A feline calicivirus nucleic acid detection kit and detection method

Abstract

This invention discloses a feline calicivirus nucleic acid detection kit and detection method. The detection kit includes: (1) a nucleic acid enrichment module and (2) a TaqMan fluorescent PCR detection module. The beneficial effects are that the sensitivity is significantly improved, the operation is simplified, the functions are integrated, the pathogenicity is classified in one step (distinguishing between highly pathogenic strains and classic strains), the stability is excellent, and the specificity is strong.

Description

Technical Field

[0001] This invention relates to the field of viral nucleic acid detection technology, and in particular to a feline calicivirus nucleic acid detection kit and detection method. Background Technology

[0002] Feline calicivirus (FCV) is one of the core pathogens causing respiratory diseases in cats. Clinically, it often presents with symptoms such as oral ulcers, high fever, cough, and runny nose. The viral genome is prone to mutation, especially mutations in the VP1 gene, which frequently lead to increased pathogenicity and altered antigenicity, posing a significant challenge to precise clinical prevention and treatment. Current FCV nucleic acid detection technologies, primarily fluorescent PCR and LAMP, still have significant technical limitations: 1) The sample processing is complex, requiring steps such as centrifugation, column purification, or magnetic bead extraction to obtain nucleic acid. This has a high operational threshold and requires numerous instruments, making it difficult to adapt to on-site testing scenarios such as grassroots veterinary stations and pet hospitals; 2) It can only achieve pathogen identification; pathogenicity assessment requires separate gene sequencing or specific analysis after detection, which is cumbersome and time-consuming. The time required is too long, which cannot meet the needs of rapid clinical decision-making; 3) It lacks a specific enrichment step for target nucleic acids, resulting in insufficient sensitivity for low viral load samples (such as early and convalescent samples, and asymptomatic samples), which easily leads to false negatives; 4) The core reagents are mostly liquid formulations, which have strict requirements for cold chain transportation and storage, poor stability, and have not formed an efficient synergistic system with nucleic acid enrichment technology; 5) Although the TaqMan probe method has the advantage of high specificity, the existing scheme does not integrate nucleic acid enrichment technology and pathogenicity assessment function, which limits the application scenarios and cannot provide a comprehensive reference for clinical treatment. Further analysis shows that the pathogenicity of FCV is directly related to the characteristic mutations of the VP1 gene. Point mutations, insertions, or deletions in the hypervariable region of this gene can lead to a significant increase in viral virulence and cause more severe clinical symptoms. In clinical practice, veterinarians often blindly prescribe antiviral drugs based solely on "FCV positive" test results. If the infection is caused by a highly pathogenic variant strain, it not only makes it difficult to control disease progression but also prolongs the course of the disease, increases treatment costs, and may even lead to the death of the cat. Furthermore, irrational drug use may accelerate the spread of highly pathogenic strains, exacerbating the FCV epidemic. Currently, FCV strain pathogenicity typing requires separate gene sequencing or multiplex PCR verification after pathogen detection, a process that takes over 24 hours and relies on specialized laboratory equipment and technicians. Grassroots institutions often struggle to obtain assessment results quickly, rendering pathogenicity assessment ineffective and unable to guide precise clinical medication and prevention. Therefore, developing a detection technology that integrates "nucleic acid enrichment, pathogen detection, and pathogenicity typing," with high reagent stability and simple, rapid operation, especially an integrated solution that simultaneously completes pathogenicity assessment and pathogen detection, has become a critical technical problem urgently needing to be solved in this field. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a feline calicivirus nucleic acid detection kit, comprising: (1) Nucleic acid enrichment module: magnetic beads modified with FCV VP1 protein-specific recombinant antibody, wherein the heavy chain variable region sequence of the recombinant antibody is shown in SEQ ID No.1 and the light chain variable region sequence is shown in SEQ ID No.2; (2) TaqMan fluorescent PCR detection module: containing qPCR Premix, FCV-specific primer pairs, and TaqMan probe. The primer pairs target the 5290~5427bp fragment of the conserved region of FCV (GenBank accession number: KT000003.1). The upstream primer sequence is shown in SEQ ID No.3, the downstream primer sequence is shown in SEQ ID No.4, and the TaqMan probe sequence is shown in SEQ ID No.5.

