CAJ | 학술논문

目的建立适用于牡蛎和粪便中快速、特异、灵敏的GⅠ、GⅡ型诺如病毒(NV)定量分型诊断方法.方法通过对GⅠ、GⅡ型NV基因组保守序列的比对分析,设计高度特异的引物和探针,建立以TaqMan探针为基础的实时聚合酶链反应方法(TaqMan Real-time RT-PCR).结果该方法对NV核酸检测高度特异,且GⅠ和GⅡ型之间无交叉反应,最低检出限达102 copies/μl.对90份新鲜牡蛎样品和37份腹泻粪便标本分别用常规RT-PCR和笔者建立的TaqMan Real-time RT-PCR进行NV检测,发现牡蛎样品中后者的检出率明显高于前者,而粪便标本中两者无明显差别.同时对阳性标本的测序分析证实结果准确可靠.结论研究中建立的TaqMan Real-time RT-PCR方法可用于海产品标本及粪便中NV定量及分型检测,可作为应对NV胃肠炎暴发的有效诊断方法.
목적 건립괄용우모려화분편중쾌속、특이、령민적GⅠ、GⅡ형낙여병독(NV)정량분형진단방법.방법 통과대GⅠ、GⅡ형NV기인조보수서렬적비대분석,설계고도특이적인물화탐침,건립이TaqMan탐침위기출적실시취합매련반응방법(TaqMan Real-time RT-PCR).결과 해방법대NV핵산검측고도특이,차GⅠ화GⅡ형지간무교차반응,최저검출한체102 copies/μl.대90빈신선모려양품화37빈복사분편표본분별용상규RT-PCR화필자건립적TaqMan Real-time RT-PCR진행NV검측,발현모려양품중후자적검출솔명현고우전자,이분편표본중량자무명현차별.동시대양성표본적측서분석증실결과준학가고.결론 연구중건립적TaqMan Real-time RT-PCR방법가용우해산품표본급분편중NV정량급분형검측,가작위응대NV위장염폭발적유효진단방법.
Objective To develop a rapid, specific and sensitive diagnostic method for quantification and typing of genogroup Ⅰ and Ⅱ norovirus in oyster shellfish and stool samples from patients who had eaten them. Methods Specific primers and probe, following large scale norovirus genome consensus analysis were designed and subsequently a TaqMan based Real-time PCR assay to detect both GⅠ and GⅡ were established. Results This method showed high specificity for norovirus nucleic acid detection, and no cross-reaction among norovirus GⅠ and GⅡ. The limit on detection of NV genomes was 102 copies/μl. A total of 90 oysters and 37 stool specimens with diarrhea were tested for norovirus by conventional reverse transcriptional PCR (RT-PCR) assay as well as the TaqMan Real-time PCR, respectively. The norovirus detection rate in oysters by TaqMan PCR was significantly higher than that by conventional RT-PCR, but no differences between the two PCR methods were found when detecting the stool samples. Reliability of the Real-time PCR for norovirus detection was further confirmed by DNA sequencing of the positive samples.Conclusion This TaqMan Real-time PCR assay was proved to be a useful method for quantification and typing for norovirus in routine monitoring of both oyster shellfish and clinical samples.This method is recommended to be an effective diagnostic method for outbreak-associated gastroenteritis due to norovirus.