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一种新型基于 CRISPR-Cas12a 的非洲猪瘟病毒检测 assay 的开发和临床应用。

Development and clinical application of a novel CRISPR-Cas12a based assay for the detection of African swine fever virus.

机构信息

State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, North Third Road, Guangzhou Higher Education Mega Center, Guangzhou, Guangdong, 510006, PR China.

Precise Genome Engineering Center, School of Life Sciences, Guangzhou University, Guangzhou, Guangdong, 510006, PR China.

出版信息

BMC Microbiol. 2020 Sep 14;20(1):282. doi: 10.1186/s12866-020-01966-6.

DOI:10.1186/s12866-020-01966-6
PMID:32928112
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7491166/
Abstract

BACKGROUND

As no treatment or effective vaccine for African swine fever virus (ASFV) is currently available, a rapid, highly sensitive diagnostic is urgently needed to curb the spread of ASFV.

RESULTS

Here we designed a novel CRISPR-Cas12a based assay for ASFV detection. To detect different ASFV genotypes, 19 crRNAs were designed to target the conserved p72 gene in ASFV, and several crRNAs with high activity were identified that could be used as alternatives in the event of new ASFV variants. The results showed that the sensitivity of the CRISPR-Cas12a based assay is about ten times higher than either the commercial quantitative PCR (qPCR) kit or the OIE-recommended qPCR. CRISPR-Cas12a based assay could also detect ASFV specifically without cross-reactivity with other important viruses in pigs and various virus genotypes. We also found that longer incubation times increased the detection limits, which could be applied to improve assay outcomes in the detection of weakly positive samples and new ASFV variants. In addition, both the CRISPR-Cas12a based assay and commercial qPCR showed very good consistency.

CONCLUSIONS

In summary, the CRISPR-Cas12a based assay offers a feasible approach and a new diagnostic technique for the diagnosis of ASFV, particularly in resource-poor settings.

摘要

背景

目前尚无治疗或有效疫苗可用于对抗非洲猪瘟病毒（ASFV），因此迫切需要一种快速、高度敏感的诊断方法来遏制 ASFV 的传播。

结果

本研究设计了一种新型基于 CRISPR-Cas12a 的 ASFV 检测方法。为了检测不同的 ASFV 基因型，我们设计了 19 个靶向 ASFV 保守 p72 基因的 crRNA，鉴定出了几个具有高活性的 crRNA，以便在出现新的 ASFV 变体时可以作为替代。结果表明，基于 CRISPR-Cas12a 的检测方法的灵敏度比商业定量 PCR（qPCR）试剂盒或 OIE 推荐的 qPCR 高约 10 倍。CRISPR-Cas12a 检测方法还可以特异性检测 ASFV，与猪体内的其他重要病毒和各种病毒基因型无交叉反应。我们还发现，延长孵育时间可以提高检测限，这可应用于改善弱阳性样本和新的 ASFV 变体的检测结果。此外，基于 CRISPR-Cas12a 的检测方法和商业 qPCR 具有非常好的一致性。

结论

综上所述，基于 CRISPR-Cas12a 的检测方法为 ASFV 的诊断提供了一种可行的方法和新技术，特别是在资源匮乏的环境下。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c76f/7491166/deb07db7dd49/12866_2020_1966_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c76f/7491166/db3762bf7c3e/12866_2020_1966_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c76f/7491166/233c68dc913c/12866_2020_1966_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c76f/7491166/6c6b931a845f/12866_2020_1966_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c76f/7491166/2c4e328da03f/12866_2020_1966_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c76f/7491166/e3791e7c8bf6/12866_2020_1966_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c76f/7491166/deb07db7dd49/12866_2020_1966_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c76f/7491166/db3762bf7c3e/12866_2020_1966_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c76f/7491166/233c68dc913c/12866_2020_1966_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c76f/7491166/6c6b931a845f/12866_2020_1966_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c76f/7491166/2c4e328da03f/12866_2020_1966_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c76f/7491166/e3791e7c8bf6/12866_2020_1966_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c76f/7491166/deb07db7dd49/12866_2020_1966_Fig6_HTML.jpg

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