[0004] As a further supplement to this technical solution, the TaqMan fluorescent PCR detection module includes a pathogenicity testing module. The pathogenicity testing module contains a specific primer pair designed for the VS-FCV gene and a TaqMan genotyping probe. The upstream primer is shown in SEQ ID No. 6, the downstream primer is shown in SEQ ID No. 7, and the TaqMan genotyping probe is shown in SEQ ID No. 8. Pathogenic mutation sites are distinguished by differences in fluorescence signals.

[0005] To further supplement this technical solution, the reagent composition of the TaqMan fluorescent PCR detection module and the pathogenicity test module is made into trehalose-mannitol composite carrier lyophilized microspheres. Each microsphere contains qPCR Premix lyophilized microspheres, 0.4 μM each of upstream and downstream primers, 0.2 μM of probe, and primer-probe protectant components. The lyophilized microspheres have a particle size of 2.2 mm ± 5%, a water content of ≤ 3%, and a dissolution time of less than 10 seconds.

[0006] As a further supplement to this technical solution, the concentration of the recombinant antibody-modified magnetic beads is 2 mg / mL, the particle size of the magnetic beads is 1 μm, and the antibody conjugation amount is 50 μg / mg of magnetic beads.

[0007] A detection method for a feline calicivirus nucleic acid detection kit includes the following steps: Includes the following steps: Step 1: Sample collection; Step 2: Recombinant antibody enrichment with magnetic beads; Step 3: Treatment with the treatment solution; Step 4: Reconstitute the lyophilized microspheres; Step 5: TaqMan fluorescent PCR amplification; Step Six: FCV Detection and Judgment; Step 7: Pathogenicity assessment; Step 8: End.

[0008] To further supplement this technical solution, the sample collection in step one specifically involves taking a cat's nasal swab or saliva sample; the recombinant antibody magnetic bead enrichment in step two specifically involves adding 150 μl of the collected sample into the recombinant antibody magnetic bead enrichment module (450 μl of sample processing solution), mixing thoroughly, and letting it stand at room temperature for 1 min to obtain a resuspension.

[0009] Further supplementing this technical solution, step three involves lysing the solution at 70℃ for 2 min; step four involves rehydrating the lyophilized microspheres; and step five involves TaqMan fluorescence PCR amplification, specifically adding 25 μl of the supernatant from the solution obtained in step three to the lyophilized microspheres, dissolving them, and then placing them in a fluorescence PCR instrument. The amplification program is as follows: reverse transcription at 50℃ for 5 min; pre-denaturation at 95℃ for 2 min; denaturation at 95℃ for 5 s; annealing and extension at 60℃ for 20 s (collecting fluorescence signals), for a total of 40 cycles.

[0010] As a further supplement to this technical solution, step six, FCV detection, is specifically defined as follows: a Ct value < 40 and the presence of a typical amplification curve indicate a positive result; the absence of a Ct value indicates a negative result.

[0011] As a further supplement to this technical solution, step seven, pathogenicity assessment, specifically involves distinguishing highly pathogenic strains (FAM signal only) from classic strains (FAM and HEX signals) by using the fluorescent signals (FAM channel / HEX channel) generated by the specific binding of the probe if the test is positive.

[0012] Application of a feline calicivirus nucleic acid detection kit in the clinical detection of feline calicivirus.

