Developmental Validation of the Quantifiler® Duo DNA Quantification Kit for Simultaneous Quantification of Total Human and Human Male DNA and Detection of PCR Inhibitors in Biological Samples*

Abstract: The Quantifiler^® Duo DNA Quantification kit enables simultaneous quantification of human DNA and human male DNA as well as detection of inhibitors of PCR in a single real-time PCR well. Pooled human male genomic DNA is used to generate standard curves for both human (ribonuclease P RNA component H1) and human male (sex determining region Y) specific targets. A shift in the cycle threshold (C_T) values for the internal positive control monitors the presence of PCR inhibitors in a sample. The assay is human specific and exhibits a high dynamic range from 0.023 to 50 ng/μL. In addition, the multiplex assay can detect as little as 25 pg/μL of human male DNA in the presence of a 1000-fold excess of human female DNA. The multiplex assay provides assessment of the DNA extract and guidance for the selection of the appropriate AmpFℓSTR^® Amplification Kit to obtain interpretable short tandem repeat profiles.     

The Effects of Differential Extraction Conditions on the Premature Lysis of Spermatozoa,

The purpose of this study was to determine the effect Proteinase K, sodium dodecyl sulfate (SDS), incubation times, and temperatures had on differential extraction efficiencies and the premature lysis of spermatozoa. The effect was measured using Quantifiler^® Duo and Identifiler? PCR Amplification kits, where the resultant male and female DNA concentrations and their ratios within the nonsperm‐ and sperm fractions (SFs) were determined. Comparisons between expected and observed ratios illustrate the quantity of female DNA in the SF increased when Proteinase K was absent during the initial incubation. Additionally, there is no indication of simultaneous sperm and epithelial cell lysis in the absence of DTT at Proteinase K concentrations ranging from 10 to 300 μg/mL. All other conditions exhibited minimal variation in DNA concentration. Therefore, despite the various protocols used for the differential lysis of cell mixtures encountered in casework, the method is robust and successful at most conditions.     

Optimization of Human mtDNA Control Region Sequencing for Forensic Applications

Sequencing mitochondrial DNA hypervariable regions I and II (HVI and HVII) is useful in forensic missing person and unidentified remains cases. Improvements in ease and sensitivity of testing will yield results from more samples in a timely fashion. Routinely, amplification of HVI and HVII is followed by Sanger sequencing using the BigDye^® Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) using 4 μL of ready reaction mix (RRM). Each sequencing reaction is then purified through column filtration before capillary electrophoresis. Using lower amounts of RRM (2 μL or 1 μL) and purification using BigDye^® XTerminator^? (Applied Biosystems) instead of columns showed no loss of sequence length and increased the quality and the sensitivity of testing, allowing HVI and HVII typing from mitochondrial genome equivalent to 125 fg of nuclear DNA, or 100 pg of HVI/HVII amplicons. Using this methodology, testing can be completed in 1 day, and the cost of testing is reduced.     

Detection of microRNAs in DNA Extractions for Forensic Biological Source Identification

Molecular‐based approaches for biological source identification are of great interest in the forensic community because of a lack of sensitivity and specificity in current methods. MicroRNAs (miRNAs) have been considered due to their robust nature and tissue specificity; however, analysis requires a separate RNA extraction, requiring an additional step in the forensic analysis workflow. The purpose of this study was to evaluate miRNA detection in blood, semen, and saliva using DNA extraction methods commonly utilized for forensic casework. RT‐qPCR analysis revealed that the tested miRNAs were consistently detectable across most tested DNA extraction methods, but detection was significantly reduced compared to RNA extracts in some biological fluids. DNase treatment was not necessary to achieve miRNA‐specific results. A previously developed miRNA panel for forensic body fluid identification was evaluated using DNA extracts, and largely demonstrated concordance with results from samples deriving from RNA extracts of semen, blood, and saliva.     

Developmental Validation of the PrepFiler™ Forensic DNA Extraction Kit for Extraction of Genomic DNA from Biological Samples*

Abstract: The PrepFiler? Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 μL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA^® paper), and touch evidence‐type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol–chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.     

Automated Quantifiler quantitative PCR setup, template normalization and PCR setup using HID EVOlution™ qPCR/STR setup on trace evidence samples

We have implemented and validated customized protocols for automated Quantifiler^® setup, template normalization and PCR setup using the Tecan HID EVOlution™ qPCR/STR setup. The protocols were validated for the Quantifiler^® human DNA quantification, AmpF?STR^® SGM Plus^® and SEfiler Plus™ PCR Amplification Kits (Applied Biosystems) according to EN/ISO 17025.     

