摘要
目的建立GI、GII型诺如病毒的常规RT-PCR和荧光定量RT-PCR检测方法,并对两种方法进行应用。方法优化筛选出最佳PCR反应体系与反应条件,并从灵敏性、特异性、临床样品检测等方面对建立的方法进行比较与评价。结果该两种方法特异性强,与札幌病毒、轮状病毒、星状病毒、腺病毒同时检测无交叉反应,同一体系内GI、GII型诺如病毒相互之间没有干扰;常规RT-PCR最低检测限为103copies/μL,荧光定量RT-PCR最低检测限为102copies/μL;对180份临床粪便样品进行检测,常规RT-PCR则检测率为5.56%(10/180),符合率达97.22%,荧光定量RT-PCR检测率为8.33%(15/180),符合率达100%;对15份阳性样品测序分析,证实均为诺如病毒。结论建立的常规RT-PCR与荧光定量RT-PCR均可用于诺如病毒的快速检测,荧光定量RT-PCR更为灵敏。
We established conventional RT-PCR and real-time RT-PCR methods for the detection of norovirus GI and GII. The PCR system and reaction conditions were optimized so that conventional RT-PCR and real-time RT-PCR method were es tablished, the sensitivity, specificity and the detection of clinical stool specimens of these two methods were compared and eval- uated. Results showed that the two methods possessed high specificity for norovirus, without any evident cross-reaction with each other or with other enteric viruses, including Sapporovirus, Rotavirus, Stellatevirus and Adenovirus; the detection limit of conventional RT-PCR was 103 copies/μL, while the detection limit of real-time RT-PCR was 10z copies/μL; the two methods were detected of 180 clinical stool specimens for norovirus; the positive rate for conventional RT-PCR was 5.56M （10/180）, the coincidence rate was 97.22M; the positive rate for real-time RT-PCR was 8.33M （15/180）, and the coincidence rate was 100%; 15 positive samples were conducted the sequencing analysis and it was confirmed that all of them were norovirus. The two methods were established in this study, which were used in the rapid detection of norovirus, while real-time RT-PCR was more sensitive than conventional RT-PCR.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2015年第7期602-606,611,共6页
Chinese Journal of Zoonoses
基金
国家"863计划"(2012AA101601)~~
