CRISPR/Cas13a combined with hybridization chain reaction for visual detection of influenza A (H1N1) virus

Principle of H1N1 influenza detection

Here, we developed a dual signal amplification strategy based on Cas13a for a visual assay of influenza H1N1. In the principle depicted in Scheme 1, the first step is the recognition of H1N1 by the colorimetric biosensor and ensured specificity of the reaction by the CRISPR/Cas13a protein system. Target RNA extracted from influenza H1N1 is bound to crRNA through base complementation pairing after entering the interior of the CRISPR/Cas13a protein. This alters the protein structure and activates the trans-cleavage ability of the CRISPR/Cas13a protein. A DNA oligonucleotide strand containing three uridine monophosphate was created (probe I). The probe was designed to serve as the substrate for trans-cleavage of Cas13a/crRNA. DNA probe I consists of an I1 strand at the 5′ end, a special RNA sequence (-UUU-) in the middle, and an I2 strand at the 3′ end. Modification of the I1 strand with biotin allows the strand to initiate the HCR. The special RNA sequence (-UUU-) is recognized and cut by the CRISPR/Cas13a system. Cas13a is activated and cleaves the RNA sequence of probe I, which separates the I1 and I2 strands. The SA-MBs are added, and the biotin interacts with SA. The uncleaved probe I and I1 strands containing biotin are adsorbed by the SA-MBs, which are then removed from the solution by magnetic separation. After adding hairpin DNA H1 and H2, the I2 chain conjugates with H1 and opens the H1 hairpin via toehold-mediated strand displacement. Hybridization of the I2-H1 complex opens the hairpin structure of H2. The opened H2 chain is complementary to the hairpin H1 chain, which results in the opening of the hairpin structure of the H1 strand, thereby initiating the HCR. Because the H1 and H2 hairpin structures are continuously opened and assembled, the G-quadruplex fragment of H1 is exposed. Subsequently, in the presence of hemin, the G-quadruplex structure folds and hemin intercalates into the structure to form a DNAzyme. The G-quadruplex DNAzyme can catalyze the redox reaction between ABTS2− and H2O2. The color of the ABTS solution changes from light green to dark green. The resulting colorimetric reaction produces a color change of the reaction solution that is visible to the naked eye, permitting the ultra-sensitive visual detection of influenza H1N1.

Scheme 1scheme 1

Schematic illustration of CRISPR/Cas13a combined with hybridization chain reaction for visual detection of influenza H1N1

Experimental feasibility analysis

To verify the feasibility of the CRISPR/Cas13a cleavage effect, the CRISPR/Cas13a cleaved product was examined by PAGE. Lanes 1–3 of Fig. 1A respectively contain bands without Cas13a, without crRNA, and without target RNA in the complete Cas13a/crRNA trans-cleavage system. Lane 4 contains the complete system. In contrast to lane 4, lanes 1–3 displayed a pronounced band at the same location. Conversely, the band of lane 4 at this location was weaker, with the appearance of new bands of cleaved probe I further down in the gel. Only in the complete system was the target RNA of influenza H1N1 able to activate the Cas13a/crRNA trans-cleavage system. In addition, the trans-cleavage ability of Cas13a was confirmed by PAGE again. As shown in Supplementary information Figure S1, a low concentration of H1N1 could activate the CRISPR/Cas13a system to cleave a high concentration of probe I, and in this range, probe I could be completely cleaved.

Fig. 1figure 1

A Nondenaturing 15% PAGE analysis of cleavage ability of Cas13a for probe I. M: 20 bp DNA ladder. Lane 1: 1 µM target RNA, 0.8 μM Cas13a, 1.25 μM I. Lane 2: 1 µM target RNA, 0.5 μM crRNA, 1.25 μM I. Lane 3: 0.5 μM crRNA, 0.8 μM Cas13a, 1.25 μM I. Lane 4: 1 µM target RNA, 0.5 μM crRNA, 0.8 μM Cas13a, 1.25 μM I. B Nondenaturing 15% PAGE analysis of the ability of magnetic beads (MBs) and the result of HCR. M: 20 bp DNA ladder. Lane 1: 2 µM H1. Lane 2: 1.5 µM H2. Lane 3: 2 µM H1, 1.5 µM H2. Lane 4: 1.25 µM I, 2 µM H1, 1.5 µM H2. Lane 5: 2 µM H1, 1.5 µM H2, 1.25 μM I, 10 μg MBs, 0.5 μM crRNA, 0.8 μM Cas13a. Lane 6: 2 µM H1, 1.5 µM H2, 1.25 μM I, 10 μg MBs, 1 µM target RNA, 0.5 μM crRNA, 0.8 μM Cas13a. C Feasibility analysis of the formation and catalysis of G-quadruplex. Negative control: 2 µM H1, 1.5 µM H2. Positive control: 1.25 µM I, 2 µM H1, 1.5 µM H2. Negative: 2 µM H1, 1.5 µM H2, 1.25 μM I, 10 μg MBs, 0.5 μM crRNA, 0.8 μM Cas13a. Positive: 2 µM H1, 1.5 µM H2, 1.25 μM I, 10 μg MBs, 1 µM target RNA, 0.5 μM crRNA, 0.8 μM Cas13a

