Quantitation of foot-and-mouth disease virus genomes in bovine tissue by competitive RT-PCR.
The sensitivity of a reverse transcription-dependent polymerase chain reaction (RT-PCR) for detecting foot-and-mouth disease virus (FMDV) genomes was quantified by use of RNA transcribed in vitro from FMDV-specific cDNA. Previously, the cDNA had been elongated by 228 base pairs. The minimum number of template molecules required to obtain the specific RT-PCR product was determined to be 10(4). This was achieved by use of 1 microg of primer for cDNA synthesis and by undertaking of at least 30 cycles of PCR. Knowing the sensitivity of the system prompted the examination of clinical samples for content of FMDV genomes. The samples were tongue and foot epithelia as well as nasal discharge, removed 11-14 days after infection from 14 cattle. They all contained FMDV genomes but not in each clinical specimen. The size difference of the products amplified from transcript and viral genome enabled the estimate by competitive RT-PCR of the number of viral genomes contained in some samples. The RNA extracted from approximately 10(7) tissue cells each was found to contain between less than 10(6) and up to 10(8) FMDV genomes, irrespective of the sample type.
Bundesforschungsanstalt für Viruskrankheiten der Tiere, Tübingen, Germany.
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W
Niedbalski
K H
KH
Adam
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O
Marquardt
D000818:Animals; D005537:Aphthovirus / Q000235:genetics / Q000302:isolation & purification*; D002417:Cattle; D018076:DNA, Complementary / Q000235:genetics; D004848:Epithelium / Q000821:virology; D005528:Foot / Q000821:virology; D016679:Genome, Viral; D017817:Nasal Lavage Fluid; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D014059:Tongue / Q000821:virology
D018076:DNA, Complementary