Chikungunya virus detection primer group and kit

Abstract

The invention relates to the technical field of biological detection, and particularly discloses a chikungunya virus detection primer group and a kit, and the primer group comprises CHIKV-F2 as shown in SEQ ID NO.1, CHIKV-R1 as shown in SEQ ID NO.2 and an RAA probe CHIKV-P1 as shown in SEQ ID NO.3. The invention further discloses a kit for detecting the chikungunya virus. The primer group provided by the invention has high specificity and high sensitivity when being used for detecting the chikungunya virus.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and in particular to a primer set and a kit for detecting chikungunya virus. Background Art

[0002] Chikungunya virus, also known as CHIKV, is an RNA virus transmitted by Aedes mosquitoes that causes chikungunya fever, a fever characterized by rash, severe joint pain, and other symptoms. First discovered in Tanzania in 1952, the virus is prevalent in tropical regions of Africa and Asia. Currently, methods for detecting chikungunya virus, including RT-PCR and nested PCR, are highly sensitive but complex and time-consuming. These methods require specialized laboratory equipment and personnel, making them difficult to use in non-laboratory settings. Therefore, there is a need to develop a highly specific and sensitive test for detecting chikungunya virus. Summary of the Invention

[0003] To develop a highly specific and sensitive product for detecting Chikungunya virus, the present invention provides a primer set and kit for detecting Chikungunya virus. The primer set provided by the present invention has high specificity and high sensitivity for detecting Chikungunya virus.

[0004] The present invention provides a Chikungunya virus detection primer set, comprising CHIKV-F2 shown in SEQ ID NO.1, CHIKV-R1 shown in SEQ ID NO.2, and RAA probe CHIKV-P1 shown in SEQ ID NO.3.

[0005] The primer set provided by the present invention has high specificity and high sensitivity for detecting Chikungunya virus.

[0006] The present invention provides a kit comprising the chikungunya virus detection primer set. The kit is used for detecting chikungunya virus in non-disease diagnosis.

[0007] Furthermore, the kit also includes an RAA reaction system, a buffer solution, a positive control, and a negative control.

[0008] Furthermore, the RAA reaction system comprises recombinase, recombinase auxiliary factor, single-strand binding protein, exonuclease, lyophilization protectant, dNTP, creatine kinase and ATP.

[0009] Furthermore, the buffer solution includes 10% by mass of PEG35000 and 280 mM magnesium acetate solution.

[0010] Furthermore, the positive control is a pEASY®-Blunt plasmid containing the Chikungunya virus gene.

[0011] Furthermore, the negative control is enzyme-free water.

[0012] Furthermore, the volumes of the components in the kit are as follows: enzyme-free water 15.9 μL, mass fraction 10% PEG35000 25.0 μL, primer CHIKV-F2 2.0 μL, primer CHIKV-R1 2.0 μL, 10 μM probe CHIKV-P1 0.6 μL, plasmid pE -CHIKV 2.0 μL, 280 mM magnesium acetate solution 2.5 μL.

[0013] The present invention also provides a non-diagnostic detection method for Chikungunya virus, comprising the following steps: The CHIKV-F2 shown in SEQ ID NO. 1, the CHIKV-R1 shown in SEQ ID NO. 2, and the RAA probe CHIKV-P1 shown in SEQ ID NO. 3 were designed; A Chikungunya virus detection kit is prepared, which includes CHIKV-F2, CHIKV-R1, RAA probe CHIKV-P1, RAA reaction system, A buffer, B buffer, positive control, and negative control; The positive control is the pEASY®-Blunt plasmid containing the Chikungunya virus gene, and the negative control is enzyme-free water; Detect the Chikungunya virus in the sample by running PCR.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The chikungunya virus detection primer set provided by the present invention has high specificity, and the RAA reaction fluorescence signal intensity is significantly higher than that of other primer-probe combinations.

[0015] The present invention only takes 15 minutes to perform fluorescence-based CHIKV detection, significantly shortening detection time and improving efficiency. In test strip-based testing, this method offers high sensitivity, ease of use, and rapid results, requiring no specialized equipment. The results meet the requirements for clinical sample testing and are particularly suitable for mobile emergency testing in resource-poor areas or at facilities such as airports and schools.

