CrRNA and CRISPR-Cas system for joint detection of CHIKV and DENV and application

Abstract

The invention provides CrRNA for combined detection of chikungunya virus (CHIKV) and dengue fever virus (DENV), the CrRNA comprises Cas12a-crRNA and Cas13a-crRNA, and the base sequences of the Cas12a-crRNA and the Cas13a-crRNA are respectively shown as SEQ ID NO.1 and SEQ ID NO.2. The invention also provides a kit for detecting the chikungunya virus (CHIKV) and the dengue fever virus (DENV). The invention also provides a CRISPR-Cas system comprising the crRNA and application of the CRISPR-Cas system, and also provides a rapid detection method for chikungunya virus and dengue virus. According to the present invention, by designing the specific Cas12a-crRNA and the specific Cas13a-crRNA, the CRISPR-Cas dual system fused with the RT-RAA is constructed, such that the efficient amplification and the accurate identification are achieved; a fluorescent probe or an immunochromatography strip is combined to read signals, sensitivity and simplicity are taken into consideration, rapid combined identification of the two viruses is achieved, the kit is adaptive to multiple scenes, and a support is provided for early diagnosis and epidemic situation prevention and control.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, and in particular to a CrRNA, CRISPR-Cas system and its application for the joint detection of chikungunya virus (CHIKV) and dengue virus (DENV). Background Technology

[0002] Arboviruses are a class of pathogens transmitted by blood-sucking arthropods such as mosquitoes and ticks. Chikungungya virus (CHIKV) and dengue virus (DENV) are among the most widespread and harmful mosquito-borne viruses globally. Both are primarily transmitted by Aedes aegypti and Aedes albopictus mosquitoes, and are prevalent in tropical and subtropical regions, showing a persistently active trend in southern provinces of my country and neighboring countries.

[0003] Chikungunya virus can cause chikungunya fever, clinically characterized by sudden high fever and severe joint pain. Joint damage in some patients can last for months or even years, severely impacting their quality of life. Since 2004, the virus has caused multiple large-scale outbreaks globally, with numerous countries and regions reporting local transmission. In my country, since the first imported case was discovered, the risk of both imported and local transmission has remained, with some southern provinces experiencing local outbreaks, highlighting its serious threat to public health.

[0004] Dengue virus comprises four serotypes, which cause dengue fever upon infection. Severe cases can progress to dengue hemorrhagic fever or dengue shock syndrome, resulting in a high mortality rate. Globally, dengue virus infects a large population, with numerous cases presenting clinical symptoms each year. Outbreaks are primarily concentrated in Southeast Asia, the Western Pacific, and the Americas. In my country's southern provinces, due to the widespread distribution of the transmission vector, local outbreaks occur almost annually, and imported cases are prevalent throughout the country. With increasing international exchanges and the impact of climate change, the risk of local transmission is further rising, and the pressure on prevention and control continues to increase.

[0005] It is noteworthy that CHIKKV and DENV are highly correlated in epidemiological characteristics, sharing not only a common transmission vector but also similar clinical symptoms (both primarily presenting with fever, rash, and joint pain), making clinical differential diagnosis extremely difficult. Recent studies have shown that cases of co-circulation and co-infection of the two viruses have emerged in some regions, further increasing the complexity of epidemic prevention and control and the difficulty of clinical diagnosis and treatment. Currently, there is no commercially available vaccine or specific treatment for chikungunya, and while some vaccines are available for dengue fever, the core of prevention and control still relies on early and accurate diagnosis and vector control. Therefore, developing a joint detection technology that can rapidly and accurately identify the two viruses has become an urgent need in the current vector-borne virus prevention and control system.

[0006] Existing laboratory diagnostic methods for CHIKV and DENV are mainly divided into three categories: First, virus isolation methods, which, as the traditional "gold standard," have high specificity but are cumbersome, time-consuming (lasting several days to weeks), and require high-level biosafety laboratories, thus failing to meet the needs of rapid diagnosis. Second, serological testing methods (such as ELISA, IFA, and neutralization tests), which rely on detecting virus-specific antibodies. However, antibody production has a 3-5 day window period and is prone to antigen cross-reaction with flaviviruses such as Zika virus, leading to false positive results. Neutralization tests, while highly specific, are complex to operate and time-consuming, limiting their clinical application. Third, molecular biological testing methods, represented by real-time quantitative PCR (qPCR), have high sensitivity and rapid detection, but require stringent laboratory conditions, operational techniques, and cold chain transportation of samples. The equipment costs are high, making it difficult to promote and apply in resource-limited scenarios such as primary healthcare institutions and on-site screening, and they are not suitable for screening unknown pathogens. In recent years, high-throughput sequencing (NGS) technology has been gradually used for virus discovery and genome tracing, with the potential to detect unknown viruses without bias. However, its high cost and complex data analysis have prevented it from being widely used in routine diagnosis.