[0013] Its beneficial effects are: (1) significantly improved sensitivity: through recombinant antibody magnetic bead enrichment + TaqMan probe specific recognition, the detection limit reaches 3 copies / μL, which is 1 to 2 orders of magnitude lower than the existing TaqMan detection technology; (2) Simplified operation: After the crude sample is directly processed, the sample is lyophilized and the microspheres are quickly reconstituted and detected. The whole process takes ≤50min, which is suitable for rapid on-site detection. (3) Functional integration: Simultaneous diagnosis and pathogenicity assessment of FCV infection are achieved without additional testing steps, and the testing efficiency is increased by 50%; the pathogenicity assessment results can directly guide clinical medication; conventional antiviral drugs (such as ribavirin) can be used for highly pathogenic strains, while alternative drugs (such as novel protease inhibitors) need to be replaced for classic strains to avoid treatment failure and transmission of classic strains caused by blind medication. (4) Excellent stability: Freeze-dried microsphere formulations can be transported and stored at room temperature without the need for a cold chain, and there is no risk of freeze-thaw loss or degradation, thus reducing logistics and storage costs; (5) High specificity: The recombinant antibody antigen-specific enrichment and TaqMan probe sequence-specific binding provide dual protection, with no cross-reaction to common pathogens such as feline herpesvirus, feline mycoplasma, and feline panleukopenia virus; (6) Clinical significance: It can dynamically monitor the epidemic trend of classic FCV strains, provide data support for farms and pet hospitals to formulate prevention and control plans, and reduce the risk of group infection. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the detection process of the reagent kit of the present invention; Figure 2 These are the results of clinical sample testing and ROC curve analysis; Figure 3 This is a fluorescence amplification curve of highly pathogenic strains and classic strains for typing; Figure 4 This is the amplification curve of the 3 copies / μL FCV standard repeated 20 times according to the present invention. Detailed Implementation

[0015] To facilitate a clearer understanding of this technical solution for those skilled in the art, the following will be described in conjunction with the appendix. Figure 1-4 The technical solution of the present invention is described in detail below: 1. Preparation of recombinant antibodies Recombinant antibodies against FCV VP1 protein were obtained by screening phage libraries. The sequences of the heavy chain variable region (as shown in SEQ ID No. 1) and the light chain variable region (as shown in SEQ ID No. 2) were verified by sequencing. After purification by prokaryotic expression, the antibody was coupled with magnetic beads to prepare an enrichment module. The coupling conditions were as follows: the antibody and magnetic beads were mixed at a ratio of 50 μg / mg in 0.1M PBS buffer (pH 7.4), incubated at 37°C for 2 h, blocked, washed 3 times, and resuspended to 2 mg / mL.

[0016] 2. Preparation of freeze-dried microspheres (1) Reagent premix: Mix 0.4 μM of upstream primer (as shown in SEQ ID No. 3), 0.4 μM of downstream primer (as shown in SEQ ID No. 4), 0.2 μM of TaqMan probe (as shown in SEQ ID No. 5), and qPCR Premix according to the single-use amount, and add trehalose-mannitol complex protectant (final concentration 10%). (2) Microsphere molding: The liquid nitrogen freeze-drying method was adopted. 15 μl / drop volume of premixed liquid was added to liquid nitrogen at a rate of 5s / drop to make 2.2 mm microspheres. The freeze-drying process was as follows: pre-freeze at -45℃ for 2h, heat up to -15℃ under vacuum of 10Pa for sublimation drying for 8h, and finally heat up to 20℃ to complete the desorption drying for 2h. The water content was controlled to be ≤3% to make freeze-dried microspheres.

[0017] (3) Preparation of lyophilized microspheres for pathogenicity assessment module: Following the above process, VP1 gene primer pairs (as shown in SEQ ID No. 6-7), typing probes (as shown in SEQ ID No. 8), and PCR reagents were mixed to prepare lyophilized microspheres.

[0018] 3. Reagent kit assembly Assemble the kit according to the following components: 100 μL / T of recombinant antibody-modified magnetic beads (2 mg / mL), 450 μL / T of sample processing solution; 1 T / tube of TaqMan fluorescent PCR detection reagent (lyophilized microspheres), packaged in a light-proof aluminum foil bag.

[0019] 4. Performance Verification (1) Sensitivity test: for 10 4 ~10 0 FCV standards were tested at copies / μL, 10 4 ~10 1 The detection rate of copies / μL was 100%, and the results are shown in Table 1. Compared with the standard method, this kit has better sensitivity, and the results are shown in Table 2.

[0020] Table 13 shows the test results of the reagent kits against FCV standards. Note: "N / A" indicates no Ct value.

[0021] Table 2. Test results of FCV standards using the standard method (DB22 / T 3111-2020) Note: "N / A" indicates no Ct value.

[0022] (2) Determination of the limit of detection: FCV standard was diluted to 5 and 3 copies / μL respectively, and 100% detection was achieved in 3 replicates. The detection results are shown in Table 3. The 3 copies / μL standard was tested 20 times, and the detection rate was 100%. Therefore, the limit of detection was determined to be 3 copies / μL.

