Simultaneous Detection of Different Zika Virus Lineages via Molecular Computation in a Point-of-Care Assay

Abstract

We have developed a generalizable "smart molecular diagnostic" capable of accurate point-of-care (POC) detection of variable nucleic acid targets. Our isothermal assay relies on multiplex execution of four loop-mediated isothermal amplification reactions, with primers that are degenerate and redundant, thereby increasing the breadth of targets while reducing the probability of amplification failure. An easy-to-read visual answer is computed directly by a multi-input Boolean OR logic gate (gate output is true if either one or more gate inputs is true) signal transducer that uses degenerate strand exchange probes to assess any combination of amplicons. We demonstrate our methodology by using the same assay to detect divergent Asian and African lineages of the evolving Zika virus (ZIKV), while maintaining selectivity against non-target viruses. Direct analysis of biological specimens proved possible, with crudely macerated ZIKV-infected Aedes aegypti mosquitoes being identified with 100% specificity and sensitivity. The ease-of-use with minimal instrumentation, broad programmability, and built-in fail-safe reliability make our smart molecular diagnostic attractive for POC use.

Keywords: boolean logic-processing nucleic acid probes; isothermal nucleic acid amplification; mosquito; mosquito surveillance; multiplex nucleic acid detection; nucleic acid computation; nucleic acid strand exchange; point-of-care diagnostics; zika virus.

Figure 1

Schematic depicting (A) loop-mediated isothermal amplification (LAMP) integrated with (B) one-, (C) two-, or (D) four-input oligonucleotide strand exchange signal transducers. LAMP uses 2 inner (FIP and BIP) and 2 outer (F3 and B3) primers along with the optional stem (SP) and loop (LP) primers to prime strand displacement DNA amplification by Bst DNA polymerase. The resulting continuous amplification (initiated by both new primer-binding and by self-priming) generates double-stranded concatameric amplicons containing single-stranded loops to which non-priming oligonucleotide strand exchange signal transducers can hybridize. 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. The 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.

Figure 2

Detection of Zika virus capsid, NS1, NS3, and NS5 genes using real-time and visually-read reverse transcription LAMP-OSD assays. Indicated copies of capsid (A), NS3 (B), NS1 (C), and NS5 (D) synthetic RNA templates were amplified by degenerate LAMP-OSD assays specific to each template. OSD fluorescence signals measured in real-time during LAMP amplification are depicted as red (10⁵ template copies), blue (10⁴ template copies), orange (10³ template copies), gray (100 template copies), and black (0 template copies; 10⁶ 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.

Figure 3

Detection of Asian and African lineage ZIKV using degenerate reverse transcription 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 in degenerate RT-LAMP-OSD assays for Zika virus capsid, NS1, NS3, and NS5 genes. OSD fluorescence signals measured at amplification 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.

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.

Figure 5

Simultaneous detection of four Zika virus genes using degenerate 4GO probes and multiplex degenerate reverse transcription LAMP (multiplex LAMP-4GO) assays. Indicated copies of capsid, NS1, NS3, and NS5 synthetic target RNA were amplified either individually (panels A–D, respectively) or as a mixture (panel E) using multiplex LAMP-4GO assays containing 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 10⁵ copies of its target RNA) traces. The x-axis depicts the duration of endpoint signal measurement. (F) Detection of Asian and African lineage ZIKV genomic RNA using degenerate multiplex LAMP-4GO assays. Genomic RNA from 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 multiplex LAMP-4GO assay for ZIKV genomic RNA. Indicated copies of an Asian lineage ZIKV genome or non-specific DENV genomes were amplified using multiplex LAMP-4GO assays. 4GO probe fluorescence, measured in real-time at 37 °C after 90 min of LAMP amplification, is depicted as blue (Asian) and black (DENV) traces with template copies indicated by open squares (2000 genomes), open circles (189 genomes), and open diamonds (2 genomes). The x-axis depicts the duration of endpoint signal measurement. For all experiments, representative results from three replicate tests are depicted.

Figure 6

Detection of ZIKV RNA using two-input 2GO probes and degenerate reverse transcription LAMP. (A) Sequence-dependent activation of 2GO probes—synthetic RNA mixtures of 10⁶ copies of CA, NS1, NS3, and NS5 RNA were amplified using individual or multiplex (Mx) degenerate LAMP assays containing either one or both CAN3.2GO and N1N5.2GO probes. 2GO probe fluorescence signals measured at amplification endpoint using LightCycler 96 real-time PCR machine are depicted as blue (LAMP with only CAN3.2GO), orange (LAMP with only N1N5.2GO), and gray (LAMP with both CAN3.2GO and N1N5.2GO) dots. LAMP primer specificities are indicated on the x-axis. (B) Visual readout of degenerate LAMP-2GO assays. Cellphone image depicts 2GO probe fluorescence at amplification endpoint in individual or multiplex ZIKV LAMP assays containing both CAN3.2GO and N1N5.2GO probes and 10⁶ 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 multiplex 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.

Figure 7

Detection of Asian and African lineage ZIKV genomes using degenerate multiplex LAMP-2GO assays. (A) Genomic RNA from DENV, or Asian or African lineage ZIKV (indicated by their GenBank accession numbers) were used as templates for amplification. 2GO probe fluorescence signals measured at amplification endpoint using LightCycler 96 real-time PCR machine are depicted as blue (Asian ZIKV), red (African ZIKV), and black (DENV) markers. (B) Detection limit of degenerate multiplex LAMP-2GO assay for ZIKV genomic RNA. Indicated copies of an Asian lineage ZIKV genome (left panel), indicated dilutions of an African ZIKV genome (right panel), and non-specific DENV genomes (“Non”) were amplified using multiplex LAMP-2GO assays. 2GO probe fluorescence was imaged at amplification endpoint using a cellphone. For all experiments, representative results from three replicate tests are depicted.

Figure 8

Detection of Zika virus-infected mosquitoes using individual- 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 NS1 and capsid LAMP-OSD assays or with multiplex 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 2 h 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.