Detection of group B and C rotaviruses by polymerase chain reaction.
We adapted the polymerase chain reaction (PCR) to detect the noncultivatable group B and C rotaviruses and introduced a simple and convenient technique to purify viral RNA from stool specimens. Double-stranded RNA present in stool extracts was purified by adsorption to hydroxyapatite and was used as the template for reverse transcription and polymerase amplification. Primer pairs specific for group B (gene 8) and group C (gene 6) rotaviruses were selected to amplify group-characteristic sizes of cDNA copies readily identifiable in ethidium bromide-stained agarose gels. These primer pairs were used separately in individual PCR assays or were pooled with a primer pair specific for group A rotavirus (gene 9) in a combined PCR assay for the simultaneous detection of all three rotavirus groups. The method was very sensitive and was used to identify both human and porcine strains of group B and C rotaviruses in stool specimens. A second PCR amplification with internal group-specific primers served to increase further the sensitivity of the test and to confirm the diagnostic results obtained in the first amplification.
Gouvea
V
V
Division of Viral and Rickettsial Diseases, Centers for Disease Control, Atlanta, Georgia 30333.
Allen
J R
JR
Glass
R I
RI
Fang
Z Y
ZY
Bremont
M
M
Cohen
J
J
McCrae
M A
MA
Saif
L J
LJ
Sinarachatanant
P
P
Caul
E O
EO
D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics; D005814:Genes, Viral; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D012401:Rotavirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000382:microbiology; D012680:Sensitivity and Specificity
D004279:DNA, Viral; D012367:RNA, Viral