Comparison of the Plexor® HY System, Quantifiler® and Quantifiler Duo® kits using the Roche LightCycler 480 System and the ABI 7900 real time PCR instrument

Bulander, Nicole; Rolf, Burkhard

doi:10.1016/j.fsigss.2009.08.154

Cited by 6 publications

(2 citation statements)

References 4 publications

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“…As reported in Table , the coefficients of variation (CVs %) of the assessments performed by the same kits in different labs are low (no more than 19%). This finding confirms that the poor concordance of the qPCR assays, using different kits, is mainly due to the different chemistry and bio‐molecular design of the kits themselves (e.g., multicopy vs. single‐copy methods, sequence and length of the probes, sequence and length of the target sequence) . In addition, also differences in the human DNA calibration standards included in the commercial kits are certainly involved .…”

Section: Resultssupporting

confidence: 53%

The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics

et al. 2014

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The role of DNA damage in PCR processivity/fidelity is a relevant topic in molecular investigation of aged/forensic samples. In order to reproduce one of the most common lesions occurring in postmortem tissues, a new protocol based on aqueous hydrolysis of the DNA was developed in vitro. Twenty-five forensic laboratories were then provided with 3.0 μg of a trial sample (TS) exhibiting, in mean, the loss of 1 base of 20, and a molecular weight below 300 bp. Each participating laboratory could freely choose any combination of methods, leading to the quantification and to the definition of the STR profile of the TS, through the documentation of each step of the analytical approaches selected. The results of the TS quantification by qPCR showed significant differences in the amount of DNA recorded by the participating laboratories using different commercial kits. These data show that only DNA quantification "relative" to the used kit (probe) is possible, being the "absolute" amount of DNA inversely related to the length of the target region (r(2) = 0.891). In addition, our results indicate that the absence of a shared stable and certified reference quantitative standard is also likely involved. STR profiling was carried out selecting five different commercial kits and amplifying the TS for a total number of 212 multiplex PCRs, thus representing an interesting overview of the different analytical protocols used by the participating laboratories. Nine laboratories decided to characterize the TS using a single kit, with a number of amplifications varying from 2 to 12, obtaining only partial STR profiles. Most of the participants determined partial or full profiles using a combination of two or more kits, and a number of amplifications varying from 2 to 27. The performance of each laboratory was described in terms of number of correctly characterized loci, dropped-out markers, unreliable genotypes, and incorrect results. The incidence of unreliable and incorrect genotypes was found to be higher for participants carrying out a limited number of amplifications, insufficient to define the correct genotypes from damaged DNA samples such as the TS. Finally, from a dataset containing about 4500 amplicons, the frequency of PCR artifacts (allele dropout, allele drop-in, and allelic imbalance) was calculated for each kit showing that the new chemistry of the kits is not able to overcome the concern of template-related factors. The results of this collaborative exercise emphasize the advantages of using a standardized degraded DNA sample in the definition of which analytical parameters are critical for the outcome of the STR profiles.

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Section: Resultssupporting

confidence: 53%

The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics

et al. 2014

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“…Other studies have demonstrated that DNA typing profiles may be obtained from samples that are below the detection limit of Quantifiler (although newer kits, including Plexor HY Õ , have been shown to have a lower detection limit). 11 Ideally, though, the quantity of DNA recovered from the evidence can be determined using the standard operating procedure so that the appropriate amount of sample can be inputted from DNA typing. Accordingly, the goal of this study was to determine which DNA extraction method yielded the highest quantity of DNA.…”

Section: Discussionmentioning

confidence: 99%

Assessing DNA recovery from chewing gum

2016

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The purpose of this study was to evaluate which DNA extraction method yields the highest quantity of DNA from chewing gum. In this study, several popular extraction methods were tested, including Chelex-100, phenol-chloroform-isoamyl alcohol (PCIA), DNA IQ, PrepFiler, and QIAamp Investigator, and the quantity of DNA recovered from chewing gum was determined using real-time polymerase chain reaction with Quantifiler. Chewed gum control samples were submitted by anonymous healthy adult donors, and discarded environmental chewing gum samples simulating forensic evidence were collected from outside public areas (e.g., campus bus stops, streets, and sidewalks). As expected, results indicate that all methods tested yielded sufficient amplifiable human DNA from chewing gum using the wet-swab method. The QIAamp performed best when DNA was extracted from whole pieces of control gum (142.7 ng on average), and the DNA IQ method performed best on the environmental whole gum samples (29.0 ng on average). On average, the QIAamp kit also recovered the most DNA from saliva swabs. The PCIA method demonstrated the highest yield with wet swabs of the environmental gum (26.4 ng of DNA on average). However, this method should be avoided with whole gum samples (no DNA yield) due to the action of the organic reagents in dissolving and softening the gum and inhibiting DNA recovery during the extraction.

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Does zero really mean nothing?—first experiences with the new PowerQuantTM system in comparison to established real-time quantification kits

Poetsch¹,

Konrad²,

Helmus³

et al. 2016

Int J Legal Med

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DNA quantification is an important step in the molecular genetic analysis of a forensic sample, hopefully providing reliable data on DNA content for a subsequent generation of reproducible STR profiles for identification. For several years, this quantification has usually been done by real-time PCR protocols and meanwhile a variety of assays are commercially available from different companies. The newest one is the PowerQuant(TM) assay by Promega Inc. which is advertised with the promise that a determined DNA concentration of 0 ng/μl in a forensic sample guarantees the impossibility to achieve true STR results, thus allowing to exclude such samples from STR analysis to save time and money. Thus, the goal of this study was to thoroughly verify the quantification step with regard to its suitability as a screening method. We have evaluated the precision and reliability of four different real-time PCR quantification assays by systematically testing DNA dilutions and forensic samples with various DNA contents. Subsequently, each sample was subjected to the Powerplex® ESX 17 fast kit to determine a reliable cutoff level for exclusion of definitely negative samples from STR analysis. An accurate quantification of different cell line DNA dilutions was not possible with any kit. However, at least the PowerQuant(TM) assay provided suitable data analyzing forensic samples, whereas in other systems up to 46 % of negative samples still displayed reliable STR analysis results. All in all, the PowerQuant(TM) assay represents a big step forward, but the evaluation of real-time PCR quantification results has still to be done with great care.

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Comparison of the Plexor® HY System, Quantifiler® and Quantifiler Duo® kits using the Roche LightCycler 480 System and the ABI 7900 real time PCR instrument

Cited by 6 publications

References 4 publications

The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics

The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics

Assessing DNA recovery from chewing gum

Does zero really mean nothing?—first experiences with the new PowerQuantTM system in comparison to established real-time quantification kits

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