Development and Evaluation of a Duo Zaire ebolavirus Real-Time RT-PCR Assay Targeting Two Regions within the Genome
Abstract
1. Introduction
2. Materials and Methods
2.1. In Silico Analysis
2.2. Mekambo EBOV RNA
2.3. EBOV RNA Transcript for LoD Calculation
2.4. RT-qPCR Assays
2.5. Specificity
2.6. Cepheid GeneXpert Open Cartridge Development
3. Results
3.1. RT-qPCR Primers and Probe Matched against EBOV Multiple Sequence Alignment: In Silico Analysis
3.2. Study Using the Mekambo EBOV RNA
3.3. LoD Calculation Using the EBOV Synthetic RNA
3.4. Specificity of the Duo Gibb + Huang Assay
3.5. Transfer of the Duo Gibb + Huang Assay onto the Flex-03 Cartridge and Validation on the GeneXpert (Cepheid)
4. Discussion
4.1. In Silico Analysis
4.2. Sensitivity of the Duo Gibb + Huang Assay
4.3. Transfer of the Duo Gibb + Huang Assay onto the Flex-03 Cartridge and Validation on the GeneXpert (Cepheid)
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Assay | Target | Amplicon | Primers/Probe | Sequence (5′-3′) |
---|---|---|---|---|
Gibb | GP | 112 bp | EBOGP1D-fwd | TGGGCTGAAAAYTGCTACAATC |
EBOGP1D-rev | GTGCCGSTATGTKCACAAAG | |||
EBOGP1DZ-Prb | FAM-CTACCAGCAGCGCCAGACGG-TAMRA | |||
Huang | NP | 80 bp | enp-F | GCAGAGCAAGGACTGATTCA |
Enp-F2 | GCAGAGCAAGGACTGATACA | |||
enp-R | ATTTTCCGTTTGATGCGAAC | |||
enp-Probe | FAMa-CAACAGCTTGGCAATCAGTTGGACA-TAMRA | |||
Panning | L | 293 bp | FiloA2.2 | AAGCCTTTCCTAGCAACATGATGGT |
FiloA2.3 | AAGCATTCCCTAGCAACATGATGGT | |||
FiloA2.4 | AAGCATTTCCTAGCAATATGATGGT | |||
FiloB | CATGTCAGTGATTATTATAACCCACCACAT | |||
Filo B-Prime | CATGTCAGTGATTATTATAAYCCACCRCAT | |||
Filo B-Ra | CATGTCAGTGACTTTTATAGCCCTCCTCAC | |||
FAMEBOSu | FAMb-TGGCACCAIACIAGTGATGATTTCGG-BHQ1 | |||
FAMEBOg | FAMb-TGGCACCACACIAGTGATGATTTTGG-BHQ1 |
Gibb et al. [9] | Huang et al. [10] | Panning et al. [11] | Standard Protocol Mekambo EBOV RNA | Standard Protocol EBOV Synthetic RNA | RealStar® Filovirus Altona Diagnostics | |
---|---|---|---|---|---|---|
Reverse Transcription | 55 °C/45 min | 50 °C/30 min | 50 °C/30 min | 50 °C/15 min | 50 °C/15 min | 55 °C/20 min |
Denaturation | 94 °C/1 min | 94 °C/5 min | 95 °C/1.5 min | 95 °C/2 min | 95 °C/2 min | 95 °C/2 min |
Cycling | 40 | 45 | 45 | 40 | 40 | 45 |
Denaturation | 94 °C/15 s | 94 °C/15 s | 95 °C/15 s | 95 °C/15 s | 95 °C/15 s | 95 °C/15 s |
Amplification | 60 °C/30 s | 60 °C/1 min | 72 °C/20 s | 60 °C/1 min | 60 °C/1 min | 72 °C/15 s |
RNA volume | 5 µL | 1–3 µL | 3 µL | 5 µL | 10 µL | 10 µL |
Total volume | 50 µL | 23–25 µL | 25 µL | 25 µL | 30 µL | 30 µL |
Primers concentration | 0.5 µM | 0.4 µM | 0.2 µM Forward 0.3 µM Reverse | 0.4 µM Gibb 0.4 µM Huang 0.2 µM Panning | 0.625 µM Gibb 0.5 µM Huang | - |
Probes concentration | 0.2 µM | 0.1 µM | 0.0667 µM | 0.16 µM Gibb and Huang | 0.25 µM Gibb | - |
0.2 µM each Panning | 0.125 µM Huang |
Genus | Virus | Acronyms | Strain (Viral Load TCID50/mL) | Reference (a) |
---|---|---|---|---|
Phlebovirus | Toscana virus | TOSV | UVE/TOSV/2014/FR/5904 (10 8,22) | 001v-02452 |
Sandfly Fever Sicilian virus | SFSV | UVE/SFSV/1943/IT/Sabin (10 6,82) | 001v-EVA77 | |
