A sequence combination, kit, method, and application for the co-detection of novel Bunyavirus and Anaplasma phagocytophila based on RPA-CRISPR.

Abstract

This invention discloses a sequence combination, kit, method, and application for the co-detection of novel Bunyavirus and Anaplasma phagocytophila based on RPA-CRISPR. It includes a first RPA primer pair and a first crRNA designed based on the conserved region of the SFTSV L fragment, a second RPA primer pair and a second crRNA designed based on the conserved region of the AP 16S rRNA gene, an optimized universal T7 helper primer, a kit prepared using this sequence combination, and a detection method for novel Bunyavirus and Anaplasma phagocytophila using this kit. The detection method of this invention combines RPA amplification with CRISPR detection to simultaneously detect novel Bunyavirus and Anaplasma phagocytophila, effectively overcoming the bottlenecks of existing technologies in co-amplification efficiency and orthogonal detection sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a sequence combination, kit, method and application for the co-detection of new Bunyavirus and Apocynophilic Anaplasma based on RPA-CRISPR. Background Technology

[0002] Fever with thrombocytopenia syndrome (SFTS) and human granulocytic anaplasmosis (HGA) are caused by different pathogens (RNA virus SFTSV and DNA bacteria Anaplasma phagocytophilum, AP). Both are clinically characterized by acute high fever, significant leukopenia, and thrombocytopenia, with severe cases progressing to multiple organ failure. Because their treatment regimens are drastically different—SFTS is primarily treated with supportive care and early antiviral therapy, while HGA is highly sensitive to doxycycline, and delayed administration significantly increases mortality—rapid etiological identification within 24 hours of symptom onset is crucial for improving clinical prognosis.

[0003] The current gold standard for laboratory diagnosis relies on real-time quantitative RT-qPCR (for RNA) or qPCR (for DNA), with a sensitivity of ≥95% and a specificity of ≥99%. However, the stringent requirements of this technology for sophisticated instruments (such as 96 / 384-well quantitative PCR instruments and nucleic acid extractors) and standardized laboratory environments make it difficult to meet the growing demands for convenient, fast, and adaptable testing.

[0004] Recombinase polymerase amplification (RPA) technology is considered a promising platform for rapid on-site diagnostics due to its ability to rapidly amplify nucleic acids within 5-20 minutes under isothermal conditions (37-42℃). In recent years, the CRISPR-Cas system has brought breakthroughs to molecular diagnostics. Specifically, the Cas12a enzyme, after specifically recognizing double-stranded DNA (dsDNA) targets, can non-specifically cleave single-stranded DNA (ssDNA) reporter molecules; while the Cas13a enzyme, after recognizing single-stranded RNA (ssRNA) targets, can non-specifically cleave ssRNA reporter molecules. The catalytic activities of these two enzymes are orthogonal and do not interfere with each other, providing a novel methodological basis for the parallel and specific detection of DNA and RNA targets in the same reaction system. Combining the rapid isothermal amplification capability of RPA with the specific recognition and signal amplification functions of the CRISPR-Cas system (RPA-CRISPR) is a cutting-edge direction for building next-generation ultrasensitive, highly specific rapid detection tools.

[0005] However, successfully applying the RPA-CRISPR technology system to the joint detection of SFTSV and AP faces two major unresolved technical bottlenecks in the design of core functional nucleic acid sequences:

[0006] The challenge of RPA co-amplification primer design: A primer set needs to be designed and validated to efficiently, specifically, and simultaneously amplify SFTSV (reverse transcription) and AP genome-specific target sequences under the same reaction tube and isothermal conditions. This primer set must meet the following requirements: (a) amplification efficiency ≥90% for both targets; (b) no significant dimerization or cross-reaction between primers or between primers themselves; and (c) amplification product length appropriate (typically 100–150 bp) to ensure effective recognition and cleavage by the subsequent CRISPR system.

[0007] The challenge of designing CRISPR co-cleaved crRNAs lies in designing highly specific and strictly orthogonal Cas13a crRNAs (targeting SFTSV) and Cas12a crRNAs (targeting AP) for the aforementioned co-amplification products. These two crRNAs must ensure that: (a) they are efficiently activated only by their respective targets, with no cross-activation between them; (b) when working in conjunction with corresponding fluorescent or chromatographic reporter systems (such as FAM-labeled ssRNA and ssDNA reporter probes), they can rapidly generate high-intensity signals with extremely low background; and (c) they possess extremely high sequence specificity to distinguish the target pathogen from its closely related species.

