A long fragment of type 1-4 dengue virus (DENV) genome armored RNA reference and preparation method and application thereof

Abstract

The application discloses a 1-4 type DENV genome long fragment armoured RNA reference product and a preparation method and application thereof, and is prepared according to the following steps: (1) constructing a prokaryotic expression skeleton vector pACYCAR (2) 1-4 type dengue virus genome long fragment cloning (3) construction of prokaryotic expression vectors pACYCAR-D1A (8.1 kb), pACYCAR-D2A (7.7 kb), pACYCAR-D3A (8.0 kb) and pACYCAR-D4A (8.1 kb) (4) inducing expression of armoured RNA (5) purifying armoured RNA. The application solves the technical problems of high biological safety risk, poor stability and insufficient gene fragment coverage of the existing dengue virus nucleic acid detection reference product, and fills the blank of the lack of high-quality reference product for dengue virus detection.

Description

Technical Field

[0001] This invention relates to the field of virus detection technology, specifically to a long fragment armored RNA reference for DENV genomes of types 1-4, its preparation method, and its application. Background Technology

[0002] Dengue virus (DENV) belongs to the genus Orthoflavivirus of the family Flaviviridae, and includes serotypes 1-4 (DENV-1, DENV-2, DENV-3, and DENV-4). The main vectors are Aedes aegypti and Aedes albopictus mosquitoes. The genome is a single-stranded positive-sense RNA, approximately 11 kb in length. The disease caused by DENV infection is called dengue fever (DF), a Class B infectious disease. Clinically, it presents with acute onset, sudden high fever, significant fatigue, loss of appetite, nausea, etc., often accompanied by severe headache, orbital pain, generalized muscle pain, and joint pain. Facial, neck, and chest flushing, and conjunctival congestion may also occur. A small number of cases are severe dengue fever, characterized by severe hemorrhage, shock, and damage to vital organs. Dengue fever is widespread in tropical and subtropical regions worldwide where the Aedes mosquito vector is present.

[0003] Specific methods for detecting the causative agent of dengue fever include viral nucleic acid detection, IgM antibody detection, neutralizing antibody detection, and virus isolation. Due to the strong serological cross-reactivity between DENV and other flaviviruses, viral nucleic acid detection is currently the primary method. Viral nucleic acid detection directly reflects the viremia phase, covering the window period from viral infection to antibody emergence. The results are crucial for determining the infectivity of suspected cases and close contacts, and for effectively controlling further viral transmission. DENV nucleic acid detection includes detecting viral-specific genomic fragments and determining the full-length or partial genome sequence. In vitro amplification of viral genomic fragments is the most sensitive, rapid, and widely used laboratory diagnostic method. Various methods for amplifying and detecting DENV-specific nucleic acids have been developed, most based on polymerase chain reaction (PCR) technology. 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 DENV 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] The core of armored RNA (AR) technology is the expression of recombinant viral envelope protein vectors in prokaryotes, enabling them to self-assemble into virus-like particles and package specific RNA fragments within them to form pseudovirus particles. Viral envelope proteins with similar functions can originate from Pseudomonas aeruginosa PRR1 or PP7 bacteriophages, filamentous bacteriophages, tobacco mosaic virus (TMV), Raul's sarcoma virus (RSV), retroviral vectors, and Escherichia coli MS2 bacteriophage, among others. MS2 bacteriophage is the most common. The MS2 bacteriophage genome is a ~3.6 kb single-stranded RNA encoding four proteins: a maturation enzyme, an envelope protein, a cleavage protein, and a replicase. A complete MS2 bacteriophage particle consists of 180 envelope protein monomers, one maturation enzyme protein, and one genomic RNA. Research has shown that cloning the MS2 maturation enzyme, envelope protein gene, and exogenous gene into a prokaryotic expression vector, and inducing the expression of bacteriophage envelope proteins to assemble into a shell, yields virus-like particles with the same morphology as wild-type bacteriophages, which encapsulate recombinant RNA molecules containing exogenous genes. This MS2-based armored RNA is actually a non-infectious MS2 derivative. Because its specific recombinant RNA sequence is packaged into MS2 pseudovirus particles by viral envelope proteins, it can effectively resist nuclease interference and can be stored long-term under very simple preservation conditions. Summary of the Invention

[0006] To address the aforementioned technical problems, the first objective of this invention is to provide a long-fragment armored RNA reference for dengue virus (DENV) genomes of types 1-4 and its preparation method. The second objective is to provide its applications. This addresses the technical problems of existing dengue virus nucleic acid detection references, such as high biosafety risks, poor stability, insufficient gene fragment coverage, and a lack of internal controls for full-process nucleic acid detection tracking, thus filling the gap in high-quality references for dengue virus detection.

[0007] To achieve the first objective mentioned above, the present invention provides the following technical solution: a method for preparing a long fragment armored RNA reference of type 1-4 DENV genome, characterized by preparation according to the following steps:

[0008] (I) Construction of the prokaryotic expression backbone vector pACYCAR

[0009] MS2 phage genomic RNA was cultured and extracted. A 1.7 kb fragment containing the MS2 maturation enzyme, envelope protein gene, and packaging recognition signal sequence was amplified by RT-PCR. This fragment was ligated to the double-digested pACYCDuet-1 plasmid, transformed into DH5α competent cells, and screened and identified to obtain the backbone vector pACYCAR.

[0010] (II) Cloning of long fragments of dengue virus genomes of types 1-4

[0011] Total RNA was extracted from inactivated DENV standard strains of types 1-4. A long genomic fragment covering the 5'UTR, capsid protein, membrane protein, and part of the outer membrane protein was amplified by RT-PCR. After TA cloning and screening, subcloned plasmids pUCmT-D1A (5.3 kb), pUCmT-D2A (4.8 kb), pUCmT-D3A (5.1 kb), and pUCmT-D4A (5.3 kb) were obtained. The inserted fragments were named D1A (2559 bp), D2A (2028 bp), D3A (2380 bp), and D4A (2579 bp), respectively.

[0012] (III) Construction of prokaryotic expression vectors pACYCAR-D1A (8.1 kb), pACYCAR-D2A (7.7 kb), pACYCAR-D3A (8.0 kb), and pACYCAR-D4A (8.1 kb)

[0013] Using seamless cloning technology, DENV-1 nt7-2420, DENV-2 nt7-2028, DENV-3 nt7-2380, and DENV-4 nt7-2400 were amplified using the long genomic fragments of types 1-4 cloned in step (II) as templates. These fragments were then ligated into linearized pACYCAR vectors amplified by reverse PCR, transformed into Top10 competent cells, and screened to obtain recombinant expression vectors pACYCAR-D1A (8.1 kb), pACYCAR-D2A (7.7 kb), pACYCAR-D3A (8.0 kb), and pACYCAR-D4A (8.1 kb).

[0014] (iv) Induced expression of armored RNA

[0015] The recombinant expression vector from step (III) was transformed into BL21(DE3) host bacteria, and expressed by IPTG to obtain engineered bacteria containing DENV armor RNA of types 1-4;

[0016] (v) Purification of armored RNA

[0017] The engineered bacterial cells were collected, washed with PBS, and sonicated. Then, they were purified by NaCl salting out, PEG6000 precipitation, and chloroform extraction to obtain the long-fragment armored RNA reference of type 1-4 DENV genome.

