Primer probe combination for serotyping of feline infectious peritonitis virus as well as application and kit of primer probe combination

Abstract

The invention relates to the technical field of virus detection, in particular to a primer probe combination for serotyping of feline infectious peritonitis virus as well as application and a kit of the primer probe combination. The primer probe combination comprises an FIPV N gene specific primer probe, an FIPVI serotype S gene specific primer probe and an FIPVII serotype S gene specific primer probe. According to the invention, through the three groups of primer probes, the FIPVI serotype strain and the FIPVII serotype strain can be subjected to serotyping identification; the primer probe combination provided by the invention can be used for identifying and diagnosing different serotypes of FIPVs at one time through a fluorescent quantitative PCR method, and has the advantages of accurate identification, high sensitivity, simplicity in operation, short time consumption and the like; and an effective technical means can be provided for rapid diagnosis of clinical feline infectious peritonitis virus and prevention of feline infectious peritonitis in China.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, in particular to a primer-probe combination for feline infectious peritonitis virus serotyping, an application thereof and a kit. Background Art

[0002] Feline infectious peritonitis (FIP) is a highly lethal infectious disease caused by feline infectious peritonitis virus (FIPV), which is difficult to diagnose clinically. FIPV, due to its monocyte / macrophage tropism, causes a systemic immune-mediated disease. Currently, clinical diagnosis relies on comprehensive assessments such as ascites analysis, tissue biopsy, and immunohistochemistry. However, these methods are highly invasive, time-consuming, and require high technical skills. Therefore, the development of rapid and specific serotype identification technologies is crucial for early diagnosis and prevention.

[0003] FIPV is prevalent worldwide. Based on the different serological reactions caused by significant sequence differences between the spike proteins, FIPV can be divided into two serotypes: FIPV I and FIPV II. Type I is dominant in Europe and the United States, while type II is more prevalent in Asia. In nature, FIPV I is more common than FIPV II and is associated with the majority (80-95%) of naturally occurring FIP cases in cats. Due to the significant differences in the antigenicity of the spike protein between the two serotypes, traditional antibody detection methods are difficult to achieve accurate typing.

[0004] The virus neutralization test (VNT) is the gold standard for early identification of FIPV, but it relies on a cell culture system, has a long operation cycle (requiring 5-7 days), and cannot distinguish FIPV from the non-pathogenic feline enteric coronavirus (FECV). In addition, serological methods have high cross-reactivity against types I and II FIPV, making specific typing difficult. Conventional RT-PCR detects FCoV by amplifying conserved sequences in the viral 3' untranslated region (UTR), but cannot distinguish FIPV from FECV and lacks specific primer design for serotyping. Some patents (such as CN107586884A) have improved the specificity of FIPV detection by designing primers targeting the spike protein gene, but it is difficult to specifically distinguish between the two serotypes. Establishing a method that can simultaneously differentiate and diagnose the two serotypes of FIPV can provide great reference value for the rapid prevention and control of FIP. Serotype identification is of great significance for vaccine development, epidemiological tracking, and the formulation of treatment strategies. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a primer-probe combination for feline infectious peritonitis virus serotyping, its application and kit, designs a primer-probe group that can directly perform feline infectious peritonitis virus serotyping, mixes the designed primer-probe group to form a primer pool, and provides a multiplex real-time fluorescence quantitative PCR kit for feline infectious peritonitis virus serotyping.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a primer probe combination for feline infectious peritonitis virus serotyping, comprising a primer probe specific to the FIPVN gene, a primer probe specific to the FIPVⅠ serotype S gene, and a primer probe specific to the FIPVⅡ serotype S gene;

[0008] The nucleotide sequence of the upstream primer of the FIPV N gene-specific primer probe is shown in SEQ ID No. 1, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 2, and the nucleotide sequence of the probe is shown in SEQ ID No. 3;

[0009] The nucleotide sequence of the upstream primer of the FIPVⅠ serotype S gene-specific primer probe is shown in SEQ ID No. 4, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 5, and the nucleotide sequence of the probe is shown in SEQ ID No. 6;

[0010] The nucleotide sequence of the upstream primer of the FIPVⅡ serotype S gene specific primer probe is shown in SEQ ID No. 7, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 8, and the nucleotide sequence of the probe is shown in SEQ ID No. 9.

