Fast, Sensitive and Specific Detection of Thailand orthohantavirus and Its Variants Using One-Step Real-Time Reverse-Transcription Polymerase Chain Reaction Assay
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethics Approval
2.2. Viral Stock and Tissue Collection
2.3. Design of Primers and Probes
2.4. Amplification of Thailand Orthohantavirus and Its Genetic Variants
3. Results
3.1. Limit of Detection of the Assays
3.2. Specificity and Sensitivity
4. Discussion and Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Name | Sequence | Position * |
---|---|---|
THAIV F S | 5’-ATG GCA ACT ATG GAR GAG TTA CAG-3’ | 46–69 |
THAIV R | 5’-ACA CTY TCM CGG TCA TGI AGT GC-3’ | 195–172 |
THAIV P S | [FAM] 5’-CAR CTT GTG GTG GCW AGR CA-3’ [BHQ1] | 94–113 |
System | LightCycler 480 Instrument II (Roche) | Applied Biosystems 7500 (Applied-Biosystems) | Pan-Hantavirus Nested RT-PCR 1 |
---|---|---|---|
RT-PCR Reagents | SensiFAST Probe One-Step kit (Bioline) | SuperScript III Platinum One-Step Kit (Invitrogen) | |
THAIV RNA Concentration (FFU/mL) 2 | Ct | Ct | |
3.45 × 105 | 21.1 | 18.7 | Positive |
3.45 × 104 | 24.6 | 22.8 | Positive |
3.45 × 103 | 27.8 | 26.9 | Positive |
3.45 × 102 | 31.5 | 30.8 | Positive |
3.45 × 101 | 34.3 | 32.9 | Positive |
3.45 | 36.4 | 36.2 | Positive |
3.45 × 10−1 | Negative | Negative | Negative |
3.45 × 10−2 | Negative | Negative | Negative |
System | LightCycler 480 Instrument II (Roche) | Applied Biosystems 7500 (Applied-Biosystems) |
---|---|---|
RT-PCR Reagents | SensiFAST Probe One-Step kit (Bioline) | SuperScript III Platinum One-Step Kit (Invitrogen) |
Virus Strain1 | ||
THAIV | Positive | Positive |
HTNV 2 | Negative | not tested |
PUUV | Negative | Negative |
SEOV 2 | Negative | not tested |
ANJZV 3 | Positive | Positive |
ANJZV 4 | Positive | Positive |
Applied Biosystems 7500 (Our Assay) | Pan-Hantavirus Nested RT-PCR (Klempa) | |
---|---|---|
Positive | Negative | |
Positive | 113 | 1 |
Negative | 0 | 341 |
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Raharinosy, V.; Heraud, J.-M.; Rabemananjara, H.A.; Telfer, S.; Rakoto, D.A.D.; Filippone, C.; Reynes, J.-M. Fast, Sensitive and Specific Detection of Thailand orthohantavirus and Its Variants Using One-Step Real-Time Reverse-Transcription Polymerase Chain Reaction Assay. Viruses 2019, 11, 718. https://doi.org/10.3390/v11080718
Raharinosy V, Heraud J-M, Rabemananjara HA, Telfer S, Rakoto DAD, Filippone C, Reynes J-M. Fast, Sensitive and Specific Detection of Thailand orthohantavirus and Its Variants Using One-Step Real-Time Reverse-Transcription Polymerase Chain Reaction Assay. Viruses. 2019; 11(8):718. https://doi.org/10.3390/v11080718
Chicago/Turabian StyleRaharinosy, Vololoniaina, Jean-Michel Heraud, Harinirina Aina Rabemananjara, Sandra Telfer, Danielle Aurore Doll Rakoto, Claudia Filippone, and Jean-Marc Reynes. 2019. "Fast, Sensitive and Specific Detection of Thailand orthohantavirus and Its Variants Using One-Step Real-Time Reverse-Transcription Polymerase Chain Reaction Assay" Viruses 11, no. 8: 718. https://doi.org/10.3390/v11080718