[0018] In the above scheme: In step (i), the MS2 phage genome was extracted using the TAKARA MiniBEST Viral RNA / DNA Extraction Kit Ver 5.0, Code No. 9766.

[0019] In the above protocol: the upstream primer for RT-PCR amplification of the MS2 1.7 kb fragment in step (I) is...

[0020] AR-Bam10F: 5'-CGCGGATCCTTTCGGGGTCCTGCTCAACTT-3',

[0021] The downstream primer was AR-Hin1789R: 5'-CCCAAGCTTGAGTTGAACTTCTTTGTTGTCTTC-3'; double digestion was performed using BamHI and HindIII, and the ligation system was incubated overnight at 16°C.

[0022] In the above scheme: the DENV standard strains of types 1-4 in step (II) are DENV-1, Hawaii, GenBank accession number KM204119; DENV-2, New Guinea C, GenBank accession number KM204118; DENV-3, H87, GenBank accession number KU050695; DENV-4, H241, GenBank accession number KR011349;

[0023] The primer combinations for RT-PCR amplification are as follows:

[0024] Den1-1F: 5'-AGTTGTTAGTCTACGTGGAC-3',

[0025] Den1-2559R: 5'-CCAATGGCYGCTGAYAGTCT-3',

[0026] Den2-1F: 5'-AGTWGTTAGTCTACGTGGAC-3',

[0027] Den2-2028R: 5'-TGGGCTGTCTTTTTCTGTGA-3',

[0028] Den3-1F: 5'-AGTTGTTAGTCTACGTGGAC-3',

[0029] Den3-2380R: 5'-GATTCCTATCGCAATGCATG-3',

[0030] Den4-1F: 5'-AGTTGTTAGTCTGTGTGGACCGACAA-3',

[0031] Den4-2579R: 5'-GGGCATTYAATATTGCAGACGCTA-3'.

[0032] The amplification conditions were: 50℃ for 30 min; 94℃ for 2 min; 94℃ for 30 sec, 48℃ / 45℃ for 30 sec, 72℃ for 2.5 min, 30 cycles; 72℃ for 10 min. Except for Den1-1F and Den1-2559R, which were annealed at 48℃, the annealing temperature for other primer combinations was 45℃.

[0033] In the above scheme, the primers used for seamless cloning in step (iii) include:

[0034] pAC-AR328F 5'-GCCACGCGATCGCTGACG-3',

[0035] pAC-AR319R 5'-ATCCAATTGAGATCTGCCATATG-3',

[0036] acD1A7f: 5'-AGATCTCAATTGGATTAGTCTGTGGACCGACAAGAACAG-3',

[0037] acD1A2420r 5'-CAGCGATCGCGTGGCCGCCTGAACCATGACTCCTAGG-3',

[0038] acD2A7f 5'-AGATCTCAATTGGATTAGTCTACGTGGACCGACAAAG-3',

[0039] acD2A2028r 5'-CAGCGATCGCGTGGCTGGGCTATCTTTTTCGTTACG-3',

[0040] acD3A7f 5'-AGATCTCAATTGGATTAGTCTACGTGGACCGACA-3',

[0041] acD3A2380r: 5'-CAGCGATCGCGTGGCGATTCCTATCGCAATGCATGA-3',

[0042] acD4A7f 5'-AGATCTCAATTGGATTAGTCTGTGTGGACGACAAGG-3',

[0043] acD4A2400r 5'-CAGCGATCGCGTGGCAGAGTGATTCCTCCAACAGCTATGC-3'.

[0044] The seamless cloning reaction was incubated in a metal bath at 50°C for 2 h. After the ligation product was transformed into Top10 competent cells, positive clones were screened using DuetUP2 and T7-ter primers. The primer sequences for DuetUP2 and T7-ter are as follows:

[0045] DuetUP2:5'-TTGTACACGGCCGCATAATC-3', T7-ter: 5'-TGCTAGTTATTGCTCAGCGG-3'

[0046] In the above scheme: the final concentration of IPTG in step (iv) is 1 mmol / L, and the induction conditions are overnight culture at 37℃ and 200 rpm with shaking.

[0047] In the above scheme: the parameters for ultrasonic disruption in step (5) 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 in an ice bath.

[0048] A method for preparing type 1-4 DENV genome long fragment armored RNA reference products yielded type 1-4 DENV genome long fragment armored RNA reference products. The DENV gene fragment lengths of the reference products are all greater than 2.0 kb, and they can withstand temperature treatments at 37℃, 25℃, 4℃, and -20℃ for 28 days. They are also resistant to DNase I, RNase A, and a combination of both. The concentrations quantified by digital PCR were 4.36 × 10⁻⁶ DENV-1 armored RNA. 4 copy / μL, DENV-2 armored RNA 1.68×10 7 copy / μL, DENV-3 armored RNA 4.39×10 7 copy / μL, DENV-4 armored RNA 4.37×10 5 copy / μL.

[0049] The application of the aforementioned type 1-4 DENV genome long fragment armored RNA reference material in the research and development and validation of DENV nucleic acid detection reagents, or in the plotting of DENV nucleic acid detection standard curves, or in the assessment of dengue virus nucleic acid detection capabilities and interlaboratory quality control in primary laboratories, or in the preparation of dengue virus nucleic acid detection kits as a positive control.

[0050] Compared with the prior art, the present invention has the following advantages:

[0051] 1. The prepared armored RNA is a non-infectious pseudovirus particle with no autonomous replication ability. It does not require high-level biosafety laboratory operation, is non-infectious, and poses no biosafety risk. It solves the biosafety risk problem of live virus reference materials and is suitable for widespread use in most laboratories in China.

[0052] 2. The developed DENV armored RNA has excellent temperature stability, is resistant to nuclease treatment, is easy to transport, and can be stored for a long time under simple conditions.

[0053] 3. The prepared DENV armored RNA of types 1-4 are all long fragments (>2.0 kb), covering the 5'UTR, capsid protein, membrane protein and some outer membrane protein genes, covering the core target region of dengue virus nucleic acid detection, which can meet the quality control requirements of multi-target detection and is superior to existing short fragment armored RNA.

[0054] 4. Absolute quantification via digital PCR ensures clear concentrations of various reference standards, allowing for the preparation of standards with different concentration gradients for plotting detection standard curves. It can also serve as a positive control and interlaboratory quality control, making it suitable for various scenarios including dengue virus nucleic acid detection reagent development, clinical testing quality control, and laboratory competency assessment.

[0055] 5. This invention constructs a backbone vector based on pACYCDuet-1, and uses seamless cloning technology to achieve efficient insertion of long DENV fragments. The induction expression and purification steps are simple, and large-scale preparation can be achieved to meet the needs of industrial production and daily use.

[0056] 6. The reference product is a virus-like particle, and its nucleic acid extraction process is highly similar to that of natural DENV in clinical samples. It can effectively simulate every step in RNA virus detection, from sample processing and nucleic acid extraction to amplification, and achieve full-process quality control from sample processing to reverse transcription. Attached Figure Description

[0057] Figure 1 The image shows the results of the temperature stability test of DENV-1 armored RNA.

