title: Rapid label-free visual assay for the detection and quantification of viral RNA using peptide nucleic acid (PNA) and gold nanoparticles (AuNPs).
abstract: A rapid label-free visual assay for the detection of viral RNA using peptide nucleic acid (PNA) probes and gold nanoparticles (AuNPs) is presented in this study. Diagnosis is a crucial step for the molecular surveillance of diseases, and a rapid visual test with high specificity could play a vital role in the management of viral diseases. In this assay, the specific agglomerative behavior of PNA with gold nanoparticles was manipulated by its complementation with viral RNA. The assay was able to detect 5-10 ng of viral RNA from various biological samples, such as allantoic fluids, cell culture fluids and vaccines, in 100 μl of test solution. The developed assay was more sensitive than a hemagglutination (HA) test, a routine platform test for the detection of Newcastle disease virus (NDV), and the developed assay was able to visually detect NDV with as little as 0.25 HA units of virus. In terms of the specificity, the test could discriminate single nucleotide differences in the target RNA and hence could provide visual viral genotyping/pathotyping. This observation was confirmed by pathotyping different known isolates of NDV. Further, the PNA-induced colorimetric changes in the presence of the target RNA at different RNA to PNA ratios yielded a standard curve with a linear coefficient of R(2)=0.990, which was comparable to the value of R(2)=0.995 from real-time PCR experiments with the same viral RNA. Therefore, the viral RNA in a given samples could be quantified using a simple visual spectrophotometer available in any clinical laboratory. This assay may find application in diagnostic assays for other RNA viruses, which are well known to undergo mutations, thus presenting challenges for their molecular surveillance, genotyping and quantification.
authors.affiliation: CIF-Bioengineering, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar 243122, India.
authors.forename: Vinay G
authors.initials: VG
authors.lastname: Joshi
authors.forename: Kantaraja
authors.initials: K
authors.lastname: Chindera
authors.forename: Arvind Kumar
authors.initials: AK
authors.lastname: Singh
authors.forename: Aditya P
authors.initials: AP
authors.lastname: Sahoo
authors.forename: Vikas D
authors.initials: VD
authors.lastname: Dighe
authors.forename: Dimpal
authors.initials: D
authors.lastname: Thakuria
authors.forename: Ashok K
authors.initials: AK
authors.lastname: Tiwari
authors.forename: Satish
authors.initials: S
authors.lastname: Kumar
mesh_terms: D005838:Genotype; D006046:Gold / Q000737:chemistry*; D053768:Metal Nanoparticles / Q000737:chemistry*; D009522:Newcastle disease virus / Q000235:genetics; D020135:Peptide Nucleic Acids / Q000138:chemical synthesis / Q000737:chemistry*; D010641:Phenotype; D012367:RNA, Viral / Q000032:analysis*; D013053:Spectrophotometry
chemical_list: D020135:Peptide Nucleic Acids; D012367:RNA, Viral; D006046:Gold
keywords: Gold nanoparticles (AuNPs); Label-free assay; Peptide nucleic acid (PNA); Viral RNA quantification; Visual RNA detection