Development of a sensitive diagnostic assay for fish nervous necrosis virus based on RT-PCR plus nested PCR
A polymerase chain reaction (PCR)-based assay to detect nervous necrosis virus (NNV) in fish was developed by using two sets of primers designed on a highly conserved region of the coat protein gene encoded by RNA2 of NNV. The first pair of primers amplified a fragment of 605 bp by one-step reverse-...
Ausführliche Beschreibung
Gespeichert in:
| Autor*in: |
Dalla Valle, L [verfasserIn] Zanella, L [verfasserIn] Patarnello, P [verfasserIn] |
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| Format: |
E-Artikel |
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| Erschienen: |
Oxford, UK: Blackwell Science Ltd ; 2000 |
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| Umfang: |
Online-Ressource |
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| Reproduktion: |
2009 ; Blackwell Publishing Journal Backfiles 1879-2005 |
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| Übergeordnetes Werk: |
In: Journal of fish diseases - Oxford [u.a.] : Wiley-Blackwell, 1978, 23(2000), 5, Seite 0 |
| Übergeordnetes Werk: |
volume:23 ; year:2000 ; number:5 ; pages:0 |
| Links: |
|---|
| DOI / URN: |
10.1046/j.1365-2761.2000.00255.x |
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NLEJ243234902 |
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| 520 | |a A polymerase chain reaction (PCR)-based assay to detect nervous necrosis virus (NNV) in fish was developed by using two sets of primers designed on a highly conserved region of the coat protein gene encoded by RNA2 of NNV. The first pair of primers amplified a fragment of 605 bp by one-step reverse-transcription (RT)-PCR, while the second pair amplified an internal segment of 255 bp by nested PCR. Addition of nested PCR increased the assay sensitivity 100-fold when carried out in a separate tube (two-step assay) and 10-fold when performed in the same tube (one-step assay). The sensitivity of the two-step assay was 104 times higher than that of virus cultivation. Nested PCR served also to confirm the specificity of the first amplification, as verified also by Southern hybridization analysis and direct sequencing. In species known to be susceptible to infection, such as European sea bass, Dicentrarchus labrax, and gilthead seabream, Sparus aurata, NNV was often detectable in brain tissue by RT-PCR alone but only by the two-step assay in blood, sperm, ovarian tissue or larvae. The same was true for sperm and ovarian tissue of shi drum, Umbrina cirrosa. NNV was also detected in the brains of Japanese red seabream, Pagrus major and brown meagre, Sciaena umbra, suggesting that these species can also be infected. No NNV was detected in samples of Artemia salina nauplii and rotifers obtained from a fish farm with an NNV outbreak. The inclusion of nested PCR in the assay appears to be necessary to screen out NNV-positive broodfish by blood sampling and testing of their larval progeny. | ||
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10.1046/j.1365-2761.2000.00255.x doi (DE-627)NLEJ243234902 DE-627 ger DE-627 rakwb Dalla Valle, L verfasserin aut Development of a sensitive diagnostic assay for fish nervous necrosis virus based on RT-PCR plus nested PCR Oxford, UK Blackwell Science Ltd 2000 Online-Ressource nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier A polymerase chain reaction (PCR)-based assay to detect nervous necrosis virus (NNV) in fish was developed by using two sets of primers designed on a highly conserved region of the coat protein gene encoded by RNA2 of NNV. The first pair of primers amplified a fragment of 605 bp by one-step reverse-transcription (RT)-PCR, while the second pair amplified an internal segment of 255 bp by nested PCR. Addition of nested PCR increased the assay sensitivity 100-fold when carried out in a separate tube (two-step assay) and 10-fold when performed in the same tube (one-step assay). The sensitivity of the two-step assay was 104 times higher than that of virus cultivation. Nested PCR served also to confirm the specificity of the first amplification, as verified also by Southern hybridization analysis and direct sequencing. In species known to be susceptible to infection, such