[0007] Therefore, there is still an urgent need to develop a new detection technology that can balance high sensitivity and specificity, is easy and rapid to operate, has low equipment dependence, and is cost-effective, in order to overcome the bottlenecks of existing methods in grassroots applications, field screening, and multiple pathogen identification. In particular, for vector-borne viruses with similar clinical symptoms and overlapping epidemic areas (such as dengue virus and chikungunya virus), developing an integrated platform capable of simultaneously detecting multiple targets will greatly improve early diagnosis efficiency, epidemic monitoring capabilities, and public health response levels, providing core technological support for vector-borne virus control in resource-limited areas.

[0008] In recent years, isothermal amplification technology has become a core technology supporting point-of-care testing (POCT) for molecular diagnostics due to its advantages of not requiring a thermal cycler and being able to rapidly amplify nucleic acids at a constant temperature. Meanwhile, CRISPR-Cas systems (such as Cas12a and Cas13a) can achieve precise identification and signal conversion of amplified products by specifically recognizing target nucleic acids and activating trans-cleavage activity. The detection platform constructed by combining these two technologies offers high sensitivity, high specificity, and convenience, providing a new direction for overcoming existing technological bottlenecks.

[0009] Therefore, there is an urgent need to develop an integrated detection solution based on the CRISPR-Cas system and isothermal amplification technology. This solution involves designing crRNA molecules that specifically recognize conserved targets of CHIKV and DENV, constructing a compatible CRISPR-Cas detection system, and achieving rapid joint identification of the two viruses. This solution must overcome the limitations of existing technologies in terms of operational complexity, equipment dependence, and detection efficiency. It should meet the needs of precise laboratory testing and be adaptable to resource-constrained scenarios such as primary healthcare and field screening. This would provide efficient technical support for the early diagnosis of CHIKV and DENV infections, rapid epidemic response, and the formulation of prevention and control strategies, filling the current technological gap in the field of joint detection of vector-borne viruses. Summary of the Invention

[0010] The technical problem to be solved by this invention is to provide a CrRNA and CRISPR-Cas system and its application for the joint detection of CHIKV and DENV. This system designs Cas12a-crRNA and Cas13a-crRNA, respectively, to specifically recognize the conserved regions of the CHIKV and DENV genomes, constructing a CRISPR-Cas dual system that integrates RT-RAA isothermal amplification technology. This achieves efficient amplification and accurate identification of the two viruses. Subsequent signal reading is achieved by combining fluorescently labeled probes or immunochromatographic test strips, ensuring both detection sensitivity and specificity while simplifying operation and reducing equipment dependence. Ultimately, this enables rapid joint identification of CHIKV and DENV, adapting to the needs of multiple scenarios in laboratories and grassroots fields. It overcomes the bottlenecks of existing technologies in terms of efficiency, cost, and applicability, providing technical support for the early diagnosis and epidemic prevention and control of these two viral infections.

[0011] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A CrRNA for the joint detection of chikungunya virus (CHIKV) and dengue virus (DENV) comprises Cas12a-crRNA and Cas13a-crRNA; the base sequence of the Cas12a-crRNA is shown in SEQ ID NO.1 and can specifically recognize the nucleic acid target of chikungunya virus; the base sequence of the Cas13a-crRNA is shown in SEQ ID NO.2 and can specifically recognize the nucleic acid target of dengue virus.

[0012] As one of the preferred embodiments of the present invention, the Cas12a-crRNA specifically recognizes the chikungunya virus nucleic acid target as the chikungunya virus E1 gene, which is suitable for the specific detection of chikungunya virus; the Cas13a-crRNA specifically recognizes the dengue virus nucleic acid target as the 3' UTR region of dengue virus, which is suitable for the specific detection of dengue virus.

[0013] As one of the preferred embodiments of the present invention, the Cas12a-crRNA is used to guide the Cas12a protein to recognize the PAM sequence containing TTTN, and to form a Cas12a / crRNA / target DNA ternary complex with the target DNA of the Chikungunya virus E1 gene amplified by RT-RAA, thereby activating the side branch cleavage activity of the Cas12a protein to cut single-stranded DNA molecules in the reaction system.

[0014] As one of the preferred embodiments of the present invention, the Cas13a-crRNA is not restricted by the PAM sequence and is used to guide the Cas13a protein to recognize and bind to the target RNA that has been amplified by RT-RAA and transcribed in vitro by T7, forming a Cas13a / crRNA / target RNA ternary complex, activating the side branch cleavage activity of the Cas13a protein to cleave single-stranded RNA molecules in the reaction system.

[0015] A CRISPR-Cas system for detecting chikungunya virus and dengue virus, comprising the aforementioned crRNA.

[0016] As one of the preferred embodiments of the present invention, it also includes Cas12a protein, Cas13a protein, signal reporter probe and RT-RAA amplification product.

[0017] As one of the preferred embodiments of the present invention, the signal reporting probe is one of the following two combinations: Combination 1: FAM-ssDNA-BHQ1 probe and ROX-ssRNA-BHQ2 probe, this combination is used for fluorescence detection; Combination 2: FAM-ssDNA-Biotin probe and Dig-ssRNA-Biotin probe, this combination is used for rapid detection with immunochromatographic strips.

