Detection of foot-and-mouth disease viral sequences in clinical specimens and ethyleneimine-inactivated preparations by the polymerase chain reaction.
The polymerase chain reaction method (PCR) has been applied to the diagnosis of foot-and-mouth disease viral RNA in tissues and, particularly, oesophageal-pharyngeal fluid (probang) samples from cattle. Using primer sets which corresponded to conserved regions of the VP1 sequence of the viral genome, it was possible to amplify sequences regardless of the serotype/strain of the virus. In comparison with infectivity assays, the PCR was generally more sensitive although there were a number of examples where only infectivity was detectable. In experiments with uninfected probang samples deliberately seeded with a dilution series of virus, the PCR proved to be approximately 10(4) times more sensitive than infectivity assays. This greater sensitivity was attributed, in part, to the ability of the PCR to amplify specifically from non-infectious RNA preparations. This enabled the identification, by sequencing, of viral RNA from chemically inactivated virus concentrates typical of those used for commercial vaccine production. Amplification of specific PCR products was also achieved with virus eluted from commercial vaccine, including preparations which had been stored for more than 10 years at 4 degrees C. The PCR technique is of considerable value, therefore, both as a complement to infectivity assays and as a powerful tool in vaccine-associated studies.
Amaral-Doel
C M
CM
AFRC Institute for Animal Health, Pirbright Laboratory, Woking, Surrey, UK.
Owen
N E
NE
Ferris
N P
NP
Kitching
R P
RP
Doel
T R
TR
D000595:Amino Acid Sequence; D000818:Animals; D005537:Aphthovirus / Q000235:genetics* / Q000302:isolation & purification; D001388:Aziridines; D001483:Base Sequence; D002213:Capsid / Q000235:genetics; D036022:Capsid Proteins; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000382:microbiology; D015342:DNA Probes; D004279:DNA, Viral / Q000235:genetics; D005536:Foot-and-Mouth Disease / Q000175:diagnosis / Q000382:microbiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D012680:Sensitivity and Specificity
D001388:Aziridines; D036022:Capsid Proteins; D015342:DNA Probes; D004279:DNA, Viral; D012367:RNA, Viral; C033132:aziridine