[0011] Preferably, the 5' end of the FIPV N gene-specific primer probe is modified with a ROX group and the 3' end is modified with MGB;

[0012] The 5' end of the FIPVⅠ serotype S gene specific primer probe is modified with a VIC group, and the 3' end is modified with an MGB group;

[0013] The 5' end of the FIPVII serotype S gene specific primer probe is modified with a FAM group, and the 3' end is modified with an MGB group.

[0014] The present invention also provides the use of the primer-probe combination described in the above technical solution in the preparation of a reagent for feline infectious peritonitis virus serotyping.

[0015] The present invention also provides a kit for feline infectious peritonitis virus serotyping, comprising the primer-probe combination described in the above technical solution.

[0016] Preferably, the primer-probe combination is mixed to form a primer pool.

[0017] Preferably, the concentration of the upstream primer of the FIPV N gene-specific primer probe in the primer pool is 100 nM, the concentration of the downstream primer is 100 nM, and the concentration of the probe is 250 nM.

[0018] Preferably, the concentration of the upstream primer of the FIPV I serotype S gene-specific primer probe in the primer pool is 300 nM, the concentration of the downstream primer is 300 nM, and the concentration of the probe is 250 nM.

[0019] Preferably, the concentration of the upstream primer of the FIPVⅡ serotype S gene-specific primer probe in the primer pool is 100 nM, the concentration of the downstream primer is 100 nM, and the concentration of the probe is 250 nM.

[0020] The present invention also provides the use of the kit described in the above technical solution in preparing a product for feline infectious peritonitis virus serotyping.

[0021] Beneficial effects of the present invention:

[0022] The present invention can perform serotyping and identification of FIPVⅠ serotype strains and FIPVⅡ serotype strains through the above three sets of primer probes; the primer probe combination provided by the present invention can be used to perform differential diagnosis of FIPV of different serotypes at one time through the fluorescent quantitative PCR method, which has the advantages of accurate identification, high sensitivity, simple operation, and short time consumption. It can provide an effective technical means for the rapid diagnosis of feline infectious peritonitis virus and the prevention of feline infectious peritonitis in clinical practice in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0024] Figure 1 The results of primer probe concentration optimization for the primer pool in Example 6 are as follows;

[0025] Figure 2 This is the annealing temperature optimization result of Example 7;

[0026] Figure 3 Standard curve drawn for the amplification of three target plasmids using the primer pool in Example 8;

[0027] Figure 4The amplification curves corresponding to the standard curves of the three target plasmids amplified by primer pool B in Example 8 are as follows;

[0028] Figure 5 This is the amplification curve of the primer pool in Example 9 when the concentration of FIPV N and FIPVⅡ-S plasmids is 0.5 copies / μl and the concentration of FIPVⅠ-S plasmid is 5 copies / μl;

[0029] Figure 6 The primer pool in Example 10 is for 3 target plasmids (20 replicates each) in 10 5 Amplification curve at the concentration of copies / μl;

[0030] Figure 7 The primer pool in Example 10 is for 3 target plasmids (20 replicates each) in 10 2 Amplification curve at the concentration of copies / μl;

[0031] Figure 8 These are the test results of a sample of FIPV79-1146 strain and a sample of FIPVⅠ serotype in Example 11 using the kit of the present invention. DETAILED DESCRIPTION

[0032] The present invention provides a primer-probe combination for feline infectious peritonitis virus serotyping, comprising an FIPV N gene-specific primer probe, an FIPV I serotype S gene-specific primer probe, and an FIPV II serotype S gene-specific primer probe; the nucleotide sequence of the upstream primer of the FIPV N gene-specific primer probe is shown as SEQ ID No.1, the nucleotide sequence of the downstream primer is shown as SEQ ID No.2, and the nucleotide sequence of the probe is shown as SEQ ID No.3; the nucleotide sequence of the upstream primer of the FIPV I serotype S gene-specific primer probe is shown as SEQ ID No.4, the nucleotide sequence of the downstream primer is shown as SEQ ID No.5, and the nucleotide sequence of the probe is shown as SEQ ID No.6; the nucleotide sequence of the upstream primer of the FIPV II serotype S gene-specific primer probe is shown as SEQ ID No.7, the nucleotide sequence of the downstream primer is shown as SEQ ID No.8, and the nucleotide sequence of the probe is shown as SEQ ID No.9.

