Pseudovirus of Nipah virus and application thereof

Abstract

The invention provides a pseudovirus of a Nipah virus and application of the pseudovirus, and belongs to the technical field of genetic engineering. The invention provides a pseudovirus of a Nipah virus and application of the pseudovirus. The pseudovirus contains a nucleic acid fragment of a Nipah virus N gene of which the nucleotide sequence is shown as SEQ ID NO.1. The invention further provides a preparation method of the pseudovirus. The pseudovirus of the Nipah virus can be used as a ribonucleic acid standard substance of the pseudovirus of the Nipah virus, and is applied to the aspects of quantity transmission, calibration, instrument testing, evaluation and control of a testing method, quality verification, quality control, production process detection and the like of a laboratory related to Nipah virus detection. The pseudovirus of the Nipah virus, as the ribonucleic acid standard substance of the pseudovirus of the Nipah virus, has the following advantages of accurate fixed value, good uniformity, stable quantity value and traceability, and can promote the improvement of the detection capability and the technical level of a national Nipah detection laboratory.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a Nipah virus pseudovirus and application thereof. Background Art

[0002] Nipah virus disease (NVD) is a zoonotic disease caused by the Nipah virus (NiV). Human infection with NiV can cause severe neurological encephalitis or respiratory disease, with a mortality rate of 40% to 70%. In pigs, NiV infection manifests as tachypnea, progressing to dyspnea and accompanied by involuntary coughing, with a mortality rate of approximately 5%.

[0003] Reverse transcription real-time quantitative polymerase chain reaction (qRT-PCR) is a commonly used method for NiV detection. Primers and probes are typically designed targeting the relatively conserved N gene of NiV. In molecular detection techniques, positive standards serve as control materials for testing experimental validity. Common positive standards include inactivated virus, naked genome, plasmids containing the target gene, and pseudoviruses. NiV is a Class I pathogenic microorganism, and cultivation must be performed in a biosafety level 4 (BSL4) laboratory. Therefore, the preparation of inactivated virus standards poses significant biosafety risks. Furthermore, viral genomic RNA is easily degraded, and plasmids lack a virus-like structure, making them inadequate for simulating the entire process of nucleic acid extraction and detection from a sample. Pseudoviruses, similar in structure to viruses, possess capsid protection, are resistant to nuclease degradation, and are stable and highly safe. To ensure the accuracy, comparability, and traceability of test results, the development of NiV pseudoviruses and their use in the preparation of NiV RNA standards are of practical significance. Summary of the Invention

[0004] The present invention provides a NiV pseudovirus and application thereof. The pseudovirus can be used to prepare a ribonucleic acid standard substance for diagnosing NiV and has the advantages of accurate determination, good uniformity and stable measurement value.

[0005] The present invention provides a NiV pseudovirus, wherein the pseudovirus contains a detection target; the detection target comprises a nucleic acid fragment of the NiVN gene whose nucleotide sequence is shown as SEQ ID NO.1.

[0006] In a preferred embodiment of the present invention, the 3' end of the nucleic acid fragment of the NiVN gene is further connected to the nucleotide sequence shown in SEQ ID NO.2; the 5' end of the nucleic acid fragment of the Nipah virus N gene is further connected to the nucleotide sequence shown in SEQ ID NO.3.

[0007] In a preferred mode of the present application, the pseudovirus uses CDH-CMV-MCS-EF1-copGFP-T2A-Puro as an expression vector.

[0008] The present application also provides a preparation method of the above-mentioned pseudovirus, comprising the following steps: co-transfecting three plasmids into an engineering cell through a three-plasmid system, culturing the transfected engineering cell, collecting the supernatant, and obtaining the pseudovirus.

[0009] In a preferred mode of the present application, the three-plasmid system comprises pCDH-CMV-MCS-EF1-copGFP-T2A-Puro, psPAX2 and pMD2.G.

[0010] In a preferred mode of the present application, the mass ratio of the pCDH-CMV-MCS-EF1-copGFP-T2A-Puro, psPAX2 and pMD2.G is 5:3.75:1.25.

[0011] In a preferred mode of the present application, the engineering cell comprises a HEK293T cell.

[0012] The present application also provides the above-mentioned pseudovirus or the pseudovirus prepared by the above-mentioned preparation method for use in the preparation of a ribonucleic acid standard substance for diagnosing NiV.

[0013] The present application also provides a kit for diagnosing NiV, which comprises the above-mentioned pseudovirus or the pseudovirus prepared by the above-mentioned preparation method.

[0014] In a preferred mode of the present application, the kit further comprises primers and probes for detecting NiV.

[0015] Beneficial effects: the present application provides a pseudovirus of NiV, which contains a detection target shown in SEQ ID NO. 1. The present application entrusts nine qualified laboratories to independently use the ddPCR method to measure the characteristic value of the pseudovirus, determines the standard value, proves that it has the advantages of good uniformity and high stability, can be used as a ribonucleic acid standard substance of NiV pseudovirus, and is used in the aspects of value transfer, calibration, test instruments, evaluation and control test methods, quality testing, quality control and production process testing of NiV detection related laboratories. The total uncertainty of the standard substance is evaluated by comprehensively considering the uncertainty introduced by uniformity, the uncertainty introduced by stability and the uncertainty introduced by the standard substance value process. The standard value of the standard substance is expressed as (6.9±1.4)×10 3copies / μL. The pseudovirus of NiV of the present application has the advantages of accurate value setting, good uniformity, stable value, and traceability as the pseudovirus ribonucleic acid standard substance of NiV, and can promote the improvement of detection capability and technical level of national Nipah detection laboratories. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is an electron microscope image of NiV pseudovirus;

