Evaluation of four commercial quantitative real-time PCR kits with inhibited and degraded samples

Holmes, Amy S.; Houston, Rachel; Elwick, Kyleen; Gangitano, David; Hughes‐Stamm, Sheree

doi:10.1007/s00414-017-1745-9

Cited by 39 publications

(19 citation statements)

References 14 publications

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“…After increasing with longer time at 95°C, the DI of the samples treated at 120°C decreased, possibly due to the high level of degradation of both targets. DI assessment has been previously shown to be useful in providing evidence of degraded DNA [18,19]. However, as mentioned above, the DI values appear to theoretically follow a parabolic shape in a graphic, in accordance with another study that assessed small and large nuclear autosomal targets [20].…”

Section: Discussionsupporting

confidence: 80%

DNA quantitation and degradation assessment: a quantitative PCR protocol designed for small forensic genetics laboratories

et al. 2020

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DNA quantitation and degradation assessment: a quantitative PCR protocol designed for small forensic genetics laboratoriesFor over 10 years, quantitative PCR (qPCR) for DNA quantitation has been reported in forensics. However, assays have not been described for small qPCR platforms. Thus, technological advancement is not always implemented in small forensic genetics laboratories. A duplex qPCR assay is reported, using a StepOne instrument and targeting a short and a long human DNA region. This study was performed according to international validation guidelines, including sensitivity, repeatability, reproducibility, precision, accuracy, contamination assessment, known and case-type samples, and degradation studies. Characterization of the genetic markers, species specificity, and population studies had already been conducted. Moreover, case-type samples were quantified, amplified using commercial kits and the number of alleles detected was recorded. Sensitivity was shown to be 10 pg/µL. Standard curve replicates demonstrated the assay is accurate, precise, as well as fairly repeatable and reproducible. The NGM Detect kit was shown to yield higher peaks than Identifiler Plus and NGM Select for degraded samples. Moreover, quality sensors were always present and proved useful. The quantification values of the large target showed a correlation with the number of alleles detected in the STR profiles for known and casework samples. The degradation index was shown to be informative, with a value of 10 or higher indicating dropout. It is suggested that after quantitation, samples with low or degraded DNA be amplified using newer amplification kits containing quality sensors to confirm that the low-quality profile was not affected by inhibition.

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Section: Discussionsupporting

confidence: 80%

DNA quantitation and degradation assessment: a quantitative PCR protocol designed for small forensic genetics laboratories

et al. 2020

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“…This variant approach had been thoroughly validated for routine analysis in our laboratory. PCR inhibition (common at samples from weapon parts, especially from inside the barrel) as indicated by the PowerQuant® data analysis tool was noted, but disregarded for the evaluation of the autosomal DNA concentrations, as an effect on the quantification is only visible at high concentrations [ 19 ].…”

Section: Methodsmentioning

confidence: 99%

A distant relationship?—investigation of correlations between DNA isolated from backspatter traces recovered from firearms, wound profile characteristics, and shooting distance

Euteneuer

Gosch

Cachée³

et al. 2020

Int J Legal Med

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In molecular ballistics, where traces originating from the use of firearms against biological targets are investigated, “backspatter” traces are of particular importance. This biological material comprising blood and tissue from the victim is propelled back from the bullet entry site towards the direction of the shooter and can consolidate and persist on the inner and outer surfaces of the firearm, from where it can be collected and analyzed. Thus, a connection between the weapon and the victim can be established solely by molecular biological trace analysis. For the criminalistic investigation of gun-related crimes, the determination of the distance between the weapon and the victim can be of critical importance in reconstructing the circumstances of a crime. In this study, we investigated possible correlations between the shooting distance and the amount of backspatter in/on the used firearm. To this purpose, we employed a previously established skull model and performed shootings in triplicates from various distances up to 50 cm with two types of handguns (pistol and revolver). Backspatter was collected from various sampling locations, and DNA contents were quantified. A post-shooting wound channel evaluation was conducted by optical and radiological evaluation. The obtained DNA yields varied considerably between replicates from the same and from different distances. In contrast, apart from contact shots, no meaningful differences were observable in wound channel evaluations. In summary, no meaningful correlation between backspatter distribution and DNA yields, the shooting distance and the condition of the wound channel could be established.

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“…Recently, several DNA quantification kits have included degradation indexes (DI). However, the size of the DNA fragments targeted as well as the calculation of DI differs between kits [34]. As DNA profiles represent the final outcome of forensic DNA analyses, we choose to use an Integrity Index (INTI) which is calculated from electropherograms.…”

Section: Discussionmentioning

confidence: 99%

Touch DNA collection – Performance of four different swabs

Comte

Baechler

Gervaix

et al. 2019

Forensic Science International: Genetics

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A collaborative study conducted by three police forensic units, a DNA laboratory, and a forensic academic institute was undertaken in order to compare the performance of four different swabs in controlled and quasioperational conditions. For this purpose, a reference swab (Prionics cardboard evidence collection kit) currently used within the police forensic units and 3 challenger swabs (COPAN 4N6FLOQSwabs™ (Genetics variety), Puritan FAB-MINI-AP and Sarstedt Forensic Swab) were used for collecting DNA traces from previously used items (referred as "touch DNA" in this article) including on 60 collars, 60 screwdrivers and 60 steering wheels obtained from volunteers. For each comparison, the surface considered was divided into two equal components; one was sampled with the reference swab and the other with one of the three challenger swabs. This lead to a total of 360 samples. Conclusions were consistent within the four operational partners. From a practical point of view, the COPAN 4N6FLOQSwabs™ (Genetics variety) was judged the most convenient to use. Furthermore, it allowed the recovery of significantly more DNA from collars (0.65 vs 0.13 ng/μL) and steering wheels (2.82 vs 1.77 ng/μL), and a similar amount of DNA from screwdrivers (0.032 vs 0.026 ng/μL) compared with the Prionics reference swab. The two other challenger swabs provided results that were not significantly different from the reference swab, except for the Puritan swab, whose performance was significantly lower for steering wheels (0.37 vs 0.58 ng/μL). As part of a conservation study, 50 μL of a blood dilution (1/4 with PBS) was deposited on a total of 105 COPAN (Genetics and Crime Scene varieties), Prionics and Sarstedt swabs. They were stored within a cupboard at room temperature. The integrity of the recovered DNA was evaluated with NGM SElect™ DNA profiles after different time-spans ranging from 1 day to 12 months by comparing the height difference of the peaks occurring at the shortest and longest loci, respectively. DNA seemed to remain stable, except when using the COPAN 4N6FLOQSwabs™ treated with an antimicrobial agent (Crime scene variety), which resulted in significant DNA degradation. Following these tests, the COPAN 4N6FLOQSwabs™ (Genetics variety), a model with a desiccant, was selected for further testing in fully operational conditions.

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Evaluation of four commercial quantitative real-time PCR kits with inhibited and degraded samples

Cited by 39 publications

References 14 publications

DNA quantitation and degradation assessment: a quantitative PCR protocol designed for small forensic genetics laboratories

DNA quantitation and degradation assessment: a quantitative PCR protocol designed for small forensic genetics laboratories

A distant relationship?—investigation of correlations between DNA isolated from backspatter traces recovered from firearms, wound profile characteristics, and shooting distance

Touch DNA collection – Performance of four different swabs

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