Developmental Validation And Forensic Investigation Of The Early Access Huaxia^TM Platinum PCR Kit

Objective: To investigate the genetic polymorphism information of the 23 autosomal STR loci included in Huaxia^TM Platinum system,to study the difference between the Eastren Han and the other population,and to evaluate the forensic efficiency of Early Access Huaxia^TM Platinum system by comparing to other available commercial STR kits.Methods: The developmental validation of Huaxia^TM Platinum PCR kit was based on Scientific Working Group on DNA Analysis methods（SWGDAM）guidelines and the Chinese National Standard（CNS）“Basic Quality Requirements of Forensic Science Human Fluorescent STR Multiplex plex PCR Testing Reagent”（GA/T815-2009）and included sensitivity,stability,precision and accuracy,repeatability and repeatability,concordance,case-type samples,stutter calculation and population studies.A total of 650 unrelated healthy individuals from the Eastren Han,Ningxia Hui and Inner Mongolia Mongolian population of China were genotyped with Huaxia^TM Platinum system.Allelic frequencies and forensic parameters of the 23 autosomal STRs were investigated.The obtained frequency distributions were compared with other previously reported population data.Pairwise genetic distances（Fst and p values）with allele frequencies for each STR locus between Eastern Han and other populations were calculated using Arlequin version 3.5.In addition,the genetic information of the Eastren Han by Huaxia^TM Platinum system were compared with other seven commercial STR kits.Results:1.Sensitivity: The sensitivity of the control DNA 007 of Huaxia^TM Platinum system was 0.125 ng to 2ng.2.Stability: In this assay,99.2% of alleles were detected which were sampled in 2007 and 81.41% of alleles were called which were sampled in 2015.Full profiles were obtained from which were sampled in 2009,2010 and 2014.For blood on gauze,about 99.4% of alleles were called.For blood on sterile filter papers,94.3% of alleles were detected.3.Precision and accuracy: Allelic ladder sizing precision study showed that the 3 × standard deviation（SD）of the allele size for multiple injections of the same sample did not exceed 0.20 bases.The SD of the average of allele size measurements was not exceed 0.15 bp.In addition,the sizing accuracy assay showed that 100 blood samples alleles（4053）tested were within ±0.50 bases of the corresponding alleles in the allelic ladder.The largest size difference was-0.41 bases,which was observed at the Penta E loci.4.Stutter calculation: In minus stutter,the stutter filters were ranged from 4.09%（Penta D）to 15.54%（D10S1043）.In plus stutter,the stutter filters were ranged from 4.20%（Penta D）to 19.46%（D10S1043）.5.Reproducibility and repeatability: The profiles were gathered from three DNA control（9947,9948 and 007）and two types of direct amplification samples（blood and buccal on FTA）were fully concordant at two laboratories.6.Case-type samples: Full and concordance profiles were determined from multiple samples by Huaxia^TM Platinum system and ForeseeTM FID23 DNA kit.7.Population study: All the 23 autosomal STR loci follow Hardy-Weinberg equilibrium（P>0.05）.A total of 282（N=500）,196（N=50）,and 229（N=100）alleles were found in Eastern Han,Ningxia Hui,and Inner Mongolia Mongolian,respectively.Power of discrimination（DP）of three populations were among 0.792⁰.986,0.782⁰.968 and 0.798⁰.980,respectively.Polymorphism information content（PIC）of three populations were among 0.559⁰.914,0.579⁰.918 and 0.566⁰.912,respectively.Combined discrimination power（CDP）of three populations were 1-4.1E-28,1-2.7E-25 and 1-1.5E-26,respectively.There were no significant difference between the Eastern Han and the other 4 Chinese groups(Mongolian,Tibetan,Yi and Ewenki.However,significant differences were observed at some STR loci between Eastern Han population and other populations.8.Comparison with other commercial STR kits: Comparing with other commercial STR kits,Huaxia^TM Platinum system contains the most number of alleles within the ladder.The value of CDP of Huaxia^TM Platinum system（1-4.1E-28）was the higest and Identifier（1-1.5E-17）and Sinofiler（1-5.2E-19）were lowest.Conclusions: The Huaxia^TM Platinum PCR kit was specially designed for Chinese and STR loci of this system are contained in the whole new CODIS loci and the Chinese National Database.The validation results demonstrated that the Huaxia^TM Platinum PCR kit is a sensitive,stable,reliable,fast and robust system in forensic genetics.The whole PCR was approximately finished within 45 minutes.In addition,Huaxia^TM Platinum system with 23 highly polymorphic STR loci in Eastren Han,Ningxia Hui and Inner Mongolia Mongolian population of China could be used for individual identification and paternity testing.Comparing with other commercial STR kits,Huaxia^TM Platinum system could provide more genetic information.In conclusion,above results demonstrated that Huaxia^TM Platinum PCR kit could be applied in forensic practice and population genetics.