Developmental validation studies on the RapidHIT™ Human DNA Identification System

The RapidHIT™ Human DNA Identification System is a fully integrated system (sample in, result out) capable of rapidly generating STR DNA profiles in around 90 min. Here we present a portion of the results from the developmental validation studies performed on the RapidHIT System.     

RapidHITTM 200系统在法医学中的应用

目的验证Rapid HIT^TM 200系统对四类法医学常见检材的检测效能,以及两次重复检测运行中模板、模板DNA和PCR产物的再利用效能。方法将口腔拭子经Rapid HIT^TM 200系统先后进行两次检验,并针对系统中产生的模板DNA和PCR产物进行首次、再次检测。将四类法医学常见检材经Rapid HIT^TM200系统检测,并针对系统运行中产生的模板、模板DNA和PCR产物进行后续检测。结果口腔拭子经该系统首次运行后STR基因座可以完全检出,再次检测过程中部分STR基因座有丢失,再次检测峰值较首次明显降低;模板DNA和PCR产物再次检测的平均STR基因座检出率均小于50%,明显低于首次检测结果。该系统对组织与口腔拭子的检测效能优于血拭子和烟蒂,首次运行后的相应检材、模板DNA、PCR产物延续后续实验流程所获得的STR基因座检出率呈递减趋势。结论Rapid HIT^TM 200系统对口腔拭子再检测效果好,但经该系统首次运行后的检材、模板DNA、PCR产物以及经该系统再次运行后的模板DNA与PCR产物不应直接用于法医学后续应用及研究。     

A comprehensive GlobalFiler™ autosomal STR reference dataset for Southern Africa

In this study a total of n = 832 autosomal DNA profiles from Southern Africa are analysed using the GlobalFiler™ STR panel. The dataset includes South Africa (SA) profiles (n = 541) produced by Ristow et al. 2016 and includes newly generated data for SA Sepedi (n = 96) and Lesotho populations (n = 195). For the newly generated (n = 291) genotypes, we report a large degree of rare and novel variation. This included (n = 7) off-ladder allele variants and (n = 7) TPOX tri-alleles. We report forensic summary statistics and genetic diversity parameters. Expected heterozygosity and observed heterozygosity ranged between (0.7– 0.9) with SE33 as the most polymorphic and TH01 the least. For SA and Lesotho genotypes the combined match probability was (1.13 ×10^-24 and 6.035 ×10^-24) and the combined paternity index (1.4 ×10⁹ and 2.44 ×10⁸) respectively. The power of exclusion (0.9999) was similar for each dataset and no significant departures from Hardy-Weinberg equilibrium (HWE) were observed after Bonferroni correction. Population comparisons were performed by MDS and neighbour-joining and population structure inferred by STRUCTURE and DAPC unsupervised clustering.     

超滤管在陈旧生物检材中的应用

目的探讨millipore超滤管滤过方法对陈旧生物检材DNA分型检验的应用价值。方法将23份陈旧血样分别剪取同样合适大小的血片3组,标为A、B、C组,磁珠法提取DNA,分别用80μL、80μL、20μL洗脱液洗脱得到模板DNA,其中A、C组模板直接扩增,B组用millipore超滤管滤过浓缩后扩增。PCR产物用AB3130x L基因分析仪检测,Gene Mapper ID V3.2软件进行自动分型。所得实验数据用SPSS软件分析处理。结果 A组没有1例样本扩出全部STR基因座,B组有18例样本扩出全部STR基因座,C组有11个样本扩出全部STR基因座。结论应用millipore超滤管滤过方法可以明显提高陈旧生物检材的DNA分型成功率。     

贵州汉族19个STR基因座多态性及法医学应用

目的调查19个常染色体STR基因座在贵州汉族人群中的等位基因分布,评估其在法医学中的应用价值。方法应用Goldeneye~(TM) DNA身份鉴定系统20A试剂盒,研究贵州520名汉族无关健康个体19个常染色体STR基因座多态性。用310型遗传分析仪进行毛细管电泳,Gene Mapper~ID v3.1进行基因分型。结果 19个常染色体STR基因座的杂合度为0.603 8~0.916 4,个体识别率为0.790 0~0.985 6,非父排除率为0.295 5~0.826 9,多态信息含量为0.553 5~0.908 9,累积个体识别率为1-1.230 0×10~(-22),累积非父排除率为0.999 999 99。贵州汉族和其他五个地域的汉族两两之间等位基因频率比较,仅贵州汉族与山东汉族、辽宁汉族、山西汉族之间存在基因频率差异具有统计学意义。结论 D19S433等19个常染色体STR基因座在贵州汉族人群中具有良好的遗传多态性,对群体遗传学和法医物证学研究有应用价值。     

