A zika virus genome segment coat rna reference and methods of making and using same

Abstract

The application discloses a Zika virus genomic fragment armoured RNA reference substance and a preparation method and application thereof, and is characterized in that the following steps are included: (1) constructing a prokaryotic expression skeleton vector pACYCAR (2) cloning a ZKV genomic fragment ZKA (nt81-2350) (3) constructing a prokaryotic expression vector pACYCAR-ZKA (4) inducing expression of armoured RNA (5) purifying the armoured RNA. The application solves the technical problems of high biological safety risk, poor stability and insufficient gene fragment coverage of existing ZKV nucleic acid detection reference substances. The application fills the blank of the lack of high-quality reference substances for ZKV detection.

Description

Technical Field

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

[0002] Arboviruses are a class of viruses transmitted by blood-sucking insects (mosquitoes, ticks, midges, black flies, sandflies, mites, lice, stink bugs, etc.). These viruses can multiply within the insects but are not pathogenic to the insects themselves. The insects transmit these viruses to humans and livestock through their bites, causing disease; therefore, arboviruses are zoonotic viruses. They include over 500 species from families such as Flaviviridae, Monovaviridae, and Bunyavirales, and can cause symptoms such as fever, hemorrhage, and encephalitis. Approximately 3,000 species of mosquitoes have been identified worldwide, of which 300 can transmit arboviruses. Arboviruses, represented by Zika virus, dengue virus, yellow fever virus, and chikungunya virus, threaten the health of approximately 3.9 billion people in tropical and subtropical regions globally.

[0003] Zika virus (ZKV) belongs to the genus Orthoflavivirus of the family Flaviviridae. It was first discovered in Uganda in 1947, and its main vectors are the Aedes aegypti and Aedes albopictus mosquitoes. The ZKV genome is a single-stranded positive-sense RNA, approximately 10.8 kb in length, and can be divided into Asian and African genotypes. Zika virus disease caused by ZKV infection is a self-limiting acute illness, clinically characterized primarily by fever, rash, conjunctivitis, or arthralgia, and rarely causes death.

[0004] The etiological detection methods for Zika virus disease include viral nucleic acid detection, IgM antibody detection, neutralizing antibody detection, and virus isolation. Due to the strong serological cross-reactivity between Zika virus and other viruses in the Flaviviridae family, 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 infected individuals and close contacts, and for effectively controlling further viral transmission. ZKV nucleic acid detection includes detecting ZKV-specific genomic fragments and determining the full-length or partial genome sequence. In vitro amplification of viral genome fragments is the most sensitive, rapid, and widely used laboratory diagnostic method. Various methods for amplifying and detecting ZKV-specific nucleic acids have been developed, most based on polymerase chain reaction (PCR) technology. Other methods include reverse transcription loop-mediated isothermal amplification (RT-LAMP) and transcription-mediated amplification (TMA), which can also be used as molecular diagnostic methods for ZKV detection in clinical samples.

[0005] 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.

[0006] 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.

[0007] 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 them to form pseudovirus particles. 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

[0008] To address the aforementioned technical problems, the present invention aims to provide a Zika virus genome fragment armored RNA reference, its preparation method, and its application. This solves the technical problems of existing ZKV 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 ZKV detection.

[0009] To achieve the first objective mentioned above, the present invention provides the following technical solution: a method for preparing a Zika virus genome fragment armored RNA reference, characterized by comprising the following steps:

[0010] (1) Construction of prokaryotic expression backbone vector pACYCAR

[0011] MS2 phage genomic RNA was cultured and extracted. A 1.7kb fragment containing the MS2 maturation enzyme, envelope protein gene and packaging recognition signal sequence was amplified by PCR. This fragment was ligated to the 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.

[0012] (2) Cloning the ZKV genome fragment ZKA (nt81-2350)

[0013] Total RNA was extracted from ZKV inactivated virus, and the genomic fragment ZKA (nt81-2350) covering capsid protein, membrane protein and some outer membrane protein genes was amplified by RT-PCR. After TA cloning and screening, the subcloned plasmid pUCmT-ZKA was obtained.

[0014] (3) Construction of prokaryotic expression vector pACYCAR-ZKA

[0015] Using seamless cloning technology, ZKA (nt81-2340) was amplified using the ZKV genome fragment cloned in step (2) as a template and then ligated with the linearized pACYCAR vector amplified by reverse PCR. After transformation into Top10 competent cells, the recombinant expression vector pACYCAR-ZKA (8.0kb) was obtained.