[0008] Currently, no publicly available literature or databases report any specific RPA primer and CRISPR-crRNA sequence combination that has been fully experimentally validated and can effectively achieve integrated detection of SFTSV and AP "co-amplification-co-splicing". Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention provides a sequence combination, kit, method, and application for the co-detection of novel Bunyavirus and Apoptosis phagocytophilia based on RPA-CRISPR. By co-optimizing the design of RPA primers and orthogonal CRISPR-crRNA core sequences, an innovative RPA-CRISPR co-detection method is constructed, which can effectively overcome the bottlenecks of existing technologies in co-amplification efficiency and orthogonal detection sensitivity. This method has significant advantages such as short detection time, high sensitivity, and simple operation, and can complete the simultaneous identification of SFTSV and AP within 40 minutes, providing a technical solution with great practical application value for rapid and accurate diagnosis.

[0010] The technical solution provided by this invention is as follows:

[0011] This invention provides a sequence combination for co-detection of novel Bunyavirus and Anaplasma phagocytophilum based on RPA-CRISPR, comprising a first RPA primer pair and a first crRNA designed based on the conserved region of the SFTSV L fragment, a second RPA primer pair and a second crRNA designed based on the conserved region of the AP 16S rRNA gene, and an optimized universal T7 helper primer.

[0012] The nucleotide sequences of the upstream and downstream primers of the first RPA primer pair are shown in SEQ ID NO.5 and SEQ ID NO.6; the nucleotide sequences of the upstream and downstream primers of the second RPA primer pair are shown in SEQ ID NO.21 and SEQ ID NO.19; and the nucleotide sequences of the first crRNA and the second crRNA are shown in SEQ ID NO.23 and SEQ ID NO.24.

[0013] Furthermore, the sequence of the universal T7 auxiliary primer is shown in SEQ ID NO.22, and its 5′ end contains a continuous guanine modification.

[0014] The present invention also provides a kit for co-detection of novel Bunyavirus and Anaplasma phagocytophilum based on RPA-CRISPR, comprising the sequence combination described above.

[0015] This invention also provides a method for co-detecting novel Bunyavirus and Anaplasma phagocytophilum based on RPA-CRISPR, based on the sequence combinations or the kits described above, the method comprising:

[0016] Total nucleic acid was extracted from the sample to be tested;

[0017] Using the extracted total nucleic acid as a template, an RPA co-amplification reaction was performed using a reaction system containing the first RPA primer pair, the second RPA primer pair, and the universal T7 auxiliary primer to obtain the co-amplification product;

[0018] The co-amplification product, the first crRNA with Cas13a enzyme and RNA reporter probe, and the second crRNA with Cas12a enzyme and DNA reporter probe are mixed to perform a CRISPR / Cas12a / Cas13a co-detection reaction.

[0019] By detecting changes in the fluorescence signals of the aforementioned RNA and DNA reporter probes, positive results for novel Bunyavirus and Anaplasma phagocytophilum were determined, respectively.

[0020] Furthermore, the reaction system for the RPA co-amplification reaction is as follows: 29.5 μL of RT-RAA lyophilized enzyme reaction particles and reconstitution buffer, 1.2 μL each of the primers shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.21, and SEQ ID NO.19 (10 μM), 2.4 μL of the primer shown in SEQ ID NO.22 (10 μM), 5 μL of nucleic acid template, and nuclease-free water to a final volume of 47.5 μL. Finally, 2.5 μL of 280 mM magnesium acetate initiator is added.

[0021] Furthermore, the reaction system for the CRISPR / Cas12a / Cas13a co-detection reaction is as follows: 1 μL of 1M HEPES buffer, 1 μL of 1M MgCl2, 2 μL of 25mM NTP, 1 μL of 50U / μL T7 RNA polymerase, 0.5 μL of 1μM LbCas12a enzyme, 0.4 μL of 10μM second crRNA, 0.5 μL of 1μM LwCas13a enzyme, 0.4 μL of 10μM first crRNA, 1 μL each of 10μM DNA reporter probe and 10μM RNA reporter probe, and 2 μL of co-amplification product, which is then brought to a final volume of 20 μL with nuclease-free water.

