Forensic validation of the PowerPlex® ESI 16 STR Multiplex and comparison of performance with AmpFlSTR® SGM Plus®.

We describe the forensic validation of Promega's PowerPlex® European Standard Investigator 16 (ESI 16) multiplex kit and compare results generated with the AmpFlSTR® SGM Plus® (SGM+) multiplex. ESI 16 combines the loci contained within the SGM+ multiplex with five additional loci: D2S441, D10S1248, D22S1045, D1S1656, and D12S391. A relative reduction in amplicon size of the SGM+ loci facilitates an increased robustness and amplification success of these amplicons with degraded DNA samples. Tests performed herein supplement ESI 16 data published previously with sensitivity, profile quality, mock casework, inhibitor and mixture study data collected in our laboratories in alignment with our internal technical and quality guidelines and those issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), the DNA Advisory Board (DAB) and the DNA working group (DNAWG) of the European Network of Forensic Science Institutes (ENFSI). Full profiles were routinely generated from a fully heterozygous single source DNA template using 62.5 pg for ESI 16 and 500 pg for SGM+. This increase in sensitivity has a consequent effect on mixture analyses and the detection of minor mixture components. The improved PCR chemistry confers enhanced tolerance to high levels of laboratory prepared inhibitors compared with SGM+ results. In summary, our results demonstrate that the ESI 16 multiplex kit is more robust and sensitive compared with SGM+ and will be a suitable replacement system for the analysis of forensic DNA samples providing compliance with the European standard set of STR loci.

Consideration of the probative value of single donor 15-plex STR profiles in UK populations and its presentation in UK courts.

The adoption of new 15 locus STR multiplex systems into UK forensic science would be facilitated by agreed guidelines for reporting the strength of DNA evidence using likelihood ratios. To facilitate such an agreement, we present an analysis of previously published UK allele frequencies for white Caucasian, Afro-Caribbean and Indo-Pakistani populations and investigate their effect on likelihood ratios for single donor profiles. We consider the implication of the five additional loci and suggest a procedure for reporting likelihood ratios for 15-plex STR profiles.

Severe Acute Respiratory Syndrome Coronavirus-2 Pneumonia in a Newborn Treated With Remdesivir and Coronavirus Disease 2019 Convalescent Plasma.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a novel pandemic virus. Mounting evidence supports the possibility of vertical transmission, which at the present time appears to be rare. We report a newborn with vertically acquired SARS-CoV-2 who developed acute respiratory failure and received remdesivir and coronavirus disease 2019 convalescent plasma.

Integrated microfluidic system for rapid forensic DNA analysis: sample collection to DNA profile.

We demonstrate a conduit for the delivery of a step change in the DNA analysis process: A fully integrated instrument for the analysis of multiplex short tandem repeat DNA profiles from reference buccal samples is described and is suitable for the processing of such samples within a forensic environment such as a police custody suite or booking office. The instrument is loaded with a DNA processing cartridge which incorporates on-board pumps and valves which direct the delivery of sample and reagents to the various reaction chambers to allow DNA purification, amplification of the DNA by PCR, and collection of the amplified product for delivery to an integral CE chip. The fluorescently labeled product is separated using micro capillary electrophoresis with a resolution of 1.2 base pairs (bp) allowing laser induced fluorescence-based detection of the amplified short tandem repeat fragments and subsequent analysis of data to produce a DNA profile which is compatible with the data format of the UK DNA database. The entire process from taking the sample from a suspect, to database compatible DNA profile production can currently be achieved in less than 4 h. By integrating such an instrument and microfluidic cartridge with the forensic process, we believe it will be possible in the near future to process a DNA sample taken from an individual in police custody and compare the profile with the DNA profiles held on a DNA Database in as little as 3 h.

An automated instrument for human STR identification: design, characterization, and experimental validation.

The microfluidic integration of an entire DNA analysis workflow on a fully integrated miniaturized instrument is reported using lab-on-a-chip automation to perform DNA fingerprinting compatible with CODIS standard relevant to the forensic community. The instrument aims to improve the cost, duration, and ease of use to perform a "sample-to-profile" analysis with no need for human intervention. The present publication describes the operation of the three major components of the system: the electronic control components, the microfluidic cartridge and CE microchip, and the optical excitation/detection module. Experimental details are given to characterize the level of performance, stability, reliability, accuracy, and sensitivity of the prototype system. A typical temperature profile from a PCR amplification process and an electropherogram of a commercial size standard (GeneScan 500™, Applied Biosystems) separation are shown to assess the relevance of the instrument to forensic applications. Finally, we present a profile from an automated integrated run where lysed cells from a buccal swab were introduced in the system and no further human intervention was required to complete the analysis.

