Amplification of all 11 RNA segments of group A rotaviruses based on reverse transcription polymerase chain reaction.
Group A rotaviruses (RVA) are a major cause of acute infantile gastroenteritis. The viral genome comprises 11 double-stranded RNA segments and the respective gene segments are classified into more than eight genotypes, according to the nucleotide sequence similarities. So far, it has been difficult to amplify full-length sequences of long RNA segments of rotaviruses by one-time only RT-PCR (especially in the genes for the viral proteins VP1, VP2, VP3 and VP4). In this study, a set of universal primers to amplify all 11 segments of RVA was designed by aligning the nucleotide sequences of the typical rotavirus strains. Using these primers and a high-fidelity and rapid DNA polymerase in a one-step reverse transcription polymerase chain reaction, almost the entire length of all 11 segments of the seven rotavirus strains Wa, DS-1, Hochi, 69M, WI61, M37 and SA11-S1 were accurately and rapidly amplified. In addition, all 11 segments of rotavirus obtained from a fecal specimen were successfully amplified. In conclusion, the method described here will be useful as an RVA detection system and protocol for complete analysis of the 11 genome sequences.
Department of Virology II, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashi-murayama, Tokyo 208-0011, Japan.
Yoshiki
Y
Fujii
Takashi
T
Shimoike
Hirotaka
H
Takagi
Kosuke
K
Murakami
Reiko
R
Todaka-Takai
YoungBin
Y
Park
Kazuhiko
K
Katayama
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics / Q000592:standards; D005243:Feces / Q000821:virology; D016679:Genome, Viral; D006801:Humans; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012401:Rotavirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D016415:Sequence Alignment
D017931:DNA Primers; D012367:RNA, Viral