as European sea bass, Dicentrarchus labrax, and gilthead seabream, Sparus aurata, NNV was often detectable in brain tissue by RT-PCR alone but only by the two-step assay in blood, sperm, ovarian tissue or larvae. The same was true for sperm and ovarian tissue of shi drum, Umbrina cirrosa. NNV was also detected in the brains of Japanese red seabream, Pagrus major and brown meagre, Sciaena umbra, suggesting that these species can also be infected. No NNV was detected in samples of Artemia salina nauplii and rotifers obtained from a fish farm with an NNV outbreak. The inclusion of nested PCR in the assay appears to be necessary to screen out NNV-positive broodfish by blood sampling and testing of their larval progeny. 2009 Blackwell Publishing Journal Backfiles 1879-2005 |2009|||||||||| Zanella, L verfasserin aut Patarnello, P verfasserin aut Paolucci, L oth Belvedere, P oth Colombo, L oth In Journal of fish diseases Oxford [u.a.] : Wiley-Blackwell, 1978 23(2000), 5, Seite 0 Online-Ressource (DE-627)NLEJ243926952 (DE-600)2020444-9 1365-2761 nnns volume:23 year:2000 number:5 pages:0 http://dx.doi.org/10.1046/j.1365-2761.2000.00255.x text/html Verlag Deutschlandweit zugänglich Volltext GBV_USEFLAG_U ZDB-1-DJB GBV_NL_ARTICLE AR 23 2000 5 0 |
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10.1046/j.1365-2761.2000.00255.x doi (DE-627)NLEJ243234902 DE-627 ger DE-627 rakwb Dalla Valle, L verfasserin aut Development of a sensitive diagnostic assay for fish nervous necrosis virus based on RT-PCR plus nested PCR Oxford, UK Blackwell Science Ltd 2000 Online-Ressource nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier A polymerase chain reaction (PCR)-based assay to detect nervous necrosis virus (NNV) in fish was developed by using two sets of primers designed on a highly conserved region of the coat protein gene encoded by RNA2 of NNV. The first pair of primers amplified a fragment of 605 bp by one-step reverse-transcription (RT)-PCR, while the second pair amplified an internal segment of 255 bp by nested PCR. Addition of nested PCR increased the assay sensitivity 100-fold when carried out in a separate tube (two-step assay) and 10-fold when performed in the same tube (one-step assay). The sensitivity of the two-step assay was 104 times higher than that of virus cultivation. Nested PCR served also to confirm the specificity of the first amplification, as verified also by Southern hybridization analysis and direct sequencing. In species known to be susceptible to infection, such as European sea bass, Dicentrarchus labrax, and gilthead seabream, Sparus aurata, NNV was often detectable in brain tissue by RT-PCR alone but only by the two-step assay in blood, sperm, ovarian tissue or larvae. The same was true for sperm and ovarian tissue of shi drum, Umbrina cirrosa. NNV was also detected in the brains of Japanese red seabream, Pagrus major and brown meagre, Sciaena umbra, suggesting that these species can also be infected. No NNV was detected in samples of Artemia salina nauplii and rotifers obtained from a fish farm with an NNV outbreak. The inclusion of nested PCR in the assay appears to be necessary to screen out NNV-positive broodfish by blood sampling and testing of their larval progeny. 2009 Blackwell Publishing Journal Backfiles 1879-2005 |2009|||||||||| Zanella, L verfasserin aut Patarnello, P verfasserin aut Paolucci, L oth Belvedere, P oth Colombo, L oth In Journal of fish diseases Oxford [u.a.] : Wiley-Blackwell, 1978 23(2000), 5, Seite 0 Online-Ressource (DE-627)NLEJ243926952 (DE-600)2020444-9 1365-2761 nnns volume:23 year:2000 number:5 pages:0 http://dx.doi.org/10.1046/j.1365-2761.2000.00255.x text/html Verlag Deutschlandweit zugänglich Volltext GBV_USEFLAG_U ZDB-1-DJB GBV_NL_ARTICLE AR 23 2000 5 0 |
