Development of a Sensitive Real-Time Fast-qPCR Based on SYBR® Green for Detection and Quantification of Chicken Parvovirus (ChPV)
Many viruses have been associated with runting and stunting syndrome (RSS). These viral infections mainly affect young chickens, causing apathy, depression, ruffled feathers, cloacal pasting, and diarrhea. Chicken Parvovirus (ChPV) is such an infection and has been detected in chickens showing signs...
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| Format: | Article |
| Language: | English |
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MDPI AG
2018-07-01
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| Series: | Veterinary Sciences |
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| Online Access: | http://www.mdpi.com/2306-7381/5/3/69 |
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| author | Luis F. Nuñez Silvana H. Santander-Parra Lucas Chaible David I. De la Torre Marcos R. Buim Alexandre Murakami Maria Lucia Zaidan Dagli Claudete S. Astolfi-Ferreira Antonio J. Piantino Ferreira |
| author_facet | Luis F. Nuñez Silvana H. Santander-Parra Lucas Chaible David I. De la Torre Marcos R. Buim Alexandre Murakami Maria Lucia Zaidan Dagli Claudete S. Astolfi-Ferreira Antonio J. Piantino Ferreira |
| author_sort | Luis F. Nuñez |
| collection | DOAJ |
| description | Many viruses have been associated with runting and stunting syndrome (RSS). These viral infections mainly affect young chickens, causing apathy, depression, ruffled feathers, cloacal pasting, and diarrhea. Chicken Parvovirus (ChPV) is such an infection and has been detected in chickens showing signs of enteric diseases worldwide. Therefore, the present study aims to develop a sensitive real-time fast-qPCR assay based on SYBR® Green for detection and quantification of ChPV. A 561-bp non-structural (NS) gene was amplified and cloned, and a pair of primers was designed based on conserved nucleotide sequences on the NS gene of ChPV, the intercalating DNA reagent SYBR® Green was employed, and the Fast mode of a thermocycler was used. The assay detects 109 to 101 copies of the genome (CG). The limit of detection (LoD) was estimated to five CG, and the limit of quantification (LoQ) was estimated at ten CG. The standard curve efficiency was 101.94%, and the melting curve showed a unique clean peak and a melting temperature of 79.3 °C. The assay was specific to amplify the ChPV NS gene, and no amplification was shown from other viral genomes or in the negative controls. A total of 141 samples were tested using the assay, of which 139 samples were found positive. The highest CG value of ChPV was 5.7 × 106 CG/uL of DNA without apparent clinical signs of enteric disturbance, and 4.6 × 106 CG/uL DNA were detected in chickens with RSS. |
| first_indexed | 2024-12-21T15:02:45Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 2306-7381 |
| language | English |
| last_indexed | 2024-12-21T15:02:45Z |
| publishDate | 2018-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Veterinary Sciences |
| spelling | doaj.art-b0327efacbfb4ab0a126c908a3ec3b152022-12-21T18:59:32ZengMDPI AGVeterinary Sciences2306-73812018-07-01536910.3390/vetsci5030069vetsci5030069Development of a Sensitive Real-Time Fast-qPCR Based on SYBR® Green for Detection and Quantification of Chicken Parvovirus (ChPV)Luis F. Nuñez0Silvana H. Santander-Parra1Lucas Chaible2David I. De la Torre3Marcos R. Buim4Alexandre Murakami5Maria Lucia Zaidan Dagli6Claudete S. Astolfi-Ferreira7Antonio J. Piantino Ferreira8Department of Pathology, School of Veterinary Medicine, University of São Paulo (USP), Av. Prof. Dr. Orlando M. Paiva, 87, CEP 05508-270 São Paulo, BrazilDepartment of Pathology, School of Veterinary Medicine, University of São Paulo (USP), Av. Prof. Dr. Orlando M. Paiva, 87, CEP 05508-270 São Paulo, BrazilDepartment of Pathology, School of Veterinary Medicine, University of