[0018] As one of the preferred embodiments of the present invention, the RT-RAA amplification product is obtained by amplification using primer pairs targeting the Chikungunya virus E1 gene and the 3' UTR region of dengue virus. The primer pairs include primer pair 1 and primer pair 2. Primer pair 1 is used to amplify the Chikungunya virus E1 gene, and its nucleotide sequence is shown in SEQ ID NO. 3 and SEQ ID NO. 4. Primer pair 2 is used to amplify the 3' UTR region of dengue virus, and its nucleotide sequence is shown in SEQ ID NO. 5 and SEQ ID NO. 6.

[0019] Application of the above-mentioned CRISPR-Cas system in the preparation of a combined detection kit for chikungunya virus and dengue virus.

[0020] A rapid detection method for chikungunya virus and dengue virus based on CRISPR, utilizing the aforementioned CRISPR-Cas system to detect the sample, includes the following specific steps: S1: Extract RNA from the sample to be tested; S2: Using the RNA as a template, the amplified product was obtained by using primer pairs targeting the E1 gene of Chikungunya virus and the 3' UTR region of dengue virus via RT-RAA technology. S3: Add the amplification product to a reaction system containing Cas12a-crRNA, Cas13a-crRNA, Cas12a protein, Cas13a protein and signal reporter probe, and detect the signal after incubation; When the signal reporting probes are FAM-ssDNA-BHQ1 and ROX-ssRNA-BHQ2, the FAM / ROX channels are monitored by a fluorescence instrument. Enhancement of fluorescence in either channel corresponds to a positive virus result. When the signal reporting probes are FAM-ssDNA-Biotin and Dig-ssRNA-Biotin, the color development of the control line and the corresponding test line is determined by colloidal gold immunochromatographic strips, indicating that the virus is positive. If the above positive signals are not present, the result is negative.

[0021] The advantages of this invention compared to the prior art are: (1) The present invention uses reverse transcription recombinase-assisted isothermal amplification (RT-RAA) technology to achieve efficient amplification of the RNA of Chikungunya virus and dengue virus. This technology does not require the cumbersome thermal cycling process in traditional PCR. It only needs to react at a constant temperature of 42℃ for 30 minutes to complete the entire amplification process. It can be achieved with only simple heating devices such as metal baths. This method has very low requirements for instruments and equipment. It does not require expensive PCR instruments or other large equipment. It has the characteristics of simple operation, short time consumption, high sensitivity, and low dependence on the professional technical background of personnel. It is particularly suitable for rapid detection of pathogens in areas with limited medical conditions, grassroots sites, or resource-scarce environments.

[0022] (2) In this invention, the designed crRNA can specifically guide the Cas12a / Cas13a protein to bind to the target nucleic acid sequence. While achieving specific recognition and cleavage of the target sequence, it can activate the trans-cleavage activity of Cas12a and Cas13a respectively, cleaving single-stranded DNA and RNA reporter molecules in the reaction system. When FAM-ssDNA-BHQ1 and ROX-ssRNA-BHQ2 are used as reporter probes, the fluorescence signal can be directly read by a portable fluorescence detection instrument to achieve quantitative or semi-quantitative analysis. When FAM-ssDNA-Biotin and Dig-ssRNA-Biotin are used as reporter probes, the results can be visually read by an immunochromatographic test strip to achieve result interpretation without instruments. Based on the above mechanism, the method established by this invention is suitable for large-scale clinical population screening. Compared with traditional PCR or ELISA methods, it not only significantly shortens the detection time (can be completed within 30-40 minutes), but also reduces the technical requirements for operators, and reliable detection can be completed without repeated professional training.

[0023] (3) This invention innovatively combines RT-RAA isothermal amplification technology with CRISPR-Cas12a / Cas13a detection system to construct a high-efficiency, multifunctional molecular diagnostic platform. This platform has significant advantages such as speed, high sensitivity, high specificity, low cost and ease of operation. It can not only meet the dual detection needs of chikungunya virus and dengue virus, but also has good versatility and can be further extended to the detection of various pathogens and gene markers such as monkeypox virus. This system effectively promotes the transformation of CRISPR detection technology from basic research to clinical application, and has strong translational innovation and significant market application potential. Attached Figure Description

[0024] Figure 1 This is an agarose gel electrophoresis image of the RT-RAA amplification products in the validation example (in the image, lane 1 is the marker, lane 2 is the CHIKV positive group, and lane 3 is the DENV positive group). Figure 2 This is a graph of the fluorescence detection results in the validation example (Figure A is a bar chart of fluorescence signals: the bar chart shows the differences in fluorescence signal intensity in the FAM and ROX channels of the CHIKV positive group, DENV positive group and template-free control NTC; Figure B is a fluorescence kinetic curve: with time as the horizontal axis and fluorescence value as the vertical axis, it shows the dynamic change trend of fluorescence signal of different sample groups with incubation time). Figure 3 The results of colloidal gold immunochromatographic strip development in the verification example (showing CHIKV respectively) + DENV + CHIKV + DENV+ (Actual colorimetric effect of the immunochromatographic strips corresponding to the negative control samples) Furthermore, it should be noted that SEQ ID NO.1 and SEQ ID NO.2 in the sequence listing of this invention are RNA sequences, and "T" in these two sequences = "U". Detailed Implementation

[0025] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Unless otherwise specified, the reagents and experimental methods used in the following embodiments are all conventional reagents or methods in the art and will not be described again.

