Anna Spas Central Forensic Laboratory of the Police Renata Zbieć-Piekarska Central Forensic Laboratory of the Police. Summary. - PDF

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Anna Spas Central Forensic Laboratory of the Police Renata Zbieć-Piekarska Central Forensic Laboratory of the Police Internal Validation of a DNA Quantification Method using the Quantifiler Human DNA Quantification

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Anna Spas Central Forensic Laboratory of the Police Renata Zbieć-Piekarska Central Forensic Laboratory of the Police Internal Validation of a DNA Quantification Method using the Quantifiler Human DNA Quantification Kit and the 7500 Real-Time PCR System with the Hid Real-Time PCR Analysis Software V 1.1 at the Biology Department of the Central Forensic Laboratory of the Police Summary The aim of the present study was the internal validation of a DNA quantification method employing Quantifiler Human DNA Quantification Kit from Applied Biosystems, used for the quantification of human total DNA, coupled with the 7500 Real-Time PCR System and the HID Real-Time PCR Analysis Software v1.1, performed at the Biology Department of the Central Forensic Laboratory of the Police. The selection of parameters relevant to the laboratory s routine casework was made based on the ENFSI DNA Working Group recommendations included in the document Recommended Minimum Criteria for the Validation of Various Aspects of the DNA Profiling Process. The assessment regarded: sensitivity, linearity, range covered by the method and precision, including intralaboratory repeatability and reproducibility. Moreover, an attempt was made to determine whether continuation of genetic analyses is rational if the 7500 Real-Time system reads are negative, i.e. no nuclear DNA is detected, while the internal positive control (IPC) results are correct. Based on conducted validation experiments, it was found that the extent to which the validated method met the predetermined acceptance criteria is satisfactory, taking into account the specificity of the tests conducted in our laboratory. However, any negative indications of 7500 Real-Time PCR system, suggesting no presence of nuclear DNA along with correct results obtained for the IPC, should be interpreted very carefully. Apparently, it is not rational to discontinue further analyses of such samples due to the availability of very sensitive new DNA amplification kits which often allow obtaining a full DNA profile. To sum up, the validation of the DNA quantification method employing the Quantifiler Human DNA Quantification Kit coupled with the 7500 Real-Time PCR System and the HID Real-Time PCR Analysis Software V1.1 was successful, therefore the method could be implemented and routinely used for the quantification of human total DNA in biological samples subject to forensic analyses conducted at the Biology Department of the Central Forensic Laboratory of the Police. Keywords internal validation, quantification of DNA, Real-Time PCR, Quantifiler Human DNA Quantification Kit Introduction Methods of molecular genetics are used in a variety of studies in the field of, e.g., ecology, evolution of organisms, oncology, forensics. Information carried by molecular markers (inherited variable alterations of DNA, RNA and proteins) allow either to draw conclusions regarding population genetics, analysis of parenthood and relationship, or to identify of individuals. Commonly used molecular biology techniques are characterized by, for instance, applicability for multiple organisms (from viruses, bacteria through humans), accuracy of the obtained results and short time of analysis. Moreover, only traces of biological material are required. Researchers in laboratories around the world constantly develop new methods intended to improve workflows and reduce the time spent on the analyses. This way, reliable information is obtained at reduced time and material consumption, as well as improved cost-effectiveness. Modern forensics follows these new trends, taking into account the increasing efficiency and throughput of the processes, along with their cost reduction. Current investigation techniques are based on advanced scientific methods, especially in the field of biology, which improves the efficiency of crime perpetrators identification. On the crime scene, PROBLEMY KRYMINALISTYKI 284(2) crime scene investigators collect trace evidence, including biological traces. Biological material that arrives in forensic biology laboratories very often contains highly degraded DNA and high levels of impurities, which may result in both small quantity and poor quality of usable DNA. The correct quantitative and qualitative assessment of DNA samples affects the final results of STR analysis. It is therefore a very important step in the analysis of biological evidence to reliably determine the quantity and quality of human DNA by assessing the potential presence of inhibitors in the analyzed sample. Initially, quantitative and qualitative DNA analysis was performed using electrophoresis on