Rapid, cheap and multiple detection of polynucleic acid is of great significance to human health, but it is still a huge challenge. Here, this paper proposes a nucleic acid detection platform named MiCaR, which combines microfluidic equipment with CRISPR-Cas12a and recombinase polymerase amplification (RPA). With only one fluorescent probe, MiCaR can simultaneously test up to 30 nucleic acid targets through microfluidic space coding. The detection limit reached 0.26 Atomol, and multiple detection only took 40 minutes. The study demonstrated the effectiveness of MiCaR by effectively detecting nine HPV subtypes targeted by the 9-valent HPV vaccine. In the detection of 100 patients at risk of HPV infection, it showed 97.8% sensitivity and 98.1% specificity.

Research ideas and results

Principle of MiCaR platform

The study showed its application in HPV subtype identification. After sampling, cervical cells were heated to induce HPV DNA release. Then, multiple RPA is directly performed without DNA extraction (Fig. 1a). The amplified products were loaded onto the microfluidic device and tested by the CRISPR-based pyrolysis assay. Readings are obtained through an automatic fluorescence imaging system. The detection of MicaR depends on the star chip (SSChip), which is designed as a central radiation network (Figure 1b). The sample is loaded into the hub hole in the center, and the spoke microchannel evenly distributes the sample into 30 marker holes. These marker holes are pre-loaded with specific Cas12a detection mixture, including Cas12a, crRNA and a fluorescent reporter. The reporter used is a fluorescent quenching agent labeled oligonucleotide named TBA11, which is a truncated form of thrombin binding inducer, and has been proved to have higher sensitivity than ordinary ssDNA reporter. If HPV DNA is matched with crRNA, the relevant wells will display bright fluorescent signals. However, when HPV DNA and crRNA do not match, a low background signal will be obtained.

Figure 1. Scheme of HPV subtype analysis strategy based on MiCaR

Note: Figure 1a is a brief overview of the steps involved in subtype analysis. The collected cervical cell samples are first melted by heat. Then, 9 HPV subtypes were amplified by RPA. The amplifiers were then tested by the CRISPR-Cas12a system on the microfluidic device, and then fluorescence imaging was performed to obtain readings. Figure 1b shows the principle of on-chip testing. Use a 30 combined star chip (SS-Chip), with a central entrance and 30 exits connected. Various Cas12a/crRNAs that identify relevant target HPV subtypes are pre-installed at the exit. After detection on the chip, the fluorescence reading of the specific outlet (i.e., spatial coding) indicates the presence of the relevant HPV subtype in the sample.

Validation of crRNAs and RPA primers for nine HPV subtypes

The best crRNAs are designed for all 9 HPV targets. Then, a matrix test is designed to verify the performance of crRNA. Next, CRISPR/Cas12a system is used to detect the products obtained through 4-plexed RPA and 9-plexed RPA. When crRNA recognizes HPV target in RPA products, Cas12a's transgenic ability is activated. Accordingly, the TBA11 reporter is cut into smaller elements. The detection results were first analyzed by denatured polyacrylamide gel electrophoresis (PAGE). The results show that the reporter of the 2-5th lane has been split (Fig. 2c). The reporter is complete in other swimlanes, indicating that the Cas12a system is not activated. Next, RPA products of 9 complexes were evaluated. The results showed that all 9 swimming lanes had small reporter fragments (Fig. 2d), indicating that 9 HPV subtypes were effectively amplified and successfully activated the Cas12a system. Some bands corresponding to the cracked reporter have different strengths (Fig. 2c, 2d), which may be due to the change in the cracking efficiency of the Cas12a system activated by different targets. The control group (without HPV template) did not activate Cas12a in the 4-plexed or 9-plexed test.

Next, the pyrolysis products detected by 4-plexed and 9-plexed were checked according to the fluorescence readings used in the subsequent chip-based experiments. Consistent with denatured PAGE results, 4 and 9 bright fluorescent signals were observed in 4-plexed and 9-plexed detection respectively (Fig. 2e). These results confirmed the excellent performance of multiple RPA and CRISPR/CAS systems, and laid a solid foundation for the subsequent analysis of HPV subtypes on chip. Based on these results, the study proposed a program for designing RPA primers and crRNA for multiple nucleic acid detection technology (Fig. 2f).

Figure 2. Comprehensive characterization of crRNA and multiple RPA performance

Note: In Fig. 2c and Fig. 2d, denatured PAGE images show the decomposition of the products of 4-plexed and 9-plexed RPA identified by 9 crRNAs. Each experiment shall be repeated at least twice independently. Figure 2e shows the characteristics of 4-plexed and 9-plexe RPA products based on fluorescence. It should be noted that the amplifiers used in Fig. 2c, Fig. 2d and Fig. 2e are from different batches of RPA detection. Figure 2f Recommended procedure for RPA primer and Cas12-crRNA design for multi-target detection.

In addition, the sensitivity of multiple RPA was investigated. A series of plasmid samples with different concentrations were prepared for 9 HPV targets. These samples were amplified and measured by the detection method based on Cas12a (Fig. 3a). The test results are shown in Fig. 3b. Except for HPV-18, the sensitivity of RPA detection of 9 compounds (one primer for each target) to all targets is 10-17 10-18 M. The low sensitivity of HPV18 may be due to the relatively low primer efficiency in the amplification process of low concentration template. In the subsequent 9-plexed RPA test, this situation was improved by using three HPV18 primers, and finally it was close to 10-18 M.

Figure 3. 9-Sensitivity test of the mixed RPA method to 9 HPV subtypes

Note: In Figure 3a, a series of samples of 9 targets with different plasmid concentrations were prepared, amplified and tested. Fig. 3b Based on Cas12a method, plasmid titration was performed after RPA.

HPV subtype analysis of patient samples using MiCaR system

In order to systematically analyze the performance of the MiCaR detection platform, the on-chip detection results and clinical detection results were statistically analyzed. The positive and negative results of all 9 subtypes were summarized in 100 sample pools, which showed that their frequencies were different. In general, MiCaR performed well in testing these patient samples, with positive predictive consistency of 97.8%, negative predictive consistency of 98.1%, sensitivity of 97.8% and specificity of 98.1%.

Isothermal amplification raw material