A chikungunya virus genome fragment armored RNA reference, its preparation method and application

Abstract

This invention discloses a chikungunya virus genome fragment armored RNA reference, its preparation method, and its application. The preparation process involves the following steps: (1) constructing the prokaryotic expression backbone vector pACYCAR; (2) cloning the CKV genome fragment CKA (nt17-2276); (3) constructing the prokaryotic expression vector pACYCAR-CKA; (4) inducing the expression of armored RNA; and (5) purifying the armored RNA. This invention solves the technical problems of high biosafety risk, poor stability, and insufficient gene fragment coverage in existing CKV nucleic acid detection references. It fills the gap in the lack of high-quality references for CKV detection.

Description

Technical Field

[0001] This invention relates to the field of virus detection technology, specifically to a chikungunya virus genome fragment armored RNA reference, its preparation method, and its application. Background Technology

[0002] Chikungunya virus (CKV) belongs to the genus Alphavirus of the family Togaviridae. It was first discovered in Tanzania, Africa in 1952. The viral genome is a single-stranded positive-sense RNA, approximately 11.8 kb in length. Based on genomic evolutionary analysis, it is currently classified into three genotypes: West African, East Central and Southern Africa, and Asian. CKV infection causes chikungunya fever, an acute infectious disease characterized by fever, rash, and joint pain. Chikungunya fever is primarily prevalent in Africa, but has since spread to Southeast Asia, South Asia, Indian Ocean islands, and the Americas. Local transmission of chikungunya fever has been reported in 119 countries and regions worldwide. In 2024, more than 460,000 suspected cases of chikungunya fever were reported globally.

[0003] The etiological detection methods for chikungunya fever include viral nucleic acid detection, IgM antibody detection, neutralizing antibody detection, and virus isolation, with viral nucleic acid detection currently being the primary method. Viral nucleic acid detection directly reflects the viremia phase, covering the window period from viral infection to antibody emergence. Its results are crucial for determining the infectivity of suspected infected individuals and close contacts, and for effectively controlling further viral transmission. CKV nucleic acid detection includes detecting CKV-specific genomic fragments and determining full-length or partial genomic sequences. In vitro amplification of viral genomic fragments is the most sensitive, rapid, and widely used laboratory diagnostic method. Other methods, such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) and transcription-mediated amplification (TMA), can also be used as molecular diagnostic methods for CKV detection in clinical samples.

[0004] Most laboratories conducting RNA virus nucleic acid testing face the challenge of lacking stable and safe viral genomic RNA references as templates and positive controls. Theoretically, inactivated viral particles, plasmid DNA, recombinant viruses, and in vitro chemically synthesized RNA can serve as sources of positive references for RNA viruses. However, due to biosafety considerations, most laboratories in China do not meet the required biosafety levels, lack the qualifications to conduct live virus-related experiments, lack suitable reference strains, and cannot provide sufficient viral cultures to meet the needs of developing diagnostic reagents and routine testing. Inactivated viral particles and chemically synthesized RNA, as RNA references, suffer from poor stability, are easily degraded, require cryogenic storage and transportation, and cannot be used in large quantities for the production of diagnostic reagents. Plasmid DNA, due to its inherent physicochemical properties, cannot simulate and control the entire process of RNA target extraction from sample RNA to reverse transcription.

[0005] Recombinant viruses or pseudoviruses containing RNA virus genome fragments can be constructed using biotechnology. These viruses have clear genetic backgrounds and are non-infectious, which not only reduces biosafety risks but also allows for large-scale in vitro replication to obtain genome fragments, making it possible to prepare stable, reliable, and low-cost positive references.

[0006] Armored RNA (AR), also known as armored RNA, pseudovirus, or armored RNA, is a technique for preparing recombinant viruses or pseudovirus particles. Its core involves expressing a recombinant viral envelope protein vector in prokaryotes, enabling it to self-assemble into virus-like particles and encapsulate specific RNA fragments within these particles to form pseudoviruses. A common example is Escherichia coli MS2 bacteriophage. Artificially constructed armored RNA is essentially a non-infectious MS2 derivative. Because its specific RNA sequence is packaged into MS2 pseudovirus particles by viral envelope proteins, it effectively resists nuclease interference and can be stored long-term under very simple preservation conditions. Summary of the Invention

[0007] To address the aforementioned technical problems, the present invention aims to provide a chikungunya virus (CKV) genomic fragment armored RNA reference, its preparation method, and its application. This solves the technical problems of existing CKV nucleic acid detection references, such as high biosafety risks, poor stability, and insufficient gene fragment coverage. It fills the gap in the availability of high-quality references for CKV detection.

[0008] To achieve the first objective mentioned above, the present invention provides the following technical solution: a method for preparing a chikungunya virus genome fragment armored RNA reference, characterized by preparation according to the following steps:

[0009] (1) Construction of prokaryotic expression backbone vector pACYCAR: MS2 phage genomic RNA was cultured and extracted. A 1.7kb fragment containing MS2 maturation enzyme, envelope protein gene and packaging recognition signal sequence was amplified by RT-PCR. The fragment was ligated with pACYCDuet-1 plasmid that had been digested with BamHI and HindIII. After transformation into DH5α competent cells, the fragment was screened and identified to obtain the backbone vector pACYCAR.

[0010] (2) Cloning the CKV genome fragment CKA (nt17-2276): Total RNA was extracted from inactivated CKV virus, and the genome fragment CKA (nt17-2276) covering the 5'UTR, non-structural protein nsp1 and part of the nsp2 gene was amplified by RT-PCR. After TA cloning and screening, the subcloned plasmid pUCmT-CKA (5.0 kb) was obtained.

[0011] (3) Construction of prokaryotic expression vector pACYCAR-CKA(nt81-2235): Using seamless cloning technology, CKA(nt81-2235) was amplified using the CKV genome fragment cloned in step (2) as a template and ligated with the linearized pACYCAR vector amplified by reverse PCR. After transformation into Top10 competent cells, the recombinant expression vector pACYCAR-CKA (8.0kb) was obtained.

[0012] (4) Induced expression of armored RNA: The recombinant expression vector from step (3) was transformed into BL21(DE3) host bacteria and induced to express by IPTG to obtain engineered bacteria containing CKV armored RNA;

[0013] (5) Purification of armored RNA: Collect engineered bacterial cells, wash with PBS, sonicate and purify by NaCl salting, PEG6000 precipitation and chloroform extraction to obtain CKV genome fragment CKA (nt81-2235) armored RNA reference.

[0014] In the above protocol: MS2 phage genome extraction was performed using the TAKARA MiniBEST Viral RNA / DNA Extraction Kit Ver 5.0, Code No. 9766.

[0015] In the above scheme: In step (1), the primers for RT-PCR amplification of the MS2 1.7kb fragment are:

[0016] Upstream primer AR-Bam10F: 5'-CGCGGATCCTTTCGGGGTCCTGCTCAACTT-3',

[0017] Downstream primer AR-Hin1789R: 5'-CCCAAGCTTGAGTTGAACTTCTTTGTTGTCTTC-3'.

[0018] The RT-PCR amplification conditions were as follows: 50℃ for 30 min; 94℃ for 2 min; 94℃ for 30 sec, 58℃ for 30 sec, 72℃ for 90 sec, for 30 cycles; 72℃ for 5 min; and the ligation system was incubated overnight in a 16℃ water bath.

[0019] In the above scheme: in step (2), the CKV strain is CHIKV-18001;

[0020] The upstream and downstream primers for RT-PCR amplification are as follows:

[0021] CHIKV-18F: 5'-CACGTAGCCTACCAGTTTCTTA-3',

[0022] CHIKV-2709R: 5'-TCCACTACAATCGGCTTGTTG-3'.

