A Novel Multiplex RT-PCR Assay for Simultaneous Detection of Dengue and Chikungunya Viruses.

Mohammed Alimul Islam,Hossam M Ashour,Md Mortuza Ali,Md Rajibur Rahman,Mohamed E El Zowalaty,Md Tanvir Rahman,Mohammad Robed Amin,Sumaiya Islam,Kouichi Morita,Mohiuddin Sharif

doi:10.3390/ijms21218281

Abstract

The goal of the study was to develop a specific, sensitive, and cost-effective molecular RT-PCR diagnostic assay for the rapid and simultaneous detection of the serotypes of dengue virus (DENV) and Chikungunya virus (CHIKV) from sera of suspected febrile patients. A single-tube, single-step multiplex RT-PCR (mRT-PCR) assay was designed for the detection of viral genomes from clinical and field samples. Specificity and sensitivity of the mRT-PCR assay were evaluated against six different combinations using two reverse transcriptases (AMV-RT and RT-Ace) and three DNA polymerases (LA-Taq, rTaq, and Tth). Among the six combinations, the AMV-RT and LA-Taq combination was more specific and sensitive than other enzyme combinations for detecting viral genomes of DENV-1, DENV-2, DENV-3, and DENV-4 (p < 0.01), and for detecting viral genomes of CHIKV (p < 0.05). The detection limits of the mRT-PCR were 10 focus forming units (FFU) for CHIKV and 1 FFU, 20 FFU, 0.1 FFU, and 10 FFU for DENV-1, DENV-2, DENV-3, and DENV-4, respectively. The primers used for the mRT-PCR did not show any cross-reactivity among the serotypes of DENV or CHIKV. Specificity and sensitivity of the newly developed mRT-PCR were validated using serum samples collected from febrile patients during dengue outbreaks in Bangladesh. The sensitivity for serotype detection of DENV and CHIKV was superior to the virus isolation method and the antigen detection method using the Dengue NS1-Ag assay. This novel mRT-PCR method can be used for molecular epidemiological surveillance of DENV and CHIKV in epidemic and endemic countries.

Highlights

Dengue virus (DENV) and Chikungunya virus (CHIKV) are arboviruses of the family Flaviviridae and Togaviridae respectively
Before being tested the specificity and sensitivity of the primer-sets designed for the multiplex RT-PCR (mRT-PCR), a single pair of the primers and sense and complementary primers for CHIKV viruses were tested by uniplex RT- PCR (uRT-PCR) in this study
Lanciotti et al developed a group-specific mRT-PCR for the detection and differentiation of the four serotypes of dengue virus (DENV) using a single-tube mRT-PCR reaction in which the sensitivity and specificity were less than the type-specific uRT-PCR. [33]

Summary

Introduction

Dengue virus (DENV) and Chikungunya virus (CHIKV) are arboviruses of the family Flaviviridae and Togaviridae respectively. Aedes mosquitoes such as Aedes aegypti and Aedes albopictus play major roles in the transmission of these viruses to humans and animals. It is estimated that billions of humans are at risk of DENV infections [4,5,6] It can weaken the immune system causing people to be vulnerable to other emerging diseases such as encephalitis [7]. CHIKV is a positive-sense RNA virus that is transmitted by the same mosquito vectors of DENV, and causes fever with similar symptoms to the classical dengue fever [8]. After its first appearance in the Newala district in Tanzania in 1953, CHIKV was transmitted to other countries [9]

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