[0017] Figure 2 is a homology analysis of S gene of each strain of NiV;

[0018] Figure 3 is the result of primer and probe concentration optimization of the ddPCR detection method; in the figure, A: the reaction concentration of primer and probe is 600 nmol·L -1 and 150 nmol·L -1 ; B: the reaction concentration of primer and probe is 750 nmol·L -1 and 250 nmol·L -1 ; C: the reaction concentration of primer and probe is 900 nmol·L -1 and 350 nmol·L -1 ;

[0019] Figure 4 is the result of annealing temperature optimization of the ddPCR detection method; in the figure, A: the annealing temperature is 55℃; B: the annealing temperature is 58℃; C: the annealing temperature is 61℃;

[0020] Figure 5 is the result of heating and cooling rate optimization of the ddPCR detection method; in the figure, A: the heating and cooling rate is 1.0℃ / S; B: the heating and cooling rate is 2.0℃ / S; C: the heating and cooling rate is 3.0℃ / S;

[0021] Figure 6 is the ddPCR amplification chart after gradient dilution of the nucleic acid of NiV pseudovirus; in the figure, A: the nucleic acid is diluted by 10 0 ; B: the nucleic acid is diluted by 10 -1 ; C: the nucleic acid is diluted by 10 -2 ; D: the nucleic acid is diluted by 10 -3 ; E: the nucleic acid is diluted by 10 -4 ;

[0022] Figure 7 is the linear graph of ddPCR gradient dilution;

[0023] Figure 8 is the technical route chart for preparing the ribonucleic acid standard substance of NiV pseudovirus;

[0024] Figure 9The physical property test result of the NiV pseudovirus ribonucleic acid standard substance;

[0025] Figure 10 The DNA residue detection result of the NiV pseudovirus ribonucleic acid standard substance; in the figure, A: the reverse transcriptase amplification result; B: the amplification result without adding reverse transcriptase;

[0026] Figure 11 The traceability diagram of the NiV pseudovirus ribonucleic acid standard substance. DETAILED DESCRIPTION

[0027] The application provides a pseudovirus of NiV, which contains a detection target; the detection target includes a nucleic acid fragment of NiV N gene as shown in SEQ ID NO. 1.

[0028] In the application, the NiV reference strain Malaysia strain (GeneBank access No. NC_002728) is selected as the NiV standard reference strain (original strain); the nucleic acid fragment of the NiV N gene is the full-length N gene with a length of 2242 nt, and the nucleotide sequence is as shown in SEQ ID NO. 1. The nucleic acid fragment of the NiV N gene in the embodiment of the application is synthesized by Shengong Biotechnology (Shanghai) Co., Ltd.

[0029] As an embodiment, for the convenience of subsequent BM seamless cloning, the nucleotide sequence as shown in SEQ ID NO. 2: 3'-acctccatagaagattctaga-5' is connected to the 3' end of the NiV N gene; and the nucleotide sequence as shown in SEQ ID NO. 3: 3'-atccttcgcggccgcggatcc-5' is further connected to the 5' end.

[0030] As an embodiment, the pseudovirus uses pCDH-CMV-MCS-EF1-copGFP-T2A-Puro as an expression vector. As an embodiment, the insertion site of the detection target on the expression vector is Xba I and BamHI. As an embodiment, the detection target (target fragment) and the vector are connected using a BM seamless cloning kit.

[0031] As an embodiment, the pseudovirus is packaged by a three-plasmid system, which includes a vector plasmid pCDH-CMV-MCS-EF1-copGFP-T2A-Puro for expressing a target gene, two auxiliary plasmids psPAX2 and pMD2.G, in the embodiment of the application, the three plasmids are purchased from System Biosciences (SBI) company, and the item number is CD513b.

[0032] As an implementation form, the mass ratio of the pCDH-CMV-MCS-EFl-copGFP-T2A-Puro, psPAX2 and pMD2.G is 5:3.75:1.25, the three plasmids are co-transfected into the engineering cells, the transfected engineering cells are cultured, and the supernatant is collected to obtain the pseudovirus.

[0033] As an implementation form, the engineering cells include HEK293T cells; the cell density of the engineering cells reaches 80%-90% of the confluence rate before transfection; the culture solution of the engineering cells is replaced with serum-free DMEM before transfection; and the required transfection complex consists of the following components: 1.25 μg of pMD2.G plasmid, 3.75 μg of pspAX2 plasmid, 5 μg of shuttle plasmid and 25 μL of LipoX Plus Reagent, in terms of 100 mm flat dishes.

[0034] The application further provides application of the pseudovirus prepared by the preparation method in the preparation of a ribonucleic acid standard substance for diagnosing NiV.

[0035] As an implementation form, the ribonucleic acid standard substance for diagnosing NiV is used in the fields of value transfer, calibration, test instrument, evaluation and control test method, quality inspection, quality control and production process detection of a related laboratory for NiV nucleic acid detection.

[0036] The application further provides a kit for diagnosing NiV, which comprises the pseudovirus prepared by the preparation method.