[0023] The amplification conditions were: 50℃ for 30 min; 94℃ for 2 min; 94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 2.5 min, 30 cycles; 72℃ for 10 min.

[0024] In the above scheme: positive clones are screened using the following primers: M13-47: 5'-AGGGTTTTCCCAGTCACG-3', M13-48: 5'-GAGCGGATAACAATTTCACAC-3'.

[0025] In the above scheme: In step (3), the primers for seamless cloning include:

[0026] pAC-AR328F: 5'-GCCACGCGATCGCTGACG-3',

[0027] pAC-AR319R: 5'-ATCCAATTGAGATCTGCCATATG-3',

[0028] acCKA70f: 5'-AGATCTCAATTGGATATCCTGTGTACGTGGAC-3',

[0029] acCKA2200r: 5'-CAGCGATCGCGTGGCAAGACTCCTATGACTGCAATTTTG-3'.

[0030] The seamless cloning reaction was incubated in a metal bath at 50°C for 2 h, and positive clones were screened using DuetUP2 and T7-ter as primers.

[0031] The primer sequences for DuetUP2 and T7-ter are as follows:

[0032] DuetUP2:5'-TTGTACACGGCCGCATAATC-3',

[0033] T7-ter: 5'-TGCTAGTTATTGCTCAGCGG-3'.

[0034] In the above scheme: In step (4), before induction, the engineered bacteria are cultured at a ratio of 1:100, shaken at 37℃ and 200 rpm for 4 h, the final concentration of IPTG is 1 mmol / L, and the induction conditions are overnight culture at 37℃ and 200 rpm.

[0035] In the above scheme: In step (5), the parameters of ultrasonic disruption are: ultrasonic power 15%, 1.5s pulse, 3s interval, total duration 30 min; final concentration of NaCl is 1 mol / L, final concentration of PEG6000 is 10% (w / v), and after overnight in an ice bath, the purified product is obtained by chloroform extraction.

[0036] A method for preparing a chikungunya virus genomic fragment armored RNA reference product is disclosed. The reference product prepared by this invention is a non-infectious pseudovirus particle, posing no biosafety risk, exhibiting excellent stability, and tolerating temperature treatments of 37℃, 25℃, 4℃, and -20℃ for 28 days, as well as treatments with DNase I and RNase A alone and in combination. The gene fragment length is approximately 2.2 kb, covering the CKV 5'UTR, nsp1, and part of the nsp2 gene; the concentration quantified by digital PCR is 7.48 × 10⁻⁶. 4 The sample size is 1 copy / μL and can be used as a positive control, standard, and quality control. It is widely used in scenarios such as R&D and verification of CKV nucleic acid detection reagents, standard curve plotting, and laboratory proficiency assessment. It solves the technical problems of high biosafety risk, poor stability, and insufficient gene fragment coverage of existing CKV nucleic acid detection reference materials.

[0037] The application of the described chikungunya virus genomic fragment armored RNA reference material in the research and validation of DENV nucleic acid detection reagents, or in the plotting of DENV nucleic acid detection standard curves, or in the assessment of the chikungunya virus nucleic acid detection capabilities and interlaboratory quality control of primary laboratories, or in the preparation of chikungunya virus nucleic acid detection kits as a positive control.

[0038] Compared with the prior art, the present invention has the following advantages:

[0039] 1. The prepared armored RNA is a non-infectious pseudovirus particle with no autonomous replication ability, no infectivity, no biosafety risk, and does not require high-level biosafety laboratory operation, making it suitable for widespread use in most laboratories in China.

[0040] 2. The reference material can withstand temperature treatment at 37℃, 25℃, 4℃, and -20℃ for 28 days, and can resist degradation by DNase I and RNase A alone and in combination. It has good stability, is easy to transport, and can be stored for a long time under simple conditions, thus solving the problems of easy degradation and low temperature dependence of traditional reference materials.

[0041] 3. Reasonable gene fragment coverage: The CKV gene fragment of the reference product is a long fragment of 2155 bp, covering the 5'UTR, nsp1 and part of the nsp2 gene, covering the core target region of CKV nucleic acid detection, which can meet the quality control requirements of multi-target detection. It is superior to existing short fragment reference products and is suitable as a template for the development of CKV nucleic acid detection system for this region.

[0042] 4. Accurate quantification and wide range of applications: Absolute quantification by digital PCR clearly shows the reference concentration to be 7.48 × 10⁻⁶. 4 With a capacity of copy / μL, it can be used to prepare standards of different concentration gradients to plot detection curves. It can also be used as a positive control and interlaboratory quality control material, suitable for multiple scenarios such as CKV nucleic acid detection reagent development, clinical testing quality control, and laboratory competency assessment.

[0043] 5. Simple preparation process and scalable production: The backbone vector is constructed based on pACYCDuet-1, and the long CKV fragment is efficiently inserted using seamless cloning technology. The induction expression and purification steps are simple and easy to operate, enabling large-scale preparation to meet the needs of industrial production and daily detection.

[0044] 6. Good simulation: The reference product is a phage-like particle, and its nucleic acid extraction process is highly similar to the nucleic acid extraction process of natural CKV in clinical samples. It can effectively simulate the RNA target to be tested and realize the whole process quality control from sample processing to reverse transcription. Attached Figure Description

[0045] Figure 1 The figure shows the temperature stability test results of the armored RNA of the CKV genome fragment CKA (nt81-2235).

[0046] Figure 2 The image shows the results of nuclease resistance testing of armored RNA from the CKV genome fragment CKA (nt81-2235).

[0047] Figure 3 This is a graph showing the serial dilution results of CKA (nt81-2235) armored RNA.

[0048] Figure 4 For CKA armored RNA (10 -4 (Dilution) Digital PCR quantification graph.

[0049] Figure 5 For CKA armored RNA (10 -4 (Dilution) Negative control digital PCR quantification graph. Detailed Implementation

[0050] The present invention will be further described below with reference to embodiments.

[0051] Example 1

[0052] Construction of a prokaryotic expression backbone vector pACYCAR

[0053] 1. MS2 phage culture

[0054] (1) Resuscitate the host bacteria. Prepare LB broth medium, revive freeze-dried Escherichia coli C-3000 (ATCC15597), and enrich at 37°C for 12 hours; streak the enrichment product onto LB agar plates and incubate at 37°C for 12-16 hours; select single colonies and inoculate with 5 ml of LB broth, and incubate overnight on a shaker at 37°C.

[0055] (2) Inoculate the overnight culture with fresh LB broth at a ratio of 1:100, and culture in a shaker at 37°C for 4-5 hours until the logarithmic growth phase. Inoculate with frozen MS2 phage (ATCC 15597-B1™) and incubate in a static incubator at 37°C overnight.

[0056] (3) Select culture tubes that are clear and transparent after overnight culture, centrifuge to remove cell debris, filter the supernatant through a 0.45μm filter, collect the filtrate and dispense it into microcentrifuge tubes, and store at -80℃ for later use;

[0057] 2. MS2 phage genome extraction

[0058] The procedure for the TAKARA MiniBEST Viral RNA / DNA Extraction Kit (Code No. 9766) is as follows:

[0059] (1) Take 200 μL of the above MS2 phage culture filtrate, add 200 μL of VGB buffer, 20 μL of proteinase K and 1.0 μL of vector RNA, mix thoroughly and incubate in a 56℃ water bath for 10 minutes, add 200 μL of anhydrous ethanol to the lysis buffer and mix thoroughly by pipetting.