[0022] Furthermore, the RPA co-amplification reaction is carried out at 39°C for 20-40 minutes, and the CRISPR / Cas12a / Cas13a reaction is carried out at 37°C. The results are interpreted by monitoring the initial fluorescence intensity threshold or the fluorescence increase threshold.

[0023] Furthermore, the criteria for interpreting the results are as follows:

[0024] If, during the detection process, the initial fluorescence intensity of the DNA reporter probe is ≥50,000 or its fluorescence increase value is >3,000, then the anaplasmophilus is considered positive.

[0025] If, during the detection process, the initial fluorescence intensity of the RNA reporter probe is ≥10,000 or its fluorescence increase value is >3,000, then the result is considered positive for the new Bunyavirus.

[0026] Furthermore, both the DNA reporter probe and the RNA reporter probe are oligonucleotide molecules labeled with a fluorescent group and a quencher group at both ends, respectively. Specifically, the DNA reporter probe is FAM-ssDNA-BHQ1, and the RNA reporter probe is ROX-ssRNA-BHQ1.

[0027] The present invention also provides the application of the above-described sequence combinations or the above-described kits in the preparation of detection products for fever with thrombocytopenia syndrome and human granulocytic anaplasmosis.

[0028] Beneficial effects

[0029] This invention utilizes optimized RPA primers to simultaneously amplify RNA virus (SFTSV) and DNA bacterial (AP) targets in the same isothermal reaction system. The designed primer combination effectively avoids primer dimers and cross-reactions, maintaining high amplification efficiency for both pathogens and solving the problem of uneven amplification efficiency for different nucleic acid types in existing combined detection methods.

[0030] This invention provides an orthogonal detection system for Cas12a / crRNA (targeting AP DNA) and Cas13a / crRNA (targeting SFTSV RNA). The two CRISPR-Cas enzymes achieve physical isolation of the signaling pathway through differences in catalytic activity, avoiding cross-activation. The crRNA used can specifically recognize the target sequence, effectively distinguishing the target pathogen from closely related species and improving detection specificity.

[0031] This invention utilizes RPA pre-amplification and CRISPR signal amplification to detect low-copy-number pathogen nucleic acids. The entire detection process can be completed in approximately 40 minutes, significantly improving detection efficiency compared to existing qPCR methods. The kit allows for reaction completion under isothermal conditions, eliminating the need for a thermal cycler or quantitative PCR instrument. Detection results can be interpreted using a portable fluorometer or lateral flow chromatography strips, making it suitable for field use or environments with limited resources.

[0032] This invention not only provides core primers, crRNA and reagent combinations, but also includes a complete operational plan from sample processing to result interpretation, providing a technical implementation method for the joint detection of SFTSV and AP.

[0033] In summary, this invention establishes a rapid, sensitive, and easy-to-operate dual-target nucleic acid detection method through the technical design of co-amplification and CRISPR detection system, effectively solving the technical problem of joint detection of pathogens with different nucleic acid types in the prior art. Detailed Implementation

[0034] Specific embodiments of the present invention are illustrated through the following examples. It should be understood that these examples are merely illustrative, and the scope of the present invention is not limited thereto. Methods without specific conditions can generally be performed with reference to known methods in the art or conditions recommended by reagent suppliers. Unless otherwise specified, the reagents used in the experiments are conventional reagents in the art and can be purchased through commercial channels.

[0035] Example 1: Construction of the detection system

[0036] To achieve efficient and balanced co-amplification of novel Bunyavirus (SFTSV) and Apocynophagocytophilia (AP) in a single reaction system, this embodiment follows the general principles of RPA primer design and, in conjunction with experimental verification, systematically screens and determines specific primer combinations suitable for "co-amplification" detection.

