Multiplex logic processing isothermal diagnostic assays for an evolving virus

Sanchita Bhadra; Miguel A. Saldaña; Hannah Grace Han; Grant L. Hughes; Andrew D. Ellington

LAMP primers for all four ZIKV targets and the four-input 4GO probe. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as red (10,000 template copies), blue (1,000 template copies), yellow (100 template copies), and black (non-specific LAMP primers with 10 5 copies of its target RNA) traces. The xaxis depicts the duration of endpoint signal measurement. (F) Detection of Asian and African lineage ZIKV genomic RNA using degenerate 4-Plex-LAMP-4GO assays. Whole cell RNA from cells infected with DENV, or Asian or African lineage ZIKV (indicated by their GenBank accession numbers) were used as templates for amplification. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian), red (African), and green (DENV) traces. The x-axis depicts the duration of endpoint signal measurement. (G) Detection of Asian and African lineage ZIKV genomic RNA using TaqMan qRT-PCR assay specific for Asian lineage ZIKV NS2b gene. Same amount of viral genomic RNA as was used in panel F were amplified and real-time measurements of assay fluorescence are depicted as blue (Asian), red (African), and green (DENV) traces. (H) Detection limit of degenerate 4-Plex-LAMP-4Go assay for ZIKV genomic RNA. Indicated copies of an Asian lineage ZIKV genome or non-specific DENV genomes were amplified using 4-Plex-LAMP-4GO assays. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian) Cellphone image depicts 2GO probe fluorescence at amplification endpoint in single-or 4-plex ZIKV LAMP assays containing both CAN3.2GO and N1N5.2GO probes and 10 6 copies of all four synthetic ZIKV RNA and a non-specific LAMP assay ('Non') containing its cognate RNA. (C) Detection limit of visually-read degenerate 4-Plex-LAMP-2GO assays. Cellphone image depicts endpoint 2GO probe fluorescence of multiplex degenerate RT-LAMP assays containing primers and indicated template RNA copies of all four ZIKV targets. The reaction without any ZIKV RNA contained a non-specific RNA and its cognate LAMP primers. For all experiments, representative results from three replicate tests are depicted. . Detection of Zika virus-infected mosquitoes using single-and multiplex degenerate reverse transcription LAMP assays. Zika virus-infected (panels A-D) and uninfected (panels E-H) Aedes aegypti mosquitoes were directly analyzed using singleplex NS1 and capsid LAMP-OSD assays or with 4-plex-LAMP-2GO assays. As a positive control, mosquitoes were tested using the A. aegypti coi LAMP-OSD assay (panels D and H). Smartphone images acquired after 2h of amplification are depicted. P: positive control; M+: mosquito analyte with LAMP primers; M-: mosquito analyte without LAMP primers; N: no template control. Results of NS2b TaqMan qRT-PCR analysis of all mosquitoes are tabulated. Figure 9 . Detection of ZIKV virions in human saliva using single-and multiplex degenerate reverse transcription LAMP assays. Asian lineage ZIKV virions spiked in human saliva -1: 2x10 6 virions/ml; 2: 2x10 5 virions/ml; 3: 2x10 4 virions/ml; 4: 10 7 non-specific virions/ml -were detected using direct singleplex analysis with degenerate NS1-specific LAMP-OSD assays or with multiplex degenerate 4-Plex-LAMP-2GO assays. Smartphone images of OSD or 2GO probe fluorescence acquired after different durations of amplification are depicted.