[0037] In one embodiment, the kit further comprises primers and probes for detecting NiV. In one embodiment, the primers comprise NP-F (3'-tagaaataatctcagacatcggaaa-5') of the nucleotide sequence shown in SEQ ID NO. 4 and NP-R (3'-cccatagacctgtcaatagtagtagc-5') of the nucleotide sequence shown in SEQ ID NO. 5; and the probe comprises NP-P (3'-FAM-tttgcccctggaggttacccattatcg-BHQ1-5') of the nucleotide sequence shown in SEQ ID NO. 6. As an embodiment, the kit is used for digital PCR (ddPCR) detection of NiV; based on 20 μL, the reaction system of the ddPCR detection is: One-step RT-ddPCR supermix 10 μL, reverse transcriptase (enzyme) 2 μL, template 2 μL, primer 1.5 μL, probe 0.5 μL and balance water; the reaction program is 50°C, 20 min; 95°C, 3 min; 94°C, 30 s, annealing temperature 58°C, 60 s, 40 cycles; heating and cooling rate 2.0°C / s.

[0038] In the present invention, the ddPCR detection method can detect specific amplification signals for NiV pseudovirus, while the detection results for other viruses are negative, indicating that the established ddPCR detection method has good specificity and repeatability, and the detection results are stable and reliable.

[0039] The absolute quantitative method for pseudoviruses described in the present invention is jointly calibrated by nine laboratories to develop a standard substance with accurate target value determination, good traceability, stability and uniformity, and high use value to meet the needs of scientific research and clinical use.

[0040] The NiV pseudovirus of the present invention is used as a RNA standard substance, which can meet the demand for standard substances in the field of public health testing. At the same time, it can overcome the problem that the inconsistent and unstable test results of disease prevention and control institutions, medical institutions, third-party testing laboratories and reagent manufacturers at all levels across the country affect the judgment of laboratory results, ensure the accuracy and reliability of NiV nucleic acid detection, and provide material support for the work of traceability, proficiency testing, quality control, method evaluation, reagent selection, etc., thereby improving NiV detection capabilities. The mass value unit of the prepared NiV pseudovirus RNA standard substance is copies / μL. There is no internationally agreed reference measurement procedure and no internationally agreed calibration product. It cannot be traced back to the SI unit in terms of measurement. Therefore, the ddPCR method and the joint determination method of multiple laboratories are used to determine the gene copies content of the standard substance. By using a confirmed absolute quantitative measurement method that meets the metrological characteristics requirements and a volume measuring instrument that has been verified / calibrated, it is ensured that the value of this standard substance can be traced back to the basic unit of entity number "one" (symbol: 1) that can be used as any basic unit of measurement system and the national legal measurement unit of volume liter (L). Establish a traceability system such as Figure 11 shown.

[0041] To further illustrate the present invention, a NiV pseudovirus and its application provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1 Construction of NiV pseudovirus

[0043] 1. Raw material screening

[0044] The NiV reference strain, Malaysian strain (GeneBank access No. NC_002728), was selected as the standard reference strain (original strain). The specific genome sequence is the full-length N gene, and the nucleotide sequence is shown in SEQ ID NO.1.

[0045] 2. Construction of NiV pseudovirus

[0046] The three-plasmid lentiviral packaging system consisting of pCDH-CMV-MCS-EF1-copGFP-T2A-Puro (expression plasmid), psPAX2 (packaging plasmid) and pMD2.G (envelope plasmid) was selected to package pseudovirus containing the NiV N gene.

[0047] 1. N gene sequence synthesis

[0048] SEQ ID NO. 1, and the sequence shown in SEQ ID NO. 2 is added at the 3' end of the N gene and the sequence shown in SEQ ID NO. 3 is added at the 5' end of the N gene according to the vector sequence, for BM seamless cloning with the vector. After the whole gene synthesis, the vector is sequenced by Sanger, and the sequence alignment is 100% consistent with the original design sequence.

[0049] 2. N gene fragment amplification

[0050] The N gene target fragment is amplified from the vector after the whole gene synthesis of the N gene using KOD enzyme, upstream primer N gene-F and downstream primer N gene-R, and recovered.

[0051] N gene-F (SEQ ID NO. 7): 3'-acctccatagaagattctagaaggaaccaagacaaacacttttgg-5';

[0052] N gene-R (SEQ ID NO. 8): 3'-atccttcgcggccgcggatcctttttcttaatgagttagtagtacaaaatagtga-5.

[0053] 3. Vector enzyme digestion

[0054] The vector pCDH-CMV-MCS-EF1-copGFP-T2A-Puro is double-digested with Xba I and BamHI; after enzyme digestion, agarose gel electrophoresis is performed, and the digested vector fragment is recovered.

[0055] 4. N gene fragment and vector ligation

[0056] The N gene target fragment recovered after PCR amplification and the vector fragment recovered after enzyme digestion are ligated using a BM seamless cloning kit.

[0057] 5. Transformation

[0058] The ligation product is transformed into commercial DH5a competent cells according to the operating instructions, and the plated plate is incubated at 37°C in an inverted incubator for 16-24 h.

[0059] 6. Identification and sequencing of positive clone plasmid

[0060] After the monoclonal colonies on the LB / Amp+ plates have grown to pinpoint size, transfer them to LB / Amp+ liquid culture medium. Incubate at 37°C in a shaker at 180 rpm for 5–7 hours until the culture medium becomes turbid. Then, pipette 2 μL of the bacterial culture as a template and perform PCR analysis using N gene-F and N gene-R. Select positive clones and send them to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing. Select the clones with the correct sequencing results.

