Direct PCR for Rapid and Safe Pathogen Detection: Laboratory Evaluation Supporting Field Use in Infectious Disease Outbreak
Abstract
1. Introduction
2. Materials and Methods: Evaluating DRDP for Extraction-Free PCR in a Field Context
The Sample Preparation and Virus Dilution for Laboratory Developed Test
3. Results
3.1. Detection Sensitivity: DRDP vs. UTM
3.2. Effect of DRDP Buffer Volume on PCR Inhibition
3.3. Omission of Initial Heat Lysis Step
3.4. Performance in a Commercial PCR Assay
4. Discussion
5. Conclusions
6. Patents
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
UTM | Universal Transportation Medium |
DRDP | DNA/RNA Defend Pro |
PCR | Polymerase Chain Reaction |
mpox | Monkey Pox Virus |
Appendix A
Virus | Forward Primer (5′→3′) | Reverse Primer (5′→3′) | Probe (5′→3′) |
---|---|---|---|
HSV-1 | CGGCCGTGTGACACTATCG | CTCGTAAAATGGCCCCTCC | CCATACCGACCACACCGACGAACC |
HSV-2 | CGCCAAATACGCCTTAGCA | GAAGGTTCTTCCCGCGAAAT | CTCGCTTAAGATGGCCGATCCCAATC |
VZV | CACGTATTTTCAGTCCTCTTCAAGTG | TTAGACGTGGAGTTGACATCGTTT | TACCGCCCGTGGAGCGCG |
DRDP Buffer in Reaction (%) | Standard Curve Slope | Calculated Efficiency (%) |
---|---|---|
15% | −3.3 | 100.5 |
20% | −3.1 | 108.8 |
25% | −3.2 | 101.4 |
Mg Added (mM) | PCR Outcome vs. UTM | Cq Shift of DRDP (+Mg) |
---|---|---|
0 | No amplification (inhibited) | no Cq (total inhibition) |
2.5 | Delayed amplification (partial inhibition) | ~5 cycles |
5 | Delayed amplification (moderate inhibition) | ~2.5 cycles |
10 | Normal amplification (inhibition fully reversed) | 0 cycles (same as UTM) |
Mg Added (mM) | PCR Outcome vs. UTM | Ct Shift of DRDP (+Mg) |
---|---|---|
0 | No amplification (inhibited) | no Ct (total inhibition) |
5 | Delayed amplifications (partial inhibition) | ~+5 |
10 | Equivalent amplification (inhibition reversed) | ~1 |
15 | Equivalent amplification (inhibition fully reversed) | ~0.7 |
20 | Equivalent amplification (inhibition fully reversed) | ~0.7 |
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Dilution | Medium | HSV-1 Cq | HSV-2 Cq | VZV Cq |
---|---|---|---|---|
1× | UTM | 18.5 ± 0.2 | 19.2 ± 0.3 | 21.0 ± 0.3 |
1× | DRDP | 18.0 ± 0.1 | 18.7 ± 0.2 | 20.5 ± 0.2 |
10× | UTM | 22.0 ± 0.3 | 23.0 ± 0.4 | 24.0 ± 0.2 |
10× | DRDP | 21.8 ± 0.2 | 22.5 ± 0.3 | 23.0 ± 0.4 |
100× | UTM | 25.5 ± 0.5 | 27.1 ± 0.6 | 27.9 ± 0.4 |
100× | DRDP | 24.3 ± 0.4 | 25.9 ± 0.5 | 26.7 ± 0.6 |
DRDP in PCR | Inhibition (No Extra Mg2+) | Cq Shift | Inhibition (Added Extra Mg2+) |
---|---|---|---|
15% | absent | <1 | n.d. |
20% | absent | <1 | n.d. |
25% | absent | <1 | n.d. |
30% | present | <=2 | none |
35% | present | failed | none |
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Brukner, I.; Oughton, M. Direct PCR for Rapid and Safe Pathogen Detection: Laboratory Evaluation Supporting Field Use in Infectious Disease Outbreak. LabMed 2025, 2, 12. https://doi.org/10.3390/labmed2030012
Brukner I, Oughton M. Direct PCR for Rapid and Safe Pathogen Detection: Laboratory Evaluation Supporting Field Use in Infectious Disease Outbreak. LabMed. 2025; 2(3):12. https://doi.org/10.3390/labmed2030012
Chicago/Turabian StyleBrukner, Ivan, and Matthew Oughton. 2025. "Direct PCR for Rapid and Safe Pathogen Detection: Laboratory Evaluation Supporting Field Use in Infectious Disease Outbreak" LabMed 2, no. 3: 12. https://doi.org/10.3390/labmed2030012
APA StyleBrukner, I., & Oughton, M. (2025). Direct PCR for Rapid and Safe Pathogen Detection: Laboratory Evaluation Supporting Field Use in Infectious Disease Outbreak. LabMed, 2(3), 12. https://doi.org/10.3390/labmed2030012