High-throughput multiplexed xMAP Luminex array panel for detection of twenty two medically important mosquito-borne arboviruses based on innovations in synthetic biology

Abstract

Mosquito-borne arboviruses are emerging world-wide as important human and animal pathogens. This makes assays for their accurate and rapid identification essential for public health, epidemiological, ecological studies. Over the past decade, many mono- and multiplexed assays targeting arboviruses nucleic acids have been reported. None has become established for the routine identification of multiple viruses in a “single tube” setting. With increasing multiplexing, the detection of viral RNAs is complicated by noise, false positives and negatives. In this study, an assay was developed that avoids these problems by combining two new kinds of nucleic acids emerging from the field of synthetic biology. The first is a “self-avoiding molecular recognition system” (SAMRS), which enables high levels of multiplexing. The second is an “artificially expanded genetic information system” (AEGIS), which enables clean PCR amplification in nested PCR formats. A conversion technology was used to place AEGIS component into amplicon, improving their efficiency of hybridization on Luminex beads. When Luminex “liquid microarrays” are exploited for downstream detection, this combination supports single-tube PCR amplification assays that can identify 22 mosquito-borne RNA viruses from the genera Flavivirus, Alphavirus, Orthobunyavirus. The assay differentiates between closely-related viruses, as dengue, West Nile, Japanese encephalitis, and the California serological group. The performance and the sensitivity of the assay were evaluated with dengue viruses and infected mosquitoes; as few as 6–10 dengue virions can be detected in a single mosquito.

Detection of chikungunya viral RNA in mosquito bodies on cationic (Q) paper based on innovations in synthetic biology

“…The design and synthesis of the primers and probes sequences were described previously (Glushakova et al, 2015) and listed in Table A1 (Appendix 1). Each RT-PCR was executed in a final volume of 20 μL according to the Invitrogen protocol for the SuperScript One-Step RT-PCR with Platinum Taq (Life Technologies).…”

“…We presented these here in the context of a series of other nucleic acid innovations, reported separately (Glushakova et al, 2015), and also shown in Figure 1 (adapted from Glushakova et al, 2015) that allow sensitive detection of viral PCR-amplicons.…”

Next-generation DNA in pathogen detection, surveillance, and CLIA-waivable diagnostics

“…Thus, FfAME and Firebird scientists developed polymerases to accept SAMRS nucleotides in templates [7]. Using AEGIS-SAMRS with conversion [9], we now offer a assay that detects 22 mosquito-borne RNA viruses in one assay [10] While PCR amplification can be used in clinics, it is not convenient at points-of-care and sampling. Thus, the cleanliness of SAMRS has been applied to helicase-dependent amplification (HDA).…”

Dengue Laboratory Diagnosis: Still Some Room for Improvement

“…Either through nucleic acid or antibody detection, the possibility of identifying multiple causal agents of disease, especially in endemic zones, is attractive. Thus, considering the geographical origin of the sample, it may be analyzed for the identification of multiple agents responsible for hemorrhagic fever syndrome included in the genera Flavivirus, Alphavirus and Orthobunyavirus, or Hantavirus, Nairovirus and Phlebovirus [103][104][105]109], or arthropod-borne viruses. The detection of flaviviral genetic material in plasma by the use of microsphere platforms has been recently reported [109].…”