A comparative study of two extraction methods routinely used for DNA recovery from simulated post coital samples

In sexual assault cases DNA profiling of spermatozoa can be of critical importance. Most methods use differential extraction of the spermatozoa to separate it from the female component. Here we have compared two commercially available differential extraction methods, the QIAamp^® DNA mini kit (Qiagen) and Differex™ with the DNA IQ^® System (Promega). Simulated postcoital samples were prepared using buccal cells from a female donor and spermatozoa from three male donors. A dilution series ranging from neat semen to a 1:1500 dilution (semen:dH₂O) was prepared and mixed with an equal volume of saliva from a female donor. Extraction efficiency was assessed using DNA concentration measured with NanoDrop 2000 and Quantifiler^® Human DNA Quantification Kit and the profile count of full, partial and mixed DNA profiles generated using SGM Plus and PowerPlex^® ESI 17. Statistical analysis was carried out using Randomisation in R, which is a robust model making no assumption of the distribution of data. Based on the amount of DNA extracted and the types of profiles no significant difference in the performance of the two extraction kits was seen. However, the processing time taken with the Differex™ System was about half than that of the QIAamp^® DNA mini kit and involved fewer liquid transfers.     

Developmental Validation of RSID™-Saliva: A Lateral Flow Immunochromatographic Strip Test for the Forensic Detection of Saliva

Abstract:  Current methods for forensic identification of saliva generally assay for the enzymatic activity of α-amylase, an enzyme long associated with human saliva. Here, we describe the R apid S tain ID entification (RSID™-Saliva), a lateral flow immunochromatographic strip test that uses two antisalivary amylase monoclonal antibodies to detect the presence of salivary amylase, rather than the activity of the enzyme. We demonstrate that RSID™-Saliva is accurate, reproducible, and highly sensitive for human saliva; RSID™-Saliva detects less than 1 μL of saliva. The sensitivity of RSID^TM-Saliva allows investigators to sample a fraction of a questioned stain while retaining the majority for DNA-STR analysis. We demonstrate that RSID™-Saliva identifies saliva from a variety of materials (e.g., cans, bottles, envelopes, and cigarette-butts) and it does not cross-react with blood, semen, urine, or vaginal fluid. RSID™-Saliva is a useful forensic test for determining which evidentiary items contain saliva and thus may yield a DNA profile.     

PuriTyper~(TM)法医学纯化试剂盒的性能验证

目的验证PuriTyperTM纯化试剂盒各项性能指标和法医学应用价值。方法收集及制备抗凝血液、常见案件检材(唾液、烟头、精液、毛发、指甲、骨骼及组织块)、斑痕样本(血斑、唾液斑、精斑)以及模拟添加抑制剂和模仿自然环境中放置的血斑。采用PuriTyperTM纯化试剂盒提取纯化并进行DNA定量,IdentifilerTM复合扩增试剂盒扩增,产物经ABI 3130遗传分析仪进行检测,Genemapper软件分析结果,对该试剂盒灵敏度、稳定性、重复性、检材适应性进行测试。结果采用该试剂盒提取0.1～40μL血液分别获得0.042～26.45ng/μL的DNA。3种斑痕样本DNA产量平行试验结果稳定。不同类型检材重复检验所获IPC的CT平均值在27.60至28.03之间。常见案件检材所得分型与已知结果均一致。结论 PuriTyperTM纯化试剂盒能够满足法医DNA检验的要求,对法医学实践具有重要的应用价值。     

The Use of Forensic Tests to Distinguish Blowfly Artifacts from Human Blood,Semen, and Saliva

This study investigated whether routinely used forensic tests can distinguish 3‐day‐old or 2‐week‐old fly artifacts, produced after feeding on human blood, semen, or saliva, from the biological fluid. Hemastix^®, Hemident^?, and Hemascein^? were unable to distinguish blood from artifacts. Hemastix^® returned false positives from negative controls. ABAcard^® Hematrace^® and Hexagon OBTI could distinguish blood from 3‐day‐old artifacts, but not 2‐week‐old artifacts. Phadebas^® and SALIgAE^® were unable to distinguish saliva from artifacts. RSID^?‐Saliva was able to distinguish saliva from 3‐day‐old artifacts, but not 2‐week‐old artifacts. Semen tests Seminal Acid Phosphatase, RSID^?‐Semen, and ABAcard^® p30 were all able to distinguish semen from 3‐day‐old artifacts, but not 2‐week‐old artifacts. The tests investigated cannot be relied upon to distinguish artifacts from biological fluids. However, if an artifact is identified by its morphology, a positive result may indicate which biological fluid the fly consumed, and this knowledge may prove useful for investigators searching for DNA.     

