A method for the rapid diagnosis of early dengue virus (DENV) infection is highly needed. Here, a prototype reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed. The assay detected DENV RNA in <20 min without the need for thermocycling amplification. The assay enabled the detection of as few as 10 copies of DENV RNA. The designed RT-RPA primers and exo probe detected the DENV genome of at least 12 genotypes of DENV circulating globally without crossreacting with other arboviruses. We assessed the diagnostic performance of the RT-RPA assay for the detection of DENV RNA in 203 serum samples of patients with clinically suspected dengue. The sera were simultaneously tested for DENV using a reverse transcription-loop-mediated isothermal amplification ( Dengue is one of the most prevalent mosquito-borne viral diseases in the tropics and subtropics. It is estimated that at least 3.6 billion people are at risk of contracting the infection (1). The spectrum of the illness of dengue ranges from mild dengue fever (DF) to severe and fatal dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) (2). Dengue viruses (DENV) are the causative agents of dengue (3). There are at least four known antigenically distinct DENV serotypes, DENV-1, DENV-2, DENV-3, and DENV-4 (4), and each serotype contains several phylogenetically distinct genotypes (5). The virus is transmitted following mosquito bites of viremic febrile patients; the viremia phase usually corresponds to the first 3 days of illness (6). Therefore, the early detection of viremic individuals is important not only for patient management but also for early and immediate implementation of appropriate vector-control measures (7).Molecular techniques to detect the presence of DENV genomic RNA sequences are gradually being accepted as routine procedures for the early detection of DENV infection. The reverse transcription-PCR (RT-PCR) and real-time quantitative RT-PCR (qRT-PCR) are the two most commonly used methods (8-12). However, the requirement for costly nucleic acid amplification instruments and the high level of skill needed for these methods have limited their use to well-equipped laboratories. The introduction of the isothermal nucleic acid amplification method, which obviates the need for any major instrumentation, could change this situation, hence allowing greater access to the nucleic acid amplification method and its wider application in regions where dengue is endemic and resources may be limited (13).We have previously demonstrated the use of reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for the detection of DENV RNA in a clinical setting (14). More recently, the recombinase polymerase amplification (RPA) assay has emerged as a novel alternative isothermal amplification method for the detection of nucleic acid (15). The RPA assay was reported to take Ͻ20 min to perform and requires a constant temperature of only 37°C to 42°C. Both the RPA and RT-RPA assays have been used for the detection of vari...

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BackgroundRecurring dengue outbreaks occur in cyclical pattern in most endemic countries. The recurrences of dengue virus (DENV) infection predispose the population to increased risk of contracting the severe forms of dengue. Understanding the DENV evolutionary mechanism underlying the recurring dengue outbreaks has important implications for epidemic prediction and disease control.ResultsWe used a set of viral envelope (E) gene to reconstruct the phylogeny of DENV-1 isolated between the periods of 1987–2011 in Malaysia. Phylogenetic analysis of DENV-1 E gene revealed that genotype I virus clade replacements were associated with the cyclical pattern of major DENV-1 outbreaks in Malaysia. A total of 9 non-conservative amino acid substitutions in the DENV-1 E gene consensus were identified; 4 in domain I, 3 in domain II and 2 in domain III. Selection pressure analyses did not reveal any positively selected codon site within the full length E gene sequences (1485 nt, 495 codons). A total of 183 (mean dN/dS = 0.0413) negatively selected sites were found within the Malaysian isolates; neither positive nor negative selection was noted for the remaining 312 codons. All the viruses were cross-neutralized by the respective patient sera suggesting no strong support for immunological advantage of any of the amino acid substitutions.ConclusionDENV-1 clade replacement is associated with recurrences of major DENV-1 outbreaks in Malaysia. Our findings are consistent with those of other studies that the DENV-1 clade replacement is a stochastic event independent of positive selection.