A Potential Single-Tube Multiplex Assay for Detecting Dengue Virus in the Field

Countries affected by dengue. By Percherie (Distribution de la dengue sur Commons) [GFDL (http://www.gnu.org/copyleft/fdl.html), CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/) or CC BY-SA 2.5-2.0-1.0 (http://creativecommons.org/licenses/by-sa/2.5-2.0-1.0)], via Wikimedia Commons
In areas of the world where the electricity is intermittent, resources are limited and transporting bulky equipment and reagents that are sensitive to temperature fluctuations is difficult, diagnosis of viruses like dengue can be challenging. If you could reduce or eliminate the need for electricity dependent equipment for diagnostic assays without sacrificing sensitivity or specificity, it would be a boon to field workers. An article published in PLOS ONE describes how researchers developed a multiplex isothermal amplification method that could assess a potential dengue infection with a visual real-time or endpoint detection in a single tube.

The gold standard assay for dengue virus infection is culturing acute-phase serum or plasma samples cultured with C6/36 mosquito cell line and isolating virus from the culture. Some of the drawbacks to this culturing method include diagnosis can take seven days or more and isolating virus from clinical samples is difficult due to the nature of the virus and low level of transient viremia. Commercially available kits use IgM- and IgG-based enzyme-linked immunosorbent assays (ELISAs) to quantitate dengue-specific antigens by capturing anti-dengue specific antibodies present in serum. However, ELISAs require appropriate collection times for accuracy, and the presence of antibodies from previous infections can lead to false positive results.

Loop-mediated isothermal amplification (LAMP) requires incubation at approximately 65°C and yields results in less than an hour. Preliminary work by other scientists had demonstrated that four tubes could be used to diagnose dengue virus (DENV) using reverse transcription LAMP (RT-LAMP). However, the small sample size and 93% detection specificity in previous studies were not ideal. Lau et al. wanted to use fewer resources so they developed primers to the 3′ noncoding region specific to all four DENV serotypes, multiplexed the primers to create a single-tube assay and optimized the resulting LAMP reaction using plasmid DNA.

The LAMP reaction was optimized for incubation time and temperature using both a dye-based endpoint detection method with a color change in the presence of DENV (violet when negative, blue when positive) and a real-time monitoring with a turbidimeter at 650nm (the reaction optical density increases when positive for DENV). Based on this work, the optimal reaction temperature was 65°C, and the incubation time for a positive result regardless of detection method was 30 minutes for DENV 1, 2 and 3 serotypes and 45 minutes for DENV 4 serotype.

To test the specificity of the primers designed to detect DENV, RNA purified from serum samples of healthy individuals and RNA samples of Japanese encephalitis virus (JEV), Chikungunya virus (CHIKV), Sindbis virus (SINV) and DENV 1–4 were added to the RT-LAMP assay and the outcome assessed using the endpoint dye. All the DENV samples were positive; all the nonDENV samples, both from healthy individuals and other viral RNA samples, were negative. These results indicate the multiplex RT-LAMP assay is specific to DENV.

Using the optimized DENV-specific assay, Lau et al. challenged their system to assess a panel of 189 suspected dengue and 24 healthy individuals. RNA was isolated from the serum of the 213 individuals and used in RT-LAMP and reverse transcription quantitative polymerase chain reaction (RT-qPCR), and the serum was tested directly in an ELISA for DENV. RT-LAMP indicated 115 samples were positive for DENV, RT-qPCR detected 98 positive RNA samples and ELISA 162 positive serum samples. Because ELISA is based on antibody detection, a previous infection can register as a false positive sample. Consequently, the DENV sensitivity and specificity determined for each method showed ELISA was highly sensitive (100%) but not as specific (52%), RT-qPCR was less sensitive (85%) but specific (100%) and RT-LAMP was both sensitive and specific (100% for both measures) based on researcher’s calculations of true and false positive and negative results for the 213 samples tested. These results demonstrated that RT-LAMP was effective in detecting dengue using viral RNA purified from serum samples from individuals.

Identifying individuals infected with DENV is an important part of disease diagnosis and surveillance. Finding a method to assess potential infections while reducing equipment and reagents needed is especially important for resource-limited countries. The multiplex single-tube RT-LAMP assay described in this PLOS ONE article is a promising method that can determine if DENV is present in a purified RNA sample using a visual dye indicator. RT-LAMP offers rapid results with little need for complex equipment. Having RT-LAMP in the field means another potential tool to use in the worldwide fight against the debilitating effects of dengue and other viruses.

Reference
Lau, Y.L., Lai, M.Y., Teoh, B.T., Abd-Jamil, J., Johari, J., Sam, S.S., Tan, K.K. and AbuBakar, S. (2015) Colorimetric detection of Dengue by single tube reverse-transcription-loop-mediated isothermal amplification. PLOS ONE 10, e0138694.

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Sara Klink

Technical Writer at Promega Corporation
Sara is a native Wisconsinite who grew up on a fifth-generation dairy farm and decided she wanted to be a scientist at age 12. She was educated at the University of Wisconsin—Parkside, where she earned a B.S. in Biology and a Master’s degree in Molecular Biology before earning her second Master’s degree in Oncology at the University of Wisconsin—Madison. She has worked for Promega Corporation for more than 15 years, first as a Technical Services Scientist, currently as a Technical Writer. Sara enjoys talking about her flock of entertaining chickens and tries not to be too ambitious when planning her spring garden.

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