lowconcentration agarose gel, where DNA was stained with ethidium bromide, a mutagenic DNA-intercalator, or other fluorescent dyes, such as SYBR Gold, SYBR Safe or Gel Red, which fluoresce under UV light. Another method was Slot-Blot hybridization with a probe specific for human DNA, which was imprecise and required a significant amount of effort. Subsequently, a non-specific method of fluorimetric DNA quantification using the Fluoroskan Ascent instrument was implemented. The method was based on an indirect calculation of DNA quantity by measuring the fluorescence of a dye intercalating into the double strand of the nucleic acid. Taking into account that in practice, biological traces are often highly degraded, difficult archival material that, apart from human DNA, also contains e.g. bacterial DNA, fluorimetric DNA quantification was not reliable. The method is non-specific for human DNA, thus the results also included DNA of other origin. Another negative aspect of this method is the type of the dye used (PicoGreen), which is a very potent mutagen due to its physicochemical (intercalating) properties. Another commonly used method is the spectrophotometric measurement of absorbance at the wavelengths of 230, 260 and 280 nm. The obtained absorbance values are automatically converted by the instrument into concentration, as well as the purity from protein and organic contaminants. NanoDrop is one of the devices that employ this technique, used for the spectrophotometric analysis of the concentration (quantity) and purity (quality) of the isolated material (DNA and RNA). The range of concentrations at which the spectrophotometric quantification is reliable is approximately 10 3,000 ng/µl, which makes this method imprecise or insufficiently sensitive in relation to forensic tests and the quantities needed for STR analysis. Only one of the latest molecular biology techniques, Real-Time PCR, has enabled precise identification and quantification of the product. By using fluorescent techniques, it is possible to monitor product quantity in each cycle of PCR conducted. The greatest advantage of the method is its high sensitivity, as well as the speed and the efficiency of the reaction. A disadvantage of Real-Time PCR is the possibility of interruption of the reaction by certain chemical compounds called inhibitors, often naturally occurring in the tested biological samples (e.g., hemoglobin, urea, humic acids and fabric dyes), as well as by organic solvents (e.g. phenol, chloroform, alcohol) and salts (e.g. chloride) used in the extraction of nucleic acids. However, this apparently negative characteristic may bring benefits to forensic analyses, providing valuable information on the quality of the tested sample that might help to optimize the amplification by removing PCR inhibitors. Applied Biosystems has met the demands of forensic laboratories which routinely deal with difficultto-manage evidence, such as biological samples, and has created three new laboratory kits: Quantifiler Human DNA Quantification Kit, Quantifiler Y Human Male DNA Quantification Kit and Quantifiler Duo DNA Quantification Kit, which, together with the 7500 Real- Time PCR System and the Hid Real-Time PCR Analysis Software V 1.1, are used for DNA quantification in identification tests conducted in forensic laboratories (Fig.1 The instrument 7500 Real-Time PCR System and Fig. 2 The inside of the instrument 7500 Real-Time PCR System; see Polish version). In accordance with the guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM) and recommendations of the standard PN-EN ISO/IEC 17025:2005 an internal validation was performed, before the routine use of new testing method in laboratories of the Biology Department of the Central Forensic Laboratory of the Police [1, 2]. Trained laboratory personnel conducted a number of experiments in order to verify the effectiveness of the new testing method in the analyses carried out by the laboratory [3, 4]. Aim of the study The main aim of the presented study was the internal validation of a DNA quantification method employing the Quantifiler Human DNA Quantification Kit from Applied Biosystems, used for quantification of human total DNA, coupled with the 7500 Real-Time PCR System and the HID Real-Time PCR Analysis Software v1.1, performed at the Biology Department of the Central Forensic Laboratory of the Police. Moreover, an attempt was made to determine whether performing further genetic analyses is rational when the 7500 Real-Time system readings are negative, i.e. no nuclear DNA is detected, while the internal positive control (IPC) results are correct. The experiment was important from the perspective of reagent consumption. 