The Recovery and Analysis of Mitochondrial DNA from Exploded Pipe Bombs*

Abstract: Improvised explosive devices (IEDs) represent one of the most common modes of arbitrarily injuring or killing human beings. Because of the heat generated by, and destruction to, an IED postconflagration, most methods for identifying who assembled the device are ineffective. In the research presented, steel pipe bombs were mock‐assembled by volunteers, and the bombs detonated under controlled conditions. The resultant shrapnel was collected and swabbed for residual cellular material. Mitochondrial DNA profiles were generated and compared blind to the pool of individuals who assembled the bombs. Assemblers were correctly identified 50% of the time, while another 19% could be placed into a group of three individuals with shared haplotypes. Only one bomb was assigned incorrectly. In some instances a contaminating profile (mixture) was also observed. Taken together, the results speak to the extreme sensitivity the methods have for identifying those who assemble IEDs, along with precautions needed when collecting and processing such evidence.     

Investigative studies into the recovery of DNA from improvised explosive device containers

Apprehending those who utilize improvised explosive devices (IEDs) is a national priority owing to their use both domestically and abroad. IEDs are often concealed in bags, boxes, or backpacks to prevent their detection. Given this, the goal of the research presented was to identify IED handlers through postblast DNA recovery from IED containers. Study participants were asked to use backpacks for 11 days, after which they served as containers for pipe bombs. Eleven postdeflagration backpack regions likely to be handled were swabbed and analyzed via mini-short tandem repeats (miniSTRs) and alleles were called blind. An experimental consensus method was examined in which profiles from all regions were considered, to help identify spurious drop-in/out. Results were correct for all loci, except one that remained ambiguous. The results show that recovering DNA from IED containers is a viable approach for aiding in the identification of those who may have been involved in an IED event.     

Rapid direct PCR for ABO blood typing

Many different molecular typing methods have been reported to complement routine serological ABO blood typing in forensics. However, these ABO genotyping methods are often time-consuming and call for an initial DNA isolation step that requires the use of expensive kits or reagents. We report here a rapid direct ABO genotyping method that eliminates the need for DNA extraction from fresh blood, hair, and body fluid stains before PCR. Using a fast PCR instrument and an optimized polymerase, the genotyping method-which employs a multiplex allele-specific primer set for the simultaneous detection of three single-nucleotide polymorphism (SNP) sites (nucleotides 261, 526, and 803)-identifies A, B, O01/O02, O03, and cis-AB01 alleles in around 70 min from sample collection to electropherogram. Not only will this ABO genotyping method be efficiently used in forensic practice for rapid screening of samples before full-blown multilocus short tandem repeat profiling, but it will also demonstrate an example of rapid direct genotyping of SNPs that offers the advantages of time- and cost-efficiency, convenience, and reduced contamination during DNA analysis.     

Analysis of forensic SNPs in the canine mtDNA HV1 mutational hotspot region

A 60 bp sequence variation hotspot in the canine mitochondrial DNA hypervariable region 1 was evaluated for its use in forensic investigations. Nineteen haplotypes containing 18 single nucleotide polymorphisms were observed among laboratory-generated and GenBank-derived domestic dog sequences representing five regional localities in the U.S. Samples from the different localities were highly variable with the levels of intra-population variability being similar among the populations studied. AMOVA further confirmed that there was no significant genetic structuring of the populations. Assays using these haplotypes were robust, canid specific and portend a rapid method for correctly excluding individual dogs as noncontributors of forensic evidence. Species-specificity of the primers was confirmed by means of in-tube polymerase chain reaction of human and cat DNA and in-silico assessment of the genomes of several animal species. Breed-specific fragments were not detected among the common haplotypes but there is evidence that this assay may be capable of differentiating domestic dog, wolf, and coyote sequences.     

A Monte Carlo simulation and sensitivity cost benefit analysis for use of nylon 4N6FLOQSwabs®

Forensic genetic laboratories are challenged with implementing innovation even if the benefits to operational performance are well demonstrated often because of internal budget constraints. A prospective cost–benefit analysis (CBA) could support justification for an increased budget by effectively demonstrating in a system-based approach the relatively small cost of increasing a laboratory budget can substantially reduce costs to society (both qualitatively and monetarily). A Monte Carlo simulation and sensitivity CBA was performed using a more expensive swab (i.e., nylon 4N6FLOQSwabs®) compared with a less costly cotton swab. Ranges of input values and tangible and intangible benefits were considered. The outcome is that the relatively small increased cost of using a nylon swab pales compared with the potential tangible and intangible benefits to the overall system. This approach provides a sounder basis for requesting additional funds to support implementation of technologies and better approximates realistic situations while accommodating uncertainty of input values.     

Genetic polymorphisms at four STR loci from the HLA region in a Venezuelan population

POPULATIONS: Whole blood samples from 74 unrelated healthy individuals were collected. The donors' sample included Venezuelan mestizos from various regions of the country, but mostly from the resident population of Caracas City. A Venezuelan mestizo is the offspring of a mating between a native Venezuelan and a person born in Europe, mainly in Spain.