[0016] (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 ZKV armored RNA;

[0017] (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 ZKV genome fragment ZKA (nt81-2340) armored RNA reference.

[0018] In the above scheme: In step (1), 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 scheme: In step (1), the upstream primer AR-Bam10F was used for RT-PCR amplification of the MS2 1.7kb fragment: 5'-CGCGGATCCTTTCGGGGTCCTGCTCAACTT-3'.

[0020] Downstream primer AR-Hin1789R: 5'-CCCAAGCTTGAGTTGAACTTCTTTGTTGTCTTC-3';

[0021] The amplification conditions were: 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.

[0022] In the above scheme: in step (2), the ZKV strain is GZDJ-1685, GenBank accession number MF099651;

[0023] The upstream and downstream primers for RT-PCR amplification are as follows:

[0024] ZIKV-81F: 5'-GGATTTGGAAACGAGAGTTTC-3',

[0025] ZIKV-2350R: 5'-TCCAAACAATGATTTGAAAGCTGCTC-3'

[0026] The amplification conditions were: 50℃ for 30 min; 94℃ for 2 min; 94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 2 min, 30 cycles; 72℃ for 10 min.

[0027] In the above scheme: In step (3), the primers for seamless cloning include:

[0028] pAC-AR328F: 5'-GCCACGCGATCGCTGACG-3',

[0029] pAC-AR319R: 5'-ATCCAATTGAGATCTGCCATATG-3',

[0030] acZKA80f: 5'-AGATCTCAATTGGATGAAACGAGAGTTTCTGGTCATG-3',

[0031] acZKA2340r: 5'-CAGCGATCGCGTGGCCAAACAATGATTTGAAAGCTGCTCC-3'; Seamless cloning reaction was incubated in a metal bath at 50℃ for 2 hours, and positive clones were screened using DuetUP2 and T7-ter as primers.

[0032] In the above scheme: In step (4), the final concentration of IPTG is 1 mmol / L, and the induction conditions are overnight culture at 37℃ and 200 rpm with shaking; before induction, the engineered bacteria are cultured at a ratio of 1:100 and cultured at 37℃ and 200 rpm for 4 hours with shaking.

[0033] In the above scheme: In step (5), the parameters of ultrasonic disruption are: ultrasonic power 15%, 1.5s pulse, 3s interval, total duration 30min; final concentration of NaCl is 1mol / L, final concentration of PEG6000 is 10% (w / v), and after overnight in an ice bath, the purified product is obtained by chloroform extraction.

[0034] A method for preparing a Zika virus genome fragment armored RNA reference product. The Zika virus genome fragment armored RNA reference product is prepared by the method described above. The ZKV gene fragment of the reference product is ZKA (nt81-2340), with a length of 2260 bp, covering capsid proteins, membrane proteins, and some outer membrane protein genes; it can withstand temperature treatment at 37℃, 25℃, 4℃, and -20℃ for 28 days, and is resistant to DNase I, RNase, and combined treatment with both; the concentration quantified by digital PCR is 5.34 × 10⁻⁶. 5 copy / μL.

[0035] The Zika virus genome fragment armored RNA reference material can be used as a template in the research and development and validation of Zika virus nucleic acid detection reagents, or as a standard in the plotting of Zika virus nucleic acid detection standard curves, or as a quality control material in the assessment of Zika virus nucleic acid detection capabilities and inter-laboratory quality control in primary laboratories, or as a positive control in the preparation of Zika virus nucleic acid detection kits.

[0036] Compared with the prior art, the present invention has the following significant advantages:

[0037] 1. High biosafety: The prepared armored RNA is a non-infectious pseudovirus particle with no autonomous replication ability. It does not require high-level biosafety laboratory operation, has no risk of infection, and has no biosafety risks. It is suitable for widespread use in most laboratories in China.

[0038] 2. Excellent stability: The reference product can withstand temperature treatment at 37℃, 25℃, 4℃, and -20℃ for 28 days, and can resist degradation by DNase I and RNase alone and in combination. It has good stability, is easy to transport, and can be stored for a long time under simple conditions, solving the problems of easy degradation and low temperature dependence of traditional reference products.

[0039] 3. Reasonable gene fragment coverage: The ZKV gene fragment of the reference product is a long fragment of 2260bp, covering capsid proteins, membrane proteins and some outer membrane protein genes, covering the core target region of ZKV nucleic acid detection, which can meet the quality control requirements of multi-target detection and is superior to existing short fragment reference products.

