An RT-PCR panel for rapid serotyping of dengue virus serotypes 1 to 4 in human serum and mosquito on a field-deployable PCR system

Abstract

BackgroundDengue fever, a mosquito-borne disease, is caused by dengue virus (DENV) which includes four major serotypes (DENV-1, -2, -3, and -4). Some serotypes cause more severe diseases than the other; severe dengue is associated with secondary infections by a different serotype. Timely serotyping can provide early warning of dengue epidemics to improve management of patients and outbreaks. A mobile insulated isothermal PCR (iiPCR) system is available to allow molecular detection of pathogens near points of need.Methodology/Principle findingsIn this study, side-by-side comparison with the CDC DENV-1-4 Real Time RT-PCR (qRT-PCR) was performed to evaluate the performance of four singleplex DENV-1–4 serotyping reverse transcription-iiPCR (RT-iiPCR) reagents for DENV subtyping on the mobile PCR system. The four RT-iiPCRs did not react with Zika virus and chikungunya virus; tests with serial dilutions of the four DENV serotypes made in human serum showed they had detection endpoints comparable to those of the reference method, indicating great analytical sensitivity and specificity. Clinical performance of the RT-iiPCR reagents was evaluated by testing 40 serum samples each (around 20 target serotype-positive and 20 DENV-negative); all four reagents had high agreement (97.5–100%) with the reference qRT-PCR. Moreover, testing of mosquitoes separately infected experimentally with each serotype showed that the four reagents detected specifically their target DENV serotypes in mosquito.Conclusions/SignificanceWith analytical and clinical performance comparable to the reference qRT-PCR assay, the four index RT-iiPCR reagents on the field-deployable PCR system can serve as a useful tool for DENV detection near points of needs.

In vitro shared transcriptomic responses of Aedes aegypti to arboviral infections: example of dengue and Rift Valley fever viruses

“…Efforts have been made to better understand the mechanisms of pathogenesis of these viruses including pathways essential for replication and to develop innovating and appropriate diagnostic tools, therapeutics and vaccines [ 15 – 17 ]. Current diagnostic methods mainly developed for the detection of specific viral molecular signatures in the host compartment by molecular based techniques [ 18 , 19 ] were adapted to trapped arthropods [ 20 – 23 ] with limitations such as (i) the need of high viral load being present in the vector at the time of the trapping, (ii) the time window in which the competent vectors exhibit a sufficient viral load, (iii) the specificity of the detection of one pathogen rather than having a larger detection system able to detect several arboviral infections at the same time. Applicability of other assays such as the Lawrence Livermore Microbial Detection Array (LLMDA) revealed the presence of mosquito-borne viruses and insect-specific viruses in field-collected mosquitoes with similar limitations [ 24 ].…”

Diagnosing the novel SARS-CoV-2 by quantitative RT-PCR: variations and opportunities

“…This is particularly important for coronaviruses, since members of this group already circulate in the human population Green, or post-amplification exonuclease-based probes, such as the TaqMan system) used for detecting and quantifying DNA amplification [37,38]. Moreover, these studies aimed to determine simple or multiplex reaction assays for virus detection and serotyping [39][40][41][42] and the development of tests for simultaneous detection of viruses [43][44][45][46].…”

Evaluation of rapid diagnostic tests to detect dengue virus infections in Taiwan

“…RT-PCR has been recommended for confirmational diagnosis of dengue virus infection, allowing for early intervention for surveillance, outbreak investigations, and clinical management. In our previous studies, reverse transcription insulated isothermal PCR (RT-iiPCR) using pan-DENV or singleplex DENV-1-4 serotyping reagents in a mobile semi-automated taco mini/Pockit system or a fully automated sample-to-answer POCKIT Central system were validated for rapid detection of dengue virus in human serum in approximately 90 minutes, reaching analytical sensitivity of 1 to 10 PFU/ ml of serum for the four serotypes of dengue virus [53][54][55]. However, the disadvantages of the nucleic acid test are that serum samples must be shipped to a central laboratory, and the experiments must be performed by well-trained medical technician to detect DENV.…”