Rapid label-free visual assay for the detection and quantification of viral RNA using peptide nucleic acid (PNA) and gold nanoparticles (AuNPs)
•Simple and easy to use visual assay for viral diagnosis.•It is rapid and label-free assay with pen-side diagnostic applications.•The assay shows high sensitivity and specificity with ability to detect single nucleotide difference at target site visually.•Method demonstrates successful quantificatio...
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| Published in: | Analytica chimica acta Vol. 795; pp. 1 - 7 |
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| Main Authors: | , , , , , , , |
| Format: | Journal Article |
| Language: | English |
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Elsevier B.V
17.09.2013
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| ISSN: | 0003-2670, 1873-4324, 1873-4324 |
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| Abstract | •Simple and easy to use visual assay for viral diagnosis.•It is rapid and label-free assay with pen-side diagnostic applications.•The assay shows high sensitivity and specificity with ability to detect single nucleotide difference at target site visually.•Method demonstrates successful quantification of viral RNA.
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 5–10ng of viral RNA from various biological samples, such as allantoic fluids, cell culture fluids and vaccines, in 100μl 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 0.25HA 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 R2=0.990, which was comparable to the value of R2=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. |
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| AbstractList | 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 5–10ng of viral RNA from various biological samples, such as allantoic fluids, cell culture fluids and vaccines, in 100μl 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 0.25HA 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 R2=0.990, which was comparable to the value of R2=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. 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 5a10 ng of viral RNA from various biological samples, such as allantoic fluids, cell culture fluids and vaccines, in 100 mu l 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 R2 = 0.990, which was comparable to the value of R2 = 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. •Simple and easy to use visual assay for viral diagnosis.•It is rapid and label-free assay with pen-side diagnostic applications.•The assay shows high sensitivity and specificity with ability to detect single nucleotide difference at target site visually.•Method demonstrates successful quantification of viral RNA. 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 5–10ng of viral RNA from various biological samples, such as allantoic fluids, cell culture fluids and vaccines, in 100μl 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 0.25HA 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 R2=0.990, which was comparable to the value of R2=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. 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. 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.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. 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 (mu)l 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 geno-typing/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. |
| Author | Dighe, Vikas D. Tiwari, Ashok K. Chindera, Kantaraja Joshi, Vinay G. Thakuria, Dimpal Kumar, Satish Sahoo, Aditya P. Singh, Arvind Kumar |
| Author_xml | – sequence: 1 givenname: Vinay G. surname: Joshi fullname: Joshi, Vinay G. organization: CIF-Bioengineering, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar 243122, India – sequence: 2 givenname: Kantaraja surname: Chindera fullname: Chindera, Kantaraja organization: CIF-Bioengineering, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar 243122, India – sequence: 3 givenname: Arvind Kumar surname: Singh fullname: Singh, Arvind Kumar organization: CIF-Bioengineering, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar 243122, India – sequence: 4 givenname: Aditya P. surname: Sahoo fullname: Sahoo, Aditya P. organization: Molecular Biology Lab, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar 243122, India – sequence: 5 givenname: Vikas D. surname: Dighe fullname: Dighe, Vikas D. organization: CIF-Bioengineering, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar 243122, India – sequence: 6 givenname: Dimpal surname: Thakuria fullname: Thakuria, Dimpal organization: CIF-Bioengineering, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar 243122, India – sequence: 7 givenname: Ashok K. surname: Tiwari fullname: Tiwari, Ashok K. organization: Molecular Biology Lab, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar 243122, India – sequence: 8 givenname: Satish surname: Kumar fullname: Kumar, Satish email: drsatishkumar_ivri@yahoo.co.in organization: CIF-Bioengineering, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar 243122, India |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/23998531$$D View this record in MEDLINE/PubMed |
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| Copyright | 2013 Elsevier B.V. Copyright © 2013 Elsevier B.V. All rights reserved. |
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| Keywords | Visual RNA detection Peptide nucleic acid (PNA) Label-free assay Viral RNA quantification Gold nanoparticles (AuNPs) |
| Language | English |
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| SubjectTerms | Avian orthoavulavirus 1 cell culture colorimetry Genotype genotyping Gold - chemistry Gold nanoparticles (AuNPs) hemagglutination Label-free assay Metal Nanoparticles - chemistry monitoring mutation nanogold Newcastle disease virus Newcastle disease virus - genetics Peptide nucleic acid (PNA) Peptide Nucleic Acids - chemical synthesis Peptide Nucleic Acids - chemistry Phenotype quantitative polymerase chain reaction RNA RNA, Viral - analysis Spectrophotometry vaccines Viral RNA quantification viruses Visual RNA detection |
| Title | Rapid label-free visual assay for the detection and quantification of viral RNA using peptide nucleic acid (PNA) and gold nanoparticles (AuNPs) |
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