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10.1046/j.1365-2761.2000.00255.x doi (DE-627)NLEJ243234902 DE-627 ger DE-627 rakwb Dalla Valle, L verfasserin aut Development of a sensitive diagnostic assay for fish nervous necrosis virus based on RT-PCR plus nested PCR Oxford, UK Blackwell Science Ltd 2000 Online-Ressource nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier A polymerase chain reaction (PCR)-based assay to detect nervous necrosis virus (NNV) in fish was developed by using two sets of primers designed on a highly conserved region of the coat protein gene encoded by RNA2 of NNV. The first pair of primers amplified a fragment of 605 bp by one-step reverse-transcription (RT)-PCR, while the second pair amplified an internal segment of 255 bp by nested PCR. Addition of nested PCR increased the assay sensitivity 100-fold when carried out in a separate tube (two-step assay) and 10-fold when performed in the same tube (one-step assay). The sensitivity of the two-step assay was 104 times higher than that of virus cultivation. Nested PCR served also to confirm the specificity of the first amplification, as verified also by Southern hybridization analysis and direct sequencing. In species known to be susceptible to infection, such as European sea bass, Dicentrarchus labrax, and gilthead seabream, Sparus aurata, NNV was often detectable in brain tissue by RT-PCR alone but only by the two-step assay in blood, sperm, ovarian tissue or larvae. The same was true for sperm and ovarian tissue of shi drum, Umbrina cirrosa. NNV was also detected in the brains of Japanese red seabream, Pagrus major and brown meagre, Sciaena umbra, suggesting that these species can also be infected. No NNV was detected in samples of Artemia salina nauplii and rotifers obtained from a fish farm with an NNV outbreak. The inclusion of nested PCR in the assay appears to be necessary to screen out NNV-positive broodfish by blood sampling and testing of their larval progeny. 2009 Blackwell Publishing Journal Backfiles 1879-2005 |2009|||||||||| Zanella, L verfasserin aut Patarnello, P verfasserin aut Paolucci, L oth Belvedere, P oth Colombo, L oth In Journal of fish diseases Oxford [u.a.] : Wiley-Blackwell, 1978 23(2000), 5, Seite 0 Online-Ressource (DE-627)NLEJ243926952 (DE-600)2020444-9 1365-2761 nnns volume:23 year:2000 number:5 pages:0 http://dx.doi.org/10.1046/j.1365-2761.2000.00255.x text/html Verlag Deutschlandweit zugänglich Volltext GBV_USEFLAG_U ZDB-1-DJB GBV_NL_ARTICLE AR 23 2000 5 0 |
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10.1046/j.1365-2761.2000.00255.x doi (DE-627)NLEJ243234902 DE-627 ger DE-627 rakwb Dalla Valle, L verfasserin aut Development of a sensitive diagnostic assay for fish nervous necrosis virus based on RT-PCR plus nested PCR Oxford, UK Blackwell Science Ltd 2000 Online-Ressource nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier A polymerase chain reaction (PCR)-based assay to detect nervous necrosis virus (NNV) in fish was developed by using two sets of primers designed on a highly conserved region of the coat protein gene encoded by RNA2 of NNV. The first pair of primers amplified a fragment of 605 bp by one-step reverse-transcription (RT)-PCR, while the second pair amplified an internal segment of 255 bp by nested PCR. Addition of nested PCR increased the assay sensitivity 100-fold when carried out in a separate tube (two-step assay) and 10-fold when performed in the same tube (one-step assay). The sensitivity of the two-step assay was 104 times higher than that of virus cultivation. Nested PCR served also to confirm the specificity of the first amplification, as verified also by Southern hybridization analysis and direct sequencing. In species known to be susceptible to infection, such as European sea bass, Dicentrarchus labrax, and gilthead seabream, Sparus aurata, NNV was often detectable in brain tissue by RT-PCR alone but only by the two-step assay in blood, sperm, ovarian tissue or larvae. The same was true for sperm and ovarian tissue of shi drum, Umbrina cirrosa. NNV was also detected in the brains of Japanese red seabream, Pagrus major and brown meagre, Sciaena umbra, suggesting that these species can also be infected. No NNV was detected in samples of Artemia salina nauplii and rotifers obtained from a fish farm with an NNV outbreak. The inclusion of nested PCR in the assay appears to be necessary to screen out NNV-positive broodfish by blood sampling and testing of their larval progeny. 2009 Blackwell Publishing Journal Backfiles 1879-2005 |2009|||||||||| Zanella, L verfasserin aut Patarnello, P verfasserin aut Paolucci, L oth Belvedere, P oth Colombo, L oth In Journal of fish diseases Oxford [u.a.] : Wiley-Blackwell, 1978 23(2000), 5, Seite 0 Online-Ressource (DE-627)NLEJ243926952 (DE-600)2020444-9 1365-2761 nnns volume:23 year:2000 number:5 pages:0 http://dx.doi.org/10.1046/j.1365-2761.2000.00255.x text/html Verlag Deutschlandweit zugänglich Volltext GBV_USEFLAG_U ZDB-1-DJB GBV_NL_ARTICLE AR 23 2000 5 0 |
