Multiplex logic processing isothermal diagnostic assays for an evolving virus

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

The previously reported ZIKV NS2b-specific TaqMan qRT-PCR assay (Supplementary Table T5 ) 33 was performed using either the Evoscript RNA Probes Master (Roche, Basel, Switzerland) or the TaqMan RNA-to-Ct 1-Step Kit (Thermo Scientific, Waltham MA, USA). Standard curves were prepared using a ten-fold dilution series of 10 5 to 10 copies of a synthetic RNA template (Supplementary Table T5 ). Reactions without any templates served as negative controls. These templates were subjected to one-step qRT-PCR in 10 µL reactions containing 800 nM each of forward (Zika4481_F) and reverse (Zika4552c_R) primers along with 200 nM of the TaqMan probe (Zika4507cTqMFAM). Amplification was performed by adding either (i) 2 µL of the 5X Evoscript RNA probes Master or (ii) 0.25 µL of the 40X TaqMan RT Enzyme Mix (Thermo Scientific) along with 5 µL of the 2X TaqMan RNA-to-Ct 1-Step master mix. Reactions were incubated for 30 min at 60 ºC followed by 10 min at 95 ºC. Subsequently 45 cycles of 15 sec at 95 ºC and 30 sec at 55 ºC were performed. TaqMan fluorescence was measured during the second step of each cycle. Reactions containing Evoscript RNA Probes Master were analyzed using the LightCycler96 qPCR machine while TaqMan RNA-to-Ct 1-Step assays were performed on the StepOnePlus real-time PCR machine (Thermo Scientific). The one-input OSD signal transducer composed of one long and one short DNA strand can hybridize to a single LAMP amplicon loop sequence leading to separation of the fluorophore (F) and quencher (Q). The OR Boolean logic processing two-input strand exchange transducer, 2GO, is composed of two labeled strands, S I and S II , and a third bridging strand S III . Either S I and/or S II can hybridize to their specific LAMP loop sequences resulting in separation of F and Q. The four-input 4GO probe composed of 5 DNA strands (S1-S5) can hybridize to any combination of up to four different LAMP amplicon loops and perform an OR Boolean operation to produce fluorescence signal. 4GO probe is denoted in terms of lettered domains (a-g), each of which represents a short fragment of DNA sequence in an otherwise continuous oligonucleotide strand. Complementarity is denoted by a single prime symbol. , NS1 (C), and NS5 (D) synthetic RNA templates were amplified by singleplex degenerate LAMP-OSD assays specific to each template. OSD fluorescence signals measured in real-time during LAMP amplification are depicted as red (10 5 template copies), blue (10 4 template copies), orange (10 3 template copies), gray (100 template copies), and black (0 template copies; 10 6 non-template RNA) traces. The x-axis depicts the duration of LAMP amplification. OSD fluorescence was also imaged at amplification endpoint using a cellphone (images depicted at the bottom of each panel). Numbers on each assay tube in these images indicate the RNA template copies used. Representative results from three replicate experiments are depicted. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/424440 doi: bioRxiv preprint endpoint using LightCycler 96 real-time PCR machine are depicted as blue (capsid), orange (NS1), gray (NS3), and yellow (NS5) bars. Representative results from three replicate experiments are depicted. Indicated copies of capsid, NS1, NS3, and NS5 synthetic target RNA were amplified either individually (panels A, B, C, and D, respectively) or as a mixture (panel E) using 4-Plex-LAMP-4GO assays containing