May 29, 2023
RNA isolation and RT-qPCR for Dengue, Chikungunya and Zika Viruses
- Maria Fernanda Avila Mejia1,
- Pei-Yun Shu2,
- Dar-Der Ji1,3
- 1International Health Program, National Yang-Ming Chiao Tung University, Taiwan, R.O.C.;
- 2Center for Diagnostics and Vaccine Development, Taiwan Centers for Disease Control, Ministry of Health and Welfare, Taiwan, R.O.C.;
- 3Department of Tropical Medicine, National Yang-Ming Chiao Tung University, Taiwan, R.O.C.
Protocol Citation: Maria Fernanda Avila Mejia, Pei-Yun Shu, Dar-Der Ji 2023. RNA isolation and RT-qPCR for Dengue, Chikungunya and Zika Viruses. protocols.io https://dx.doi.org/10.17504/protocols.io.bcwyixfw
Manuscript citation:
Mejía MFÁ, Shu PY, Ji DD. Accuracy of Dengue, Chikungunya, and Zika diagnoses by primary healthcare physicians in Tegucigalpa, Honduras. BMC Infect Dis. 2023 Jun 1;23(1):371. doi: 10.1186/s12879-023-08346-1. PMID: 37264307; PMCID: PMC10233517.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 25, 2020
Last Modified: May 29, 2023
Protocol Integer ID: 33464
Keywords: Dengue, Chikungunya, Zika, filter paper, RT-qPCR,
Funders Acknowledgements:
Ministry of Science and Technology, Taiwan, R.O.C.
Grant ID: MOST 107-2320-B-010-009
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Abstract
This protocol is for dried blood spot (DBS) sample collection, the extraction of viral RNA from dried blood spots, and the following RT-qPCR setup for the detection of Dengue, Chikungunya, and Zika viruses.
Materials
MATERIALS
double distilled water (ddH2O)
QIAamp Viral RNA Mini KitQiagenCatalog #52904
Bunsen burner
Quantinova SYBR Green RT-PCR KitQiagenCatalog #208154
6-mm Paper Punch
Applied Biosystems StepOnePlus™ Real-Time PCR SystemThermo Fisher ScientificCatalog #4376600
Whatman® 903 protein saver cardsMerck MilliporeSigma (Sigma-Aldrich)Catalog #WHA10531018
Before start
During RT-qPCR preparation, it is important to avoid DNA cross-contamination. Use an area exclusively for RNA work.
Clean the working area and all equipment with 10% bleach.
Dispose of contaminated sharps and waste appropriately
Blood Sample Collection
Blood Sample Collection
1. Wear gloves and label the filter paper to be used before collecting DBS.
2. Clean the finger from which sample will be collected with 75% alcohol.
3. Fingerprick with a small lancet after alcohol dries off. Apply gentle pressure to the finger and allow a large drop of to collect at the puncture site.
4. Remember NOT to press the filter paper against the puncture wound, rather allow the blood to drop and saturate the full circle. Avoid smering the blood spot.
5. After the blood spots are completely on the filter paper, allow it to dry before placing it in a sealed plastic bag.
6. Store the filter paper in the sealed plastic bag with a desiccant package and then store it in closed box. Avoid exposure to the sun.
RNA Viral Extraction
RNA Viral Extraction
1. Use the paper puncher to punch a 6-mm punch in the dried blood spot. Sterilize the paper puncher with fire and make a clean punch after each use to avoid cross-contamination.
2. Extract Viral RNA utilizing the QIAamp Viral RNA Mini Kit according to the manufacturer's instructions..
RT-qPCR
RT-qPCR
3. Prepare the RT-qPCR master mix using the Quantinova SYBR Green RT-PCR Kit for the three arboviruses. Three final volumes of 25 µl of PCR mixes contained 5 µL of RNA extract with individual DENV, CHIKV, or ZIKV primer pairs at a concentration of 0.3 µM, 0.3 µM, and 0.4 µM, respectively.
Primers
| Primer | Sequence 5' to 3' | Gen Bank Reference No. | |
| DENV-F | CAA TAT GCT GAA ACG CGA GAG AAA | AF038403 | |
| DENV-R1-3 | CCC CAT CTA ACC AAT ATT CCT GCT | AF180817, AF038403, M93130 | |
| DENV-R4 | CCC CAT CTG TTC AGT ATC CCT GCT | M14931 | |
| CHIKV-F | AAG CTY CGC GTC CTT TAC CAA G | EU192142, EU192143 | |
| CHIKV-R | CCA AAT TGT CCY GGT CTT CCT | EU192142, EU192143 | |
| ZIKV-F | GCA ACA TGG CGG AGG TAA GAT | KU321639 | |
| ZIKV-R | GCT CTY GGT GAA TTR GGC GT | KU321639 |
Master Mix
| Dengue | Chikungunya | Zika | ||||
| Master Mix | µL | Master Mix | µL | Master Mix | µL | |
| RNA Sample | 5.0 | RNA Sample | 5.0 | RNA Sample | 5.0 | |
| ddH20 | 3.75 | ddH20 | 4.5 | ddH20 | 4.0 | |
| SYBR green | 12.5 | SYBR green | 12.5 | SYBR green | 12.5 | |
| RT Mix | 0.25 | RT Mix | 0.25 | RT Mix | 0.25 | |
| Primer-F (0.3 µM) | 0.75 | Primer-F (0.3 µM) | 0.75 | Primer-F (0.4 µM)) | 1.0 | |
| Primer-R1 (0.3 µM) | 0.75 | Primer-R (0.3 µM) | 0.75 | Primer-R (0.4 µM) | 1.0 | |
| Primer-R2 (0.3 µM) | 0.75 | Rox | 1.25 | Rox | 1.25 | |
| Rox | 1.25 | |||||
| Total | 25 | 25 | 25 | |||
4. Process the RT-qPCR in the Applied Biosystems StepOnePlus™ Real-Time PCR System with the following conditions:
| 1. RT | 1 cycle | |
| 50 °C for 30 min | ||
| 2. Pre-incubation | 1 cycle | |
| 95 °C for 15 min | ||
| 3. 3-step amplification | 45 cycles | |
| 95 °C for 15 s | ||
| 55 °C for 30 s | ||
| 72 °C for 20 s | ||
| 77 °C for 20 s | ||
| 4. Melting | 1 cycle | |
| 95 °C for 60 s | increase in temperature (1°C/30 s) to 90 °C | |
| 60 °C for 30 s | ||
| 97 °C for 1 s |
5. A sample was considered positive if the fluorescence curve crossed the threshold line within 40 cycles (Cq<40); and if the melting curve for DENV was around 80.25°C for DENV-1, 81.75°C for DENV-2, 80.,5°C for DENV-3 and DENV-4 for 83°C, for CHIKV and ZIKV was between 81°C and 82°C. Send positive samples for sequencing.