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10.1046/j.1365-2761.2000.00255.x doi (DE-627)NLEJ243234902 DE-627 ger DE-627 rakwb Dalla Valle, L verfasserin aut Development of a sensitive diagnostic assay for fish nervous necrosis virus based on RT-PCR plus nested PCR Oxford, UK Blackwell Science Ltd 2000 Online-Ressource nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier A polymerase chain reaction (PCR)-based assay to detect nervous necrosis virus (NNV) in fish was developed by using two sets of primers designed on a highly conserved region of the coat protein gene encoded by RNA2 of NNV. The first pair of primers amplified a fragment of 605 bp by one-step reverse-transcription (RT)-PCR, while the second pair amplified an internal segment of 255 bp by nested PCR. Addition of nested PCR increased the assay sensitivity 100-fold when carried out in a separate tube (two-step assay) and 10-fold when performed in the same tube (one-step assay). The sensitivity of the two-step assay was 104 times higher than that of virus cultivation. Nested PCR served also to confirm the specificity of the first amplification, as verified also by Southern hybridization analysis and direct sequencing. In species known to be susceptible to infection, such as European sea bass, Dicentrarchus labrax, and gilthead seabream, Sparus aurata, NNV was often detectable in brain tissue by RT-PCR alone but only by the two-step assay in blood, sperm, ovarian tissue or larvae. The same was true for sperm and ovarian tissue of shi drum, Umbrina cirrosa. NNV was also detected in the brains of Japanese red seabream, Pagrus major and brown meagre, Sciaena umbra, suggesting that these species can also be infected. No NNV was detected in samples of Artemia salina nauplii and rotifers obtained from a fish farm with an NNV outbreak. The inclusion of nested PCR in the assay appears to be necessary to screen out NNV-positive broodfish by blood sampling and testing of their larval progeny. 2009 Blackwell Publishing Journal Backfiles 1879-2005 |2009|||||||||| Zanella, L verfasserin aut Patarnello, P verfasserin aut Paolucci, L oth Belvedere, P oth Colombo, L oth In Journal of fish diseases Oxford [u.a.] : Wiley-Blackwell, 1978 23(2000), 5, Seite 0 Online-Ressource (DE-627)NLEJ243926952 (DE-600)2020444-9 1365-2761 nnns volume:23 year:2000 number:5 pages:0 http://dx.doi.org/10.1046/j.1365-2761.2000.00255.x text/html Verlag Deutschlandweit zugänglich Volltext GBV_USEFLAG_U ZDB-1-DJB GBV_NL_ARTICLE AR 23 2000 5 0 |
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Development of a sensitive diagnostic assay for fish nervous necrosis virus based on RT-PCR plus nested PCR |
| abstract |
A polymerase chain reaction (PCR)-based assay to detect nervous necrosis virus (NNV) in fish was developed by using two sets of primers designed on a highly conserved region of the coat protein gene encoded by RNA2 of NNV. The first pair of primers amplified a fragment of 605 bp by one-step reverse-transcription (RT)-PCR, while the second pair amplified an internal segment of 255 bp by nested PCR. Addition of nested PCR increased the assay sensitivity 100-fold when carried out in a separate tube (two-step assay) and 10-fold when performed in the same tube (one-step assay). The sensitivity of the two-step assay was 104 times higher than that of virus cultivation. Nested PCR served also to confirm the specificity of the first amplification, as verified also by Southern hybridization analysis and direct sequencing. In species known to be susceptible to infection, such as European sea bass, Dicentrarchus labrax, and gilthead seabream, Sparus aurata, NNV was often detectable in brain tissue by RT-PCR alone but only by the two-step assay in blood, sperm, ovarian tissue or larvae. The same was true for sperm and ovarian tissue of shi drum, Umbrina cirrosa. NNV was also detected in the brains of Japanese red seabream, Pagrus major and brown meagre, Sciaena umbra, suggesting that these species can also be infected. No NNV was detected in samples of Artemia salina nauplii and rotifers obtained from a fish farm with an NNV outbreak. The inclusion of nested PCR in the assay appears to be necessary to screen out NNV-positive broodfish by blood sampling and testing of their larval progeny. |
