Primer and probe combination for detecting multiple RNA viruses capable of infecting sf9 cells, and use thereof

Abstract

The present invention relates to the technical field of exogenous RNA virus detection and control of biological products. Provided are a primer and probe combination for detecting multiple RNA viruses capable of infecting Sf9 cells, and the use thereof. Provided is a primer and probe combination capable of simultaneous detection of multiple RNA viruses, which are capable of infecting Sf9 cells, in insect cells and biological products thereof. The primer and probe combination contains seven forward primers, seven reverse primers, and six probes; can cover multiple RNA viruses capable of infecting Sf9 cells; has the advantages of high sensitivity, good specificity, high applicability, a small sample amount, etc.; and can be widely used in the safety evaluation of exogenous RNA viruses in insect cells and biological products thereof.

Description

Primer probe combination for detecting multiple RNA viruses capable of infecting Sf9 cells and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of exogenous RNA virus detection control of biological products, and particularly relates to a primer probe combination for detecting multiple RNA viruses capable of infecting Sf9 cells and application thereof. BACKGROUND

[0002] The baculovirus-insect cell expression system is an exogenous gene expression system taking baculovirus as a carrier and insect cells as a host. The system has the advantages of easy screening, high expression level and post-translational modification, etc. As one of the widely used eukaryotic expression systems today, it has been successfully used in the development process of human and animal biological products. The insect cells commonly used for production include Spodoptera frugiperda ovary cell line (Sf9 / Sf21) and Trichoplusia ni ovary cell line (Hi-Five).

[0003] As an important production cell matrix, the detection of exogenous viruses of Sf9 cells is one of the keys to prevent biological safety problems. According to the quality requirements of biological technology products in the “People's Republic of China Pharmacopoeia” and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), the virus safety of biological products needs to be evaluated; the U.S. Food and Drug Administration (FDA) also clearly states that insect cells used for the production of biological products need to be tested for species-specific viruses. However, there is no regulation or guidance document that clearly indicates the specific virus species that Sf9 cells need to be tested.

[0004] At present, there are documents that have clearly reported that the RNA viruses capable of infecting Sf9 cells include sf-Rhabdovirus, St Louis encephalitis virus, Japanese B encephalitis virus, Dengue-1 and 2, and Chikungunya virus. Although some patents and documents have mentioned that some of these RNA viruses can be detected by qPCR method. However, some methods cannot comprehensively cover the RNA viruses capable of infecting Sf9 cells, and some methods have not verified the applicability of Sf9 cells and their biological products.

[0005] Therefore, there is an urgent need for a comprehensive and exogenous RNA virus qPCR detection technology suitable for the quality control of Sf9 cells and their biological products. SUMMARY

[0006] The application provides a primer probe combination for detecting a plurality of RNA viruses that can infect Sf9 cells and its application. The provided primer probe combination has a wide coverage, high sensitivity, strong specificity, and is not affected by cell matrix interference, and is suitable for Sf9 cells and their biological product quality control. The application is specifically implemented by the following technologies.

[0007] In the first aspect of the application, a primer probe combination for detecting a plurality of RNA viruses that can infect Sf9 cells is provided, comprising the following primers and detection probes:

[0008] The upstream primer sfRV-F and the downstream primer sfRV-R for detecting Spodoptera frugiperda virus have nucleotide sequences as shown in SEQ ID NO. 1-2; the detection probe sfRV-P has a nucleotide sequence as shown in SEQ ID NO. 3;

[0009] The upstream primer SLEV-F and the downstream primer SLEV-R for detecting St. Louis encephalitis virus have nucleotide sequences as shown in SEQ ID NO. 4-5; the detection probe SLEV-P has a nucleotide sequence as shown in SEQ ID NO. 6;

[0010] The upstream primer JEV-F and the downstream primer JEV-R for detecting Japanese encephalitis virus have nucleotide sequences as shown in SEQ ID NO. 7-8; the detection probe JEV-P has a nucleotide sequence as shown in SEQ ID NO. 9;

[0011] The upstream primers DEN-F1 and DEN-F2 and the downstream primers DEN-R1 and DEN-R2 for detecting dengue virus have nucleotide sequences as shown in SEQ ID NO. 10-13, respectively; the detection probes DEN-P1 and DEN-P2 have nucleotide sequences as shown in SEQ ID NO. 14-15, respectively;

[0012] The upstream primers CHIKV-F1 and CHIKV-F2 and the downstream primers CHIKV-R1 and CHIKV-R2 for detecting chikungunya virus have nucleotide sequences as shown in SEQ ID NO. 16-19, respectively; the detection probe CHIKV-P has a nucleotide sequence as shown in SEQ ID NO. 20.