[0033] Table 1 FIPV primer probe sequences

[0034]

[0035] In the present invention, the 5' end of the FIPV N gene-specific primer probe is preferably modified with a ROX group, and the 3' end is preferably modified with MGB; the 5' end of the FIPVⅠ serotype S gene-specific primer probe is preferably modified with a VIC group, and the 3' end is preferably modified with MGB; the 5' end of the FIPVⅡ serotype S gene-specific primer probe is preferably modified with a FAM group, and the 3' end is preferably modified with MGB.

[0036] The present invention also provides the use of the primer-probe combination described in the above technical solution in the preparation of a reagent for feline infectious peritonitis virus serotyping.

[0037] The present invention also provides a kit for feline infectious peritonitis virus serotyping, comprising the primer-probe combination described in the above technical solution. In the present invention, the primer-probe combination is preferably mixed to form a primer pool. In the present invention, the concentration of the upstream primer of the FIPV N gene-specific primer probe in the primer pool is preferably 100nM, the concentration of the downstream primer is preferably 100nM, and the concentration of the probe is preferably 250nM. In the present invention, the concentration of the upstream primer of the FIPVⅠ serotype S gene-specific primer probe in the primer pool is preferably 300nM, the concentration of the downstream primer is preferably 300nM, and the concentration of the probe is preferably 250nM. In the present invention, the concentration of the upstream primer of the FIPVⅡ serotype S gene-specific primer probe in the primer pool is preferably 100nM, the concentration of the downstream primer is preferably 100nM, and the concentration of the probe is preferably 250nM.

[0038] The present invention also provides the use of the kit described in the above technical solution in preparing a product for feline infectious peritonitis virus serotyping.

[0039] In order to further illustrate the present invention, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1

[0041] 1. Design of FIPV-specific primer probes. 69 FIPV full genome sequences from the past five years in my country were downloaded from Genbank, and their N and S genes were extracted respectively. The sequence alignment software MEGA was used to perform multiple sequence alignment on the two genes. Then, the N gene was selected to design primer probes in the regions that were consistent and conserved between the FIPVⅠ serotype strain and the FIPVⅡ serotype strain. The S gene was selected to design primer probes for the two serotypes in the regions that were inconsistent between the FIPVⅠ serotype strain and the FIPVⅡ serotype strain but conserved within the serotype. Specifically, the above primer probes are shown in Table 1, and the primer probes were synthesized by Shanghai Jierui Biotechnology Co., Ltd.

[0042] Table 1 FIPV primer probe sequences

[0043]

[0044]

[0045] 2. Standard plasmid synthesis. Based on the three pairs of designed primer probes, the corresponding target fragments were synthesized and cloned into the pUC57 plasmid vector to obtain three standard plasmids. The standard plasmids were synthesized by Suzhou Hongxun Biotechnology Co., Ltd.

[0046] 3. Nucleic acid extraction and amplification kits. The nucleic acid extraction kit was purchased from Suzhou Youyi Landi Biotechnology Co., Ltd.: UE Body Fluid Viral DNA / RNA Mini-Prep Kit; the real-time fluorescence quantitative PCR kit was purchased from Yisheng Biotechnology Co., Ltd.: ⅢOne Step RT-qPCR Probe Kit. Specific extraction and amplification steps were performed according to the kit instructions.

[0047] 4. Verification of the effectiveness and specificity of a single pair of primer probes. The three synthesized standard plasmids were diluted to 10,000 copies / μl respectively, and the UE body fluid virus DNA / RNA small-scale preparation kit was used to extract 2 FIPVⅡ serotype strains (FIPV DF2, FIPV 79-1146) nucleic acids, 1 feline parvovirus (FPV) nucleic acid, 1 feline calicivirus (FCV) nucleic acid, 1 feline herpesvirus (FHV) nucleic acid, 1 feline leukemia virus (FeLV) nucleic acid, 1 feline immunodeficiency virus (FIV) nucleic acid, and 1 feline rotavirus (FRV) nucleic acid. Use 3 pairs of primer probe groups respectively, using qPCR detection was performed using the One Step RT-qPCR Probe Kit. The qPCR system is shown in Table 2.

