Multiplex CRISPR-Cas Assay for Rapid, Isothermal and Visual Detection of White Spot Syndrome Virus (WSSV) and Enterocytozoon hepatopenaei (EHP) in Penaeid Shrimp

Abstract

White spot syndrome virus (WSSV) and Enterocytozoon hepatopenaei (EHP) represent the most economically destructive pathogens in the current shrimp industry. WSSV causes white spot disease (WSD) responsible for rapid shrimp mortality, while EHP stunts growth and therefore reduces overall productivity. Despite the importance of timely disease detection, current diagnostic methods for WSSV and EHP are typically singleplex, and those offering multiplex detection face issues such as complexity, low field compatibility and/or low sensitivity. Here, we introduce an orthogonal, multiplex CRISPR-Cas assay for concomitant detection of WSSV and EHP. This method combines recombinase polymerase amplification (RPA) for target DNA enrichment with Cas12a and Cas13a enzymes for fluorescent detection. This assay produces distinct fluorescent colours for different diagnostic outcomes, allowing naked eye visualisation without ambiguity. Further validation reveals that the assay detects as few as 20 and 200 copies of target DNA from EHP and WSSV, respectively, while producing no false positives with DNA from other shrimp pathogens. Moreover, the assay excellently agrees with established PCR methods in evaluation of clinical samples. Requiring only 37°C and less than an hour to complete, multiplex CRISPR-Cas assay presents a promising tool for onsite diagnostics, offering high accuracy while saving time and resources.

Keywords: CRISPR diagnostics; Cas12a; Cas13a; Enterocytozoon hepatopenaei (EHP); penaeid shrimp; white spot syndrome virus (WSSV).

FIGURE 1

Schematic illustrating the principle of multiplex CRISPR‐Cas assay. Three different scenarios are displayed: Single infection with EHP (‘+EHP’), single infection with WSSV (‘+WSSV’) and co‐infection (‘+EHP + WSSV’). The DNA extract is first pre‐amplified using recombinase polymerase amplification (RPA). The forward primer for WSSV contains a T7 promoter (arrow) to allow subsequent in vitro transcription. This step takes 20 min at 37°C Then, the amplification product is transferred to another tube containing reagents for T7 in vitro transcription, Cas12a assay and Cas13a assay. If the specimen contains only EHP, the RPA product will directly serve as the substrate for Cas12a and trigger the trans‐cleavage of the ssDNA‐linked ROX‐quencher (RQ) reporter, resulting in red fluorescence. In the case of WSSV, the RPA product must first be transcribed into RNA, which is then cleaved by Cas13a and activates the trans‐cleavage of the ssRNA‐linked FAM‐quencher (FQ) reporter, producing green fluorescence. Co‐infected samples will generate both red and green fluorescence, therefore appearing yellow. The transcription and CRISPR detection step combined takes 30 min at 37°C.

FIGURE 2

Screening for optimal Cas13a guide RNA. (A) Fluorescence obtained from Cas13a reactions using WSSV01, WSSV02 or WSSV03 as guide RNA, measured by a microplate reader. + and – signify reactions containing target RNA and negative controls containing no target RNA, respectively. All + reactions contained 1 nM of target RNA. (B) Reaction vials from (A) when exposed to blue light. (C) Fluorescence from Cas13a reactions using WSSV01 as guide RNA and containing serially diluted target RNA (10¹–10⁻⁶ nM). (D) Reaction vials from (C) when exposed to blue light. All experiments were conducted in triplicate. Bar and error bar represent average fluorescence and standard deviation, respectively. Asterisks indicate statistically significant difference from the negative control containing no target RNA according to the Student's t‐test: ***p < 0.001; ****p < 0.0001. n.s. indicates no statistically significant difference.

FIGURE 3

Preliminary evaluation of the multiplex CRISPR‐Cas assay. The vp28 and ptp2 genes on respective plasmids (2 × 10⁴ copies) were individually amplified by RPA. For brevity, these RPA products are referred to as ‘vp28 amplicon’ and ‘ptp2 amplicon’, respectively. (A) Fluorescence from the multiplex CRISPR‐Cas reactions performed with vp28 amplicon alone, ptp2 amplicon alone and both. The signal was measured in FAM and ROX channels, which correspond to WSSV and EHP, respectively. The experiment was performed in triplicate. Bar and error bar represent average fluorescence and standard deviation, respectively. The reaction vials were also exposed to blue light and the image was captured with iPhone XR using the default settings (B) and Xiaomi Redmi Note 8 using manual settings described in Section 2.7 (C).

FIGURE 4

Analytical sensitivity of multiplex CRISPR‐Cas. Samples contained 2–2 × 10⁶ copies of pVP28 and pPTP2 DNA in a 1:1 ratio. (A) Fluorescence analysed by a microplate reader. n = 3. Bar and error bar represent average fluorescence and standard deviation, respectively. Circles represent individual data points. (B) Reactions from (A) visualised by exposure to blue light. (C) Multiplex CRISPR‐Cas performed on samples containing 200 copies of one target and 2 × 10⁶ copies of the other. (D) Reactions from (C) visualised by exposure to blue light.

FIGURE 5

Analytical specificity of multiplex CRISPR‐Cas. The experiment was performed in triplicate and visualised using a microplate reader (A) and blue light exposure (B). Bar and error bar represent average fluorescence and standard deviation, respectively. Healthy: Shrimp free from WSSV and EHP; AHPND: Vibrio parahaemolyticus strain causing acute hepatopancreatic necrosis disease; IHHNV: Infectious hypodermal and haematopoietic necrosis virus; DHPV: Decapod hepanhamaparvovirus 1.

FIGURE 6

Results of clinical sample evaluation using multiplex CRISPR‐Cas. The experiment was performed in triplicate. Bar and error bar represent average fluorescence and standard deviation, respectively. The images of blue light‐exposed vials are shown below the bar plot. −ve: Negative control (water). +ve: Positive control (a mixture of pVP28 and pPTP2, which are plasmids harbouring the vp28 gene from WSSV and the ptp2 gene from EHP, at 2 × 10⁶ copies each).