[0026] Example 1 This embodiment presents a CrRNA for the combined detection of CHIKV and DENV.

[0027] The core of CRISPR technology lies in CrRNA, therefore the choice of CrRNA is directly related to the effectiveness of the final detection method.

[0028] In this embodiment, Cas12a-crRNA and Cas13a-crRNA were designed for conserved regions of the chikungunya virus and dengue virus genomes, respectively. The base sequence of Cas12a-crRNA is shown in SEQ ID NO.1 and is used for specific detection of the chikungunya virus E1 gene; the base sequence of Cas13a-crRNA is shown in SEQ ID NO.2 and is used for specific detection of the 3' UTR region of dengue virus.

[0029] Specifically, the Cas12a-crRNA is used to guide the Cas12a protein to recognize the PAM sequence containing TTTN, and to form a Cas12a / crRNA / target DNA ternary complex with the target DNA of the Chikungunya virus E1 gene amplified by RT-RAA, thereby activating the side branch cleavage activity of the Cas12a protein to cut any single-stranded DNA molecule in the reaction system.

[0030] The Cas13a-crRNA is not restricted by the PAM sequence and is used to guide the Cas13a protein to recognize and bind to the target RNA that has been amplified by RT-RAA and transcribed in vitro by T7, forming a Cas13a / crRNA / target RNA ternary complex, activating the side branch cleavage activity of the Cas13a protein to cleave any single-stranded RNA molecule in the reaction system.

[0031] Example 2 This embodiment describes a CRISPR-Cas system for detecting CHIKV and DENV, comprising Cas12a protein, Cas12a-crRNA, Cas13a protein, Cas13a-crRNA, FAM-ssDNA-BHQ1 probe, ROX-ssRNA-BHQ2 probe, and RT-RAA amplification product.

[0032] The RT-RAA amplification product is obtained by amplification using primer pairs targeting the Chikungunya virus E1 gene and the 3' UTR region of dengue virus. The primer pairs include primer pair 1 and primer pair 2. Primer pair 1 is used to amplify the Chikungunya virus E1 gene and includes primers E1-F4 and E1-R5, with nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. Primer pair 2 is used to amplify the 3' UTR region of dengue virus and includes primers UTR-RAA-F2 and UTR-RAA-R1, with nucleotide sequences as shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0033] The RT-RAA amplification system (50 μL) is as follows: 25 μL A buffer; 13.5 μL nuclease-free water; lyophilized enzyme powder tubes (A buffer + nuclease-free water, mainly used to dilute and dissolve the lyophilized enzyme); 1 μL of 10 μM primer E1-F4 (targeting the Chikungunya virus E1 gene); 1 μL of 10 μM primer UTR-RAA-F2 (targeting dengue virus 3' UTR); 1 μL of 10 μM primer E1-R5 (targeting the Chikungunya virus E1 gene, used in conjunction with primer E1-F4); 1 μL of 10 μM primer UTR-RAA-R1 (targeting dengue virus 3' UTR, used in conjunction with primer UTR-RAA-F2); 5 μL of template RNA, the template being derived from the sample to be tested.

[0034] The complete CRISPR-Cas dual-system lysis reaction system (20 μL) is as follows: Cas12a protein (100 nM) 2 μL; Cas12a-crRNA (25 nM) 0.5 μL; FAM-ssDNA-BHQ1 probe (500 nM) 1 μL; Cas13a protein (100 nM) 2 μL; Cas13a-crRNA (50 nM) 1 μL; ROX-ssRNA-BHQ2 probe (500 nM) 1.5 μL; RNase inhibitor (40 U / μL) 0.5 μL; T7 RNA polymerase (1.25 U / μL) 0.5 μL; NTP Mix (1.25 mM) 1 μL; 10× buffer 2 μL; nuclease-free water 6 μL; RT-RAA amplification product 2 μL.

[0035] Example 3 The CRISPR-Cas system in this embodiment is basically the same as that in Example 2, except that the signal reporter probe adopts a combination of "FAM-ssDNA-Biotin probe and Dig-ssRNA-Biotin probe", and the CRISPR-Cas dual-system lysis reaction system is divided into two parts: the bottom of the PCR tube and the cap of the PCR tube, with a total system volume of 20 μL.

[0036] The complete CRISPR-Cas dual-system cleavage reaction system is as follows: 1. PCR tube bottom portion (total volume 10μL): Cas12a protein (100nM) 2μL, Cas12a-crRNA (25nM) 0.5μL, 10×buffer 2μL, FAM-ssDNA-Biotin probe (400nM) 0.8μL, RT-RAA amplification product 2μL, nuclease-free water (ddH2O) 2.3μL.