[0048] Table 2 qPCR system

[0049]

[0050] RT-qPCR amplification was performed using a SLAN-96S fluorescent quantitative PCR instrument from Shanghai Hongshi Medical Technology Co., Ltd. The RT-qPCR amplification program was as follows: reverse transcription at 50°C for 15 minutes, pre-denaturation at 95°C for 30 seconds, denaturation at 95°C for 30 seconds, and extension at 60°C for 30 seconds, for 45 cycles. Fluorescence signals from the ROX, VIC, and FAM channels were collected during the extension period.

[0051] Results: Each pair of primer probes had an amplification curve when the corresponding target nucleic acid was added, with a CT value between 20-29. No amplification was observed when non-target nucleic acid was added. The results are shown in Table 3, indicating that each primer probe had good effectiveness and specificity.

[0052] Table 3 Results of validation of effectiveness and specificity of single primer-probe pairs

[0053]

[0054] The value represents the amplification curve CT value -: RT-qPCR no amplification curve

[0055] 5. Verification of primer pool validity and specificity: The three pairs of primer probes were mixed to form a primer pool. The primer composition is shown in Table 4.

[0056] Table 4 RT-qPCR primer pool

[0057]

[0058] Using the same nucleic acid as in step 4, three additional plasmid mixtures were prepared as positive plasmid controls and tested using an RT-qPCR amplification kit. The RT-qPCR system is shown in Table 5.

[0059] Table 5 RT-qPCR system

[0060]

[0061] RT-qPCR amplification was performed using the SLAN-96S fluorescence quantitative PCR instrument from Shanghai Hongshi Medical Technology Co., Ltd. The amplification procedure was the same as in step 4.

[0062] Results: When the primer pools were added with nucleic acid containing the target fragment, amplification curves were obtained with CT values ​​between 19 and 29. When nucleic acid containing no target fragment was added, no amplification occurred. The results are shown in Table 6, indicating that both primer pools had good effectiveness and specificity.

[0063] Table 6 Results of primer pool validity and specificity verification

[0064]

[0065] The value represents the amplification curve CT value -: RT-qPCR no amplification curve

[0066] 6. Optimization of the optimal working concentration of primers and probes

[0067] 6.1 Optimization of the optimal working concentration of primer pool and probe

[0068] (1) Optimization of FIPV NF / FIPV NR primer concentration

[0069] The primer mixture containing FIPV N-F / FIPV N-R was prepared with working concentrations of 100 nM, 150 nM, 200 nM, 250 nM, and 300 nM, respectively. The concentrations of other primers and probes were fixed, and the plasmid containing the target fragment was amplified as the template. Each concentration condition was detected in triplicate. The RT-qPCR reaction system is shown in Table 7.

[0070] Table 7. PRRSV2-F / PRRSV2-R primer concentration optimization

[0071]

[0072] The reaction conditions were the same as in Step 4.

[0073] (2) FIPV I-S-F / FIPV I-S-R, FIPV II-S-F / FIPV II-S-R primer concentration optimization

[0074] The primer concentration optimization method for FIPV N-F / FIPV N-R was used to prepare each primer pair with working concentrations of 100 nM, 150 nM, 200 nM, 250 nM, and 300 nM, respectively. Then, the plasmid containing the target fragment was amplified according to the method of fixing the concentrations of other primers and probes.

[0075] (3) FIPV N-P, FIPV I-S-P, FIPV II-S-P probe concentration optimization

[0076] Similar to the primer concentration optimization method, the probes were prepared with working concentrations of 50 nM, 100 nM, 150 nM, 200 nM, and 250 nM, respectively. Then, the plasmid containing the target fragment was amplified according to the method of fixing the concentrations of other primers and probes.

