基于RAA-CRISPR/Cas13a的鲤春病毒血症病毒快速检测方法的建立

Establishment of a Rapid Detection Method for Spring Viremia of Carp Virus Based on RAA-CRISPR/Cas13a

中文关键词: 鲤春病毒血症病毒  恒温扩增  CRISPR  重组酶介导等温核酸扩增技术

英文关键词: Spring Viremia of Carp Virus  Constant Temperature Amplification  Clustered Regularly Interspaced Short Palindromic Repeats  Recombinase-Aided Amplification

中文摘要:

      鲤春病毒血症病毒(Spring viraemia ofcarp virus,SVCV)是一种高致病性病原,对鲤科鱼类造成巨大危害,被世界动物卫生组织列为必须通报疫病。2002年,鲤春病毒血症(Spring Viremia of Carp,SCV)在中国首次检出后,迅速在全国蔓延,给我国鲤鱼养殖业造成巨大威胁。因此,我国农业部将其列为二类动物疫病。迄今为止,早期、快速、准确的诊断仍然是控制其传播和流行的重要手段。本研究基于CRISPR/Cas13a系统结合重组酶介导等温核酸扩增技术(recombinase-aided amplification, RAA),通过对GenBank 上登录的SVCV全基因序列比对,针对SVCV聚合酶L基因高度保守的区域设计了一组RAA扩增引物和相应crRNA。建立了一种可用于SVCV现场快速检测的RAA-CRISPR/Cas13a的检测方法,可以覆盖SVCV的4种不同基因型(Ia-Id)。按照世界动物卫生组织推荐的检测方法验证程序,经试验验证该检测方法有较好的重复性,最低检出限为 115 copies/ μL,与其他病原之间不发生交叉反应；通过对实验室分离保存的60份来自进出境水生动物疫病监测、国内重大水生动物疫病监测,以及攻毒实验的样本进行检测,RAA-CRISPR/Cas13a检测结果与实时荧光RT-PCR、套式RT-PCR和病毒分离培养结果完全一致,诊断灵敏度和诊断特异性均达到100%；用本研究建立的方法在三个不同实验室开展实验室间比对,在相同条件下对二十份样本进行检测,三个实验室的检测结果一致。结果表明本研究建立的方法重现性良好。这是首次采用CRISPR-Cas13a系统进行SVCV检测的研究,对SVCV快速诊断和防控具有重要现实意义。

英文摘要:

      Spring viremia of carp virus (SVCV) is a highly pathogenic pathogen, causing great harm to cyprinid fishes, and has been listed as a notifiable disease by the World Organization for Animal Health. Spring viremia of common carp (Cyprinus carpio) is a viral infectious disease of fish, which is highly concern in the world. The disease has been prevalent in Europe, Asia, and North America. In 2002, Spring Viremia of Carp (SCV) was detected for the first time in China, and spread rapidly throughout the country, posing a huge threat to the carp farming industry in China. It was listed as a second-class animal disease. So far, early, rapid, and accurate diagnosis is still an important means to control its spread and prevalence. At present, most of the commonly used detection of SVCV requires specific amplification equipment and temperature cycles to complete the detection, which is prone to false negatives. It has high requirements for, equipment, and personnel, and a long detection cycle. It is difficult to complete fast and effective daily testing. The recombinase-aided amplification (RAA) is a rapid amplification technique of nucleic acid at constant temperature, which can achieve nucleic acid amplification at constant temperature and is easy to operate. In recent years, it has been reported that regular clustered interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) systems, such as CRISPR-Cas12 and CRISPR-Cas13, are combined with isothermal amplification technology to detect RNA viruses., to improve the specificity and sensitivity of detection further. The notable characteristics of this technology are constant temperature, fast reaction speed, and miniaturization, and it is suitable for rapid diagnosis and regular monitoring in the field. As an SVC international reference laboratory recognized by WOAH, this research team has been cooperating with the SVC reference laboratory in the UK to carry out the screening, comparison, and optimization of nucleic acid rapid detection. Based on CRISPR/Cas13a system and recombinase-mediated isothermal nucleic acid amplification technology, a group of RAA amplification primers and corresponding crRNA primers were designed for the highly conserved region of SVCV polymerase L gene by aligning the whole gene sequence of SVCV registered on GenBank, and a preliminary RAA-CRISPR/Cas13a detection was established for the rapid detection of SVCV in the field. It can cover 4 different genotypes of SVCV (Ia-Id). According to the detection and verification procedures recommended by the World Organization for Animal Health, it has been confirmed that some of the detection is reproducible, the minimum detection concentration is 115 copies/μL, and it does not cross-react with other pathogens; Through the detection of 60 samples isolated and stored in the laboratory from the monitoring of inbound and outbound aquatic animal diseases, the monitoring of major aquatic animal diseases in my country, and the challenge experiment, the RAA-CRISPR/Cas13a detection results are consistent with real-time fluorescent RT-PCR, nested The results of RT-PCR and virus isolation and culture are consistent, and the diagnostic sensitivity and diagnostic specificity are both 100%; In the same condition, twenty samples were tested in three different laboratories, and the results of the three laboratories were consistent. The results show that the reproducibility of the study is good. This is the first study of SVCV detection using the CRISPR-Cas13a system, which has important practical significance for the rapid diagnosis and prevention of SVCV.