Primer probe combination for detecting vesicular virus and application of primer probe combination

Abstract

The invention discloses a primer probe combination for detecting a vesicular virus and application of the primer probe combination, and relates to the technical field of detection of the vesicular virus. Aiming at the detection of the vesicular virus, the invention provides a set of primer probe combination which comprises forward primers F1, F2 and F3, reverse primers R1 and R2, and detection probes Probe1 and Probe2. The invention also correspondingly provides a product for detecting the vesicular virus and an application method for detecting the vesicular virus in the fields of biological product detection and the like which do not aim at disease diagnosis and treatment. The primer probe combination is high in detection sensitivity (up to 20 copies / microliter) of the vesicular virus, strong in specificity, good in universality and strong in durability, and is not interfered by a cell matrix.

Description

Technical Field

[0001] This invention relates to the field of vesicular virus detection technology, and particularly to primer-probe combinations for detecting vesicular viruses. Background Technology

[0002] Insect cells are the host cells for baculovirus-insect cell eukaryotic expression systems. Due to their high protein expression efficiency and diverse post-translational modifications, they can express both single target proteins and multiple exogenous proteins simultaneously, making them widely used in the production of recombinant proteins and vaccines. Commonly used insect cell lines include the fall armyworm ovary cell line (Sf9, Sf21) and the white armyworm ovary cell line (Tn, High Five / Hi-5).

[0003] Vesicular viruses are large, double-stranded DNA viruses that are highly infectious and pathogenic to cells of noctuid moths and belong to the category of exogenous factors in insect cells. The four currently identified vesicular viruses, such as fall armyworm vesicular virus (SfAV-1a), white armyworm vesicular virus (TnAV-2a, 6b, 2c, 2d), tobacco leafhopper vesicular virus (HvAV-3c, 3e, 3f, 3g, 3h, 3i, 3j), and beet armyworm vesicular virus (SeAV-5a), can all infect Sf and Tn cells.

[0004] According to Section 5.2.3 of the European Pharmacopoeia and the quality requirements for biotechnology products set forth by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), screening for exogenous factors is required for cell substrates used in the production of bioproducts. Therefore, a safety assessment of vesicular viruses, one of the exogenous factors in insect cells used in production and their bioproducts, is necessary to achieve sensitive, accurate, and rapid detection.

[0005] Currently, the detection of vesicular viruses is either qPCR detection targeting a specific strain of HvAV or multiple strains of HvAV and its similar strain SeAV. However, there are no clear reports on the detection of SfAV vesicular virus derived from sf cells and TnAV vesicular virus derived from Tn cells. Furthermore, no technology has been found that can simultaneously detect these four vesicular viruses (SfAV, TnAV, HvAV, and SeAV) and meet the requirements for biopharmaceutical detection in terms of specificity, sensitivity, and matrix robustness. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention, through extensive experimentation, has discovered and provides a qPCR primer-probe combination capable of simultaneously detecting four vesicular viruses: SfAV, TnAV, HvAV, and SeAV. This combination exhibits good versatility, high sensitivity, and strong specificity for vesicular virus detection, and is unaffected by matrix interference. Specifically, this is achieved through the following techniques.

[0007] A primer-probe combination for detecting vesicular viruses includes forward primers F1, F2, and F3, reverse primers R1 and R2, and detection probes Probe1 and Probe2. The nucleotide sequences of the forward primers F1, F2, and F3 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; the nucleotide sequences of the reverse primers R1 and R2 are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively; and the nucleotide sequences of the detection probes Probe1 and Probe2 are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively.

[0008] A product for detecting vesicular viruses, the product comprising the primer-probe combination of claim 1.

[0009] Furthermore, it also includes a positive control, which is a mixture of standard plasmids containing at least one target sequence of SfAV, TnAV, and HvAV.

[0010] Optionally, the above-mentioned standard plasmid is at least one of pUC57-SfAV, pUC57-TnAV, and pUC57-HvAV, that is, a recombinant plasmid containing the target sequence of SfAV, TnAV, or HvAV respectively.

[0011] The standard plasmids pUC57-SfAV, pUC57-TnAV, and pUC57-HvAV were obtained by recombining the detection target sequences of SfAV, TnAV, and HvAV on the basic plasmid pUC57. Other suitable basic plasmids can also be used to prepare the corresponding recombinant plasmids.

[0012] Further optionally, the nucleotide sequences of the standard plasmids pUC57-SfAV, pUC57-TnAV and pUC57-HvAV are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0013] Furthermore, the product is a test kit, test strip, or test chip.

