Advertisement

The best possible result from the minimum available

Published:September 24, 2019DOI:https://doi.org/10.1016/j.fsigss.2019.09.060

      Abstract

      In accordance with the Italian DNA legislation (DPR 7 April 2016, n. 87) a number of markers lower than seven are not considered usable for inclusion in the Italian forensic DNA database. For this reason, if the forensic DNA analysis performed in our laboratory do not provide acceptable results for a number greater than or equal to seven, the profile is not indicated in the final report. Thus, having indications about the possible success of an analysis before executing it, is a crucial point in the validation process of the accreditated method used in our laboratory.
      To achieve this goal, the quantification of extracts before typing plays a fundamental role. Especially when touched objects need to be examined tens or hundreds of nanograms may be present, but also very few or no cell can be present on the object. As such, quantification of every sample can ensure the maximum efficiency and prevent repeat analyses, over-amplified samples or completely useless examination.
      Quantifiler® Trio DNA quantification kit was validated in our laboratory according the guidelines approved by the ENFSI and always used before STR amplification of forensic casework DNA samples. Our attention has focused in particular on the definition of a minimum threshold at which it is useless to carry out DNA typing defining correlation of the negative results of the quantification by the absence of genetic profiles, as a result of DNA typing. Moreover, the validation of the Savant™ SPD131DDA SpeedVac™ Concentrator to get the maximum possible yield from DNA extracts was also investigated.

      Keywords

      1. Introduction

      The challenge in analyzing inhibited, degraded or low template samples can benefit by quantification before performing the STR analysis. The Quantifiler® Trio Kit uses the TaqMan® system process for quantitative, real-time PCR amplification of multiple-copy loci in an assay providing much greater detection sensitivity than kits that use single-copy assay targets. This kit enables to simultaneously obtain a quantitative and qualitative assessment of total human and human male DNA. Using a set of primers to amplify and detected two autosomal multiple-copy target loci, known as the small autosomal (SA) and large autosomal (LA), the assay is able to detect until few human DNA molecules, providing a quality index (DI) to detect the presence of degraded DNA along with PCR inhibitors. A Y chromosome target (Y) is also included in the kit that allows the quantification of a sample's human male genomic DNA component. Moreover, an Internal PCR Control (IPC) is used to evaluate the hypothetical inhibition in the DNA extracts.
      The introduction of a new kit in a ISO17025 forensic laboratory requires an internal validation procedure. ENFSI DNA Working Group has agreed upon the minimum validation criteria as laid down in a document published in 2010 [
      Recommended Minimum Criteria for the Validation of Various Aspects of the DNA Profiling Process.
      ]. For DNA quantification the minimum parameters to be validated are:
      • -
        Repeatability : five replicates of the standard DNA.
      • -
        Reproducibility : five replicates of the standard (as in the repeatability test) quantified by another person.
      • -
        Sensitivity (limit of detection): a series of five dilutions tested in three replicates.
      • -
        Determination of the link between quantification results and the genetic profile (no quantification / DNA = no profile) due to a different sensitivity between quantification and PCR multiplexes or possible inhibition.
      Here, the results of the Quantifiler® Trio Kit validation process in combination with a QuantStudio™ 6 Flex Real-Time PCR Systems (Thermo Fischer Scientific, USA) are shown and the performance range of the system for forensic applications is evaluated. The performance of a DNA concentrator was also investigated, when the quantification results were negative.

      2. Material and methods

      Quantifiler® THP DNA Standard provided by the kit was used to prepare different DNA dilutions: 50 ng/ul (Std.1), 5 ng/ul (Std.2), 0.5 ng/ul (Std.3), 0.05 ng/ul (Std.4) and 0.005 ng/μl (Std. 5). Every dilution was run in triplicate. Quantifiler® THP DNA Standard was also used for setting the standard curve.
      Data repeatability, reproducibility and sensitivity were evaluated by two different operators as suggested by ENFSI recommendations. Standard curves obtained in duplicate from fifteen different experiments were reviewed in order to establish Slope, Y-intercept and R2 values. The correlation between the quantification results and the genetic profile was lastly verified by using PowerPlex® Fusion 6C System (Promega Corporation, USA). In real casework the standard extraction method provides a 40 μl final volume of DNA from forensic samples. We used the DNA Concentrator in simulated forensic extract when the final concentration was between 0 to 0.005 ng/μl to verify the increase in final concentration and possible contamination.