[0077] (4) Primer pool primer and probe concentration optimization results

[0078] After each sample was subjected to RT-qPCR detection, the amplification curve CT value and fluorescence signal intensity Rn were obtained. The values of three replicate wells were averaged, and the above primer and probe concentration optimization results were statistically summarized as shown in Table 8. Figure 1 The method for selecting the best primer or probe concentration was to preferentially select the primer or probe concentration with the smallest CT value. If the CT values were the same, the primer or probe concentration with the largest Rn value was selected. Finally, the optimized working concentrations of each primer and probe were as follows:

[0079] FIPV N-F: 100 nM, FIPV N-R: 100 nM, FIPV N-P: 250 nM;

[0080] FIPVⅠ-SF: 300nM, FIPVⅠ-SR: 300nM, FIPVⅠ-SP: 250nM;

[0081] FIPVⅡ-SF: 100nM, FIPVⅡ-SR: 100nM, FIPVⅡ-SP: 250nM.

[0082] 7. Annealing temperature optimization

[0083] Prepare 500 μl of primer pool according to the optimal concentration of primers and probes optimized in step 6, as shown in Table 8.

[0084] Table 8 Primer pool preparation after concentration optimization

[0085]

[0086] The 10 5 The standard plasmids (100 copies / μl) were mixed and used as templates. RT-qPCR was performed using a SLAN-96S instrument. Three replicates were tested for each condition. The optimal annealing temperatures of primer pool A and primer pool B were screened. The reaction system was the same as in step 5. The reaction procedure is shown in Table 9.

[0087] Table 9 Annealing temperature optimization reaction program

[0088]

[0089] Results: The CT values ​​of the three replicate wells after the experiment were averaged and the annealing temperature optimization results were statistically summarized, as follows: Figure 2 As shown in FIG, the annealing temperature with the lowest CT value and the highest Rn value was selected as the optimal annealing temperature. Finally, the optimal annealing temperature was selected to be 64°C.

[0090] According to the above optimization results of primer and probe concentration and annealing temperature, the final reaction system and reaction procedure of the FIPV serotyping kit were determined. The primer pool was prepared as shown in step 7, the reaction system was consistent with step 5, and the reaction procedure was consistent with step 7, wherein the annealing temperature was selected to be 60°C, and the mixed standard plasmids (each plasmid concentration was about 10 4 copies / μl) was used as the positive control of this kit.

[0091] 8. Establishment of Standard Curve

[0092] The three standard plasmids with known copy numbers were diluted 10-fold in series and RT-qPCR was performed using the kit prepared by the present invention and SLAN-96S instrument. Each concentration of plasmid was tested in triplicate. The standard curve was drawn using Graphpad Prism8 software with the CT value of the amplification curve as the ordinate and the logarithm of the template copy number as the abscissa. The primer pool standard curve is shown in Figure 1. Figure 3-Figure 4 The slopes of the standard curves for the primer pool amplification of FIPV N, FIPV I-S, and FIPV II-S plasmids were -3.104, -3.386, and -3.284, respectively. The amplification efficiencies were 109.97%, 97.39%, and 101.61%, respectively. 2 All of them were greater than 0.99, indicating that the primer pool had a high amplification efficiency.

[0093] 9. Sensitivity test

[0094] The standard plasmid was diluted to 10 copies / μl, 5 copies / μl, 1 copies / μl, and 0.5 copies / μl, and then mixed to obtain a mixed plasmid. The mixed plasmid was used as a template, and 12 replicates were detected at each concentration to determine the detection limit of the kit prepared by the present invention for each serotype of FIPV. The detection rate of the lowest detection repeat experiment reaching more than 95% was determined as the detection limit for the genotype or lineage strain. The detection results of plasmids at various concentrations are shown in Table 10, which shows that the detection limit of the kit prepared by the present invention for FIPV N and FIPVⅡ-S genes can reach 0.5 copies / μl; the detection limit for FIPVⅠ-S gene can reach 5 copies / μl, which proves the high sensitivity of the kit. The amplification curves when the concentrations of FIPV N and FIPVⅡ-S plasmids are 0.5 copies / μl and the concentration of FIPVⅠ-S plasmids are 5 copies / μl are shown in the attached figure. Figure 5 .

