HNB-RT-LAMP Coupled with CRISPR/Cas12a for the Simultaneous Detection of Enteroviruses and Enterovirus A71 in a Single Tube

Figures

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  Figure 1. HNB-LAMP-CRISPR principle.

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  Figure 2. Feasibility Testing. (A) Design of the LAMP primer set and crRNA using the VP1 gene sequence of the EV-A71. (B) Specific sequences of the primers and crRNA are shown, with arrows indicating the 5′ to 3′ direction. The crRNA is designed downstream of the PAM site (5′-TTTG-3′). (C) HNB-RT-LAMP colorimetric and gel electrophoresis analyses were conducted using the standalone EV-A71 primers set. (D) Multiplex HNB-RT-LAMP colorimetric and gel electrophoresis analyses were conducted using the mixed primer set for EV-A71 and universal EVs. (E) CRISPR/Cas12a detection was performed on the amplification products of the multiplex RT-LAMP using the mixed primer set for EV-A71 and EVs. Observation was made under a blue light transilluminator, and fluorescence intensity was analyzed using a microplate reader. The difference in fluorescence intensity between the EV-A71 group and the NTC group was statistically significant (P < 0.001, ****), whereas other EVs showed no significant difference from the NTC (P > 0.05, ns). The experiment was carried out three times.

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  Figure 3. One-tube strategy for screening EVs and genotyping EV-A71. (A) Protocol of “One-Tube” strategy. At the bottom of the tube is the HNB-RT-LAMP reagent, with the CRISPR/Cas12a reagent added on top. The sample is added to the HNB-RT-LAMP reagent for RT-LAMP, enabling the screening of EVs. By mixing HNB-RT-LAMP and CRISPR/Cas12a reagents, the CRISPR/Cas12a assay is carried out to screen for EV-A71 positive samples. The entire procedure is performed in a single tube. (B) Principle of visual colorimetric detection. For EV samples, after RT-LAMP amplification, the precipitation of Mg2P2O7 results in a blue color change under natural light, while samples without EVs appear violet as the RT-LAMP amplification does not occur in them. The EV-A71 positive samples emit green fluorescence under blue light, resulting from the trans-cleavage activity of CRISPR/Cas12a, which generates fluorescent signals. Samples with other EVs appear red due to the fluorescence characteristics of HNB under blue light. (C) Colorimetric results of EV-A71, other EVs, and NTC samples after the HNB-RT-LAMP reaction under natural light and blue light excitation. (D) Colorimetric results of EV-A71 and other EV samples…

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  Figure 4. Sensitivity testing. (A) Sensitivity testing of consecutive dilutions of the EV 5′-UTR region using HNB-RT-LAMP. (B) Sensitivity testing of consecutive dilutions of the EV-A71 VP1 gene using HNB-LAMP-CRISPR. (C) Emission spectrum of consecutive dilutions of the EV-A71 VP1 gene using HNB-LAMP-CRISPR under 590 nm excitation. (D) Sensitivity testing of the EV-A71 VP1 gene using conventional RT-LAMP-CRISPR. The experiment was carried out three times.

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  Figure 5. Clinical sample testing of HNB-LAMP-CRISPR. (A) HNB-LAMP-CRISPR results of NP swab samples from 94 suspected HFMD patients under 12 years old. (B) Positivity rates of EVs and EV-A71 Based on HNB-LAMP-CRISPR. (C) Sensitivity and specificity of HNB-LAMP-CRISPR in detecting EVs are compared to RT-qPCR. (D) Sensitivity and specificity of HNB-LAMP-CRISPR in detecting EV-A71 compared to RT-qPCR.