[0014] This invention also provides a method for detecting vesicular viruses that is not intended for disease diagnosis and treatment, using the primer-probe combination provided by this invention, or using corresponding products for detection.

[0015] Furthermore, the genome of the sample to be tested is extracted as a template for amplification, and the CT value is read; When the following conditions are met simultaneously: (1) R of the standard curve 2 ≥0.99; (2) The positive control was positive, with a Ct value <35 and a clear amplification curve; (3) The negative control is negative, with no Ct value or no normal amplification curve.

[0016] If the Ct value of the test sample is <37 and there is a clear amplification curve, it is judged as positive; otherwise, it is negative.

[0017] It should be noted that the claim of "not for the purpose of disease diagnosis and treatment" in this invention refers to the detection of vesicular viruses in non-human or animal bodies, including but not limited to vesicular virus contamination in environments / scenarios such as air, water, soil, biological products, surfaces of objects / instruments, waste liquids, and laboratories. In other words, the above-mentioned technical means provided by this invention can be used in any situation where it is not directly used for the diagnosis or treatment of vesicular virus-related diseases / symptoms in humans or animals.

[0018] Of course, the technical means of this invention can also be considered for the diagnosis or treatment of diseases / symptoms related to vesicle viruses in humans or animals.

[0019] Compared with the prior art, the advantages of the present invention are as follows: The present invention provides a qPCR primer and probe combination and detection system that can simultaneously detect four vesicular viruses, SfAV, TnAV, HvAV and SeAV, with good versatility; it is not affected by the insect cell genome in the detection sample, has strong durability and good specificity; the detection limit can reach 20 copies / μl, with high sensitivity; it can be used for rapid detection and safety assessment of vesicular viruses in production insect cells and their biological products. Attached Figure Description

[0020] Figure 1-4 The specificity, sensitivity, specificity, and universality of the seven primer groups in Example 1 were screened. Figure 5 This is for the specificity and universality testing of the four-primer mixture system in Example 1; Figure 6 This is to verify the sensitivity of the four-primer mixture system in Example 1; Figure 7 For the specificity and universality detection of the five-primer mixture system in Example 1; Figure 8 This is to verify the sensitivity of the five-primer mixture system in Example 1; Figure 9 For matrix robustness testing of the five-primer mixture system in Example 1; Figure 10 The standard curves for TnAV, SfAV, and HvAV amplification in Example 1; Figure 11 This is a specificity detection of the vesicular virus qPCR detection method in Example 2; Figure 12 This is a robustness test of the vesicular virus qPCR detection method in Example 2. Detailed Implementation

[0021] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] The terminology used in this invention employs definitions readily known or commonly understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar or equivalent to those described herein may be used in the practice or testing of this invention, this document specifically describes multiple embodiments of the methods and materials.

[0023] The terms “nucleic acid,” “oligonucleotide,” and “polynucleotide” are used interchangeably and refer to single-stranded and / or double-stranded polymers of nucleotide monomers, including but not limited to 2’-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by phosphodiester bonds or nucleotide analogs. Nucleotide monomers in nucleic acids may be referred to as “nucleotide residues.” Nucleic acids may consist entirely of deoxyribonucleotides, entirely of ribonucleotides, or a chimeric mixture thereof, and may include nucleotide analogs. A nucleotide monomer unit may contain any of the nucleotides described herein, including but not limited to nucleotides and / or nucleotide analogs. Nucleic acids typically range in size from a few nucleotide residues to several thousand nucleotide residues. “Oligonucleotide” generally refers to a relatively short polymer of nucleotides, such as 1–80. Unless otherwise indicated, whenever a nucleic acid sequence is presented, it should be understood that the nucleotides are arranged in a 5’ to 3’ sequence from left to right. Unless otherwise specified, “A” represents deoxyadenosine, “C” represents deoxycytidine, “G” represents deoxyguanosine, “T” represents deoxythymidine, and “U” represents deoxyuridine.

[0024] According to the conventions of the art, the length of nucleic acids can be expressed as bases, base pairs (abbreviated "bp"), nucleotides / nucleotide residues (abbreviated "nt"), or kilobases ("kb"). Where the context permits, the terms "base," "nucleotide," and "nucleotide residue" can describe single-stranded or double-stranded polynucleotides. When the term is applied to double-stranded molecules, it is used to refer to the entire length and should be understood as equivalent to the term "base pair."

[0025] The term "primer" and its variants generally refer to oligonucleotides that act as the starting point for DNA synthesis in a PCR reaction. Primers are typically about 15 to about 35 nucleotides in length and hybridize to regions complementary to the target sequence.

