Use of an internal standard in a TaqMan nested reverse transcription-polymerase chain reaction for the detection of bovine viral diarrhoea virus.
Vet Microbiol
; 96(4): 357-66, 2003 Nov 07.
Article
en En
| MEDLINE
| ID: mdl-14599783
ABSTRACT
The aim of this study was to improve molecular methods for the detection of bovine viral diarrhoea virus (BVDV). A single-tube nested reverse-transcriptase polymerase chain reaction (nRT-PCR) employing the 5'-3'-exonuclease assay (TaqMan) system was optimised for use with bulk milk, semen and whole blood samples. An artificial template (mimic) was engineered to provide in-tube validation of negative samples by demonstrating the absence of substances inhibitory to RT or PCR. This mimic was constructed by disrupting the BVDV amplicon at the TaqMan probe site by inserting a 295bp fragment of human genomic DNA. The mimic amplicon was discriminated from the BVDV RT-PCR products using a second TaqMan probe, with a different fluorochrome specific for the inserted DNA. This new method was more sensitive than BVDV antigen ELISA methods and the existing RT-PCR method used in the laboratory for detection of BVDV in bulk milk. Furthermore, RNA extracted by robotic methods has proved suitable for use in this assay. This TaqMan nRT-PCR will be a valuable method for the detection of BVDV in a variety of biological matrices including milk and semen.
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Banco de datos:
MEDLINE
Asunto principal:
Diarrea Mucosa Bovina Viral
/
Virus de la Diarrea Viral Bovina
/
Polimerasa Taq
/
Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
Tipo de estudio:
Diagnostic_studies
Límite:
Animals
Idioma:
En
Revista:
Vet Microbiol
Año:
2003
Tipo del documento:
Article
País de afiliación:
Reino Unido