[0016] Short reaction time: RAA technology can complete reactions in a short time, such as 15 minutes at 39°C. Suitable for on-site testing: RAA technology is easy to operate and does not require complex equipment. RAA-nfo, combined with disposable nucleic acid test strips, only requires a constant temperature water bath for testing, making it suitable for rapid testing in non-laboratory environments or on-site. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is a comparison of the fluorescence signal intensities of the RAA reactions of the primer-probe combinations in Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4, where NTC stands for negative, i.e., the pEASY®-Blunt plasmid of the Chikungunya virus gene in the reaction system is replaced with enzyme-free water.

[0019] Figure 2 This is a comparison chart of the fluorescence signal intensity of the RAA reaction of the primer-probe combination in Comparative Example 1, Comparative Example 5, Comparative Example 6 and Comparative Example 7.

[0020] Figure 3 This is the specific detection result of the detection kit in Example 1 for detecting Chikungunya virus.

[0021] Figure 4 This is the sensitivity test result of the detection kit in Example 1 for detecting Chikungunya virus.

[0022] Figure 5 The figure compares the detection results of Chikungunya virus using different primer and probe combinations in the strip-type RAA test. In the figure, 1 represents the primer-probe group F1R1-P2 of Comparative Example 5; 2 represents the primer-probe combination F1R2-P2 of Comparative Example 1; 3 represents the primer-probe combination F2R1-P2 of Comparative Example 7; 4 represents the primer-probe combination F2R2-P2 of Comparative Example 6; 5 represents the negative control; 6 represents the primer-probe combination F1R1-P1 of Comparative Example 3; 7 represents the primer-probe combination F1R2-P1 of Comparative Example 2; 8 represents the primer-probe combination F2R1-P1 of Example 1; 9 represents the primer-probe combination F2R2-P1 of Comparative Example 4; and 10 represents the negative control.

[0023] Figure 6 This figure shows the specific detection of Chikungunya virus by the primer-probe combination in Example 1 in the RAA test strip. In the figure, CHIKV represents Chikungunya virus, JEV represents Japanese encephalitis virus, ADV represents adenovirus, and NC represents a negative control without any virus.

[0024] Figure 7The sensitivity test of the primer-probe combination in Example 1 for Chikungunya virus in the test strip type RAA test. In the figure, numbers 1 to 7 represent samples of pEASY®-Blunt plasmid containing different concentrations of Chikungunya virus gene, where 1 represents a concentration of 11.8×10 5 copies / µL; 2 means the concentration is 11.8×10 4 copies / µL; 3 means the concentration is 11.8×10 3 copies / µL; 4 means the concentration is 11.8×10 2 copies / µL; 5 means the concentration is 11.8×10 1 copies / µL; 6 means the concentration is 11.8×10 0 copies / µL; 7 means the concentration is 11.8×10 0 ×1 / 2 copies / µL; 8 represents the negative control. DETAILED DESCRIPTION

[0025] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0026] Example 1: A Chikungunya virus detection primer and detection kit.

[0027] 1. Design of specific primers for Chikungunya virus Chikungunya virus-specific primers CHIKV-F2 shown in SEQ ID NO.1 and CHIKV-R1 shown in SEQ ID NO.2 were designed and obtained. At the same time, the RAA probe CHIKV-P1 labeled with THF residues shown in SEQ ID NO.3 was obtained. The reporter fluorescent dye labeled with the probe was FAM, and the labeled fluorescence quencher group was BHQ-1.

[0028] SEQ ID NO.1: 5'-CGTGGTCCGTTAGTTGGGCAGTAACTTTAA-3'; SEQ ID NO.2: 5'-CTACCACCCCTACAGGCAGCACAGGAAGAT-3'; SEQ ID NO. 3: 5'-TCGGAGTCTCTATTATTCCAGCACCAGCTC (FAM-dT) Thf (BHQ1-dT) CCTCAAGTTGTTCCAC-3' (C3 Spacer).