Flavivirus | Japanese encephalitis virus | JEV | UVE/JEV/2009/LA/CNS769 (10 5,57) | 001v-02217 |
Saint-Louis encephalitis virus | SLEV | UVE/SLEV/UNK/US/MSI-7 (10 4,82) | 001v-EVA128 | |
Tick-borne encephalitis virus | TBEV | UVE/TBEV/2013/FR/32.11 WT-PCR (10 8,82) | 001v-02352 | |
West-Nile virus | WNV | UVE/WNV/2008/US/R94224 (10 7,32) | 001v-02224 | |
Yellow Fever virus | YFV | UVE/YFV/UNK/XX/French neurotropic R94224 (10 7,32) | 001v-02226 | |
Usutu virus | USUV | UVE/USUV/1959/ZA/SAAR-1776 (10 5,32) | 001v-EVA138 | |
Murray Valley virus | MVEV | UVE/MVEV/UNK/AU/3329 (10 4,32) | 001v-EVA145 | |
Zika virus | ZIKV | UVE/ZIKV/1947/UG/MR766 (10 4,32) | 001v-EVA143 | |
Dengue virus | DENV-1 | UVE/DENV-1/2013/NC/CNR_17132 (10 7,57) | 001v-03151 | |
Alphavirus | Venezuelan equine encephalitis virus | VEEV | UVE/VEEV/UNK/XX/TC83 vaccine (10 9,42) | 001v-EVA1459 |
Western equine encephalitis virus | WEEV | UVE/WEEV/UNK/XX/47a (10 8,32) | 001v-EVA1479 | |
Eastern equine encephalitis virus | EEEV | UVE/EEEV/1999/XX/H178_99 (10 7,82) | 001v-EVA1480 | |
O’nyong-nyong virus | ONNV | UVE/ONNV/UNK/SN/Dakar 234 (10 4,22) | 001v-EVA1044 | |
Chikungunya virus | CHIKV | UVE/CHIKV/2017/FR/45625-26 (10 6,16) | 001v-03433 | |
Semliki Forest virus | SFV | UVE/SFV/UNK/XX/1745 (10 4,42) | 001v-02468 | |
Sindbis virus | SINV | UVE/SINV/UNK/EG/Egypt 339 (10 4,32) | 001v-02469 | |
Filovirus | Marburg virus | MBGV | Popp | n/a |
Marburg virus | MBGV | Musoke | n/a | |
Nairovirus | Crimean-Congo hemorrhagic fever virus | CCHF | Unk | n/a |
Replicate | RNA Copies/Reaction | ||||
---|---|---|---|---|---|
16.8 | 12.6 | 8.4 | 4.2 | 1.7 | |
Flex-C#1 | 37.1 | 38.7 | 39.6 | negative | negative |
Flex-C#2 | 36.3 | 36.0 | 38.9 | 38.0 | negative |
Flex-C#3 | 36.4 | 36.9 | 39.2 | negative | negative |
Flex-C#4 | 38.4 | 37.6 | 37.9 | negative | negative |
Flex-C#5 | 36.2 | 38.2 | negative | 36.7 | negative |
Flex-C#6 | 36.5 | 39.3 | negative | 38.0 | negative |
Flex-C#7 | 39.7 | 37.8 | negative | negative | negative |
Flex-C#8 | 36.1 | 36.2 | negative | negative | negative |
Mean | 37.1 (1.3) | 37.6 (1.2) | 38.9 (0.6) | 37.6 (0.6) | - |
GIBB et al. [9] | Replicate | RNA Copies/µL | ||||||
1 | 0.75 | 0.5 | 0.2 | 0.1 | 0.075 | 0.05 | ||
1 | 36.57 | 36.66 | 37.28 | 38.76 | >40 | >40 | >40 | |
2 | 36.59 | 35.99 | 37.25 | 38.43 | >40 | >40 | >40 | |
3 | 37.02 | 36.62 | 36.98 | >40 | 38.36 | >40 | >40 | |
4 | 36.47 | 37.07 | >40 | 38.27 | >40 | >40 | >40 | |
5 | 38.47 | 36.17 | >40 | 38.27 | >40 | >40 | >40 | |
6 | 37.49 | >40 | 38.54 | >40 | 38.54 | >40 | >40 | |
7 | >40 | 38.53 | 37.59 | >40 | 38.52 | 39.85 | >40 | |
Mean Ct (SD) | 37.10 (0.7) | 36.84 (0.8) | 37.53 (0.5) | 38.43 (0.2) | 38.47 (0.1) | 39.85 | >40 | |
HUANG et al. [10] | Replicate | RNA Copies/µL | ||||||
1 | 0.75 | 0.5 | 0.2 | 0.1 | 0.075 | 0.05 | ||
1 | 37.59 | >40 | 38.14 | >40 | 38.60 | >40 | >40 | |
2 | 37.92 | 38.02 | >40 | 40 | >40 | >40 | >40 | |
3 | 37.24 | 38.42 | 39.62 | >40 | 39.09 | >40 | >40 | |
4 | 37.30 | 38.48 | >40 | >40 | >40 | 37.95 | >40 | |
