Multiplexed Diagnosis of Four Serotypes of Dengue Virus by Real-time RT-PCR
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Original Article
Multiplexed Diagnosis of Four Serotypes of Dengue Virus by Real-time RT-PCR Jiyeon Kim1,2 & Eung-Soo Hwang
1,2,3,
*
Received: 25 May, 2020 / Accepted: 20 August, 2020 / Published online: 16 November, 2020 ⒸThe Korean BioChip Society and Springer 2020
Abstract The number of people at risk for dengue viral infection is about 50% of the world's population. Dengue viral infection is classified as dengue fever and severe dengue depending on the severity. Severe dengue may be associated with infection by the different serotype from the primary infected serotype. The circulating epidemic serotypes have changed continuously and periodically. It is very important to diagnose dengue viral infection as early as possible to be treated adequately. The purpose of this study was the development of a simultaneous detection method for all the serotypes of dengue virus in a single sample. The consensus primers and four different fluoresceintagged probes that specifically binds to each serotype of dengue virus were designed from the multiple alignment data of full-length dengue viral sequences, which were retrieved from GenBank. They were located between the 5’ untranslated region and the capsid gene of the dengue viruses. Each serotype of dengue viruses isolated in Korea was detected correctly with the established multiplex real time RT-PCR at the level of 5-10 copies/reaction. Other RNA viruses such as the Japanese encephalitis virus, Zika virus, yellow fever virus, and Chikungunya virus were not amplified with this method. The results of the inter-assays and intraassays were within the acceptable variation ranges. Conclusively, the developed method can detect each 1 Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, 03080, Republic of Korea 2 Global Center for Infectious Diseases, Seoul National University College of Medicine, Seoul, 03080, Republic of Korea 3 Institute of Endemic Diseases, Seoul National University Medical Research Center, Seoul, 03080, Republic of Korea *Correspondence and requests for materials should be addressed to E.S. Hwang ( [email protected])
serotype of dengue virus specifically and quantitatively even when different serotypes of dengue virus are present in a sample. Keywords: Dengue virus, Multiplex PCR, Diagnosis
Introduction Dengue virus (DENV) is the causative agent of dengue fever and sever dengue disease which are prevalent in tropical and subtropical countries1. DENV is transmitted mostly by the Egyptian dendritic mosquito (Aedes aegypti) and the white line mosquito (Aedes albopictus)2,3. Dengue fever was prevalent in nine countries before 1970, but in recent decades the epidemic area has been gradually spreading to more than 100 countries including Africa, the Americas, and Southeast Asia4-6. Nearly 400 million people suffer from DENV infection annually, which is one of the 17 diseases prioritized by the World Health Organization. DENV is a single positive-stranded RNA virus in the family Flaviviridae. There are four antigenicall
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