[0058] Figure 2 The image shows the results of the temperature stability test of DENV-2 armored RNA.

[0059] Figure 3 The image shows the results of the temperature stability test of DENV-3 armored RNA.

[0060] Figure 4 The image shows the results of the temperature stability test of DENV-4 armored RNA.

[0061] Figure 5 The image shows the results of the DENV-1 RNA armor RNA nuclease resistance test.

[0062] Figure 6 The image shows the results of the DENV-2 RNA armor RNA nuclease resistance test.

[0063] Figure 7 The image shows the results of the DENV-3 RNA armor RNA nuclease resistance test.

[0064] Figure 8 The image shows the results of the DENV-4 RNA armor RNA nuclease resistance test.

[0065] Figure 9 This is a graph showing the serial dilution results of DENV-1 armored RNA.

[0066] Figure 10 Figure showing the results of serial dilutions of DENV-2 armored RNA.

[0067] Figure 11 Figure showing the results of serial dilutions of DENV-3 armored RNA.

[0068] Figure 12 Figure showing the results of serial dilutions of DENV-4 armored RNA.

[0069] Figure 13 Digital PCR quantification of DENV-1 armored RNA.

[0070] Figure 14 This is a digital PCR quantification diagram of the DENV-1 armored RNA negative control.

[0071] Figure 15 This is a digital PCR quantification diagram of DENV-2 armored RNA.

[0072] Figure 16 This is a digital PCR quantification diagram of the DENV-2 armored RNA negative control.

[0073] Figure 17 Digital PCR quantification of DENV-3 armored RNA.

[0074] Figure 18 This is a digital PCR quantification graph of the DENV-3 armored RNA negative control.

[0075] Figure 19 Digital PCR quantification of DENV-4 armored RNA.

[0076] Figure 20 This is a digital PCR quantification diagram of the DENV-4 armored RNA negative control. Detailed Implementation

[0077] The present invention will be further described below with reference to embodiments.

[0078] (I) Construction of armored RNA vectors for dengue virus types 1-4 (DENV)

[0079] 1. MS2 phage culture

[0080] (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, incubate at 37°C for 12-16 hours, select single colonies and inoculate into 5 ml of LB broth, and incubate overnight on a shaker at 37°C;

[0081] (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.

[0082] (3) Select a culture tube that has been cultured overnight and is clear and transparent. Centrifuge to remove cell debris, filter the supernatant through a 0.45 μm filter, collect the filtrate and dispense it into microcentrifuge tubes, and store at -80℃ for later use.

[0083] 2. MS2 phage genome extraction

[0084] The procedure for the TAKARA MiniBEST Viral RNA / DNA Extraction Kit (Code No. 9766) is as follows:

[0085] (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.

[0086] (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.

[0087] (3) Add 500 μL of RWA buffer to the centrifuge column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0088] (4) Add 700 μL of RWB buffer to the centrifuge column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate;

[0089] (5) Repeat step 4;

[0090] (6) Place the centrifuge column on the collection tube and centrifuge at 12,000 rpm for 2 min.

[0091] (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 Spin Column membrane, let 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.

[0092] 3. RT-PCR amplification of a 1.7 kb-MS2 fragment

[0093] (1) Primers for RT-PCR amplification of the MS2 (1.7 kb) fragment

[0094] 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:

[0095] Upstream primer AR-Bam10F: 5'-CGCGGATCCTTTCGGGGTCCTGCTCAACTT-3',

[0096] Downstream primer AR-Hin1789R: 5'-CCCAAGCTTGAGTTGAACTTCTTTGTTGTCTTC-3'.

[0097] Primers were fitted with BamHI recognition sites (GGATCC) and HindIII recognition sites (AAGCTT), respectively.

[0098] (2) RT-PCR amplification

[0099] 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:

[0100] 1) 2×1 Step Buffer (Dye Plus) 25 μL;

[0101] 2)AR-Bam10F (10 μM) 2 μL;

[0102] 3) AR-Hin1789R (10 μM) 2 μL;

[0103] 4) Template RNA 2 μL;

[0104] 5) PrimeScript 1Step Enzyme Mix 2 μL;

[0105] 6)RNase Free dH2O up to 50 μL.

[0106] 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.

[0107] After the amplification reaction was completed, 1% agarose gel electrophoresis was performed, and a 1.7 kb fragment was observed as a positive result.

[0108] (3) Gel recovery of amplification products

[0109] The amplified DNA fragments were purified and recovered using the SanPrep column-gel extraction kit (product catalog: B518131) from Sangon Biotech (Shanghai) Co., Ltd., following the instructions:

[0110] 1) Separate the target fragment from other bands 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.

[0111] 2) Add Buffer B2 at a ratio of 1:3 (i.e., 100 mg of gel to 300 μL of liquid), and place in a 50°C water bath for 5-10 minutes until the gel block dissolves.

[0112] 3) Transfer the solution to the adsorption column, centrifuge at 8000g for 30 sec, and discard the liquid; add 300 μL of Buffer B2 to the adsorption column, centrifuge at 9000g for 30 sec, and discard the liquid; add 500 μL of washing buffer, centrifuge at 9000g for 30 sec, and discard the liquid; add another 500 μL of washing buffer to wash once; centrifuge at 9000g for 1 min.

[0113] 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.

[0114] 4. Preparation of plasmid pACYCDuet-1 vector DNA (4.0 kb)

[0115] 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.

[0116] 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:

[0117] (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;

[0118] (2) Take 5 mL of bacterial culture, centrifuge at 8,000g for 2 min at room temperature, collect the bacterial cells in 1.5 mL microcentrifuge tubes, and pour out or aspirate the culture medium.

[0119] (3) Add 250 μL of Buffer P1 to the bacterial pellet and aspirate or shake until the bacterial cells are completely resuspended;

[0120] (4) Add 250 μL of buffer P2, immediately and gently invert the centrifuge tube 5-10 times to mix, and let stand at room temperature for 2-4 min;

[0121] (5) Add 350 μL of buffer P3 and immediately gently invert the centrifuge tube 5-10 times to mix thoroughly;

[0122] (6) Centrifuge at the maximum speed (≥ 12,000 g) for 7 min, carefully transfer all the supernatant into the adsorption column, and centrifuge at 9,000 g for 30 sec. Discard the liquid in the collection tube and place the adsorption column into the same collection tube.

[0123] (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.

[0124] (8) Repeat step 7 once.

[0125] (9) Place the empty adsorption column and collection tube into a centrifuge and centrifuge at 9,000g for 1 min.

[0126] (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.

[0127] 5. Construction of the prokaryotic expression backbone vector pACYCAR (5.7 kb)

[0128] (1) Restriction enzyme digestion of pACYCDuet-1 plasmid DNA (4.0 kb) and insert MS2 fragment (1.7 kb). The pACYCDuet1 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.

[0129] (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):

[0130] 1) 2 μL of 10×T4 DNA ligase buffer;

[0131] 2) Double enzyme digestion and recovery of vector pACYCDuet-15 μL;

[0132] 3) Double enzyme digestion to recover 5 μL of the inserted MS2 fragment;

[0133] 4) 1 μL of T4 DNA ligase;

[0134] 5) Add ddH2O to 20 μL.