[0060] (2) Place the centrifuge column on the collection tube, transfer the solution into the centrifuge column, centrifuge at 12,000 rpm for 2 min, and discard the filtrate.

[0061] (3) Add 500 μL of RWA buffer to the centrifuge column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0062] (4) Add 700 μL of RWB buffer to the centrifuge column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate;

[0063] (5) Repeat step 4;

[0064] (6) Place the centrifuge column on the collection tube and centrifuge at 12,000 rpm for 2 min.

[0065] (7) Place the centrifuge column on a new 1.5 mL ribonuclease-free collection tube, add 50 μL of ribonuclease-free distilled water to the center of the centrifuge column membrane, let it stand at room temperature for 5 min, centrifuge at 12,000 rpm for 2 min to elute RNA, and store at -80℃ for later use.

[0066] 3. RT-PCR amplification of the MS2 (1.7 kb) fragment

[0067] (1) Primers for RT-PCR amplification of the MS2 (1.7 kb) fragment

[0068] Primers designed based on the MS2 phage genome sequence (GenBank accession number: NC_001417) to amplify fragments containing the MS2 maturation enzyme, envelope protein gene, and packaging recognition signal sequence are as follows:

[0069] Upstream primer AR-Bam10F: 5'-CGCGGATCCTTTCGGGGTCCTGCTCAACTT-3',

[0070] Downstream primer AR-Hin1789R: 5'-CCCAAGCTTGAGTTGAACTTCTTTGTTGTCTTC-3'.

[0071] The primers were fitted with BamHI recognition sites (GGATCC) and HindIII recognition sites (AAGCTT) for restriction endonucleases, respectively. The primer oligonucleotides were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0072] (2) RT-PCR amplification

[0073] Following the instructions for the TAKARA PrimeScript™ One Step RT-PCR Kit Ver.2 (Dye Plus) Code No. RR057A, prepare the following 50 μL reaction mixture:

[0074] 1) 2×1 Step Buffer (Dye Plus) 25 μL;

[0075] 2)AR-Bam10F (10 μM) 2 μL;

[0076] 3) AR-Hin1789R (10 μM) 2 μL;

[0077] 4) Template RNA 2 μL;

[0078] 5) PrimeScript 1Step Enzyme Mix 2 μL;

[0079] 6)RNase Free dH2O up to 50 μL.

[0080] The amplification conditions were: 50℃ for 30 min; 94℃ for 2 min; 94℃ for 30 sec, 58℃ for 30 sec, 72℃ for 90 sec, 30 cycles; 72℃ for 5 min.

[0081] After the amplification reaction was completed, 1% agarose gel electrophoresis was performed, and a 1.7 kb fragment was observed as a positive result.

[0082] (3) Gel recovery of amplification products

[0083] The amplified DNA fragments were purified and recovered using the SanPrep column-gel extraction kit (product catalog number: B518131) from Sangon Biotech (Shanghai) Co., Ltd., following the instructions:

[0084] 1) Separate the target fragment from other impurities using 1% agarose gel electrophoresis, cut off the agarose gel block containing the target fragment, place it in a 1.5 mL centrifuge tube, and weigh it;

[0085] 2) Add Buffer B2 at a ratio of 1:3 (i.e., 100 mg of gel to 300 μL of liquid), and incubate in a 50°C water bath for 5-10 minutes until the gel block dissolves;

[0086] 3) Transfer the solution to the adsorption column, centrifuge at 8000g for 30 seconds, and discard the liquid; add 300 μL of Buffer B2 to the adsorption column, centrifuge at 9000g for 30 seconds, and discard the liquid; add 500 μL of washing buffer, centrifuge at 9000g for 30 seconds, and discard the liquid; add another 500 μL of washing buffer to wash once; centrifuge at 9000g for 1 min.

[0087] 4) Place the adsorption column in a new centrifuge tube, add 35 μL of eluent to the center of the adsorption membrane, let stand at room temperature for 1-2 min, and centrifuge at 9000g for 1 min. Take a small amount of eluent for electrophoresis to observe the purity and concentration of the fragments, and store it in a -20℃ refrigerator for later use.

[0088] 4. Preparation of plasmid pACYCDuet-1 vector DNA (4.0 kb)

[0089] The pACYCDuet-1 plasmid (4.0 kb) is designed for the co-expression of two target genes. This vector encodes two multiple cloning sites (MCS-1, MCS-2) and carries the P15A replicon, the lacI gene, and the chloramphenicol resistance gene. Genes inserted into MCS-1 can be sequenced using ACYCDuetUP1 and DuetDOWN1 primers, and genes inserted into MCS-2 can be sequenced and identified using the DuetUP2 primer with the T7 terminator. The pACYCDuet-1 plasmid (4.0 kb) was purchased from Chongqing Gram Biotechnology Co., Ltd.

[0090] The pACYCDuet-1 vector DNA (4.0 kb) was extracted using a kit from Sangon Biotech (Shanghai) Co., Ltd. (SanPrep Column-Based Plasmid DNA Mini-Extraction Kit, Product Catalog No.: B518191). The procedure is as follows:

[0091] (1) Inoculate the target strain into LB medium containing chloramphenicol and culture it in a shaker at 37°C for 12-16 h with full shaking;

[0092] (2) Take 5 mL of bacterial culture, centrifuge at 8,000 g for 2 min at room temperature to collect all the bacterial cells into a 1.5 mL microcentrifuge tube, and pour out or aspirate the culture medium.

[0093] (3) Add 250 μL of Buffer P1 to the bacterial pellet and aspirate or shake until the bacterial cells are completely resuspended;

[0094] (4) Add 250 μL Buffer P2, immediately and gently invert the centrifuge tube 5-10 times to mix, and let stand at room temperature for 2-4 min;

[0095] (5) Add 350 μL Buffer P3 and immediately gently invert the centrifuge tube 5-10 times to mix thoroughly;

[0096] (6) Centrifuge at the maximum speed (≥ 12,000g) for 7 min, carefully transfer all the supernatant into the adsorption column, and centrifuge at 9,000g for 30 sec. Discard the liquid in the collection tube and place the adsorption column into the same collection tube.

[0097] (7) Add 500 μL of washing buffer to the adsorption column and centrifuge at 9,000g for 30 sec. Discard the liquid in the collection tube and place the adsorption column into the same collection tube.

[0098] (8) Repeat step 7 once.

[0099] (9) Place the empty adsorption column and collection tube into a centrifuge and centrifuge at 9,000g for 1 min.

[0100] (10) Add 50 μL of elution buffer to the center of the adsorption membrane, let stand at room temperature for 1-2 min, and centrifuge at 9,000g for 1 min. Store the obtained plasmid DNA solution at -20℃ or use it for subsequent experiments.

[0101] 5. Construction of the prokaryotic expression backbone vector pACYCAR (5.7 kb)

[0102] (1) Restriction enzyme digestion of pACYCDuet-1 plasmid DNA (4.0 kb) and insert MS2 fragment (1.7 kb). The pACYCDuet-1 plasmid double digestion system (10×buffer K 20 μL, BamHI 5 μL, Hind III 5 μL, plasmid DNA 60 μL, add ddH2O to a total volume of 200 μL) was mixed and placed in a water bath at 37℃ overnight. The approximately 4 kb vector fragment was recovered by agarose gel electrophoresis for later use. The insert MS2 fragment double digestion system (10×buffer K 4 μL, BamHI 2 μL, Hind III 2 μL, recover 20 μL of amplified fragment DNA, add ddH2O to a total volume of 40 μL) was mixed and placed in a water bath at 37℃ overnight. The approximately 1.7 kb insert fragment was recovered by agarose gel electrophoresis for later use.