[0037] 2. PCR tube cap portion (total volume 10μL): Cas13a protein (100nM) 2μL, Cas13a-crRNA (50nM) 1μL, NTP Mix (25mM) 1μL, 10×buffer 2μL, RNase inhibitor (40U / μL) 0.5μL, T7 RNA polymerase (50U / μL) 0.5μL, RT-RAA amplification product (consistent with the product used at the bottom of the PCR tube) 2μL, Dig-ssRNA-Biotin probe (400nM) 0.8μL, nuclease-free water (ddH2O) 0.2μL.

[0038] Example 4 This embodiment provides a CHIKV and DENV combined detection kit (fluorescence detection), which includes the Cas12a protein, Cas12a-crRNA, Cas13a protein, Cas13a-crRNA, FAM-ssDNA-BHQ1 probe, ROX-ssRNA-BHQ2 probe, as well as primers and reagents required for the RAA amplification system and the CRISPR-Cas dual-system lysis reaction system, as described in Example 2.

[0039] Example 5 This embodiment presents a combined detection kit for chikungunya virus and dengue virus (rapid detection using immunochromatographic strips), which includes the Cas12a protein, Cas12a-crRNA, Cas13a protein, Cas13a-crRNA, FAM-ssDNA-Biotin probe and Dig-ssRNA-Biotin probe, as well as primers and reagents required for the RAA amplification system and the CRISPR-Cas dual-system lysis reaction system, as described in Example 3.

[0040] Example 6 This embodiment presents a combined CHIKV and DENV detection method for indoor fluorescence detection. It utilizes the CRISPR-Cas system (adapted to a fluorescence signal reporting system) from Example 2 to detect the sample. This method is suitable for indoor settings such as laboratories and medical institutions, and includes the following specific steps: I. RNA Extraction: RNA was extracted from the sample to be tested (such as serum, plasma, saliva or tissue homogenate) using a commercial nucleic acid extraction kit, following the instructions of the kit. II. RT-RAA Primer Design and Amplification: 1. Primer design: Primer pair 1 (E1-F4 / E1-R5) was designed targeting the conserved region of the E1 gene of chikungunya virus, and primer pair 2 (UTR-RAA-F2 / UTR-RAA-R1) was designed targeting the conserved region of the 3' UTR region of dengue virus. The nucleotide sequences are shown in SEQ ID NO.3~6, respectively. The primers were prepared and purified using conventional nucleic acid synthesis techniques.

[0041] 2. Construction of amplification system: According to the 50 μL RT-RAA amplification system formula described in Example 2, add Abuffer, nuclease-free water, lyophilized enzyme powder, primer pair 1 (E1-F4 / E1-R5), primer pair 2 (UTR-RAA-F2 / UTR-RAA-R1), and the RNA template extracted in step 1 in sequence, mix gently, and then centrifuge briefly.

[0042] 3. Amplification reaction: The above amplification system was placed in a metal bath at 42℃ and reacted at a constant temperature for 30 minutes. After the reaction, RT-RAA amplification products containing target DNA of the Chikungunya virus E1 gene and target DNA of the 3' UTR region of dengue virus were obtained.

[0043] 4. Separate the amplification products by agarose gel electrophoresis, observe and record the results using a gel imaging system; if a specific band appears at the expected size and position, it is determined to be positive for RT-RAA amplification and used for subsequent CRISPR-Cas detection reaction.

[0044] III. CRISPR-Cas Dual-System Detection Response 1. Reaction system construction: According to the CRISPR-Cas dual-system lysis reaction system formulation described in Example 2, Cas12a protein, Cas12a-crRNA, FAM-ssDNA-BHQ1 probe, Cas13a protein, Cas13a-crRNA, ROX-ssRNA-BHQ2 probe, RNase inhibitor, T7 RNA polymerase, NTP Mix, 10× buffer and nuclease-free water were added in sequence. After mixing, 2 μL of the RT-RAA amplification product from step 2 was added and gently mixed again.

[0045] 2. Incubation reaction: The reaction system is incubated at 37°C for 20 minutes to allow the Cas protein to form a ternary complex with crRNA and target nucleic acid and activate trans-cleavage activity to cleave the corresponding reporter probe.

[0046] IV. Signal Detection and Result Determination The incubated reaction system was placed in a real-time quantitative PCR instrument to monitor the changes in fluorescence signals in the FAM and ROX channels. If the fluorescence signal in the FAM channel is significantly enhanced (compared to the negative control), the sample is considered positive for Chikungunya virus. If the ROX channel fluorescence signal is significantly enhanced (compared to the negative control), the sample is considered positive for dengue virus. If there is no significant enhancement in the fluorescence signals of both channels, consistent with the negative control, then the sample is determined to be negative for both viruses. The experiment included a negative control containing no template RNA and a positive control containing known positive RNA to eliminate the interference of false positives and false negatives.

