In our study, we designed and used a hardware device— lateral flow chromatographic test strips. To better showcase our products, the following descriptions have been provided.
For nutritional deficiency-type epidermolysis bullosa (DEB), current prenatal diagnostic methods for detecting fetal gene mutations face notable limitations. Invasive techniques such as amniocentesis carry a significant risk of miscarriage. Non-invasive prenatal testing (NIPT), while safer, is expensive and exhibits limited specificity when targeting single-gene mutations. Moreover, both approaches lack rapid and intuitive result interpretation, which compromises detection efficiency.
To address these issues, we have developed a novel non-invasive detection method based on the Cas12a protein system. By constructing a plasmid containing the wild-type Cas12a gene, we successfully expressed and purified the Cas12a protein. Specific crRNAs were designed to guide Cas12a to precisely recognize and cleave target DNA sequences harboring pathogenic mutations. In the presence of mutant DNA, Cas12a is activated and initiates collateral (non-specific) cleavage activity, cutting a fluorescent reporter probe and releasing a detectable fluorescence signal, establishing the basis of the fluorescence detection mode.
Additionally, a lateral flow assay was developed based on antigen-antibody interaction principles. RPA amplification and Cas12a cleavage reaction are performed on the DNA of the specimen to be tested. The test results can be obtained by analyzing the cleavage product using a test strip. As the sample migrates with the chromatographic buffer, it binds to the control line (C line) to confirm test validity. If a specific mutation is present, it further binds to the test line (T line), producing a dual-band visual signal (C line and T line) for intuitive result interpretation.
Cell-free fetal DNA (cfDNA) is extracted non-invasively from maternal peripheral blood, thereby avoiding the risks associated with invasive procedures and enhancing the safety of prenatal diagnosis. The high specificity of Cas12a-crRNA targeting enables selective binding to mutant DNA sequences, and its collateral cleavage activity amplifies the detection signal. This significantly improves the detection sensitivity for low-abundance fetal mutations and reduces the likelihood of false positives and false negatives.
Our dual-mode detection platform—quantitative fluorescence readout and qualitative lateral flow strip—simplifies the workflow and result interpretation. It eliminates the need for complex instruments, enables rapid and visualized result reporting, and reduces dependence on laboratory infrastructure, making the method accessible for use in primary and community-level healthcare settings.
By enabling early and accurate identification of fetal gene mutations, this method supports clinical decision-making, allowing timely genetic counseling, interventions, or reproductive planning. Ultimately, it contributes to reducing the birth rate of fetuses with severe genetic disorders and improving overall public health outcomes.
Existing diagnostic methods for dystrophic epidermolysis bullosa (DEB) rely on expensive specialized equipment and skilled personnel, leading to high detection costs. This increases the burden on patients and society while lowering the disease detection rate, particularly in economically and resource-limited areas. Lateral flow immunoassay test strips are a portable, easy-to-store, and rapid (2-minute) diagnostic tool. They are paper-based, low-cost (0.2 $/test strip), highly specific due to antigen-antibody binding, compact, easy to carry and distribute, with simple components and processing, and pose no social or biological hazards. In this study, we incorporated two-step signal amplification through RPA and Cas12a cleavage, significantly enhancing sensitivity compared to conventional protein-based test strips. In conclusion, the lateral flow immunoassay test strip we designed offers rapid, equipment-free detection with advantages including portability, speed, high specificity, and sensitivity. It offers an effective diagnostic tool for DEB field screening, providing a more convenient and cost-effective diagnostic approach for society. This can help address diagnostic challenges in resource-poor areas, reduce the healthcare burden, and improve early disease detection rates, particularly in remote regions.
The test strips are assembled by layering the sample pad, conjugate pad, and absorbent pad sequentially on a nitrocellulose membrane mounted to an adhesive backing. Each layer overlaps the next by 2 mm, and the final assembly is cut into 3 mm-wide strips for use.
The backing plate is used to fix the components in place. The sample pad is the loading area for the analyte (which contains intact/broken Biotin-ssDNA-FAM probe Cas12a cleavage products). The conjugate pad is sprayed with colloidal gold-labeled anti-FAM rabbit antibodies, which contain color-forming components and can bind to specific elements within the analyte. Streptavidin and goat anti-rabbit secondary antibodies are sequentially fixed onto the NC membrane by stripe lines, defined as the Control line (C line) and Test line (T line), respectively. The absorbent pad has a strong water-absorption capacity to ensure proper chromatography.