[0103] (2) Double digestion of pACYCDuet-1 plasmid DNA (4.0 kb) and insertion of MS2 fragment (1.7 kb) for ligation. Preparation of ligation reaction system (20 μL):

[0104] 1) 2 μL of 10×T4 DNA ligase buffer;

[0105] 2) Double enzyme digestion and recovery of vector pACYCDuet-15 μL;

[0106] 3) Double enzyme digestion to recover 5 μL of the inserted MS2 fragment;

[0107] 4) 1 μL of T4 DNA ligase;

[0108] 5) Add ddH2O to 20 μL.

[0109] After thorough mixing, the mixture was incubated overnight in a 16°C water bath. The ligation product was transformed into competent host cells DH5α, plated on LB agar plates containing chloramphenicol, and incubated overnight at 37°C.

[0110] (3) Positive clone screening: Select obvious single colonies on the plate, inoculate with 5 mL of chloramphenicol-containing LB medium, and incubate overnight at 37°C and 200 rpm. Collect 100 μL of overnight culture medium at 8,000 g for 1 min and discard the supernatant. Resuspend the bacterial cells in 100 μL of ddH2O, place in a 100°C metal bath for 5 min, then at 12,000 g for 2 min, and use the supernatant as a DNA template for clone screening. Using primers ACYCDuetUP1 (5'-GATTATGCGGCCGTGTACAA-3') and DuetDOWN1 (5'-GATCTCGACGCTCTCCCT-3'), follow the TAKRA Premix Taq kit. TM (Ex Taq TM Version 2.0 plus dye), Code No. RR902A. Prepare a 25 μL reaction mixture according to the instructions.

[0111] 1) Premix Taq (Ex Taq Version 2.0 plus dye) 12.5 μL;

[0112] 2)ACYCDuetUP1 (10μM) 1 μL;

[0113] 3) DuetDOWN1 (10μM) 1 μL;

[0114] 4) 2 μL of template;

[0115] 5) Add sterile water to 25 μL.

[0116] The amplification reaction conditions were: 95℃ for 4 min; 95℃ for 30 sec, 52℃ for 30 sec, 72℃ for 90 sec, 30 cycles; 72℃ for 5 min). After the reaction was completed, the product was subjected to 1% agarose gel electrophoresis. A fragment of approximately 1.7 kb in the amplified product was observed to be a suspected positive. Plasmid DNA was extracted from the suspected positive clone.

[0117] (4) Sequencing Verification: The small amount of plasmid DNA prepared above was sent to the Chongqing Sequencing Department of Sangon Biotech (Shanghai) Co., Ltd., and commercial SANGER sequencing was performed using ACYCDuetUP1 primers and DuetDOWN1 primers, respectively. The recombinant plasmid with correct sequencing was named pACYCAR (5.7 kb). The plasmid pACYCAR was transformed into DH5α host bacteria to prepare 25% glycerol bacteria, which were stored at -80℃ for later use.

[0118] The inserted fragment sequence is as follows:

[0119] MS2-1.7K_11-1789

[0120]

[0121] 6. Cloning of the CKV genome fragment CKA (nt17-2276)

[0122] The strain information is as follows: CKV isolate CHIKV-18001 was obtained from the Microbiology Laboratory of Chongqing Center for Disease Control and Prevention. For biosafety reasons, the introduced strain was an inactivated virus culture medium (with added viral RNA extract).

[0123] (1) Extraction of total viral RNA

[0124] The following are the operating steps using the kit (QIAGEN RNeasy Mini Kit, QIAGEN, Cat# 74104):

[0125] 1) Mix 600 μL of viral culture medium containing Buffer RLT by vortexing, add an equal volume of 70% ethanol, and mix with a pipette.

[0126] 2) Transfer 700 μL of the mixture to an RNeasy spin column placed on a 2 mL collection tube, gently cap the centrifuge tube, centrifuge for 15 seconds at ≥8000g, and discard the outflowing liquid; repeat this step, add the remaining mixture to the centrifuge column, centrifuge, and discard the liquid;

[0127] 3) Add 700 μL of Buffer RW1 to the RNeasy spin column, gently cap the centrifuge tube, centrifuge for 15 seconds at ≥8000g, discard the overflow liquid; reuse the collection tube.

[0128] 4) Add 500 μL of Buffer RPE to the RNeasy spin column, gently cap the centrifuge tube, centrifuge for 15 seconds at ≥8000g, discard the overflow liquid; reuse the collection tube.

[0129] 5) Add 500 μL of Buffer RPE to the RNeasy spin column, gently cap the centrifuge tube, centrifuge for 2 min at ≥8000g, and discard the outflowing liquid;

[0130] 6) Carefully transfer the RNeasy spin column to a new 1.5 mL collection tube, add 50 μL of RNase-free water to the center of the centrifuge column membrane, gently cap the centrifuge tube, centrifuge for 1 min at ≥8000g, and collect the elution liquid, which is the total viral RNA.

[0131] (2) RT-PCR amplification of the CKV genome fragment CKA (nt17-2276)

[0132] Based on the CKV reference strain S27 genome sequence information (GenBank accession No. NC_004162) in the NCBI database, the following subcloning primers were designed:

[0133] CHIKV-18F: 5'-CACGTAGCCTACCAGTTTCTTA-3',

[0134] CHIKV-2709R: 5'-TCCACTACAATCGGCTTGTTG-3'.

[0135] The primer oligonucleotides cover the 5' untranslated region (5'UTR), non-structural protein nsp1, and part of the non-structural protein nsp2 genes of the S27 strain. The primer oligonucleotides were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0136] Amplification of the chikungunya virus genomic fragment CKA (nt17-2276). Using genomic RNA extracted from the CKV isolate CHIKV-18001 as a template, and selecting CHIKV-18F / CHIKV-2709R as upstream and downstream primers respectively, a 50 μL reaction volume was prepared according to the instructions of the TAKARA PrimeScript™ One Step RT-PCR Kit Ver.2 (Dye Plus) CodeNo.RR057A.

[0137] 1) 2×1 Step Buffer (Dye Plus) 25 μL;

[0138] 2) CHIKV-18F (10μM) 2 μL;

[0139] 3) CHIKV-2709R (10 μM) 2 μL;

[0140] 4) Template RNA 2 μL;

[0141] 5) PrimeScript 1Step Enzyme Mix 2 μL;

[0142] 6)RNase Free dH2O up to 50 μL.

[0143] The amplification conditions were: 50℃ for 30 min; 94℃ for 2 min; 94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 2.5 min, 30 cycles; 72℃ for 10 min.

[0144] After the amplification reaction was completed, 1% agarose gel electrophoresis was performed. Amplification products with a length of approximately 2000-3000 bp were considered potentially positive. The amplification products were then recovered and purified by agarose gel electrophoresis.

[0145] (3) CKV genome fragment CKA (nt17-2276)TA clone.

[0146] Prepare the following ligation system (10 μL) according to the instructions of Sangon Biotech (Shanghai) Co., Ltd.'s T vector PCR product cloning kit (product catalog number: B522213):

[0147] 1) pUCm-T Vector (50 ng / μL) 1μL;

[0148] 2)10×Ligation Buffer 1 μL;

[0149] 3) 50% PEG4000 1 μL;

[0150] 4) T4 DNA Ligase 1 μL;

[0151] 5) Insert fragment (recovered amplification product) 4 μL;

[0152] 6) Add sterilized ddH2O to a final volume of 10 μL.