[0047] Example 7 This embodiment presents a rapid detection method for CHIKV and DENV using immunochromatographic strips under low-resource outdoor conditions. Utilizing the CRISPR-Cas system (a visual detection system adapted to biotin-labeled probes and immunochromatographic strips) from Example 3, it enables rapid on-site screening of samples. This method requires no complex instruments and is suitable for low-resource scenarios such as primary healthcare institutions, field sites, and port screenings. The specific steps include: I. RNA Extraction: Same as Example 6.

[0048] II. RT-RAA Primer Design and Amplification: Same as in Example 6, RT-RAA amplification products were obtained.

[0049] III. CRISPR-Cas Dual-System Detection Response 1. Reaction system construction: Follow the "PCR tube bottom + PCR tube cap" separate tube formula described in Example 3, adapted for simplified on-site operation: First, add 2 μL of Cas12a protein (100 nM), 0.5 μL of Cas12a-crRNA (25 nM), 2 μL of 10× buffer, 0.8 μL of FAM-ssDNA-Biotin probe (400 nM), 2 μL of RT-RAA amplification product, and 2.3 μL of nuclease-free water to the bottom of the PCR tube in sequence, and gently invert twice to mix. Next, add the following to the PCR tube cap in sequence: 2 μL of Cas13a protein (100 nM), 1 μL of Cas13a-crRNA (50 nM), 1 μL of NTPMix (25 mM), 2 μL of 10× buffer, 0.5 μL of RNase inhibitor (40 U / μL), 0.5 μL of T7 RNA polymerase (50 U / μL), 2 μL of RT-RAA amplification product, 0.8 μL of Dig-ssRNA-Biotin probe (400 nM), and 0.2 μL of nuclease-free water. Then, tighten the cap and set aside.

[0050] 2. Incubation reaction: Place the PCR tube (including the split-tube system) in a portable incubator at 37°C and incubate for 10 minutes. Then, centrifuge (if a centrifuge is not available, manually invert the PCR tube 5-6 times to fully mix the cap and bottom components) to completely fuse the components in the cap and bottom. After mixing, continue incubating at 37°C for 20 minutes to activate the trans-cleavage activity of Cas12a / Cas13a protein, complete the cleavage of the biotinylated probe, and generate a cleavage product that can bind to the immunochromatographic strip.

[0051] IV. Coupling reaction between the cleavage product and the colloidal gold immunochromatographic strip 1. Product processing: Add 20 μL of nuclease-free water (ddH2O) to the cleavage product obtained in step 3, adjust the total reaction volume to 50 μL, and mix gently for later use.

[0052] 2. Immunochromatographic strip detection: Colloidal gold immunochromatographic strips (model JY0308, CRISPR dual-target test strip) produced by Nanjing Wobo Biotechnology Co., Ltd. are used. The principle is as follows: the T1 line of the test strip is coated with goat anti-mouse secondary antibody, the T2 line is coated with goat anti-rabbit secondary antibody, and the control line (C line) is coated with streptavidin. The labeled probe in the cleavage product can bind to the gold nanoparticle complex on the coupling pad of the test strip, flow with the chromatography solvent and be captured by the antibody or streptavidin on the corresponding line, and develop color through the accumulation of gold nanoparticles.

[0053] Procedure: Add 50 μL of the processed cutting product to the sample well of the immunochromatographic strip, or vertically insert the lower end of the test strip into the product solution (the liquid level should not exceed the mark line of the sample well), let it stand at room temperature for 3-5 minutes, and observe the color development of the bands.

[0054] V. Result Interpretation (Based on Colloidal Gold Immunochromatographic Strip Color Development) The results are determined based on the colorimetric combination of the T1 line, T2 line, and control line (C line): If a red band appears on the T1 line, the T2 line does not show color, and a blue band appears on the control line, it indicates that only the Cas12a protein-mediated cleavage reaction occurs in the system, and the sample is determined to be positive for Chikungunya virus. If a blue band appears on the T2 line, the T1 line does not show any color, and a red band appears on the control line, it indicates that only the Cas13a protein-mediated cleavage reaction occurs in the system, and the sample is determined to be positive for dengue virus. If a red band appears on the T1 line, a blue band appears on the T2 line, and a purple band appears on the control line, it indicates that Cas12a and Cas13a protein-mediated cleavage reactions occur simultaneously in the system, and the sample is determined to be positive for both chikungunya virus and dengue virus. If neither the T1 nor T2 line shows color, and only the control line shows a purple band: this indicates that no cleavage reaction has occurred in the system, and the sample is determined to be negative for both viruses. If the control line (C line) does not develop color: Regardless of whether the T1 / T2 lines develop color, the test is considered invalid (test strip failure or improper operation), and a retest is required.

[0055] VI. On-site comparison setup Simultaneously set up a negative control (reaction system without template RNA) and a positive control (reaction system containing known CHIKV / DENV positive RNA), and operate according to the above steps: the negative control should only show color at the control line, and the positive control should show color at the T line of the corresponding target plus the control line, to verify the effectiveness of the detection system.

