Marburg virus detection standard substance as well as preparation system and application thereof

Abstract

The invention provides a Marburg virus detection standard substance as well as a preparation system and application thereof, and belongs to the technical field of biological detection. The armored RNA standard substance containing the Marburg virus NP gene segment, provided by the invention, contains the NP gene segment, can be used for nucleic acid detection of the Marburg virus taking the NP gene as a target spot, and is used for quality control of detection methods and reagents such as common PCR (Polymerase Chain Reaction), fluorescent quantitative PCR, digital PCR, LAMP (Loop-Mediated Isothermal Amplification) and the like. The armored RNA standard substance provided by the invention has the advantages of high purity, no plasmid DNA residue and good quality control performance. The armored RNA virus-like particle simulates the structure of a natural virus, and can participate in links such as nucleic acid extraction and amplification as an animal body or human body blood sample containing Marburg virus, so that the whole-course quality control of the whole detection process is realized, and the authenticity and reliability of the detection result are ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and particularly relates to a Marburg virus detection reference material, a preparation system thereof and an application thereof. Background Art

[0002] Marburg virus disease is a severe human fatal infectious disease with a fatality rate as high as 88%. Marburg virus and Ebola virus belong to the same family of filoviruses and are transmitted through physical contact, body fluid contact, etc. Since Marburg virus was first discovered in 1967, outbreaks or epidemics have occurred in a total of 11 countries in Europe, Africa and the Americas so far. Although there have been no reports or serological evidence of Marburg virus infection in China and its surrounding areas, with the strengthening of economic cooperation between China and African regions, frequent international transactions and personnel flows, the risk of importation is increasing continuously. At present, technical methods or standards for detecting viruses using nucleic acid detection technology have been established both at home and abroad. However, there are few reports on the preparation of positive reference standards.

[0003] In virus nucleic acid detection, random errors in the operation of experimental personnel, differences in temperature between wells of the thermal cycler, residues of inhibitors after specimen nucleic acid extraction, concentration of the target nucleic acid to be amplified, efficiency of reverse transcription, and reagent problems, etc. may all affect the efficiency of PCR amplification, thus resulting in deviation of results, or even false negative and false positive results. Therefore, quality control products and reference standards must be used in the nucleic acid detection of RNA viruses to ensure the reliability of results. Due to the potential infectious risk of natural viruses and the instability of their RNA molecules, currently used quality control products such as cDNA, plasmids, naked RNA, and in vitro transcribed RNA are difficult to meet the test requirements. Therefore, the development of stable and non-bio-infectious quality control substances and reference standards is of great significance not only for the RT-PCR or real-time fluorescence RT-PCR detection of RNA viruses, but also for the evaluation of commercial virus RT-PCR or real-time fluorescence RT-PCR detection kits. Summary of the Invention

[0004] The purpose of the present invention is to provide a Marburg virus detection reference material, a preparation system thereof and an application thereof. The reference material has characteristics such as stability, ability to simulate the structure of Marburg virus, good biological safety, and resistance to RNase, and can be used for the quality control of Marburg virus nucleic acid detection methods and kits.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a Marburg virus detection reference material, wherein the Marburg virus detection reference material includes a virus-like particle shell formed by self-assembly of the capsid protein of bacteriophage MS2 and a Marburg virus NP gene fragment encapsulated therein;

[0007] The nucleotide sequence of the Marburg virus NP gene fragment is shown in SEQ ID NO:2.

[0008] The present invention also provides a recombinant vector expressing the above Marburg virus detection reference substance, starting from an initial vector and comprising the following components:

[0009] Component a: the Marburg virus NP gene sequence with the nucleotide sequence shown in SEQ ID NO:2;

[0010] Component b: the phage MS2 amplification sequence including the 5′ non-coding region sequence, the maturase protein gene, the capsid protein gene, the packaging site and a partial start site sequence of the replicase gene.

[0011] Preferably, the initial vector is the pACYCDuet-1 plasmid.

[0012] Preferably, the nucleotide sequence of the phage MS2 amplification sequence is shown in SEQ ID NO:1.

