Application of RT-PCR designed from the sequence of the local SRSV strain to the screening in viral gastroenteritis outbreaks.
The Yuri strain of small round structured virus (SRSV) was cloned from a fecal specimen in which virus particles were observed by electron microscopy. The most common RT-PCR protocol in Japan, however, using 35/36 and NV81/NV82/SM82 nested primer pairs, could not detect the SRSV genome in this specimen. Nucleotide and amino acid sequence analysis revealed that the Yuri strain is genetically close to the genotype II of SRSV. A novel procedure using primer sets designed from the nucleotide sequence of the Yuri strain was applied to the screening of 119 stool samples obtained from subjects with sporadic diarrhea and 46 samples obtained during seven foodborne gastroenteritis outbreaks. Using this novel procedure, PCR bands were detected in 44% and 52% of the samples, respectively. These detection rates were approximately twice those obtained with the 35/36 and NV81/NV82/SM82 nested primers. In particular, more than 40% of positive samples could be detected by using only the Yuri primer sets. Furthermore, three improvements were made in the RNA preparation, cDNA synthesis, and amplification steps to save materials and time. The background, or extra bands, in the amplification reaction resulting from DNA in the fecal specimens was completely removed by DNase I treatment just before cDNA synthesis. Random nonamers were used as universal primers in the reverse transcription. No difference in sensitivity or specificity was noted in the final results when either random nonamers or specific primers were used. The use of a preamplification step under low stringent conditions before standard amplification under highly stringent conditions compensated for any mismatched bases in the primers with respect to the target sequences. Thus our novel procedure using Yuri primer sets may be useful for the screening of SRSV in the recent SRSV outbreaks in Japan.
Department of Microbiology, Akita Prefectural Institute of Public Health, Akita, Japan.
H
H
Saito
S
S
Saito
K
K
Kamada
S
S
Harata
H
H
Sato
M
M
Morita
Y
Y
Miyajima
D000595:Amino Acid Sequence; D001483:Base Sequence; D017250:Caliciviridae Infections / Q000175:diagnosis* / Q000453:epidemiology / Q000821:virology; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000737:chemistry; D003851:Deoxyribonucleases / Q000378:metabolism; D003967:Diarrhea / Q000175:diagnosis / Q000821:virology; D004196:Disease Outbreaks; D005243:Feces / Q000821:virology; D005260:Female; D005759:Gastroenteritis / Q000175:diagnosis* / Q000453:epidemiology / Q000821:virology; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009663:Norwalk virus / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D018076:DNA, Complementary; D012367:RNA, Viral; D003851:Deoxyribonucleases