Figure 1. Structure diagram of the test strip.
This test strip is designed based on the trans-cleavage activity of Cas12a and specific antigen-antibody interactions, converting the molecular signal of ssDNA probes cleaved by Cas12a into visible color changes. First, Cas12a is expressed and purified. A crRNA is designed to match the mutated sequence of the gene encoding type VII collagen (COL7A1), which is associated with dystrophic epidermolysis bullosa (DEB). Guided by the crRNA, Cas12a cleaves the mutated sequence and trans-cleaves the ssDNA probes in the system, while no cleavage occurs in the presence of non-mutated sequences. In other words, cleavage of the ssDNA probe only happens when the mutated sequence is detected, and the ssDNA probe remains intact in the absence of a mutation.
Based on this principle, the ssDNA probes are labeled at both ends, with the labels corresponding to antibodies on the hardware—the test strip. Specifically, the ends of the ssDNA probes are labeled with biotin and FAM. On the test strip, the colorimetric agent is colloidal gold particles, and the conjugate pad contains colloidal gold-labeled anti-FAM rabbit antibodies (which can bind to FAM). The C line consists of streptavidin (which can bind to biotin at multiple sites), and the T line contains goat anti-rabbit antibodies (secondary antibodies that can bind to anti-FAM rabbit antibodies).
In the absence of a mutated sequence, Cas12a trans-cleavage does not occur, and the ssDNA probe remains intact with FAM and biotin at the two ends. The intact probe binds to the gold-labeled anti-FAM antibody in the conjugate pad, forming an AuNPs-anti-FAM antibody complex-FAM-ssDNA-Biotin complex. This complex is completely captured at the C line by binding with streptavidin, and the colloidal gold particles produce a color change at the C line. The complex does not continue to chromatograph, and no color is observed at the T line.
In the presence of a mutated sequence, Cas12a trans-cleavage occurs, cleaving the ssDNA probe into two fragments, each containing FAM and biotin at one end. The FAM-labeled fragment binds to the colloidal gold-labeled anti-FAM rabbit antibody. This complex does not contain components that can bind to streptavidin at the C line, so it does not get fixed at the C line during chromatography. The complex continues to chromatograph and is eventually captured at the T line by secondary antibodies that can bind to anti-FAM rabbit antibodies. As this complex contains colloidal gold nanoparticles, the T line will display color. Meanwhile, the biotin-labeled fragment is captured by streptavidin at the C line, but since there is no colorimetric agent, no color change occurs at the C line.
It is important to note that due to the ideal condition of 100% cleavage efficiency, in practical scenarios, both the intact ssDNA probe (which would produce a color change at the C line) and the cleaved ssDNA fragment (which would produce a color change at the T line) are present in the system when a mutated sequence is detected. Therefore, the presence of a mutated sequence results in color changes at both the C and T lines simultaneously. The principle diagram is presented in Figure 2.
Figure 2. Schematic diagram of the test strip detection principle.
Intended Use:
Cas12a-based prenatal mutation detection test strip for dystrophic epidermolysis bullosa (DEB).
Main Components:
PVC backing, nitrocellulose membrane, glass fiber, and filter paper pad.
Packaging Specification:
100 Tests.
Storage Conditions and Shelf Life:
- Store at room temperature for a shelf life of 12 months, or at 2–8°C for a shelf life of 18 months. Keep dry and away from light. The performance of the test strips remains stable within the shelf life, even after opening.
- See label for the date of manufacture and expiration date.
The samples to be tested undergo DNA extraction, RPA, and Cas12a enzymatic cleavage reactions (containing ssDNA probes labeled with Biotin and FAM at both ends), and the products are then analyzed using test strips.
In the RPA-Cas12a system, the bypass cleavage activity of Cas12a is triggered only in the presence of target DNA (complementary to crRNA), at which point the ssDNA is cleaved into fragments containing Biotin at one end and FAM at the other. Fragments containing only the FAM end can bind to colloidal gold-labeled anti-FAM antibodies on the binding pad, which are then captured by anti-rabbit secondary antibodies on the T line, causing the T line to color. If no mutated sequence is present, the intact ssDNA probe binds to the gold-labeled antibody, and the resulting complex is captured by streptavidin on the C line, causing the C line to color. In summary, a colored T line indicates the presence of a mutated gene, resulting in a positive result; a colored C line alone indicates that no mutated sequence was detected, resulting in a negative result.