2 PROBLEMY KRYMINALISTYKI 284(2) 2014 Materials and methods In the conducted experiments, the pooled human male genomic DNA standard of known profile and baseline concentration [200 ng/µl], provided with the Quantifiler Human DNA Quantification Kit, was used. Quantitative and qualitative DNA analysis was performed via Real-Time PCR using the Quantifiler Human DNA Quantification Kit from Applied Biosystems coupled with the 7500 Real-Time PCR System and the HID Real-Time PCR Analysis Software v1.1. Samples were amplified via multiplex PCR on the GeneAmp PCR System 9700 thermal cycler from Applied Biosystems, using kit reagents: AmpFISTR NGM PCR and AmpFISTR Y-filer PCR from Applied Biosystems and ESI 17 Power Plex from Promega. PCR products were separated using the ABI PRISM 3130XL capillary DNA sequencer from Applied Biosystems coupled with data collection software. The separation was run in the 10 Genetic Analyzer Buffer containing EDTA. The products were separated in 36-cm capillaries filled with the denaturing medium POP4TM from Applied Biosystems. Result analysis was carried out using the GeneMapper ID-X 1.1 expert software from Applied Biosystems. Description of the validated method The validated method is specific for human DNA due to the use of the TaqMan MGB probe. The used oligonucleotides contain a fluorescent reporter dye, FAM (hybridizing with the target human DNA) or VIC (hybridizing with the internal positive control), at the 5 terminus and a fluorescence quencher at the 3 terminus. After binding to a complementary sequence, the probe is degraded during the elongation step by the AmpliTaq Gold polymerase, endowed with 5 -exonuclease activity, which causes the fluorochrome to separate from the quencher. This leads to fluorescent light emission. The initial quantity of DNA in the sample is measured based on the increase in fluorescence. The stronger the fluorescence, the higher the number of copies of DNA, which is monitored in each amplification cycle. After a certain number of cycles, the level of fluorescence exceeds a predefined CT threshold indicating that the reaction kinetics has entered the exponential phase of product generation. The CT value is used for the calculation of the quantity of DNA present in the mixture at the onset of the reaction. To this end, a series of DNA standard dilutions is prepared, and the program calculates the CT values of standard quantification, which subsequently are used to draw the standard curve. After the amplification, the CT values of the tested samples are extrapolated onto the curve, which provides information on DNA concentration in the sample. The Hid Real-Time PCR Analysis Software V1.1 generates a regression line by computing the best fitting data of standard quantity. The formula that describes the CT value is (1): CT = m [log (Qty)] + b (1), where: m slope, b Y-intercept, Qty initial quantity of DNA. The resulting data are analyzed according to the following values: R2 defined as the degree of match between the standard curve regression line and the individual quantification results of tested samples. The value 1.00 indicates a perfect match between the regression curve and the value data points. R2 0.99 indicates a very close match between the standard curve and quantification values of tested samples. If R2 0.98, the correctness of microplate settings (e.g., values of standard quantity entered during the creation of document for the plate, correctness of dilution of the standards) should be checked, followed by error elimination and repeated analysis. Curve slope, indicating the efficiency of amplification for the experiment. It is important to note that slope values differ for each set of reagents: Quantifiler Human, Quantifiler Y, Quantifiler Duo, and are presented in Table 1; Y-intercept indicating the expected CT value for a sample of the quantity = 1 (e.g., 1 ng/µl). CT values of the internal positive control (IPC) are measured to check the quality and correctness of amplification, as well as the presence of inhibitors in the tested sample [5]. Description of the validated kit The Quantifiler Human DNA Quantification Kit from Applied Biosystems is used for the quantification of human total DNA. The kit amplifies a fragment of genomic DNA, htert (human telomerase reverse transcriptase gene), of 62 bp. The kit includes: mix of primers, reaction mix, DNA standard (pooled human male genomic DNA, male DNA sequence of a known profile). The mix of primers contains: pairs of synthesized primers, internal positive control (IPC) and two probes labeled with the reporter dyes FAM and VIC. The internal positive control (IPC) indicates a correct amplification. Based on the value of IPC CT, it can be inferred if the sample actually does not contain DNA or contains an inhibitor. The probe with the FAM dye hybridizes with target human DNA, while the probe with VIC dye hybridizes with the internal positive control. The reaction mix contains: AmpliTaq Gold polymerase, dntp, passive ROX dye and buffer. PROBLEMY KRYMINALISTYKI 284(2) Table 1 Slop values specific for each reagent kits recommended by the manufacturer Kit In order to minimize the chance of differences in the tests results, kits with the same serial number were used for validation tests. Results The selection of parameters relevant for routine tests was made based on the ENFSI DNA Working Group recommendations included in the document Recommended Minimum Criteria for the Validation