[0056] Verification Example This validation example is used to verify the feasibility of the CRISPR-based rapid detection methods for CHIKV and DENV described above. The indoor fluorescence detection method of Example 6 and the outdoor low-resource-condition immunochromatographic strip detection method of Example 7 are validated respectively. Simultaneously, the RT-RAA amplification efficiency and the specific cleavage capability of the CRISPR-Cas system are verified. The specific steps are as follows: I. Preparation of Verification Materials 1. Samples and reagents: Samples to be tested: CHIKV positive RNA (known concentration, derived from serum of clinically confirmed cases), DENV positive RNA (DENV-1 type, known concentration), CHIKV / DENV double positive RNA (artificially mixed sample), negative RNA (RNA extracted from serum of healthy individuals).

[0057] Core reagents: CRISPR-Cas fluorescence detection system of Example 2 (containing FAM-ssDNA-BHQ1 / ROX-ssRNA-BHQ2 probes), CRISPR-Cas tube-type system of Example 3 (containing FAM-ssDNA-Biotin / Dig-ssRNA-Biotin probes), JY0308 colloidal gold immunochromatographic strips from Nanjing Wobo Biotechnology Co., Ltd., commercial RNA extraction kit, lyophilized enzyme powder and A buffer required for RT-RAA amplification.

[0058] 2. Instruments and equipment: Indoor validation: Real-time PCR instrument (for fluorescence signal monitoring), agarose gel electrophoresis instrument and gel imaging system (for identification of RT-RAA amplification products); Outdoor simulation verification: portable metal bath (42℃ / 37℃ constant temperature), manual centrifuge tube rack (replace centrifuge).

[0059] II. Verification Steps and Results 1. RT-RAA amplification efficiency verification (corresponding to step two of Examples 6 / 7) Using CHIKV-positive RNA, DENV-positive RNA, double-positive RNA, and negative RNA as templates, the reaction was constructed according to the 50 μL RT-RAA amplification system formulation in Example 2, and the reaction was carried out at 42℃ for 30 minutes. After amplification, 10 μL of the amplification product was taken for 1.5% agarose gel electrophoresis (120V, 20 minutes), and the bands were observed using a gel imaging system.

[0060] The results are as follows Figure 1 As shown: Both the CHIKV positive group and the DENV positive group showed specific bands at the expected size positions (approximately 150 bp for the CHIKV E1 gene target and approximately 228 bp for the DENV 3' UTR target).

[0061] The above results demonstrate that the RT-RAA primer pairs (SEQ ID NO. 3~6) designed in this invention can efficiently amplify two viral targets without non-specific amplification, and the amplification efficiency meets the requirements for subsequent CRISPR detection.

[0062] 2. Validation of the indoor fluorescence detection method (corresponding to Example 6) Take the above RT-RAA amplification products (taking CHIKV-positive and DENV-positive RT-RAA amplification products as examples), construct the reaction according to the 20 μL CRISPR-Cas dual-system fluorescent lysis reaction system in Example 2, add the samples on ice and centrifuge to mix; place in a real-time fluorescence quantitative PCR instrument, incubate at 37℃ for 20 minutes, monitor the fluorescence signals of FAM channel and ROX channel in real time, and set up a template-free control (NTC, only the system has no template RNA).

[0063] The results are as follows Figure 2 As shown: In the CHIKV positive group, only the FAM channel (CHIKV target) showed a significant increase in fluorescence signal (fluorescence value ≥1000), in the DENV positive group, only the ROX channel (DENV target) showed a significant increase in fluorescence signal (fluorescence value ≥110000), and in the NTC group, there was no signal enhancement; The above results demonstrate that the fluorescence detection method of the present invention can specifically distinguish between CHIKV and DENV infections, with no false positives / false negatives, and has high detection sensitivity.

[0064] 3. Validation of the outdoor immunochromatographic strip detection method (corresponding to Example 7) Take the above RT-RAA amplification product and construct the reaction according to the "PCR tube bottom + PCR tube cap" separate tube system in Example 3. Incubate at 37°C for 10 minutes, manually invert and mix the tube cap and tube bottom components, and continue incubation for 20 minutes. Add 20 μL of nuclease-free water to the cleavage product, adjust the volume to 50 μL, and drop it into the sample well of JY0308 type immunochromatographic strip. Let it stand at room temperature for 3-5 minutes and observe the band color development.

[0065] The results are as follows Figure 3 As shown: CHIKV positive group (CHIKV) + T1 line (red) shows color, T2 line does not show color, and control line (blue) shows color. DENV positive group (DENV) + T2 line (blue) shows color, T1 line does not show color, and control line (red) shows color. Double positive group (CHIKV) + DENV + ): T1 line (red) and T2 line (blue) both show color, and the quality control line (purple) shows color; Negative control group: Only the control line (purple) was colored; the T1 and T2 lines were not colored.

