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Rapid and sensitive detection of ostreid herpesvirus 1 in oyster samples by real-time PCR
- Source :
- Journal of Virological Methods (0166-0934) (Elsevier), 2008-05, Vol. 149, N. 2, P. 269-276
- Publication Year :
- 2008
- Publisher :
- Elsevier, 2008.
-
Abstract
- Herpes and herpes-like virus infections have been reported in various marine mollusc species associated with high mortality rates. Following the characterisation and genome sequencing of ostreid herpesvirus 1 (OsHV-1), specific diagnostic tools have been developed based on conventional PCR techniques or in situ hybridisation. We have now developed a real-time PCR assay for rapid, sensitive and quantitative detection of OsHV-1, and compared it with a conventional PCR technique described previously. The new assay utilised SYBR((R)) Green chemistry with specific primers C(9)/C(10) targeting the C region. The melt curve analysis of OsHV-1 DNA or DNA extracted from infected material showed only one melting temperature peak (75.75+/-0.1 degrees C). The assay had a detection limit of 4 copies/microL of viral genomic DNA and a dynamic range of 5 logs. Using infected oyster samples as template, the assay was about 100-fold more sensitive than single PCR method using C(2)/C(6) primers. The assay was applied successfully for rapid diagnosis (100 min) and quantitation of OsHV-1 in different developmental stages of Crassostrea gigas. Although it already exists a competitive PCR method to quantify OsHV-1 DNA, quantitative data that will emerge in future using the new sensitive and reliable assay will illuminate aspects of pathogenesis, in particular the viral loads in asymptomatic oysters and the kinetics of infection in specific target tissues.
- Subjects :
- Oyster
Ostreid herpesvirus 1
Diamines
SYBR® Green
Polymerase Chain Reaction
Sensitivity and Specificity
Melting curve analysis
DNA sequencing
law.invention
03 medical and health sciences
chemistry.chemical_compound
law
Virology
biology.animal
Animals
Transition Temperature
Benzothiazoles
Crassostrea
Organic Chemicals
Herpesviridae
Polymerase chain reaction
DNA Primers
030304 developmental biology
0303 health sciences
biology
Herpesvirus
Herpesviridae Infections
04 agricultural and veterinary sciences
Molecular biology
genomic DNA
Real time PCR
Real-time polymerase chain reaction
chemistry
Crassostrea gigas
Quinolines
040102 fisheries
0401 agriculture, forestry, and fisheries
Viral load
DNA
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Journal :
- Journal of Virological Methods (0166-0934) (Elsevier), 2008-05, Vol. 149, N. 2, P. 269-276
- Accession number :
- edsair.doi.dedup.....44f6e3c2cb8ae783fb6855a4c00f27d2