Getting Ahead: Extraction of DNA from Skeletonized Cranial Material and Teeth

Between 1990 and 2018, the Defense POW/MIA Accounting Agency submitted 2177 cranial elements and 1565 teeth to the Armed Forces Medical Examiner System—Armed Forces DNA Identification Laboratory for DNA testing. In an effort to identify missing United States service members, materials were recovered from wartime losses inclusive of World War II, the Korean War, and Southeast Asia. Using four different DNA extraction protocols, DNA testing was performed using mitochondrial DNA Sanger sequencing, modified AmpFlSTR^® Yfiler?, AmpFlSTR^® MiniFiler?, PowerPlex^® Fusion, or Next Generation Sequencing. This paper aims to provide optimal strategies for the DNA testing of skeletonized cranial materials. Cranial elements produced the most consistent results in Sanger sequencing using an organic purification; however, teeth were most successful for the same platform with an inorganic purification. The inverse is true for STR testing of cranial bones. Of the cranial elements, the temporal provided the most consistent results.     

Validation of Reduced Reagent Volumes in the Implementation of the Quantifiler® Trio Quantification Kit

The Quantifiler^® Trio Quantification Kit has been developed to quantify the total amount of amplifiable and human male DNA in samples and to estimate the extent of DNA degradation. To minimize the cost of DNA quantification, we evaluated kit performance using a reduced volume of reagents (1/10‐volume) using DNA samples of varying types and concentrations. Our results demonstrated concordance between the manufacturer's method and the low‐volume method for DNA quantification, DNA degradation index estimation, and human male DNA quantification. We confirmed the practical utility of the low‐volume method with 109 casework samples by evaluating short tandem repeat (STR) profiling success with respect to DNA quantity and quality. We also defined a cutoff value for DNA quantity to ensure reliable STR results. Using a reduced volume of reagents, 10 times more reactions per kit are possible; accordingly, this method reduces the cost of DNA quantification, while maintaining performance.     

The NucleoSpin DNA Clean-up XS kit for the concentration and purification of genomic DNA extracts: An alternative to microdialysis filtration

Traditionally, DNA extracts from biological evidence items have been concentrated and rinsed using microdialysis filtration units, including the Centricon^® and Microcon^® centrifugal filter devices. As an alternative to microdialysis filtration, we present an optimized method for using NucleoSpin^® XS silica columns to concentrate and clean-up aqueous extracts from the organic extraction of DNA from biological samples. The method can be used with standard organic extraction and dithiothreitol (DTT)-based differential extraction methods with no modifications to these methods prior to the concentration and clean-up step. Extracts from laboratory-prepared bloodstains, saliva and semen stains have been successfully amplified with both qPCR and STR assays. Finally, the total time to process a set of samples with the NucleoSpin^® XS column is approximately 30 min vs. approximately 1.5 h with the Centricon^® YM-100 filter device.     

The use of Polilight in the detection of seminal fluid, saliva, and bloodstains and comparison with conventional chemical-based screening tests

Biological stains can be difficult to detect at crime scenes or on items recovered from crime scenes. The use of a versatile light source may assist in their detection. The ability of Polilight to locate potential semen, saliva, and blood stains on a range of substrates and at different dilutions was tested. We also tested the use of Polilight in comparison with conventional chemical-based presumptive screening tests such as acid phosphatase (AP), Phadebas, and luminol, often used in casework for detecting potential semen, saliva, and blood stains, respectively. The Polilight was able to locate stains that were not apparent to the naked eye. The color of the material on which a stain is deposited can have an effect on the detectibility of the stain. The Polilight was found to be comparable with the AP and Phadebas tests in terms of its sensitivity. In a comparative study between the AP test and Polilight on 40 casework exhibits, one false-negative result was observed when using the Polilight. On a series of mock casework exhibits it was determined that the Polilight can be used successfully to locate saliva stains for DNA analysis. The sensitivity of luminol for detecting potential bloodstains was greater than that of Polilight; however the Polilight has particular application in instances where a bloodstain may have been concealed with paint. Overall, the Polilight is a relatively safe, simple, noninvasive, and nondestructive technique suitable for use in forensic casework.     