São Paulo (USP), Av. Prof. Dr. Orlando M. Paiva, 87, CEP 05508-270 São Paulo, BrazilDepartment of Pathology, School of Veterinary Medicine, University of São Paulo (USP), Av. Prof. Dr. Orlando M. Paiva, 87, CEP 05508-270 São Paulo, BrazilBiological Institute, Av. Gaspar Ricardo, 1700, CEP 17690-000 Bastos, BrazilDepartment of Pathology, School of Veterinary Medicine, University of São Paulo (USP), Av. Prof. Dr. Orlando M. Paiva, 87, CEP 05508-270 São Paulo, BrazilDepartment of Pathology, School of Veterinary Medicine, University of São Paulo (USP), Av. Prof. Dr. Orlando M. Paiva, 87, CEP 05508-270 São Paulo, BrazilDepartment of Pathology, School of Veterinary Medicine, University of São Paulo (USP), Av. Prof. Dr. Orlando M. Paiva, 87, CEP 05508-270 São Paulo, BrazilDepartment of Pathology, School of Veterinary Medicine, University of São Paulo (USP), Av. Prof. Dr. Orlando M. Paiva, 87, CEP 05508-270 São Paulo, BrazilMany viruses have been associated with runting and stunting syndrome (RSS). These viral infections mainly affect young chickens, causing apathy, depression, ruffled feathers, cloacal pasting, and diarrhea. Chicken Parvovirus (ChPV) is such an infection and has been detected in chickens showing signs of enteric diseases worldwide. Therefore, the present study aims to develop a sensitive real-time fast-qPCR assay based on SYBR® Green for detection and quantification of ChPV. A 561-bp non-structural (NS) gene was amplified and cloned, and a pair of primers was designed based on conserved nucleotide sequences on the NS gene of ChPV, the intercalating DNA reagent SYBR® Green was employed, and the Fast mode of a thermocycler was used. The assay detects 109 to 101 copies of the genome (CG). The limit of detection (LoD) was estimated to five CG, and the limit of quantification (LoQ) was estimated at ten CG. The standard curve efficiency was 101.94%, and the melting curve showed a unique clean peak and a melting temperature of 79.3 °C. The assay was specific to amplify the ChPV NS gene, and no amplification was shown from other viral genomes or in the negative controls. A total of 141 samples were tested using the assay, of which 139 samples were found positive. The highest CG value of ChPV was 5.7 × 106 CG/uL of DNA without apparent clinical signs of enteric disturbance, and 4.6 × 106 CG/uL DNA were detected in chickens with RSS.http://www.mdpi.com/2306-7381/5/3/69chickenparvovirusreal timeqPCRSYBR® Green |
| spellingShingle | Luis F. Nuñez Silvana H. Santander-Parra Lucas Chaible David I. De la Torre Marcos R. Buim Alexandre Murakami Maria Lucia Zaidan Dagli Claudete S. Astolfi-Ferreira Antonio J. Piantino Ferreira Development of a Sensitive Real-Time Fast-qPCR Based on SYBR® Green for Detection and Quantification of Chicken Parvovirus (ChPV) Veterinary Sciences chicken parvovirus real time qPCR SYBR® Green |
| title | Development of a Sensitive Real-Time Fast-qPCR Based on SYBR® Green for Detection and Quantification of Chicken Parvovirus (ChPV) |
| title_full | Development of a Sensitive Real-Time Fast-qPCR Based on SYBR® Green for Detection and Quantification of Chicken Parvovirus (ChPV) |
| title_fullStr | Development of a Sensitive Real-Time Fast-qPCR Based on SYBR® Green for Detection and Quantification of Chicken Parvovirus (ChPV) |
| title_full_unstemmed | Development of a Sensitive Real-Time Fast-qPCR Based on SYBR® Green for Detection and Quantification of Chicken Parvovirus (ChPV) |
| title_short | Development of a Sensitive Real-Time Fast-qPCR Based on SYBR® Green for Detection and Quantification of Chicken Parvovirus (ChPV) |
| title_sort | development of a sensitive real time fast qpcr based on sybr r green for detection and quantification of chicken parvovirus chpv |
| topic | chicken parvovirus real time qPCR SYBR® Green |
| url | http://www.mdpi.com/2306-7381/5/3/69 |
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