[0013] The present invention also provides a recombinant expression strain comprising the above-mentioned recombinant vector.

[0014] The present invention also provides a method for preparing the above Marburg virus detection reference substance, comprising the following steps:

[0015] Transform the above recombinant vector into a target strain, and obtain the Marburg virus detection reference substance after induced expression.

[0016] Preferably, the induced expression is achieved by IPTG induction.

[0017] Preferably, the concentration of IPTG is 0.3 - 0.8 mmol / L.

[0018] Preferably, after the induced expression, an extraction and purification treatment is further included, and the extraction and purification treatment comprises the following steps:

[0019] Collect and lyse the bacteria obtained by the induced expression, sequentially degrade bacterial DNA, RNA and plasmid DNA, remove bacterial debris, precipitate and recover phage particles, and recover the aqueous phase containing phage particles after removing impurities in the phage suspension again;

[0020] Perform centrifugation purification and concentration treatment on the aqueous phase containing phage particles to obtain the Marburg virus detection reference substance after extraction and purification.

[0021] The present invention also provides the use of the above-mentioned Marburg virus detection standard substance, recombinant vector, recombinant expression strain or preparation method of Marburg virus detection standard substance in preparing Marburg virus detection kits, Marburg virus detection positive control substances or Marburg virus detection quality control substances.

[0022] Beneficial effects of the present invention:

[0023] The present invention provides an armored RNA standard material containing a Marburg virus NP gene fragment. The NP gene fragment contained within this armored RNA can be used for nucleic acid detection of Marburg virus targeting the NP gene and for quality control of detection methods and reagents such as conventional PCR, fluorescent quantitative PCR, digital PCR, and LAMP. The armored RNA standard material provided by the present invention is high in purity, free of plasmid DNA residue, and exhibits excellent quality control performance. The NP gene fragment contained within this armored RNA standard material is encapsulated by the capsid protein of bacteriophage MS2, making it resistant to RNase degradation and more stable and easier to store than in vitro transcribed naked RNA. The armored RNA virus-like particles mimic the structure of the natural virus and can be used in nucleic acid extraction and amplification, similar to animal or human blood samples containing Marburg virus, enabling full quality control throughout the entire detection process and ensuring the authenticity and reliability of test results. This armored RNA is non-infectious, biosafe, and easy to prepare and transport. Compared to Marburg virus particles, it is more suitable for widespread application in nucleic acid detection laboratories. The armored RNA standard material was quantified using fluorescent quantitative RT-PCR and can be used for quantitative analysis of test samples and evaluation of detection methods and reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is an agarose gel electrophoresis diagram of the RT-PCR amplification product of the Marburg virus NP gene, in which: M: DL 1000bp Marker; 1: reverse transcription product PLV-Marb-NP / cDNA; 2: unreverse transcribed PLV-Marb-NP / RNA; 3: virus particle solution;

[0025] Figure 2 This is the SDS-PAGE electrophoresis diagram of the armored RNA qualitative virus-like particles of the Marburg virus NP gene fragment. In the figure: M: 10-180kDa protein marker; 1: recombinant bacteria without IPTG; 2: recombinant bacteria induced by IPTG;

[0026] Figure 3 The results of real-time fluorescence quantitative RT-PCR detection of Marburg virus were obtained by detecting 10-fold serial dilutions of in vitro transcribed RNA. DETAILED DESCRIPTION

[0027] The technical solution provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0028] In the embodiment, a qualitative reference material of armored RNA containing the Marburg virus NP gene fragment is provided. The prokaryotic expression vector of this reference material is plasmid pACYCDuet-1-MS2-NP. The expression vector is transformed into an expression strain and induced to express to obtain a qualitative virus-like particle of armored RNA containing the Marburg virus NP gene fragment.

