Accurate detection of Newcastle disease virus using proximity‐dependent DNA aptamer ligation assays
Detecting viral antigens at low concentrations in field samples can be crucial for early veterinary diagnostics. Proximity ligation assays (PLAs) in both solution and solid‐phase formats are widely used for high‐performance protein detection in medical research. However, the affinity reagents used,...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2021-04-01
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| Series: | FEBS Open Bio |
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| Online Access: | https://doi.org/10.1002/2211-5463.13117 |
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| author | Boutheina Marnissi Khouloud Khalfaoui Tonge Ebai Felipe Marques Souza de Oliveira Abdeljelil Ghram Masood Kamali‐Moghaddam Issam Hmila |
| author_facet | Boutheina Marnissi Khouloud Khalfaoui Tonge Ebai Felipe Marques Souza de Oliveira Abdeljelil Ghram Masood Kamali‐Moghaddam Issam Hmila |
| author_sort | Boutheina Marnissi |
| collection | DOAJ |
| description | Detecting viral antigens at low concentrations in field samples can be crucial for early veterinary diagnostics. Proximity ligation assays (PLAs) in both solution and solid‐phase formats are widely used for high‐performance protein detection in medical research. However, the affinity reagents used, which are mainly poly‐ and monoclonal antibodies, play an important role in the performance of PLAs. Here, we have established the first homogeneous and solid‐phase proximity‐dependent DNA aptamer ligation assays for rapid and accurate detection of Newcastle disease virus (NDV). NDV is detected by a pair of extended DNA aptamers that, upon binding in proximity to proteins on the envelope of the virus, are joined by enzymatic ligation to form a unique amplicon that can be sensitively detected using real‐time PCR. The sensitivity, specificity, and reproducibility of the assays were validated using 40 farm samples. The results demonstrated that the developed homogeneous and solid‐phase PLAs, which use NDV‐selective DNA aptamers, are more sensitive than the sandwich enzymatic‐linked aptamer assay (ELAA), and have a comparable sensitivity to real‐time reverse transcription PCR (rRT‐PCR) as the gold standard detection method. In addition, the solid‐phase PLA was shown to have a greater dynamic range with improved lower limit of detection, upper‐ and lower limit of quantification, and minimal detectable dose as compared with those of ELAA and rRT‐PCR. The specificity of PLA is shown to be concordant with rRT‐PCR. |
| first_indexed | 2024-12-20T08:55:59Z |
| format | Article |
| id | doaj.art-a428a097918d41d49ad0ee4198143363 |
| institution | Directory Open Access Journal |
| issn | 2211-5463 |
| language | English |
| last_indexed | 2024-12-20T08:55:59Z |
| publishDate | 2021-04-01 |
| publisher | Wiley |
| record_format | Article |
| series | FEBS Open Bio |
| spelling | doaj.art-a428a097918d41d49ad0ee41981433632022-12-21T19:46:00ZengWileyFEBS Open Bio2211-54632021-04-011141122113110.1002/2211-5463.13117Accurate detection of Newcastle disease virus using proximity‐dependent DNA aptamer ligation assaysBoutheina Marnissi0Khouloud Khalfaoui1Tonge Ebai2Felipe Marques Souza de Oliveira3Abdeljelil Ghram4Masood Kamali‐Moghaddam5Issam Hmila6Laboratory of Epidemiology and Veterinary Microbiology Institut Pasteur of TunisUniversity Tunis El Manar Tunis TunisiaLaboratory of Epidemiology and Veterinary Microbiology Institut Pasteur of TunisUniversity Tunis El Manar Tunis TunisiaDepartment of Immunology, Genetics and Pathology Science for Life Laboratory Uppsala University SwedenDepartment of Immunology, Genetics and Pathology Science for Life Laboratory Uppsala University SwedenLaboratory of Epidemiology and Veterinary Microbiology Institut Pasteur of TunisUniversity Tunis El Manar Tunis TunisiaDepartment of Immunology, Genetics and Pathology Science for Life Laboratory Uppsala University SwedenLaboratory of Epidemiology and Veterinary Microbiology Institut Pasteur of TunisUniversity Tunis El Manar Tunis TunisiaDetecting viral antigens at low concentrations in field samples can be crucial for early veterinary diagnostics. Proximity ligation assays (PLAs) in both solution and solid‐phase formats are widely used for high‐performance protein detection in medical research. However, the affinity reagents used, which are mainly poly‐ and monoclonal antibodies, play an important role in the performance of PLAs. Here, we have established the first homogeneous and solid‐phase proximity‐dependent DNA aptamer ligation assays for rapid and accurate detection of Newcastle disease virus (NDV). NDV is detected by a pair of extended DNA aptamers that, upon binding in proximity to proteins on the envelope of the virus, are joined by enzymatic ligation to form a unique amplicon that can be sensitively detected using real‐time PCR. The sensitivity, specificity, and reproducibility of the assays were validated using 40 farm samples. The results demonstrated that the developed homogeneous and solid‐phase PLAs, which use NDV‐selective DNA aptamers, are more sensitive than the sandwich enzymatic‐linked aptamer assay (ELAA), and have a comparable sensitivity to real‐time reverse transcription PCR (rRT‐PCR) as the gold standard detection method. In addition, the solid‐phase PLA was shown to have a greater dynamic range with improved lower limit of detection, upper‐ and lower limit of quantification, and minimal detectable dose as compared with those of ELAA and rRT‐PCR. The specificity of PLA is shown to be concordant with rRT‐PCR.https://doi.org/10.1002/2211-5463.13117aptamersNewcastle disease virusproximity ligation assaysrRT‐PCRsandwich ELAA |
| spellingShingle | Boutheina Marnissi Khouloud Khalfaoui Tonge Ebai Felipe Marques Souza de Oliveira Abdeljelil Ghram Masood Kamali‐Moghaddam Issam Hmila Accurate detection of Newcastle disease virus using proximity‐dependent DNA aptamer ligation assays FEBS Open Bio aptamers Newcastle disease virus proximity ligation assays rRT‐PCR sandwich ELAA |
| title | Accurate detection of Newcastle disease virus using proximity‐dependent DNA aptamer ligation assays |
| title_full | Accurate detection of Newcastle disease virus using proximity‐dependent DNA aptamer ligation assays |
| title_fullStr | Accurate detection of Newcastle disease virus using proximity‐dependent DNA aptamer ligation assays |
| title_full_unstemmed | Accurate detection of Newcastle disease virus using proximity‐dependent DNA aptamer ligation assays |
| title_short | Accurate detection of Newcastle disease virus using proximity‐dependent DNA aptamer ligation assays |
| title_sort | accurate detection of newcastle disease virus using proximity dependent dna aptamer ligation assays |
| topic | aptamers Newcastle disease virus proximity ligation assays rRT‐PCR sandwich ELAA |
| url | https://doi.org/10.1002/2211-5463.13117 |
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