[0095] Table 10 Sensitivity test results

[0096]

[0097] 10. Repeatability Experiment

[0098] As attached Figure 6 -Attached Figure 7 As shown, the standard plasmids were diluted to 10 5 copies / μl, 10 2copies / μl, then mixed to obtain mixed plasmids, and 20 repeats were detected for each concentration, to evaluate the repeatability of the kit prepared by the application. The amplification CT value of each sample was obtained after detection, and the average CT, standard deviation (SD), and coefficient of variation (CV) were calculated. The repeatability experiment results showed that for the template of 10 5 copies / μl, the amplification CT value was between 20-22, and the coefficient of variation was 1.55%-2.07%; for the template of 10 2 copies / μl, the amplification CT value was between 30-32, and the coefficient of variation was 1.71%-2.06%, indicating that the kit prepared by the application has high repeatability.

[0099] Table 11 Repeatability experiment results

[0100]

[0101] 11. Clinical sample detection

[0102] To verify the detection effect of the kit prepared by the application on clinical RNA samples, 59 cat anal swabs and ascites samples from an animal hospital were detected, and the FIPV 79-1146 strain preserved in the laboratory was used as a positive control. RNA extraction was performed according to the method described in step 2, and then the kit prepared by the application was used for RT-qPCR detection using the SLAN-96S instrument.

[0103] Results: Among the 59 samples, 2 samples were detected as FIPV positive, with CT values between 27-34, and both of the 2 samples were FIPV I serotype strains, achieving one-step identification of FIPV serotypes. The CT values of the 2 samples detected as FIPV positive are shown in Table 12, and the amplification curves of the positive control FIPV 79-1146 strain and sample K1 are shown in Figures 1 and 2, respectively. Figure 8

[0104] Table 12 Clinical sample detection results

[0105]

[0106] The numerical value represents the CT value of the amplification curve: RT-qPCR has no amplification curve

[0107] Although the above embodiment has made a detailed description of the application, it is only a part of the embodiments of the application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which are within the protection scope of the application.​

Claims

1. A primer-probe combination for serotyping of feline infectious peritonitis virus, characterized in that: Including FIPV N gene specific primer probe, FIPVⅠ serotype S gene specific primer probe and FIPVⅡ serotype S gene specific primer probe; The nucleotide sequence of the upstream primer of the FIPV N gene-specific primer probe is shown in SEQ ID No. 1, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 2, and the nucleotide sequence of the probe is shown in SEQ ID No. 3; The nucleotide sequence of the upstream primer of the FIPVⅠ serotype S gene-specific primer probe is shown in SEQ ID No. 4, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 5, and the nucleotide sequence of the probe is shown in SEQ ID No. 6; The nucleotide sequence of the upstream primer of the FIPVⅡ serotype S gene specific primer probe is shown in SEQ ID No.7, the nucleotide sequence of the downstream primer is shown in SEQ ID No.8, and the nucleotide sequence of the probe is shown in SEQ ID No.

9.

2. The primer-probe combination according to claim 1, characterized in that The 5' end of the FIPV N gene-specific primer probe is modified with a ROX group, and the 3' end is modified with MGB; The 5' end of the FIPVⅠ serotype S gene specific primer probe is modified with a VIC group, and the 3' end is modified with an MGB group; The 5' end of the FIPVII serotype S gene specific primer probe is modified with a FAM group, and the 3' end is modified with an MGB group.

3. Application of the primer-probe combination according to claim 1 or 2 in the preparation of a reagent for feline infectious peritonitis virus serotyping.

4. A kit for serotyping of feline infectious peritonitis virus, characterized in that: Comprising the primer-probe combination according to claim 1 or 2.

5. The kit according to claim 4, characterized in that The primer-probe combination is mixed to form a primer pool.

6. The kit according to claim 5, characterized in that The concentration of the upstream primer of the FIPV N gene-specific primer probe in the primer pool is 100 nM, the concentration of the downstream primer is 100 nM, and the concentration of the probe is 250 nM.

7. The kit according to claim 5, characterized in that The concentration of the upstream primer of the FIPVⅠ serotype S gene specific primer probe in the primer pool is 300 nM, the concentration of the downstream primer is 300 nM, and the concentration of the probe is 250 nM.

8. The kit according to claim 5, wherein The concentration of the upstream primer of the FIPVⅡ serotype S gene specific primer probe in the primer pool is 100 nM, the concentration of the downstream primer is 100 nM, and the concentration of the probe is 250 nM.

9. Use of the kit according to any one of claims 5 to 8 in the preparation of a product for feline infectious peritonitis virus serotyping.

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