[0061] 7. Endotoxin-free plasmid extraction

[0062] 100 μL of the bacterial suspension of the selected positive clone was inoculated into 150-200 mL of LB / Amp+ liquid culture medium, and cultured in a shaker at 37°C and 180 rpm for 8-12 h. The plasmid was extracted using a Promega plasmid extraction kit; the concentration of the extracted plasmid was determined using an ultra-micro nucleic acid protein detector, and the plasmid was stored at -20°C for later use.

[0063] 8. Fake virus packaging

[0064] Day 1: 293T cells were passaged into 100 mm dishes using antibiotic-free DMEM-10% FBS for transfection and cultured in a 37°C, 5% CO2 incubator.

[0065] The next day: When the cell density reaches about 80-90% confluence, transfection can be performed.

[0066] Lipofectamine transfection: Preheat OptiMEM in a 37°C water bath. Bring LipoX Plus Reagent to room temperature before use and shake well before use. Replace the culture medium with serum-free DMEM before transfection. The transfection complex required for transfection of a 100 mm dish includes: 1.25 μg of pMD2.G plasmid, 3.75 μg of pspAX2 plasmid, 5 μg of pCDH-CMV-MCS-EF1-copGFP-T2A-Puro plasmid, and 25 μL of LipoX Plus Reagent.

[0067] Medium change: 6 h after transfection, replace with fresh complete medium containing 10% fetal bovine serum (FBS).

[0068] Cell collection: 48 h after transfection, collect the supernatant containing pseudovirus particles into a 15 mL centrifuge tube and centrifuge at 150 × g for 3 min to collect the supernatant.

[0069] 9. Pseudovirus Purification

[0070] Eliminate residual DNA: Use universal nuclease treatment. Add 0.1 μL of Benonase per 1 mL of crude viral extract and incubate at 37°C in a water bath for 1 hour to remove the cellular genome and residual plasmid DNA from the viral fluid. Centrifuge at 600 × g, 4°C for 10 minutes, and collect the supernatant.

[0071] Column purification: Purification was performed according to the operating instructions of the lentivirus purification column UFC910096;

[0072] The lentiviral sample liquid obtained by column purification was filtered through a 0.45 μm needle filter, aliquoted and stored at -80°C.

[0073] 10. Taking electron microscope photos of fake viruses

[0074] The purified virus solution was centrifuged at 80,000 g for 2 h, the supernatant was discarded, the pellet was resuspended in 100 μL PBS and negatively stained with 2% phosphotungstic acid, and the image was obtained using a Thermo HT7800 electron microscope, in which the typical pseudovirion morphology was observed ( Figure 1 ).

[0075] 11. Sanger sequencing to verify the integrity of the pseudovirus sequence

[0076] Using the extracted pseudovirus nucleic acid as a template, amplification was performed using primers N gene-F and N gene-R at either end of the target gene sequence. The amplified product was sent to Beijing Liuhe BGI Genomics Co., Ltd. for Sanger sequencing. Sequence alignment of the sequenced full-length gene fragment with the designed fragment showed 100% consistency, indicating successful pseudovirus packaging.

[0077] Example 2 Establishment of NiV Digital PCR (ddPCR) Detection Method

[0078] A ddPCR detection method was established using the NiV pseudovirus expressed in Example 1. The primers and probes used were the same primers and probes used for fluorescent quantitative PCR detection of the N gene in NiV disease diagnostic technology (NY / T 1469-2007). The ddPCR method was optimized, including primer and probe concentrations, annealing temperature, and heating and cooling rates. The linear detection range and minimum detection limit of the optimized ddPCR method were determined, and its specificity and repeatability were evaluated.

[0079] 1. Primer and probe design

[0080] Download multiple viral N gene sequences for sequence comparison. For comparison results, see Figure 2As can be seen, the N gene is conserved across all viruses. Therefore, primers and probes used for fluorescent quantitative PCR detection of the N gene in NiV disease diagnosis technology (NY / T 1469-2007) were selected. The specific nucleotide sequences are SEQ ID No. 4 to SEQ ID No. 6.

[0081] 2. Optimization of anti-primer and probe concentrations for ddPCR

[0082] 200 μL of NiV pseudovirus was used as template to extract nucleic acid and set up 20 μL ddPCR reaction system, i.e., fixed One-step RT-ddPCR mix 10 μL, reverse transcriptase 2 μL, template 2 μL, and three primer and probe combinations (600-150 nmol·L -1 , 750-250nmol·L -1 , 900-350nmol·L -1 ) Fill the volume to 20 μL with ddH2O and perform ddPCR. Taking into account the copy number detection results and the distribution of positive and negative droplets, the optimal probe and primer concentrations were selected. The reaction procedure was 50°C for 20 minutes, 95°C for 3 minutes, 94°C for 30 seconds, and 55°C for 60 seconds, for 40 cycles, with a ramp rate of 2.0°C / s.

[0083] The results of reaction system optimization are shown in Figure 3 Among the three primer and probe combinations, the distribution of positive and negative droplets was 750-250 nmol·L -1 The group had the best effect, and the N gene copy number was 7.8×10 2 copies / μL、7.06×10 3 copies / μL and 3.05×10 3 copies / μL, of which 750 nmol·L -1 primers and 250 nmol·L -1 The probe reaction concentration, that is, the upper and lower primers (10 μmol·L -1 ) were added in an amount of 0.75 μL each, and the probe (10 μmol·L -1 ) was added at a concentration of 0.5 μL, the highest gene copy number was measured, so this concentration was selected as the final primer and probe reaction concentration.