[0037] 1. Preparation of experimental materials and target sequences

[0038] 1.1 Experimental Materials

[0039] (1) Positive control: SFTSV L fragment pseudovirus (Fubai Ao (Suzhou) Biomedical Technology Co., Ltd.); AP16S rDNA plasmid (Sangon Biotech (Shanghai) Co., Ltd.);

[0040] (2) Negative control: Nuclease-free water (Solepro);

[0041] (3) Nucleic acid extraction: Qiagen AllPrep DNA / RNA Mini Kit (Qiagen);

[0042] (4) RPA reagent: TwistAmp® Basic RT;

[0043] (5) CRISPR reagents: LbCas12a (Gentech), LwCas13a (Gentech), RNase inhibitor (Novizan), T7 RNA polymerase (Sangon Biotech), NTP (Sangon Biotech), HEPES (Sangon Biotech), MgCl2 (Sangon Biotech), FAM-ssDNA-BHQ1 reporter probe: 5′-FAM-TTATT-BHQ1-3′ (Sangon Biotech), ROX-ssRNA-BHQ1 reporter probe: 5′-ROX-rUrUrUrUrU-BHQ1-3′ (Sangon Biotech);

[0044] (6) Instrument: Fluorescence detector (Roche).

[0045] 1.2 Target Sequence Preparation

[0046] 1.2.1 Target sequence acquisition and conserved region screening

[0047] Genomic sequences of the novel Bunyavirus (SFTSV) and Anaplasma phagocytophilum (AP) isolated from different geographical origins and time periods were systematically obtained and organized from public databases such as NCBI GenBank. Multiple sequence alignment software was used to compare and analyze the collected sequences, comprehensively evaluating sequence conservation, intraspecific consistency, and interspecific specificity. The analysis identified highly conserved and specific regions in the SFTSV L fragment and the AP 16S rRNA gene, which were then used as target sequences for designing detection elements.

[0048] 1.2.2 Construction of positive control samples

[0049] The conserved region of SFTSV L was located and sent to Fubai Ao (Suzhou) Biomedical Technology Co., Ltd. for RNA pseudovirus synthesis. The sequence is as follows (SEQ ID NO.25):

[0050] CTGGACGCCACATCGGGAGGAACTCTCAGCCACTCTGTCATGGTGTACTGGGGGGATAGGAAGAAGTATCAGGCCTTATTGAACAGGATGGGCCTTCCTGAGGACTGGGTGGAGCAGATAGATGAGAATCCTGGAGTCCTTTACAGGAGAGCTGCCAACAAAAAGGAACTACTCTTGAAACTGGCAGAGAAGGTCCATTCACCAGGTGTGACT AGCAGCCTGAGTAAAGGGCATGTAGTGCCTCGGGTGGTGTCAGCAGGAGTGTACCTTCTCTCACGCCACTGCTTTCGCTTTAGTTCAAGTATCCATGGAAGGGGCTCAGCACAGAAGGCTAGTCTTATAAAACTGCTGATGATGTCTTCTATTTCTGCCATGAAGCACGGGGGCTCACTAAACCCTAATCAGGAGCGAATGCTCTTCCCTCAGG

[0051] The conserved region of the AP 16S rRNA was located and sent to Sangon Biotech (Shanghai) Co., Ltd. for plasmid synthesis. The sequence is as follows (SEQ ID NO.26):

[0052] TGGAACTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCTATGCCGCGTGAGTGAGGAAGGCCTTAGGGTTGTAAAACTCTTTCAGTAGGGAAGATAATGACGGTACCTACAGAAGAAGTCCCGGCAAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGGGCAAGCGTTGTTCGGAATTATTGGGCGTAAAGGGCATGTAGGCGGTTCGGTAAGTTAAAGGTGAAATGCCAGGGCTTAACCCTGGAGCTGCTTTTAATACTGCCAGACTAGAGTCCGGGAGAGGATAGCGGAATTCCTAGTGTAGAGGTGAAATTCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTATCTGGTCCGGTACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAGTGCTGAATGTGGGGATTTTTTATCTCTGTGTTGTAGCTAACGCGTTAAGCACTCCGCCTGGGGACTACGGTCGCAAGACTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTGACATGGAGATTAGATCCTTCTTAACGGAAGGGCGCAGTTCGGCTGGATCTCGCACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGTAACCCTCATCCTTAGTTGCCAGCGGGTTAAGCCGGGC

[0053] 1.3 Construction of PCR detection system

[0054] To compare the detection performance of this invention with that of conventional methods, a PCR detection system was also established. The PCR detection system was constructed with reference to the literature "Establishment and Application of TaqMan Probe Real-Time Fluorescent Quantitative Reverse Transcription-Polymerase Chain Reaction Method for Detecting the L Gene of Novel Bunyavirus" and "Prevalence of anaplasma phagocytophilum in ixodesricinus ticks determined by polymerase chain reaction with two pairs of primers detecting 16S rRNA and anka genes".