- According to the number of samples to be tested, take the corresponding number of test strips and mark them on the absorbent pad. Each test strip is for single-sample, single-use testing.
- Using a micropipette, add 5 μL of Cas12a reaction product to the sample pad.
- Place the test strip into 70 μL of 1×PBS buffer solution (containing 0.01% Tween-20) for chromatography (use as soon as possible), ensuring not to touch the NC membrane.
- Add 80 μL of diluted digestion product to the sample application area of the test strip. Read the results after two minutes.
The results can be interpreted according to Figure 3.
- Positive Results: Both the control line and test line appear, or only a strong test line appears.
- Negative Results: Only the control line appears, with no test line.
- Invalid Results: No visible color bands appear, or only a faint test line appears.
Figure 3. Result interpretation diagram.
- This product is for research use only. It is a single-use item and cannot be reused.
- Use the test strip as soon as possible after opening. Do not use if the strip has changed color; results should be interpreted within the specified time.
- Do not touch the test area of the strip with your hands to avoid contamination.
Standard plasmids containing c.520G (non-mutated sequence) and c.520G>A (mutated sequence), as well as c.6745C (non-mutated sequence) and c.6745C>T (mutated sequence), were used as test samples for the feasibility analysis experiment.
As shown in Figure 4, the test strips for the non-mutated sequences, c.520G and c.6745C, only showed a control line (C line), which is consistent with the negative control, indicating a negative result. In contrast, the test strips for the mutated sequences, c.520G>A and c.6745C>T, displayed both the control (C line) and test (T line) lines, indicating a positive result.
This demonstrates that the test strip, in combination with the Cas12a reaction, can effectively differentiate between mutated and non-mutated sequences, confirming its feasibility.
Figure 4: Feasibility of the test strip combined with Cas12a reaction for single-nucleotide mutation detection
A gradient dilution of standard plasmids containing c.520G>A (mutated sequence) and c.6745C>T (mutated sequence) was used as the test sample to perform the sensitivity analysis. The plasmid concentrations for the analysis were as follows: 108, 105, 104, 103, 102, 101, and 100 copies/µL, with a plasmid concentration of 10^8 copies/µL containing non-mutated sequences used as the negative control.
As shown in Figure 5, high-concentration plasmid samples resulted in strong test (T) lines and weak control (C) lines. As the plasmid concentration decreased to a certain level, the T line disappeared, and the C line gradually became the strongest.
For the mutated sequences c.520G>A and c.6745C>T, the minimum plasmid concentrations that produced a visible T line on the test strip were 103 and 104 copies/µL, respectively. Therefore, the detection limits of this test strip method for the c.520G>A and c.6745C>T mutations were 103 copies/µL (equivalent to 10-5 ng/µL) and 104 copies/µL (equivalent to 10-4 ng/µL).
Figure 5: Detection sensitivity of the test strip combined with Cas12a reaction for single-nucleotide mutation detection
Samples containing c.520G (non-mutated sequence) and c.520G>A (mutated sequence), as well as c.6745C (non-mutated sequence) and c.6745C>T (mutated sequence), were randomly mixed with other samples from unrelated diseases to create random blind samples. This was done to evaluate the analytical capability of the test strip on actual samples.
As shown in Figure 6, some test strips displayed both T lines and C lines, while others only displayed the C line. Specifically, for the detection of c.520G>A, test strips numbered 2, 3, 7, 10, 11, and P showed both T and C lines, while the remaining strips only showed C lines. For c.6745C>T, test strips numbered 2, 3, 7, 10, 11, and P showed both T and C lines, while the others only showed C lines.
All samples were structurally validated by other methods, and the detection results were consistent. This demonstrates that the test strip method can accurately distinguish mutated sequences from complex samples without interference, indicating its excellent applicability and practical capability.
Figure 6: Analytical capability of the test strip combined with Cas12a reaction for random samples
The detection standard is based on the appearance of a strong test line (T line) or the simultaneous appearance of both the control line (C line) and the test line (T line).
All samples to be tested underwent DNA extraction, RPA amplification, and Cas12a cleavage reactions (using Biotin-ssDNA-FAM probes). A 5 μL sample of the final product was used for test strip analysis.