[0153] Mix well and incubate overnight at 16°C. Transform DH5α competent cells with the ligation product, plate on LB agar plates containing ampicillin, and incubate overnight at 37°C.

[0154] (4) Screening of positive clones of CKV genome fragment CKA (nt17-2276)

[0155] Select a single, clearly visible colony from the plate, inoculate it with 5 mL of LB broth containing ampicillin, and incubate overnight at 37°C with a shaker at 200 rpm. Collect 100 μL of culture medium at 8,000 g for 1 min to collect the bacterial cells, discarding the supernatant. Resuspend the bacterial cells in 100 μL of ddH2O, place in a 100°C metal bath for 5 min, then at 8,000 g for 2 min. Use the supernatant as a DNA template for cloning screening. Possibly positive clones are screened using the following primers (M13-47: 5'-AGGGTTTTCCCAGTCACG-3', M13-48: 5'-GAGCGGATAACAATTTCACAC-3'). Prepare a 25 μL reaction mixture using the TAKARA Premix Taq™ (Ex Taq™ Version 2.0 plus dye) kit, Code No. RR902A.

[0156] 1) Premix Taq (Ex Taq Version 2.0 plus dye) 12.5 μL;

[0157] 2) Template DNA 2 μL;

[0158] 3) M13-47 (10 μM) 1 μL;

[0159] 4) M13-48 (10 μM) 1 μL;

[0160] 5) Add sterile water to 25 μL.

[0161] Amplification parameters: 94℃ for 4 min; 94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 2.5 min, 30 cycles; 72℃ for 5 min.

[0162] Amplification products with a fragment close to 2000-3000 bp are considered suspected positive.

[0163] A small amount of plasmid DNA was prepared and sent to the Chongqing Sequencing Department of Sangon Biotech (Shanghai) Co., Ltd. Commercial SANGER sequencing was performed using M13-47 and M13-48 primers. The correctly sequenced plasmid was named pUCmT-CKA (5.0 kb). The pUCmT-CKA (5.0 kb) plasmid was transformed into DH5α host bacteria to prepare 25% glycerol-containing bacteria, which were then stored at -80℃ for later use.

[0164] Sequencing results of the cloned CKV genome fragment CKA (nt17-2276) showed that the full-length cloned CKA fragment was 2260 bp, corresponding to the CKV reference strain S27 genome sequence (GenBank accession No. NC_004162) nt17-2276.

[0165] The sequence of the cloned CKV genome fragment CKA (nt17-2276) is as follows:

[0166] >CKA

[0167]

[0168] 7. Construction of the prokaryotic expression vector pACYCAR-CKA (nt81-2235)

[0169] Using an in-fusion cloning strategy, the vector pACYCAR (5.7 kb) and the inserted CKV genomic fragment CKA (nt81-2235) were amplified separately. Utilizing the homologous sequences of 15-25 nt at the ends of the vector and the inserted fragment, the mixture was reacted at 50°C for 5-60 minutes with T5 exonuclease, DNA polymerase, and DNA ligase, and then directly transformed into competent cells.

[0170] (1) Primer design

[0171] Primers were designed using the TAKARA online tool based on the vector pACYCAR (5.7 kb) and the inserted CKV genome fragment CKA (nt81-2235) sequence:

[0172] pAC-AR328F: 5'-GCCACGCGATCGCTGACG-3',

[0173] pAC-AR319R:5'-ATCCAATTGAGATCTGCCATATG-3',

[0174] acCKA70f: 5'-AGATCTCAATTGGATATCCTGTGTACGTGGAC-3',

[0175] acCKA2200r: 5'-CAGCGATCGCGTGGCAAGACTCCTATGACTGCAATTTTG-3'.

[0176] The primer oligonucleotides were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0177] (2) Reverse amplification of the full-length pACYCAR (5.7 kb) plasmid

[0178] The pACYCAR (5.7 kb) plasmid was digested with FseI to obtain a linear vector DNA template. Using pAC-AR328F and pAC-AR319RF as primers, the following reverse PCR system (200 μL) was prepared according to the TaKaRa Premix Taq™ (Ex Taq™ Version 2.0 plusdye) kit (Code No. RR902A):

[0179] 1)Premix Taq (Ex Taq Version 2.0 plus dye) 100 μL;

[0180] 2) Template DNA 2 μL,

[0181] 3) pAC-AR328F (10 μM) 8 μL;

[0182] 4) pAC-AR319R (10 μM) 8 μL;

[0183] 5) Add sterile water to 200 μL.

[0184] Mix well, dispense 50 μL into 0.2 mL thin-walled PCR tubes, and perform PCR amplification.

[0185] The amplification conditions were: 94℃ for 4 min; 94℃ for 30 sec, 47℃ for 30 sec, 72℃ for 5 min, 30 cycles; 72℃ for 10 min.

[0186] After amplification, the amplification products were combined and observed by 1% agarose gel electrophoresis. Products with a size close to 5.7 kb were considered positive. The amplification products were then recovered and purified.

[0187] (3) PCR amplification of the CKV genome fragment CKA (nt81-2235)

[0188] Using pUCmT-CKA subcloned plasmid DNA (5 kb) diluted 1:20 as a model, and acCKA70f and acCKA2200r as primers, the plasmid was prepared according to the TaKaRa Premix Taq kit. TM (Ex Taq TM Prepare PCR system (100 μL) using Version 2.0 plus dye (Code No. RR902A):

[0189] 1)Premix Taq (Ex Taq Version 2.0 plus dye) 50 μL;

[0190] 2) Template DNA 4 μL;

[0191] 3) acCKA70f (10 μM) 4 μL;

[0192] 4) acCKA2200r (10 μM) 4 μL;

[0193] 5) Add sterile water to 100 μL.

[0194] Mix well and aliquot into 0.2 mL thin-walled PCR tubes at 50 μL / tube for amplification. Amplification conditions: 94℃ for 4 min; 94℃ for 30 sec, 56℃ for 30 sec, 72℃ for 2 min, 30 cycles; 72℃ for 10 min.

[0195] After amplification, the amplification products were combined and observed by 1% agarose gel electrophoresis. Products with a size close to 2.2 kb were considered positive. The amplification products were then recovered and purified.

[0196] (4) Seamless cloning of pACYCAR (5.7 kb) plasmid and CKV genome fragment CKA (nt81-2235)

[0197] 1) Seamless cloning reaction and transformation

[0198] Following the instructions of the TAKARA In-Fusion® Snap Assembly Master Mix (Code No. 638948) kit, the procedure was as follows: Add 4 μL of 5×In-Fusion Snap Assembly Master Mix to a 0.2 mL thin-walled PCR tube, recover and purify 4 μL of the linear vector pACYCAR (5.7 kb) plasmid, and recover and purify 3 μL of the CKV genomic fragment CKA (nt81-2235). Add deionized water to a final volume of 20 μL. Mix well and incubate at 50°C for 2 h, then immediately place on ice. Transfer the mixture to 100 μL of competent cells (top10), gently mix, and incubate on ice for 30 min. Heat shock at 42°C for 45 sec, then continue incubation on ice for 2 min. Add 500 μL of SOC medium preheated to 37°C and incubate at 37°C for 1 h using a shaker at 180 rpm. Spread on LB agar plates containing chloramphenicol and incubate overnight at 37°C.