[0029] In the prokaryotic expression vector pACYCDuet-1-MS2-NP plasmid, the packaging sequence is a sequence containing the 5′ non-coding region sequence of the MS2 phage genome, the maturase protein gene, the capsid protein gene, the packaging site, and the partial starting point of the replicase gene as shown in SEQ ID NO.1, and the Marburg virus NP gene sequence is as shown in SEQ ID NO.2:

[0030] Phage MS2 amplification sequence (including 5′ non-coding region sequence, maturase protein gene, capsid protein gene, packaging site, and partial starting site sequence of replicase gene), SEQ ID NO.1:

[0031]

[0032] Marburg virus NP gene fragment, SEQ ID NO.2:

[0033]

[0034] Obtaining and Identification of Armored RNA Virus Particles Containing Marburg Virus NP Gene Fragment in Example 1

[0035] 1. Materials

[0036] DH5α competent cells and E. coli BL21(DE3) competent cells were purchased from Tiangen Biochemical Technology Co., Ltd. and Beijing Dongfang Shunke Biotechnology Co., Ltd. respectively, and the pACYCDuet-1 plasmid was purchased from Beijing Tianjingsha. The following sequences and primer syntheses were all completed by Sangon Biotech (Shanghai) Co., Ltd.

[0037] 2. Methods

[0038] 2.1 Amplification of MS2 Phage Sequence

[0039] Referring to the MS2 phage (NC_001417.2) sequence in GenBank, primers were designed. The amplified product was located at positions 81 - 1740 bp. The upstream primer MS-F added a BamHⅠ restriction site and protective bases (numbered Seq3) at the 5′ end, and the downstream primer MS-R2 added a HandⅢ restriction site and protective bases (numbered Seq4) at the 5′ end, as shown in Table 1; The phage MS2 was stored in this laboratory. After activation and culture, the RNA of the culture was extracted as a template. The TianGen one-step RT-PCR kit was used to amplify the target fragment of phage MS2. The amplification conditions were as follows: reverse transcription of viral nucleic acid into cDNA at 42℃ for 30 min; pre-denaturation at 95℃ for 3 min; denaturation at 94℃ for 30 sec, annealing at 65℃ for 30 sec, extension at 72℃ for 30 sec, for 37 cycles; supplementary extension at 72℃ for 5 min.

[0040] Table 1 PCR Primer Sequences

[0041]

[0042] 2.2 Synthesis of Marburg Virus Target Sequence

[0043] Referring to the Marburg virus (JX458838.1) sequence in GenBank, the NP fragment of Marburg virus was designed. A KpnI restriction site and a C-variant pac site (GGGGTACCacatgaggatcacccatgt) consisting of 19 bases of MS2 were added to the 5′ end of the seq2 sequence, and a PacI restriction site and a C-variant pac site (acatgaggatcacccatgtTTAATT) were added to the 3′ end

[0044] acatgaggatcacccatgtTTAATT

[0045] AAGG. The sequence and primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and a pUC-57 plasmid containing the target fragment was provided, named pUC-57-NP.

[0046] 2.3 Double digestion of the phage amplified sequence and plasmid vector

[0047] The above phage amplified product was recovered by gel extraction, and then it and the plasmid pACYCDuet-1 were respectively subjected to double digestion. The digestion system was: 5 μL of the phage amplified product recovered by gel extraction; 2 μL of 10×M buffer; 1 μL of BamHI; 1 μL of HindⅢ; add ddH2O to a total volume of 20 μL. For plasmid pACYCDuet-1: 5 μL; 2 μL of 10×M buffer; 1 μL of BamHI; 1 μL of HindⅢ; add ddH2O to a total volume of 20 μL. After digestion at 37°C for 1 h each, one-tenth volume of loading buffer was added, and the digested products were recovered by gel extraction.

[0048] 2.4 Construction of plasmid vector pACYCDuet-1-MS

[0049] The above gel-extracted and digested products were ligated. The ligation system was: 8 μL of pACYCDuet-1; 2 μL of the phage sequence double-digested product; 2 μL of 10×T4 DNA ligase buffer; 1 μL of T4 DNA ligase; add ddH2O to a total volume of 20 μL. Ligation was carried out overnight at 16°C. Then, the transformed competent cells DH5α were transformed, and the transformed bacterial culture solution was spread on an LB plate containing ampicillin and cultured at 37°C for 12 h. A single colony was picked into 20 μL of sterile water, and after mixing, 2 μL of the diluted bacterial solution was used as a template for PCR identification. The reaction system was: 2 μL of the bacterial solution; 1 μL of primer MS-F (10 μM); 1 μL of primer MS-R2 (10 μM); 12.5 μL of 2×Taq PCRMix; add ddH2O to a total volume of 25 μL. The reaction conditions were: pre-denaturation at 95°C for 3 min; denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 60 sec, for 35 cycles; additional extension at 72°C for 5 min.

