Highly Sensitive CRISPR/Cas12a-Based Fluorescence Detection of Porcine Reproductive and Respiratory Syndrome Virus,ACS Synthetic Biology

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Highly Sensitive CRISPR/Cas12a-Based Fluorescence Detection of Porcine Reproductive and Respiratory Syndrome Virus
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2021-09-20 , DOI: 10.1021/acssynbio.1c00103
Siyuan Liu _{1,

2} , Dagang Tao ₃ , Yuying Liao ₄ , Yalan Yang ₂ , Shouzhang Sun ₅ , Yulan Zhao ₂ , Peng Yang _{2,

6} , Yijie Tang ₂ , Bin Chen ₁ , Yonggang Liu ₅ , Shengsong Xie ₃ , Zhonglin Tang _{2,

7,

8,

9}

Affiliation

College of Animal Science & Technology, Hunan Agricultural University, Changsha 410128, PR China.
Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture & Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518000, PR China.
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education & Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, PR China.
Guangxi Veterinary Research Institute, Nanning 530001, PR China.
Faculty of Animal Science and Technology, Yunnan Agricultural University, Yunnan 650201, PR China.
School of Life Sciences, Henan University, Kaifeng 475004, PR China.
State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China.
Kunpeng Institute of Modern Agriculture at Foshan, Foshan 528226, PR China.
GuangXi Engineering Research Center for Resource Development of Bama Xiang Pig, Bama 547500, PR China.

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease of swine that is caused by PRRS virus (PRRSV). In this study, we established a fluorescence assay for highly sensitive detection of PRRSV through integration of the reverse transcription–recombinase polymerase amplification (RT-RPA)-coupled Cas12a system with an optical property of single stranded DNA–fluorescently quenched (ssDNA-FQ) reporter. This technique can achieve isothermal and visual detection of PRRSV in 25 min. In particular, the assay reaction can be completed in a single tube. The limit of sensitivity for PRRSV detection was single copy without cross-reactivity of other porcine viruses. Correlation between 11 PRRSV clinical samples measured by the quantitative reverse transcription polymerase chain reaction (RT-qPCR) and CRISPR/Cas12a assay was determined; the result showed that our results were highly accurate. To sum up, this study developed a visual, sensitive, and specific method of nucleic acid detection based on a CRISPR-Cas12a technique for the on-site detection of PRRSV.

中文翻译：

基于 CRISPR/Cas12a 的猪繁殖和呼吸综合征病毒的高灵敏度荧光检测

猪繁殖与呼吸综合征（PRRS）是由PRRS病毒（PRRSV）引起的一种重要的猪疾病。在本研究中，我们通过整合逆转录-重组酶聚合酶扩增 (RT-RPA) 耦合 Cas12a 系统与单链 DNA 荧光淬灭 (ssDNA-FQ) 的光学特性，建立了一种用于高灵敏度检测 PRRSV 的荧光测定法。记者。该技术可在25分钟内实现PRRSV等温目测检测。特别地，测定反应可以在单个试管中完成。PRRSV 检测的灵敏度极限是单拷贝，没有其他猪病毒的交叉反应。确定了通过定量逆转录聚合酶链反应 (RT-qPCR) 和 CRISPR/Cas12a 测定法测量的 11 个 PRRSV 临床样本之间的相关性；结果表明我们的结果非常准确。综上所述，本研究开发了一种基于CRISPR-Cas12a技术的视觉、灵敏、特异性的核酸检测方法，用于PRRSV的现场检测。

更新日期：2021-10-15

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