[0023] Table 3. Sensitivity Detection Limit Detection Results Note: "N / A" indicates no Ct value.

[0024] Table 4. Detection results of the lowest detection limit of this product's reagent kit. Note: "N / A" indicates no Ct value.

[0025] (3) Specificity test: The genomes of feline herpesvirus, feline mycoplasma, feline panleukopenia virus, saliva and oral and nasal swabs from healthy cats were tested, and no Ct value or specific amplification curve was found.

[0026] (4) Pathogenicity assessment and verification: Ten FCV strains isolated clinically (6 highly pathogenic strains and 4 classical strains) were tested. The highly pathogenic strains showed fluorescence signals only in the FAM channel, while the classical strains showed fluorescence signals in both the FAM and HEX channels. The typing accuracy reached 100%. Reference strain information is shown in Table 5.

[0027] Table 5. Reference Information on Highly Pathogenic and Classical FCV Strains (5) Clinical correlation: The conventional antiviral drugs were ineffective in treating the four classic strains of the diseased cats. The symptoms were relieved within 72 hours after the alternative drugs were changed. The six highly pathogenic strains of the diseased cats were cured after conventional treatment, which confirmed the consistency between the typing results and the treatment effect. Clinical sample test: The clinical background information of 100 samples were tested and compared with the standard method of fluorescent PCR. The positive concordance rate was 99.0% and the negative concordance rate was 98.2%. Among the positive samples, the classic strains accounted for 32%, which was consistent with the local FCV epidemic data.

[0028] (6) Stability test: After 21 months of storage at room temperature, the properties, detection sensitivity and specificity of the freeze-dried microspheres did not change significantly. After reconstitution, they remained stable at 2~8℃ and 30℃ for 72 hours.

[0029] (7) Enrichment efficiency verification: Samples were serially diluted using FCV standard (GenBank accession number: MN123456.1) and enriched using the recombinant antibody magnetic beads of this invention, conventional polyclonal antibody magnetic beads (magnetic beads of the same particle size + rabbit anti-FCV polyclonal antibody), antibody-free blank magnetic beads, and commercially available FCV immunomagnetic beads, respectively. The amount of nucleic acid after enrichment was detected by a micro-spectrophotometer, and the enrichment efficiency was calculated as (concentration after enrichment / initial concentration) × 100%. The results are shown in Table 6. The enrichment efficiency of the magnetic beads of this invention was significantly higher than that of the control group across the entire concentration range, especially at low concentrations (≤10² copies / μL), which confirmed the capture advantage of the recombinant antibody magnetic beads for low-concentration samples; the enrichment efficiency of the antibody-free blank magnetic beads was consistently <10%.

[0030] Table 6. Enrichment efficiency of different magnetic beads for FCV The detection process of this invention is as follows: Figure 1 The process includes: sample collection (30s) → recombinant antibody magnetic bead enrichment (1min) → processing solution treatment (2min) → lyophilized microsphere reconstitution (10s) → fluorescent PCR amplification (40 cycles) → pathogenicity assessment and interpretation.

[0031] Criteria for determining positive and negative thresholds: The Youden index of the ROC curve is used to determine the positive and negative thresholds of the data. The Youden index = sensitivity + specificity - 1. The value with the highest Youden index corresponds to the positive or negative threshold. Sample test results and software analysis results are shown below. Figure 2 When the cut-off value for positive and negative results is 40, the sensitivity and specificity are the highest, both reaching 100%, the area under the ROC curve is 1, and the Youden index is 1. Therefore, 40 was chosen as the cut-off value for positive and negative results of the kit.

[0032] like Figure 3 As shown, the channel signals are as follows: for highly pathogenic strains, only the FAM channel shows a specific amplification curve, while for classic strains, both the FAM and HEX channels show specific amplification curves with no cross-signal interference.

[0033] The above technical solutions only embody the preferred technical solutions of the present invention. Any modifications that may be made by those skilled in the art to certain parts thereof embody the principles of the present invention and fall within the protection scope of the present invention.

