Amplification of rhinovirus specific nucleic acids from clinical samples using the polymerase chain reaction.
We describe a novel method for the detection of human rhinoviruses in clinical samples, using the polymerase chain reaction. Two synthetic oligonucleotide primers were produced that bind in the 5' noncoding region of all rhinovirus serotypes tested, about 350 nucleotides apart, and were used to prime polymerase chain reaction amplification of the intervening stretch of DNA. The product of this reaction, which can be clearly visualized by gel electrophoresis, is a discrete 380 bp band, the occurrence of which is diagnostic of the presence of a rhinovirus in the clinical sample analysed. The technique, which is rapid, sensitive, and reliable, has been used successfully for all the different rhinovirus serotypes tested to date in our laboratory. However, the sensitivity of detection is greatly dependent on the inclusion of both tRNA and vanadyl complexes during the viral RNA extraction process. Using this technique, under optimal conditions, we were able to detect virus in clinical samples with titres as low as TCID50 10(2.5).
Gama
R E
RE
Department of Biology, University of Essex, Colchester, England.
Horsnell
P R
PR
Hughes
P J
PJ
North
C
C
Bruce
C B
CB
al-Nakib
W
W
Stanway
G
G
D001483:Base Sequence; D004279:DNA, Viral / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase; D005784:Gene Amplification; D006801:Humans; D009693:Nucleic Acid Hybridization; D010850:Picornaviridae Infections / Q000235:genetics*; D012229:Rhinovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D012703:Serotyping
D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase