Pathogen detection is crucial for human, animal, and environmental health; crop protection; and biosafety. Current culture-based methods have long turnaround times and lack sensitivity. Nucleic acid amplification tests offer high specificity and sensitivity. However, their cost and complexity remain a significant hurdle to their applications in resource-limited settings. Thus, point-of-need molecular diagnostic platforms that can be used by minimally trained personnel are needed. The nuclease protection assay (NPA) is a nucleic acid hybridization–based technique that does not rely on amplification, can be paired with other methods to improve specificity, and has the potential to be developed into a point-of-need device. In traditional NPAs, hybridization of an anti-sense probe to the target sequence is followed by single-strand nuclease digestion. The double-stranded target-probe hybrids are protected from nuclease digestion, precipitated, and visualized using autoradiography or other methods. We have developed a paper-based nuclease protection assay (PB-NPA) that can be implemented in field settings as the detection approach requires limited equipment and technical expertise. The PB-NPA uses a lateral flow format to capture the labeled target-probe hybrids onto a nitrocellulose membrane modified with an anti-label antibody. A colorimetric enzyme-substrate pair is used for signal visualization, producing a test line. The nuclease digestion of non-target and mismatched DNA provides high specificity while signal amplification with the reporter enzyme-substrate provides high sensitivity. We have also developed an on-chip sample pretreatment step utilizing chitosan-modified paper to eliminate possible interferents from the reaction and preconcentrate nucleic acids, thereby significantly reducing the need for auxiliary equipment.
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Tran NK, Wisner D, Albertson T, Cohen S, Greenhalgh D, Galante J, et al. Quantitative point-of-care pathogen detection in septicemia. Point Care. 2008;7:107–10.
Vidic J, Vizzini P, Manzano M, Kavanaugh D, Ramarao N, Zivkovic M, Radonic V, Knezevic N, Giouroudi I, Gadjanski I. Point-of-Need DNA Testing for detection of foodborne pathogenic bacteria. Sensors. 2019; https://doi.org/10.3390/s19051100.
Neethirajan S, Kobayashi I, Nakajima N, Wu D, Nandagopal S, Lin F. Microfluidics for food, agriculture and biosystems industries. Lab Chip. 2011;11:1574–86.
Koedrith P, Thasiphu T, Weon JI, Boonprasert R, Tuitemwong K, Tuitemwong P. Recent trends in rapid environmental monitoring of pathogens and toxicants: potential of nanoparticle-based biosensor and applications. Sci World J. 2015. https://doi.org/10.1155/2015/510982.
McLeish C, Nightingale P. Biosecurity, bioterrorism and the governance of science: the increasing convergence of science and security policy. Res Policy. 2007;37:1635–54.
Woolhouse ME, Haydon DT, Antia R. Emerging pathogens: the epidemiology and evolution of species jumps. Trends Ecol Evol. 2005;20:238–44.
Wade W. Unculturable bacteria—the uncharacterized organisms that cause oral infections. J Roy Soc Med. 2002;95:81–3.
Rodriguez NM, Wong WS, Liu L, Dewar R, Klapperich CM. A fully integrated paperfluidic molecular diagnostic chip for the extraction, amplification, and detection of nucleic acids from clinical samples. Lab Chip. 2016;16:753–63.
Connelly JT, Rolland JP, Whitesides GM. “Paper machine” for molecular diagnostics. Anal Chem. 2015;87:7595–601.
B.D. Systems, (2013), 510(k) summary BD ProbeTec™Trichomonas vaginalis (TV) Qx amplified DNA assay, (https://www.accessdata.fda.gov/cdrh_docs/reviews/K130268.pdf) accessed 29 January, 2020.
Kim HJ, Tong Y, Tang W, Quimson L, Cope VA, Pan X, et al. A rapid and simple isothermal nucleic acid amplification test for detection of herpes simplex virus types 1 and 2. J ClinVirol. 2011;50:26–30.
Chang CC, Chen CC, Wei SC, Lu HH, Liang YH, Lin CW. Diagnostic devices for isothermal nucleic acid amplification. Sensors. 2012;12:8319–37.
Stults JR, Snoeyenbos-West O, Methe B, Lovley DR, Chandler DP. Application of the 5′ fluorogenic exonuclease assay (TaqMan) for quantitative ribosomal DNA and rRNA analysis in sediments. Appl Environ Microbiol. 2001;67:2781–9.
Filer JE, Channon RB, Henry CS, Geiss BJ. A nuclease protection ELISA assay for colorimetric and electrochemical detection of nucleic acids. Anal Methods. 2019;11:1027–34.
Cai Q, Li R, Zhen Y, Mi T, Yu Z. Detection of two Prorocentrum species using sandwich hybridization integrated with nuclease protection assay. Harmful Algae. 2006;5:300–9.
Yu Z, Tiezhu M, Zhigang Y. Detection of Phaeocystis globosa using sandwich hybridization integrated with nuclease protection assay (NPA-SH). J Environ Sci. 2008;20:1481–6.
Zhen Y, Yu Z, Cai Q, Mi T, Li R. Detection of two diatoms using sandwich hybridization integrated with nuclease protection assay (NPA-SH). Hydrobiologia. 2007;575:1–11.
Park M, Park SY, Hwang J, Jung SW, Lee J, Chang M, et al. Integration of the nuclease protection assay with sandwich hybridization (NPA-SH) for sensitive detection of Heterocapsa triquetra. Acta Oceanol Sin. 2018;37:107–12.
Labbe A, Pillai D, Hongvangthong B, Vanisaveth V, Pomphida S, Inkathone S, et al. The performance and utility of rapid diagnostic assays for Plasmodium falciparum malaria in a field setting in the Lao People's Democratic Republic. Ann Trop Med Parasit. 2001;95:671–7.