Claims

1. A feline calicivirus nucleic acid detection kit, characterized by, include: (1) Nucleic acid enrichment module: magnetic beads modified with FCV VP1 protein-specific recombinant antibody, wherein the heavy chain variable region sequence of the recombinant antibody is shown in SEQ ID No.1 and the light chain variable region sequence is shown in SEQ ID No.2; (2) TaqMan fluorescent PCR detection module: containing qPCR Premix, FCV-specific primer pairs, and TaqMan probe. The primer pairs target the 5290~5427bp fragment of the conserved region of FCV (GenBank accession number: KT000003.1). The upstream primer sequence is shown in SEQ ID No.3, the downstream primer sequence is shown in SEQ ID No.4, and the TaqMan probe sequence is shown in SEQ ID No.

5.

2. The cat calicivirus nucleic acid detection kit according to claim 1, characterized by, The TaqMan fluorescent PCR detection module includes a pathogenicity testing module, which contains specific primer pairs designed for the VS-FCV gene and TaqMan genotyping probes. The upstream primer is shown in SEQ ID No. 6, the downstream primer is shown in SEQ ID No. 7, and the TaqMan genotyping probe is shown in SEQ ID No.

8. Pathogenic mutation sites are distinguished by differences in fluorescence signals.

3. The cat calicivirus nucleic acid detection kit according to claim 2, characterized by, The reagent composition of the TaqMan fluorescent PCR detection module and the pathogenicity test module is used to prepare trehalose-mannitol composite carrier lyophilized microspheres. Each microsphere contains qPCR Premix lyophilized microspheres, 0.4 μM each of upstream and downstream primers, 0.2 μM of probe, and primer-probe protectant components. The lyophilized microspheres have a particle size of 2.2 mm ± 5%, a water content of ≤ 3%, and a dissolution time of less than 10 seconds.

4. The cat calicivirus nucleic acid detection kit according to claim 3, characterized by, The concentration of the recombinant antibody-modified magnetic beads is 2 mg / mL, the particle size is 1 μm, and the antibody conjugation amount is 50 μg / mg of magnetic beads.

5. The method according to any one of claims 1 to 4, wherein the method is characterized in that, Includes the following steps: Step 1: Sample collection; Step 2: Recombinant antibody enrichment with magnetic beads; Step 3: Treatment with the treatment solution; Step 4: Reconstitute the lyophilized microspheres; Step 5: TaqMan fluorescent PCR amplification; Step Six: FCV Detection and Judgment; Step 7: Pathogenicity assessment; Step 8: End.

6. The method for detecting feline calicivirus nucleic acid according to claim 5, wherein The first step, sample collection, specifically involves taking a cat's nasal swab or saliva sample; the second step, recombinant antibody magnetic bead enrichment, specifically involves adding 150 μl of the collected sample into the recombinant antibody magnetic bead enrichment module (450 μl of sample processing solution), mixing thoroughly, and letting it stand at room temperature for 1 min to obtain a resuspension.

7. The method for detecting feline calicivirus nucleic acid according to claim 6, characterized in that, The specific steps of step three are: lysis at 70℃ for 2 min; step four are: reconstitution of the lyophilized microspheres; and step five are: TaqMan fluorescence PCR amplification, which involves adding 25 μl of the supernatant from the solution obtained in step three to the lyophilized microspheres, dissolving them, and then placing them in a fluorescence PCR instrument. The amplification program is as follows: reverse transcription at 50℃ for 5 min; pre-denaturation at 95℃ for 2 min; denaturation at 95℃ for 5 s and annealing extension at 60℃ for 20 s (collecting fluorescence signals), for a total of 40 cycles.

8. The method for detecting feline calicivirus nucleic acid according to claim 7, characterized in that, The specific determination of FCV detection in step six is ​​as follows: a Ct value < 40 and the presence of a typical amplification curve indicate a positive result; the absence of a Ct value indicates a negative result.

9. The method for detecting feline calicivirus nucleic acid according to claim 8, characterized in that, The pathogenicity assessment in step seven specifically involves, if the test is positive, distinguishing between highly pathogenic strains (FAM signal only) and classic strains (FAM and HEX signals) by using the fluorescent signals (FAM channel / HEX channel) generated by the specific binding of the probe.

10. The application of the feline calicivirus nucleic acid detection kit according to any one of claims 1-4 in the clinical detection of feline calicivirus.

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