Method for evaluating copy number of specific nucleic acid in quantitative pseudovirus antigen standard

Abstract

The invention belongs to the technical field of biology, and particularly relates to an evaluation method for the copy number of specific nucleic acid in a quantitative pseudovirus antigen standard substance, and the evaluation method comprises the following steps: (1) providing a pseudovirus antigen standard substance; (2) treating a pseudovirus antigen standard substance by DNA enzyme; (3) extracting nucleic acid of the treated pseudovirus antigen standard substance; (4) preparing a micro-droplet type digital PCR reaction system; (5) carrying out micro-droplet treatment on the reaction system; (6) performing micro-droplet digital PCR amplification on each reaction system subjected to micro-droplet treatment, and calculating the nucleic acid concentration of the pseudovirus antigen standard substance according to an amplification result; the invention also provides a primer probe composition for detecting rabies virus pseudovirus based on the disclosed method. According to the provided method and composition, accurate evaluation of the copy number of specific nucleic acid in a pseudovirus antigen standard substance is realized through the optimal primer and probe combination.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for evaluating the copy number of specific nucleic acid in a quantitative pseudovirus antigen standard. Background Art

[0002] Pseudovirus antigen standards are important tools for the development and quality control of vaccines and antiviral drugs. The accurate determination of the number of nucleic acid copies is crucial for the potency evaluation and batch consistency of vaccines and antiviral drugs. At present, commonly used nucleic acid quantification methods (such as qPCR) have problems such as insufficient sensitivity and strong standard curve dependence, which makes it difficult to meet the needs of high-precision quantification. Therefore, it is of great significance to develop a highly sensitive and accurate quantitative method.

[0003] Digital PCR is a new technology that has emerged in recent years. By processing samples into tiny units, the absolute number of target molecules to be tested as low as a single copy can be determined. Digital PCR can directly count the number of target molecules without relying on any calibrants or external standards, and is an absolute quantitative PCR technology.

[0004] However, there are few reports on the application of digital PCR in the quantification of specific nucleic acid copy number of pseudovirus antigen standards. Summary of the invention

[0005] The object of the present invention is to provide a method for evaluating the copy number of specific nucleic acid in a quantitative pseudovirus antigen standard, which comprises the following steps: (1) Providing pseudovirus antigen standards; (2) DNA enzyme treated pseudovirus antigen standard; (3) Extracting nucleic acid from the treated pseudovirus antigen standard; (4) Preparing a reaction system for droplet digital PCR; (5) Performing micro-droplet treatment on the reaction system; (6) Performing droplet digital PCR amplification on each reaction system that has been treated with droplets, and calculating the nucleic acid concentration of the pseudovirus antigen standard based on the amplification results.

[0006] As used herein, pseudovirus antigen standards are virus-like particles (VLPs) or pseudovirus particles constructed by molecular biology techniques that contain key antigenic components of viruses (such as surface proteins) but do not have the ability to replicate.

[0007] In some embodiments, the pseudovirus antigen standard is a rabies virus pseudovirus.

[0008] In some embodiments, the pseudovirus antigen standard comprises a rabies virus-specific target gene sequence.

[0009] In some embodiments, the pseudovirus antigen standard comprises a viral coat protein, such as VSV-G.

[0010] In some embodiments, the pseudovirus antigen standard is prepared as follows: cloning the rabies virus-specific target gene sequence by PCR amplification; connecting the gene sequence obtained by the above cloning to a lentiviral expression vector by homologous recombination to obtain a recombinant expression plasmid; co-transfecting the recombinant expression plasmid with an auxiliary plasmid into cells and then packaging pseudovirus particles to obtain pseudovirus particles that have packaged the target gene and are secreted in the supernatant.

[0011] In some embodiments, the lentiviral expression vector is FV115, and the framework structure of the vector is CMV-MCS-3Flag-Ubi-ZSGreen-IRES-Puromycin In some embodiments, the helper plasmid is psPAX2, pMD2G.

[0012] In some embodiments, the rabies virus-specific target gene sequence is shown as SEQ ID NO: 1.

[0013] In some embodiments, the reaction system of the droplet digital PCR comprises amplification primers and probes targeting the rabies virus-specific target gene.

[0014] In some embodiments, the sequences of the amplification primers are shown in SEQ ID NO:2 and SEQ ID NO:3.

[0015] In some embodiments, the sequence of the probe is shown in SEQ ID NO:4.

[0016] In some embodiments, the droplet digital PCR reaction system further comprises a reaction buffer, a reverse transcriptase, a TAQ DNA polymerase, and nuclease-free water.

[0017] In some embodiments, both ends of the probe sequence are labeled with a fluorescent group and a quencher group, respectively.

[0018] In some embodiments, the fluorescent group is at least one of FAM, HEX, VIC, TET, ROX and CY5; the quenching group is at least one of TAMRA, MGB, BHQ1, BHQ2 and BHQ3.

