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

摘要:

为了解决目前巢式RT-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及假阴性等问题，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RT-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RT-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。

Abstract:

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 RT-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.