Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS).
A one step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of viral hemorrhagic septicaemia virus (VHS). A set of six primers were designed, based on the G-protein sequence of the VHS virus serotypes (He, F1, 23.75, Klapmolle and Rindsholm). The assay was optimised to amplify VHS RNA by incubation at 63 degrees C for only 1h, and required only a simple water bath or heating block to provide a constant temperature of 63 degrees C. RT-LAMP amplification products were detected by visual inspection using SYBR Green I stain and had a ladder-like appearance when electrophoresed on an agarose gel. The detection limit of the RT-LAMP assay was found to be similar to the commonly used RT-PCR method: both methods detected VHS RNA at a dilution of 10(6). The assay was evaluated using clinical samples and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for VHS virus.
Soliman
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H
Institute of Zoology, Fish Biology and Fish Diseases, Faculty of Veterinary Medicine, University of Munich, Kaulbachstr. 37, 80539 Munich, Germany.
El-Matbouli
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M
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel / Q000662:veterinary; D005399:Fishes; D005784:Gene Amplification; D031941:Hemorrhagic Septicemia, Viral / Q000175:diagnosis* / Q000821:virology; D008969:Molecular Sequence Data; D029141:Novirhabdovirus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards / Q000662:veterinary*; D012680:Sensitivity and Specificity; D012703:Serotyping / Q000662:veterinary; D014759:Viral Envelope Proteins / Q000032:analysis / Q000235:genetics
D017931:DNA Primers; C096089:G protein, viral hemorrhagic septicemia virus; D012367:RNA, Viral; D014759:Viral Envelope Proteins