[0135] 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.

[0136] (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. Take 100 μL of overnight culture medium, centrifuge at 8,000 g for 1 min to collect the bacterial cells, and discard the supernatant. Add 100 μL of ddH2O to resuspend the bacterial cells, place in a 100°C metal bath for 5 min, centrifuge at 8,000 g for 2 min, and take the supernatant as the DNA template for clone screening. Using primers ACYCDuetUP1 (5'-GATTATGCGGCCGTGTACAA-3') and DuetDOWN1 (5'-GATCTCGACGCTCTCCCT-3'), prepare a 25 μL reaction system according to the instructions of the TAKARA Premix Taq™ (Ex Taq™ Version 2.0 plus dye) kit, Code No. RR902A:

[0137] 1)Premix Taq (Ex Taq Version 2.0 plus dye) 12.5 μL;

[0138] 2)ACYCDuetUP1 (10μM) 1 μL,

[0139] 3) DuetDOWN1 (10μM) 1 μL;

[0140] 4) 2 μL of template;

[0141] 5) Add sterile water to 25 μL.

[0142] 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.

[0143] (4) Sequencing Verification: The plasmid DNA was extracted from the small-scale preparation of suspected positive clones and sent to the Chongqing Sequencing Department of Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification. and Commercial SANGER sequencing was performed, and the correctly sequenced recombinant plasmid was named pACYCAR (5.7 kb). The pACYCAR plasmid was transformed into DH5α host bacteria to prepare 25% glycerol bacteria, which were then stored at -80℃ for later use.

[0144] The inserted fragment sequence is as follows:

[0145] MS2-1.7K_11-1789

[0146]

[0147] 6. Cloning of long-fragment genomes of dengue virus types 1-4 (DENV)

[0148] The strain information is as follows: DENV standard strains of types 1-4 were obtained from the Fujian Provincial Center for Disease Control and Prevention, namely: DENV-1, Hawaii, GenBank accession number KM204119; DENV-2, New Guinea C, GenBank accession number KM204118; DENV-3, H87, GenBank accession number KU050695; DENV-4, H241, GenBank accession number KR011349. For biosafety reasons, the introduced strains were inactivated virus cultures (with added viral RNA extraction solution).

[0149] (1) Extraction of total viral RNA

[0150] Viral total RNA was extracted using a kit (QIAGEN RNeasy Mini Kit, QIAGEN, Cat#74104), and the procedure is as follows:

[0151] a) Mix 600 μL of viral culture medium containing Buffer RLT by vortexing, add an equal volume of 70% ethanol, and mix by pipetting.

[0152] b) 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 ≥8000 g, and discard the outflowing liquid; repeat this step to centrifuge the remaining mixture and discard the liquid.

[0153] c) Add 700 μL of buffer RW1 to the RNeasy spin column, gently cap the centrifuge tube, incubate for 15 seconds at ≥8000g, discard the outflowing liquid; reuse the collection tube.

[0154] d) Add 500 μL of buffer RPE to the RNeasy spin column, gently cap the centrifuge tube, incubate for 15 seconds at ≥8000g, discard the outflowing liquid; reuse the collection tube.

[0155] e) Add 500 μL of buffer RPE to the RNeasy spin column, gently cap the centrifuge tube, incubate for 2 min at ≥8000g, and discard the outflowing liquid.

[0156] f) 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, and collect the elution liquid at ≥8000g for 1 min.

[0157] (2) RT-PCR amplification of long fragments of dengue virus (DENV) genome of types 1-4

[0158] Based on the genome sequence information of four DENV type 1-4 standard strains in the NCBI database, subcloning primers were designed as follows:

[0159] Den1-1F: 5'-AGTTGTTAGTCTACGTGGAC-3',

[0160] Den1-2559R: 5'-CCAATGGCYGCTGAYAGTCT-3',

[0161] Den2-1F: 5'-AGTWGTTAGTCTACGTGGAC-3',

[0162] Den2-2028R: 5'-TGGGCTGTCTTTTTCTGTGA-3',

[0163] Den3-1F: 5'-AGTTGTTAGTCTACGTGGAC-3',

[0164] Den3-2380R: 5'-GATTCCTATCGCAATGCATG-3',

[0165] Den4-1F: 5'-AGTTGTTAGTCTGTGTGGACCGACAA-3',

[0166] Den4-2579R: 5'-GGGCATTYAATATTGCAGACGCTA-3'.

[0167] The long genome fragments of DENV types 1-4 cover the 5' untranslated region (5'UTR), capsid protein, membrane protein, and part of the envelope protein genes of the DENV strain. The cloning fragments of DENV types 1-4 are 2559 bp, 2028 bp, 2380 bp, and 2579 bp, respectively. Primer oligonucleotides were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0168] Long genomic fragments of DENV types 1-4 were amplified. Genomic RNA extracted from standard DENV strains of types 1-4 was used as templates, and specific primers were selected for each type (combinations as follows: Den1-1F / Den1-2559R, Den2-1F / Den2-2028R, Den3-1F / Den3-2380R, Den4-1F / Den4-2579R). The amplification was performed using the TAKARA PrimeScript one-step pre-dyed RT-PCR kit. TM™ One Step RT-PCR Kit Ver.2 (Dye Plus) Code No. RR057A. Instructions for use: Prepare a 50 μL reaction mixture.

[0169] 1) 2×1 Step Buffer (Dye Plus) 25 μL;

[0170] 2) P1 (10 μM) 2 μL;

[0171] 3) P2 (10 μM) 2 μL;

[0172] 4) Template RNA 2 μL;

[0173] 5) PrimeScript 1Step Enzyme Mix 2 μL;

[0174] 5)RNase Free dH2O up to 50 μL.

[0175] The amplification conditions were: 50℃ for 30 min; 94℃ for 2 min; 94℃ for 30 sec, 48℃ / 45℃ for 30 sec, 72℃ for 2.5 min, 30 cycles; 72℃ for 10 min. Except for Den1-1F / Den1-2559R, which had an annealing temperature of 48℃, all other primer combinations were annealed at 45℃.

[0176] After the amplification reaction was completed, 1% agarose gel electrophoresis was performed. A 2-3 kb fragment observed in the amplified product was considered a suspected positive result. The amplified product was then recovered and purified by agarose gel electrophoresis.

[0177] (3) TA cloning of long fragments of the DENV genome of types 1-4

[0178] 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):

[0179] 1) pUCm-T Vector (50 ng / μL) 1μL;

[0180] 2)10×Ligation Buffer 1 μL;

[0181] 3) 50% PEG4000 1 μL;

[0182] 4) T4 DNA Ligase 1 μL;

[0183] 5) Insert fragment (recovered amplification product) 4 μL;

[0184] 6) Add sterilized ddH2O to a final volume of 10 μL.

[0185] 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.