Engler K, Efstratiou A, Norn D, Kozlov R, Selga I, Glushkevich T, et al. Immunochromatographic strip test for rapid detection of diphtheria toxin: description and multicenter evaluation in areas of low and high prevalence of diphtheria. J Clin Microbiol. 2002;40:80–3.
Aveyard J, Mehrabi M, Cossins A, Braven H, Wilson R. One step visual detection of PCR products with gold nanoparticles and a nucleic acid lateral flow (NALF) device. Chem Commun. 2007:4251–3.
Cordray MS, Richards-Kortum RR. A paper and plastic device for the combined isothermal amplification and lateral flow detection of Plasmodium DNA. Malar J. 2015. https://doi.org/10.1186/s12936-015-0995-6.
Jaroenram W, Kiatpathomchai W, Flegel TW. Rapid and sensitive detection of white spot syndrome virus by loop-mediated isothermal amplification combined with a lateral flow dipstick. Mol Cell Probes. 2009;23:65–70.
Kersting S, Rausch V, Bier FF, Nickisch-Rosenegk Mv. Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis. Malar J. 2014; https://doi.org/10.1186/1475-2875-13-99.
Jauset-Rubio M, Svobodová M, Mairal T, McNeil C, Keegan N, Saeed A, et al. Ultrasensitive, rapid and inexpensive detection of DNA using paper based lateral flow assay. Sci Rep. 2016. https://doi.org/10.1038/srep37732.
Bonner J, Kung G, Bekhor I. A method for the hybridization of nucleic acid molecules at low temperature. Biochemistry. 1967;6:3650–3.
Zhang DY, Chen SX, Yin P. Optimizing the specificity of nucleic acid hybridization. Nat Chem. 2012;4:208–14.
McConaughy BL, Laird C, McCarthy BJ. Nucleic acid reassociation in formamide. Biochemistry. 1969;8:3289–95.
Rehan M, Younus H. Effect of organic solvents on the conformation and interaction of catalase and anticatalase antibodies. Int J Biol Macromol. 2006;38:289–95.
Caruccio L, Byrne K, Procter J, Stroncek D. A novel method using formamide for the elution of antibodies from erythrocytes. Vox Sang. 2002;83:63–9.
Desai NA, Shankar V. Single-strand-specific nucleases. FEMS Microbiol Rev. 2003;26:457–91.
Rimsza LM, Wright G, Schwartz M, Chan WC, Jaffe ES, Gascoyne RD, et al. Accurate classification of diffuse large B-cell lymphoma into germinal center and activated B-cell subtypes using a nuclease protection assay on formalin-fixed, paraffin-embedded tissues. Clin Cancer Res. 2011;17:3727–32.
Martel RR, Botros IW, Rounseville MP, Hinton JP, Staples RR, Morales DA, et al. Multiplexed screening assay for mRNA combining nuclease protection with luminescent array detection. Assay Drug Dev Technol. 2002;1:61–71.
Till BJ, Burtner C, Comai L, Henikoff S. Mismatch cleavage by single-strand specific nucleases. Nucleic Acids Res. 2004;32:2632–41.
Shahidi F, Abuzaytoun R. Chitin, chitosan, and co-products: chemistry, production, applications, and health effects. Adv Food Nutr Res. 2005;49:93–137.
Layek B, Singh J. Chitosan for DNA and gene therapy. In: Jennings JA, Bumgradner JD. Chitosan based biomaterials Volume 2. Amsterdam: Elsevier; 2017. pp. 209–244.
Bozkir A, Saka OM. Chitosan–DNA nanoparticles: effect on DNA integrity, bacterial transformation and transfection efficiency. J Drug Target. 2004;12:281–8.
Chen X, Cui DF. Microfluidic devices for sample pretreatment and applications. Microsyst Technol. 2009;15:667–76.
Díaz-González M, de la Escosura-Muñiz A, González-García MB, Costa-García A. DNA hybridization biosensors using polylysine modified SPCEs. Biosens Bioelectron. 2008;23:1340–6.
Byrnes S, Bishop J, Lafleur L, Buser J, Lutz B, Yager P. One-step purification and concentration of DNA in porous membranes for point-of-care applications. Lab Chip. 2015;15:2647–59.
Schlappi TS, McCalla SE, Schoepp NG, Ismagilov RF. Flow-through capture and in situ amplification can enable rapid detection of a few single molecules of nucleic acids from several milliliters of solution. Anal Chem. 2016;88:7647–53.
Kidd-Ljunggren K, Holmberg A, Bläckberg J, Lindqvist B. High levels of hepatitis B virus DNA in body fluids from chronic carriers. J Hosp Infect. 2006;64:352–7.
Shike H, Shimizu C, Kanegaye J, Foley JL, Burns JC. Quantitation of adenovirus genome during acute infection in normal children. Pediatr Infect Dis J. 2005;24:29–33.
The authors would like to thank Dr. John Wydallis for technical assistance. We also would like to thank the members of the Henry and Geiss labs for the helpful comments and discussions.
This work was supported by Colorado State University to CSH, DSD, and BJG and the National Institutes of Health (R01 AI132668) to BJG.
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Noviana, E., Jain, S., Hofstetter, J. et al. Paper-based nuclease protection assay with on-chip sample pretreatment for point-of-need nucleic acid detection. Anal Bioanal Chem 412, 3051–3061 (2020). https://doi.org/10.1007/s00216-020-02569-w
- Nuclease protection assay
- Nucleic acid detection
- Paper-based device
- Lateral flow assay