,and consistent typing results were obtained when different tissues of the same individual were repeatedly tested for the same sample.No specific amplification peak was found when common animal samples were tested.3.In this study,the population genetics of the fluorescence detection system constructed by compound amplification was investigated.A total of 259 alleles were detected from 16 autosomal STR loci in 145 samples.The heterozygosity of 16autosomal intermediate genes ranged from 0.531（FCA391,FCA732）to 0.876（F85）,with an average heterozygosity of 0.687.The polymorphic information content ranged from 0.664（FCA441）to 0.952（F85）,with an average of 0.790.The random matching probability distribution ranged from 0.012（F85）to 0.133（FCA732）,with an average of 0.034.The individual recognition rate of 16 loci was≥0.867（FCA732）,and the exclusion probability of paternity was≥0.216（FCA391,FCA732）.The cumulative individual recognition probability was 1-3.59×10^-20,the cumulative non-father exclusion probability was 1-6.35×10^-5,and the cumulative random matching probability was 3.61×10^-20.ConclusionThe validation results indicated the robustness and reliability of the new STR multiplex system,and it can be used in forensic cases such as individual and paternity identification of Felis catus.