[0084] 3. Optimization of annealing temperature for ddPCR method

[0085] According to the above 20 μL ddPCR reaction system, three annealing temperatures of 55℃, 58℃ and 61℃ were set respectively, and ddPCR experiment was carried out. Considering the copy number detection results and the positive and negative droplet distribution, the optimal annealing temperature of the reaction was determined.

[0086] The results are shown in Figure 4 At the annealing temperatures of 55℃ and 61℃, there was no significant difference in the dispersion effect of positive and negative droplets, and the copy numbers were 7.04×10 3 copies / μL and 7.08×10 3 copies / μL respectively; at the annealing temperature of 58℃, the positive droplets were more concentrated than those at the annealing temperatures of 55℃ and 61℃, and the copy number was also higher, which was 7.5×10 2 copies / μL; therefore, the annealing temperature was set to 58℃.

[0087] 4. Optimization of the heating and cooling rate of ddPCR method

[0088] According to the above 20 μL ddPCR reaction system, the reaction program was selected to optimize the annealing temperature, and the other reaction programs were unchanged, only the heating and cooling rate was changed, three heating and cooling rates of 1.0℃ / S, 2.0℃ / S and 3.0℃ / S were set, ddPCR experiment was carried out, and the best heating and cooling rate of the reaction was determined.

[0089] The results are shown in Figure 5 , the amplification results of three heating and cooling rates of 1.0℃ / S, 2.0℃ / S and 3.0℃ / S were 6.8×10 3 copies / μL, 6.9×10 3 copies / μL and 6.4×10 3 copies / μL respectively, the difference of the fluorescence signal value of positive and negative droplets at the heating and cooling rate of 2.0℃ / S was larger than that at the other two heating and cooling rates, and the detected copy number was the highest, so the heating and cooling rate of 2.0℃ / S was selected.

[0090] 5. Determination of the dynamic range and sensitivity of the ddPCR detection method

[0091] Take 200 μL of pseudovirus, extract nucleic acid, and perform 10-fold serial dilution of the extracted nucleic acid. Take 10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 dilutions for ddPCR detection, and each concentration is repeated 3 times. The ddPCR results are established by gradient dilution to form a linear graph, and the R 2 = 0.999, the linear relationship is good, and the quantitative linear dynamic range is 5.17×10 0-6.82 x 10 4 copies / μL, the lowest detection limit was 5.17 copies / μL, and the results are shown in Table 1. Figure 6 、 Figure 7 and Table 1.

[0092] Table 1. Results of ddPCR detection of 10-fold dilution samples (copies / μL)

[0093] Theoretical concentration Detection 1 Detection 2 Detection 3 AVG SD RSD <![CDATA[10 0 ]]> 7.03E+04 6.95E+04 6.90E+04 6.96E+04 6.44E+02 0.9％ <![CDATA[10 -1 ]]> 7.41E+03 7.47E+03 8.08E+03 7.65E+03 3.70E+02 4.8％ <![CDATA[10 -2 ]]> 6.51E+02 6.93E+02 7.07E+02 6.84E+02 2.92E+01 4.3％ <![CDATA[10 -3 ]]> 5.19E+01 6.07E+01 6.13E+01 5.80E+01 5.26E+00 9.1％ 10 -4 ]] 4.60E+00 5.00E+00 5.70E+00 5.10E+00 5.57E-01 10.9％

[0094] 6. Specific detection of the ddPCR detection method

[0095] In designing the primers and probes, the conservation of the target was taken into consideration, and in addition, the primer and probe sequences used were from the NiV disease diagnostic technique (NY / T 1469-2007), the specificity of which has been verified, and therefore no further verification is required here.

[0096] 7. Reproducibility detection of the ddPCR detection method

[0097] Two tubes of NiV pseudovirus were taken, and the nucleic acid was extracted for ddPCR detection, with 3 detections per tube. The results of each detection are shown in Table 2, and the coefficient of variation between the detection results was calculated to be 3.4%, indicating that the ddPCR method established has good reproducibility.

[0098] Table 2. Reproducibility detection of the ddPCR method

[0099]

[0100] Example 3

[0101] According to the technical route shown in Figure 8 , a NiV pseudovirus ribonucleic acid standard substance was developed

[0102] I. Preparation of the NiV pseudovirus ribonucleic acid standard substance and initial homogeneity test

[0103] 1. Preparation of the standard substance

[0104] 200 μL of the purified pseudovirus stock solution was placed in a clean 500 mL beaker, and 199.8 mL of virus preservation solution was added to dilute it 1000-fold to obtain 200 mL of a candidate standard substance. After mixing with a magnetic stirrer for 30 min, the initial homogeneity test was performed.

[0105] 2. Initial homogeneity test

[0106] The homogeneity of the mixed sample was preliminarily detected according to JJF 1343-2022 "Standard Material Valuation and Homogeneity Stability Evaluation". Three samples of 200 μL were taken from the upper, middle and lower parts of the mixed sample. The nucleic acid of the sample was extracted, and the ddPCR method was used for amplification. Each sample was analyzed for 3 times, and the difference in genome content of the 9 times of data was investigated. The copy number of the sample to be tested and the calculation results are shown in Table 3. The virus nucleic acid content of the samples taken from different parts has no significant difference, and the homogeneity is good, which can be subpackaged.

[0107] Table 3 Copy number and results of homogeneity preliminary detection (x 10 3 copies / μL)

[0108]

[0109] II. Subpackaging

[0110] Subpackaging was carried out in a class II biosafety cabinet in a ten-thousand-level working area. Subpackaging was carried out in 1.5 mL cryogenic tubes, 650 μL / tube, a total of 300 tubes. After subpackaging, the rubber ring cover was sealed and stored in a -80°C environment.

