Detection and serotyping of rotaviruses in stool specimens by using reverse transcription and polymerase chain reaction amplification.
Direct rotavirus serotyping (VP7, G type) in stool specimens was carried out by reverse transcription and polymerase chain reaction amplification (RT-PCR) and compared to serotyping by enzyme immunoassay with monoclonal antibodies (EIA-MAb). The methods used for double-stranded (ds) RNA extraction, RT-PCR amplification, and the primers used were modified from previous reports [Gouvea et al.: Journal of Clinical Microbiology 29:519-523, 1990; Gentsch et al.: Journal of Clinical Microbiology, 1992]. For samples that were positive by both methods, the serotypes obtained were identical, however RT-PCR typing was found to be considerably more sensitive (70.4% samples serotyped) than EIA-MAb (35.6% of samples serotyped). The overall sensitivities for detection of rotavirus in stool samples by latex agglutination, enzyme immunoassay, electron microscopy, polyacrylamide gel electrophoresis, and RT-PCR were essentially the same. The results confirm that RT-PCR typing (genotyping) is extremely valuable for G typing of samples which cannot be typed by EIA-MAb. We also developed a PCR confirmation technique for serotypes 1, 2, and 4.
National Institute of Health, Tokyo, Japan.
H
H
Ushijima
H
H
Koike
A
A
Mukoyama
A
A
Hasegawa
S
S
Nishimura
J
J
Gentsch
D000208:Acute Disease; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D005243:Feces / Q000382:microbiology*; D005759:Gastroenteritis / Q000382:microbiology*; D006801:Humans; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000145:classification* / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000382:microbiology*; D012703:Serotyping; D014158:Transcription, Genetic