[0029] In the sequence listing, the sequence of the RAA probe CHIKV-P1 shown in SEQ ID NO. 3 is recorded as: 5'-TCGGAGTCTCTATTATTCCAGCACCAGCTCNCCTCAAGTTGTTCCAC-3' (C3 Spacer), where (C3 Spacer) indicates that a C3 spacer group is added to the 3' end, and N represents: (FAM-dT)Thf(BHQ1-dT); (FAM-dT)Thf(BHQ1-dT) indicates that FAM is labeled at a specific position of deoxythymidine through a chemical bond to form a fluorescently labeled nucleotide; (BHQ1-dT) indicates that BHQ1 is labeled on another deoxythymidine to quench the fluorescence signal of FAM; Thf represents a spacer group used to connect (FAM-dT) and (BHQ1-dT).

[0030] The primer-probe combination of Example 1 was named F2R1-P1.

[0031] 2. Chikungunya virus detection kit The Chikungunya virus detection kit includes the above-mentioned primer probe combination F2R1-P1, RAA reaction system, A Buffer, B Buffer, positive control and negative control.

[0032] The fluorescent RAA reaction system includes recombinase, recombinase cofactor, single-strand binding protein, nuclease, lyoprotectant, dNTP, creatine kinase and ATP; A buffer is 10% PEG35000 by mass; B buffer is 280mM magnesium acetate; the positive control is the pEASY®-Blunt plasmid containing the chikungunya virus gene; and the negative control is enzyme-free water.

[0033] The RAA reaction system used in the present invention is: a fluorescent nucleic acid amplification reagent (RAA method) without the positive control and the negative control. The fluorescent nucleic acid amplification reagent (RAA method) was purchased from Hangzhou Zhongce Biotechnology Co., Ltd., product number: S002ZC.

[0034] Comparative Example 1: A primer and kit for detecting Chikungunya virus.

[0035] Chikungunya virus-specific primers CHIKV-F1 shown in SEQ ID NO.4 and CHIKV-R2 shown in SEQ ID NO.5 were designed and obtained, and the RAA probe CHIKV-P2 shown in SEQ ID NO.6 was also obtained. The probe-labeled reporter fluorescent dye was FAM, and the labeled fluorescence quencher group was BHQ-1.

[0036] SEQ ID NO.4: 5'-GGTACTCCCCCACGACGTGGTCCGTTAGTT-3'; SEQ ID NO.5: 5'-AGGCAGCACAGGAAGATGTCCAGGTCGAAA-3'; SEQ ID NO. 6: 5'-CCTCTCGGAGTCCTATTATTCCAGCACCAGC(FAM-dT)Thf(BHQ1-dT)TATCCTCAAGTTGTTC-3'.

[0037] In the sequence listing, the sequence of the RAA probe CHIKV-P1 shown in SEQ ID NO. 6 is recorded as: 5'-CCTCTCGGAGTCTCTATTATTCCAGCACCAGCNTATCCTCAAGTTGTTC-'; wherein, N represents (FAM-dT)Thf(BHQ1-dT); (FAM-dT) in (FAM-dT)Thf(BHQ1-dT) indicates that FAM is labeled at a specific position of deoxythymidine through a chemical bond to form a fluorescently labeled nucleotide; (BHQ1-dT) indicates that BHQ1 is labeled on another deoxythymidine to quench the fluorescence signal of FAM; Thf represents a spacer group used to connect (FAM-dT) and (BHQ1-dT).

[0038] The primer-probe combination of Comparative Example 1 was named F1R2-P2.

[0039] 2. Chikungunya virus detection kit The components of the Chikungunya virus detection kit include the above-mentioned primer probe combination F1R2-P2, RAA reaction system, A Buffer, B Buffer, positive control and negative control.

[0040] The fluorescent RAA reaction system includes recombinase, recombinase cofactor, single-strand binding protein, nuclease, lyoprotectant, dNTP, creatine kinase and ATP; A buffer is 10% PEG35000 by mass; B buffer is 280mM magnesium acetate; the positive control is the pEASY®-Blunt plasmid containing the chikungunya virus gene; and the negative control is enzyme-free water.

[0041] Comparative Example 2: A primer and kit for detecting Chikungunya virus.