[0199] 2) Screening for positive clones

[0200] Select distinct single colonies from the plate, inoculate with 5 mL of chloramphenicol-containing LB broth, and incubate overnight at 37°C with a shaker at 200 rpm. Collect 100 μL of culture medium at 8,000 g for 1 min to collect the bacterial cells, discarding the supernatant. Resuspend the bacterial cells in 100 μL of ddH2O, place in a 100°C metal bath for 5 min, then at 8,000 g for 2 min, and use the supernatant as a DNA template for cloning screening. Identify the inserted CKV genomic fragment CKA (nt81-2235) using primers DuetUP2:5'-TTGTACACGGCCGCATAATC-3' and T7-ter:5'-TGCTAGTTATTGCTCAGCGG-3'. Use the TaKaRa Premix Taq kit. TM(Ex Taq TM The reaction mixture (Version 2.0 plus dye) (Code No. RR902A) was prepared as follows: 12.5 μL Premix Taq (Ex Taq Version 2.0 plus dye), 2 μL template DNA, 1 μL DuetUP2 (10 μM), 1 μL T7-ter (10 μM), and sterile water to a final volume of 25 μL. Amplification conditions: 94℃ for 4 min; 94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 2 min, 30 cycles; 72℃ for 10 min. After amplification, the product size was observed by 1% agarose gel electrophoresis. Products with a size close to 2.2 kb were considered potentially positive clones.

[0201] 3) Large-scale preparation of positive clone plasmid DNA.

[0202] Take 1 mL of the suspected positive clone culture, transfer it to 100 mL of chloramphenicol-containing LB liquid medium, and incubate overnight at 37°C with a shaker at 180 rpm. Extract plasmid DNA. The procedure is as follows: Use the UNlQ-500 column-based plasmid DNA extraction kit (product catalog number: B511243) from Sangon Biotech (Shanghai) Co., Ltd.:

[0203] Centrifuge 100 mL of overnight culture at 4,000 rpm for 10 min, collect all cells, and discard or aspirate the culture medium.

[0204] Add 10 mL of Buffer P1 to the bacterial pellet and aspirate or shake until the bacterial cells are completely suspended.

[0205] Add 10 mL of Buffer P2, immediately and gently invert the centrifuge tube 5-10 times to mix, and let stand at room temperature for 2-4 minutes.

[0206] Add 14 mL of Buffer P3, immediately invert 5-10 times, and let stand at room temperature for 5 minutes.

[0207] Centrifuge at 4,000 rpm for 15 min.

[0208] Carefully transfer all the supernatant into the adsorption column, incubate at room temperature for 5 min, and centrifuge at 4,000 rpm for 10 min.

[0209] Discard the liquid in the collection tube and place the adsorption column into the same collection tube.

[0210] Add 5 mL Wash Solution to the adsorption column and centrifuge at 4,000 rpm for 5 min.

[0211] Discard the liquid in the collection tube and place the adsorption column into the same collection tube.

[0212] Repeat the washing process once.

[0213] Place the empty adsorption column and collection tube into a centrifuge and centrifuge at 4,000 rpm for 10 min.

[0214] Place the adsorption column into a clean 50 mL centrifuge tube, add 2 mL of Elution Buffer to the center of the adsorption membrane, incubate at room temperature for 2 min, and centrifuge at 4,000 rpm for 5 min. Store the obtained plasmid DNA solution at -20℃ or use it for subsequent experiments.

[0215] 4) Sequencing verification of the inserted CKV genome fragment CKA (nt81-2235).

[0216] A large number of plasmid DNA samples of suspected positive clones were sent to the Chongqing Sequencing Department of Sangon Biotech (Shanghai) Co., Ltd. for commercial SANGER sequencing using primers DuetUP2 and T7-ter. The clone with the correct insertion of the CKA fragment was named pACYCAR-CKA (8.0 kb).

[0217] CKV genome fragment CKA (nt81-2235) armored RNA expression and purification

[0218] 1. BL21(DE3) host bacteria induce expression

[0219] (1) Transform the prokaryotic expression vector pACYCAR-CKA (8.0 kb) into the BL21 (DE3) host bacteria, pick a single colony on a chloramphenicol resistance plate, inoculate it into 5 mL of LB liquid medium containing chloramphenicol, and incubate overnight at 37°C and 200 rpm on a shaker.

[0220] (2) Transfer the culture to 100 mL of LB liquid medium containing chloramphenicol at a ratio of 1:100 and culture at 37℃ with shaking at 200 rpm for 4 h;

[0221] (3) Add IPTG inducer to a final concentration of 1 mmol / L and incubate overnight at 37°C with shaking at 200 rpm.

[0222] 2. Purification of CKV genomic fragment CKA (nt81-2235) armored RNA

[0223] (1) Collect bacterial cells in 50 mL centrifuge tubes in portions, centrifuge at 4000 rpm for 10 minutes; resuspend the bacterial cells in 30 mL of 1×PBS buffer, centrifuge at 4000 rpm for 10 minutes, and collect the bacterial cells. Repeat the washing process twice.

[0224] (2) Ultrasonic disruption. Add 10 mL of 1× ultrasonic treatment solution (5 mmol / L MgSO4, 0.1 mol / L NaCl, 50 mmol / L Tris-HCl, pH 8.0) to the bacterial cells to resuspend the bacteria; set the ultrasonic power to 15%, 1.5s pulse, 3s interval, for a total of 30 min.

[0225] After sonication, place the centrifuge tube into a centrifuge and centrifuge at 4000 rpm for 30 min. Transfer the supernatant to a new 15 mL centrifuge tube.

[0226] (3) Add 0.585 g of solid NaCl to the supernatant after centrifugation to a final concentration of 1 mol / L, shake to dissolve NaCl, add PEG6000 to a final concentration of 10% (w / v), shake vigorously on a shaker at 37℃ for 10 min, and incubate overnight on ice.

[0227] (4) Centrifuge the mixture at 4000 rpm for 30 min and discard the supernatant. Add 5 mL of 1×PBS to the centrifuge tube to suspend the precipitate, add an equal volume of chloroform, and shake on a shaker at 37℃ for 10 min. Centrifuge at 4000 rpm for 15 min, and carefully aspirate the upper aqueous phase into a new 15 mL centrifuge tube. This is the purified CKV armored RNA, which should be stored in a freezer at -80℃.

[0228] Experimental Example 1

[0229] Stability verification of CKV genome fragment CKA (nt81-2235) armored RNA

[0230] Take 1 mL of purified CKA (nt81-2235) armored RNA sample stored at -80℃, dilute it with 1×PBS in a 15 mL centrifuge tube at a ratio of 1:10, aliquot it into 1.5 mL microcentrifuge tubes at a ratio of 200 μL / tube, and store it in a -80℃ freezer for later use.

[0231] 1. Temperature stability test

[0232] (1) Treatment at different temperatures

[0233] Take 16 aliquots of CKA armored RNA samples and place 4 tubes at 37℃, 25℃, 4℃, and -20℃ respectively. At 1 d, 7 d, 14 d, and 28 d, retrieve one tube and place it in a -80℃ refrigerator for testing.

[0234] (2) Extraction of CKA armored RNA

[0235] CKA armor RNA was extracted using the TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0 (Code No. 9766) from TAKARA, starting with 200 μL of liquid sample and ending with 50 μL of elution.

[0236] (3) Detection of CKA armored RNA primer-probe

[0237] The U.S. Centers for Disease Control and Prevention (CDC) recommended a real-time fluorescent RT-PCR system for CKV in 2016, with the following primers and probes:

[0238] Upstream primer CHK-F: 5'-ACCATCGGTGTTCCATCTAAAG -3',

[0239] Downstream primer CHK-R: 5'-GCCTGGGCTCATCGTTATT-3'

[0240] Fluorescent probe CHK-P: 5'-FAM-ACAGTGGTTTCGTGTGAGGGCTAC-BHQ1-3'.

