Highly sensitive detection of white spot syndrome virus with an RPA-CRISPR combined one-pot method

A recombinase polymerase amplification and Pyrococcus furiosus Argonaute combined method for ultra‐sensitive detection of white spot syndrome virus in shrimp

“…Assays solely dependent on isothermal amplifications could be affected by amplification interfering factors from the on-site environment. Therefore, RPA and CRISPR combined assays were developed to increase the reliability of on-site detections, achieving a sensitivity of 10 copies (Wang et al, 2023). Considering the lab-running nested PCR method showed a sensitivity of two copies (Natividad et al, 2008), there was room for on-site detection assays to improve the sensitivity.…”

“…AF173993) was selected as the detection target as has been established previously (Wang et al, 2023;Xia et al, 2014). A 757-bp fragment of the VP28 gene was amplified with the primer pair WSSV-PCR-757 FP (5′-TTGCC AAT TGT CCT GTT ACG TACTCTG-3′) and WSSV-PCR-757 RP (5′-ACGAT TTA TTT ACT CGG TCT CAGTGCC-3′) and was inserted into the pMD18T vector with the standard molecular cloning technique as reported previously (Wang et al, 2023). The plasmid was verified by sequencing (General Biology Co. Ltd.) and used as the standard plasmid for the VP28 gene fragment in this study.…”

“…aquatic disease, on-site detection, PfAgo, RPA, WSSV As there is currently no effective treatment for this disease, developing early detection technologies for WSSV is of great significance for the prevention of WSD (Hossain et al, 2022) (Jang et al, 2009;Jaroenram et al, 2009;Mendoza-Cano & Sánchez-Paz, 2013;Mrotzek et al, 2010;Natividad et al, 2008;Xia et al, 2014;Zhang et al, 2022). For convenient detection in the field, isothermal amplification technologies, LAMP and RPA, with the aid of CRISPR-Cas nuclease, have provided on-site detection tools for WSSV (Chaijarasphong et al, 2019;Chen et al, 2021;Wang et al, 2023).…”

“…Many nucleic acid detection technologies have been used for WSSV detection, including polymerase chain reaction (PCR), quantitative PCR (qPCR), nested PCR, loop‐mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), with a detection sensitivity range of 2–200 copies of the viral genome (Jang et al, 2009; Jaroenram et al, 2009; Mendoza‐Cano & Sánchez‐Paz, 2013; Mrotzek et al, 2010; Natividad et al, 2008; Xia et al, 2014; Zhang et al, 2022). For convenient detection in the field, isothermal amplification technologies, LAMP and RPA, with the aid of CRISPR‐Cas nuclease, have provided on‐site detection tools for WSSV (Chaijarasphong et al, 2019; Chen et al, 2021; Wang et al, 2023).…”

“…By using a recombinase, T4 uvsX, the system realized low‐temperature strand‐opening and achieved potent DNA amplification capacity (Piepenburg et al, 2006). Here, a fragment of gene VP28 of WSSV was selected as the detection target as had been established previously (Wang et al, 2023; Xia et al, 2014). This fragment was amplified by RPA, and the amplicon was used as the substrate for Pf Ago cleavage after a DNA purification step.…”