RNA isolation and RT-qPCR for Dengue, Chikungunya and Zika Viruses
Maria Fernanda Avila Mejia, Pei-Yun Shu, Dar-Der Ji
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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.
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
Steps
Blood Sample Collection
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Wear gloves and label the filter paper to be used before collecting DBS.
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Clean the finger from which sample will be collected with 75% alcohol.
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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.
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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.
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After the blood spots are completely on the filter paper, allow it to dry before placing it in a sealed plastic bag.
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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
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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.
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Extract Viral RNA utilizing the QIAamp Viral RNA Mini Kit according to the manufacturer's instructions..
RT-qPCR
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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
| A | B | C |
|---|---|---|
| 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
| A | B | C | D | E | F |
|---|---|---|---|---|---|
| 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 |
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Process the RT-qPCR in the Applied Biosystems StepOnePlus™ Real-Time PCR System with the following conditions:
| A | B |
|---|---|
| 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 |
- 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.
