Rapid, sensitive, and visual detection of mandarin fish ranavirus and infectious spleen and kidney necrosis virus using an RPA-CRISPR/Cas12a system

Abstract

Iridoviruses are large cytoplasmic icosahedral viruses that contain dsDNA. Among them, mandarin fish ranavirus (MRV) and infectious spleen and kidney necrosis virus (ISKNV) are particularly notable due to their high contagiousness and pathogenicity. These viruses pose a significant threat to fish aquaculture, resulting in substantial annual economic losses for the fish farming industry. Therefore, the development of novel, rapid virus detection technologies is essential for the prevention and control of ISKNV and MRV diseases. In this study, we developed a rapid, sensitive, and visual detection method for MRV and ISKNV using the recombinase polymerase amplification (RPA)-CRISPR/Cas12a system. This method can detect as low as 1 copy/μL of MRV and 0.1 copy/μL of ISKNV, demonstrating excellent specificity and reproducibility. The detection can be performed at a constant temperature of 37-39°C, eliminating the need for complex equipment. A 30-min RPA amplification followed by a 15-min CRISPR/Cas reaction is sufficient for detecting most samples. For low-concentration samples, extending the CRISPR/Cas reaction time to 60 min improves result visibility. The designed RPA reaction system is capable of performing reverse transcription of RNA, allowing for the detection of mRNA transcribed from the MCP gene of MRV and ISKNV in the sample. Furthermore, two probes were identified that can be observed without the need for excitation light. In conclusion, a field-suitable detection method for ISKNV and MRV has been established, providing a powerful tool for the prompt diagnosis of these aquatic pathogens and aiding in the prevention and control of ISKNV and MRV diseases.

Keywords: CRISPR/Cas12a; ISKNV; RPA; iridovirus; ranavirus.

Figure 1

Screening of RPA primers and crRNA. Nucleic acid agarose gel electrophoresis results are presented, showing amplification of (A) MRV templates and (B) ISKNV templates using various RPA primers. For MRV templates, the following primer pairs were used: 1, MRV-RPA-F1/MRV-RPA-R1; 2, MRV-RPA-F1/MRV-RPA-R2; 3, MRV-RPA-F1/MRV-RPA-R3; 4, MRV-RPA-F2/MRV-RPA-R1; 5, MRV-RPA-F2/MRV-RPA-R2; 6, MRV-RPA-F2/MRV-RPA-R3; 7, MRV-RPA-F3/MRV-RPA-R1; 8, MRV-RPA-F3/MRV-RPA-R2; 9, MRV-RPA-F3/MRV-RPA-R3. “+” represents a positive amplification result, while “−” represents a negative result. Similarly, for ISKNV templates, the primer pairs used were: 1, ISKNV-RPA-F1/ISKNV-RPA-R1; 2, ISKNV-RPA-F1/ISKNV-RPA-R2; 3, ISKNV-RPA-F1/ISKNV-RPA-R3; 4, ISKNV-RPA-F2/ISKNV-RPA-R1; 5, ISKNV-RPA-F2/ISKNV-RPA-R2; 6, ISKNV-RPA-F2/ISKNV-RPA-R3; 7, ISKNV-RPA-F3/ISKNV-RPA-R1; 8, ISKNV-RPA-F3/ISKNV-RPA-R2; 9, ISKNV-RPA-F3/ISKNV-RPA-R3. The detection results for (C) MRV and (D) ISKNV using various crRNAs are compared. The y-axis indicates the relative fluorescence intensity, while the x-axis indicates the various types of crRNA added to the samples.

Figure 2

Optimization of the reaction system. (A,B) Detection of Cas12a protein and crRNA concentrations for MRV and ISKNV, respectively. (C,D) Comparison of detection results using different concentrations of fluorescent probes for MRV and ISKNV, respectively. The x-axis represents the volumes of 10 μM Cas12a protein and crRNA used, while the y-axis indicates the relative fluorescence intensity.

Figure 3

RNA detection capability and probe optimization. Validation of the ability to detect RNA targets (treated with DNase) using (A) the MRV detection system and (B) the ISKNV detection system. The x-axis represents the types of templates used, and the y-axis represents the relative fluorescence intensity. (C) Detection of positive samples using various types of fluorescent probes at a final concentration of 10 μM, observed under natural light.

Figure 4

Sensitivity of the CRISPR/Cas12a detection method. (A,B) Fluorescence intensity after a 15-minCRISPR/Cas12a reaction with varying concentrations of MRV and ISKNV standards. (C,D) Fluorescence intensity after a 60-min CRISPR/Cas12a reaction with different concentrations of MRV and ISKNV standards. The x-axis represents the standard sample concentration, and the y-axis indicates the relative fluorescence intensity. NC indicates DEPC water. Significant differences are indicated by asterisks (*). Ns, not significant (p > 0.05); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 5

Specificity of the CRISPR/Cas12a detection method. Validation of the specificity for the (A) MRV detection system and (B) ISKNV detection system using different templates. The x-axis represents the types of templates used, and the y-axis indicates the relative fluorescence intensity. NC indicates DEPC water.

Figure 6

Reproducibility of the CRISPR/Cas12a detection method. Reproducibility validation of the (A) MRV detection system and (B) ISKNV detection system using positive DNA templates. Each sample was tested six times. The x-axis represents the templates used and their respective replicates, while the y-axis represents the relative fluorescence values, as well as NC indicates DEPC water.

Figure 7

Detection results of actual samples. (A) Gel electrophoresis results of nested PCR products for MRV detection in the samples; (B) RPA-CRISPR/Cas12a detection results for MRV in the samples; (C) Gel electrophoresis results of nested PCR products for ISKNV detection in the samples; (D) RPA-CRISPR/Cas12a detection results for ISKNV in the samples. The numbers 1–8 represent the samples, “+” represents the positive control, and “−” represents the negative control.

Figure 8

Schematic diagram of the RPA-CRISPR/Cas12a detection method of MRV and ISKNV.