Subsequently, the activated Cas13a/crRNA trans-cleavage system cleaved the DNA probe I to produce the I1 and I2 chains. The feasibility of the detection system was verified by PAGE. The I2 strand commenced the subsequent HCR to generate H1–H2 double helices (Fig. 1B). Lanes 1 and 2 show that H1 and H2 were both in monomeric form. Lane 3 displays the situation when H1 and H2 coexist. Lane 4 shows a nicked double-helix structure formed when DNA probes I, H1, and H2 coexist. Lane 5 and 6 display the co-mixture of H1 and H2 after adding the Cas13a/crRNA system and MBs in the absence (lane 5) and presence (lane 6) of H1N1 target RNA. As anticipated, high molecular weight bands with extremely low electrophoretic mobility were generated in lanes 4 and 6. In contrast, these bands were absent in lanes 3 and 5. Compared to lanes 1 and 2, the H1 and H2 bands were less prominent in lanes 4 and 6, and were absent in lanes 3 and 5. The findings demonstrate the successful initiation of HCR and verifies the separation capability of the MBs for further development of the colorimetric biosensor.

G-quadruplex formation, catalysis, and the overall feasibility of the protocol were validated using colorimetry. The products in each of the above lanes were incubated with hemin, followed by the addition of hemin and ABTS2−. As shown in Fig. 1C, in positive samples and positive control samples, the colorless mixture turned green. Only these samples displayed a significant increase in absorption intensity. The absorbance of the positive sample was almost indistinguishable from that of the positive control sample.

Optimization of experimental conditions

To maximize the detection of influenza H1N1 viruses, pivotal assay factors that needed to be optimized were the concentration of Cas13a, CRISPR/Cas13a cleavage temperature, the concentration ratio of Na+ to K+, the concentration of probe I, the concentration ratio of hairpin H1 and H2, the concentration of hemin solution, CRISPR/Cas13a cleavage time, and HCR time. The ratio of I/I0, in which I is the absorbance in the presence of the target RNA and I0 is the absorbance in the absence of target RNA, was used to evaluate the performance of the assay parameters.

The concentration of Cas13a/crRNA affected the degree of the trans-cleavage of probe I, which influenced the amplification efficiency of subsequent HCRs. As shown in Fig. 2A, the I/I0 value varied with the concentration of Cas13a/crRNA and tended to be stable when the concentration of Cas13a/crRNA was ≥ 0.8 µM. This concentration was determined to be the optimum concentration of Cas13a/crRNA. The I/I0 value changed with the difference Cas13a/crRNA trans-cleavage temperature (Fig. 2B). When the temperature was 37 ℃, the value of I/I0 was maximum. When the temperature value exceeded 37 ℃, there was a subsequent decrease in the value of I/I0. The findings indicated that a temperature that was too high would affect the activity of the Cas13a protein. Insufficient probe I was not conducive to HCRs, while excess probe I increased the background signal. The I/I0 value gradually increased from 0.25 to 1.75 µM with increasing probe I concentration (Fig. 2C). When the concentration of probe I exceeded 1.25 μM, there was only a small and negligible increase in absorbance. Considering the cost, 1.25 μM was selected as the optimum amount of primer probe I added. The effect of the concentration between hemin and G-quadruplex was also monitored. The I/I0 value initially increased and then decreased (Fig. 2D). The I/I0 value reached a maximum when the concentration of hemin was 10 µM. The concentration was selected as the optimum concentration of hemin. The I/I0 value increased as the Cas13a/crRNA trans-cleavage time increased from 10 to 40 min (Fig. 2E). When the trans-cleavage time reached 30 min, further increases in time led to a smooth increase in I/I0 followed by a slight decrease. Thirty minutes was identified as the best digestion time. The I/I0 value increased with increasing HCR time and reached a peak at 40 min (Fig. 2F). Forty minutes was sufficient for HCR to fully react and was chosen.

