Looking into the Quantification of Forensic Samples with Real-Time PCR
<p>Diagrammatic representation of the forensic genetic analysis process used by the Forensic Genetic Unit of AOU Careggi. The method is accredited according to the ISO/IEC 17025 standard by the Italian Accreditation Body, Accredia (Lab nr. 1268) with the denomination “DNA typing for human identification, mixed stains, Y-STR, paternity and kinship testing (genetic profile)”.</p> "> Figure 2
<p>IPC value in real experiments on casework samples. Complete inhibition was observed in only 2 out of 757 DNA samples. The synthetic internal PCR control (IPC) template DNA is present at a consistent concentration across all reactions on a plate. Therefore, the IPC Ct should be relatively constant in typical reactions if PCR inhibitors and/or higher concentrations of DNA are not present in the extract. The use of the IPC system helps us distinguish between true negative sample results and reactions affected by the presence of PCR inhibitors, the assay setup, or a chemistry or instrument failure.</p> "> Figure 3
<p>(<b>a</b>) Linearity for the standard curve for the Quantifiler™ HP and Trio Kit is from 5 pg/μL to 100 ng/μL as declared by the manufacturer. Correlation between expected and observed quantification of DNA control. (<b>b</b>) Percentage correlation between expected and observed quantification of DNA control. Each point on the graph was obtained from the average quantification value of the five points used for constructing the standard curve in a typical quantification reaction (50, 5, 0.5, 0.05, and 0.005 ng/µL) measured in quintuplicate. Linearity for the standard curve for the Quantifiler™ HP and Trio Kit is from 5 pg/μL to 100 ng/μL as declared by the manufacturer.</p> "> Figure 4
<p>(<b>a</b>) The standard curve is a graph of the Ct of quantification standard reactions plotted against the starting quantity of the standards. The software calculates the regression line by calculating the best fit with the quantification standard data points. The regression line formula has the form Ct = m [log (Qty)] + b, where m is the slope, b is the Y–intercept, and Qty is the starting DNA quantity. The slope indicates the PCR amplification efficiency for the assay. A slope of 3.3 indicates 100% amplification efficiency. The figure shows the slopes determined in twenty different experiments. (<b>b</b>) R<sup>2</sup> value—measure of the closeness of fit between the standard curve regression line and the individual Ct data points of quantification standard reactions. A value of 1.00 indicates a perfect fit between the regression line and the data points. The figure shows the R<sup>2</sup> observed in twenty different experiments. (<b>c</b>) The Y–intercept indicates the expected Ct value for a sample with Qty = 1 (for example, 1 ng/μL). The figure shows the Y-intercept results in twenty different experiments for small, Y, and large probes.</p> "> Figure 4 Cont.
<p>(<b>a</b>) The standard curve is a graph of the Ct of quantification standard reactions plotted against the starting quantity of the standards. The software calculates the regression line by calculating the best fit with the quantification standard data points. The regression line formula has the form Ct = m [log (Qty)] + b, where m is the slope, b is the Y–intercept, and Qty is the starting DNA quantity. The slope indicates the PCR amplification efficiency for the assay. A slope of 3.3 indicates 100% amplification efficiency. The figure shows the slopes determined in twenty different experiments. (<b>b</b>) R<sup>2</sup> value—measure of the closeness of fit between the standard curve regression line and the individual Ct data points of quantification standard reactions. A value of 1.00 indicates a perfect fit between the regression line and the data points. The figure shows the R<sup>2</sup> observed in twenty different experiments. (<b>c</b>) The Y–intercept indicates the expected Ct value for a sample with Qty = 1 (for example, 1 ng/μL). The figure shows the Y-intercept results in twenty different experiments for small, Y, and large probes.</p> "> Figure 5