[0042] 1. Develop RAA primers and probes based on RAA primer design principles The other steps are basically the same as those in Example 1, except that: CHIKV-F1 shown in SEQ ID NO.4 and CHIKV-R2 shown in SEQ ID NO.5 were used as a specific primer pair, and CHIKV-P1 shown in SEQ ID NO.3 was used as a probe.

[0043] The primer-probe combination of Comparative Example 2 was named F1R2-P1.

[0044] 2. Chikungunya virus detection kit The components of the Chikungunya virus detection kit include the above-mentioned primer probe combination F1R2-P1, RAA reaction system, A Buffer, B Buffer, positive control and negative control.

[0045] The fluorescent RAA reaction system includes recombinase, recombinase cofactor, single-strand binding protein, nuclease, lyoprotectant, dNTP, creatine kinase and ATP; A buffer is 10% PEG35000 by mass; B buffer is 280mM magnesium acetate; the positive control is the pEASY®-Blunt plasmid containing the chikungunya virus gene; and the negative control is enzyme-free water.

[0046] Comparative Example 3: A primer and kit for detecting Chikungunya virus.

[0047] The other steps are basically the same as those in Example 1, except that: CHIKV-F1 shown in SEQ ID NO.4 and CHIKV-R1 shown in SEQ ID NO.2 were used as a specific primer pair, and CHIKV-P1 shown in SEQ ID NO.3 was used as a probe.

[0048] The primer-probe combination of Comparative Example 3 was named F1R1-P1.

[0049] 2. Chikungunya virus detection kit The components of the Chikungunya virus detection kit include the above-mentioned primer probe combination F1R1-P1, RAA reaction system, A Buffer, B Buffer, positive control and negative control.

[0050] The fluorescent RAA reaction system includes recombinase, recombinase cofactor, single-strand binding protein, nuclease, lyoprotectant, dNTP, creatine kinase and ATP; A buffer is 10% PEG35000 by mass; B buffer is 280mM magnesium acetate; the positive control is the pEASY®-Blunt plasmid containing the chikungunya virus gene; and the negative control is enzyme-free water.

[0051] Comparative Example 4: A primer and kit for detecting Chikungunya virus.

[0052] The other steps are basically the same as those in Example 1, except that: CHIKV-F2 shown in SEQ ID NO.1 and CHIKV-R2 shown in SEQ ID NO.5 were used as a specific primer pair, and CHIKV-P1 shown in SEQ ID NO.3 was used as a probe.

[0053] The primer-probe combination of Comparative Example 4 was named F2R2-P1.

[0054] 2. Chikungunya virus detection kit The components of the Chikungunya virus detection kit include the above-mentioned primer probe combination F2R2-P1, RAA reaction system, A Buffer, B Buffer, positive control and negative control.

[0055] The fluorescent RAA reaction system includes recombinase, recombinase cofactor, single-strand binding protein, nuclease, lyoprotectant, dNTP, creatine kinase and ATP; A buffer is 10% PEG35000 by mass; B buffer is 280mM magnesium acetate; the positive control is the pEASY®-Blunt plasmid containing the chikungunya virus gene; and the negative control is enzyme-free water.

[0056] Comparative Example 5: A primer and kit for detecting Chikungunya virus.

[0057] The other steps are basically the same as those in Example 1, except that: CHIKV-F1 shown in SEQ ID NO.4 and CHIKV-R1 shown in SEQ ID NO.2 were used as a specific primer pair, and CHIKV-P2 shown in SEQ ID NO.6 was used as a probe.

[0058] The primer-probe combination of Comparative Example 5 was named F1R1-P2.

[0059] 2. Chikungunya virus detection kit The components of the Chikungunya virus detection kit include the above-mentioned primer probe combination F1R1-P2, RAA reaction system, A Buffer, B Buffer, positive control and negative control.

[0060] The fluorescent RAA reaction system includes recombinase, recombinase cofactor, single-strand binding protein, nuclease, lyoprotectant, dNTP, creatine kinase and ATP; A buffer is 10% PEG35000 by mass; B buffer is 280mM magnesium acetate; the positive control is the pEASY®-Blunt plasmid containing the chikungunya virus gene; and the negative control is enzyme-free water.