[0040] 4. Absolute quantification by digital PCR confirmed the reference concentration to be 5.34 × 10⁻⁶. 5 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 ZKV nucleic acid detection reagent development, clinical testing quality control, and laboratory competency assessment.

[0041] 5. A backbone vector was constructed based on pACYCDuet-1, and the ZKV long fragment was 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.

[0042] 6. The reference standard is a phage-like particle. Its nucleic acid extraction process is highly similar to that of natural ZKV in clinical samples. It can effectively simulate the RNA target to be tested and achieve full-process quality control from sample processing to reverse transcription. Attached Figure Description

[0043] Figure 1 The image shows the temperature stability test results of ZKA armored RNA (nt81-2340).

[0044] Figure 2 The image shows the results of the nuclease resistance test for ZKA armored RNA (nt81-2340).

[0045] Figure 3 This image shows the serial dilution results of ZKA armored RNA (nt81-2340).

[0046] Figure 4 ZKA armored RNA (10 -5 (Dilution) Digital PCR quantification graph.

[0047] Figure 5 ZKA armored RNA (10 -5 (Dilution) Negative control digital PCR quantification graph. Detailed Implementation

[0048] The present invention will be further described below with reference to embodiments.

[0049] 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.

[0050] Example 1

[0051] 1. MS2 phage culture

[0052] (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.

[0053] (2) Inoculate the overnight culture with fresh LB broth at a ratio of 1:100, and incubate at 37°C and 180 rpm for 4-5 hours until the logarithmic growth phase. Inoculate with frozen MS2 phage (ATCC 15597-B1™) and incubate at 37°C overnight.

[0054] (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.

[0055] 2. MS2 phage genome extraction

[0056] The procedure for the TAKARA MiniBEST Viral RNA / DNA Extraction Kit (Code No. 9766) is as follows:

[0057] (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.

[0058] (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.

[0059] (3) Add 500 μL of RWA buffer to the centrifuge column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0060] (4) Add 700 μL of RWB buffer to the centrifuge column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate;

[0061] (5) Repeat step 4;

[0062] (6) Place the centrifuge column on the collection tube and centrifuge at 12,000 rpm for 2 min.

[0063] (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.

[0064] 3. RT-PCR amplification of the MS2 (1.7 kb) fragment

[0065] (1) Primers for RT-PCR amplification of the MS2 (1.7 kb) fragment

[0066] Based on the MS2 phage genome sequence (GenBank accession number: NC_001417), primers were designed as follows to amplify fragments containing the MS2 maturation enzyme, envelope protein gene, and packaging recognition signal sequence:

[0067] Upstream primer AR-Bam10F: 5'-CGCGGATCCTTTCGGGGTCCTGCTCAACTT-3',

[0068] Downstream primer AR-Hin1789R: 5'-CCCAAGCTTGAGTTGAACTTCTTTGTTGTCTTC-3'.

[0069] 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.

[0070] (2) RT-PCR amplification

[0071] 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:

[0072] 1) 2×1 Step Buffer (Dye Plus) 25 μL;

[0073] 2)AR-Bam10F (10 μM) 2 μL;

[0074] 3) AR-Hin1789R (10 μM) 2 μL;

[0075] 4) Template RNA 2 μL;

[0076] 5) PrimeScript 1Step Enzyme Mix 2 μL;

[0077] 6)RNase Free dH2O up to 50 μL.

[0078] 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.

[0079] After the amplification reaction was completed, 1% agarose gel electrophoresis was performed, and a 1.7 kb fragment was observed as a positive result.

[0080] (3) Gel recovery of amplification products

[0081] 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:

[0082] 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;

[0083] 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;

[0084] 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.

[0085] 4) Place the adsorption column in a new centrifuge tube, add 35 μL of elution buffer 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 elution buffer for electrophoresis to observe the purity and concentration of the fragments, and store it in a -20℃ refrigerator for later use.

[0086] 4. Preparation of plasmid pACYCDuet-1 vector DNA (4.0 kb)

[0087] 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.

[0088] The pACYCDuet-1 vector DNA (4.0 kb) was extracted using the SanPrep column-based plasmid DNA mini-extraction kit from Sangon Biotech (Shanghai) Co., Ltd., product catalog number: B518191. The procedure is as follows:

[0089] (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;

[0090] (2) Take 5 mL of bacterial culture, centrifuge at 8,000g 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.

