A primer probe combination for feline infectious peritonitis virus serotyping and application and kit thereof

Abstract

The present application relates to the technical field of virus detection, in particular to a primer probe combination for feline infectious peritonitis virus serotyping, application and kit thereof. The primer probe combination comprises FIPV N gene specific primer probe, FIPV I serotype S gene specific primer probe and FIPV II serotype S gene specific primer probe. The three groups of primer probes can be used for serotyping identification of FIPV I serotype strain and FIPV II serotype strain. The primer probe combination can be used for differential diagnosis of different serotypes of FIPV by fluorescence quantitative PCR method, and has the advantages of accurate identification, high sensitivity, simple operation, short time consumption and the like, and can provide effective technical means for rapid diagnosis of feline infectious peritonitis virus in China and prevention of feline infectious peritonitis.

Description

TECHNICAL FIELD

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

[0002] Feline infectious peritonitis (FIP) is a highly fatal infectious disease caused by feline infectious peritonitis virus (FIPV) infection, and the clinical diagnosis is difficult. FIPV has a single cell / macrophage affinity, leading to systemic immune-mediated diseases. At present, the clinical diagnosis relies on comprehensive evaluation, 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 technology is of great importance for early diagnosis and prevention and control.

[0003] FIPV is prevalent worldwide. According to the different serological responses caused by the significant sequence differences between the Spike proteins, FIPV can be divided into two serotypes, FIPV I and FIPV II, of which 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 most (80-95%) naturally occurring FIP cases in cats. Due to the significant differences in the antigenicity of the Spike proteins between the two serotypes, it is difficult to achieve accurate typing using traditional antibody detection methods.

[0004] Virus neutralization test (VNT) is the gold standard for early identification of FIPV, but it relies on cell culture systems, has a long operation period (5-7 days), and cannot distinguish FIPV from non-pathogenic feline enteric coronavirus (FECV). In addition, the serological method has high cross-reactivity for type I and type II FIPV, making it difficult to achieve specific typing. Conventional RT-PCR detects FCoV by amplifying the conserved sequence of the 3' untranslated region (UTR) of the virus, but cannot distinguish FIPV from FECV, and lacks specific primer design for serotype typing. Some patents (such as CN107586884A) improve the specificity of FIPV detection by designing primers targeting the Spike protein gene, but it is difficult to specifically distinguish the two serotypes. The establishment of a method that can simultaneously identify 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 treatment strategy development. SUMMARY

[0005] In order to solve the above problems, the application provides a primer probe combination for feline infectious peritonitis virus serotyping and application and kit thereof, a primer probe combination capable of directly performing feline infectious peritonitis virus serotyping is designed, the designed primer probe combination is mixed to form a primer pool, and a multiplex real-time fluorescent quantitative PCR kit for feline infectious peritonitis virus serotyping is provided.

[0006] In order to achieve the above purpose, the application provides the following technical scheme:

[0007] The application provides a primer probe combination for feline infectious peritonitis virus serotyping, which comprises FIPVN gene specific primers and probes, FIPV I serotype S gene specific primers and probes and FIPV II serotype S gene specific primers and probes.

[0008] The nucleotide sequence of the upstream primer of the FIPV N gene specific primers and probes 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 FIPV I serotype S gene specific primers and probes 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 FIPV II serotype S gene specific primers and probes 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 probe of the FIPV N gene specific primers and probes is modified by using a ROX group, and the 3' end is modified by using MGB.

[0012] The 5' end of the probe of the FIPV I serotype S gene specific primers and probes is modified by using a VIC group, and the 3' end is modified by using MGB.

[0013] The 5' end of the probe of the FIPV II serotype S gene specific primers and probes is modified by using a FAM group, and the 3' end is modified by using MGB.

[0014] The application also provides application of the primer probe combination in the preparation of a reagent for feline infectious peritonitis virus serotyping.

[0015] The application further provides a kit for feline infectious peritonitis virus serotyping, comprising the primer probe combination in the technical scheme.

[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 FIPV II 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 application further provides application of the kit in the technical scheme in preparation of a product for feline infectious peritonitis virus serotyping.

[0021] The application has the following beneficial effects:

[0022] The three groups of primer probes can be used for serotyping identification of FIPV I serotype strains and FIPV II serotype strains; the primer probe combination provided by the application can be used for differential diagnosis of different serotypes of FIPV by a fluorescence quantitative PCR method, and has the advantages of accurate identification, high sensitivity, simple operation, short time consumption, etc., and can provide an effective technical means for rapid diagnosis of feline infectious peritonitis virus in China and prevention of feline infectious peritonitis. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced.

[0024] Figure 1 The table is the primer probe concentration optimization result in example 6;

[0025] Figure 2 The table is the annealing temperature optimization result in example 7;

[0026] Figure 3 The table is the standard curve drawn by amplification of three target plasmids in the primer pool in example 8;

[0027] Figure 4The amplification curve of the three plasmids corresponding to the standard curve of the primer pool B in Example 8 is amplified;

[0028] Figure 5 The amplification curve of the primer pool in Example 9 for FIPV N and FIPV II-S plasmid concentration of 0.5 copies / μl, and FIPV I-S plasmid concentration of 5 copies / μl;

[0029] Figure 6 The amplification curve of the primer pool in Example 10 for three target plasmids (20 repeats each) at a concentration of 10 5 copies / μl;

[0030] Figure 7 The amplification curve of the primer pool in Example 10 for three target plasmids (20 repeats each) at a concentration of 10 2 copies / μl;

[0031] Figure 8 The detection results of the kit of the present application for one FIPV 79-1146 strain and one FIPV I serotype sample in Example 11. DETAILED DESCRIPTION

[0032] The present application provides a primer probe combination for feline infectious peritonitis virus serotyping, comprising FIPV N gene specific primer probe, FIPV I serotype S gene specific primer probe and 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 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 FIPV I 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 FIPV II 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.