[0061] Comparative Example 6: A primer and kit for detecting Chikungunya virus.

[0062] The other steps are basically the same as those in Example 1, except that: CHIKV-F2 shown in SEQ ID NO.1 and CHIKV-R2 shown in SEQ ID NO.5 were used as a specific primer pair, and CHIKV-P2 shown in SEQ ID NO.6 was used as a probe.

[0063] The primer-probe combination of Comparative Example 6 was named F2R2-P2.

[0064] 2. Chikungunya virus detection kit The components of the Chikungunya virus detection kit include the above-mentioned primer probe combination F2R2-P2, RAA reaction system, A Buffer, B Buffer, positive control and negative control.

[0065] The fluorescent RAA reaction system includes recombinase, recombinase cofactor, single-strand binding protein, nuclease, lyoprotectant, dNTP, creatine kinase and ATP; A buffer is 10% PEG35000 by mass; B buffer is 280mM magnesium acetate; the positive control is the pEASY®-Blunt plasmid containing the chikungunya virus gene; and the negative control is enzyme-free water.

[0066] Comparative Example 7: A primer and kit for detecting Chikungunya virus.

[0067] The other steps are basically the same as those in Example 1, except that: CHIKV-F2 shown in SEQ ID NO.1 and CHIKV-R1 shown in SEQ ID NO.2 were used as a specific primer pair, and CHIKV-P2 shown in SEQ ID NO.6 was used as a probe.

[0068] The primer-probe combination of Comparative Example 7 was named F2R1-P2.

[0069] 2. Chikungunya virus detection kit The components of the Chikungunya virus detection kit include the above-mentioned primer probe combination F2R1-P2, RAA reaction system, A Buffer, B Buffer, positive control and negative control.

[0070] The fluorescent RAA reaction system includes recombinase, recombinase cofactor, single-strand binding protein, nuclease, lyoprotectant, dNTP, creatine kinase and ATP; A buffer is 10% PEG35000 by mass; B buffer is 280mM magnesium acetate; the positive control is the pEASY®-Blunt plasmid containing the chikungunya virus gene; and the negative control is enzyme-free water.

[0071] 1. The kits of Example 1 and Comparative Examples 1 to 7 were used to perform real-time fluorescence RAA reaction detection on Chikungunya virus. The detection method comprises the following steps: To the reaction tube containing the reaction system, add 25.0 µL of A Buffer, 2.0 µL of 10 µM upstream primer, 2.0 µL of 10 µM downstream primer, 0.6 µL of 10 µM probe, 2.0 µL of positive template, and 15.9 µL of enzyme-free water. Then, add B Buffer to the tube cap, cap the tube, centrifuge, mix by inverting, centrifuge for 10 seconds, and immediately detect. React at 39°C for 15 minutes on the instrument RAA-F1620, and collect FAM fluorescence. The positive template is the pEASY®-Blunt plasmid containing the Chikungunya virus gene, with a copy number of 11.8×10 5 copies / µL.

[0072] The results are as follows Figures 1 and 2 As shown, the fluorescence signal of the combination using the RAA probe CHIKV-P1 shown in SEQ ID NO. 3 is significantly higher than that using the CHIKV-P2 probe shown in SEQ ID NO. 6. Comparison of different primer-probe combinations shows that the fluorescence signal intensity using the primer-probe combination F2R1-P1 in Example 1 of the present invention is significantly higher than that of Comparative Examples 1 to 7.

[0073] Specificity Detection The detection kit in Example 1 was used to detect different viruses, and cDNAs of Chikungunya virus (CHIKV), Japanese encephalitis virus (JEV), dengue virus (DENV), and adenovirus (ADV) were selected for real-time fluorescence RAA detection. Specificity was determined by analyzing the differences in fluorescence signal responses of different samples.