[0055] 2. Construction and optimization of RPA detection system

[0056] The specific reaction configuration of the detection system used in this experiment is as follows: The total reaction volume is 50 μL, containing 29.5 μL of RT-RAA lyophilized enzyme reaction particles and reconstitution buffer (RPA reagent: TwistAmp® Basic RT). Then, 2.4 μL / primer (stock solution concentration 10 μM) is added. The primers can be selected from any two or more of primers 1, 2, 3, 4, or 5, depending on the target detection requirements, to achieve a final concentration of 0.2 μM for each primer in the reaction system. Next, 5 μL of nucleic acid template and an appropriate amount of enzyme-free water are added, bringing the total volume to 47.5 μL. Finally, 2.5 μL of 280 mM magnesium acetate initiator is added and mixed thoroughly to start the amplification reaction. After incubation at 39℃ for 20 min, the products are purified and the amplification efficiency of each primer pair is evaluated by agarose gel electrophoresis.

[0057] 2.1 Preliminary Construction of Single RPA Detection System and Primer Performance Verification

[0058] First, multiple pairs of candidate RPA primers (SFTSV-F / R series and AP-F / R series) were independently designed for the conserved regions of the SFTSV L fragment and the AP 16S rRNA gene. All primer designs strictly adhered to the following principles: length 30-35 nt, GC content between 30% and 70%, and their potential secondary structures, dimer formation risk, and sequence specificity were evaluated using bioinformatics tools (such as PrimerExplorer V5 and NCBI BLAST). Primers that met the amplification efficiency requirements underwent a second round of detection to confirm the detection limit. The sequence listing used in the experiment is as follows:

[0059] Table 1 RPA primers

[0060]

[0061] Table 2 Confirmation of SFTSV RPA and AP RPA detection primers

[0062]

[0063]

[0064] The SFTSVF5+SFTSVR1 primers showed the best detection sensitivity. Two pairs of primers, APF1+APR4 and APF3+APR4, were obtained from the conserved region of the AP 16S rRNA gene. These primers were retained for the next stage of compatibility screening using co-amplification.

[0065] 2.2 Validation of “co-amplification” primer combinations

[0066] The core objective of this stage is to confirm the detection performance of the dual amplification system. The specific steps are as follows: The qualified SFTSV primer pairs (SFTSVF5 / SFTSVR1) selected in the previous stage are combined with AP primer pairs (such as APF1 / APR4 / APF3 / APR4), and the SFTSV and AP templates are detected separately. Visual evaluation: When non-target primer sets are present in the system, will they significantly inhibit the amplification efficiency of the target template (e.g., due to non-specific binding between primers competing for enzymes or raw materials), or will they trigger non-target amplification?

[0067] Table 3 Detection performance of the dual amplification system

[0068]

[0069] Observations revealed that primer combinations that met the singleton amplification performance requirements exhibited poor multiplex amplification performance, failing to meet detection needs. The SFTSVF5 / SFTSVR1 / APF1 / APR4 combination showed superior detection performance, but its detection sensitivity still fell short of requirements.

[0070] 3. Further optimization of the multiple RAA reaction system

[0071] To further improve the amplification efficiency and detection sensitivity of the multiplex amplification system, the primer system was structurally optimized again: while keeping the SFTSV detection primers unchanged, a T7 promoter sequence (APF1-T7) was introduced into the 5′ end of the upstream primer APF1 for the AP target, making the upstream recombination efficiency of the two primers similar; at the same time, an additional sequence-optimized T7 helper primer COM-T7-5G, with 5 consecutive guanine (G) bases modified at its 5′ end, was added to the reaction system. After the first round of recombination amplification, COM-T7-5G can continue recombination as the main primer, forming a groove-like reaction effect, improving the amplification stability and overall signal amplification capability in the multiplex system. The amount of each primer in the amplification system was also optimized in this part. The final confirmed RAA detection was performed in a separate 50 μL reaction system. The RT-RAA amplification system consisted of 29.5 μL of RT-RAA lyophilized enzyme reaction particles and reconstitution buffer, followed by primer components: 1.2 μL of SFTSVF5 primer (10 μM), 1.2 μL of SFTSVF1 primer (10 μM), 1.2 μL of APR4 primer (10 μM), 1.2 μL of APF1-T7 primer (10 μM) with a T7 promoter sequence at the 5′ end, and 2.4 μL of optimized T7 auxiliary primer COM-T7-5G (10 μM). Then, 5 μL of nucleic acid template was added, and 5.8 μL of nuclease-free water was added to bring the total volume to 47.5 μL. Finally, 2.5 μL of 280 mM magnesium acetate initiator was added, and the mixture was inverted and mixed thoroughly before starting the amplification reaction. The primer sequences used in this experiment are as follows:

