文章摘要
贾添慧,董蕾,王永杰,喻勇新.牡蛎中GⅡ型诺如病毒巢式RT-PCR检测方法的优化与评价[J].上海海洋大学学报,2021,30(2):239-246
牡蛎中GⅡ型诺如病毒巢式RT-PCR检测方法的优化与评价
Updating and evaluation of nested RT-PCR for specific detection of genogroup Ⅱ norovirus in oysters
投稿时间:2020-03-02  修订日期:2020-04-16
DOI:10.12024/jsou.20200302944
中文关键词: 牡蛎  GⅡ型诺如病毒  巢式RFPCR  特异性  阳性率
英文关键词: oysters  genogroupⅡ norovirus  nested RT-PCR  specificity  positive rate
基金项目:“十三五”国家重点研发计划重点专项(2017YFC1600703);国家自然科学基金(31601570)
作者单位E-mail
贾添慧 上海海洋大学 食品学院, 上海 201306  
董蕾 上海海洋大学 食品学院, 上海 201306  
王永杰 上海海洋大学 食品学院, 上海 201306
农业农村部水产品贮藏保鲜质量安全风险评估实验室, 上海 201306 
 
喻勇新 上海海洋大学 食品学院, 上海 201306
农业农村部水产品贮藏保鲜质量安全风险评估实验室, 上海 201306 
yxyu@shou.edu.cn 
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中文摘要:
      为了解决目前巢式PCR法检测牡蛎样品中GⅡ型诺如病毒时存在的非特异性扩增、假阴性等问题,对NCBI数据库中在线获得的所有GⅡ型诺如病毒序列进行系统分析,重新设计出针对GⅡ型诺如病毒检测的通用型巢式PCR引物(NG2OF/NG2OR),并从基因型覆盖度、特异性、灵敏度和实际样品检测的稳定性几个方面对新设计的引物进行评估。结果显示:新引物的特异性好,只对GⅡ型诺如病毒进行有效扩增,且不受牡蛎样品本底的影响;新引物灵敏度高,最低可检测至26.4个拷贝;在对92个牡蛎样品进行检测时,新引物的阳性检出率为28.3%,比经典引物高出7.5%。因此,新建立的GⅡ型诺如病毒巢式PCR检测方法在检测牡蛎样品时特异性更强,灵敏度更高,为牡蛎中诺如病毒的检测提供了一种新的、可靠的技术手段。
英文摘要:
      New primers (NG2OF/NG2OR) were designed based on analysis of all GⅡ norovirus sequences available online to solve the problem of non-specific amplification and false negative in detecting GⅡ norovirus in oysters by performing the present nested PCR method. The primers are evaluated for their coverage, specificity, sensitivity and stability. The results show that:New primers were specifically targeted for GⅡ norovirus and not affected by the original background of the oyster samples. The assay successfully detects GⅡ norovirus from norovirus contaminated oysters with the sensitivity of 26.4 noroviruses. The assay has a 28.3% positive rate as 92 oyster samples are detected, which is 7.5% higher than the typical method. Thus, the new nested PCR method, with a higher sensitivity, is more efficient in the detection of oyster samples. It provided a new and reliable assay for the detection of GⅡ norovirus in oysters.
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