[0091] (3) Add 250 μL of Buffer P1 to the bacterial pellet and aspirate or shake until the bacterial cells are completely resuspended;

[0092] (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;

[0093] (5) Add 350 μL Buffer P3 and immediately gently invert the centrifuge tube 5-10 times to mix thoroughly;

[0094] (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.

[0095] (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.

[0096] (8) Repeat step 7 once.

[0097] (9) Place the empty adsorption column and collection tube into a centrifuge and centrifuge at 9,000g for 1 min.

[0098] (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.

[0099] 5. Construction of the prokaryotic expression backbone vector pACYCAR (5.7 kb)

[0100] (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, recovered amplified fragment DNA 20 μL, 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.

[0101] (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):

[0102] 1) 2 μL of 10×T4 DNA ligase buffer;

[0103] 2) Double enzyme digestion and recovery of vector pACYCDuet-15 μL;

[0104] 3) Double enzyme digestion to recover 5 μL of the inserted MS2 fragment;

[0105] 4) 1 μL of T4 DNA ligase;

[0106] 5) Add ddH2O to 20 μL.

[0107] 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.

[0108] (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.

[0109] 1) Premix Taq (Ex Taq Version 2.0 plus dye) 12.5 μL;

[0110] 2)ACYCDuetUP1 (10μM) 1 μL;

[0111] 3) DuetDOWN1 (10μM) 1 μL;

[0112] 4) 2 μL of template;

[0113] 5) Add sterile water to 25 μL.

[0114] 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.

[0115] (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.

[0116] The inserted fragment sequence is as follows:

[0117] MS2-1.7K_11-1789

[0118]

[0119] 6. Cloning of the ZKV genome fragment ZKA (nt81-2350)

[0120] The strain information is as follows: ZKV isolate GZDJ-1685 was obtained from the Vector-borne Disease Research Laboratory of the Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention. Its full-length polyprotein coding sequence can be found in GenBank accession number MF099651. For biosafety reasons, the introduced strain was an inactivated virus culture medium (with added viral RNA extract).

[0121] (1) Extraction of total viral RNA

[0122] The following are the operating steps using the kit (QIAGEN RNeasy Mini Kit, QIAGEN, Cat# 74104):

[0123] 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.

[0124] 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;

[0125] 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.

[0126] 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.

[0127] 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;

[0128] 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.

[0129] (2) RT-PCR amplification of the ZKV genomic fragment ZKA (nt81-2350)

[0130] Based on the ZKV reference strain MR-766 sequence information in the NCBI database (GenBank accession No. KX377335), the following subcloning primers were designed, covering the capsid, membrane, and envelope proteins of the MR-766 strain, with genomic sequence positions nt81-2350 and a full length of 2270 bp.

[0131] ZIKV-81F: 5'-GGATTTGGAAACGAGAGTTTC-3',

[0132] ZIKV-2350R: 5'-TCCAAACAATGATTTGAAAGCTGCTC-3'.

[0133] The primer oligonucleotides were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0134] Zika virus genomic fragment ZKA (nt81-2350) was amplified. Using genomic RNA extracted from ZKV isolate GZDJ-1685 as a template, ZIKV-81F / ZIKV-2350R were selected as upstream and downstream primers, respectively. 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.

[0135] 1) 2×1 Step Buffer (Dye Plus) 25 μL;

[0136] 2)ZIKV-81F (10μM) 2 μL;

[0137] 3)ZIKV-2350R (10 μM) 2 μL;

[0138] 4) Template RNA 2 μL;

[0139] 5) PrimeScript 1Step Enzyme Mix 2 μL;

[0140] 5)RNase Free dH2O up to 50 μL.

[0141] The amplification conditions were: 50℃ for 30 min; 94℃ for 2 min; 94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 2 min, 30 cycles; 72℃ for 10 min.

[0142] After the amplification reaction was completed, 1% agarose gel electrophoresis was performed. A positive result was indicated by a fragment of approximately 2270 bp in the amplified product. The amplified product was then recovered and purified by agarose gel electrophoresis.

[0143] (3) Zika virus genome fragment ZKA(nt81-2350)TA clone

[0144] 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):

[0145] 1) pUCm-T Vector (50 ng / µL) 1µL;

[0146] 2)10×Ligation Buffer 1 µL;

[0147] 3) 50% PEG4000 1 µL;

[0148] 4) T4 DNA Ligase 1 µL;

[0149] 5) Insert fragment (recovered amplification product) 4 µL;

[0150] 6) Add sterilized ddH2O to a final volume of 10 µL.

