Abstract
Early-stage detection is essential for effective treatment of pediatric virus infections. In traditional immuno-PCR, a single antibody recognition event is associated with one to three DNA tags, which are subsequently amplified by PCR. In this protocol, we describe a nanoparticle-amplified immuno-PCR assay that combines antibody recognition of traditional ELISA with a 50-fold nanoparticle valence amplification step followed by amplification by traditional PCR. The assay detects a respiratory syncytial virus (RSV) surface fusion protein using a Synagis antibody bound to a 15 nm gold nanoparticle co-functionalized with thiolated DNA complementary to a hybridized 76-base Tag DNA. The Tag DNA to Synagis ratio is 50 to 1. The presence of virus particles triggers the formation of a “sandwich” complex comprised of the gold nanoparticle construct, virus, and a 1 μm antibody-functionalized magnetic particle used for extraction. Virus-containing complexes are isolated using a magnet, DNA tags released by heating to 95 °C, and detected via real-time PCR. The limit of detection of the nanoparticle-amplified immuno-PCR assay was compared to traditional ELISA and traditional RT-PCR using RSV-infected HEp-2 cell extracts. Nanoparticle-amplified immuno-PCR showed a ∼4,000-fold improvement in the limit of detection compared to ELISA and a fourfold improvement in the limit of detection compared to traditional RT-PCR. Nanoparticle-amplified immuno-PCR offers a viable platform for the development of an early-stage diagnostics requiring an exceptionally low limit of detection.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Robert CW (2003) Review of epidemiology and clinical risk factors for severe respiratory syncytial virus (RSV) infection. J Pediatr 143:112–117
Shay DK, Holman RC, Newman RD, Liu LL, Stout JW, Anderson LJ (1999) Bronchiolitis-associated hospitalizations among US children, 1980–1996. J Am Med Assoc 282:1440–1446
Falsey AR, Walsh EE (2005) Respiratory syncytial virus infection in elderly adults. Drugs Aging 22:577–587
Falsey AR, Hennessey PA, Formica MA, Cox C, Walsh EE (2005) Respiratory syncytial virus infection in elderly and high-risk adults. N Engl J Med 352:1749–1759
Hall CB (2001) Respiratory syncytial virus and parainfluenza virus. N Engl J Med 344:1917–1928
Moore E, Barber J, Tripp R (2008) Respiratory syncytial virus (RSV) attachment and nonstructural proteins modify the type I interferon response associated with suppressor of cytokine signaling (SOCS) proteins and IFN-stimulated gene-15 (ISG15). Virol J 5:116
Tripp RA (2004) Pathogenesis of respiratory syncytial virus infection. Viral Immunol 17:165–181
Chin J, Magoffin RL, Shearer LA, Schieble JH, Lennette EH (1969) Field evaluation of a respiratory syncytial virus vaccine and a trivalent parainfluenza virus vaccine in a pediatric population. Am J Epidemiol 89:449–463
Kapikian AZ, Mitchell RH, Chanock RM, Shvedoff RA, Stewart CE (1969) An epidemiologic study of altered clinical reactivity to respiratory syncytial (RS) virus infection in children previously vaccinated with an inactivated RS virus vaccine. Am J Epidemiol 89:405–421
Kim HW, Canchola JG, Brandt CD, Pyles G, Chanock RM, Jensen K, Parrott RH (1969) Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. Am J Epidemiol 89:422–434
Kaur J, Tang RS, Spaete RR, Schickli JH (2008) Optimization of plasmid-only rescue of highly attenuated and temperature-sensitive respiratory syncytial virus (RSV) vaccine candidates for human trials. J Virol Methods 153:196–202
Committee on Infectious Diseases, Committee on Fetus and Newborn (1998) Prevention of respiratory syncytial virus infections: indications for the use of palivizumab and update on the use of RSV-IGIV. Pediatrics 102:1211–1216
Meissner HC, Bocchini JA Jr, Brady MT, Hall CB, Kimberlin DW, Pickering LK (2009) The role of immunoprophylaxis in the reduction of disease attributable to respiratory syncytial virus. Pediatrics 124:1676–1679
Committee on Infectious Diseases (1996) Reassessment of the indications for ribavirin therapy in respiratory syncytial virus infections. Pediatrics 97:137–140
Goodrich JS, Miller MB (2007) Comparison of Cepheid’s analyte-specific reagents with BD directigen for detection of respiratory syncytial virus. J Clin Microbiol 45:604–606
Perkins SM, Webb DL, Torrance SA, El Saleeby C, Harrison LM, Aitken JA, Patel A, DeVincenzo JP (2005) Comparison of a real-time reverse transcriptase PCR assay and a culture technique for quantitative assessment of viral load in children naturally infected with respiratory syncytial virus. J Clin Microbiol 43:2356–2362
Chomczynski P (1992) Solubilization in formamide protects RNA from degradation. Nucleic Acids Res 20:3791–3792
Hargrove JL, Schmidt FH (1989) The role of mRNA and protein stability in gene expression. FASEB J 3:2360–2370
Mahony JB (2008) Detection of respiratory viruses by molecular methods. Clin Microbiol Rev 21:716–747
Bentzen EL, House F, Utley TJ, Crowe JE, Wright DW (2005) Progression of respiratory syncytial virus infection monitored by fluorescent quantum dot probes. Nano Lett 5:591–595
Adler M, Schulz S, Fischer R, Niemeyer CM (2005) Detection of Rotavirus from stool samples using a standardized immuno-PCR (“Imperacer”) method with end-point and real-time detection. Biochem Biophys Res Commun 333:1289–1294
Barletta J, Bartolome A (2007) Immuno-polymerase chain reaction as a unique molecular tool for detection of infectious agents. Expert Opin Med Diagn 1:267–288
Stowers CC, Haselton FR, Boczko EM (2010) An analysis of quantitative PCR reliability through replicates using the Ct method. J Biomed Sci Eng 3:459–469
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Perez, J.W., Adams, N.M., Zimmerman, G.R., Haselton, F.R., Wright, D.W. (2013). Detecting Respiratory Syncytial Virus Using Nanoparticle-Amplified Immuno-PCR. In: Rosenthal, S., Wright, D. (eds) NanoBiotechnology Protocols. Methods in Molecular Biology, vol 1026. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-468-5_8
Download citation
DOI: https://doi.org/10.1007/978-1-62703-468-5_8
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-467-8
Online ISBN: 978-1-62703-468-5
eBook Packages: Springer Protocols