[0013] In the above detection probes provided by the application, a fluorescent group is connected to the 5' segment of the nucleotide sequence, and a quenching group is connected to the 3'.

[0014] The fluorescent group and the quenching group are both common groups in the art.

[0015] Optionally, the fluorescent group can be selected from, but not limited to, FAM, TET, NED, ROX, CY3, CY5, VIC, JOE, HEX, Texas RED or LC RED460.

[0016] Optionally, the quenching group can be selected from, but not limited to, MGB, TAMRA, NFQ, ECLIPSE, DABCYL, BHQ1 or BHQ2.

[0017] In a second aspect of the present application, a product for detecting a plurality of Sf9 cell RNA viruses is provided, comprising the primer probe combination.

[0018] Further, the product further comprises a plasmid standard of the RNA virus.

[0019] Still further, the detection sequence of the plasmid standard in the product is shown in SEQ ID NO. 21-26, respectively.

[0020] Still further, the product is a detection kit, a detection test strip or a detection chip.

[0021] In a third aspect of the present application, a method for detecting a plurality of Sf9 cell RNA viruses without the purpose of disease diagnosis and treatment is provided, comprising the following steps:

[0022] The plasmid standard of the RNA virus is used as a positive control, and nuclease-free water is used as a negative control; nucleic acid of a sample to be tested is extracted and reverse transcribed.

[0023] The primer probe combination or the product is used for qPCR, and the results are determined according to the amplification curve and the Ct value.

[0024] Further, the reaction procedure of the qPCR in the method is as follows: 95℃, 5min; 95℃, 10s, 57℃, 30s, 40 cycles.

[0025] Still further, the reaction system of the qPCR in the method is as follows: 4.4 μL of nuclease-free water, 10 μL of qPCR reaction solution, 0.2 μL of 20 μM upstream primer, 0.2 μL of 20 μM downstream primer, 0.2 μL of 10 μM detection probe, and 5 μL of template.

[0026] The claim in the above-described detection method of this invention that it is "not for the purpose of disease diagnosis and treatment" refers to the detection of RNA virus infection in non-insect animals, including but not limited to RNA virus contamination in environments / scenarios such as biological products, air, water, soil, surfaces of objects / instruments, waste liquids, and laboratories. In other words, the above-described technical means provided by this invention can be used in any application scenario that is not directly used for the diagnosis or treatment of RNA virus-related diseases / symptoms in animals.

[0027] Of course, the technical means of this invention can also be considered for detecting RNA viruses in insects and other animals, or for diagnosing / treating diseases / symptoms related to RNA viruses in animals.

[0028] In a fourth aspect, the present invention provides an application of the primer-probe combination or product described above in the detection of RNA viruses in biological products or pharmaceuticals.

[0029] Compared with existing technologies, the advantages of this invention are as follows: The primer and probe combination provided by this invention has a wide coverage and can detect a variety of RNA viruses that can infect Sf9 cells, namely fall armyworm rhabdovirus, St. Louis encephalitis virus, Japanese encephalitis virus, dengue virus, and chikungunya virus; it has high sensitivity, with 100% detection of plasmid standards of each RNA virus at 20 copies / μL; it has good specificity, with each RNA virus plasmid standard showing a positive S-type amplification curve in the corresponding qPCR system, a negative NTC amplification curve, and a negative Sf9 cell genome amplification curve; it is not affected by cell matrix interference and is suitable for the quality control of Sf9 cells and their biological products. Attached Figure Description

[0030] Figures 1-6 show the linear range of the RNA virus systems that can infect Sf9 cells in Example 2.

[0031] Figure 7-12 shows the specificity verification of each RNA virus system that can infect Sf9 cells in Example 3.

[0032] Figures 13-18 show the applicability verification of various RNA virus systems that can infect Sf9 cells in Example 5. Detailed Implementation

[0033] The technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0034] Embodiment 1: Design of primer probe combination

[0035] In the embodiments of the present application, RNA viruses that can infect Sf9 cells are summarized, and primer probe combinations for qPCR detection are designed for these viruses. The RNA viruses that can infect Sf9 cells are shown in Table 1 below.