[0151] 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.

[0152] (4) Screening of Zika virus genome fragment ZKA (nt81-2350) positive clones

[0153] 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):

[0154] 1) Premix Taq (Ex Taq Version 2.0 plus dye) 12.5 μL;

[0155] 2) Template DNA 2 μL;

[0156] 3) M13-47 (10 μM) 1 μL;

[0157] 4) M13-48 (10 μM) 1 μL;

[0158] 5) Add sterile water to 25 μL.

[0159] Amplification parameters: 94℃ for 4 min; 94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 2 min, 30 cycles; 72℃ for 5 min.

[0160] Amplification products with a fragment close to 2270 bp are considered positive.

[0161] 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-ZKA (5.0 kb). The plasmid pUCmT-ZKA (5.0 kb) was transformed into DH5α host bacteria to prepare 25% glycerol-containing bacteria, which were stored at -80℃ for later use.

[0162] The sequence of the cloned Zika virus genome fragment ZKA (nt81-2350) is as follows:

[0163] ZKA

[0164]

[0165] 7. Construction of the prokaryotic expression vector pACYCAR-ZKA (8.0 kb)

[0166] Using an in-fusion cloning strategy, the vector pACYCAR (5.7 kb) and the insert fragment ZKA (nt81-2340) were amplified separately. Utilizing the homologous sequences of 15-25 nt at the ends of the vector and the insert fragment, 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.

[0167] (1) Primer design

[0168] Primers were designed using the TAKARA online tool based on the vector pACYCAR (5.7 kb) and the insert fragment ZKA (nt81-2340) sequence:

[0169] pAC-AR328F: 5'-GCCACGCGATCGCTGACG-3',

[0170] pAC-AR319R: 5'-ATCCAATTGAGATCTGCCATATG-3',

[0171] acZKA80f: 5'-AGATCTCAATTGGATGAAACGAGAGTTTCTGGTCATG-3',

[0172] acZKA2340r: 5'-CAGCGATCGCGTGGCCAAACAATGATTTGAAAGCTGCTCC-3'.

[0173] The primer oligonucleotides were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0174] (2) Reverse amplification of the full-length pACYCAR (5.7 kb) plasmid

[0175] The pACYCAR (5.7KB) 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):

[0176] 1)Premix Taq (Ex Taq Version 2.0 plus dye) 100 μL;

[0177] 2) Template DNA 2 μL;

[0178] 3) pAC-AR328F (10 μM) 8 μL;

[0179] 4) pAC-AR319R (10 μM) 8 μL;

[0180] 5) Add sterile water to 200 μL.

[0181] Mix well, dispense 50 μL into 0.2 mL thin-walled PCR tubes, and perform PCR amplification.

[0182] 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.

[0183] 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.

[0184] (3) PCR amplification of the ZKV genomic fragment ZKA (nt81-2340)

[0185] Using pUCmT-ZKA subcloned plasmid DNA (5 kb) diluted 1:20 as a template, and acZKA80f and acZKA2340r as primers, a PCR system (100 μL) was prepared according to the TaKaRa Premix Taq™ (Ex Taq™ Version 2.0 plus dye) kit (Code No. RR902A):

[0186] 1)Premix Taq (Ex Taq Version 2.0 plus dye) 50 μL;

[0187] 2) Template DNA 4 μL;

[0188] 3) acZKA80f (10 μM) 4 μL;

[0189] 4) acZKA2340r (10 μM) 4 μL;

[0190] 5) Add sterile water to 100 μL.

[0191] 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.

[0192] 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.

[0193] (4) Seamless cloning of pACYCAR plasmid (5.7 kb) and ZKV genome fragment ZKA (nt81-2340).

[0194] 1) Seamless cloning reaction and transformation of host bacteria

[0195] 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 plasmid (5.7 kb), recover and purify 3 μL of the insert fragment ZKA (nt81-2340), and 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 on a shaker at 180 rpm. Spread on LB agar plates containing chloramphenicol and incubate overnight at 37°C.

