Development of a fast and sensitive RT-qPCR assay based on SYBR® green for diagnostic and quantification of Avian Nephritis Virus (ANV) in chickens affected with enteric disease
Abstract Background Enteric viruses are among the most prominent etiological agents of Runting-Stunting Syndrome (RSS). The Avian Nephritis Virus (ANV) is an astrovirus associated with enteric diseases in poultry, whose early diagnosis is essential for maintaining a good poultry breeding environment...
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| Format: | Article |
| Language: | English |
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BMC
2024-01-01
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| Series: | BMC Veterinary Research |
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| Online Access: | https://doi.org/10.1186/s12917-024-03881-8 |
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| author | Anthony Loor-Giler Sara Castillo-Reyes Silvana Santander-Parra Manuel Caza Nikolaos C. Kyriakidis Antonio J. Piantino Ferreira Luis Nuñez |
| author_facet | Anthony Loor-Giler Sara Castillo-Reyes Silvana Santander-Parra Manuel Caza Nikolaos C. Kyriakidis Antonio J. Piantino Ferreira Luis Nuñez |
| author_sort | Anthony Loor-Giler |
| collection | DOAJ |
| description | Abstract Background Enteric viruses are among the most prominent etiological agents of Runting-Stunting Syndrome (RSS). The Avian Nephritis Virus (ANV) is an astrovirus associated with enteric diseases in poultry, whose early diagnosis is essential for maintaining a good poultry breeding environment. ANV is an RNA virus that rapidly mutates, except for some conserved regions such as ORF1b. Therefore, the approach of a diagnostic method based on fast-RT-qPCR using SYBR® Green that focuses on the amplification of a fragment of ORF1b is presented as a feasible alternative for the diagnosis of this viral agent. In this study, the proposed assay showed a standard curve with an efficiency of 103.8% and a LoD and LoQ of 1 gene viral copies. The assay was specific to amplify the ORF 1b gene, and no amplification was shown from other viral genomes or in the negative controls. 200 enteric (feces) samples from chickens (broilers) and laying hens with signs of RSS from Ecuadorian poultry flocks were examined to validate the proposed method. Results Using our method, 164 positive results were obtained out of the total number of samples run, while the presence of viral RNA was detected in samples collected from one day to 44 weeks old in both avian lines. Conclusions Our study presents a novel, rapid, robust, and sensitive molecular assay capable of detecting and quantifying even low copy numbers of the ANV in commercial birds, therefore introducing a handy tool in the early diagnosis of ANV in enteric disease outbreaks in poultry. |
| first_indexed | 2024-03-07T14:56:59Z |
| format | Article |
| id | doaj.art-e96203206731484c8b4e5e25bb273919 |
| institution | Directory Open Access Journal |
| issn | 1746-6148 |
| language | English |
| last_indexed | 2024-03-07T14:56:59Z |
| publishDate | 2024-01-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Veterinary Research |
| spelling | doaj.art-e96203206731484c8b4e5e25bb2739192024-03-05T19:24:54ZengBMCBMC Veterinary Research1746-61482024-01-0120111110.1186/s12917-024-03881-8Development of a fast and sensitive RT-qPCR assay based on SYBR® green for diagnostic and quantification of Avian Nephritis Virus (ANV) in chickens affected with enteric diseaseAnthony Loor-Giler0Sara Castillo-Reyes1Silvana Santander-Parra2Manuel Caza3Nikolaos C. Kyriakidis4Antonio J. Piantino Ferreira5Luis Nuñez6Facultad de Ingeniería y Ciencias Aplicadas, Carrera de Ingeniería en Biotecnología, Universidad de Las Américas (UDLA)Facultad de Ciencias de La Salud, de Las Américas (UDLA)Facultad de Ciencias de La Salud, de Las Américas (UDLA)Facultad de Ciencias de La Salud, de Las Américas (UDLA)Facultad de Medicina, Cancer Research Group, Universidad de Las Américas (UDLA)Department of Pathology, School of Veterinary Medicine, University of Sao PauloFacultad de Ciencias de La Salud, de Las Américas (UDLA)Abstract Background Enteric viruses are among the most prominent etiological agents of Runting-Stunting Syndrome (RSS). The Avian Nephritis Virus (ANV) is an astrovirus associated with enteric diseases in poultry, whose early diagnosis is essential for maintaining a good poultry breeding environment. ANV is an RNA virus that rapidly mutates, except for some conserved regions such as ORF1b. Therefore, the approach of a diagnostic method based on fast-RT-qPCR using SYBR® Green that focuses on the amplification of a fragment of ORF1b is presented as a feasible alternative for the diagnosis of this viral agent. In this study, the proposed assay showed a standard curve with an efficiency of 103.8% and a LoD and LoQ of 1 gene viral copies. The assay was specific to amplify the ORF 1b gene, and no amplification was shown from other viral genomes or in the negative controls. 200 enteric (feces) samples from chickens (broilers) and laying hens with signs of RSS from Ecuadorian poultry flocks were examined to validate the proposed method. Results Using our method, 164 positive results were obtained out of the total number of samples run, while the presence of viral RNA was detected in samples collected from one day to 44 weeks old in both avian lines. Conclusions Our study presents a novel, rapid, robust, and sensitive molecular assay capable of detecting and quantifying even low copy numbers of the ANV in commercial birds, therefore introducing a handy tool in the early diagnosis of ANV in enteric disease outbreaks in poultry.https://doi.org/10.1186/s12917-024-03881-8RT-qPCREnteric virusesANVSYBR Greendiagnosis |
| spellingShingle | Anthony Loor-Giler Sara Castillo-Reyes Silvana Santander-Parra Manuel Caza Nikolaos C. Kyriakidis Antonio J. Piantino Ferreira Luis Nuñez Development of a fast and sensitive RT-qPCR assay based on SYBR® green for diagnostic and quantification of Avian Nephritis Virus (ANV) in chickens affected with enteric disease BMC Veterinary Research RT-qPCR Enteric viruses ANV SYBR Green diagnosis |
| title | Development of a fast and sensitive RT-qPCR assay based on SYBR® green for diagnostic and quantification of Avian Nephritis Virus (ANV) in chickens affected with enteric disease |
| title_full | Development of a fast and sensitive RT-qPCR assay based on SYBR® green for diagnostic and quantification of Avian Nephritis Virus (ANV) in chickens affected with enteric disease |
| title_fullStr | Development of a fast and sensitive RT-qPCR assay based on SYBR® green for diagnostic and quantification of Avian Nephritis Virus (ANV) in chickens affected with enteric disease |
| title_full_unstemmed | Development of a fast and sensitive RT-qPCR assay based on SYBR® green for diagnostic and quantification of Avian Nephritis Virus (ANV) in chickens affected with enteric disease |
| title_short | Development of a fast and sensitive RT-qPCR assay based on SYBR® green for diagnostic and quantification of Avian Nephritis Virus (ANV) in chickens affected with enteric disease |
| title_sort | development of a fast and sensitive rt qpcr assay based on sybr r green for diagnostic and quantification of avian nephritis virus anv in chickens affected with enteric disease |
| topic | RT-qPCR Enteric viruses ANV SYBR Green diagnosis |
| url | https://doi.org/10.1186/s12917-024-03881-8 |
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