Detection of Porcine Circovirus (PCV) Using CRISPR-Cas12a/13a Coupled with Isothermal Amplification

重组酶聚合酶扩增 猪圆环病毒 环介导等温扩增 清脆的 生物 聚合酶链反应 病毒学 分子生物学 圆环病毒 计算生物学 病毒 遗传学 DNA 基因
作者
Huijuan Wang,Gang Zhou,Huiming Liu,Ruqun Peng,Tingli Sun,Sujuan Li,Mingjie Chen,Ying-si Wang,Qingshan Shi,Xiao‐Bao Xie
出处
期刊:Viruses [MDPI AG]
卷期号:16 (10): 1548-1548 被引量:1
标识
DOI:10.3390/v16101548
摘要

The impact of porcine circovirus (PCV) on the worldwide pig industry is profound, leading to notable economic losses. Early and prompt identification of PCV is essential in managing and controlling this disease effectively. A range of detection techniques for PCV have been developed and primarily divided into two categories focusing on nucleic acid or serum antibody identification. The methodologies encompass conventional polymerase chain reaction (PCR), real-time fluorescence quantitative PCR (qPCR), fluorescence in situ hybridization (FISH), loop-mediated isothermal amplification (LAMP), immunofluorescence assay (IFA), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA). Despite their efficacy, these techniques are often impeded by the necessity for substantial investment in equipment, specialized knowledge, and intricate procedural steps, which complicate their application in real-time field detections. To surmount these challenges, a sensitive, rapid, and specific PCV detection method using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12a/13a coupled with isothermal amplification, such as enzymatic recombinase amplification (ERA), recombinase polymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP), has been developed. This novel method has undergone meticulous optimization for detecting PCV types 2, 3, and 4, boasting a remarkable sensitivity to identify a single copy per microliter. The specificity of this technique is exemplary, with no observable interaction with other porcine viruses such as PEDV, PRRSV, PRV, and CSFV. Its reliability has been validated with clinical samples, where it produced a perfect alignment with qPCR findings, showcasing a 100% coincidence rate. The elegance of merging CRISPR-Cas technology with isothermal amplification assays lies in its on-site testing without the need for expensive tools or trained personnel, rendering it exceptionally suitable for on-site applications, especially in resource-constrained swine farming environments. This review assesses and compares the process and characteristics inherent in the utilization of ERA/LAMP/RPA-CRISPR-Cas12a/Cas13a methodologies for the detection of PCV, providing critical insights into their practicality and effectiveness.
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