A Custom qPCR Assay to Simultaneously Quantify Human and Microbial DNA
Abstract
:1. Introduction
2. Materials and Methods
2.1. qPCR Assays
2.2. Reproducibility Study
2.3. Bacterial DNA Amplification
2.4. Dataset Creation
2.5. DNA Extraction of Surface Swabs
2.6. Data Analysis
3. Results and Discussion
3.1. Multiplex qPCR Assay Development and Reproducibility Study
3.2. Bacterial DNA Amplification
3.3. Dataset Creation
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Assay | Quantifiler® HP | Bacterial Single-Plex | Multiplex |
---|---|---|---|
Number of Targets | Two | One | Three |
Master Mix components | HP Reaction and Primer Mixes | 10× buffer containing Mg2+, dNTPs, BSA, Hot Start Taq, bacterial primers and probe, PCR grade water | HP Reaction and Primer Mixes, bacterial primers and probe, PCR grade water |
Final volume | 20 μL | 30 μL | 25 μL |
Cycling parameters | Holding stage at 95 °C for 2 min, 40 cycles of 95 °C for 9 s, 60 °C for 30 s | Initial stage at 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s, 52 °C for 1 min, 72 °C for 1 min | Holding stage at 95 °C for 2 min, 40 cycles of 95 °C for 9 s, 60 °C for 30 s |
Sample Type | N | Description |
---|---|---|
Keyboard | 8 | Enter, shift, “e,” “r,” “t,” “a,” “s,” “d,” “f,” “g,” “h,” “j,” “k,” “l,” ctrl, and fn keys |
Computer Mouse | 8 | Sides, top, and roller |
Smart Watch | 10 | Entire band, back of screen |
Elevator button | 4 | Buttons and immediate outside area |
Cleaning supplies | 4 | Upper 1/3 of handle |
Sebum deposit | 10 | Forehead of donor was rubbed; fingertip was placed on desktop |
Dispenser | 4 | Pump of each one hand sanitizer dispenser and three hand soap dispensers |
Fridge Handle | 2 | Entirety of handle of fridge |
Dresser Drawer | 2 | Entirety of handle for each drawer of dresser |
Light Switch | 3 | Entirety of switch, not panel |
TV Remote | 2 | Front, back, side, and in between buttons |
Sample Type | N | Average Human DNA Quantity (ng/μL) | Standard Deviation | Avg Bacterial DNA Quantity (ng/μL) | Standard Deviation | Average IPC (CT Value) |
---|---|---|---|---|---|---|
Keyboard | 8 | 0.0075 | 0.006 | 2.21 × 10−7 | 1.40 × 10−7 | 29.7 |
Mouse | 8 | 0.076 | 0.183 | 2.52 × 10−7 | 1.42 × 10−7 | 29.5 |
Smart Watch | 10 | 0.357 | 0.426 | 1.14 × 10−5 | 1.28 × 10−5 | 29.7 |
Elevator button | 4 | 0.012 | 0.011 | 2.13 × 10−7 | 1.39 × 10−7 | 29.7 |
Cleaning supplies | 4 | 0.007 | 0.005 | 4.40 × 10−7 | 5.15 × 10−7 | 29.6 |
Sebum deposit | 10 | 0.059 | 0.060 | 4.76 × 10−7 | 5.23 × 10−7 | 29.8 |
Dispensers | 4 | 0.016 | 0.013 | 4.71 × 10−7 | 3.43 × 10−7 | 29.4 |
Fridge Handle | 2 | 0.039 | 0.049 | 8.34 × 10−8 | 9.52 × 10−8 | 29.9 |
Dresser Drawer | 2 | 0.056 | 0.020 | 2.45 × 10−7 | 1.22 × 10−7 | 29.8 |
Light Switch | 3 | 0.097 | 0.057 | 2.83 × 10−7 | 1.79 × 10−7 | 29.8 |
TV Remote | 2 | 0.165 | 0.040 | 3.98 × 10−7 | 4.63 × 10−7 | 29.7 |
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Foster, M.; McElhoe, J.A.; Holland, M.M. A Custom qPCR Assay to Simultaneously Quantify Human and Microbial DNA. Genes 2024, 15, 1129. https://doi.org/10.3390/genes15091129
Foster M, McElhoe JA, Holland MM. A Custom qPCR Assay to Simultaneously Quantify Human and Microbial DNA. Genes. 2024; 15(9):1129. https://doi.org/10.3390/genes15091129
Chicago/Turabian StyleFoster, Miriam, Jennifer A. McElhoe, and Mitchell M. Holland. 2024. "A Custom qPCR Assay to Simultaneously Quantify Human and Microbial DNA" Genes 15, no. 9: 1129. https://doi.org/10.3390/genes15091129