[0111] III. Test of properties of NiV pseudovirus ribonucleic acid standard material

[0112] 1. Physical property test

[0113] The standard material was a colorless transparent liquid, as shown in Figure 9 .

[0114] 2. Inspection of DNA contamination

[0115] ddPCR was performed with and without reverse transcriptase to detect DNA residues in the pseudovirus solution.

[0116] Take 100 μL of NiV pseudovirus ribonucleic acid standard material, extract nucleic acid. Prepare the reaction solution with reverse transcriptase: 8 μL of 2x one step ddPCRmix, 2 μL of reverse transcriptase, 1.8 μL of upstream primer, 1.8 μL of downstream primer, 0.7 μL of probe, 2 μL of total nucleic acid of pseudovirus and 4 μL of RNase Free dH2O, mix well and centrifuge at 3000 rpm for a short time. Prepare the reaction solution without reverse transcriptase: 8 μL of 2x one step ddPCRmix, 1.8 μL of upstream primer, 1.8 μL of downstream primer, 0.7 μL of probe, 2 μL of pseudovirus nucleic acid and 4 μL of RNase Free dH2O, mix well and centrifuge at 3000 rpm for a short time, and detect according to the ddPCR method described in Example 2.

[0117] The results are shown in Figure 10As shown, the ddPCR reaction system without reverse transcriptase has no detection results, and the ddPCR reaction system with reverse transcriptase is normally amplified, so it can be considered that the pseudovirus does not contain DNA residues.

[0118] 3. Other virus tests

[0119] The laboratory for virus product testing of the China Institute for Control of Animal Disease and Drug Administration (hereinafter referred to as the laboratory) has preserved pathogens such as porcine reproductive and respiratory syndrome virus, pseudorabies virus, classical swine fever virus, small ruminant pestivirus, inactivated African swine fever virus, inactivated bovine dermatophilus virus, Schmallenberg virus pseudovirus, NiV pseudovirus, and bovine viral diarrhea / mucosal disease virus. In order to control the quality of the standard material, it is necessary to detect whether the above-mentioned viruses are mixed in the standard material.

[0120] Take the NiV pseudovirus ribonucleic acid standard material, extract the nucleic acid, and then perform porcine reproductive and respiratory syndrome virus (method source: GB_T 18090-2023 Porcine Reproductive and Respiratory Syndrome Diagnostic Method), pseudorabies virus (method source: Chapter 2.1.2 WOAH Terrestrial Animal Diagnostic Tests and Vaccines Manual - Seventh Edition Multiple Animal Co-infection Diseases - Pseudorabies (Aujeszky's Disease)), classical swine fever virus (method source: T / CVMA5-2018 Real-time Fluorescent PCR Detection Method for African Swine Fever Virus), small ruminant pestivirus (method source: Small Ruminant Pestivirus Diagnostic Technology GB / T27982-2011), inactivated African swine fever virus (method source: African Swine Fever Diagnostic Technology GB / T21675-2022), inactivated bovine dermatophilus virus (method source: Bovine Dermatophilus Diagnostic Technology GB / T39602-2020), Schmallenberg virus (method source: Schmallenberg Disease Quarantine Technical Specification SN / T 4661-2016), and bovine viral diarrhea / mucosal disease virus (method source: Bovine Viral Diarrhea / Mucosal Disease Diagnostic Technology Specification GBT_18637-2018) nucleic acid detection. The results show that, in addition to the NiV specific primer detection result being positive, the detection results of other pathogens are negative. It is indicated that there is no contamination of other viruses in the NiV pseudovirus ribonucleic acid standard material.

[0121] Four, uniformity evaluation of NiV pseudovirus ribonucleic acid standard material

[0122] According to the requirements of JJF1343-2022 "Standard Material Valuation and Uniformity, Stability Evaluation" in China, the uniformity of the NiV ribonucleic acid standard material was evaluated, and the detection method used the ddPCR method established in Example 2.

[0123] A total of 300 tubes of NiV RNA standard material were prepared for this batch. Fifteen tubes (numbers: 295, 276, 235, 226, 201, 192, 188, 165, 138, 107, 095, 079, 068, 037, and 005) were randomly selected. Each tube was sampled three times, with 200 μL sampled each time, and three replicate measurements were performed.

[0124] Viral nucleic acid was extracted and the nucleic acid content of NiV RNA standard material was detected by ddPCR method. The homogeneity test was performed by variance analysis (F test). The specific results are shown in Tables 4 and 5. According to the degrees of freedom and the given significance level α, the critical value can be found in Tables 4 and 5 to calculate F. <F α (F (0.05,14,30) =2.04) showed that there was no statistical difference between the groups and the samples were uniform.

[0125] Table 4 Measurement data of uniformity evaluation (×10 3 copies / μL)

[0126]

[0127]

[0128] Table 5 Uniformity evaluation results

[0129]

[0130] 5. Stability testing of NiV pseudoviral RNA standard materials

[0131] According to the requirements of my country's JJF1343-2022 "Value Determination, Uniformity and Stability Evaluation of Standard Materials", NiV ribonucleic acid standard materials are tested for long-term stability, short-term stability, and stability after repeated freeze-thaw after opening the bottle.

[0132] 1. Stability testing plan

[0133] The stability of this batch of standard substances was tested using the ddPCR method established in Example 2.