| abstractGer |
A polymerase chain reaction (PCR)-based assay to detect nervous necrosis virus (NNV) in fish was developed by using two sets of primers designed on a highly conserved region of the coat protein gene encoded by RNA2 of NNV. The first pair of primers amplified a fragment of 605 bp by one-step reverse-transcription (RT)-PCR, while the second pair amplified an internal segment of 255 bp by nested PCR. Addition of nested PCR increased the assay sensitivity 100-fold when carried out in a separate tube (two-step assay) and 10-fold when performed in the same tube (one-step assay). The sensitivity of the two-step assay was 104 times higher than that of virus cultivation. Nested PCR served also to confirm the specificity of the first amplification, as verified also by Southern hybridization analysis and direct sequencing. In species known to be susceptible to infection, such as European sea bass, Dicentrarchus labrax, and gilthead seabream, Sparus aurata, NNV was often detectable in brain tissue by RT-PCR alone but only by the two-step assay in blood, sperm, ovarian tissue or larvae. The same was true for sperm and ovarian tissue of shi drum, Umbrina cirrosa. NNV was also detected in the brains of Japanese red seabream, Pagrus major and brown meagre, Sciaena umbra, suggesting that these species can also be infected. No NNV was detected in samples of Artemia salina nauplii and rotifers obtained from a fish farm with an NNV outbreak. The inclusion of nested PCR in the assay appears to be necessary to screen out NNV-positive broodfish by blood sampling and testing of their larval progeny. |
| abstract_unstemmed |
A polymerase chain reaction (PCR)-based assay to detect nervous necrosis virus (NNV) in fish was developed by using two sets of primers designed on a highly conserved region of the coat protein gene encoded by RNA2 of NNV. The first pair of primers amplified a fragment of 605 bp by one-step reverse-transcription (RT)-PCR, while the second pair amplified an internal segment of 255 bp by nested PCR. Addition of nested PCR increased the assay sensitivity 100-fold when carried out in a separate tube (two-step assay) and 10-fold when performed in the same tube (one-step assay). The sensitivity of the two-step assay was 104 times higher than that of virus cultivation. Nested PCR served also to confirm the specificity of the first amplification, as verified also by Southern hybridization analysis and direct sequencing. In species known to be susceptible to infection, such as European sea bass, Dicentrarchus labrax, and gilthead seabream, Sparus aurata, NNV was often detectable in brain tissue by RT-PCR alone but only by the two-step assay in blood, sperm, ovarian tissue or larvae. The same was true for sperm and ovarian tissue of shi drum, Umbrina cirrosa. NNV was also detected in the brains of Japanese red seabream, Pagrus major and brown meagre, Sciaena umbra, suggesting that these species can also be infected. No NNV was detected in samples of Artemia salina nauplii and rotifers obtained from a fish farm with an NNV outbreak. The inclusion of nested PCR in the assay appears to be necessary to screen out NNV-positive broodfish by blood sampling and testing of their larval progeny. |
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| title_short |
Development of a sensitive diagnostic assay for fish nervous necrosis virus based on RT-PCR plus nested PCR |
| url |
http://dx.doi.org/10.1046/j.1365-2761.2000.00255.x |
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| author2 |
Zanella, L Patarnello, P Paolucci, L Belvedere, P Colombo, L |
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Zanella, L Patarnello, P Paolucci, L Belvedere, P Colombo, L |
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| doi_str |
10.1046/j.1365-2761.2000.00255.x |
| up_date |
2024-07-06T04:47:06.220Z |
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