[0074] Detection Procedure: To the reaction tube, add 25.0 µL of A Buffer, 2.0 µL of 10 μM CHIKV-F2, 2.0 µL of 10 μM CHIKV-R1, 0.6 µL of 10 μM CHIKV-P1, 2.0 µL of the positive template, and 15.9 µL of enzyme-free water. Add B Buffer to the cap of the tube, cap the tube, and centrifuge for 10 seconds, mixing by inversion. Immediately test the tube at 39°C in an RAA-F1620 instrument for 15 minutes, and collect FAM fluorescence. The positive template is the pEASY®-Blunt plasmid containing the chikungunya virus gene, or cDNA from Japanese encephalitis virus, dengue virus, or adenovirus.

[0075] The results are as follows Figure 3 As shown, JEV, DENV and ADV are all close to the NTC baseline, and there is no significant difference in fluorescence intensity, while Chikungunya virus: CHIKV produces a high-intensity fluorescence signal during detection, which is significantly higher than the baseline level. The detection kit in Example 1 has a high specificity for detecting Chikungunya virus.

[0076] 3. Sensitivity Testing Detection steps: Add 25.0 µL of A Buffer, 2.0 µL of 10 μM CHIKV-F2, 2.0 µL of 10 μM CHIKV-R1, 0.6 µL of 10 μM CHIKV-P1, 2.0 µL of positive template, and 15.9 µL of enzyme-free water to the reaction tube in sequence. Then add B Buffer to the tube cap, close the tube cap, and centrifuge. Mix by inverting and centrifuging for 10 seconds. Detect immediately. React at 39°C on the RAA-F1620 instrument for 15 minutes, and collect FAM fluorescence.

[0077] Different concentrations of pEASY®-Blunt plasmid containing Chikungunya virus gene were used as positive templates. The concentrations of positive templates were 11.8×10 5 copies / µL; 11.8×10 4 copies / µL; 11.8×10 3 copies / µL; 11.8×10 2 copies / µL; 11.8×10 1 copies / µL; 11.8×10 0 copies / µL; 5.9 copies / µL.

[0078] The results are as follows Figure 4 As shown, when the positive template concentration was 11.8×10 0copies / µL, the fluorescence intensity detected was higher than the NTC baseline level, and the lowest detection concentration was 11.8×10 0 copies / µL, that is, the detection limit is 11.8×10 0 copies / µL.

[0079] The criteria for judging the results of real-time fluorescence RAA reaction are as follows: using a fluorescence detector for detection, taking the obtained cumulative fluorescence value as the signal intensity, and analyzing and judging according to the following criteria.

[0080] Negative judgment standard: the fluorescence amount is less than or equal to twice the fluorescence amount of the negative control. The negative control here refers to the fluorescence value of the negative control group using DEPC water as a template.

[0081] Positive judgment standard: the fluorescence amount is greater than 2 times the fluorescence amount of the negative control. The negative control here refers to the fluorescence value of the negative control group using DEPC water as a template.

[0082] The results are as follows Figure 5 As shown, the primer-probe combination in Example 1 is optimal.

[0083] The specific steps for constructing the pEASY®-Blunt plasmid containing the Chikungunya virus gene are as follows: 1. Extraction of viral nucleic acid CHIKV and other viruses were extracted by preparing reaction systems according to the operating instructions of the nucleic acid extraction kit, and RNA viruses were reverse transcribed. All products were stored at -20°C for future use.

[0084] 2. Construction of recombinant plasmid Target gene identification: 45 CHIKV full genome sequences were downloaded from the NCBI database. Multiple sequence alignment was performed using MAGA software to screen for conserved regions of CHIKV. Based on the RAA primer probe design principles, Primer 5 and Beacon Designer software were used to collaboratively design primer probes for the real-time fluorescence RAA CHIKV detection assay. The selected primer probes and conserved regions were then compared using Nucleotide Blast analysis in the NCBI database to determine their species specificity and type homology to ensure the absence of nonspecific binding with other viruses.

[0085] PCR-F shown in SEQ ID NO.7 and PCR-R shown in SEQ ID NO.8 were designed upstream and downstream of the target fragment region in the CHIKV RAA experiment. The kit 2× TransStart® FastPfu Fly PCR SuperMix to amplify CHKV nucleic acid fragments and insert the target nucleic acid fragments pEASY® -blunt plasmid, used to construct the pEASY®-Blunt plasmid containing the Chikungunya virus gene.