[0026] The term "forward primer" refers to an oligonucleotide annealed to a specific strand of the target DNA, typically the antisense strand. The term "reverse primer" refers to an oligonucleotide annealed to the opposite strand of the target DNA, typically the sense strand. It should be understood that when the designation of the sense and antisense strands is interchanged, the corresponding names of the forward and reverse primers may also be interchanged.

[0027] The term "probe" and its variants (e.g., detection probe) refer to oligonucleotides that hybridize with target nucleic acids in a PCR reaction.

[0028] The terms "target sequence," "target nucleic acid," or "target" are used interchangeably and refer to the portion of a nucleic acid sequence to be amplified, detected, or amplified and detected, which may be annealed or hybridized with a probe or primer under hybridization, annealing, or amplification conditions. The term "hybridization" refers to the base-pairing interaction between two nucleic acids that results in the formation of a double helix. 100% complementarity of the two nucleic acids across their full length is not required for hybridization to occur.

[0029] The term "sample" refers to any composition containing or presumed to contain nucleic acids. Samples can be derived from biological sources ("biological samples"), such as tissues (e.g., biopsy samples), extracts or cultures, and biological or physiological fluids. For example, samples may include skin, plasma, serum, cerebrospinal fluid, lymph, synovial fluid, urine, tears, blood cells, organs, and tumors. Samples may also include samples from in vitro cultures established from cells taken from an individual or immobilized samples, such as formalin-fixed paraffin-embedded tissue (FFPET) and nucleic acid isolates derived therefrom.

[0030] Unless otherwise specified, all reagents and instruments used in the following specific embodiments can be purchased through conventional channels. Unless otherwise specified, the initial concentrations of the reagents or reaction raw materials used are those of the purchased raw materials.

[0031] Example 1: Screening of target genes and primer pairs Based on the conservation analysis of various proteins of vesicular viruses, this invention initially targets their main capsid protein (cap) and DNA polymerase (pol). Nucleic acid sequences of the corresponding proteins of each virus were searched, and sequence alignment analysis revealed that SfAV has 65% identity with HvAV and SeAV, and approximately 48% identity with TnAV.

[0032] Therefore, in this embodiment, using SfAV and HvAV as reference sequences, a total of 5 sets of universal primer pairs were designed to target the major capsid proteins (cap-p1, cap-p2, cap-p3, cap-p4 and cap-p5), and 2 sets of universal primer pairs were designed to target DNA polymerases (pol-p6 and p7).

[0033] Subsequently, reference plasmids for the corresponding fragments of SfAV and HvAV viruses were constructed, namely pUC57-SfAV-cap, pUC57-SfAV-pol, pUC57-HvAV-cap, and pUC57-HvAV-pol. Specificity testing was then performed (each primer pair detected 10-10 of the corresponding reference plasmid for the SfAV virus). 6 10 5 and 10 4 The system must be able to effectively detect the corresponding plasmid and have a single solubility peak. Sensitivity (i.e., the detection efficiency of each primer pair for the corresponding reference plasmid of SfAV virus within 10^- ... 4 10 3 10 2 The system and nuclease-free water must be able to effectively detect 10 copies / systems of the corresponding plasmid. 2 The primer pairs should exhibit specificity (each primer pair should detect 100 ng of sf9 cell genome without showing a significant amplification curve) and universality (each primer pair should detect 10 copies / system of the corresponding reference plasmid of HvAV virus). 5 10 4 10 3 and 10 2 The system must be able to effectively detect the corresponding plasmid and have no non-specific dissolution peaks for screening.

[0034] The results are as follows Figure 1-4 As shown, the details are as follows: (1) Specificity (primer dimer) screening revealed that primer pair cap-p2 had a non-specific dissolution peak, while primer pair cap-p4 only had a 10-fold dissolution peak. 6 The copies / system exhibits a specific dissolution peak, and plasmids of other concentrations cannot be detected. (2) Sensitivity screening revealed that primer pair pol-7 could not distinguish 10 2 The copy / system and nuclease-free water have poor sensitivity; (3) Specificity (Sf genome) screening revealed that primer pairs cap-p1 and pol-6 showed obvious amplification curves with the sf9 cell genome, indicating poor specificity; (4) Universality (SfAV and HvAV detection) screening revealed that primer pair cap-p3 could only recognize 10% of the corresponding plasmids of HvAV virus. 5 The copy / system has low plasmid content, which cannot be detected, and its universality is not up to standard.

[0035] Therefore, only the primer pair cap-p5 has good specificity, sensitivity, exclusivity and universality, which meets the screening requirements.