5 | 38.35 | 37.39 | >40 | >40 | >40 | 39.98 | >40 | |
6 | 37.89 | >40 | 38.07 | >40 | >40 | 39.98 | >40 | |
7 | 37.48 | 37.48 | 39.13 | 39.31 | 39.92 | >40 | >40 | |
Mean Ct (SD) | 37.68 (0.4) | 37.96 (0.5) | 38.74 (0.7) | 39.66 | 39.20 (0.5) | 39.30 (1.0) | >40 | |
DUO GIBB + HUANG (this study) | Replicate | RNA Copies/µL | ||||||
1 | 0.75 | 0.5 | 0.2 | 0.1 | 0.075 | 0.05 | ||
1 | NT | 32.14 | 33.04 | 34.89 | 36.40 | 32.98 | >40 | |
2 | NT | 33.01 | 32.78 | 34.93 | 36.24 | 33.17 | >40 | |
3 | NT | 32.87 | 33.10 | 34.03 | >40 | 33.80 | 38.27 | |
4 | NT | 32.83 | 33.20 | 34.88 | >40 | 33.48 | >40 | |
5 | NT | 32.77 | 36.61 | 34.67 | 37.45 | > 40 | >40 | |
6 | NT | 32.28 | 33.53 | 35.63 | 35.34 | 32.95 | >40 | |
7 | NT | 32.71 | 32.74 | >40 | 35.43 | >40 | >40 | |
8 | NT | 32.59 | 32.84 | 34.64 | 35.18 | 33.41 | >40 | |
9 | NT | 31.98 | 33.40 | 34.04 | 35.39 | 33.97 | >40 | |
10 | NT | 32.44 | 33.12 | 35.12 | >40 | 33.01 | >40 | |
11 | NT | 33.11 | 33.33 | 34.67 | 35.88 | 33.83 | >40 | |
12 | NT | 32.27 | 32.73 | 35.36 | 35.36 | 33.56 | >40 | |
13 | NT | 32.19 | 33.45 | >40 | 38.37 | 36.71 | >40 | |
14 | NT | 32.41 | 33.04 | 34.29 | >40 | 34.74 | >40 | |
15 | NT | 33.06 | 33.29 | 34.48 | 36.04 | 34.18 | >40 | |
16 | NT | 32.47 | 32.41 | 35.69 | >40 | >40 | 34.65 | |
17 | NT | 32.07 | 32.93 | 34.70 | >40 | 33.18 | >40 | |
18 | NT | 32.22 | 33.15 | >40 | 35.86 | 32.29 | >40 | |
19 | NT | 34.00 | 33.46 | 36.39 | 35.78 | 36.10 | >40 | |
20 | NT | 32.38 | 34.32 | >40 | 33.92 | 33.17 | >40 | |
21 | NT | 33.12 | 33.05 | 34.57 | 36.72 | >40 | > 40 | |
22 | NT | 32.44 | 32.55 | 34.31 | >40 | >40 | >40 | |
23 | NT | 33.08 | 32.23 | >40 | > 40 | >40 | >40 | |
Mean Ct (SD) | 32.63 (0.5) | 33.23 (0.8) | 34.85 (0.6) | 35.96 (1.0) | 33.80 (1.1) | 36.46 (1.8) |
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Share and Cite
Thirion, L.; Charrel, R.N.; Boehmann, Y.; Corcostegui, I.; Raoul, H.; de Lamballerie, X. Development and Evaluation of a Duo Zaire ebolavirus Real-Time RT-PCR Assay Targeting Two Regions within the Genome. Microorganisms 2019, 7, 652. https://doi.org/10.3390/microorganisms7120652
Thirion L, Charrel RN, Boehmann Y, Corcostegui I, Raoul H, de Lamballerie X. Development and Evaluation of a Duo Zaire ebolavirus Real-Time RT-PCR Assay Targeting Two Regions within the Genome. Microorganisms. 2019; 7(12):652. https://doi.org/10.3390/microorganisms7120652
Chicago/Turabian StyleThirion, Laurence, Remi N. Charrel, Yannik Boehmann, Iban Corcostegui, Hervé Raoul, and Xavier de Lamballerie. 2019. "Development and Evaluation of a Duo Zaire ebolavirus Real-Time RT-PCR Assay Targeting Two Regions within the Genome" Microorganisms 7, no. 12: 652. https://doi.org/10.3390/microorganisms7120652
APA StyleThirion, L., Charrel, R. N., Boehmann, Y., Corcostegui, I., Raoul, H., & de Lamballerie, X. (2019). Development and Evaluation of a Duo Zaire ebolavirus Real-Time RT-PCR Assay Targeting Two Regions within the Genome. Microorganisms, 7(12), 652. https://doi.org/10.3390/microorganisms7120652