Development of a nucleic acid detection method based on the CRISPR-Cas13 for point-of-care testing of bovine viral diarrhea virus-1b.

IF 2.7 3区 农林科学 Q1 AGRICULTURE, DAIRY & ANIMAL SCIENCE
Journal of Animal Science and Technology Pub Date : 2024-07-01 Epub Date: 2024-07-31 DOI:10.5187/jast.2024.e77
Sungeun Hwang, Wonhee Lee, Yoonseok Lee
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引用次数: 0

Abstract

Bovine viral diarrhea (BVD) is a single-stranded, positive-sense ribonucleic acid (RNA) virus belonging to the genus Pestivirus of the Flaviviridae family. BVD frequently causes economic losses to farmers. Among bovine viral diarrhea virus (BVDV) strains, BVDV-1b is predominant and widespread in Hanwoo calves. Reverse-transcription polymerase chain reaction (RT-PCR) is an essential method for diagnosing BVDV-1b and has become the gold standard for diagnosis in the Republic of Korea. However, this diagnostic method is time-consuming and requires expensive equipment. Therefore, Clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems have been used for point-of-care (POC) testing of viruses. Developing a sensitive and specific method for POC testing of BVDV-1b would be advantageous for controlling the spread of infection. Thus, this study aimed to develop a novel nucleic acid detection method using the CRISPR-Cas13 system for POC testing of BVDV-1b. The sequence of the BVD virus was extracted from National Center for Biotechnology Information (NC_001461.1), and the 5' untranslated region, commonly used for detection, was selected. CRISPR RNA (crRNA) was designed using the Cas13 design program and optimized for the expression and purification of the LwCas13a protein. Madin Darby bovine kidney (MDBK) cells were infected with BVDV-1b, incubated, and the viral RNA was extracted. To enable POC viral detection, the compatibility of the CRISPR-Cas13 system was verified with a paper-based strip through collateral cleavage activity. Finally, a colorimetric assay was used to evaluate the detection of BVDV-1b by combining the previously obtained crRNA and Cas13a protein on a paper strip. In conclusion, the CRISPR-Cas13 system is highly sensitive, specific, and capable of nucleic acid detection, making it an optimal system for the early point-of-care testing of BVDV-1b.

开发基于 CRISPR-Cas13 的核酸检测方法,用于牛病毒性腹泻病毒-1b 的床旁检测。
牛病毒性腹泻(BVD)是一种单链正义核糖核酸(RNA)病毒,属于黄病毒科佩斯病毒属。BVD 经常给农民造成经济损失。在牛病毒性腹泻病毒 (BVDV) 株系中,BVDV-1b 是主要株系,在汉和犊牛中广泛存在。反转录聚合酶链反应(RT-PCR)是诊断 BVDV-1b 的基本方法,在大韩民国已成为诊断的金标准。然而,这种诊断方法耗时且需要昂贵的设备。因此,簇状规则间隔短回文重复序列-Cas(CRISPR-Cas)系统已被用于病毒的床旁检测(POC)。开发一种灵敏而特异的方法用于 BVDV-1b 的 POC 检测将有利于控制感染的传播。因此,本研究旨在利用CRISPR-Cas13系统开发一种新型核酸检测方法,用于BVDV-1b的POC检测。研究人员从美国国家生物技术信息中心(NC_001461.1)提取了BVD病毒的序列,并选择了常用于检测的5'非翻译区。利用 Cas13 设计程序设计了 CRISPR RNA(crRNA),并对 LwCas13a 蛋白的表达和纯化进行了优化。用 BVDV-1b 感染马丁达比牛肾(MDBK)细胞,培养并提取病毒 RNA。为了实现 POC 病毒检测,通过附带的裂解活性,用纸基条带验证了 CRISPR-Cas13 系统的兼容性。最后,通过在纸条上结合之前获得的 crRNA 和 Cas13a 蛋白,使用比色法评估了 BVDV-1b 的检测效果。总之,CRISPR-Cas13 系统具有高灵敏度、特异性和核酸检测能力,是进行 BVDV-1b 早期床旁检测的最佳系统。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of Animal Science and Technology
Journal of Animal Science and Technology Agricultural and Biological Sciences-Food Science
CiteScore
4.50
自引率
8.70%
发文量
96
审稿时长
7 weeks
期刊介绍: Journal of Animal Science and Technology (J. Anim. Sci. Technol. or JAST) is a peer-reviewed, open access journal publishing original research, review articles and notes in all fields of animal science. Topics covered by the journal include: genetics and breeding, physiology, nutrition of monogastric animals, nutrition of ruminants, animal products (milk, meat, eggs and their by-products) and their processing, grasslands and roughages, livestock environment, animal biotechnology, animal behavior and welfare. Articles generally report research involving beef cattle, dairy cattle, pigs, companion animals, goats, horses, and sheep. However, studies involving other farm animals, aquatic and wildlife species, and laboratory animal species that address fundamental questions related to livestock and companion animal biology will also be considered for publication. The Journal of Animal Science and Technology (J. Anim. Technol. or JAST) has been the official journal of The Korean Society of Animal Science and Technology (KSAST) since 2000, formerly known as The Korean Journal of Animal Sciences (launched in 1956).
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