[0066] The above results show that the immunochromatographic strip detection method of the present invention does not require complex instruments, the results can be clearly determined by the naked eye, and are completely consistent with the fluorescence detection results. It is suitable for outdoor low-resource scenarios, and its specificity and accuracy meet the detection requirements.

[0067] III. Verification Conclusion Through multi-dimensional verification of RT-RAA amplification efficiency, indoor fluorescence detection, and outdoor immunochromatographic strip detection, it is confirmed that the CRISPR-Cas system and corresponding detection methods of the present invention (Examples 6 and 7) can efficiently, specifically, and accurately detect chikungunya virus and dengue virus. It can meet the needs of precise laboratory testing and is also suitable for outdoor low-resource field screening. The technical solution is highly feasible and can be used for subsequent reagent kit development and clinical sample testing.

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A CrRNA for the joint detection of chikungunya virus and dengue virus, characterized in that, It includes Cas12a-crRNA and Cas13a-crRNA; the base sequence of the Cas12a-crRNA is shown in SEQ ID NO.1, which can specifically recognize the nucleic acid target of chikungunya virus; the base sequence of the Cas13a-crRNA is shown in SEQ ID NO.2, which can specifically recognize the nucleic acid target of dengue virus.

2. The CrRNA according to claim 1, characterized in that, The Cas12a-crRNA specifically recognizes the chikungunya virus nucleic acid target as the chikungunya virus E1 gene, which is suitable for the specific detection of chikungunya virus; the Cas13a-crRNA specifically recognizes the dengue virus nucleic acid target as the 3' UTR region of dengue virus, which is suitable for the specific detection of dengue virus.

3. The CrRNA according to claim 1, characterized in that, The Cas12a-crRNA is used to guide the Cas12a protein to recognize the PAM sequence containing TTTN, and to form a Cas12a / crRNA / target DNA ternary complex with the target DNA of the Chikungunya virus E1 gene amplified by RT-RAA, thereby activating the side branch cleavage activity of the Cas12a protein to cut single-stranded DNA molecules in the reaction system.

4. The CrRNA according to claim 1, characterized in that, The Cas13a-crRNA is not restricted by the PAM sequence and is used to guide the Cas13a protein to recognize and bind to the target RNA that has been amplified by RT-RAA and transcribed in vitro by T7, forming a Cas13a / crRNA / target RNA ternary complex, activating the side branch cleavage activity of the Cas13a protein to cleave single-stranded RNA molecules in the reaction system.

5. A CRISPR-Cas system for detecting chikungunya virus and dengue virus, characterized in that, Includes the crRNA described in any one of claims 1 to 4.

6. The CRISPR-Cas system according to claim 5, characterized in that, It also includes Cas12a protein, Cas13a protein, signal reporter probe, and RT-RAA amplification products.

7. The CRISPR-Cas system according to claim 6, characterized in that, The signal reporting probe is one of the following two combinations: Combination 1: FAM-ssDNA-BHQ1 probe and ROX-ssRNA-BHQ2 probe, this combination is used for fluorescence detection; Combination 2: FAM-ssDNA-Biotin probe and Dig-ssRNA-Biotin probe, this combination is used for rapid detection with immunochromatographic strips.

8. The CRISPR-Cas system according to claim 6, characterized in that, The RT-RAA amplification product was obtained by amplification using primer pairs targeting the Chikungunya virus E1 gene and the 3' UTR region of dengue virus. The primer pairs include primer pair 1 and primer pair 2. Primer pair 1 is used to amplify the Chikungunya virus E1 gene, and its nucleotide sequence is shown in SEQ ID NO. 3 and SEQ ID NO.

4. Primer pair 2 is used to amplify the 3' UTR region of dengue virus, and its nucleotide sequence is shown in SEQ ID NO. 5 and SEQ ID NO.

6.

9. The application of the CRISPR-Cas system as described in any one of claims 5 to 8 in the preparation of a combined detection kit for chikungunya virus and dengue virus.

10. A rapid detection method for chikungunya virus and dengue virus based on CRISPR, characterized in that, The CRISPR-Cas system according to any one of claims 5 to 8 is used to detect the sample to be tested, comprising the following specific steps: S1: Extract RNA from the sample to be tested; S2: Using the RNA as a template, the amplified product was obtained by using primer pairs targeting the E1 gene of Chikungunya virus and the 3' UTR region of dengue virus via RT-RAA technology. S3: Add the amplification product to a reaction system containing Cas12a-crRNA, Cas13a-crRNA, Cas12a protein, Cas13a protein and signal reporter probe, and detect the signal after incubation; When the signal reporting probes are FAM-ssDNA-BHQ1 and ROX-ssRNA-BHQ2, the FAM / ROX channels are monitored by a fluorescence instrument. Enhancement of fluorescence in either channel corresponds to a positive virus result. When the signal reporting probes are FAM-ssDNA-Biotin and Dig-ssRNA-Biotin, the color development of the control line and the corresponding test line is determined by colloidal gold immunochromatographic strips, indicating that the virus is positive. If the above positive signals are not present, the result is negative.

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