Anal Chim Acta. 2020 Jan 6;1093:123-130. doi: 10.1016/j.aca.2019.09.060. Epub 2019 Sep 25. Label-free peptide nucleic acid biosensor for visual detection of multiple strains of influenza A virus suitable for field applications.

Kumar N1, Bhatia S2, Pateriya AK2, Sood R2, Nagarajan S3, Murugkar HV3, Kumar S4, Singh P5, Singh VP2.
Author information

1 Diagnostics & Vaccines Section, ICAR-National Institute of High Security Animal Diseases, Bhopal, 462022, India. Electronic address: navyog.yadav84@gmail.com. 2 Diagnostics & Vaccines Section, ICAR-National Institute of High Security Animal Diseases, Bhopal, 462022, India. 3 Avian Diseases Section, ICAR-National Institute of High Security Animal Diseases, Bhopal, 462022, India. 4 Central Instrumentation Facility- Bioengineering, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, 243122, India. 5 Central Instrumentation Facility- Bioengineering, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, 243122, India; Biophysics and Electron Microscopy Section, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, 243122, India.

Abstract

Accurate and rapid diagnosis of Influenza A viruses (IAVs) is challenging because of multiple strains circulating in humans and animal populations, and the emergence of new strains. In this study, we demonstrate a simple and rapid strategy for visual detection of multiple strains of IAVs (H1 to H16 subtypes) using peptide nucleic acid (PNA) as a biosensor and unmodified gold nanoparticles (AuNPs) as a reporter. The design principle of the assay is based on the color change on account of free PNA-induced aggregation of AuNPs in the presence of non-complementary viral RNA sequence and vice-versa. The assay could detect IAV RNA with a visual limit of detection of 2.3 ng. The quantification of RNA with a considerable accuracy on a simple spectrophotometer was achieved on plotting the PNA-induced colorimetric changes (absorption ratio of A640/A520) in the presence of a varying concentration of complementary RNA. As a proof-of-concept, the visual assay was validated on 419 avian clinical samples and receiver operating characteristic (ROC) curve analysis showed a high diagnostic specificity (96.46%, 95% CI = 93.8 to 98.2) and sensitivity (82.41%, 95% CI = 73.9 to 89.1) when RT-qPCR was used as reference test. Hence, the simplicity, rapidity, and universality of this strategy make it a potential candidate visual assay for clinical diagnosis and surveillance of IAVs, especially in the resource-limited settings. The proposed strategy establishes new avenues for developing a simple and rapid diagnostic system for viral infections and biomolecules.
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KEYWORDS:

Gold nanoparticles; Influenza A viruses; Label-free visual assay; Peptide nucleic acid

PMID: 31735205 DOI: 10.1016/j.aca.2019.09.060