Development and validation of a real-time TaqMan PCR assay for the detection of betanodavirus in clinical specimens

Panzarin, Valentina; Patarnello, Pierpaolo; Mori, Antonio; Rampazzo, E.; Cappellozza, Elisabetta; Bovo, G.; Cattoli, Giovanni

doi:10.1007/s00705-010-0701-5

Cited by 63 publications

(59 citation statements)

References 29 publications

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“…Nodavirus screening was performed by qRT-PCR using the Applied Bio system 7500 machine and reaction conditions were realized according the validated protocol of Panzarin et al [21] with a slight modification of hybridization and extension duration which was adaptable to the 7500 machine. Oligonucleotides were able to target and amplify a conserved 69-bp-long region of the viral genome localized in the RNA2 strand, encoding the coat protein (CP).…”

Section: Genome Extraction and Virus Detection By Amplificationmentioning

confidence: 99%

Nodaviruses in Wild Fish Population Collected Around Aquaculture Cage Sites from Coastal Areas of Tunisia

Cherif¹,

Amdouni²

2017

Fish Aquac J

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This report describes the viral epidemiology of wild fish adjacent to cage farms within the Tunisian coasts and is focused on viral nervous necrosis virus (VNNV). A total of 92 apparently healthy wild marine fish were collected near aquaculture facilities in five different coastal areas of Tunisia. The brains and eyes of fish were examined by quantitative real time reverse transcriptase-polymerase chain reaction (qRT-PCR) to detect the nodavirus coat protein gene of. A total of 57 out of 92 (61.9%) samples were positive for nodavirus by qRT-PCR. This finding indicates that carrier fish occur at a considerable level in populations of wild marine fish. Samples from 13 fish species were found to be positive to the virus genome: Sarpa salpa, Trachurs trachurus, Boobs boops, Sardinella aurita, Diplodus vulgaris, Diplodus puntazzo Liza aurata, Diplodue sargus, Sparus aurata, Sardina pilchardus, Spicara maena, Spondyliosoma cantharus, and Diplodus annularis. The partial sequences of the RNA2 coat protein gene of these strains were identical with RGNNV type previously identified within farmed sea bass and sea bream species in Tunisia, with a homology >97%. With respect to the proximity of the sampling sites to the coast and to rearing facilities, results analysis can suggest that these viruses may be indigenous to Tunisian coastal waters.

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Section: Genome Extraction and Virus Detection By Amplificationmentioning

confidence: 99%

Nodaviruses in Wild Fish Population Collected Around Aquaculture Cage Sites from Coastal Areas of Tunisia

Cherif¹,

Amdouni²

2017

Fish Aquac J

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“…Briefly, paraffin sections were trypsinized at 37°C for 30 min, pre-incubated with normal horse serum for 20 min, and incubated with a rabbit polyclonal hyperimmune serum Panzarin et al (2010). Briefly, total RNA was extracted from 100 µl of sample using the NucleoSpin ® RNA II kit (MachereyNagel) according to the manufacturer's instructions.…”

Section: Samplingmentioning

confidence: 99%

Viral encephalopathy and retinopathy outbreak in freshwater fish farmed in Italy

Bovo¹,

Gustinelli²,

Quaglio³

et al. 2011

Dis. Aquat. Org.

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Viral encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN), is a neuropathological condition affecting > 40 species of fish. Although VER affects mainly marine fish, the disease has also been detected in certain species reared in freshwater environments. There are relatively few reports concerning the disease in freshwater species, and there is not much information on clinical signs. Nevertheless, the most common clinical findings reported from affected freshwater species are consistent with the typical signs observed in marine species. In this paper we describe the main clinical signs and the laboratory results associated with the detection of a betanodavirus in hybrid striped bass × white bass (Morone saxatilis × Morone chrysops) and largemouth bass Micropterus salmoides, reared in a freshwater environment. We also detected the virus by realtime PCR and isolated it in cell culture from a batch of pike-perch Sander lucioperca farmed in the same system.

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“…RNA and DNA extraction using the QIAamp viral RNA and DNA mini kit (QIAGEN), respectively. The RT-q-PCR was carried out with the Superscript III Reverse Transcriptase (Invitrogen) according to protocols for random hexamer primed cDNA synthesis as described by Panzarin (2010) 11 and the OIE manual (2013) 12 for VNNV detection. For GIV detection, the primer pair (GIV-F, TCAACGCCTGGCTCACTC and GIV-R, CCTTCGCACATGTTAAACTG) was designed by the alignment of 13 di®erent GIV major capsid protein DNA sequences for PCR ampli¯cation, and the TaqMan probe was designed by the Roche Universal Probe Library Assay Design Center from the National Center for Biotechnology Information (NCBI) database for RT-q-PCR.…”

Section: Rt-q-pcr Analysismentioning

confidence: 99%

A BIVALENT INACTIVATED VACCINE OF VIRAL NERVOUS NECROSIS VIRUS AND GROUPER IRIDOVIRUS APPLIED TO GROUPER BROODFISH (EPINEPHELUS COIOIDES) REDUCES THE RISK OF VERTICAL TRANSMISSION

Huang

Cheng²,

et al. 2017

Taiwan Vet J

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Forty-one brood¯sh of orange-spotted groupers (Epinephelus coioides) were selected to evaluate the e®ectiveness of a viral nervous necrosis virus (VNNV) and grouper iridovirus (GIV) inactivated bivalent vaccine in grouper brood¯sh. Real-time quantitative PCR analysis showed that a detection rate of 10.5% (2/19) was found in egg specimens of VNNV and GIV, which carried approximately 1780 copies of GIV viral DNA in the egg specimens from brood¯sh before vaccination. This con¯rmed the vertical transmission route of GIV in brood¯sh. A signi¯cant increase of the anti-VNNV serum antibody titer was more than 50% in the high titer level (1:1810 to 1:5120) and 45% in the moderate titer level (1:452 to 1:1280), which were higher than those of the anti-GIV display, with 50% (10/20) in a titer of 1:57 to 1:320 and 40% (8/20) in a titer of 1:452 to 1:1280 one month after the vaccination. This result showed that the VNNV is a highly antigenic virus and can e®ectively induce neutralizing antibodies better than GIV. In addition, the VNNV and GIV viral copy numbers were 97.1 and 1780 copies per g host egg DNA from the brood¯sh before vaccination, respectively. One month after the vaccination, the viral genomes of VNNV and GIV were undetectable in egg specimens. The results show that immunization can induce the production of speci¯c protective neutralizing antibodies, and the infective antigens can thereby be eliminated by the immunity.The results demonstrate that the speci¯c antibodies of GIV and VNNV induced by vaccination can reduce the risk of vertical transmission of VNNV and GIV in grouper brood¯sh.

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Development and validation of a real-time TaqMan PCR assay for the detection of betanodavirus in clinical specimens

Cited by 63 publications

References 29 publications

Nodaviruses in Wild Fish Population Collected Around Aquaculture Cage Sites from Coastal Areas of Tunisia

Nodaviruses in Wild Fish Population Collected Around Aquaculture Cage Sites from Coastal Areas of Tunisia

Viral encephalopathy and retinopathy outbreak in freshwater fish farmed in Italy

A BIVALENT INACTIVATED VACCINE OF VIRAL NERVOUS NECROSIS VIRUS AND GROUPER IRIDOVIRUS APPLIED TO GROUPER BROODFISH (EPINEPHELUS COIOIDES) REDUCES THE RISK OF VERTICAL TRANSMISSION

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