[0072] Table 4 Detection Primer Sequences

[0073]

[0074] Table 5 Detection performance of the dual amplification system

[0075]

[0076] 4. Design and optimization of crRNA

[0077] For the optimized SFTSV and AP RPA amplification products, highly specific spacer sequences were designed and screened. These spacer sequences were fused with optimized Cas13a or Cas12a crRNA backbone sequences, and crRNA was obtained through in vitro transcription or chemical synthesis. All designs were bioinformatically analyzed to ensure intraspecific conservation and interspecific specificity, and experimentally validated to avoid primer dimers and cross-reactivity.

[0078] 5. Verification and determination of orthogonality of crRNA

[0079] The obtained crRNAs were combined with their corresponding Cas enzymes (Cas12a or Cas13a) and reporter systems, respectively. Cross-activation tests and sensitivity titration experiments were used to verify their strict orthogonality (i.e., specific activation only by the corresponding target, with no cross-reaction) and high detection sensitivity. Finally, a set of crRNA combinations that could independently and efficiently drive signal generation in a dual-target coexistence system was determined, constituting the core sequence of the detection system.

[0080] 6. Construction of CRISPR / Cas12a / Cas13a co-detection system

[0081] The CRISPR assay reaction system consists of a separate 20 μL volume, and all operations must be performed on ice to ensure enzyme activity. The reaction system contains the following components: 1 μL HEPES (1M), 1 μL MgCl2 (1M), 2 μL NTP (25mM), 1 μL T7 RNA polymerase (50U / μL), 0.5 μL LbCas12a enzyme (1μM), 0.4 μL Cas12a crRNA targeting AP (10μM), 0.5 μL LwCas13a enzyme (1μM), 0.4 μL Cas13a crRNA targeting SFTSV (10μM), and 2 μL template RPA amplification product, 1 μL each of 10 μM single-stranded DNA fluorescent reporter probe and 10 μM single-stranded RNA fluorescent reporter probe. The system is then brought to a final volume of 20 μL with nuclease-free water. The negative control is prepared by replacing the template RPA amplification product with an equal volume of nuclease-free water and following the same RPA amplification and shearing procedure.

[0082] 7. Fluorescence signal detection

[0083] After briefly centrifuging and mixing the prepared CRISPR detection reaction solution, transfer it to the corresponding reaction well / tube of a real-time quantitative PCR instrument or a dedicated portable fluorescence detector. Set the reaction temperature to 37°C and immediately start the reaction and signal acquisition program. The instrument acquires the fluorescence intensity of the FAM channel (corresponding to the Cas12a / AP system) and the ROX channel (corresponding to the Cas13a / SFTSV system) synchronously or sequentially at a frequency of 1 minute / time. Judgment criteria: 1) Initial fluorescence judgment: If the initial fluorescence intensity Rn0(FAM) ≥ 50,000, or the initial fluorescence intensity Rn0(ROX) ≥ 10,000, then the channel is directly interpreted as positive. 2) Fluorescence intensity change judgment: If the increase in fluorescence intensity (ΔRn) of any channel (FAM or ROX) during the amplification process is > 3,000, ΔRn = Rn 30 -Rn0 indicates that the channel is positive.

[0084] The key improvement of this invention lies in the addition of a T7 promoter sequence to the 5′ end of the RPA upstream primer designed for the Cas12a detection system. Simultaneously, a sequence-optimized T7 primer with five consecutive guanine (G) bases modified at its 5′ end is added to the reaction system. This combined strategy significantly improves the efficiency and stability of the dual-target parallel amplification system, ensuring efficient and reliable nucleic acid amplification in each channel under multiplex detection conditions.

