Evaluation and application of reverse transcription loop-mediated isothermal amplification for detection of noroviruses.
A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of norovirus (NV) was developed. In order to design primer sets for the detection of a wide range of NVs, NVs were categorized into three groups, that is, genogroup I (GI), prevalent GII, and minor GII; three sets of primers were developed for each group. Clinical specimens of patients suffering from enteric RNA viruses, such as NV, group A and C rotavirus, and sapovirus were examined using these primer sets. Various genotypes of NVs were detected in clinical specimens from patients infected with NV where no false positive reaction was observed with other enteric RNA viruses. Additionally, 88 samples of acute gastroenteritis outbreaks were analyzed by an RT-LAMP assay and compared with the results of routine RT-PCR. The results of the RT-LAMP assay corresponded well to that of RT-PCR. These findings suggest the practical application of the RT-LAMP assay for the detection of NVs in clinical specimens. Consequently, the RT-LAMP system and conventional detection kits (NVGI and NVGII detection kits; Eiken Chemical Co., Ltd., Japan) were compared. The detection rate of the prevalent and minor GII primer sets was similar to that of the conventional NVGII kit, while the detection rate of the GI primer set is different because it can detect several genotypes better than the conventional NVGI kit. This is an initial report that the RT-LAMP system is able to detect NVs in clinical specimens within a wide range.
Yoda
Tomoko
T
Department of Infectious Diseases, Osaka Prefectural Institute of Public Health, Osaka, Japan. yoda@iph.pref.osaka.jp
Suzuki
Yasuhiko
Y
Yamazaki
Kenji
K
Sakon
Naomi
N
Kanki
Masashi
M
Aoyama
Ikuko
I
Tsukamoto
Teizo
T
D017250:Caliciviridae Infections / Q000175:diagnosis*; D017931:DNA Primers / Q000235:genetics; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000821:virology*; D006801:Humans; D025202:Molecular Diagnostic Techniques; D008969:Molecular Sequence Data; D029322:Norovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D012367:RNA, Viral