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. 2018 Dec 14;10(12):714. doi: 10.3390/v10120714

Figure 4.

Figure 4

Simultaneous detection of four Zika virus genes using multiplex reverse transcription degenerate LAMP-OSD (multiplex LAMP-OSD) assay. (A) Real-time multiplex LAMP-OSD—synthetic RNA mixtures containing indicated copies of each of the four ZIKV synthetic RNA templates (CA, NS1, NS3, and NS5) were amplified using multiplex LAMP-OSD assays containing 21 degenerate primers and 4 degenerate OSD probes for simultaneous LAMP amplification and sequence-specific detection of all four ZIKV targets. OSD fluorescence signals measured in real-time during LAMP amplification are depicted as blue (10,000 copies each of CA, NS1, NS3, and NS5 RNA) and orange (0 ZIKV RNA; 106 copies of DENV RNA) traces. The x-axis depicts the duration of LAMP amplification. (B) Endpoint multiplex LAMP-OSD assay with visual detection—synthetic RNA mixtures containing indicated copies of each of the four ZIKV synthetic RNA templates (CA, NS1, NS3, and NS5) were amplified using degenerate multiplex LAMP-OSD assays. OSD fluorescence was imaged after 90 min of amplification using a cellphone. Numbers above each assay tube indicate the RNA template copies used. The reaction with ‘0’ ZIKV RNA received 106 copies of DENV RNA. (C-F) Performance of individual assays in the multiplex LAMP-OSD system—synthetic RNA mixtures containing indicated copies of each of the four ZIKV synthetic RNA templates (CA, NS1, NS3, and NS5) were amplified using multiplex LAMP-OSD assays containing LAMP primers for all four targets but only one type of OSD for either capsid (C), NS1 (D), NS3 (E), or NS5 (F) amplicons. OSD fluorescence signals measured in real-time during LAMP amplification are depicted as blue (10,000 copies each of CA, NS1, NS3, and NS5 RNA) and orange (0 ZIKV RNA; 106 copies of DENV RNA) traces. The x-axis depicts the duration of LAMP amplification. (G) Detection of Asian and African lineage ZIKV genomic RNA using degenerate multiplex LAMP-OSD assays. Genomic RNA from DENV, CHIKV, or Asian or African lineage ZIKV (indicated by their GenBank accession numbers) were used as templates for amplification. Real-time OSD fluorescence signals are depicted as blue (Asian), red (African), black (CHIKV), and green (DENV) traces. The x-axis depicts the duration of LAMP amplification. For all experiments, representative results from three replicate tests are depicted.