[0036] Table 1: Types of RNA viruses that can infect Sf9 cells

[0037] In the embodiments of the present application, biological analysis technology is used to classify and analyze all nucleic acid information of the RNA virus types shown in Table 1 in Genebank, and to extract the conserved regions that can cover specific types of RNA viruses and their variants or subtypes to the greatest extent for primer and probe design.

[0038] Finally, the primer probe combinations for qPCR detection of each RNA virus are selected as shown in Table 2 below.

[0039] Table 2: Primer probe combinations for RNA viruses that can infect Sf9 cells

[0040] Embodiment 2: Linear range of primer probe combination for detecting RNA viruses that can infect Sf9 cells

[0041] 1. The plasmid standard of each virus was diluted 10 times from 2x10 6 copies / μL in 6 gradients, respectively 2x10 6 , 2x10 5 , 2x10 4 , 2x10 3 , 2x10 2 and 2x10 1 copies / μL, then 5 μL was taken for qPCR reaction to draw the standard curve.

[0042] The plasmid standard is pUC57-sfRV, pUC57-SLEV, pUC57-JEV, pUC57-DEN1, pUC57-DEN2 and pUC57-CHIKV. The conserved region gene sequence of the plasmid standard is shown in Table 3 below.

[0043] Table 3. Detection sequences (target sequences) of standard plasmids

[0044] 2. The settings for each virus qPCR reaction system are shown in Table 4 below. The qPCR reaction program is: 95℃ for 5 min; 95℃ for 10 s, 57℃ for 30 s, for 40 cycles. The FAM fluorescence channel is selected.

[0045] Table 4. qPCR reaction system for Sf9 cell-infectable RNA viruses

[0046] The experimental results for each virus are shown in Figures 1-6. It can be seen that the corresponding standard plasmids for each virus have a 2×10⁻⁶ ppm. 6 -2×10 1 All six gradients (copies / μL) could be detected, and the correlation coefficient R was [value missing]. 2 ≥0.99, indicating a good linear relationship.

[0047] Example 3: Validation of the specificity of primer-probe combination detection for Sf9 cell-infecting RNA viruses

[0048] This embodiment validates the specificity of the detection method for RNA viruses that can infect Sf9 cells. Using the qPCR system and method of Example 2, plasmid standards (positive), Sf9 cell genome, and nuclease-free water (NTC) of each virus were detected. The concentration of the RNA virus plasmid standard was 2 × 10⁻⁶. 5 The number of Sf9 cells from which the genome was extracted was 1 × 10^9 copies / μL. 6 .

[0049] The detection results are shown in Figures 7-12. All RNA virus plasmid standards showed positive S-type amplification curves in their respective qPCR systems, while NTC showed no amplification (negative), and Sf9 cell genome showed no amplification curve (negative). This indicates that the primer-probe combination provided by this invention has good specificity.

[0050] Example 4: Detection Limit of Primer-Probe Combination for Plasmids Infecting Sf9 Cell RNA Viruses

[0051] This example evaluates the detection limit of a method for detecting RNA viruses that can infect Sf9 cells. Using the qPCR system and method of Example 2, plasmid standards at 20 copies / μL were tested for each virus; three independent replicates were performed, 8 replicates per well, for a total of 24 replicates. At least 23 wells must be positive, i.e., a detection rate of 95%.

[0052] The detection results are shown in Table 5-10. All RNA virus plasmid standards with 20 copies / μL were detected at 100%, indicating high sensitivity.

[0053] Table 5. Detection limits and Ct values ​​of plasmid standards for insect RNA virus SfRV

[0054] Table 6. Detection limits and Ct values ​​of plasmid standards for insect RNA virus SLEV

[0055] Table 7. Detection limits and Ct values ​​of plasmid standards for insect RNA virus JEV

[0056] Table 8. Detection limits and Ct values ​​of plasmid standards for insect RNA virus DEN1

[0057] Table 9. Detection limits and Ct values ​​of plasmid standards for insect RNA virus DEN2.

[0058] Table 10. Limit of Detection (LOD) and Ct values ​​for plasmid standards of insect RNA virus CHIKV

[0059] Example 5: Validation of the applicability of primer-probe combination for detecting RNA viruses infecting Sf9 cells

[0060] This embodiment validates the applicability of the detection method for RNA viruses that can infect Sf9 cells, in order to evaluate the interference of the cell matrix on the detection system.

