Development of a one-step duplex RT-qPCR for the quantification of phocine distemper virus.
J Wildl Dis
; 51(2): 454-65, 2015 Apr.
Article
en En
| MEDLINE
| ID: mdl-25647591
ABSTRACT
Worldwide, stranded marine mammals and the network personnel who respond to marine mammal mortality have provided much of the information regarding marine morbillivirus infections. An assay to determine the amount of virus present in tissue samples would be useful to assist in routine surveying of animal health and for monitoring large-scale die-off events. False negatives from poor-quality samples prevent determination of the true extent of infection, while only small amounts of tissue samples or archived RNA may be available at the time of collection for future retrospective analysis. We developed a one-step duplex real-time reverse transcriptase-quantitative-PCR assay (RT-qPCR) based on Taqman probe technology to quantify phocine distemper virus (PDV) isolated from an outbreak in harbor (Phoca vitulina concolor) and gray seals (Halichoerus grypus) along the northeast US coast in 2006. The glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene was selected to assess RNA quality. This duplex assay is specific for PDV and sensitive through a range of 10(0) to 10(9) copies ds-plasmid DNA. For the GAPDH target, the reaction in duplex amplified 10(0) to 10(9) copies of ds-plasmid DNA and was detectable in multiple seal species. This assay reduced the likelihood of false negative results due to degradation of tissues and well-to-well variability while providing sensitive and specific detection of PDV, which would be applicable in molecular epidemiologic studies and pathogen detection in field and laboratory investigations involving a variety of seal species.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Contexto en salud:
4_TD
Problema de salud:
4_measles
Asunto principal:
Virus del Moquillo Focino
/
Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
/
Phocidae
/
Moquillo
Tipo de estudio:
Diagnostic_studies
Límite:
Animals
Idioma:
En
Revista:
J Wildl Dis
Año:
2015
Tipo del documento:
Article
País de afiliación:
Estados Unidos