Fig. 2figure 2

Optimization of experimental conditions. A Cas13a protein concentration. B Restriction enzyme digestion temperature. C Probe I concentration. D Hemin concentration. E Restriction enzyme digestion time. F HCR time

The concentration ratio of Na+ and K+, and of hairpin H1 and H2, was also optimized. Formation and stabilization of G-quadruplexes depended on cations. In the presence of monovalent cations such as Na+, K+, NH4+, Rb+, and others, guanylate-rich DNA or RNA could form a stable G-quadruplex structure. Of these cations, K+ and Na+ are ubiquitous in cells, so the concentration ratio of Na+ and K+ in the buffer was optimized. When the concentration ratio of Na+ to K+ was 1:10, the absorbance value of the sample was the highest (see Supplementary information Figure S2A). The amount of G-quadruplex that formed was closely related to hairpin H1 and H2. Therefore, the concentration ratio of these hairpins was optimized. The absorbance value increased with increasing concentration ratio. When the concentration ratio of hairpin H1 and H2 reached 2:1.5, the absorbance value was highest. Further increases of the hairpin H2 concentration produced significantly decreased absorbance (see Supplementary information Figure S2B). Therefore, the 2:1.5 concentration ratio of hairpin H1 to H2 was chosen as the optimal condition for the experiment.

Sensitivity

The linear range and sensitivity of the developed sensing system for influenza H1N1 detection were measured using the optimized experimental conditions. The susceptive response relationships between the concentration of the influenza H1N1 target RNA probe and the variance of absorption intensity indicated variable absorption intensity with increasing concentration of target RNA extracted from influenza H1N1 (101, 102, 103, 104, and 105 pM) (Fig. 3A). Over the range of 101 ~ 105 pM, good linear correlation from signal change and lg (target RNA concentration) was observed (Fig. 3B). The regression equation was I/I0 = 1.057 log10C − 0.1560 (R2 = 0.912). The limit of detection was calculated as 0.152 pM based on 3σ/slope, where σ is the standard deviation and k represents the slope of the line. Compared with several CRISPR/Cas detection systems, in terms of time and sensitivity, our detection method was comparable or superior (see Supplementary information Table S2).

Fig. 3figure 3

A Concentration of H1N1 target RNA from 100 nM to 10 pM, and the intensity of colorimetric change. B Analysis of the linear relationship between the concentration of H1N1 target RNA (100 nM ~ 10 pM) and the intensity of colorimetric change

Specificity

An excellent virus method has the ability to distinguish influenza viruses based on their subtype and high similarity sequence. The specificity of the colorimetric biosensor was evaluated by four influenza viruses (H3N2, H5N1, H9N2, and H7N9) and other three types of probes for base mutation (M1, M2, and M3 represent one base mutation, two base mutations, and three base mutations, respectively). As shown in Fig. 4, at the same concentration (100 nM), the altered signal intensity about influenza viruses in the same system and similar sequences did not obviously differ from that of the background (no addition of target RNA); only H1N1 RNA produced a prominent altered signal. These results indicated that the detection method has good specificity, with good resolution of even a single base mutation.

Fig. 4figure 4

Selectivity of the proposed method using different influenza virus RNA targets and different probes for base mutation. The concentration of each target RNA was 100 nM, and the other conditions remained the same

Analysis of spiked serum samples

Influenza H1N1 virus can be detected in serum following infection. To assess the practical feasibility of the developed method, different concentrations of H1N1 target RNA probe (1, 10, and 100 nM) were added to 10 × diluted serum. The H1N1 assay was then performed. The recovery for the 1, 10, and 100 nM concentrations of H1N1 target RNA probe was 83.90%, 104.50%, and 104.77%, respectively. The relative standard deviation ranged from 0.27 to 6.95% (Table 1). These findings suggested the potential value and practicability of this colorimetric biosensor to monitor influenza H1N1 in spiked biological samples.

Table 1 Recovery test of H1N1 target RNA detection in tenfold diluted serum samples (n = 3)Real-sample analysis

We further examined the applicability and specificity of the colorimetric biosensor in detecting target RNA in complicated RNA extracts. To verify the accuracy of this sensing method, the colorimetric biosensor was used to detect influenza H1N1 in allantoic fluid of chicken embryo samples. The total RNA of the virus at this concentration was extracted from 1.0 × 108 copies/mL virus titer, according to this experimental method. As shown in Fig. 5, the results of the experimental group (infected group) and the control group (uninfected group) were significantly different. This result demonstrates that this experimental approach could accurately capture target RNAs in complex RNA samples. Therefore, the assay presented in this experiment is a promising tool for the detection of H1N1 influenza virus in complex matrices.

Fig. 5figure 5

Absorbance values of infected and uninfected groups. C represents the infected group and A represents the uninfected group. H1N1 influenza virus titer (C1: 1.0 × 108 copies/mL, C2: 1.0 × 107 copies/mL) in allantoic fluid samples

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