RPA-CRISPR is an in vitro nucleic acid detection platform based on recombinase polymerase amplification and CRISPR-mediated cleavage of the FAM-BHQ RNA reporter (Fig. 1A). The RNA reporter (5'-FAM-UUUUU) and its cleavage product were validated by electrophoresis (Fig. 1B). To specifically detect the viral DNA of ASFV, three crRNAs were validated and applied in RPA-CRISPR (Fig. 1C). The twofold gradient dilutions of crRNA and CRISPR-lwCas13a protein were used for phalanx titration test to determine the optimal concentrations. In this study, 1.626 ng/μL of CRISPR-lwCas13a protein and 2.63 ng/μL of crRNA were determined to be the optimal concentrations for development of RPA-CRISPR detection of ASFV (Fig. 1D, 1E).
Figure 1. Schematic diagram and system optimization of RPA-CRISPR. A Schematic diagram in the whole process of RPA-CRISPR detection. B Analysis of RNA reporter cleavage by acrylamide denaturing electrophoresis. C Validation of crRNAs. D Two-fold gradient dilution of CRISPR-lwCas13a protein was used for determining the optimal concentrations. E Two-fold gradient dilution of crRNA was used for determining the optimal concentrations.
RPA-CRISPR is a specific nucleic acid detection method with attomolar sensitivity. To determine the detection limit, tenfold serial dilutions of ASFV P72 plasmid and viral genomic DNA were used for sensitivity assay. P72 plasmid was diluted in sterile water to determine the methodological sensitivity. HUDSON-treated blood suspension containing ASFV genomic DNA was used for sensitivity test. The results indicated that the detection limit was as low as a single copy/μL (100 copy/μL) no matter when RPACRISPR targeted P72 plasmid in water solutions (Fig. 2A) or genomic DNA in blood suspension (Fig. 2B).
Figure 2. Validation of sensitivity and specificity of qPCR-based RPACRISPR. A tenfold serial dilutions of ASFV VP72 plasmid in ddH2O were used for determining the sensitivity of RPA-CRISPR. B tenfold serial dilutions of ASFV genomic DNA in blood suspension were used for determining the sensitivity of RPA-CRISPR. ASFV viral DNA and control viral DNA or RNA (PCV-2 ZJ/C, PCV-2 SH, CSFV-C, PRRSV JAX1-R, PRRSV R98, PRV Bartha-K61, PRV HB- 98, TGEV + PEDV) were dissolved in sterile water C and blood suspension collected from the SPF pigs D to determine specificity of RPA-CRISPR, respectively. The blood suspension collected from the SPF pigs was used as the negative control. E ASFV viral DNA was separately added in different tissue suspensions to be analyzed by RPA-CRISPR. Tissue suspension from the SPF pigs was included as a negative control.
Viral genomic DNA or RNA was extracted from ASFV positive blood samples and commercial vaccines of other porcine viruses (PCV-2 ZJ/C, PCV-2 SH, CSFV-C, PRRSV JAX1-R, PRRSV R98, PRV Bartha-K61, PRV HB-98, TGEV and PEDV) and was dissolved in either sterile water or blood suspension collected from specific-pathogen-free (SPF) pigs. ASFV genomic DNA and other control viral DNA or RNA were used for determining the specificity of RPA-CRISPR. The results indicated that RPA-CRISPR accurately distinguished viral DNA of ASFV from control viral DNA or RNA with no cross-reactivity in either water or blood suspension (Fig. 2C, 2D). ASFV viral DNA (50 ng) was separately added into 1 mL of different tissue suspensions (heart, liver, lung, spleen, kidney and blood) to validate whether cellular components from different tissues affect RPA-CRISPR reaction. Each type of tissue suspension containing no ASFV viral DNA was included as a negative control. The results indicated that RPA-CRISPR could accurately distinguish the ASFV viral DNA from negative control in different tissue suspensions (Fig. 2E).
Since the specificity of RPA-CRISPR depends on the number of mismatches on crRNA target region, sequences of ASFV different genotype strains were compared to identify the mismatches. The results indicated that no mismatch could be found at the target regions of all crRNAs on ASFV P72 gene (genotype Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅴ and ⅩⅩ) (Table 1). The genotype Ⅸ and Ⅹ could not be detected by crRNA-1, while genotype Ⅶ, Ⅸ and Ⅹ could not be identified by crRNA-3. crRNA-2 could be used for detecting all the genotypes analyzed in this study (Table 1).
Isolate name GenBank no Country Genotype Mismatches cr1a cr2b cr3c E75 FN557520.1 Spain Ⅰ 0 0 0 Georgia 2007/1 NC_044959.1 Georgia Ⅱ 0 0 0 Wuhan 2019–1 MN393476.1 China Ⅱ 0 0 0 Krasnodar 2012 KJ195685.1 Russia Ⅱ 0 0 0 Pig/HLJ/2018 MK333180.1 China Ⅱ 0 0 0 Warmbaths AY261365.1 South Africa Ⅰ/Ⅲ 0 0 0 Warthog AY261366.1 Namibia Ⅳ 0 0 0 Tengani 62 AY261364.1 Malawi Ⅴ 0 0 0 Mkuzi 1979 AY261362.1 South Africa Ⅰ/Ⅶ 0 0 2 Ken06.Bus NC_044946.1 Kenya Ⅸ 2 0 2 Ken05/Tk1 KM111294.1 Kenya Ⅹ 2 0 2 Pretoriuskop/96/4 AY261363.1 South Africa ⅩⅩ/Ⅰ 0 0 0 a, b, c crRNA-1, crRNA-2 and crRNA-3, respectively
Table 1. Analysis of mismatches on crRNA targeted regions of P72 genes from different genotype strains.