[0036] Subsequently, based on the above experimental results, the sequence of primer pair cap-p5 at the corresponding position in TnAV was synthesized and mixed with primer pair cap-p5 in an equal proportion as primers for detecting vesicular viruses. After testing, it was found that this four-primer mixture system has strong specificity, can simultaneously detect three vesicular viruses, and can effectively distinguish them by the solubility peak, fully meeting the expected design. The results are as follows: Figure 5 As shown. However, as Figure 6 As shown, when validating the sensitivity (20 copies / μl) of this four-primer mixture system, HvAV occasionally exhibited non-specific amplification at this sensitivity (marked by the blue curve, with its main dissolution peak appearing at 70-75℃), which may affect the interpretation of trace virus detection results in real sample testing. A series of optimizations, such as adjusting primer storage conditions and changing the qPCR-mix, did not significantly improve the results.

[0037] Subsequently, an HvAV-specific forward primer was added to the four-primer mixture system. Testing and verification revealed that, as... Figure 7 , 8 As shown, this five-primer mixture system meets the design requirements in terms of specificity, versatility, and sensitivity. However, matrix robustness verification was performed by extracting untreated packaging pseudoviruses (adv-SfAV, adv-TnAV, adv-HvAV) and those treated with 1×10⁻⁶ cells. 6 The nucleic acids of the packaging pseudoviruses (adv-SfAV + sf9, adv-TnAV + sf9, adv-HvAV + sf9) in sf9 cells were analyzed; the difference in Ct values ​​(ΔCt) between cells with and without the five primer mixture was calculated after qPCR. SfAV To assess the impact of the cell matrix on virus detection, the calculation formula is as follows: ; Ultimately, it was discovered that ΔCt SfAV =2.1, ΔCt TnAV =1.86, ΔCt HvAV =2.5, the result is shown in Figure 9 A lag of approximately 2-2.5 CTs, i.e., ΔCt > 2, indicates that 1 × 10 6 A single sf9 cell can inhibit viral detection. Changing the nucleic acid extraction kit or qPCR-mix did not significantly improve the situation; reducing the number of cells can effectively alleviate the matrix inhibition effect, but this is obviously not suitable for real sample testing.

[0038] To address the matrix insufficiency issue of the aforementioned five-primer mixture system, different specific probes were added to the system, and these were used as primer-probe combinations for system screening and optimization. This embodiment ultimately provides primer-probe combinations capable of simultaneously detecting four vesicular viruses: fall armyworm vesicular virus (SfAV), white armyworm vesicular virus (TnAV), tobacco bud armyworm vesicular virus (HvAV), and beet armyworm vesicular virus (SeAV), as shown in Table 1 below.

[0039] Table 1 Primer-Probe Combinations

[0040] The numbering rule for the degenerate bases in the above primer-probe combination is as follows: R is base A or G, and W is base A or T.

[0041] The detection sequences for the standard plasmids used to detect the four types of vesicular viruses are shown in Table 2 below.

[0042] Table 2. Detection sequences (target sequences) of standard plasmids

[0043] Since the SeAV primer and probe regions are completely identical to those of HvAV, there is no need to synthesize and detect standard plasmids separately.

[0044] The standard plasmids pUC57-SfAV, pUC57-TnAV, and pUC57-HvAV were serially diluted to 2 × 10⁻⁶ m². 1 -2×10 6 Copies / μl are used as standards.

[0045] The reaction system for real-time PCR is shown in Table 3 below.

[0046] Table 3 qPCR reaction system

[0047] The quantitative PCR amplification program was as follows: digestion at 37℃ for 2 min; denaturation at 95℃ for 5 min; annealing at 95℃ for 10 s and extension at 59℃ for 30 s per cycle, for a total of 40 cycles.

[0048] Example 2: Validation of the sensitivity, specificity, and robustness of the primer-probe combination The primer-probe combination finally screened in Example 1 was subjected to verification tests to assess its universality, sensitivity, specificity, and robustness.

[0049] 1. Universality Testing

[0050] Using the primer-probe combination and detection method obtained in Example 1, qPCR detection was performed on the serially diluted standard plasmids pUC57-SfAV, pUC57-TnAV, and pUC57-HvAV. The experimental results (standard curve) are shown below. Figure 10 As shown.

[0051] from Figure 10 It can be seen that all gradients of pUC57-SfAV, pUC57-TnAV, and pUC57-HvAV can be detected, and the correlation coefficient RV is high. 2 ≥0.99, indicating a good linear relationship.

[0052] 2. Sensitivity Verification To evaluate the detection capability of the primer-probe combination in Example 1 for four vesicular viruses, this example performed three independent replicate detections on 20 copies / μl of the standard plasmids pUC57-SfAV, pUC57-TnAV, and pUC57-HvAV, for a total of 24 wells. At least 23 wells were required to be positive, i.e., a detection rate of 95%.