[0134] The packaged standard substances were first stored in a -80°C refrigerator, and the storage conditions were -20°C. Two tubes of standard substances were extracted each time and stored in a refrigerator at a specified temperature of -20°C for the specified time. The storage time was 0, 1, 2, 4, and 6 months. Each tube of sample was tested 3 times, and ddPCR was used for detection. The mean was calculated and stability analysis was performed.

[0135] Analysis of the stability of the standard substance at -20°C storage conditions revealed a value less than T(0.95,5)*S(β1). Therefore, it is considered that the slope is not significantly different and no instability is observed, thus meeting the requirements of practical measurements. This standard substance is considered stable at -20°C for six months. The results are shown in Table 6.

[0136] Table 6-20℃ long-term stability data and conclusions of standard substances (×10 3 copies / μL)

[0137]

[0138]

[0139] 2. Short-term stability test

[0140] 1) Short-term stability test of standard substances at 4°C

[0141] Ten tubes of standard substances were stored in a refrigerator at 4°C. Two tubes of standard substances were extracted each time on days 0, 1, 2, 4, and 7. Each tube of sample was tested three times using ddPCR. The mean was calculated and a short-term stability analysis at 4°C was performed.

[0142] The stability of the standard substance under 4℃ storage conditions was analyzed (Table 7). The results were less than t (0.95，3) *s(β1), so it is considered that there is no significant difference in the slope and no instability is observed, which meets the needs of actual measurement. It can be assumed that this standard substance is stable at 4°C for 7 days.

[0143] Table 7 Short-term stability test and conclusion of 4℃ standard substances (×10 3 copies / μL)

[0144]

[0145] 2) Short-term stability test of standard substances at 25°C

[0146] Synchronous stability testing was used to investigate the stability of use at room temperature. Eight tubes of standard substances were stored in a 25°C environment. Two tubes of standard substances were extracted each time at 0, 12, 24, and 36 hours. Each tube of sample was tested three times, for a total of six data points. The test data were summarized and statistically tested.

[0147] The results are shown in Tables 8 and 9. The detection results of the standard substance stored at 25°C for 12, 24 and 36 hours are subjected to T test statistical analysis with the detection results of the standard substance stored for 0 hour. The P values of the standard substance stored for 12 and 24 hours are greater than 0.05, and thus it is considered that there is no significant difference between the detection results of the standard substance stored for 12 and 24 hours and the detection results of the standard substance stored for 0 hour. In addition, the P value of the detection results of the standard substance stored for 36 hours is less than 0.05, and thus it is considered that there is a significant difference between the detection results of the standard substance stored for 36 hours and the detection results of the standard substance stored for 0 hour. It is considered that the standard substance can be stable for 24 hours at 25°C.

[0148] Table 8 Short-term stability test of 25°C standard substance (x 10 3 copies / μL)

[0149]

[0150] Table 9 Statistical results of short-term stability test of 25°C standard substance

[0151]

[0152] 3. Repeated freeze-thaw test after opening the bottle

[0153] 1) Opening the bottle and thawing stability experiment:

[0154] Two tubes of the standard substance are taken out, and the detection is performed after the third time of opening the bottle and thawing. Each time, the standard substance is balanced at room temperature, and after thawing, the standard substance is stored at -20°C again. The opening and thawing of the bottle is performed three times for each tube, and the detection data of the first time of opening the bottle and thawing is used as a control group. The detection is performed by using the ddPCR method. The stability of the standard substance after opening the bottle and thawing is analyzed.

[0155] The detection results of the standard substance after opening the bottle and thawing three times and the detection results of the standard substance after opening the bottle and thawing one time are subjected to T test statistical analysis. The P value is greater than 0.05, and thus it is considered that there is no significant difference between the detection results of the standard substance after opening the bottle and thawing three times and the detection results of the standard substance after opening the bottle and thawing one time. It is considered that the standard substance can be opened and thawed three times. The results are shown in Tables 10 and 11.

[0156] Table 10 copies number of opening the bottle and thawing experiment (x 10 3 copies / μL)

[0157]

[0158] Table 11 Statistical results of opening the bottle and thawing stability test of the standard substance

[0159]

[0160] In summary, the standard substance can be stable for 6 months at -20°C, can be stable for 7 days at 4°C, can be stable for 24 hours at 25°C, and can be opened and thawed three times.

[0161] VI. Determination of the Value of NiV Pseudoviral RNA Reference Material

[0162] ddPCR was used to perform absolute quantification of the standard substances, and (copies / μL) was selected as the unit of measurement.

[0163] Determination method: Nine qualified laboratories independently used the ddPCR method to determine the characteristic values ​​of standard substances.

[0164] 1. Selection of calibration laboratory

[0165] The organizing unit invited nine laboratories capable of nucleic acid calibration to participate in this collaborative calibration of reference materials. See Table 12 for the laboratory numbers, names, locations, and accreditation categories.

[0166] All participating laboratories have passed proficiency testing and possess the capability to detect NiV pseudoviruses, as well as biosafety laboratories qualified for NiV pseudovirus detection. During the calibration process, all instruments used have undergone calibration and verification by metrology departments or inter-laboratory instrument comparisons, meeting certification and accreditation requirements. The reagents and consumables used have also undergone performance evaluation, ensuring the accuracy and reliability of the values ​​obtained from the collaborative calibration of reference materials.