[0186] (4) Screening of positive clones with long genomic fragments of DENV type 1-4

[0187] Select distinct single colonies from the plate, inoculate 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 clone selection. Possible positive clones are screened using the following primers (M13-47: 5'-AGGGTTTTCCCAGTCACG-3', M13-48: 5'-GAGCGGATAACAATTTCACAC-3'), using the TAKARA Premix Taq kit. TM ™ (Ex Taq TM Prepare a 25 μL reaction system using™ Version 2.0 plus dye (Code No. RR902A):

[0188] 1)Premix Taq (Ex Taq Version 2.0 plus dye) 12.5 μL;

[0189] 2) Template DNA 2 μL;

[0190] 3) M13-47 (10 μM) 1 μL;

[0191] 4) M13-48 (10 μM) 1 μL;

[0192] 5) Add sterile water to 25 μL.

[0193] 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.

[0194] Amplified fragments of 2-3 kb were considered suspected positive. Small-scale 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 clones were named pUCmT-D1A (5.3 kb), pUCmT-D2A (4.8 kb), pUCmT-D3A (5.1 kb), and pUCmT-D4A (5.3 kb), respectively; the inserted fragments were named D1A (2559 bp), D2A (2028 bp), D3A (2380 bp), and D4A (2579 bp), respectively. The four subclones were transformed into DH5α host bacteria to prepare 25% glycerol-containing bacteria, which were stored at -80℃ for later use.

[0195] The long genome sequences of cloned DENV types 1-4 are as follows:

[0196] >D1A (DENV-1, Hawaii, KM204119, nt3-2564)

[0197]

[0198] >D2A(DENV-2, New Guinea C, KM204118,nt1-2028)

[0199]

[0200] >D3A (DENV-3,H87, KU050695, nt1-2380)

[0201]

[0202] >D4A(DENV-4, H241, KR011349, nt1-2572)

[0203]

[0204] 7. Construction of prokaryotic expression vectors pACYCAR-D1A (8.1 kb), pACYCAR-D2A (7.7 kb), pACYCAR-D3A (8.0 kb), and pACYCAR-D4A (8.1 kb).

[0205] Using an in-fusion cloning strategy, the vector pACYCAR (5.7 kb) and insert fragments D1A (2414 bp), D2A (2022 bp), D3A (2374 bp), and D4A (2394 bp) were amplified separately. Utilizing the homologous sequences of 15-25 nt at the ends of the vector and insert fragments, the cells were directly transformed into competent cells at 50°C for 5-60 minutes under the action of T5 exonuclease, DNA polymerase, and DNA ligase.

[0206] (1) Primer design

[0207] Based on the vector pACYCAR (5.7 kb) and the DENV insert sequences and sequence sites of types 1-4: DENV-1 genome fragment sequence position nt7-2420 (2414 bp), DENV-2 genome fragment sequence position nt7-2028 (2022 bp), DENV-3 genome fragment sequence position nt7-2380 (2374 bp), and DENV-4 genome fragment sequence position nt7-2400 (2394 bp), primers were designed using the TAKARA online tool as follows:

[0208] pAC-AR328F: 5'-GCCACGCGATCGCTGACG-3',

[0209] pAC-AR319R: 5'-ATCCAATTGAGATCTGCCATATG-3',

[0210] acD1A7f: 5'-AGATCTCAATTGGATTAGTCTGTGGACCGACAAGAACAG-3',

[0211] acD1A2420r: 5'-CAGCGATCGCGTGGCCGCCTGAACCATGACTCCTAGG-3',

[0212] acD2A7f: 5'-AGATCTCAATTGGATTAGTCTACGTGGACCGACAAAG-3',

[0213] acD2A2028r: 5'-CAGCGATCGCGTGGCTGGGCTATCTTTTTCGTTACG-3',

[0214] acD3A7f: 5'-AGATCTCAATTGGATTAGTCTACGTGGACCGACA-3',

[0215] acD3A2380r: 5'-CAGCGATCGCGTGGCGATTCCTATCGCAATGCATGA-3',

[0216] acD4A7f: 5'-AGATCTCAATTGGATTAGTCTGTGTGGACGACAAGG-3',

[0217] acD4A2400r: 5'-CAGCGATCGCGTGGCAGAGTGATTCCTCCAACAGCTATGC-3'.

[0218] The primer oligonucleotides were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0219] (2) Reverse amplification of the full-length pACYCAR (5.7 kb) plasmid

[0220] Linear vector DNA templates were obtained by digesting pACYCAR plasmid with FseI. Using pAC-AR328F and pAC-AR319RF as primers, the DNA was extracted according to the TaKaRa Premix Taq kit. TM ™ (Ex Taq TM Prepare a 200 μL reverse PCR system using™ Version 2.0 plus dye (Code No. RR902A):

[0221] 1)Premix Taq (Ex Taq Version 2.0 plus dye) 100 μL;

[0222] 2) Template DNA 2 μL,

[0223] 3) pAC-AR328F (10 μM) 8 μL;

[0224] 4) pAC-AR319R (10 μM) 8 μL;

[0225] 5) Add sterile water to 200 μL.

[0226] Mix well, dispense 50 μL into 0.2 mL thin-walled PCR tubes, and perform PCR amplification.

[0227] 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.

[0228] 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.

[0229] (3) PCR amplification of long fragments of the DENV genome of types 1-4

[0230] Using 1:20 diluted DENV subcloning plasmid DNA of types 1-4, pUCmT-D1A (5.3 kb), pUCmT-D2A (4.8 kb), pUCmT-D3A (5.1 kb), and pUCmT-D4A (5.3 kb), as templates, matched upstream and downstream primers were selected and applied 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):

[0231] 1)Premix Taq (Ex Taq Version 2.0 plus dye) 50 μL;

[0232] 2) Template DNA 4 μL;

[0233] 3) Upstream primer (10 μM) 4 μL;

[0234] 4) Downstream primer (10 μM) 4 μL;

[0235] 5) Add sterile water to 100 μL.

[0236] Mix well and aliquot into 0.2 mL thin-walled PCR tubes at 50 μL / tube for PCR 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.

[0237] After amplification, the amplification products were combined and observed by 1% agarose gel electrophoresis. Products with a size of approximately 2-3 kb were considered potentially positive. The amplification products were then recovered and purified.

[0238] (4) Type 1-4 DENV genome fragments were seamlessly cloned with pACYCAR plasmid (5.7 kb).

[0239] 1) Seamless cloning reaction and transformation of host bacteria

[0240] Following the instructions of the TAKARA In-Fusion Snap Assembly Master Mix kit (Code No. 638948), 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 plasmid (5.7 kb). Recover and purify 3 μL of the DENV long fragments of types 1-4 (D1A, 2414 bp; D2A, 2022 bp; D3A, 2374 bp; D4A, 2394 bp). Add deionized water to a final volume of 20 μL. Mix well and incubate at 50°C for 2 h. 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 on a shaker at 180 rpm. Spread the mixture onto LB agar plates containing chloramphenicol and incubate overnight at 37°C.