<p>The relative Bland–Altman plot. The mean of the relatives’ differences is (–0.02 ± 0.02).</p> "> Figure 6
<p>The relative Bland–Altman plot for quantity values lower than 0.05 ng/μL. The mean of the relative difference for these values is 0.02 with a confidence interval of 0.02.</p> "> Figure 7
<p>The Figure shows raw data amplification results obtained by using DNA samples from casework obtained from subungual grooves from a known individual. Each sample was obtained by rubbing a swab into the subungual sulcus. 4 = left ring finger; 7 = right index finger; 8-1 = medium right; 9 = right ring finger. The concentration indicated in the boxes for each sample was that before the concentration phase.</p> "> Figure 8
<p>Amplification results using Globalfiler™ PCR Amplification kit. The figure shows the electropherograms for the same samples as in <a href="#genes-15-00759-f007" class="html-fig">Figure 7</a>, compared to the reference DNA from a saliva sample of the donor (only the blue channel is shown). Although some “drop-in” and “drop-out” phenomena were observed in some loci, the donor’s genetic profile is evident for almost all DNA markers.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. DNA Extraction
2.2. Analytical Phase of the Quantification Method
2.3. Using Quantifiler™ Trio to Quantify Dilutions of DNA Standards
2.4. Reanalysis of the QuantiFiler™ Trio
Standard Versus Sample/Sample Versus Standard
2.5. Concentration of Extracts from Forensic Samples
2.6. Amplification
3. Results
3.1. Extraction Method
3.2. Performance of the Quantifiler™ Trio Kit
3.3. Standard Versus Sample/Sample Versus Standard Results
3.4. Genotyping of Concentrated Samples
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
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Standard | Concentration (ng/µL) | Volume | Dilution Factor |
---|---|---|---|
St. 1 | 50.000 | 10 µL [100 ng/µL stock] + 10 µL QuantiFiler™ THP DNA dilution buffer | 2X |
St. 2 | 5.000 | 10 µL [Std. 1] + 90 µL QuantiFiler™ THP DNA dilution buffer | 10X |
St. 3 | 0.500 | 10 µL [Std. 2] + 90 µL QuantiFiler™ THP DNA dilution buffer | 10X |
St. 4 | 0.050 | 10 µL [Std. 3] + 90 µL QuantiFiler™ THP DNA dilution buffer | 10X |
St. 5 | 0.005 | 10 µL [Std. 4] + 90 µL QuantiFiler™ THP DNA dilution buffer | 10X |
Theoretical Input in ng/µL | ||||||
---|---|---|---|---|---|---|
50 | 5 | 0.5 | 0.05 | 0.005 | ||
probe | ||||||
Large autosomal | average | 50.504 | 5.435 | 0.510 | 0.053 | 0.005 |
standard deviation | 5.503 | 0.227 | 0.028 | 0.003 | 0.001 | |
% difference (BIAS) | 1.008 | 8.692 | 2.077 | 5.188 | 6.312 | |
Small autosomal | average | 41.664 | 4.963 | 0.498 | 0.055 | 0.006 |
standard deviation | 13.068 | 0.318 | 0.090 | 0.003 | 0.001 | |
% difference (BIAS) | −16.672 | −0.730 | −0.309 | 10.492 | 15.734 | |
Y | average | 41.237 | 4.934 | 0.474 | 0.057 | 0.005 |
standard deviation | 7.154 | 0.517 | 0.062 | 0.009 | 0.001 | |
% difference (BIAS) | −17.526 | −1.321 | −5.145 | 13.536 | 1.384 |
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Ricci, U.; Ciappi, D.; Carboni, I.; Centrone, C.; Giotti, I.; Petti, M.; Alice, B.; Pelo, E. Looking into the Quantification of Forensic Samples with Real-Time PCR. Genes 2024, 15, 759. https://doi.org/10.3390/genes15060759
Ricci U, Ciappi D, Carboni I, Centrone C, Giotti I, Petti M, Alice B, Pelo E. Looking into the Quantification of Forensic Samples with Real-Time PCR. Genes. 2024; 15(6):759. https://doi.org/10.3390/genes15060759
Chicago/Turabian StyleRicci, Ugo, Dario Ciappi, Ilaria Carboni, Claudia Centrone, Irene Giotti, Martina Petti, Brogi Alice, and Elisabetta Pelo. 2024. "Looking into the Quantification of Forensic Samples with Real-Time PCR" Genes 15, no. 6: 759. https://doi.org/10.3390/genes15060759