To compare RPA-CRISPR with the traditional PCR method, a TaqMan qPCR-based method was established according to the previous studies (King et al. 2003). The tenfold serial dilution of ASFV P72 plasmid was used for constructing standard curve and determining the sensitivity of TaqMan qPCR. A standard curve with R2 of 0.994 was constructed for quantitative detection of viral DNA copy numbers (Fig. 3A). The detection limit of qPCR was 101 copies/μL when ASFV P72 plasmid was used as PCR template (Fig. 3B). In specificity test, viral genomic DNA and RNA of ASFV and other porcine viruses were used for qPCR detection. The results indicated that qPCR assay could accurately distinguish ASFV viral DNA from control viral DNA or RNA with no cross-reactivity (Fig. 3C).
Figure 3. Sensitivity and specificity validation of qPCR. A tenfold serial dilutions of ASFV VP72 plasmid were used for constructing the standard curve of qPCR. B tenfold serial dilutions of ASFV VP72 plasmid were used for sensitivity validation of qPCR. C ASFV viral DNA and control viral DNA or RNA (PCV-2 ZJ/C, PCV-2 SH, CSFV-C, PRRSV JAX1-R, PRRSV R98, PRV Bartha-K61, PRV HB-98, TGEV + PEDV) were dissolved in sterile water for determining specificity of qPCR.
To validate the repeatability, variation coefficients of intra-assay and inter-assay were analyzed by using the Ct value obtained from qPCR assay of 10 folds serially diluted plasmid (5 × 107 copies/μL – 5 × 105 copies/μL). The results indicated that the intra-assay (Table 2) and interassay (Table 3) variation coefficients were less than 3%. Therefore, this qPCR assay exhibited a robust repeatability both in intra-assay and inter-assay.
Plasmid concentration (copies/μL) Ct value sample 1 Ct value sample 2 Ct value sample 3 Standard deviation(SD) Variation coefficient(CV) 5 × 107 12.11 12.20 12.21 0.055 0.45% 5 × 106 15.36 15.40 15.46 0.050 0.32% 5 × 105 18.57 18.57 18.65 0.046 0.24%
Table 2. Intra-assay variation coefficient of qPCR.
Plasmid concentration (copies/μL) Ct value sample 1 Ct value sample 2 Ct value sample 3 Standard deviation(SD) Variation coefficient(CV) 5 × 107 12.09 12.47 12.75 0.331 2.66% 5 × 106 15.36 15.58 16.26 0.469 2.98% 5 × 105 18.73 19.21 19.26 0.296 1.53%
Table 3. Inter-assay variation coefficient of qPCR.
In this lateral flow strip-based RPA-CRISPR assay, RNA reporter was labeled with a 5' FAM and a 3' biotin for detecting the collateral cleavage effect. The detection product of RPA-CRISPR was tenfold diluted and then was added into sample pad on the strip for determining the detection result (Fig. 4A). The concentration of the streptavidin was previously reduced to make both Test (T) line and Control (C) line appear as two consistent bands for negative control when preparing strips in the method section. Under this condition, the result judgment standard of this strip was depended on the degree of elimination on T line. Briefly, a high-intensity band was observed at C line, but not at T line of the strip due to the cleavage of reporter RNA induced by ASFV positive samples. In contrast, in ASFV negative samples, high-intensity bands were observed at both C and T lines, since the colloidal gold conjugated anti-FAM antibody was immobilized on T line by not-cleaved reporter RNA. The intensity ratio of T to C line was calculated to reveal significant difference between ASFV weakly positive specimen and negative control (Fig. 4B).
Figure 4. Schematic diagram of detection process, sensitivity and specificity of lateral flow strip-based RPA-CRISPR. A Detection process of lateral flow strip-based RPA-CRISPR. B Detection method of lateral flow strip-based RPA-CRISPR. C Sensitivity of lateral flow strip-based RPA-CRISPR with visual observation. D Intensity ratio of T line and C line in sensitivity test of flow strip-based RPA-CRISPR. E Specificity of lateral flow strip-based RPA-CRISPR.
Since the collateral cleavage effect was determined by using a lateral flow strip instead of a qPCR system, sensitivity and specificity of this lateral flow strip-based RPACRISPR assay needed to be further evaluated. Lateral flow strip-based RPA-CRISPR could detect as low as 102 copies/μL with visual observation (Fig. 4C). The T/C line intensity ratio obtained by an HR8000 immuno-quantitative detector indicated that the plasmid at concentration of 101 copies/μL (Fig. 4D) was ASFV weakly positive. Specificity test results revealed that the lateral flow stripbased RPA-CRISPR could accurately distinguish ASFV viral DNA from control viral DNA or RNA with no crossreactivity (Fig. 4E).
In order to validate the reliability of clinical sample detection, the results of qPCR-based RPA-CRISPR, lateral flow strip-based RPA-CRISPR and qPCR were compared. Total 27 ASFV positive specimens and 25 ASFV negative specimens were applied in all 3 different assays mentioned above. The results of qPCR-based RPA-CRISPR and lateral flow strip-based RPA-CRISPR were compared with those of qPCR. The coincidence rates of ASFV positive samples and ASFV negative samples were calculated. The results demonstrated that the detection accuracy of qPCR and qPCR-based RPA-CRISPR exhibited a coincidence rate as high as 100% for both all ASFV positive and negative samples (Fig. 5A). The lateral flow strip-based RPA-CRISPR assay displayed 8 false-negative results obtained from the 27 detected ASFV positive samples with the coincidence rate of 70.3%, compared with qPCR (Fig. 5A). Notably, the results of the 25 ASFV negative samples by using three methods were completely identical with coincidence rate of 100% (Fig. 5B).