[0241] 2) Screening for positive clones

[0242] 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 bacterial cells from 100 μL of 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 8,000 g for 2 min. Use the supernatant as a DNA template for cloning screening. Identify the inserted fragments (D1A, 2414 bp; D2A, 2022 bp; D3A, 2374 bp; D4A, 2394 bp) using primers DuetUP2:5'-TTGTACACGGCCGCATAATC-3' and T7-ter:5'-TGCTAGTTATTGCTCAGCGG-3'. Use the TaKaRa PremixTaq kit. TM ™ (Ex Taq TM Prepare the reaction system (25 μL) using Ex Taq (Ex Taq Version 2.0 plus dye) (Code No. RR902A): 12.5 μL Premix Taq, 2 μL template DNA, 1 μL DuetUP2 (10 μM), 1 μL T7-ter (10 μM), and add sterile water to 25 μL.

[0243] Amplification conditions: 94℃ for 4 min; 94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 2.5 min, 30 cycles; 72℃ for 10 min. After amplification, the product size was observed by 1% agarose gel electrophoresis. Products of 2-3 kb were suspected positive clones.

[0244] 3) Large-scale preparation of positive clone plasmid DNA

[0245] Take 1 mL of the suspected positive clone culture and inoculate it into 100 mL of chloramphenicol-containing LB liquid medium. Incubate overnight at 37°C with a shaker at 180 rpm to 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.

[0246] Centrifuge 100 mL of overnight culture at 4,000 rpm for 10 min, collect the cells, and discard or aspirate the culture medium.

[0247] Add 10 mL of Buffer P1 to the bacterial pellet and aspirate or shake until the bacterial cells are completely suspended.

[0248] 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.

[0249] Add 14 mL of Buffer P3, immediately invert 5-10 times, and let stand at room temperature for 5 minutes.

[0250] Centrifuge at 4,000 rpm for 15 min.

[0251] 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.

[0252] Discard the liquid in the collection tube and place the adsorption column into the same collection tube.

[0253] Add 5 mL Wash Solution to the adsorption column and centrifuge at 4,000 rpm for 5 min.

[0254] Discard the liquid in the collection tube and place the adsorption column into the same collection tube.

[0255] Repeat the washing process once.

[0256] Place the empty adsorption column and collection tube into a centrifuge and centrifuge at 4,000 rpm for 10 min.

[0257] 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 suspected positive plasmid DNA solution at -20℃ or use it for subsequent experiments.

[0258] 4) Sequencing verification of DENV inserts of types 1-4: D1A (2414 bp), D2A (2022 bp), D3A (2374 bp), and D4A (2394 bp).

[0259] A large number of plasmid DNA samples from 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. Clones that correctly inserted the DENV genome fragments of types 1-4 were named pACYCAR-D1A (8.1 kb), pACYCAR-D2A (7.7 kb), pACYCAR-D3A (8.0 kb), and pACYCAR-D4A (8.1 kb), respectively, and inserted the D1A (2414 bp), D2A (2022 bp), D3A (2374 bp), and D4A (2394 bp) fragments, respectively.

[0260] (II) Expression and purification of long-fragment armored RNA of DENV genome of types 1-4 (DENV-1 nt7-2420, DENV-2 nt7-2028, DENV-3 nt7-2380, DENV-4 nt7-2400)

[0261] 1. BL21(DE3) host bacteria induce expression

[0262] (1) The prokaryotic expression vectors pACYCAR-D1A (8.1 kb), pACYCAR-D2A (7.7 kb), pACYCAR-D3A (8.0 kb), and pACYCAR-D4A (8.1 kb) were transformed into BL21 (DE3) host bacteria, and single colonies were picked from chloramphenicol resistance plates and inoculated into 5 mL LB liquid medium containing chloramphenicol. The culture was carried out overnight at 37°C and 200 rpm.

[0263] (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;

[0264] (3) Add IPTG inducer to a final concentration of 1 mmol / L and incubate overnight at 37°C with shaking at 200 rpm.

[0265] 2. Purification of DENV armored RNA of types 1-4

[0266] (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.

[0267] (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.

[0268] 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.

[0269] (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.

[0270] (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 DENV armored RNA of type 1-4, which should be stored in a freezer at -80℃.

[0271] (III) Stability verification of DENV-1, DENV-2, DENV-3, and DENV-4 armored RNA of types 1-4

[0272] Take 1 mL of purified DENV armored RNA samples of types 1-4 stored in a -80℃ freezer, dilute with 1×PBS in a 1:10 ratio in a 15 mL centrifuge tube, aliquot 200 μL / tube into 1.5 mL microcentrifuge tubes, and store in a -80℃ freezer for later use.

[0273] 1. Temperature stability test

[0274] (1) Treatment at different temperatures

[0275] For each of the above-mentioned aliquoted DENV armored RNA samples of types 1-4, 16 tubes were taken. Four tubes were placed at 37℃, 25℃, 4℃, and -20℃ respectively. One tube was retrieved at 1 day, 7 days, 14 days, and 28 days and placed in a -80℃ refrigerator for testing.

[0276] (2) DENV armored RNA extraction

[0277] DENV armored RNA of types 1-4 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.

[0278] (3) Primers and probes for detecting DENV genomic fragments in DENV armored RNA of types 1-4

[0279] The U.S. Centers for Disease Control and Prevention (CDC) recommended the following primer-probe combination for real-time fluorescent RT-PCR of dengue virus types 1-4 in 2016:

[0280] Upstream primer DENV-F: 5'-TAGTCTRCGTGGACCGACAAG-3',

[0281] Downstream primer 1 DENV-R1: 5'-CAGTTGACACRCGGTTTCTC-3',

[0282] Downstream primer 2 DENV-R2 5'-GGGTTGATACGCGGTTTCTC-3',

[0283] Fluorescent probe DENV-P: 5'-FAM-CGYCTWTCAATATGCTGAAACGCG-BHQ1-3'.

[0284] The target region of this system is the 5'UTR gene (nt5-175, KM204119, KM204118, KU050695, KR011349) of dengue virus type 1-4 standard strains, which is suitable as a primer-probe for RT-qPCR detection of the prepared DENV armored RNA. Primers and probe oligonucleotides were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0285] (4) Real-time fluorescence RT-PCR detection of DENV genomic fragments in DENV armored RNA of types 1-4

[0286] 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):

[0287] 1)One Step PrimeScript III RT-qPCR Mix (2×) 10 μL;

[0288] 2) RNase-Free H2O 6.4 μL;

[0289] 3) DENV-F (10 μM) 0.4 μL;

[0290] 4)DENV-R1 (10 μM) 0.4 μL;

[0291] 5)DENV-R2 (10 μM) 0.4 μL;

[0292] 6)DENV-P (10 μM) 0.4 μL;

[0293] 7) Template 2 μL.

[0294] 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.

[0295] (5) Temperature stability test results of DENV armored RNA of types 1-4 ( Figure 1-4 )

[0296] The results showed that DENV genomic fragments of DENV types 1-4 (DENV-1, DENV-2, DENV-3, DENV-4) 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). The DENV genomic RNA of DENV types 1-4 (DENV-1, DENV-2, DENV-3, DENV-4) was stable under temperature conditions.