[0086] SEQ ID NO.7: 5'-CTGACCTGCTGCTGTCTATGG-3'; SEQ ID NO. 8: 5'-GCATCGCAATGTGGTGTAGC-3'.

[0087] pEASY ®-Blunt vector was purchased from Beijing Quanshijin Biotechnology Co., Ltd., catalog number: CB111.

[0088] 3. Plasmid transformation Heat shock method was used to transform competent cells with the pEASY®-Blunt plasmid containing the Chikungunya virus gene: (1) Add 50 μL of Trans1-T1 competent cells, flick to mix, and place on ice for 25 min. (2) Place in a 42°C water bath for 30 s, then immediately place on ice for 2 min. (3) Add 250 μL of LB medium equilibrated to room temperature, incubate at 37°C at 200 rpm for 1 h. (4) Mix 8 μL of 500 μM IPTG and 20 mg / ml X-gal, spread evenly on the prepared plate, and place in a 37°C incubator for 30 min. (5) To obtain more clones, centrifuge the bacterial solution in (4) at 1,500 × g for 1 min, discard part of the supernatant, retain 150 μL, flick to suspend the cells, wait for IPTG and X-gal to be absorbed, take all the bacterial solution and spread evenly on the plate, incubate at 200 rpm at 37°C in an incubator overnight, and amplify the product. (6) Pick a white single colony and incubate it in the resistant bacterial solution for 12 h.

[0089] 4. Plasmid Extraction The DNA was extracted according to the instructions of the AxyGEN plasmid DNA mini kit.

[0090] 5. Sequencing purchased from Beijing Quanshijin Biotechnology Co., Ltd. pEASY The M13F and M13R fragments included with the ®-Blunt SimpleCloning Kit were sent to Shanghai Sangon Biotech (Shanghai) Co., Ltd. for bidirectional sequencing. Sequencing results were used to verify the successful construction of the recombinant plasmid by comparing it to the NCBI database. Sequencing results in this experiment confirmed the successful construction of the recombinant plasmid.

[0091] 6. Calculation of recombinant plasmid copy number The copy number of the successfully constructed plasmid was calculated according to the formula.

[0092] The formula is: copy number (copies / μL) = (6.02×10 23 × concentration (ng / μL) × 10 -9 Calculate the concentration of the recombinant plasmid standard using the above formula (copies / μL) / (660 × DNA length). Store the extracted plasmid standard at -20°C.

[0093] Example 2: Chikungunya virus test strip type RAA detection was performed using the primer probe combination in Example 1.

[0094] 1. Primers and probes The base sequences used in the fluorescence detection method and the test strip detection method are consistent. The primers used in the test strip detection of this embodiment have modified groups added to their sequences in order to react with the components in the test strip.

[0095] Development and preparation of primers: Common PCR primers were designed upstream and downstream of the target fragment region of CHIKV RAA experiment amplification. PCR primers were used to amplify CHKV nucleic acid fragments. The products could be directly cloned into pEASY ®-Blunt vector, and the plasmid was extracted after pure culture of Escherichia coli engineered bacteria as a positive control. The primers included CHIKV-F2 shown in SEQ ID NO.1, CHIKV-R1-nfo shown in SEQ ID NO.9, and CHIKV-P1-nfo shown in SEQ ID NO.10. All primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0096] SEQ ID NO. 9: 5'Biotin-CTACCACCCCTACAGGCAGCACAGGAAGAT-3'.

[0097] In the sequence listing, the sequence of CHIKV-R1-nfo shown in SEQ ID NO. 9 is recorded as: 5'-NCTACCACCCCTACAGGCAGCACAGGAAGAT-3', wherein N represents a 5'-linked Biotin tag.

[0098] CHIKV-P1-nfo: 5`6-FAM-TCGGAGTCTCTATTATTCCAGCACCAGCTCT(idSp)TCCTCAAGTTGTTCCAC-3' (C3 spacer). (C3 Spacer) indicates that a C3 spacer group is added to the 3' end.