循环次数与体系对DNA检测灵敏度的影响

目的探讨通过增加PCR循环次数和缩小扩增体系改善DNA检测灵敏度的可行性。方法将10例无血缘关系健康志愿者精液DNA样本分别定量为50、40、30、25、20、15、10 pg/μL,分别用10、5、3μL的体系和28、30、32、34、36次循环进行扩增,用3130遗传分析仪检测15个常染色体STR基因座。结果 28次循环下,3μL体系可对40pg/μL及以上样本正确分型。20pg/μL及以上样本在10、5、3μL体系中,34次循环均可正确分型,增加到36次循环,出现非特异性谱带,无法正确分型。结论增加循环次数和缩小扩增体系在一定程度上可以提高DNA检测的灵敏度。     

Reduction of Powerplex® Y23 Reaction Volume for Genotyping Buccal Cell Samples on FTATM Cards

PowerPlex^® Y23 is a novel kit for Y‐STR typing that includes new highly discriminating loci. The Israel DNA Database laboratory has recently adopted it for routine Y‐STR analysis. This study examined PCR amplification from 1.2‐mm FTA punch in reduced volumes of 5 and 10 μL. Direct amplification and washing of the FTA punches were examined in different PCR cycle numbers. One short robotically performed wash was found to improve the quality and the percent of profiles obtained. The optimal PCR cycle number was determined for 5 and 10 μL reaction volumes. The percent of obtained profiles, color balance, and reproducibility were examined. High‐quality profiles were achieved in 90% and 88% of the samples amplified in 5 and 10 μL, respectively, in the first attempt. Volume reduction to 5 μL has a vast economic impact especially for DNA database laboratories.     

Internal Validation of Human Mitochondrial DNA Quantification Using Real‐Time PCR

The quantity of mitochondrial DNA (mtDNA) template added for amplification and subsequent dye terminator reactions is critical for obtaining quality sequence data. Validation of a human mtDNA real‐time quantitative PCR (qPCR) assay demonstrated its high degree of reproducibility and precision as well as an extremely sensitive threshold of detection (0.0001 pg/μL or approximately six human mtDNA copies/μL). A study of 35 nonprobative bone and teeth evidence samples revealed that 20 pg of mtDNA template is recommended for successful HV1 and HV2 sequence analysis; however, as little as 0.013 pg can generate a full mtDNA profile when using enhanced amplification reactions. The assay can also detect PCR inhibition and is useful for identifying samples that may benefit from re‐purification. Overall, the assay is an excellent method to quantify mtDNA and is useful for determining the best analytical approach for successful sequencing.     

Could Secondary DNA Transfer Falsely Place Someone at the Scene of a Crime?,

The occurrence of secondary DNA transfer has been previously established. However, the transfer of DNA through an intermediary has not been revisited with more sensitive current technologies implemented to increase the likelihood of obtaining results from low‐template/low‐quality samples. This study evaluated whether this increased sensitivity could lead to the detection of interpretable secondary DNA transfer profiles. After two minutes of hand to hand contact, participants immediately handled assigned knives. Swabbings of the knives with detectable amounts of DNA were amplified with the Identifiler^® Plus Amplification Kit and injected on a 3130xl. DNA typing results indicated that secondary DNA transfer was detected in 85% of the samples. In five samples, the secondary contributor was either the only contributor or the major contributor identified despite never coming into direct contact with the knife. This study demonstrates the risk of assuming that DNA recovered from an object resulted from direct contact.     

Evaluation of Commercial Kits for Dual Extraction of DNA and RNA from Human Body Fluids, ,

STR typing of DNA evidence can identify the donor with a high power of discrimination but cannot identify the tissue origin of a body‐fluid stain. Using RNA to attribute a crime scene stain to a particular tissue may aid in reconstruction efforts. With blood from 10 donors, four DNA and RNA coextraction kits were evaluated by measuring yields and STR and mRNA profiles. T tests indicated some significant differences in kit performance. The Zymo Research ZR‐Duet^? kit performed best based on average DNA (41.4 ng) and mRNA (4.07 ng) yields and was the only kit to provide complete DNA/RNA profiles for all samples. The consistency of this kit was challenged by data from additional blood and saliva donors. Further testing is advised before a superior kit is unequivocally chosen. Stand‐alone DNA or RNA purification generally offers higher yield, but coextraction may still allow successful STR profiling and tissue source identification.