Advertisement

Validation of Quantifiler® Trio DNA Quantification kit in forensic samples

  • N. Gouveia
    Affiliations
    Forensics Genetic and Biology Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal

    Faculty of Sciences, University of Lisbon, Lisbon, Portugal
    Search for articles by this author
  • P. Brito
    Affiliations
    Forensics Genetic and Biology Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal

    CENCIFOR, Forensic Sciences Centre, Coimbra, Portugal
    Search for articles by this author
  • A. Serra
    Affiliations
    Forensics Genetic and Biology Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal
    Search for articles by this author
  • F. Balsa
    Affiliations
    Forensics Genetic and Biology Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal

    CENCIFOR, Forensic Sciences Centre, Coimbra, Portugal
    Search for articles by this author
  • L. Andrade
    Affiliations
    Forensics Genetic and Biology Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal

    CENCIFOR, Forensic Sciences Centre, Coimbra, Portugal
    Search for articles by this author
  • M. São Bento
    Affiliations
    Forensics Genetic and Biology Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal

    CENCIFOR, Forensic Sciences Centre, Coimbra, Portugal
    Search for articles by this author
  • P. Cunha
    Affiliations
    Forensics Genetic and Biology Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal
    Search for articles by this author
  • V. Bogas
    Affiliations
    Forensics Genetic and Biology Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal

    CENCIFOR, Forensic Sciences Centre, Coimbra, Portugal
    Search for articles by this author
  • V. Lopes
    Affiliations
    Forensics Genetic and Biology Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal

    CENCIFOR, Forensic Sciences Centre, Coimbra, Portugal
    Search for articles by this author
  • M.J. Porto
    Correspondence
    Corresponding author. Fax: +351239826132.
    Affiliations
    Forensics Genetic and Biology Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal

    CENCIFOR, Forensic Sciences Centre, Coimbra, Portugal
    Search for articles by this author
Published:September 10, 2015DOI:https://doi.org/10.1016/j.fsigss.2015.09.010

      Abstract

      The new quantification kit known as Quantifiler® Trio is introduced as more robust and sensitive compared to the Quantifiler® Duo, providing additional information in the analysis of forensic samples. Validation studies were designed to evaluate the sensitivity of the Quantifiler® Trio kit and its performance with mixture and degraded samples.

      Keywords

      1. Introduction

      Forensic samples often have low quantity and/or degraded DNA, may contain PCR inhibitors and, particularly in sexual assault samples, a high quantity of female DNA compared to male DNA. These factors can make it difficult to decide whether to continue with autosomal STR analysis, which STR kit to use and how much DNA to add to the reaction [
      • Liu J.
      Direct qPCR quantification using the Quantifiler® Trio DNA quantification kit.
      ,
      • Vernarecci S.
      • et al.
      Quantifiler® Trio Kit and forensic samples management: a matter of degradation.
      ]. The Quantifiler® Trio DNA Quantification kit helps overcome these obstacles through enhanced targets that have been designed to determine the quantity of DNA in highly compromised forensic samples, enabling to assess the level of DNA degradation through a new degradation index functionality which was not possible to achieve with the Quantifiler® Duo [
      • Applied Biosystems
      Quantifiler® Trio DNA Quantification Kit User Guide.
      ].
      The aim of this study is to test the sensitivity of the Quantifiler® Trio kit and its performance with mixture and degraded samples.

      2. Materials and methods

      The standard curve was generated through tenfold serial dilutions with the 5 standard concentrations ranging from 50 ng/μL to 0.005 ng/μL.
      The sensitivity of the Quantifiler® Trio was tested using serial dilutions of 6 DNA controls to obtain concentrations up to a minimum value of 0.005 ng/μL, which represent the smallest point of the standard curve.
      In order to test the detection limit of minor contributor, mixture samples containing 1 ng/μL of male DNA and 1 ng/μL of female DNA were prepared. The ratio of male to female DNA was 1:1, 1:2, 1:5, 1:10, 1:15, 1:20, 1:40 and 1:50.
      Casework samples were studied to evaluate the impact of different levels of degradation on genetic profiles determined.
      All of the samples were quantified according to the manufacturer’s instructions on the 7500 Real-Time PCR System and analyzed using the HID Real-Time PCR Analysis Software v1.2. GlobalFiler® PCR Amplification kit was used in amplified samples, normalized to a standard concentration of around 1 ng/μL.

