Multiplexed kit based on Luminex technology and achievements in synthetic biology discriminates Zika, chikungunya, and dengue viruses in mosquitoes

Abstract

Background The global expansion of dengue (DENV), chikungunya (CHIKV), and Zika viruses (ZIKV) is having a serious impact on public health. Because these arboviruses are transmitted by the same mosquito species and co-circulate in the same area, a sensitive diagnostic assay that detects them together, with discrimination, is needed. Methods We present here a diagnostics panel based on reverse transcription-PCR amplification of viral RNA and an xMap Luminex architecture involving direct hybridization of PCRamplicons and virus-specific probes. Two DNA innovations (“artificially expanded genetic information systems”, AEGIS, and “self-avoiding molecular recognition systems”, SAMRS) increase the hybridization sensitivity on Luminex microspheres and PCR specificity of the multiplex assay compared to the standard approach (standard nucleotides). Results The diagnostics panel detects, if they are present, these viruses with a resolution of 20 genome equivalents (DENV1), or 10 (DENV3–4, CHIKV) and 80 (DENV2, ZIKV) genome equivalents per assay. It identifies ZIKV, CHIKV and DENV RNAs in a single infected mosquito, in mosquito pools comprised of 5 to 50 individuals, and mosquito saliva (ZIKV, CHIKV, and DENV2). Infected mosquitoes and saliva were also collected on a cationic surface (Q-paper), which binds mosquito and viral nucleic acids electrostatically. All samples from infected mosquitoes displayed only target-specific signals; signals from non-infected samples were at background levels. Conclusions Our results provide an efficient and multiplex tool that may be used for surveillance of emerging mosquito-borne pathogens which aids targeted mosquito control in areas at high risk for transmission. Electronic supplementary material The online version of this article (10.1186/s12879-019-3998-z) contains supplementary material, which is available to authorized users.

Vector competence of Aedes albopictus populations for chikungunya virus is shaped by their demographic history

“…If we consider the historical dynamics of Ae. albopictus expansion and the temporal and spatial history of CHIKV evolution and spread, it appears that they are bound by convergent adaptive evolution 39,48,49 . Indeed, the historical sequential divergence and admixture of Ae.…”

Optimization of cationic (Q)-paper for detection of arboviruses in infected mosquitoes

“…Samples of saliva from five non-infected mosquitoes were used as negative controls. Adsorbed RNA was then eluted from Q-papers as described above and analyzed either by monoplexed Luminex-based assay that targets CHIKV alone using single CHIKV-specific probe (Figure 5B) or that targets a variety of arboviruses (DENV serotypes 1–4, ZIKV and CHIKV) in an assay having by a six-fold multiplexed capability (Figure 6) (Glushakova et al 2018). All oligonucleotides used in the multiplexed assay are presented in Appendix B (Table B.1).…”

“…The time of saliva collection was estimated based on the highest CHIKV titers expectorated in saliva (Dubrulle et al 2009, Alto et al 2017). The procedure for collecting infected saliva was described previously (Glushakova et al 2018). Briefly, mosquitoes were deprived of sucrose (24 hours) and transferred to tubes fitted with a lid with honey-soaked Q-paper (1 cm diameter).…”

Advances in the differential molecular diagnosis of vesicular disease pathogens in swine

“…Due to its high-throughput characteristics, Luminex technology enables multiplex detection of pathogens. A variety of commercial kits based on Luminex technology for different pathogens have also been developed, which provides convenience for the rapid diagnosis of different pathogens, especially those with similar symptoms ( Krunic et al, 2011 ; Gray and Coupland, 2014 ; Glushakova et al, 2019 ).…”