[0196] 2) Screening for positive clones

[0197] 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 insert fragment ZKA (nt81-2340) using primers DuetUP2:5'-TTGTACACGGCCGCATAATC-3' and T7-ter:5'-TGCTAGTTATTGCTCAGCGG-3'. Use the TaKaRa Premix Taq kit. TM (Ex TaqTM Prepare the reaction mixture (25 μL) using Premix Taq (Ex Taq Version 2.0 plus dye) (Code No. RR902A): 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 add 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, observe the product size using 1% agarose gel electrophoresis. Products with a size close to 2.2 kb are considered potentially positive clones.

[0198] 3) Large-scale preparation of positive clone plasmid DNA

[0199] 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.:

[0200] a) Centrifuge 100 mL of overnight culture at 4,000 rpm for 10 min, collect all cells, and discard or aspirate the culture medium.

[0201] b) Add 10 mL of Buffer P1 to the bacterial pellet and aspirate or shake until the bacterial cells are completely suspended.

[0202] c) 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 min.

[0203] d) Add 14 mL of Buffer P3, immediately invert 5-10 times, and let stand at room temperature for 5 min.

[0204] e) Centrifuge at 4,000 rpm for 15 min.

[0205] f) 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.

[0206] g) Discard the liquid in the collection tube and place the adsorption column into the same collection tube.

[0207] h) Add 5 mL Wash Solution to the adsorption column and centrifuge at 4,000 rpm for 5 min.

[0208] i) Discard the liquid in the collection tube and place the adsorption column into the same collection tube.

[0209] j) Repeat the washing step once.

[0210] k) Place the empty adsorption column and collection tube into a centrifuge and centrifuge at 4,000 rpm for 10 min.

[0211] 1) 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.

[0212] 4) Sequencing verification of the inserted ZKV genomic fragment ZKA (nt81-2340)

[0213] 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. The clone with the correct insertion of the ZKA fragment was named pACYCAR-ZKA (8.0 kb).

[0214] 8. Expression and purification of ZKV genomic fragment ZKA (nt81-2340) armored RNA

[0215] (1) BL21(DE3) host bacteria induce expression

[0216] 1) Transform the prokaryotic expression vector pACYCAR-ZKA (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.

[0217] 2) Transfer the culture to 100 mL of LB liquid medium containing chloramphenicol at a ratio of 1:100 for expansion culture, and culture at 37℃ with shaking at 200 rpm for 4 h;

[0218] 3) Add IPTG inducer to a final concentration of 1 mmol / L, and incubate overnight at 37°C with shaking at 200 rpm.

[0219] (2) Purification of ZKV genomic fragment ZKA (nt81-2340) armored RNA

[0220] 1) Collect bacterial cells in 50 mL centrifuge tubes in fractions, 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.

[0221] 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.

[0222] 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.

[0223] 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.

[0224] 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°C 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 ZKV armored RNA, which should be stored at -80°C.

[0225] Experimental Example 1

[0226] Stability verification of ZKV genome fragment ZKA (nt81-2340) armored RNA

[0227] Take 1 mL of purified ZKV genomic fragment ZKA (nt81-2340) 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.

[0228] 1. Temperature stability test

[0229] (1) Treatment at different temperatures

[0230] Four tubes of ZKA armored RNA samples 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.

[0231] (2) ZKA ​​armored RNA extraction

[0232] ZKA 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 a final elution volume of 50 μL.

[0233] (3) Detection of ZKV armored RNA primer-probe

[0234] The U.S. Centers for Disease Control and Prevention (CDC) recommended the ZKV real-time fluorescent RT-PCR system in 2016. The primers and probes are as follows:

[0235] Upstream primer ZK-F: 5'-CCGCTGCCCAACACAAG-3',

[0236] Downstream primer ZK-R: 5'-CCACTAACGTTCTTTTGCAGACAT-3',

[0237] Fluorescent probe ZK-P: 5'-FAM-AGCCTACCTTGACAAGCAGTCAGACACTCAA-BHQ1-3'.

[0238] The target region of this system is the E gene of ZKV (1193-1268, KX377335), which is suitable as a primer-probe for RT-qPCR detection of the prepared ZKA armored RNA. The primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0239] (4) Real-time fluorescence RT-PCR detection of ZKV genomic fragments in ZKA armored RNA (nt81-2340)

[0240] 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):

[0241] 1)One Step PrimeScript III RT-qPCR Mix (2×) 10 μL;

[0242] 2) RNase-Free H2O 6.8 μL;

[0243] 3) ZK-F (10 μM) 0.4 μL;

[0244] 4) ZK-R (10 μM) 0.4 μL;

[0245] 5) ZK-P (10 μM) 0.4 μL;

[0246] 6) Template 2 μL.