[0167] Table 12 Participating in the fixed value laboratory

[0168]

[0169] 2. Determination of characteristic values

[0170] Each of the nine laboratories tested two tubes of standard reference material, repeated the test three times on each tube, performed statistical analysis on the experimental data, and returned the experimental data to the research and development unit for confirmation.

[0171] 1) Instrument status

[0172] All equipment, including ddPCR instruments and pipettes, was calibrated before use to ensure the accuracy, validity, and traceability of the results. The verification of the characteristic values ​​of the reference materials was conducted by qualified testing institutions, and the ddPCR instruments used for the verification underwent instrument quality inspection.

[0173] 2) Characteristic value combined fixed value test results

[0174] Table 13 Results of cooperative evaluation of NiV pseudoviral RNA reference materials

[0175]

[0176] 3) Statistical processing of fixed value data

[0177] According to the national conditions and international advanced experience of test analysis, the processing method of experimental results mainly refers to JJF 1343-2022 "Standard Material Value, Homogeneity and Stability Evaluation", and the data obtained by detection are subjected to overall data normal distribution test, laboratory data suspicious value test and group data precision test.

[0178] From the statistical results, it can be seen that the measurement data of each group is equal-precision data, and the average values between groups have no significant difference, so the standard value of the standard material can be represented by the arithmetic mean.

[0179]

[0180] 3. Uncertainty evaluation

[0181] For standard materials, the uncertainty of the value results is composed of three parts, which are the uncertainty introduced by the homogeneity of the standard material, the uncertainty introduced by the stability of the standard material and the uncertainty brought by the value process of the standard material. The value uncertainty and the uncertainty introduced by homogeneity evaluation and stability detection are superimposed according to the square and square root method to give the combined standard uncertainty, denoted as u CRM . The uncertainty obtained by multiplying the factor (the factor is called the inclusion factor, denoted as k) is called the expanded uncertainty or the total uncertainty, denoted as U. The calculation of the three parts of uncertainty refers to JJF 1343-2022 "Standard Material Value, Homogeneity and Stability Evaluation". After calculation, the combined relative standard uncertainty of the standard material is:

[0182]

[0183] The relative expanded uncertainty is obtained by multiplying the relative standard uncertainty of the standard material by the inclusion factor k, and k=2.

[0184] U rei(CRM) = k*u rel(CRM) = 2 x 0.099 = 20%

[0185] 4. Expression of standard material value results

[0186] According to JJF 1343-2022 "Standard Material Value, Homogeneity and Stability Evaluation", the standard material value results are expressed as: standard value ± expanded uncertainty.

[0187] Standard value = 6.9 x 10 3 (copies / μL)

[0188] Expanded uncertainty = standard value x relative expanded uncertainty = 6.9 x 10 3 x 0.2 = 1.4 x 10 3(copies / μL) Table 14 standard material mass value results

[0189] name Results (x10 3 copies / μL))]]> Expanded uncertainty U(k=2) 1.4 Standard value 6.9 Standard value ± expanded uncertainty 6.9±1.4

[0190] In summary, the present application prepares a NiV pseudovirus containing the N gene sequence of the NiV, which can be used to prepare the NiV pseudovirus ribonucleic acid standard material. The ddPCR method is used, multiple laboratories cooperate to value, and statistical analysis is performed on the data to determine the quantity value and uncertainty of the NiV pseudovirus ribonucleic acid standard material. The ribonucleic acid standard material prepared by the NiV pseudovirus of the present application can be used in the fields of quantity value transfer, calibration, test instruments, evaluation and control test methods, quality testing, quality control and production process testing of related laboratories for NiV pathogenic microorganism nucleic acid detection.

[0191] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. A pseudovirus of Nipah virus, characterized in that The pseudovirus contains a detection target; the detection target includes a nucleic acid fragment of the Nipah virus N gene whose nucleotide sequence is shown in SEQ ID NO.

1.

2. The pseudovirus according to claim 1, wherein The 3' end of the nucleic acid fragment of the Nipah virus N gene is further connected to the nucleotide sequence shown in SEQ ID NO.2; the 5' end of the nucleic acid fragment of the Nipah virus N gene is further connected to the nucleotide sequence shown in SEQ ID NO.

3.

3. The pseudovirus according to claim 1, wherein The pseudovirus uses CDH-CMV-MCS-EF1-copGFP-T2A-Puro as an expression vector.

4. The method for preparing a pseudovirus according to any one of claims 1 to 3, characterized in that: The following steps are involved: The three plasmids are co-transfected into the engineered cells through a three-plasmid system, the transfected engineered cells are cultured, and the supernatant is collected to obtain the pseudovirus.

5. The preparation method according to claim 4, characterized in that The three-plasmid system includes pCDH-CMV-MCS-EF1-copGFP-T2A-Puro, psPAX2 and pMD2.G.

6. The preparation method according to claim 5, characterized in that The mass ratio of pCDH-CMV-MCS-EF1-copGFP-T2A-Puro, psPAX2 and pMD2.G is 5:3.75:1.

25.

7. The preparation method according to claim 4, characterized in that The engineered cells include HEK293T cells.

8. Use of the pseudovirus according to any one of claims 1 to 3 or the pseudovirus prepared by the preparation method according to any one of claims 4 to 7 in the preparation of Nipah virus pseudovirus RNA standard material.

9. A kit for diagnosing Nipah virus, characterized in that: The kit comprises the pseudovirus according to any one of claims 1 to 3 or the pseudovirus prepared by the preparation method according to any one of claims 4 to 7.

10. The kit according to claim 9, characterized in that The kit also includes primers and probes for detecting Nipah virus.

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