[0241] The target region of this system is the CKV nsp1 gene (856-961, NC_004162), making it suitable as a primer-probe for RT-qPCR detection of CKV armored RNA samples. The primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0242] (4) Real-time fluorescence RT-PCR detection of CKV genome fragments in CKA armored RNA

[0243] According to TAKRA's One Step PrimeScript TM III. Preparation of the real-time fluorescence RT-PCR reaction system (20 μL) using the RT-qPCR Mix kit (Code No. RR600A):

[0244] 1)One Step PrimeScript III RT-qPCR Mix (2×) 10 μL;

[0245] 2) RNase-Free H2O 6.8 μL;

[0246] 3) CHK-F (10 μM) 0.4 μL;

[0247] 4) CHK-R (10 μM) 0.4 μL;

[0248] 5) CHK-P (10 μM) 0.4 μL;

[0249] 6) Template 2 μL.

[0250] Amplification conditions: 52℃ for 5 min, 95℃ for 10 sec; 95℃ for 5 sec, 60℃ for 31 sec (fluorescence signal acquisition), 40 cycles. The amplification reaction was performed on a Thermo Fisher Scientific 7500 Real-Time PCR System.

[0251] (5) Temperature stability test results of CKV genome fragment CKA (nt81-2235) armored RNA ( Figure 1 )

[0252] The results showed that the CKV genome fragment CKA (nt81-2235) armored RNA could be detected by real-time fluorescent RT-PCR at different temperatures (37℃, 25℃, 4℃, -20℃) and different times (1 d, 7 d, 14 d, 28 d), indicating that the CKV genome fragment CKA (nt81-2235) armored RNA was temperature-stable.

[0253] 2. Nuclease resistance test for CKV genomic fragment CKA (nt81-2235)

[0254] (1) Nuclease-resistant treatment

[0255] Take 1 mL of purified CKV genomic fragment CKA (nt81-2235) armored RNA sample stored at -80℃, dilute it 1:10 in a 15 mL centrifuge tube with 1×PBS, and aliquot 200 μL / tube into 1.5 mL microcentrifuge tubes. Take 4 tubes of the above 200 μL / tube sample and process them as follows:

[0256] 1) Add 5 μL of DNase I (5 U / μL) + 10 μL of DNase I buffer (10×) to tube 1;

[0257] 2) Add 5 μL of RNase (10 mg / mL) to tube 2;

[0258] 3) Add 5 μL of DNase I (5 U / μL) + 5 μL of RNase (10 mg / mL) + 10 μL of DNase I buffer (10×) to tube 3.

[0259] 4) Tube 4 served as an untreated control.

[0260] Place the four sample tubes in a 37°C water bath for 30 minutes.

[0261] (2) Real-time fluorescence RT-PCR detection of CKV genomic fragment in CKA (nt81-2235) armored RNA

[0262] CKA armor RNA was extracted using the TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0 (Code No. 9766) from TAKARA, starting with 200 μL of liquid sample and ending with 50 μL of elution.

[0263] Prepare a 20 μL real-time fluorescence RT-PCR reaction system according to the TAKARA One Step PrimeScript™ III RT-qPCR Mix kit (Code No. RR600A):

[0264] 1)One Step PrimeScript III RT-qPCR Mix (2×) 10 μL;

[0265] 2) RNase-Free H2O 6.8 μL;

[0266] 3) CHK-F (10 μM) 0.4 μL;

[0267] 4) CHK-R (10 μM) 0.4 μL;

[0268] 5) CHK-P (10 μM) 0.4 μL;

[0269] 6) Template 2 μL.

[0270] Amplification conditions: 52℃ for 5 min, 95℃ for 10 sec; 95℃ for 5 sec, 60℃ for 31 sec (fluorescence signal acquisition), 40 cycles. The amplification reaction was performed on a Thermo Fisher Scientific 7500 Real-Time PCR System.

[0271] (3) Results of nuclease resistance test of armored RNA of CKV genome fragment CKA (nt81-2235) Figure 2 )

[0272] The results showed that the armored RNA of the CKV genome fragment CKA (nt81-2235) could be detected by real-time fluorescent RT-PCR after treatment with DNase and RNase, indicating that the armored RNA of the CKV genome fragment CKA (nt81-2235) is resistant to nucleases.

[0273] 3. Serial dilution of CKV genome fragment CKA (nt81-2235) armored RNA

[0274] (1) Series of dilution treatments

[0275] Take 200 μL of purified CKA armored RNA sample stored at -80℃ and extract CKA armored RNA according to the TAKARA TaKaRaMiniBEST Viral RNA / DNA Extraction Kit Ver.5.0 (Code No. 9766). The final elution volume is 50 μL. The extract is then serially diluted 10-fold with RNase-free water (10... -1 Up to 10 -8 ), RT-qPCR was used to detect the CKV genome fragment in CKA (nt81-2235) armored RNA.

[0276] (2) Real-time fluorescence RT-PCR detection of CKV genomic fragments in CKA armored RNA (nt81-2235)

[0277] Prepare a 20 μL real-time fluorescence RT-PCR reaction system according to the TAKARA One Step PrimeScript™ III RT-qPCR Mix kit (Code No. RR600A):

[0278] 1)One Step PrimeScript III RT-qPCR Mix (2×) 10 μL;

[0279] 2) RNase-Free H2O 6.8 μL;

[0280] 3) CHK-F (10 μM) 0.4 μL;

[0281] 4) CHK-R (10 μM) 0.4 μL;

[0282] 5) CHK-P (10 μM) 0.4 μL;

[0283] 6) Template 2 μL.

[0284] Amplification conditions: 52℃ for 5 min, 95℃ for 10 sec; 95℃ for 5 sec, 60℃ for 31 sec (fluorescence signal acquisition), 40 cycles. The amplification reaction was performed on a Thermo Fisher Scientific 7500 Real-Time PCR System.

[0285] (3) Serial dilution results of CKV genome fragment CKA (nt81-2235) armored RNA ( Figure 3 )

[0286] The results showed that the armored RNA of the CKV genome fragment CKA (nt81-2235) was serially diluted, and the lowest dilution detectable by real-time fluorescent RT-PCR was 10. -6 .

[0287] Experimental Example 2

[0288] Digital PCR quantification of CKV genome fragment CKA (nt81-2235) armored RNA

[0289] 1. Method

[0290] Absolute quantification of the prepared CKA (nt81-2235) armored RNA was performed using Stilla digital PCR (French). The optimal dilution for digital PCR detection of the CKV armored RNA was 10⁻⁶. -4 .

[0291] 2 Digital PCR Primers and Probes

[0292] Digital PCR quantification of CKA (nt81-2235) armored RNA samples was performed using the primer-probe CHK-F / CHK-R / CHK-P primer-probe system recommended by the US Centers for Disease Control and Prevention (CDC). The primers and probe oligonucleotides were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0293] 3. Digital PCR Quantitative Experiment

[0294] (1) Digital PCR reaction system

[0295] The sodium fluorescein salt (catalog number 140007050) and the highly sensitive one-step reverse transcriptase premix (2×) (catalog number 15000701K) used in the detection were both produced by Beijing Aipubai Biotechnology Co., Ltd.