[0099] In the sequence listing, the CHIKV-P1-nfo shown in SEQ ID NO. 10 is recorded as: 6-NTCGGAGTCTCTATTATTCCAGCACCAGCTCTNTCCTCAAGTTGTTCCAC-3', wherein the first N indicates that the 6-FAM label is connected to the 5' end, and the second N indicates that the idSp group is connected at the position.

[0100] 2. Test kit The kit includes the RAA reaction system, A Buffer, B Buffer, positive controls, and negative controls. The RAA reaction system contains recombinase, single-strand binding protein, strand-displacing DNA polymerase, and lyophilized exonuclease powder; A Buffer is 10% PEG 35000; B Buffer is magnesium acetate solution; the positive control is the pEASY®-Blunt plasmid containing the chikungunya virus gene, and the negative control is enzyme-free water.

[0101] The disposable nucleic acid test strips used in the test strip detection process were purchased from Hangzhou Zhongce Biotechnology Co., Ltd., product number: R103ZC.

[0102] The CHIKV test strip type RAA-nfo reaction system is shown in Table 1.

[0103] Table 1 CHIKV RAA-nfo reaction system

[0104] 3. Specificity detection In order to study the specificity of the RAA reaction system, cDNAs of Chikungunya virus (CHIKV), Japanese encephalitis (JEV), dengue virus (DENV), and adenovirus (ADV) were selected for strip-type RAA detection.

[0105] The results are as follows Figure 6 As shown, the primer-probe combination in Example 1 of the present invention is specific to Chikungunya virus in the test strip type RAA detection.

[0106] 4. Sensitivity testing The obtained pEASY®-Blunt plasmid containing the Chikungunya virus gene was used as a template for dilution of the concentration system, and 11.8×10 5 copies / µL, 11.8×10 4 copies / µL, 11.8×10 3 copies / µL, 11.8×10 2 copies / µL, 11.8×10 1 copies / µL, 11.8×100 copies / µL and 11.8×10 0 The sensitivity test of RAA-nfo combined with disposable paper strip method was performed for the plasmid with a concentration of ×1 / 2 copies / µL. The reaction system was first added according to the RAA-nfo reaction to react. After the reaction was completed, the disposable nucleic acid test paper was used for detection within 15 minutes.

[0107] The results are as follows Figure 7 As shown in the figure, the detection limit is 11.8 copies / µL, which can be observed by naked eye.

[0108] The present invention developed a real-time fluorescence RAA CHIKV detection kit. Using this kit, CHIKV can be rapidly detected within 15 minutes, with a sensitivity of 11.8 copies / μL and no amplification for JEV, DENV, and ADV, demonstrating good specificity. Studies using RAA-nfo combined with disposable nucleic acid test strips have also shown a sensitivity of 11.8 copies / μL, with no amplification for JEV and ADV.

[0109] Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts become known.

[0110] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. A primer set for detecting Chikungunya virus, characterized in that: It includes CHIKV-F2 shown in SEQ ID NO.1, CHIKV-R1 shown in SEQ ID NO.2 and RAA probe CHIKV-P1 shown in SEQ ID NO.

3.

2. A kit, characterized in that The kit comprises the Chikungunya virus detection primer set according to claim 1.

3. The kit according to claim 2, wherein The kit further comprises an RAA reaction system, a buffer, a positive control and a negative control.

4. The kit according to claim 3, wherein The reaction system includes recombinase, recombinase auxiliary factor, single-strand binding protein, nuclease, lyophilization protectant, dNTP, creatine kinase and ATP.

5. The kit according to claim 3, characterized in that The buffer solution includes 10% by mass of PEG35000 and 280 mM magnesium acetate solution.

6. The kit according to claim 5, characterized in that The positive control was the pEASY®-Blunt plasmid containing the Chikungunya virus gene.

7. The kit according to claim 6, characterized in that The negative control was enzyme-free water.

8. The kit according to claim 7, characterized in that The volumes of the components in the kit are as follows: 15.9 μL of enzyme-free water, 25.0 μL of 10% PEG35000, 2.0 μL of primer CHIKV-F2, 2.0 μL of primer CHIKV-R1, 0.6 μL of probe CHIKV-P1, 2.0 μL of plasmid-CHIKV, and 2.5 μL of 280 mM magnesium acetate solution.

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