This report is the results of the internship project that took place in Indonesia International Institute of Life Sciences (i3L). The title for this project is "Design of a new diagnostic tool for dengue serotypes" with the aim of designing a diagnostic tool using LAMP primer capable of distinguishing the different serotypes between DENV1, DENV2, DENV3, DENV4 of distinguish dengue. Last but not least, I would like to thank the Indonesia International Institute of Life Sciences (i3L) for giving me the chance to do my internships here regardless of the pandemic situations.
LAMP primer design for DENV1 which consists of 10 primers required for LAMP (F3, B3, FIP and BIP). Thus, in this study we decided to design a diagnostic tool for dengue serotypes using loop amplification-mediated isothermals (LAMP), which is known to be able to detect various diseases such as dengue in this case. Thus, this study was undertaken to design a LAMP primer that can be used to detect dengue serotypes.
The Indonesian International Institute of Life Sciences (i3L) was established in 2014 as an elite higher education institution, categorically in the field of life sciences. While the business school offers 4 major programs such as Creative Digital Marketing, International Business Management, International Applied Accounting and Life Sciences Business and Entrepreneurship.
Project Description
Internship Project
- Project Background
- Scope of the Project
- Objectives/Aims
- Problem Formulation and Proposed Solution
- Materials and Methods 1 Materials preparations
- Methods
Recently, Indonesia announced that they have approved vaccines for dengue called “Qdenga” manufactured by Takeda Pharmaceutical Company (López-Medina et al., 2022). Since the cases found in Jakarta and Surabaya, the incidence of people infected with dengue has since increased and spread to more provinces around Indonesia and now it is endemic in all regions of Indonesia (Kraemer et al., 2015). In 1968, to prevent and control the progress of the dengue cases, the Ministry of Health (MoH) Indonesia developed a program called the dengue prevention and control programs on a national scale in collaboration with the Directorate General for Control of Communicable Diseases (Harapan et al., 2019).
Although these measurements have been practiced in the neighborhood, dengue has also spread and expanded its incidence and geographic area, leading to a hyperendemic with DENV serotypes in circulation (Eram et al., 1979; Hotta et al., 1970). Not to mention that people grouped into DF types are not registered in the surveillance systems, which makes the registration of people infected with dengue supposed to be higher compared to the reported cases in the surveillance systems (Karyanti et al ., 2014). Studies show that among the four different dengue serotypes (DENV1, DENV2, DENV3 and DENV4), each one indicates how severe the dengue fever is when infected. Vicente et al., 2016) Therefore, early detection of the serotypes can help administer the best treatment and prevent the high incidence of severe dengue outbreaks.
The sequences of the dengue serotypes whole genome were obtained from the biological databases called the National Center for Biotechnology Information (NCBI). Another alignment of the target gene (NS3) was also performed using bioinformatics tool called T-Coffee.
Findings
Result
The whole genome sequence mini-map was shown in Figure 3 with DENV3 as the reference shows in gray color and shows the red color referring to the polymorphism site. LAMP primer design for DENV1 which consists of the necessary primer for LAMP (F3, B3, FIP and BIP). According to the WHO, it can be estimated that 50 million cases are reported annually worldwide and that approximately half a million people with DHF require hospitalization and that 90% of reported cases were in children (World Health Organization, 2011).
Another supporting statement that dengue cases mostly occur in children is where a clinical study was conducted in the western part of Uttar Pradesh where the results of the study show that children in group III (7 to 12 years old and group IV (3 up to 18 years old ), which can be said that most cases occur in children compared to adults (Kumar et al., 2020). For these reasons, practical measures have been practiced preventing transmission and controlling the mosquito population. Some hygiene measures such as since the use of insecticides and larvicides and the removal of breeding sites have been practiced to control and prevent the spread and transmission of diseases (Thomas et al., 2003).
A study conducted in 2021 indicates that the dengue serotypes most commonly affecting humans are DENV3, followed by DENV2 and DENV1 (Simo Tchetgna et al., 2021). It has the advantages of being easy to perform, cheaper, and a whole blood sample is required for this test (Goncalves et al., 2018). The sensitivity of LAMP also suggests an immediate possibility to prevent any cross-contamination that could lead to erroneous amplification (Li et al., 2011).
To further confirm the positive results, it can also undergo additional procedures such as the use of agarose gel electrophoresis, real-time fluorescent dyes, fluorescent dyes or UV light (Soroka et al., 2021). Following the selection of the target gene available in the sequence, there are 3 different structural proteins (capsid, membrane and envelope) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) (Barrero & Mistchenko, 2004) ) (Chawla et al., 2014). NS3 in control of helicase and protease activity in the C-terminal and N-terminal domain (Swarbrick et al., 2017).
NS5 also has its own role in RNA methyltransferase activity and RNA-dependent RNA polymerase activity (Zou et al., 2011). This conserved region was then used as the template for the forward primer of the multiplex primer due to the limited possibility to design a universal primer for that target. Additionally, a degenerate primer was also a considered method to design the primer, but it is known to be sometimes limited by the assay design limitation and usually does not account for possible new mutations (Kline et al., 2021).
Conclusion and Recommendation
Self Reflection
LAMP primer design for DENV2 consisting of the necessary primer for LAMP (F3, B3, FIP and BIP). LAMP primer design for DENV4 consisting of the necessary primer for LAMP (F3, B3, FIP and BIP). Immunocapture pairs with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for rapid detection of type 1 dengue virus.
Epidemiology of dengue hemorrhagic fever in Indonesia: Analysis of five decades of data from the National Disease Surveillance. BMC Research Notes, 12 (1), 350. Changing incidence of Dengue Hemorrhagic Fever in Indonesia: A 45-year registry-based analysis. BMC Infectious Diseases. Simultaneous detection and differentiation of dengue virus, Japanese encephalitis virus, and West Nile virus serotypes 1–4 by a combined reverse transcription loop-mediated isothermal amplification assay.
The NS3 helicase from dengue virus specifically recognizes the viral RNA sequence to ensure optimal replication. Nucleic Acids Research. Molecular evolution of dengue viruses: Contributions of phylogenetics to understanding prominent arboviral history and epidemiology. Comprehensive guidelines for the prevention and control of dengue and dengue hemorrhagic fever (Rev. and expanded. ed).
Mutagenesis of dengue virus protein NS2A revealed a novel domain responsible for virus-induced cytopathic effect and interactions between NS2A and NS2B transmembrane segments. Journal of Virology, 91(12), e01836-16.