[0019] The present invention also provides a primer-probe combination for detecting rabies virus pseudovirus, characterized in that the rabies virus pseudovirus comprises a rabies virus-specific target gene sequence as shown in SEQ ID NO: 1, the primer-probe combination comprises primers as shown in SEQ ID NO: 2 and SEQ ID NO: 3 and a probe as shown in SEQ ID NO: 4, and both ends of the probe sequence are respectively labeled with a fluorescent group and a quenching group.

[0020] In some embodiments, the fluorescent group is at least one of FAM, HEX, VIC, TET, ROX and CY5; the quenching group is at least one of TAMRA, MGB, BHQ1, BHQ2 and BHQ3.

[0021] Advantageous Effects of the Invention The present invention specifically develops specific pseudovirus antigen standards, adopts droplet digital PCR (ddPCR) technology, avoids the problem of traditional fluorescent quantitative PCR (qPCR) relying on standard curves, and improves the accuracy and repeatability of detection. The method of the present invention is suitable for the detection of low copy number nucleic acids, and is combined with a specific rabies virus-specific target gene sequence (SEQ ID NO: 1) and optimized specific primers and probes (SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4) to detect rabies virus pseudoviruses and improve detection specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the results of determining the value of the pseudovirus antigen standard of the present invention. DETAILED DESCRIPTION

[0023] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0024] Example 1: Preparation of pseudovirus antigen standards The LN34 gene of rabies virus (RV) was synthesized by chemical synthesis (the sequence is shown in SEQ ID NO: 1), and restriction sites of XbaI and NotI and protective bases were inserted upstream and downstream; Through seamless cloning, the CMV-MCS-3Flag-Ubi-ZSGreen-IRES-Puromycin vector plasmid (FV115) and the LN34 target gene fragment were double-digested and then ligated to obtain an enzyme-ligated product; Using Amp as the prokaryotic amplification screening marker and Puromycin as the eukaryotic screening marker, the enzyme-linked product was transformed into the competent E. coli DH5α and inoculated into 5 mL of LB medium without resistance, and the bacteria were shaken at 200 rpm for 4 hours. Then 100 μL of the bacterial solution was diluted and spread on the LB plate containing Amp resistance, and the positive clones were screened at 37°C for overnight inversion culture. The positive clones were expanded and cultured in LB medium containing Amp resistance; the plasmid was extracted by alkaline lysis method, and the inserted fragment was sequenced and verified, and the result was consistent with SEQ ID NO: 1; The primers used for the sequencing are as follows: Forward primer: 5′-ACGCTTAACAACAAAACCAGA-3′; Reverse primer: 5′-CTGATATTTTGCTCAACCTGT-3′; The obtained LN34 gene vector plasmid was co-transfected with auxiliary plasmids psPAX2 and pMD2G to prepare pseudoviral particles; the specific transfection method is as follows: a. After HEK293T cells were revived, they were subcultured for 2 generations using DMEM medium containing 10% FBS until the cells were stable, then the cells were trypsinized and placed in a culture dish to culture until the cell confluence reached 70% for transfection; b. 4 h before transfection, remove the serum-containing medium, wash the cells with sterile PBS, and then replace with Opti-MEM medium to starve the cells; c. Take 10 μg of vector plasmid, 5 μg of psPAX2 plasmid, and 5 μg of pMD2G plasmid, adjust the volume to 500 μL with Opti-MEM medium, and let stand at room temperature for 5 minutes to obtain a plasmid mixture; d. Mix 50 μL of PEI (polyethyleneimine) transfection reagent with 450 μL of Opti-MEM medium, let stand at room temperature for 5 minutes, add the plasmid mixture to the PEI diluent, mix by inversion, let stand at room temperature for 10 minutes, then add the cells and shake the culture dish to ensure even distribution; e. 8 h after transfection, remove the culture medium and replace it with fresh serum-containing culture medium; f. Observe the expression of green fluorescent protein under an inverted microscope 48 hours after transfection. After successful transfection, collect the culture medium, filter it through a 0.22μm filter membrane and collect it in a sterile ultracentrifuge tube, centrifuge it at 80000g for 4 hours, discard the supernatant, resuspend the precipitate with virus store buffer, and store it at -80℃, which is the pseudovirus antigen standard.

[0025] Example 2: Evaluation of the number of copies of specific nucleic acid of pseudovirus antigen standard DNA enzyme was added to the pseudovirus antigen standard to remove free DNA in the virus solution. After removing the DNA residue, nucleic acid was extracted. Viral nucleic acid was extracted by centrifugation. 140 μl of the standard was equilibrated to room temperature, treated with virus lysis solution, anhydrous ethanol and washing buffer in sequence, and finally washed with 70 μl of enzyme-free water to obtain the RNA of the standard by centrifugation. The digital PCR system was prepared by mixing the RNA of the standard as a template, adding reverse transcriptase, high-fidelity DNA polymerase, PCR reaction buffer and rabies virus-specific primers and probes. The generated mixture was added to the PCR plate and amplified according to the predetermined program. The sequences of the primers used are shown in SEQ ID NO: 2 (5'-TCCGGATGATGTATGCTCCT-3') and SEQ ID NO: 3 (5'-ATAGCTGGTCCAGTCTTCCG-3') and the sequence of the probe is shown in SEQ ID NO: 4 (5'-FAM-ACTTGGCAGCAGCAATGCAGTTCT-TAMRA-3').