[0296] Prepare the reaction mixture (25 μL) according to the instructions:

[0297] 1) MasterMix 12.5 μL;

[0298] 2) 10× fluorescein sodium salt (1 μM) 2.5 μL;

[0299] 3) CHK-F (10 μM) 2.5 μL;

[0300] 4)CHK-R (10 μM) 2.5 μL;

[0301] 5) CHK-P (5 μM) 1.25 μL;

[0302] 6) Add H2O to bring the volume to 22.5 μL.

[0303] The above reagents were shaken to mix well, and dispensed into 22.5 μL tubes. 2.5 μL of diluted CKV armor RNA to be tested was added to each tube and mixed well. 25 μL of the mixture was loaded onto a digital PCR amplification chip (Saphhire Chips for the Naica Crystal Digital PCR System, Ref # C14012).

[0304] (2) Digital PCR amplification:

[0305] Digital PCR amplification was performed using a STILLA TECHNOLOGIES digital PCR instrument (Ref # H14000) from France. The amplification program is as follows:

[0306] 1) Zone: 40℃, AP950

[0307] 2) Amplification conditions: reverse transcription at 50℃ for 10 min, pre-denaturation at 95℃ for 3 min; cycling at 95℃ for 15 sec, 60℃ for 30 sec, for 45 cycles.

[0308] 3) Pressure relief: 25℃

[0309] 4. Digital PCR Quantitative Data Reading of CKV Genome Fragment CKA (nt81-2235) Armored RNA

[0310] (1) CKA (nt81-2235) armored RNA (10 -4 ) Digital PCR quantification results ( Figure 4-5 )

[0311] (2) Table 1 shows the results of digital PCR quantification of CKA (nt81-2235) armored RNA. The concentration of Chikungunya CKA armored RNA was 7.48×104 (copy / μL).

[0312] Table 1. Quantitative results of CKV armored RNA by digital PCR

[0313]

[0314] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a chikungunya virus genome fragment armored RNA reference, characterized in that, Prepare according to the following steps: (1) Construction of prokaryotic expression backbone vector pACYCAR: MS2 phage genomic RNA was cultured and extracted. A 1.7kb fragment containing MS2 maturation enzyme, envelope protein gene and packaging recognition signal sequence was amplified by RT-PCR. The fragment was ligated with pACYCDuet-1 plasmid that had been digested with BamHI and HindIII. After transformation into DH5α competent cells, the fragment was screened and identified to obtain the backbone vector pACYCAR. (2) Cloning the CKV genome fragment CKA (nt17-2276): Total RNA was extracted from inactivated CKV virus, and the genome fragment CKA (nt17-2276) covering the 5'UTR, non-structural protein nsp1 and part of the nsp2 gene was amplified by RT-PCR. After TA cloning and screening, the subcloned plasmid pUCmT-CKA (5.0 kb) was obtained. (3) Construction of prokaryotic expression vector pACYCAR-CKA: Using seamless cloning technology, CKA (nt81-2235) was amplified using the CKV genome fragment cloned in step (2) as a template and ligated with the linearized pACYCAR vector amplified by reverse PCR. After transformation into Top10 competent cells, the recombinant expression vector pACYCAR-CKA (8.0kb) was obtained. (4) Induced expression of armored RNA: The recombinant expression vector from step (3) was transformed into BL21(DE3) host bacteria and induced to express by IPTG to obtain engineered bacteria containing CKV armored RNA; (5) Purification of armored RNA: Collect engineered bacterial cells, wash with PBS, sonicate and purify by NaCl salting, PEG6000 precipitation and chloroform extraction to obtain CKV genome fragment CKA (nt81-2235) armored RNA reference.

2. The method for preparing the armored RNA reference of the chikungunya virus genome fragment according to claim 1, characterized in that: MS2 phage genome extraction was performed using the TAKARA MiniBEST ViralRNA / DNA Extraction Kit Ver 5.0, Code No. 9766.

3. The method for preparing the armored RNA reference of the chikungunya virus genome fragment according to claim 1 or 2, characterized in that: In step (1), the primers for RT-PCR amplification of the MS2 1.7kb fragment are: Upstream primer AR-Bam10F: 5'-CGCGGATCCTTTCGGGGTCCTGCTCAACTT-3', Downstream primer AR-Hin1789R: 5'-CCCAAGCTTGAGTTGAACTTCTTTGTTGTCTTC-3'; The RT-PCR amplification conditions were: 50℃ for 30 min; 94℃ for 2 min; 94℃ for 30 sec, 58℃ for 30 sec, 72℃ for 90 sec, 30 cycles; 72℃ for 5 min; and the ligation system was incubated overnight in a 16℃ water bath.

4. The method for preparing the armored RNA reference of the chikungunya virus genome fragment according to claim 3, characterized in that: In step (2), the CKV strain is CHIKV-18001; The upstream and downstream primers for RT-PCR amplification are as follows: CHIKV-18F: 5'-CACGTAGCCTACCAGTTTCTTA-3', CHIKV-2709R: 5'-TCCACTACAATCGGCTTGTTG-3'; Amplification conditions: 50℃ for 30 min; 94℃ for 2 min; 94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 2.5 min, 30 cycles; 72℃ for 10 min.

5. The method for preparing the armored RNA reference of the chikungunya virus genome fragment according to claim 4, characterized in that: Positive clones were screened using the following primers: M13-47: 5'-AGGGTTTTCCCAGTCACG-3', M13-48: 5'-GAGCGGATAACAATTTCACAC-3'.

6. The method for preparing the armored RNA reference of the chikungunya virus genome fragment according to claim 5, characterized in that: In step (3), the primers for seamless cloning include pAC-AR328F: 5'-GCCACGCGATCGCTGACG-3', pAC-AR319R: 5'-ATCCAATTGAGATCTGCCATATG-3', acCKA70f: 5'-AGATCTCAATTGGATATCCTGTGTACGTGGAC-3', acCKA2200r: 5'-CAGCGATCGCGTGGCAAGACTCCTATGACTGCAATTTTG-3'; The seamless cloning reaction was incubated in a metal bath at 50°C for 2 h, and positive clones were screened using DuetUP2 and T7-ter as primers. The primer sequences for DuetUP2 and T7-ter are as follows: DuetUP2:5'-TTGTACACGGCCGCATAATC-3', T7-ter: 5'-TGCTAGTTATTGCTCAGCGG-3'.

7. The method for preparing the armored RNA reference of the chikungunya virus genome fragment according to claim 6, characterized in that: In step (4), the engineered bacteria were cultured at a ratio of 1:100 before induction, and cultured at 37℃ and 200 rpm for 4 h with shaking. The final concentration of IPTG was 1 mmol / L, and the induction conditions were overnight culture at 37℃ and 200 rpm with shaking.

8. The method for preparing the armored RNA reference of the chikungunya virus genome fragment according to claim 7, characterized in that: In step (5), the ultrasonic disruption parameters are: ultrasonic power 15%, 1.5s pulse, 3s interval, total duration 30 min; final NaCl concentration is 1 mol / L, final PEG6000 concentration is 10% (w / v), and the purified product is obtained by chloroform extraction after overnight ice bath.

9. A chikungunya virus genome fragment armored RNA reference prepared by the method of any one of claims 1-8.

10. The application of the chikungunya virus genome fragment armored RNA reference as described in claim 9 as a template for the research and validation of CKV nucleic acid detection reagents, or as a standard for plotting CKV nucleic acid detection standard curves, or as a quality control for assessing the chikungunya virus nucleic acid detection capabilities of primary laboratories and for inter-laboratory quality control, or as a positive control in the preparation of chikungunya virus nucleic acid detection kits.

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