Development of visual detection of African swine fever virus using CRISPR/LwCas13a lateral flow strip based on structural protein gene D117L

IF 2.7 2区 农林科学 Q3 MICROBIOLOGY
Desheng Zhang , Sen Jiang , Nengwen Xia , Jiajia Zhang , Anjing Liu , Dafu Deng , Chenyang Zhang , Yuxin Sun , Nanhua Chen , Xilong Kang , Zhiming Pan , Wanglong Zheng , Jianzhong Zhu
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Abstract

African swine fever virus (ASFV) is a large double stranded DNA arbovirus that is highly contagious and seriously endangers domestic and wild pigs. In the past decade, African swine fever (ASF) has spread in many countries in the Caucasus, Russian Federation, Eastern Europe and Asia, causing significant losses to the pig industry. At present, there is a lack of effective vaccine and treatment for ASF. Therefore, the rapid and accurate detection is crucial for ASF prevention and control. In this study, we have developed a portable lateral flow strip (LFS) detection mediated by recombinase polymerase amplification (RPA) and CRISPR/LwCas13a, which is performed at 37 ℃ and visualized by eyes without the need for complex instruments. This RPA-LwCas13a-LFS is based on the ASFV structural protein p17 gene (D117L), with a detection sensitivity up to 2 gene copies. This method is highly specific and has no cross reactivity to 7 other pig viruses. In the detection of two batches of 100 clinical samples, the p17 (D117L) RPA-LwCas13a-LFS had 100% coincidence with conventional quantitative PCR (qPCR). These findings demonstrate the potential of this simple, rapid, sensitive, and specific ASFV detection method for on-site ASFV detection.

利用基于结构蛋白基因 D117L 的 CRISPR/LwCas13a 侧流条带开发非洲猪瘟病毒的可视检测方法
非洲猪瘟病毒(ASFV)是一种大型双链 DNA 虫媒病毒,具有高度传染性,严重危害家猪和野猪。近十年来,非洲猪瘟(ASF)在高加索、俄罗斯联邦、东欧和亚洲的许多国家蔓延,给养猪业造成了重大损失。目前,非洲猪瘟缺乏有效的疫苗和治疗方法。因此,快速准确的检测对预防和控制 ASF 至关重要。在这项研究中,我们开发了一种由重组酶聚合酶扩增(RPA)和CRISPR/LwCas13a介导的便携式侧流条带(LFS)检测方法,该方法在37 ℃下进行,无需复杂仪器,眼睛即可观察。这种 RPA-LwCas13a-LFS 方法基于 ASFV 结构蛋白 p17 基因(D117L),检测灵敏度可达 2 个基因拷贝。该方法特异性强,与其他 7 种猪病毒无交叉反应。在对两批 100 份临床样本的检测中,p17 (D117L) RPA-LwCas13a-LFS 与传统定量 PCR (qPCR) 的吻合率为 100%。这些发现证明了这种简单、快速、灵敏、特异的 ASFV 检测方法在现场 ASFV 检测中的潜力。
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来源期刊
Veterinary microbiology
Veterinary microbiology 农林科学-兽医学
CiteScore
5.90
自引率
6.10%
发文量
221
审稿时长
52 days
期刊介绍: Veterinary Microbiology is concerned with microbial (bacterial, fungal, viral) diseases of domesticated vertebrate animals (livestock, companion animals, fur-bearing animals, game, poultry, fish) that supply food, other useful products or companionship. In addition, Microbial diseases of wild animals living in captivity, or as members of the feral fauna will also be considered if the infections are of interest because of their interrelation with humans (zoonoses) and/or domestic animals. Studies of antimicrobial resistance are also included, provided that the results represent a substantial advance in knowledge. Authors are strongly encouraged to read - prior to submission - the Editorials (''Scope or cope'' and ''Scope or cope II'') published previously in the journal. The Editors reserve the right to suggest submission to another journal for those papers which they feel would be more appropriate for consideration by that journal. Original research papers of high quality and novelty on aspects of control, host response, molecular biology, pathogenesis, prevention, and treatment of microbial diseases of animals are published. Papers dealing primarily with immunology, epidemiology, molecular biology and antiviral or microbial agents will only be considered if they demonstrate a clear impact on a disease. Papers focusing solely on diagnostic techniques (such as another PCR protocol or ELISA) will not be published - focus should be on a microorganism and not on a particular technique. Papers only reporting microbial sequences, transcriptomics data, or proteomics data will not be considered unless the results represent a substantial advance in knowledge. Drug trial papers will be considered if they have general application or significance. Papers on the identification of microorganisms will also be considered, but detailed taxonomic studies do not fall within the scope of the journal. Case reports will not be published, unless they have general application or contain novel aspects. Papers of geographically limited interest, which repeat what had been established elsewhere will not be considered. The readership of the journal is global.
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