[0247] 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.

[0248] (5) Temperature stability test results of ZKA armored RNA (nt81-2340) Figure 1 )

[0249] The results showed that ZKA armored RNA (nt81-2340) 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 ZKA armored RNA (nt81-2340) was temperature-stable.

[0250] 2. ZKA armored RNA (nt81-2340) nuclease resistance test

[0251] (1) Nuclease-resistant treatment

[0252] Take 1 mL of purified ZKA armored RNA (nt81-2340) 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 aliquots and process them as follows:

[0253] 1) Add 5 μL of DNase I (5 U / μL) + 10 μL of DNase I buffer (10×) to tube 1;

[0254] 2) Add 5 μL of RNase (10 mg / mL) to tube 2;

[0255] 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.

[0256] 4) Tube 4 served as an untreated control.

[0257] Place the four sample tubes in a 37°C water bath for 30 minutes.

[0258] (2) Real-time fluorescent RT-PCR detection of ZKA armored RNA (nt81-2340)

[0259] ZKA 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 a final elution volume of 50 μL.

[0260] 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):

[0261] 1)One Step PrimeScript III RT-qPCR Mix (2×) 10 μL;

[0262] 2) RNase-Free H2O 6.8 μL;

[0263] 3) ZK-F (10 μM) 0.4 μL;

[0264] 4) ZK-R (10 μM) 0.4 μL;

[0265] 5) ZK-P (10 μM) 0.4 μL;

[0266] 6) Template 2 μL.

[0267] 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.

[0268] (3) Results of nuclease resistance test for ZKA armored RNA (nt81-2340) Figure 2 )

[0269] The results showed that ZKA armored RNA (nt81-2340) could be detected by real-time fluorescent RT-PCR after treatment with DNase and RNase, indicating that ZKA armored RNA (nt81-2340) is resistant to nucleases.

[0270] 3. ZKA armored RNA (nt81-2340) serial dilutions

[0271] (1) Series of dilution treatments

[0272] Take 200 μL of purified ZKV armored RNA sample stored at -80℃ and extract ZKV armored RNA using 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 ZKV genomic fragment in ZKA armored RNA (nt81-2340).

[0273] (2) Real-time fluorescence RT-PCR detection of ZKV genomic fragment in ZKA armored RNA (nt81-2340)

[0274] 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):

[0275] 1)One Step PrimeScript III RT-qPCR Mix (2×) 10 μL;

[0276] 2) RNase-Free H2O 6.8 μL;

[0277] 3) ZK-F (10 μM) 0.4 μL;

[0278] 4) ZK-R (10 μM) 0.4 μL;

[0279] 5) ZK-P (10 μM) 0.4 μL;

[0280] 6) Template 2 μL.

[0281] 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.

[0282] (3) Results of serial dilutions of ZKA armored RNA (nt81-2340) Figure 3 )

[0283] The results showed that for ZKA armored RNA (nt81-2340) serial dilutions, the lowest dilution detectable by real-time fluorescent RT-PCR was 10. -8 .

[0284] Experimental Example 2

[0285] Digital PCR quantification of ZKV genomic fragment ZKA (nt81-2340) armored RNA

[0286] 1. Method

[0287] The prepared ZKV armored RNA (nt81-2340) was absolutely quantified using Stilla digital PCR (French). The optimal dilution for digital PCR detection was 10⁻⁶. -5 .

[0288] 2. Digital PCR primers and probes

[0289] Digital PCR quantification of ZKV armored RNA was performed using the ZKV real-time fluorescent RT-PCR primer-probe system recommended by the US Centers for Disease Control and Prevention (CDC): ZK-F / ZK-R / ZK-P. The primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0290] 3. Digital PCR Quantitative Experiment

[0291] (1) Digital PCR reaction system

[0292] 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.

[0293] Prepare the reaction mixture (25 μL) according to the instructions:

[0294] 1) MasterMix 12.5 μL;

[0295] 2) 10× fluorescein sodium salt (1 μM) 2.5 μL;

[0296] 3) ZK-F (10 μM) 2.5 μL;

[0297] 4) ZK-R (10 μM) 2.5 μL;

[0298] 5) ZK-P (5 μM) 1.25 μL,

[0299] 6) Add H2O to bring the volume to 22.5 μL.

