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

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.

COVID‐19 and dengue infection in Bangladesh: A case of coinfection where hemoptysis as first presentation

“…COVID‐19 is a novel illness with many unknown features, unusual presentations such as severe hemoptysis should also arouse concern regarding the diagnosis of COVID‐19, particularly in pandemic situations in dengue‐endemic settings. Additional viral outbursts, such as hepatitis viruses, Chikungunya viruses, Epstein‐Barr virus (EBV), influenza virus incidences, and so on, may impact the prevalence of viral coinfection with COVID‐19, thus various anti‐COVID assessments must be considered in the future 20,21 …”

Development of Highly Sensitive Sandwich ELISA for the Early-Phase Diagnosis of Chikungunya Virus Utilizing rE2-E1 Protein

“… 48 Owing to the lack of antiviral treatments and vaccines for CHIKV, a specific detection method is urgently required. Several methods for detecting chikungunya and dengue viruses have been described, including immunofluorescence technology, plaque assay, viral isolation, and multiplex RT-PCR; 49 however, the difficulty of carrying out these procedures has led to the use of more practical methods, such as ELISA and RT-PCR. 50 For CHIKV detection, the RT-PCR test is extremely specific and sensitive, but the chemicals and equipment are too expensive for general use.…”

Evaluation of DNA-Launched Virus-Like Particle Vaccines in an Immune Competent Mouse Model of Chikungunya Virus Infection

“…Human infections with CHIKV are of increasing concerns in tropical and subtropical regions of the world and have the potential to significantly impact quality of life. New diagnostic test have been developed to differentiate CHIKV infections from other common mosquito borne viruses such as DENV and ZIKV that circulate in these regions [ 22 , 23 ]; and can be utilized to target vaccination campaigns to areas that need it most. To this end, next generation vaccines which are both safe and effective are desperately needed to prevent infection and chronic disease associated with CHIKV worldwide.…”