[0297] 2. Type 1-4 DENV armored RNA nuclease resistance test

[0298] (1) Nuclease-resistant treatment

[0299] Take 1 mL of purified DENV armored RNA sample stored at -80℃ and dilute it with 1×PBS at a ratio of 1:10 in 15 mL centrifuge tubes. Aliquot the diluted sample into 200 μL / tube microcentrifuge tubes. Take four tubes of each of the DENV armored RNA types 1-4 and aliquot them into 200 μL / tube microcentrifuge tubes. Proceed as follows:

[0300] 1) Add 5 μL of DNase I (5 U / μL) + 10 μL of DNase I buffer (10×) to tube 1;

[0301] 2) Add 5 μL of RNase (10 mg / mL) to tube 2;

[0302] 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;

[0303] 4) Tube 4 served as an untreated control.

[0304] The 16 sample tubes were placed in a 37°C water bath for 30 minutes.

[0305] (2) Real-time fluorescent RT-PCR detection of DENV armored RNA of types 1-4

[0306] Viral 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 a final elution volume of 50 μL.

[0307] 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):

[0308] 1)One Step PrimeScript III RT-qPCR Mix (2×) 10 μL;

[0309] 2) RNase-Free H2O 6.4 μL;

[0310] 3) DENV-F (10 μM) 0.4 μL;

[0311] 4)DENV-R1 (10 μM) 0.4 μL;

[0312] 5)DENV-R2 (10 μM) 0.4 μL;

[0313] 6)DENV-P (10 μM) 0.4 μL;

[0314] 7) Template 2 μL.

[0315] 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.

[0316] (3) Results of nuclease resistance test for DENV armored RNA of types 1-4 ( Figure 5-8 )

[0317] The results showed that DENV genomic fragments could be detected by real-time fluorescent RT-PCR after treatment with DNase and RNase for DENV armor RNA of types 1-4 (DENV-1, DENV-2, DENV-3, DENV-4), indicating that DENV armor RNA of types 1-4 (DENV-1, DENV-2, DENV-3, DENV-4) is resistant to nucleases.

[0318] 3.1-4 type DENV armored RNA serial dilution

[0319] (1) Series of dilution treatments

[0320] Take 200 μL of purified DENV armored RNA sample stored at -80℃ and extract viral RNA using the TAKARA TaKaRa MiniBEST 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 detection of DENV fragments.

[0321] (2) Real-time fluorescence RT-PCR detection of DENV genomic fragments in DENV armored RNA of types 1-4

[0322] 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):

[0323] 1)One Step PrimeScript III RT-qPCR Mix (2×) 10 μL;

[0324] 2) RNase-Free H2O 6.4 μL;

[0325] 3) DENV-F (10 μM) 0.4 μL;

[0326] 4)DENV-R1 (10 μM) 0.4 μL;

[0327] 5)DENV-R2 (10 μM) 0.4 μL;

[0328] 6) DENV-P (10 μM) 0.4 μL;

[0329] 7) Template 2 μL.

[0330] 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.

[0331] (3) Results of serial dilutions of DENV armored RNA of types 1-4 ( Figure 9-12 )

[0332] The results showed that the lowest dilution detectable by real-time fluorescent RT-PCR for DENV-1, DENV-2, DENV-3, and DENV-4 armored RNA of DENV types 1-4 (DENV-1, DENV-2, DENV-3, and DENV-4) was 10. -5 10 -8 10 -7 10 -5 .

[0333] (V) Digital PCR quantification of DENV-1, DENV-2, DENV-3, and DENV-4 armored RNA of types 1-4

[0334] 1. Method

[0335] Absolute quantification of DENV armored RNA of types 1-4 was performed using Stilla digital PCR (French). The optimal dilution for digital PCR detection of the tested DENV armored RNA of types 1-4 was: DENV-1 10... -2.5 DENV-2 10 -6 DENV-3 10 -5.5DENV-4 10 -3 .

[0336] 2. Digital PCR primers and probes

[0337] Digital PCR quantification of DENV armored RNA samples of types 1-4 was performed using the primer-probe system for real-time fluorescent RT-PCR of DENV types 1-4 recommended by the US Centers for Disease Control and Prevention (CDC), DENV-F / DENV-R1 / DENV-R2 / DENV-P. The primers and probe oligonucleotides were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0338] 3. Digital PCR Quantitative Experiment

[0339] (1) Digital PCR reaction system:

[0340] 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.

[0341] Prepare the reaction mixture (25 μL) according to the instructions:

[0342] 1) MasterMix 12.5 μL;

[0343] 2) 10× fluorescein sodium salt (1 μM) 2.5 μL;

[0344] 3) DENV-F (20 μM) 1.25 μL;

[0345] 4) DENV-R1 (20 μM) 1.25 μL;

[0346] 5)DENV-R2 (20 μM) 1.25 μL;

[0347] 6) DENV-P (5 μM) 1.25 μL;

[0348] 7) Add H2O to bring the volume to 22.5 μL.

[0349] The above reagents were shaken to mix well, and dispensed into 22.5 μL tubes. 2.5 μL of diluted DENV armored RNA of type 1-4 to be tested was added to each tube and mixed well. 25 μL of the mixture was then loaded onto a digital PCR amplification chip (Saphhire Chips for the Naica Crystal Digital PCR System, Ref # C14012).

[0350] (2) Digital PCR amplification:

[0351] Digital PCR amplification was performed using a STILLA TECHNOLOGIES digital PCR instrument (Ref # H14000) from France. The amplification program is as follows:

[0352] 1) Zone: 40℃, AP950

[0353] 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.

[0354] 3) Pressure release: 25℃.

[0355] 4. Reading of digital PCR quantitative data for DENV armored RNA of types 1-4

[0356] (1) Quantitative results of digital PCR of DENV armored RNA of types 1-4

[0357] 1) DENV-1 armored RNA (10 -2.5 Digital PCR quantification Figure 13 .

[0358] 2) DENV-1 armored RNA (10 -2.5 Negative control digital PCR quantification Figure 14 .

[0359] 3) DENV-2 armored RNA (10 -6 Digital PCR quantification Figure 15 .

[0360] 4) DENV-2 armored RNA (10 -6 Negative control digital PCR quantification Figure 16 .

[0361] 5) DENV-3 armored RNA (10 -5.5 Digital PCR quantification Figure 17 .

[0362] 6) DENV-3 armored RNA (10 -5.5 Negative control digital PCR quantification Figure 18 .

[0363] 7) DENV-4 armored RNA (10 -3 Digital PCR quantification Figure 19 .

[0364] 8) DENV-4 armored RNA (10 -3 Negative control digital PCR quantification Figure 20 .

[0365] (2) Table 1 shows the quantitative results of digital PCR for DENV armored RNA of types 1-4. The concentrations of DENV armored RNA were 4.36 × 10⁻⁴ for DENV-1 armored RNA. 4 copy / μL, DENV-2 armored RNA 1.68×10 7 copy / μL, DENV-3 armored RNA 4.39×10 7 copy / μL, DENV-4 armored RNA 4.37×10 5 copy / μL.