[0050] 2.5 Double digestion of plasmid pUC-57-NP and plasmid vector pACYCDuet-1-MS

[0051] The synthetic sequence pUC-57-NP and the plasmid pACYCDuet-1-MS were respectively double digested. The digestion system for the synthetic sequence pUC-57-NP was as follows: 10 μL of the synthetic sequence pUC-57-NP, 5 μL of 10× NEB buffer, 1 μL of Kpn I, 1 μL of PacI, and ddH2O was added to a total volume of 50 μL. For the plasmid pACYCDuet-1-MS, it was 10 μL of the plasmid pACYCDuet-1-MS, 5 μL of 10× NEB buffer, 1 μL of KpnI, 1 μL of PacI, and ddH2O was added to a total volume of 50 μL. After digestion at 37°C for 2 h, one-tenth volume of loading buffer was added, and the digested products were recovered by gel extraction.

[0052] 2.6 Construction of the plasmid vector pACYCDuet-1-MS-NP

[0053] The above gel-extracted digested products were ligated. The ligation system was as follows: 14 μL of pACYCDuet-1-MS, 2 μL of the double-digested product of Marburg virus, 2 μL of 10× T4 DNA ligase buffer, 0.5 μL of T4 DNA ligase, and ddH2O was added to a total volume of 20 μL. Ligation was carried out overnight at 16°C. Then, it was transformed into competent E. coli cells. The transformed bacterial culture solution was spread on an LB plate containing chloramphenicol and cultured at 37°C for 12 h. A single colony was picked and resuspended in 20 μL of sterile water. After mixing, 2 μL of the diluted bacterial solution was taken as a template for PCR identification. The reaction system was: 2 μL of the bacterial solution, 1 μL of primer MARV-F (10 μM) (numbered Seq3), 1 μL of MARV-R (10 μM) (numbered Seq4), 12.5 μL of 2× Taq PCR Mix, and ddH2O was added to a total volume of 25 μL. The reaction conditions were: pre-denaturation at 95°C for 3 min; denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 60 sec, for 35 cycles; and final extension at 72°C for 5 min. The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing identification.

[0054] 2.7 Induced expression and purification of virus-like particles

[0055] The recombinant plasmid pACYCDuet-1-MS-NP was transformed into E. coli BL21(DE3) competent cells and spread on an agar plate containing 25 mg / mL chloramphenicol. After overnight incubation, single colonies were picked and cultured in LB liquid medium overnight. Then, 1 mL of the culture was taken and added to 99 mL of LB medium, and cultured at 37 °C with a rotation speed of 220 r / min until the OD600nm value of the bacterial solution reached 0.6. IPTG with a concentration of 0.5 mmol / L was added for induction culture for 5 h. The bacteria were collected by centrifugation at 12000 r / min. Then, the bacteria were lysed using a bacterial lysate without ultrasonic treatment. Subsequently, DNase I and RNase A were added to a final concentration of 1 μg / mL each, and the mixture was placed at room temperature for 30 minutes to degrade bacterial DNA, RNA, and plasmid DNA. Solid sodium chloride was added to a final concentration of 1 mol / L (29.2 g of solid sodium chloride was added to every 500 mL of the culture solution), and it was stirred to dissolve. After ice-bathing for 1 hour, centrifugation was performed at 4 °C at 11000 g for 10 minutes to remove bacterial debris. The supernatant was poured into a clean flask; solid polyethylene glycol (PEG 8000) was added to a final concentration of 10% w / v (i.e., 50 g was added to every 500 mL of the supernatant), and it was dissolved at room temperature. After ice-bathing for at least 1 hour to form precipitates of phage particles, centrifugation was performed at 4 °C at 11000 g for 10 minutes to recover the precipitated phage particles. The supernatant was discarded, and the centrifuge tube was tilted for 5 minutes to allow the remaining liquid to flow out fully from the phage precipitate. The remaining liquid was aspirated with a pipette; the phage precipitate was resuspended with physiological saline (8 mL of physiological saline solution was added to every 500 mL of the supernatant obtained in step 3), the centrifuge tube wall was rinsed thoroughly, DNase I was added to a final concentration of 1 μg / mL, an equal volume of chloroform was added, and it was shaken for 30 seconds to remove polyethylene glycol and bacterial debris in the phage suspension. Centrifugation was performed at 4 °C at 3000 g for 15 minutes to recover the aqueous phase containing phage particles; the aqueous phase containing phage was transferred to an ultrafiltration centrifuge tube (Millipore MLtra-15 10KD) and centrifuged at 4000 r / min for 1 h for purification and concentration.