[0300] The above reagents were shaken to mix well, and dispensed into 22.5 μL tubes. 2.5 μL of diluted ZKV armored RNA 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).

[0301] (2) Digital PCR amplification

[0302] Digital PCR amplification was performed using a STILLA TECHNOLOGIES digital PCR instrument (Ref # H14000) from France. The amplification program is as follows:

[0303] 1) Zone: 40℃, AP950

[0304] 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.

[0305] 3) Pressure relief: 25℃

[0306] 4. Reading of ZKA (nt81-2340) armored RNA digital PCR quantitative data

[0307] (1) ZKV genome fragment ZKA (nt81-2340) armored RNA (10 -5 (Dilution) Digital PCR quantification results () Figure 4-5 )

[0308] (2) Table 1 shows the quantitative results of ZKA armored RNA digital PCR. The concentration of ZKA armored RNA was 5.34 × 10⁻⁶. 5 (copy / μL).

[0309] Table 1. Quantitative results of ZKA armor RNA by digital PCR

[0310]

[0311] 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 Zika virus genome fragment armored RNA reference, characterized in that, Includes the following steps: (1) Construction of prokaryotic expression backbone vector pACYCAR MS2 phage genomic RNA was cultured and extracted. A 1.7kb fragment containing the MS2 maturation enzyme, envelope protein gene and packaging recognition signal sequence was amplified by PCR. This fragment was ligated with the 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 ZKV genome fragment ZKA (nt81-2350) Total RNA was extracted from ZKV inactivated virus, and the genomic fragment ZKA (nt81-2350) covering capsid protein, membrane protein and some outer membrane protein genes was amplified by RT-PCR. After TA cloning and screening, the subcloned plasmid pUCmT-ZKA was obtained. (3) Construction of prokaryotic expression vector pACYCAR-ZKA Using seamless cloning technology, ZKA (nt81-2340) was amplified using the ZKV genome fragment cloned in step (2) as a template and then ligated with the linearized pACYCAR vector amplified by reverse PCR. After transformation into Top10 competent cells, the recombinant expression vector pACYCAR-ZKA (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 ZKV 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 ZKV genome fragment ZKA (nt81-2340) armored RNA reference.

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

3. The method for preparing the Zika virus genome fragment armored RNA reference according to claim 1 as described in claim 2, characterized in that: In step (1), the upstream primer AR-Bam10F was used for RT-PCR amplification of the MS2 1.7kb fragment: 5'-CGCGGATCCTTTCGGGGTCCTGCTCAACTT-3'. Downstream primer AR-Hin1789R: 5'-CCCAAGCTTGAGTTGAACTTCTTTGTTGTCTTC-3'; The amplification conditions were: 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.

4. The method for preparing the Zika virus genome fragment armored RNA reference according to any one of claims 1-3, characterized in that: In step (2), the ZKV strain is GZDJ-1685, GenBank accession number MF099651; The upstream and downstream primers for RT-PCR amplification were ZIKV-81F: 5'-GGATTTGGAAACGAGAGTTTC-3' and ZIKV-2350R: 5'-TCCAAACAATGATTTGAAAGCTGCTC-3', respectively. The amplification conditions were: 50℃ for 30 min; 94℃ for 2 min; 94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 2 min, for 30 cycles; 72℃ for 10 min.

5. The method for preparing the Zika virus genome fragment armored RNA reference according to claim 4, characterized in that, In step (3), the primers for seamless cloning include: pAC-AR328F: 5'-GCCACGCGATCGCTGACG-3', pAC-AR319R: 5'-ATCCAATTGAGATCTGCCATATG-3', acZKA80f: 5'-AGATCTCAATTGGATGAAACGAGAGTTTCTGGTCATG-3', acZKA2340r: 5'-CAGCGATCGCGTGGCCAAACAATGATTTGAAAGCTGCTCC-3'; Seamless cloning reaction was incubated in a metal bath at 50℃ for 2 hours, and positive clones were screened using DuetUP2 and T7-ter as primers.

6. The method for preparing the Zika virus genome fragment armored RNA reference according to claim 5, characterized in that: In step (4), the final concentration of IPTG is 1 mmol / L, and the induction conditions are overnight culture at 37℃ and 200 rpm with shaking. Before induction, the engineered bacteria are cultured at a ratio of 1:100 and cultured at 37℃ and 200 rpm for 4 hours with shaking.

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

8. A Zika virus genome fragment armored RNA reference prepared by the method of any one of claims 1-7.

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

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