[0085] This invention successfully screened and identified a set of core functional nucleic acid sequence combinations capable of efficiently, specifically, and orthogonally performing "co-amplification-co-detection" of novel Bunyavirus (SFTSV) and Anaplasma phagocytosolicum (AP). This combination contains two pairs of specific primers for co-amplification, and two highly specific crRNAs for activating the cleavage activities of Cas12a and Cas13a enzymes, respectively. Specific sequence information is shown in Table 6.

[0086] Table 6. Detection primer-crRNA sequences

[0087]

[0088] Example 2: Sensitivity Confirmation of the Detection System

[0089] To determine the sensitivity of the RPA-CRISPR combined detection system established in this invention, in vitro transcribed RNA (mimicking viral RNA) containing a conserved sequence of the SFTSV L fragment and plasmid DNA (mimicking bacterial DNA) containing a conserved sequence of the AP 16S rRNA gene were serially diluted 10-fold using nuclease-free water.

[0090] Select covering 10 3 copies / μL, 10 2 copies / μL, 10 1 Four concentration gradients of nucleic acid templates were used, at concentrations of 1 copy / μL and 1 copy / μL, with nuclease-free water serving as a negative control. RPA-CRISPR assays were performed; the experimental procedure is as follows:

[0091] 1. RPA Co-amplification: RPA co-amplification was performed in a 50 μL reaction system. The reaction system contained 29.5 μL of RT-RAA lyophilized enzyme reaction particles and reconstitution buffer, and amplification primer components (stock solution concentration 10 μM, specific primer combination as described above in the optimized system), 5 μL of nucleic acid template, and nuclease-free water to a final volume of 47.5 μL. Finally, 2.5 μL of 280 mM magnesium acetate initiator was added, mixed well, and the reaction was started. The amplification reaction was incubated at 39℃ for 20 min.

[0092] 2. CRISPR detection reaction: Add 1 μL of HEPES buffer (1M), 1 μL of MgCl2 (1M), and 2 μL of NTP mixture (25mM) to the reaction system; add T7 phage-derived DNA-dependent RNA polymerase (T7 RNA polymerase) to make the final concentration approximately 1 U / μL.

[0093] Subsequently, Cas enzyme-crRNA complexes were added: 0.5 μL of LbCas12a nuclease (1 μM) and 0.4 μL of Cas12a crRNA (10 μM); and 0.5 μL of LwCas13a nuclease (1 μM) and 0.4 μL of Cas13a crRNA (10 μM). Simultaneously, 1 μL each of a 10 μM single-stranded DNA fluorescent reporter probe and a 10 μM single-stranded RNA fluorescent reporter probe were added to the system to respond to the trans-cleavage activities of Cas12a and Cas13a, respectively.

[0094] Add 5 μL of co-amplification product to the above detection system, then add nuclease-free water to make up to 20 μL. After thorough mixing, place the system in a PCR instrument and react at 37 ℃. Set the instrument to collect fluorescence signals once per minute for 30 consecutive cycles for real-time fluorescence monitoring. Record the initial fluorescence signal intensity and the endpoint fluorescence signal intensity, and calculate ΔRn.

[0095] The detection results (as shown in Table 7) indicate that the detection rate was 100% (10 / 10) when the SFTSV RNA concentration was 10^1 copies / μL and 90% (9 / 10) when the RNA concentration was 10^0 copies / μL. The detection rates of the control PCR method were 40% and 0%, respectively. Similarly, the detection rate was 100% (10 / 10) when the APDNA template concentration was 10^1 copies / μL and 100% (10 / 10) when the RNA concentration was 10^0 copies / μL. The detection rates of the control PCR method were 50% and 0%, respectively. The detection sensitivity of this method is superior to that of previously reported PCR systems.

[0096] Table 7 Detection Sensitivity Results

[0097]

[0098]

[0099] Note: Bold numbers in the table indicate that Rn0 > tangent value, which is considered positive; all other values ​​are judged based on ΔRn.

