title: 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.
authors: Foundation for Applied Molecular Evolution (FfAME), 720 SW 2nd Avenue, Suite 201, Gainesville, FL 32601, United States.
authors: Lyudmyla G
authors: LG
authors: Glushakova
authors: Foundation for Applied Molecular Evolution (FfAME), 720 SW 2nd Avenue, Suite 201, Gainesville, FL 32601, United States.
authors: Andrea
authors: A
authors: Bradley
authors: Foundation for Applied Molecular Evolution (FfAME), 720 SW 2nd Avenue, Suite 201, Gainesville, FL 32601, United States.
authors: Kevin M
authors: KM
authors: Bradley
authors: Florida Medical Entomology Laboratory, University of Florida, 200 9th Street SE, Vero Beach, FL 32962, United States.
authors: Barry W
authors: BW
authors: Alto
authors: Foundation for Applied Molecular Evolution (FfAME), 720 SW 2nd Avenue, Suite 201, Gainesville, FL 32601, United States.
authors: Shuichi
authors: S
authors: Hoshika
authors: Firebird Biomolecular Sciences LLC, 13709 Progress Boulevard, Box 7, Alachua, FL 32615, United States.
authors: Daniel
authors: D
authors: Hutter
authors: Foundation for Applied Molecular Evolution (FfAME), 720 SW 2nd Avenue, Suite 201, Gainesville, FL 32601, United States.
authors: Nidhi
authors: N
authors: Sharma
authors: Foundation for Applied Molecular Evolution (FfAME), 720 SW 2nd Avenue, Suite 201, Gainesville, FL 32601, United States.
authors: Zunyi
authors: Z
authors: Yang
authors: Firebird Biomolecular Sciences LLC, 13709 Progress Boulevard, Box 7, Alachua, FL 32615, United States.
authors: Myong-Jung
authors: MJ
authors: Kim
authors: Foundation for Applied Molecular Evolution (FfAME), 720 SW 2nd Avenue, Suite 201, Gainesville, FL 32601, United States; Firebird Biomolecular Sciences LLC, 13709 Progress Boulevard, Box 7, Alachua, FL 32615, United States. Electronic address: manuscripts@ffame.org.
authors: Steven A
authors: SA
authors: Benner
mesh_terms: D000818:Animals; D001103:Arboviruses / Q000235:genetics / Q000302:isolation & purification*; D009033:Culicidae / Q000821:virology*; D005260:Female; D057166:High-Throughput Screening Assays / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D058615:Synthetic Biology / Q000379:methods*
keywords: Artificially expanded genetic information system (AEGIS); Dengue virus; Luminex direct hybridization assay; Reverse-transcription PCR; Self-avoiding molecular recognition system (SAMRS)