      3. Results

      DNA quantities obtained with the Quantifiler® Trio were very similar to the expected quantities for both controls and dilutions (Table 1).
      Table 1Quantification results of controls and respective dilutions obtained with Quantifiler® Trio.
      SamplesExpected quantity (ng/μL)Measured quantity (ng/μL)SamplesExpected quantity (ng/μL)Measured quantity (ng/μL)
      Control 1107.6326Control 2108.8824
      Control 1 Dil-10.20.1372Control 2 Dil-10.20.1122
      Control 1 Dil-20.020.0131Control 2 Dil-20.020.0133
      Control 1 Dil-30.010.0064Control 2 Dil-30.010.0092
      Control 1 Dil-40.0050.0038Control 2 Dil-40.0050.0028
      Control 30.10.0863Control 40.10.0728
      Control 3 Dil-10.050.0433Control 4 Dil-10.050.0261
      Control 3 Dil-20.0250.0178Control 4 Dil-20.0250.0128
      Control 3 Dil-30.01250.0071Control 4 Dil-30.01250.0071
      Control 3 Dil-40.006250.0026Control 4 Dil-40.006250.0063
      Control 521.6023Control 622.8847
      Control 5 Dil-10.20.1504Control 6 Dil-10.20.1477
      Control 5 Dil-20.020.0136Control 6 Dil-20.020.0127
      Control 5 Dil-30.010.0080Control 6 Dil-30.010.0052
      Control 5 Dil-40.0050.0036Control 6 Dil-40.0050.0029
      Results for the mixture samples are summarized in Table 2. The measured male to female DNA ratio was very close to the expected values for all ratios tested. Amplification results showed that it was possible to obtain a complete female profile in all ratios, whereas a male profile was not visible from a ratio greater than 1:20.
      Table 2Quantification and amplification results of mixture samples obtained with Quantifiler® Trio and GlobalFiler® kits, respectively. The concentration of female DNA can be approximated by subtracting the male DNA concentration from the human DNA concentration.
      Expected M:F ratioQuantifiler® Trio KitGlobalFiler® Kit
      Human DNA (ng/μL)Male DNA (ng/μL)Female DNA (ng/μL)Measured M:F ratioFemale profileMale profile
      1:10.98870.52470.46401:0.88CompleteComplete
      1:21.05630.36900.68731:1.86CompleteComplete
      1:51.04920.17620.87301:4.96CompleteComplete
      1:101.09410.09550.99861:10.46Complete23/24
      1:151.08350.05771.02581:17.79Complete19/24
      1:201.03800.04650.99151:21.33Complete14/24
      1:400.67430.01910.65521:34.22Complete
      1:500.88810.01570.87241:55.43Complete
      Table 3 contains results for the casework samples. Samples A and B, C and D, E and F have similar concentrations but different degradation index, while samples G and H have different concentrations but similar level of degradation. For a similar concentration, a higher degradation index resulted in a lower quality of genetic profile. On the other hand, more concentrated samples allowed a more complete profile where the level of degradation was similar. Finally, samples I and J are an example of the impact of extreme degradation in obtaining genetic profiles.
      Table 3Quantification and amplification results of casework samples obtained with Quantifiler® Trio and GlobalFiler® kits, respectively. (a – Degradation index was not calculated due to a significant degradation of the samples.)
      SamplesQuantifiler® Trio KitGlobalFiler® Kit
      Human DNA (ng/μL)Degradation

      Index
      Profile
      A0.03562.3012Complete
      B0.034614.553915/24
      C0.01281.0668Complete
      D0.017214.687015/24
      E0.02402.1259Complete
      F0.022311.53308/24
      G0.04057.414413/24
      H0.08736.982821/24
      I0.2094a12/24
      J0.0290a6/24

      4. Discussion and conclusion

      Sensitivity studies showed that the DNA quantities obtained with Quantifiler® Trio were very similar to the expected quantities for each control and dilution. The measured male to female ratios were in agreement with the expected ratios revealing the ability of the Quantifiler® Trio to detect small quantities of male DNA in the presence of high amounts of female DNA. Furthermore, it was possible to conclude that a male profile was not visible from a ratio greater than 1:20 and so Y-STR analysis is recommended in these samples. Also, the results demonstrated that the degradation index information can be used to adjust the quantity of DNA in PCR and predict the quality of the expected profiles.
      All of these results showed that Quantifiler® Trio is more informative and sensitive in comparison with Quantifiler® Duo.

      Conflict of interest

      None.

      References

        • Liu J.
        Direct qPCR quantification using the Quantifiler® Trio DNA quantification kit.
        Forensic Sci. Int. Genet. 2014; 13: 10-19
        • Vernarecci S.
        • et al.
        Quantifiler® Trio Kit and forensic samples management: a matter of degradation.
        Forensic Science International: Genetics. 2015; 16: 77-85
        • Applied Biosystems
        Quantifiler® Trio DNA Quantification Kit User Guide.
        Life Technologies, 2014