Developmental validation of the PowerPlex® ESX 16 and PowerPlex® ESX 17 Systems.

We describe the developmental validation study performed on the PowerPlex(®) ESX 16 (European Standard Extended 16) and the PowerPlex(®) ESX 17 Systems, part of a suite of four new DNA profiling kits developed by Promega in response to the ENFSI and EDNAP groups' call for new STR multiplexes for Europe. The PowerPlex(®) ESX 16 System combines the 11 loci compatible with the UK National DNA Database, contained within the AmpFlSTR(®) SGM Plus(®) PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to incorporate these five new loci as mini- and midi-STRs while maintaining the loci found in the AmpFlSTR(®) SGM Plus(®) kit as standard size. The PowerPlex(®) ESX 17 System amplifies the same loci as the PowerPlex(®) ESX 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpFlSTR(®) SGM Plus(®) kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex(®) ESX 16 and ESX 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5 pg of a fully heterozygous single source DNA template. In mixture analysis, a range of 52-95% of unique minor contributor alleles was observed at 19:1 mixture ratios where only 25 pg of the minor component was present. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of information obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors.

D-dimer assays for the identification of menstrual blood.

A method to reliably distinguish menstrual blood from blood in the normal circulation (peripheral blood) would be of considerable use in the forensic analysis and interpretation of evidence in sexual offence investigations. Previous attempts to address this issue have explored microscopy, lactate dehydrogenase isozyme identification, mRNA and miRNA profiling, and identification of the products of fibrinolysis. Here, four assays for D-dimer, a terminal degradation product of fibrinolysis, are evaluated for their specificity and sensitivity in detection of menstrual blood. In addition the effect of exercise, and sample storage upon D-dimer detection was investigated. Comparison of different assays revealed significant differences in results given. Nevertheless, no positive results for D-dimer were obtained using peripheral blood, mixtures of peripheral blood with semen, or peripheral blood taken from donors after moderate exercise. D-dimer was found to be detectable in 100% of menstrual blood samples after 1 week at room temperature and also in samples stored long-term (>3 years) at -20 °C. D-dimer may be an effective, simple to use tool for the presumptive identification of menstrual blood identification.

Analysis and interpretation of mixed profiles generated by 34 cycle SGM Plus(®) amplification.

Interpretation rules for standard 28 cycle PCR have been described previously for the analysis of mixed STR profiles. In this study the same guidelines are applied to 200 mixtures derived from pairs of known donors combined in ratios of 1:1, 2:1 and 5:1 which have been profiled in duplicate with SGM Plus(®) at total inputs ranging from 1ng to 50pg. The paired profiles were distributed among 35 FSS (Forensic Science Service) reporting officers trained in low copy number (LCN) interpretation who analysed them blind following standard casework procedures. Based upon the results from initial duplicate 34 cycle PCR reactions, the reporting officers made appropriate decisions regarding the benefits of processing the reserved third aliquot. Using the combined results, 49 consensus profiles were successfully resolved into major and minor contributor peaks. This demonstrates the reliability of the interpretation rules used in standard 28 cycle SGM Plus analysis when applied to 34 cycle generated profiles by trained and experienced reporting officers. No minor contributor peaks were assigned to a major profile in the final reported results. Those profiles which did not show sufficiently marked and consistent differentiation into major and minor peaks would have been correctly resolved if the profile of one contributor (e.g. the "victim") was known.

Developmental validation of the PowerPlex(®) ESI 16 and PowerPlex(®) ESI 17 Systems: STR multiplexes for the new European standard.

In response to the ENFSI and EDNAP groups' call for new STR multiplexes for Europe, Promega(®) developed a suite of four new DNA profiling kits. This paper describes the developmental validation study performed on the PowerPlex(®) ESI 16 (European Standard Investigator 16) and the PowerPlex(®) ESI 17 Systems. The PowerPlex(®) ESI 16 System combines the 11 loci compatible with the UK National DNA Database(®), contained within the AmpFlSTR(®) SGM Plus(®) PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to reduce the amplicon size of the loci found in the AmpFlSTR(®) SGM Plus(®) kit. This design facilitates increased robustness and amplification success for the loci used in the national DNA databases created in many countries, when analyzing degraded DNA samples. The PowerPlex(®) ESI 17 System amplifies the same loci as the PowerPlex(®) ESI 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpFlSTR(®) SGM Plus(®) kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex(®) ESI 16 and ESI 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5pg of a fully heterozygous single source DNA template. This high level of sensitivity was found to impact on mixture analyses, where 54-86% of unique minor contributor alleles were routinely observed in a 1:19 mixture ratio. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of data obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors.