DETECTION OF DENGUE VIRUS AMPLIFIED BY REVERSE-TRANSCRIPTION
HELICASE-DEPENDENT AMPLIFICATION
Eishika Dissanayake1,2,3,*, Photchanathorn Prombun4, Nuntaya Pornmun5, Wansika
Kiatpathomchai4,6, Prida Malasit3,5, Prapat Suriyaphol2,3,#
1
Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj
Hospital, Mahidol University, Thailand
2
Division of Bioinformatics and Data Management for Research, Office for Research and
Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand
3
Center for Emerging and Neglected Infectious Diseases (CENID), Mahidol University,
Thailand
4
Center of Excellence for Shrimp Molecular Biology and Biotechnology (CENTEX Shrimp),
Faculty of Science, Mahidol University, Thailand
5
Dengue Hemorrhagic Fever Research Unit, Office for Research and Development, Faculty
of Medicine Siriraj Hospital, Mahidol University, Thailand
6
National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science
and Technology Development Agency, Thailand
*e-mail: eishika@gmail.com, #e-mail: prapat.sur@mahidol.ac.th
Abstract
Dengue virus (DV) is a mosquito-borne virus causing a heavy disease burden.
Polymerase chain reaction (PCR) based methods allow rapid, specific detection, but are
limited by the need for thermal cycling. Reverse-transcription Helicase-dependent
Amplification (RT-HDA) is an isothermal technique which allows amplification at a constant
temperature making it more suitable for field and bed-side testing. In this study we optimized
conditions for amplifying DV serotype 1 strain Hawaii (DV1) and DV serotype 3 strain H87
(DV3) using RT-HDA. We also tested the use of colloidal nanogold particles (AuNPs) and a
lateral flow dipstick (LFD) for the detection of the RT-HDA product.
We designed serotype-specific primers for DV1 and DV3 and optimized NaCl and
MgSO4 levels to obtain maximum sensitivity of the RT-HDA reaction. When optimized the
sensitivity was 104 FFU/ml and 102 FFU/ml for DV1 and DV3 respectively. We then
hybridized the DV1 RT-HDA product with a AuNP probe functionalized with a DNA
sequence complementary to the DV1 amplicon. Any visible color change of the AuNPs with
the addition of MgSO4 from red to blue/purple was recorded and analyzed spectroscopically.
The DV3 reverse primer was labeled with biotin and a FITC-labeled probe complementary to
the DV3 RT-HDA product was designed. RT-HDA was performed with the biotin-labeled
primer followed by FITC probe hybridization. The complex was detected using the
Milenia® GenLine HybriDetect LFD.
The DV1 product hybridized with the AuNP probe remained red giving an absorption
peak at 520 nm with MgSO4 addition. The negative control and blank turned blue-purple
with MgSO4 shifting the peak to 600 nm. The color change was seen with 105 FFU/ml of
DV1 RNA. The DV3 product was detectable with the LFD upto 104 FFU/ml.
This study is the proof of concept that DV can be amplified using RT-HDA and
detected by the naked eye using AuNPs or LFD. With optimization and improvement it is
possible to detect the amplicon with both AuNPs and LFD with greater sensitivity.
Keywords: reverse-transcription helicase-dependent amplification, dengue virus, gold
nanoparticles, lateral flow dipstick