[0366] Table 1. Quantitative results of DENV armored RNA type 1-4 by digital PCR

[0367]

[0368] 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 long-fragment armored RNA reference of type 1-4 DENV genome, characterized in that, Prepare according to the following steps: (I) Construction of the prokaryotic expression backbone vector pACYCAR MS2 phage genomic RNA was cultured and extracted. A 1.7 kb fragment containing the MS2 maturation enzyme, envelope protein gene and packaging recognition signal sequence was amplified by RT-PCR. The fragment was ligated with the double-digested pACYCDuet-1 plasmid, transformed into DH5α competent cells and screened and identified to obtain the backbone vector pACYCAR. (II) Cloning of long fragments of dengue virus genomes of types 1-4 Total RNA was extracted from inactivated DENV standard strains of types 1-4. A long genomic fragment covering the 5'UTR, capsid protein, membrane protein, and part of the outer membrane protein was amplified by RT-PCR. After TA cloning and screening, subcloned plasmids pUCmT-D1A (5.3 kb), pUCmT-D2A (4.8 kb), pUCmT-D3A (5.1 kb), and pUCmT-D4A (5.3 kb) were obtained. The inserted fragments were named D1A (2559 bp), D2A (2028 bp), D3A (2380 bp), and D4A (2579 bp), respectively. (III) Construction of prokaryotic expression vectors pACYCAR-D1A (8.1 kb), pACYCAR-D2A (7.7 kb), pACYCAR-D3A (8.0 kb), and pACYCAR-D4A (8.1 kb) Using seamless cloning technology, DENV-1 nt7-2420, DENV-2 nt7-2028, DENV-3 nt7-2380, and DENV-4 nt7-2400 were amplified using the long genomic fragments of types 1-4 cloned in step (II) as templates. These fragments were then ligated into linearized pACYCAR vectors amplified by reverse PCR, transformed into Top10 competent cells, and screened to obtain recombinant expression vectors pACYCAR-D1A (8.1 kb), pACYCAR-D2A (7.7 kb), pACYCAR-D3A (8.0 kb), and pACYCAR-D4A (8.1 kb). (iv) Induced expression of armored RNA The recombinant expression vector from step (III) was transformed into BL21(DE3) host bacteria, and expressed by IPTG to obtain engineered bacteria containing DENV armor RNA of types 1-4; (v) Purification of armored RNA The engineered bacterial cells were collected, washed with PBS, and sonicated. Then, they were purified by NaCl salting out, PEG6000 precipitation, and chloroform extraction to obtain the long-fragment armored RNA reference of type 1-4 DENV genome.

2. The method for preparing the DENV genome long fragment armored RNA reference of type 1-4 according to claim 1, characterized in that: In step (1), the MS2 phage genome was extracted using the TAKARA TAKARAMiniBEST Viral RNA / DNA Extraction Kit Ver 5.0, Code No. 9766.

3. The method for preparing the DENV genome long fragment armored RNA reference of type 1-4 according to claim 2, characterized in that: In step (I), the upstream primer for RT-PCR amplification of the MS2 1.7 kb fragment was: AR-Bam10F: 5'-CGCGGATCCTTTCGGGGTCCTGCTCAACTT-3', and the downstream primer was: AR-Hin1789R: 5'-CCCAAGCTTGAGTTGAACTTCTTTGTTGTCTTC-3'. Double digestion was performed using BamHI and HindIII, and the ligation system was incubated overnight at 16°C.

4. The method for preparing the DENV genome long fragment armored RNA reference of type 1-4 according to claim 1, characterized in that: In step (II), the DENV standard strains of types 1-4 are DENV-1, Hawaii, GenBank accession number KM204119; DENV-2, New Guinea C, GenBank accession number KM204118; DENV-3, H87, GenBank accession number KU050695; DENV-4, H241, GenBank accession number KR011349; The primer combinations for RT-PCR amplification are as follows: Den1-1F: 5'-AGTTGTTAGTCTACGTGGAC-3', Den1-2559R: 5'-CCAATGGCYGCTGAYAGTCT-3', Den2-1F: 5'-AGTWGTTAGTCTACGTGGAC-3', Den2-2028R: 5'-TGGGCTGTCTTTTTCTGTGA-3', Den3-1F: 5'-AGTTGTTAGTCTACGTGGAC-3', Den3-2380R: 5'-GATTCCTATCGCAATGCATG-3', Den4-1F: 5'-AGTTGTTAGTCTGTGTGGACCGACAA-3', Den4-2579R: 5'-GGGCATTYAATATTGCAGACGCTA-3'; The amplification conditions were: 50℃ for 30 min; 94℃ for 2 min; 94℃ for 30 sec, 48℃ / 45℃ for 30 sec, 72℃ for 2.5 min, 30 cycles; 72℃ for 10 min. Except for Den1-1F and Den1-2559R, which were annealed at 48℃, the annealing temperature for other primer combinations was 45℃.

5. The method for preparing the DENV genome long fragment armored RNA reference according to claim 1, characterized in that: The primers used for seamless cloning in step (iii) include: pAC-AR328F 5'-GCCACGCGATCGCTGACG-3', pAC-AR319R 5'-ATCCAATTGAGATCTGCCATATG-3, acD1A7f: 5'-AGATCTCAATTGGATTAGTCTGTGGACCGACAAGAACAG-3', acD1A2420r 5'-CAGCGATCGCGTGGCCGCCTGAACCATGACTCCTAGG-3', acD2A7f 5'-AGATCTCAATTGGATTAGTCTACGTGGACCGACAAAG-3', acD2A2028r 5'-CAGCGATCGCGTGGCTGGGCTATCTTTTTCGTTACG-3', acD3A7f 5'-AGATCTCAATTGGATTAGTCTACGTGGACCGACA-3', acD3A2380r: 5'-CAGCGATCGCGTGGCGATTCCTATCGCAATGCATGA-3', acD4A7f 5'-AGATCTCAATTGGATTAGTCTGTGTGGACGACAAGG-3', acD4A2400r 5'-CAGCGATCGCGTGGCAGATGTGATTCCTCCAACAGCTATGC-3'; The seamless cloning reaction was incubated in a metal bath at 50°C for 2 h. After the ligation product was transformed into Top10 competent cells, positive clones were screened using DuetUP2 and T7-ter primers. The primer sequences for DuetUP2 and T7-ter are as follows: DuetUP2:5'-TTGTACACGGCCGCATAATC-3', T7-ter: 5'-TGCTAGTTATTGCTCAGCGG-3'.

6. The method for preparing the DENV genome long fragment armored RNA reference of type 1-4 according to claim 5, characterized in that: In step (iv), the final concentration of IPTG is 1 mmol / L, and the induction conditions are overnight culture at 37°C with shaking at 200 rpm.

7. The method for preparing the DENV genome long fragment armored RNA reference of type 1-4 according to claim 6, characterized in that: The parameters for ultrasonic disruption in step (5) are: ultrasonic power 15%, 1.5s pulse, 3s interval, total duration 30min; final NaCl concentration 1 mol / L, final PEG6000 concentration 10% (w / v), and purified product obtained by chloroform extraction after overnight in an ice bath.

8. A type 1-4 DENV genome long fragment armored RNA reference prepared by the method of any one of claims 1-7.

9. The application of the type 1-4 DENV genome long fragment armored RNA reference as described in claim 8 in the research and development and verification of DENV nucleic acid detection reagents, or in the plotting of DENV nucleic acid detection standard curves as a standard, or in the assessment of dengue virus nucleic acid detection capabilities and interlaboratory quality control in primary laboratories, or in the preparation of dengue virus nucleic acid detection kits as a positive control.

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