[0056] 2.8 PCR Identification of Virus-Like Particles

[0057] To verify whether the prepared armored RNA particles contain the Marburg virus NP gene fragment, the RNA inside the capsid of the armored RNA particles was extracted using a kit, and then reverse transcription was performed at 50 °C for 1 h using primers. PCR identification was performed on the reverse transcription products, nucleic acids without reverse transcription, and armored RNA particles without nucleic acid extraction. The primers are shown as Seq5 and Seq6. The PCR amplification products were detected by 1.5% agarose gel electrophoresis, and the results are shown in Figure 1 ).

[0058] 2.9 Protein Electrophoresis Identification of Virus-Like Particles

[0059] Take 1 mL each of the uninduced bacterial solution and the recombinant bacterial solution induced by IPTG, centrifuge at 12,000 r / min for 1 min to collect the bacterial cell precipitate, dissolve it in 30 μL of PBS buffer, add an equal volume of protein loading buffer, mix well, boil for 6 min, centrifuge, take 10 μL of the supernatant for each sample to load the sample, and perform SDS-PAGE electrophoresis analysis( Figure 2 ).

[0060] Example 2 Preparation of an armored RNA reference material containing the Marburg virus NP gene fragment

[0061] 1. Determination of the value of the armored RNA reference material containing the Marburg virus NP gene fragment

[0062] Determine the copy number of the NP gene fragment contained in the armored RNA virus-like particles obtained in Example 1. The determination method is as follows: Digest the plasmid pUC-57-NP with restriction enzymes, purify and recover it, and then use it as a template according to the Ribo MAX TM Large Scale RNAProducion System-T7 kit instructions for in vitro transcription. The transcription product is digested with DNase to remove the DNA template therein, and then the RNA is re-extracted with TRIzol to obtain a high-purity in vitro transcribed RNA solution of the NP fragment. By measuring the absorbance values at 260 nm and 280 nm of the in vitro transcribed RNA, calculate its copy number through the molecular weight, and then prepare an external standard of the in vitro transcribed RNA by 10-fold serial dilution. Using the fluorescence RT-PCR method in the standard, detect the in vitro transcribed RNA with known concentration prepared and the RNA extracted from the armored RNA particles. The primers and probes are shown as Seq7-9. Take the Ct values obtained from the detection of the serially diluted in vitro transcribed RNA as quantitative parameters, perform linear regression on the logarithms of the template copy numbers contained in different dilutions, and obtain a regression equation( Figure 3 ), and calculate the copy number of the armored RNA nucleic acid through the formula.

[0063] 2. Dilution, aliquoting and storage of the reference material

[0064] According to the above determination results, dilute the armored RNA virus-like particles obtained in Example 1 with physiological saline to 10 6 copies / mL, mix well, aliquot into cryotubes, 0.2 mL per tube, and store at -80 °C.