[0053] The results are shown in Table 4 below. All pUC57-SfAV, pUC57-TnAV, and pUC57-HvAV plasmids at 20 copies / μl were detected with 100% accuracy. In summary, the results of Example 1 demonstrate that the primer-probe combinations, standard plasmids, and corresponding detection methods provided for detecting four vesicular viruses can accurately and sensitively reflect the actual amplification of SfAV, TnAV, HvAV, and SeAV (the primer-probe sequences for SeAV are identical to those for HvAV, so the verification results can be referenced to HvAV). The methods exhibit good versatility and high sensitivity.

[0054] Table 4. Sensitivity validation of the vesicular virus qPCR detection method

[0055] 3. Specificity verification To evaluate whether the primer-probe combination in Example 1 would produce non-specific amplification of nucleic acids in commonly used insect cells, this example used the TaKaRa virus nucleic acid extraction kit to extract the genomes of the fall armyworm ovary cell line Sf9 and the white armyworm ovary cell line Hi5, and detected them using the qPCR method in Example 1. The template-free control (NTC) was RNase-free water, and the positive control was the pUC57-TnAV plasmid standard from Example 1 (pUC57-SfAV or pUC57-HvAV could also be used).

[0056] The results are as follows Figure 11As shown, the template-free control (NTC) was negative, while the positive control exhibited S-type amplification, indicating the effectiveness of the qPCR detection system. Neither the Sf9 nor Hi5 cell genome samples showed amplification curves, thus indicating negative results. This demonstrates that the primer-probe combination provided in Example 1 possesses good specificity for detecting vesicular viruses.

[0057] 4. Durability verification Cells are often present in real samples. To assess the impact of insect cells on virus extraction and detection, a viral nucleic acid extraction kit (Simgen, 4002050) was used to extract packaged pseudoviruses (adv-SfAV, adv-TnAV, adv-HvAV) without added cells and those with added cells (1×10⁻⁶ cells). 6 Packed pseudoviruses (adv-SfAV+sf9, adv-TnAV+sf9, adv-HvAV+sf9) were collected from sf9 cells and detected using the qPCR method described in Example 1. The positive control was the pUC57-TnAV plasmid standard.

[0058] The results are as follows Figure 12 As shown, both the packaged pseudoviruses with and without sf9 cells exhibited distinct amplification curves, ΔCt SfAV =0.03, ΔCt TnAV =0.12, ΔCt HvAV =1.42, ΔCt is less than 2, which indicates that 1×10 6 The presence of a single sf9 cell does not affect the extraction and detection of vesicular viruses in the sample. This indicates that the primer-probe combination provided in Example 1 has good anti-interference properties against the sf9 cell genome and can be used for the detection of vesicular viruses in insect cells and their biological products.

[0059] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A primer-probe combination for detecting vesicular viruses, characterized in that, It includes forward primers F1, F2, and F3, reverse primers R1 and R2, and detection probes Probe1 and Probe2; the nucleotide sequences of forward primers F1, F2, and F3 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; the nucleotide sequences of reverse primers R1 and R2 are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively; and the nucleotide sequences of detection probes Probe1 and Probe2 are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively.

2. A product for detecting vesicular viruses, characterized in that, The product comprises the primer-probe combination as described in claim 1.

3. The product for detecting vesicular viruses according to claim 2, characterized in that, It also includes a positive control, which is a mixture of standard plasmids containing at least one target sequence of SfAV, TnAV, and HvAV.

4. The product for detecting vesicular viruses according to claim 3, characterized in that, The standard plasmid is at least one of pUC57-SfAV, pUC57-TnAV, and pUC57-HvAV.

5. The product for detecting vesicular viruses according to claim 4, characterized in that, The nucleotide sequences of the standard plasmids pUC57-SfAV, pUC57-TnAV, and pUC57-HvAV are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.

6. The product for detecting vesicular viruses according to claim 2, characterized in that, The products mentioned are test kits, test strips, or test chips.

7. A method for detecting vesicular viruses not for the purpose of disease diagnosis and treatment, characterized in that, The detection is performed using the primer-probe combination as described in claim 1, or the product as described in any one of claims 2-6.

8. The method according to claim 7, characterized in that, The genome of the sample to be tested was extracted and used as a template for amplification, and the CT values ​​were read. R satisfies the standard curve 2 ≥0.99; a positive control is positive, with a Ct value <35 and a clear amplification curve; a negative control is negative, with no Ct value or no normal amplification curve; if the test sample has a Ct value <37 and a clear amplification curve, it is considered positive, otherwise it is negative.

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