      3. Results and discussion

      Systematic underestimation of DNA concentration (>16%) was observed at 50 ng/μl for both SA and Y targets, while the LA target showed the actual concentration (˜1%). At 5 ng/μl, the DNA concentration slightly differed from the expected one for all targets (<1%), while at lower values the DNA appeared coinciding with the expected concentration. No inhibition was observed for all samples. Degradation Index was always <1. The standard curve slopes ranged between −3.1 and -3.4, indicating an optimal 100% PCR amplification efficiency (average LA= -3.34, SA= -3.26, Y= -3.28: Fig. 1). The Y-interceptors average showed a target-depending variation, with 24.61 for LA, 26.23 for SA and 25.89 for Y. R2 average value never decreased <0.993; 0.990 was only observed in one single experiment for the SA target. No reproducible results were observed by amplifying DNA samples with 0.002 ng/μl or less and no DNA profiles were obtained when real time quantification gave negative results. In some concentrated samples a significant increase in concentration was observed without contamination.
      Fig. 1
      Fig. 1Slope’s trend for the targets in fifteen standard curves.

      4. Conclusion

      The definition of a priori threshold is mandatory in the ISO17025 validation process and it provides a crucial support in evaluating the analyses workflow with an objective point of view. The validation process of the quantification system showed that the standard curve for the Quantifiler® Trio Kit is not linear in full range from 5 pg/μl to 50 ng/μl. At high DNA concentrations, the SA and Y probes appeared to be inaccurate. Thus, when a high DNA quantity is suspected in a sample, only LA target results should be considered for estimating the effective quantity of DNA; in these cases a DNA dilution prior to quantification is desirable. High DNA levels could in fact inhibit the amplification, mimicking the absence of genetic material [
      • Martins C.
      • Lima G.
      • Carvalho M.R.
      • Cainé L.
      • Porto M.J.
      DNA quantification by real-time PCR in different forensic samples.
      ]. In the present study, the concentration threshold for the PCR amplification was 0.003 ng/μl. Below this value no reproducible DNA profiles were obtained, also using the maximum template volume in the reaction (15 μl). This quantification value probably does not correlate with the diploid human genome equivalents in the template, but proves to be an efficient indicator for assessing the success of a low DNA template analysis [
      • Caragine T.
      • Mikulasovich R.
      • Tamariz J.
      • Bajda E.
      • Sebestyen J.
      • Baum H.
      • Prinz M.
      Validation of testing and interpretation protocols for low template DNA samples using AmpFlSTR Identifiler.
      ], before conducting an experiment. In these cases, however, it is possible to use a DNA concentrator to reduce the final volume by increasing the concentration of the extract.

      Declaration of Competing Interest

      None.

      References

      1. Recommended Minimum Criteria for the Validation of Various Aspects of the DNA Profiling Process.
        2010 (available in)
        • Martins C.
        • Lima G.
        • Carvalho M.R.
        • Cainé L.
        • Porto M.J.
        DNA quantification by real-time PCR in different forensic samples.
        Forensic Sci. Int.: Genetics Supplement Series. 2015; 5: e545-e546
        • Caragine T.
        • Mikulasovich R.
        • Tamariz J.
        • Bajda E.
        • Sebestyen J.
        • Baum H.
        • Prinz M.
        Validation of testing and interpretation protocols for low template DNA samples using AmpFlSTR Identifiler.
        Croat. Med. J. 2009; 50: 250-267