[0065] 3. Verification of the reference material

[0066] 1. Homogeneity test

[0067] Randomly select 10 reference materials, extract RNA, perform real-time fluorescence RT-PCR detection, and conduct statistical analysis on the obtained Ct values. The coefficient of variation of the results is 3.5%, all less than 5%, indicating that the distribution of virus-like particles in the sample is uniform and consistent.

[0068] 2. Stability test

[0069] Short-term stability investigation: To investigate the stability of the reference material during short-term transportation, three temperatures of 4°C, 25°C (room temperature), and 37°C were set respectively. At the time points of the 3rd day, 7th day, and 14th day, randomly select 3 tubes, and perform 3 replicates for each tube. Extract RNA, and use the fluorescence quantitative RT-PCR method to detect the Ct value of the reference material. Analyze the results by F-test. The result is p > 0.5, indicating that the properties of the reference material are stable at different temperatures and times.

[0070] Long-term stability investigation: The long-term stability was investigated under the storage conditions of -20°C and -80°C. At the 1st month, 2nd month, 3rd month, and 6th month, randomly select 3 tubes, and perform 3 replicates for each tube. Extract RNA, and use the fluorescence quantitative RT-PCR method to determine the Ct value of the reference material to investigate its long-term storage stability under the conditions of -20°C and -80°C. Through analysis of variance, the result is p > 0.5, indicating that after storing at -20°C and -80°C for 6 months, the nucleic acid content in the sample remains basically unchanged, that is, the properties of the reference material are stable. According to GB / T15000.3-2023 "General Guidelines for the Work of Reference Samples - Part 3: Evaluation of the Certification, Homogeneity and Stability of Reference Samples", linear fitting analysis was performed on the long-term stability data.

[0071] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A Marburg virus detection reference material, characterized in that, The Marburg virus detection reference material includes a virus-like particle shell formed by self-assembly of the capsid protein of bacteriophage MS2 and a Marburg virus NP gene fragment encapsulated therein; The nucleotide sequence of the Marburg virus NP gene fragment is as shown in SEQ ID NO:

2.

2. The recombinant vector expressing the Marburg virus detection reference material according to claim 1, characterized in that, Starting from the initial vector, it includes the following components: Component a: The Marburg virus NP gene sequence with the nucleotide sequence as shown in SEQ ID NO:2; Component b: The amplification sequence of bacteriophage MS2 including the 5′ non-coding region sequence, the maturase protein gene, the capsid protein gene, the packaging site, and the partial start site sequence of the replicase gene.

3. The recombinant vector according to claim 2, characterized in that, The initial vector is the pACYCDuet-1 plasmid.

4. The recombinant vector according to claim 2, wherein The nucleotide sequence of the bacteriophage MS2 amplification sequence is as shown in SEQ ID NO:

1.

5. A recombinant expression strain containing the recombinant vector according to any one of claims 2 to 4.

6. The preparation method of the Marburg virus detection reference material according to claim 1, characterized in that, It includes the following steps: Transform the recombinant vector according to any one of claims 2 to 4 into the target strain, and obtain the Marburg virus detection reference material after induced expression.

7. The preparation method according to claim 6, characterized in that, The induced expression is achieved by IPTG induction.

8. The preparation method according to claim 6, characterized in that, The concentration of the IPTG is 0.3 to 0.8 mmol / L.

9. The preparation method according to claim 6, characterized in that, After the induced expression, it further includes an extraction and purification treatment, and the extraction and purification treatment includes the following steps: Collect and lyse the bacteria obtained by the induced expression, sequentially degrade bacterial DNA, RNA, and plasmid DNA, remove bacterial debris, precipitate and recover the phage particles, and recover the aqueous phase containing phage particles after removing impurities in the phage suspension again; Perform centrifugal purification and concentration treatment on the aqueous phase containing phage particles to obtain the Marburg virus detection reference material after extraction and purification.

10. The application of the Marburg virus detection reference material according to claim 1, the recombinant vector according to any one of claims 2 to 4, the recombinant expression strain according to claim 5, or the preparation method of the Marburg virus detection reference material according to any one of claims 6 to 9 in the preparation of a Marburg virus detection kit, a Marburg virus detection positive control, or a Marburg virus detection quality control product.

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