According to guidelines of the quality assurance standards for the forensic DNA testing laboratories, prior to introducing an existing DNA analysis procedure, reliability of the procedure has to be demonstrated by carrying out internal validation. In order to introduce a sensitive and accurate DNA quantification method in our laboratory, a validation study of the 7500 Real Time PCR System (Applied Biosystems) using the QUANTIFILER Human DNA Quantification Kit (Applied Biosystems) was performed. Here we report the results and the experience we have gained during this internal validation study.
Accurate DNA quantification in forensic casework samples is a crucial contributing factor to successful STR genotyping. Advances in forensic DNA analysis have resulted in the advent of Real Time PCR as a reliable and sensitive DNA quantification method using small sample amount. The ABI 7500 Real Time PCR Sequence Detection System is a robust, promising tool to perform DNA quantification, with high automation feasibility.
2. Materials and methods
2.1 Sources and preparation of genomic DNA used in testing
Human genomic DNA was extracted from single source and casework samples using a commercial purification kit (Nucleospin® by Macherey-Nagel). In total, 47 samples were studied in order to evaluate reproducibility, precision and accuracy, sensitivity and performance of mixture samples.
2.2 Quantifiler human kit assays
Assays were performed according to the protocol specified in the User's Manual. Duplicate reactions of each quantification standard and triplicate reactions of each analysis sample were run.
2.3 PowerPlex® (Promega Corp.) PCR Amplification Kits genotyping assays
STR analysis was performed using the standard protocol for the PowerPlex® 16 and PowerPlex® Y, PCR Amplification Kits. PowerPlex® reactions were run on an ABI PRISM 3100 Genetic Analyzer and analyzed with GeneMapper® v.3.1 (ABI).
2.4 DNA quantification of DNA samples of known concentrations
Standard DNA sample for Quantiblot, (ABI) and the 9948 male DNA sample (Promega), were tested in triplicate in three different sets using the Quantifiler system, in order to assess probable differences between given and experimentally determined quantities.
2.5 Reproducibility, precision and accuracy studies
Thirty-seven single source and casework samples and one standard DNA sample supplied by ABI, were tested in triplicate reactions in each plate. The assay was repeated twice.
2.6 Mixture studies
Mixture ratios of 1:1, 1:0.5, 1:0.1 and 1:0.05 female to male, male to female, and male to male were prepared up to a total DNA concentration of 2 ng/μL. Each specific ratio was also subjected in serial dilutions so that the total DNA concentration of the mixture would be 1 ng/μL, 0.5 ng/μL, 0.25 ng/μL, 0.125 ng/μL, 0.06 ng/μL and 0.03 ng/μL. Triplicate reactions were set for every sample of different ratio and concentration (112 samples in total).
2.7 Sensitivity studies and stochastic effects
Serial dilutions of eight samples were set so that various concentrations (0.25, 0.06, 0.03, 0.015) were tested down to 0.007 ng/μL. Each sample was tested in triplicate reactions. Eight reagent blanks, coming from various DNA purification protocols, were also tested to evaluate possible stochastic effects.
3.1 DNA quantification of samples of known DNA concentration
Our data showed small to medium differences between the Quantiblot standard DNA concentrations given by suppliers and those experimentally determined with Quantifiler. Different concentrations – depending on the batch – were also determined for the second DNA standard used in our study.
3.2 Precision and accuracy
The accuracy and precision of the Quantifiler kit assays is a function of the measurement of CT in amplification reactions. An assessment of this parameter measurement was made by running triplicate reactions of the quantification standard dilutions typically used in the Quantifiler kit assays, repeated in two different sets. The results of the CT precision experiments are shown in Table 1. Our results provide a useful indication of CT stability, which was consistent from reaction to reaction and day to day.
Table 1Quantifiler kit CT run to run precision results
|DNA concentration (ng/μL)||Mean CT||S.D. CT|
Our results demonstrate stability of measured CT values and consequent quantification for thirty-eight samples examined.
The following step of this study was the setting up of STR amplification reactions using the DNA amounts determined by Quantifiler kit assays. All samples tested, produced full, balanced profiles with no off scale allele peaks or ambiguous stutter bands (data not shown).
3.4 Mixture studies
The main goal of the mixture study validation was to determine the ability (limits) of the system to detect the minor contributor by STR genotyping of a mixed stain. Our autosomal STR results claim that mixed stains with ratio equal to or less than 1:0.1 could be considered as single source DNA stains with stutter bands or increased background depending on the initial DNA concentration (see Fig. 1). STR amplification of the Y chromosome, in samples containing male DNA as a minor contributor, succeeded in producing Y STR male profile in samples of DNA concentration 2–0.25 ng/μL (see Fig. 2). Samples with less DNA failed to produce full Y STR profile.
The results of sensitivity testing showed that all concentrations of each DNA tested gave positive assay results and the resulting plots of CT vs. DNA concentration showed all data points to be located along a straight line, indicating valid results (Fig. 3). Samples of 16 pg/μL DNA concentration gave correct STR genotype for all samples tested. Samples with less than 16 pg/μL DNA concentration failed to give full DNA profiles and drop outs were also observed.
Quantification of all the reagent blanks showed that they did not contain detectable DNA.
The internal validation study that was conducted for the evaluation of Quantifiler Human DNA Quantification Kit (Applied Biosystems) has demonstrated that it is a reliable system with high sensitivity, which gives accurate and reproducible results.
Accepted: December 17, 2007
Received in revised form: December 17, 2007
Received: August 25, 2007
© 2008 Published by Elsevier Inc.