Low-cost detection of norovirus using paper-based cell-free systems and synbody-based viral enrichment

Abstract

Provided herein are methods and systems for low-cost, low-equipment detection of pathogens in biological sample. In particular, provided herein is a low-cost method for detecting norovirus that provides reliable, visible test with femtomolar, attomolar, and zeptomolar detection limits and that uses materials suitable for deployment of the methods in the field.

We claim: 
     
         1 . A method of detecting a target pathogen nucleic acid in a sample, the method comprising the steps of:
 (a) contacting a biological sample obtained from a subject to a pathogen detection agent under conditions that promote binding of the pathogen detection agent to the target pathogen if present in the sample;   (b) isolating nucleic acids from pathogen bound by the pathogen detection agent;   (c) amplifying the isolated nucleic acids using isothermal amplification; and   (d) contacting the amplified nucleic acid to a toehold switch, wherein the toehold switch encodes at least a portion of a reporter protein and comprises one or more single-stranded toehold sequence domains that are complementary to a target pathogen nucleic acid or the reverse complement thereof, wherein the contacting occurs under conditions that allow translation of the coding domain in the presence of the target nucleic acid but not in the absence of the target nucleic acid, and detecting the reporter protein as an indicator that the target pathogen nucleic acid is present in the amplified nucleic acids.   
     
     
         2 . The method of  claim 1 , wherein the pathogen detection agent is a norovirus detection agent and the target pathogen nucleic acid is norovirus RNA. 
     
     
         3 . The method of  claim 1 , wherein target pathogen nucleic acid is detected at concentrations in a range of zeptomoles/liter (zM). 
     
     
         4 . The method of  claim 1 , wherein target pathogen nucleic acid is detected at concentration between about 270 zM to about 270 aM. 
     
     
         5 . The method of  claim 1 , wherein the pathogen detection agent is a synbody. 
     
     
         6 . The method of  claim 5 , wherein the synbody comprises biotin. 
     
     
         7 . The method of  claim 6 , wherein the biotin-containing synbody is bound to a streptavidin-coated magnetic bead. 
     
     
         8 . The method of  claim 6 , wherein isolating comprises a magnetic capture assay. 
     
     
         9 . The method of  claim 1 , wherein the toehold switch encodes at least a portion of lacZ. 
     
     
         10 . The method of  claim 1 , wherein the toehold switch encodes lacZα and the amplified nucleic acids are contacted under conditions which promote formation of a lacZ tetramer. 
     
     
         11 . The method of  claim 10 , wherein lacZω is provided on a substrate to which the amplified nucleic acids are contacted. 
     
     
         12 . A synthetic norovirus-specific toehold switch sensor comprising a fully or partially double-stranded stem domain, a loop domain, a toehold domain, and at least a portion of a coding sequence of a reporter gene, wherein the toehold domain and at least a portion of the stem domain are complementary to a target norovirus RNA sequence. 
     
     
         13 . The toehold switch sensor of  claim 12 , comprising a RNA sequence selected from SEQ ID NOs:1-12. 
     
     
         14 . A kit for detecting a pathogen-associated nucleic acid, comprising a plurality of preserved test articles, a pathogen detection agent, a plurality of toehold switches that encode at least a portion of a reporter protein and comprise one or more single-stranded toehold sequence domains that are complementary to a target pathogen nucleic acid or the reverse complement thereof, and an electronic optical reader. 
     
     
         15 . The kit of  claim 14 , wherein the pathogen detection agent is a synbody. 
     
     
         16 . The kit of  claim 14 , wherein the plurality of preserved test articles comprises one or more selected from preserved paper test articles and preserved test tube articles. 
     
     
         17 . The kit of  claim 14 , further comprising instructions for performing the method of  claim 1 .