[0100] Example 3: Specificity Confirmation of the Detection System

[0101] To systematically evaluate the specificity of the detection system, this study selected multiple pathogens with similar clinical symptoms or taxonomical relationships to SFTSV and AP for testing, including dengue virus, influenza B virus, hantavirus, Rickettsia rickettsia, Orientia scrub typhus, Ehrlich aspergillus chafing, Anaplasma phagocytophilum, Leptospira, and Bartonella. Genomic DNA from healthy individuals and nuclease-free water were used as controls. All non-target samples were required to produce negative results to ensure no cross-reactivity of the detection system. Specific test specimens were primarily obtained through outsourcing or artificial synthesis.

[0102] Table 8 Specific detection results

[0103]

[0104] The RPA-CRISPR system of this invention was used to detect all the aforementioned non-target samples. The results showed that all non-target samples were negative. No specific signal growth was observed in real-time fluorescence detection. These results indicate that the primer and crRNA combination designed in this invention has high specificity and no cross-reactivity with the aforementioned non-target pathogens.

[0105] Example 4: Detection of unknown samples using the kit of the present invention

[0106] Serum samples were collected from patients with suspected fever with thrombocytopenia syndrome (SFTS) and human granulocytic anaplasmosis (HGA) and healthy individuals. After centrifugation, total nucleic acid was extracted using a commercial nucleic acid extraction kit as a template for testing.

[0107] (1) Detection group: The serum samples were tested using the reaction system confirmed in Example 2 of this invention;

[0108] (2) Comparison method group: Simultaneous detection was performed using the PCR method reported in the literature;

[0109] (3) Blank control: No template control (NTC), using nuclease-free water instead of template;

[0110] (4) Positive control: Synthetic pseudovirus / plasmid was used as a control;

[0111] (5) The test results are shown in Table 9.

[0112] Table 9 Detection results of unknown samples

[0113]

[0114]

[0115] Results analysis: Using 73 clinical samples, total nucleic acid was extracted and tested. The kit of this invention and PCR detection were performed simultaneously. Each experiment included positive and negative controls. The results showed that the detection results of both methods were negative for all 73 samples, which was consistent with the PCR results. Therefore, the kit of this invention has strong stability and reliable detection results.

[0116] This invention achieves high sensitivity and high specificity in detection performance through optimized primer design and reaction system. Experimental data show that the detection method achieves a limit of detection of 1-10 copies / μL for the target pathogen, which is superior to existing PCR detection systems. Furthermore, no non-specific amplification was observed in cross-reactivity tests with various pathogens exhibiting similar clinical symptoms or taxonomically related pathogens (including dengue virus, influenza B virus, Rickettsia rickettsia, Ehrlich. chaffi), indicating its excellent detection specificity.

[0117] It should be noted that the embodiments and descriptions described herein are intended to illustrate the technical principles and implementation methods of the invention, and do not constitute a limitation on the scope of protection of the invention. Those skilled in the art should understand that various adjustments, substitutions, or improvements can be made to the invention without departing from its design principles and technical intent, and all such modifications fall within the scope of protection covered by the claims and their equivalents.

Claims

1. A sequence combination for co-detection of novel Bunyavirus and Anaplasma phagocytophilum based on RPA-CRISPR, characterized in that, It includes a first RPA primer pair and a first crRNA designed based on the conserved region of the SFTSV L fragment, a second RPA primer pair and a second crRNA designed based on the conserved region of the AP 16S rRNA gene, and an optimized universal T7 helper primer. The nucleotide sequences of the upstream and downstream primers of the first RPA primer pair are shown in SEQ ID NO.5 and SEQ ID NO.6; the nucleotide sequences of the upstream and downstream primers of the second RPA primer pair are shown in SEQ ID NO.21 and SEQ ID NO.19; the nucleotide sequences of the first crRNA and the second crRNA are shown in SEQ ID NO.23 and SEQ ID NO.24; and the sequence of the universal T7 helper primer is shown in SEQ ID NO.22, with its 5′ end containing continuous guanine modification.

2. A kit for co-detection of novel Bunyavirus and Anaplasma phagocytophilum based on RPA-CRISPR, characterized in that, Includes the sequence combination described in claim 1.

3. The use of the sequence combination of claim 1 or the kit of claim 2 in the preparation of a detection product for fever with thrombocytopenia syndrome and human granulocytic anaplasmosis.

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