[0061] In this embodiment, a viral nucleic acid extraction kit (Simgen, 4002050) was used to extract packaged pseudoviruses without added cells and those with added 1×10⁻⁶ cells. 6 The nucleic acid of the packaging pseudovirus from Sf9 cells was extracted; the cDNA was reverse transcribed using a reverse transcription kit (Takara, RR036A), and detected using the qPCR system and method described in Example 1. Positive controls were the corresponding plasmid standards for each virus (2 × 10⁻⁶). 5 The negative control was nuclease-free water (copies / μL). The difference in Ct values ​​between the cells with and without added cells was calculated to assess the interference of the cell matrix on the virus extraction and detection system. The applicability assessment required a Ct difference of less than 2, meaning the cell matrix did not interfere with the detection and was suitable for the cell sample. The detection results are shown in Figures 13-18, demonstrating that the primer-probe combination provided by this invention was not affected by the Sf9 cell matrix during detection, exhibiting excellent applicability.

[0062] The above detailed description describes the implementation of the present application, but the present application is not limited to the specific details in the above implementation. Within the scope of the claims and technical concepts of the present application, various simple modifications and changes can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

Claims

1. A primer probe combination for detecting a plurality of RNA viruses that can infect Sf9 cells, characterized in that, The primer probe combination comprises the following primers and detection probes: The upstream primer sfRV-F and the downstream primer sfRV-R for detecting Spodoptera frugiperda nucleopolyhedrovirus, the nucleotide sequences of which are shown in SEQ ID NO. 1-2; the detection probe sfRV-P, the nucleotide sequence of which is shown in SEQ ID NO. 3; The upstream primer SLEV-F and the downstream primer SLEV-R for detecting St. Louis encephalitis virus, the nucleotide sequences of which are shown in SEQ ID NO. 4-5; the detection probe SLEV-P, the nucleotide sequence of which is shown in SEQ ID NO. 6; The upstream primer JEV-F and the downstream primer JEV-R for detecting Japanese encephalitis virus, the nucleotide sequences of which are shown in SEQ ID NO. 7-8; the detection probe JEV-P, the nucleotide sequence of which is shown in SEQ ID NO. 9; The upstream primer DEN-F1 and DEN-F2 and the downstream primer DEN-R1 and DEN-R2 for detecting dengue virus, the nucleotide sequences of which are shown in SEQ ID NO. 10-13, respectively; the detection probe DEN-P1 and DEN-P2, the nucleotide sequences of which are shown in SEQ ID NO. 14-15, respectively; The upstream primer CHIKV-F1 and CHIKV-F2 and the downstream primer CHIKV-R1 and CHIKV-R2 for detecting chikungunya virus, the nucleotide sequences of which are shown in SEQ ID NO. 16-19, respectively; the detection probe CHIKV-P, the nucleotide sequence of which is shown in SEQ ID NO.

20.

2. A product for detecting a plurality of infectable Sf9 cell RNA viruses, characterized in that, The primer probe combination of claim 1.

3. The product for detecting a plurality of infectious Sf9 cell RNA viruses according to claim 2, characterized by, The plasmid standard of the RNA virus is also included.

4. The product for detecting a plurality of infectious Sf9 cell RNA viruses according to claim 3, characterized by, The detection sequences of the plasmid standard are shown in SEQ ID NO. 21-26, respectively.

5. The product for detecting a plurality of infectious Sf9 cell RNA viruses according to claim 2, characterized by, The product is a detection kit, a detection test strip or a detection chip.

6. A method for detecting a plurality of RNA viruses that can infect Sf9 cells, which is not for the purpose of diagnosis and treatment of diseases, characterized by, The steps comprise: The plasmid standard of the RNA virus is used as a positive control, and nuclease-free water is used as a negative control; nucleic acid of a sample to be tested is extracted and reverse transcribed; qPCR is performed using the primer probe combination of claim 1 or using the product of any one of claims 2-5, and the results are determined according to the amplification curve and the Ct value.

7. The method of claim 6, wherein the method is not for the purpose of diagnosis and treatment of disease. The reaction program of qPCR is: 95℃, 5min; 95℃ 10s, 57℃ 30s, cycle 40 times.

8. The method of claim 7, wherein the method is not for the purpose of diagnosis and treatment of disease. The reaction system of qPCR is: nuclease-free water 4.4μL, qPCR reaction solution 10μL, upstream primer 20μM 0.2μL, downstream primer 20μM 0.2μL, detection probe 10μM 0.2μL, template 5μL.

9. The primer probe combination of claim 1 or the product of any one of claims 2-5 for detecting the RNA virus that can infect Sf9 cells in biological products or pharmaceutical products.

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