Development And Validation Of 9-Dye Fluorenscent Multiplex STR Amplification System

Objective:Autosomal STR loci（short tandem repeats,STR）are widely applied for personal identification,kinship testing and human genetics research owing to their high diversities and abundance.Multi-dye fluorescence-labeled STR multiplex amplification typing technique based on capillary electrophoresis platform（CE）is highly sensitive,accurate,and automated.It is the most commonly used method in forensic laboratories.Currently,commercial 5-dye and 6-dye autosomal STR kits are available to meet the needs of most cases.However,for degraded specimens affected by factors such as temperature,humidity,microorganisms,DNA in the specimens is prone to degradation and large fragments may be lost,making it difficult to obtain reliable typing results with a single conventional commercial STR kit.The mini STR technique,which shortens the amplicon lengths,can significantly improve the typing success ratio of degraded specimens.However,affected by the fluorescent signal channel（up to 6-dye）,the number of mini STR loci that can be accommodated in the same system is limited.Thus,mini STR has not yet reached its maximum value.In addition,it is necessary to increase the number of included loci to reduce the probability of random matching between unrelated individuals.Therefore,in this study,we selected 40 loci and attempted to construct a 9-dye fluorescent STR multiplex amplification system,including more mini STR and STR loci with better polymorphism in Chinese population,in order to obtain more valuable information and increase the reliability of results in a single detection.Methods:1.Construction of a 9-dye fluorescent multiplex amplification system.39 autosomal STR loci and 1 sex determining maker Amelogenin were selected according to the set screening conditions（core loci in major international and domestic DNA databases,loci with high polymorphism in Chinese population,etc.）,and primers were designed based on the mini STR concept.The 40 loci were divided into 8 groups and labeled with FAM,TET,HEX,NED,NH598,NH618,NH635,and NH650 fluorescent dyes on the 5′end of the foward primer of each locus.The 9-dye fluorescent STR multiplex amplification system was constructed through repeated optimization of the composite amplification system（primer design,adjustment of primer concentration for each locus,buffer system and PCR reaction conditions）.The amplification products were detected by electrophoresis using a domestic GA118-24B genetic analyzer,and the electrophoresis data was analyzed and interpreted by the GAMarker DNA typing expert system.All alleles were named according to the nomenclature rules proposed by the International Society of Forensic Genetics（ISFG）,and panel and bin files were compiled.2.Validation of the multiplex amplification system and its forensic applications.According to the requirements of the national standard GB/T 37226-2018,the sensitivity,mixed samples,species specificity,stability,anti-inhibition,accuracy,degraded samples,and detection capabilities of different types of forensic samples were studied for the constructed 9-dye fluorescent-labeled STR multiplex amplification system.The detection results of different types of forensic samples were compared with those of DNATyper^TM25 and DNATyper^TM30 kits to verify their adaptability and typing consistency.Based on the allelic frequency of STR loci in the Chinese Han population,the relevant forensic genetic parameters of the 9-dye fluorescent-labeled STR multiplex amplification system,DNATyper^TM25 and DNATyper^TM30 kits were calculated to evaluate the application value of the system in forensic sciences.Results:1.Construction of a 9-dye fluorescent multiplex amplification system.In this study,a 9-dye fluorescent STR multiplex amplification system was successfully constructed,including Amelogenin,D5S818,D21S11,D7S820,CSF1PO,D2S1338,D3S1358,v WA,D8S1179,D16S539,Penta D,TPOX,TH01,D22S1045,D18S51,FGA,D6S1043,D13S317,D12S391,Penta E,D2S441,D1S1656,D19S433,D10S1248,D6S477,D1S1627,D8S1132,D15S659,D17S974,D6S1017,D4S2408,D9S2157,D6S474,D3S3045,D19S253,D20S482,D14S1434,D11S4463 and D10S1435（28 loci<300 bp）.All loci showed peak height balance and no nonspecific amplification.2.Validation of the multiplex amplification system and its forensic applications.The 9-dye fluorescent STR multiplex amplification system developed in this study has a sensitivity of 0.125 ng and shows tolerance to common PCR inhibitors.No specific amplification peaks were observed in species specificity testing.The minimum detection ratio of two-gender mixture is 1:4.The system can be used to detect different types of case samples and produces consistent results when compared to other similar commercial kits.Degraded sample testing shows that the system detects more loci in a single run than other kits.The precision testing results show that the deviation of all average allele fragment migrations is less than 0.15 bp,within the resolution range of the genetic analyzer.For the Chinese Han population,the total discrimination power（TDP）of the autosomal STR loci in the 9-dye fluorescent STR multiplex amplification system,DNATyper^TM25,and DNATyper^TM30 kits are 1-7.85×10^-45,1-1.33×10^-22,and 1-1.99×10^-35,respectively.The cumulative power of exclusion in duos（CPE_D）of the autosomal STR loci are 0.999999999667104,0.999941236134284 and 0.99999997909338,and the cumulative power of exclusion in trios（CPE_T）of the autosomal STR loci are0.99999999999999972968,0.999999948566468 and 0.999999999999661,respectively.Conclusions:In this study,the 9-dye fluorescent labeling technology was used for the first time to make the multiplex amplification reagents.Through the performance evaluation and experimental verification,the system exhibited high sensitivity,good species specificity,and accurate and stable typing results.With 28 mini STR incorporated,it can effectively improve the detection efficiency of trace degraded samples.The system can obtain genetic information from 40 loci in a single detection,with high system efficiency and compatibility with other autosomal STR kits,which can meet the requirements of routine case sample testing and DNA database construction,and provide a new technical solution for the development of domestic DNA testing reagents.