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.
ACS Synth Biol. 2021 Oct 15;10(10):2499-2507. doi: 10.1021/acssynbio.1c00103. Epub 2021 Sep 20.
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.
猪繁殖与呼吸综合征（PRRS）是由猪繁殖与呼吸综合征病毒（PRRSV）引起的一种严重危害养猪业的重要疾病。本研究建立了一种通过将逆转录-重组酶聚合酶扩增（RT-RPA）与 Cas12a 系统的光学性质（单链 DNA-荧光猝灭（ssDNA-FQ）报告基因）相结合，用于高灵敏度检测 PRRSV 的荧光检测方法。该技术可在 25 分钟内实现 PRRSV 的等温可视化检测。特别是，该检测反应可以在单个管中完成。检测 PRRSV 的最低灵敏度可达单拷贝，与其他猪病毒无交叉反应。通过定量逆转录聚合酶链反应（RT-qPCR）和 CRISPR/Cas12a 检测的 11 份 PRRSV 临床样本的相关性确定；结果表明，该方法具有较高的准确性。总之，本研究开发了一种基于 CRISPR-Cas12a 技术的用于现场检测 PRRSV 的可视化、灵敏和特异的核酸检测方法。