of Various Aspects of the DNA Profiling Process. The assessed parameters included: sensitivity, linearity, range covered by the method and precision, including intralaboratory repeatability and reproducibility [2]. The obtained results are discussed below. Sensitivity tests Recommended slope values Średnia wartość slope Quantifiler Human 2.9 do Quantifiler Y 3.0 do Quantifiler Duo 3.0 do Sensitivity tests were carried out to determine the range of human DNA concentrations that are reliably measured by the 7500 Real-Time PCR system with the HID Real-Time PCR Analysis Software v1.1 using the Quantifiler Human DNA Quantification Kit. In the validation experiment, 10 dilutions of the DNA standard at the initial concentration of 200 ng/µl were prepared. Dilutions A to E were measured three times, while dilutions F to J were measured six times. A diagram of the DNA standard dilutions is presented in Table 2. The adopted acceptance criterion for sensitivity was the range of concentrations recommended by the manufacturer, i.e. 23 pg/µl to 50 ng/µl [7, 8]. The experiment indicated that obtained results of human DNA quantification within the range of 11.5 pg/ µl to 75 ng/µl are similar to the quantities of tested DNA. Analysis based on the median values led to the conclusion that the scatter of the results is relatively small, indicating that obtained values are similar. At 5.7 pg/µl and below, indications of the instrument are not repeatable. Summary of the results is shown in Table 3 and Figure 3 (The sensivity of the method for the range of dilutions/samples A-D; see Polish version) and Figure 4 (The sensivity of the method for the range of dilutions/samples E-J; see Polish version) Based on the sensitivity tests, it was found that the range of sensitivity of the method experimentally determined at the Central Forensic Laboratory of the Police is between 11.5 pg/µl and 75 ng/µl DNA and meets the current criteria for acceptance. Linearity tests The tested method of DNA quantification is characterized by a linear correlation between the initial quantity of DNA template and CT value, i.e. the cycle in which product generation enters the exponential phase. The CT value of the breakpoint cycle depends on the reaction kinetics and initial DNA concentration. Usually, the CT value indicates the moment in which product generation enters the exponential phase. In order to determine the linearity of the method, the researchers used the above results obtained in the experiment aimed to establish the sensitivity range. The linearity of the method was assessed using linear correlation coefficient R. The linear correlation coefficient R is a measure of a linear correlation between two variables (e.g., DNA concentration and CT). The closer the linear correlation coefficient R Dilution scheme of DNA standard at a concentration of 200 ng/µl Table 2 Dilution Obtainde DNA concentration [ng/µl] Dilution scheme No. of repetitions dil. A µl std. (200 ng/µl) µl H 2 dil. B µl std.1 (50 ng/µl) + 30 µl H 2 dil. C 5 10 µl dil. B (20 ng/µl) + 30 µl H 2 dil. D 1 10 µl dil. C (5 ng/µl) + 40 µl H 2 dil. E µl dil. D (1 ng/µl) + 90 µl H 2 dil. F µl std. 7 (0,068 ng/µl) µl H 2 dil. G µl dil. H (0,023 ng/µl) µl H 2 dil. H µl dil. I (0,0115 ng/µl) µl H 2 dil. I µl dil. J (0,00575 ng/µl) µl H 2 dil. J µl dil. K (0,00288 ng/µl) µl H 2 4 PROBLEMY KRYMINALISTYKI 284(2) 2014 Table 3 Sensivity of method mediane calculated concentration of DNA and desired concentration of DNA in the analyzed samples Sample A DNA [ng/μl] concentration used Obtained DNA [ng/μl] concentration Mediane calculated of DNA [ng/μl] CT Mediane CT CT IPC A A B B B C C C D D D E E E F F F F F F G G G G G G H H H H H Undetermined H I I I Undetermined I I I Undetermined J Undetermined J Undetermined J J J Undetermined J Undetermined Mediana CT IPC PROBLEMY KRYMINALISTYKI 284(2) Table 4 Values of correlation coefficient R and nature / character of the linear relationship Values of corelation coefficient R Linear relationship 0.2 no linear relationship weak relationship moderate relationship quite strong relationship 0.9 very quite strong relationship gets to 1, the stronger the linear correlation is. Table 4 presents the criteria for linear correlation coefficient assessment. The adopted acceptance criterion was correlation coefficient R 0.9, which would indicate a very strong linear correlation between the initial quantity of DNA template and the CT value. The results of linearity test obtained experimentally are presented graphically in Figure 5 (The linearity test of the method; see Polish version). The graph shows the area with a linear correlation between median calculated DNA concentration and median CT value. For the full range of DNA concentrations, samples A to J, the linear correlation coefficient R obtained in the experiments was: In view of the above, it should be noted that the target acceptance criterion for the linearity of the method was met within the full range of concentrations. Range of measurement Based on a comprehensive
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