[0026] The reaction system and amplification program of digital PCR are shown in Tables 1 and 2. The numerical reading is performed using a reader and QuantaSoft software. The instrument is run by setting parameters such as ABS, One-Step RT-ddPCR Kit for Probes, FAM channel, and HEX / VIC channel. The results are observed and the RNA value of the standard is obtained based on the droplet signal of the FAM / HEX channel. Table 3 shows the evaluation results of 18 randomly selected pseudovirus antigen standards, with each unit measured three times.

[0027] Table 1: PCR reaction system

[0028] Table 2: PCR reaction program

[0029] Table 3: Pseudovirus antigen standard determination results (×10 4 copy / ml)

[0030] The results in Table 3 are as follows Figure 1 shown.

[0031] Example 3: Reliability of the method for evaluating the number of copies of specific nucleic acid of pseudovirus antigen standard The rabies LN34 gene pseudovirus RNA standard material (GBW(E)091127) and different primer and probe sequences were used to verify the copy number evaluation method of the pseudovirus antigen standard specific nucleic acid. The results showed that the evaluation method using primers of SEQ ID NO: 2 and 3 and probe of SEQ ID NO: 4 had the best reliability.

[0032] Table 4: Sequence information of different primer-probe combinations

[0033] Table 5: Reliability of the method for evaluating the copy number of specific nucleic acid of pseudovirus antigen standards

[0034] From the results in Table 5, it can be seen that the primer and probe combination of SEQ ID NO: 2-4 achieves the best reliability.

[0035] Example 4: Method for evaluating the number of copies of specific nucleic acid of pseudovirus antigen standard Determination of the uniformity of pseudovirus antigen standard According to the method of Example 1, 200 tubes of pseudovirus antigen standard for pathogen nucleic acid detection were prepared, with a specification of 150 μl / tube. According to JJF 1343-2022 "Value determination and uniformity and stability evaluation of standard substances", 11 packaging units (tubes) were randomly selected for nucleic acid extraction for each standard substance, and different primer probe sets were used to perform digital PCR detection according to the evaluation method of Example 2; the data obtained by the detection were subjected to the F test to determine whether there were differences between and within tubes, and the measurement results are shown in Table 6.

[0036] Table 6: Homogeneity determination of rabies pseudovirus standards

[0037] From the data in Table 6, it can be seen that different primer-probe sets produced different results for the uniformity of the same sample, among which the F values ​​of the results of combinations 1, 2 and 4 were all less than F 0.05(10，11) , indicating that there is no significant difference in the uniformity of the standard products. However, it can be seen from the results that the uniformity measured by combination 1 is significantly better and the difference is lower. In addition, the F value of combination 3 is greater than F 0.05(10，11) , and the results showed that there were significant differences in the uniformity of the standards, which shows that the selection of a specific primer probe set is very important. Once a combination with poor detection effect is selected, it is easy to cause incorrect evaluation results of pseudovirus standards.

[0038] It should be noted that the preferred embodiments of the present invention are given in the specification of the present invention, but the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not intended to be additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the present invention specification; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims

1. A method for evaluating the copy number of specific nucleic acid in a quantitative pseudovirus antigen standard, characterized in that: The following steps are involved: (1) Providing pseudovirus antigen standards; (2) DNA enzyme treated pseudovirus antigen standard; (3) Extracting nucleic acid from the treated pseudovirus antigen standard; (4) Preparing a reaction system for droplet digital PCR; (5) Performing micro-droplet treatment on the reaction system; (6) performing droplet digital PCR amplification on each reaction system treated with droplets, and calculating the nucleic acid concentration of the pseudovirus antigen standard according to the amplification results; The pseudovirus antigen standard contains a rabies virus-specific target gene sequence; The rabies virus-specific target gene sequence is shown in SEQ ID NO: 1; The reaction system of the droplet digital PCR comprises amplification primers and probes for the rabies virus-specific target gene; The sequences of the amplification primers are shown in SEQ ID NO: 2 and SEQ ID NO: 3; The sequence of the probe is shown in SEQ ID NO:

4.

2. The method according to claim 1, characterized in that The reaction system of the droplet digital PCR also includes a reaction buffer, a reverse transcriptase, a TAQ DNA polymerase and nuclease-free water.

3. The method according to claim 2, characterized in that Both ends of the probe sequence are labeled with a fluorescent group and a quenching group respectively.

4. The method according to claim 3, characterized in that The fluorescent group is at least one of FAM, HEX, VIC, TET, ROX and CY5; the quenching group is at least one of TAMRA, MGB, BHQ1, BHQ2 and BHQ3.

5. A primer-probe composition for detecting rabies virus pseudovirus, characterized in that: The rabies virus pseudovirus comprises a rabies virus-specific target gene sequence as shown in SEQ ID NO: 1, the primer-probe composition comprises primers as shown in SEQ ID NO: 2 and SEQ ID NO: 3 and a probe as shown in SEQ ID NO: 4, and both ends of the probe sequence are respectively labeled with a fluorescent group and a quenching group.

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