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

• Published in: Journal of virological methods 2015-03, Vol.214, p.60-74
• Main Authors: Glushakova, Lyudmyla G., Bradley, Andrea, Bradley, Kevin M., Alto, Barry W., Hoshika, Shuichi, Hutter, Daniel, Sharma, Nidhi, Yang, Zunyi, Kim, Myong-Jung, Benner, Steven A.
• Format: Article
• Language: English
• Subjects: Alphavirus; Animals; Arboviruses - genetics; Arboviruses - isolation & purification; Artificially expanded genetic information system (AEGIS); Culicidae; Culicidae - virology; Dengue virus; Female; Flavivirus; High-Throughput Screening Assays - methods; Luminex direct hybridization assay; Nucleic Acid Hybridization - methods; Orthobunyavirus; Polymerase Chain Reaction - methods; Reverse-transcription PCR; Self-avoiding molecular recognition system (SAMRS); Sensitivity and Specificity; Synthetic Biology - methods
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2015.01.003

•The 22-fold multiplexed panel for mosquito-borne arboviruses detection is developed.•The panel differentiates viruses from genera Flavivirus, Alphavirus, Orthobunyavirus.•Our innovative strategy bases on unnatural DNA base pairs, SAMRS and AEGIS.•Our strategy exhibits high fidelity and efficiency in PCR and Luminex hybridization.•The measuring capability of the panel is about 6–10 virions per infected mosquito. 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.