[0033] Table 1 FIPV primer probe sequence

[0034]

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

[0036] The present application also provides the use of the primer probe combination in the preparation of a reagent for the serotyping of feline infectious peritonitis virus.

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

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

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

[0040] Example 1

[0041] 1. FIPV specific primer probe design. Download 69 FIPV full genome sequences in China in the past 5 years from Genbank, and extract the N gene and S gene respectively. Use sequence alignment software MEGA to perform multiple sequence alignment on the two genes. Then select the region consistent and conserved between FIPV I serotype strain and FIPV II serotype strain of N gene to design primer probe. Select the region inconsistent between FIPV I serotype strain and FIPV II serotype strain of S gene, but conserved within the serotype, to design primer probe for the two serotypes respectively. Specifically, the above primer probe is shown in Table 1, and the primer probe is synthesized by Shanghai Jerui Biological Technology Co., Ltd.

[0042] Table 1 FIPV primer probe sequence

[0043]

[0044]

[0045] 2. Standard plasmid synthesis. According to the designed 3 pairs of primers and probes, the corresponding target fragments were synthesized and cloned into pUC57 plasmid vector to obtain 3 standard plasmids, which were synthesized by Suzhou Hongxun Biotechnology Co., Ltd.

[0046] 3. Nucleic acid extraction and amplification kit. The kit for nucleic acid extraction was purchased from Suzhou Youyilan Di Biotechnology Co., Ltd.: UE body fluid virus DNA / RNA small amount preparation kit; the kit for real-time fluorescent quantitative PCR was purchased from Yixing Biotechnology Co., Ltd.: ⅢOne Step RT-qPCR Probe Kit. The specific extraction and amplification steps were carried out according to the above kit instructions.

[0047] 4. Effectiveness and specificity verification of single pair of primers and probes. The synthesized 3 standard plasmids were diluted to 10000 copies / μl respectively, and 2 FIPV Ⅱ 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, 1 feline rotavirus (FRV) nucleic acid were extracted using UE body fluid virus DNA / RNA small amount preparation kit. qPCR detection was carried out using 3 pairs of primers and probes respectively, using ⅢOne Step RT-qPCR Probe Kit. The specific extraction and amplification steps were carried out according to the above kit instructions.

[0048] Table 2 qPCR system

[0049]

[0050] RT-qPCR amplification was carried out using SLAN-96S fluorescent quantitative PCR instrument of Shanghai Hongshi Medical Technology Co., Ltd., and the RT-qPCR amplification program was as follows: 50℃ reverse transcription for 15min; 95℃ pre-denaturation for 30s; 95℃ denaturation for 30s, 60℃ extension for 30s, amplification for 45 cycles. The fluorescence signals of ROX, VIC and FAM channels were collected during extension.

[0051] Results: Each pair of primer probe has amplification curve when the corresponding nucleic acid is added, the CT value is between 20-29, no amplification when non-target nucleic acid is added, the results are shown in Table 3, which shows that each primer probe has good effectiveness and specificity.

[0052] Table 3 verification results of effectiveness and specificity of single pair of primer probe

[0053]

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

[0055] 5. Verification of effectiveness and specificity of primer pool. Three pairs of primer probes are mixed to form a primer pool, and 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 plasmid mixtures were prepared as positive plasmid controls, and RT-qPCR amplification kit was used for detection, and the RT-qPCR system is shown in Table 5.

[0059] Table 5 RT-qPCR system

[0060]

[0061] Using SLAN-96S fluorescent quantitative PCR instrument of Shanghai Hongshi Medical Technology Co., Ltd. to perform RT-qPCR amplification, the amplification procedure is the same as step 4.

[0062] Results: The primer pool has amplification curve when the nucleic acid containing the target fragment is added, the CT value is between 19-29, no amplification when the nucleic acid without the target fragment is added, the results are shown in Table 6, which shows that the two primer pools have good effectiveness and specificity.

[0063] Table 6 verification results of effectiveness and specificity of primer pool

[0064]

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

[0066] 6. Optimization of optimal working concentration of primer probe

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

[0068] (1) FIPV N-F / FIPV N-R primer concentration optimization

[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] FIPV I-S-F: 300 nM, FIPV I-S-R: 300 nM, FIPV I-S-P: 250 nM;

[0081] FIPV II-S-F: 100 nM, FIPV II-S-R: 100 nM, FIPV II-S-P: 250 nM.

[0082] 7. Annealing temperature optimization

[0083] The primer pool 500 μl was prepared according to the optimal concentration of the primer probe optimized in step 6, as shown in Table 8.

[0084] Table 8 Preparation of primer pool after concentration optimization

[0085]

[0086] 10 copies / μl of the standard plasmid containing the target fragment were mixed as a template, and RT-qPCR was performed using SLAN-96S instrument, 3 repeats were detected for each condition, and the optimal annealing temperature of primer pool A and primer pool B was screened, the reaction system was the same as step 5, and the reaction program was as shown in Table 9. 5 copies / μl of the standard plasmid containing the target fragment were mixed as a template, and RT-qPCR was performed using SLAN-96S instrument, 3 repeats were detected for each condition, and the optimal annealing temperature of primer pool A and primer pool B was screened, the reaction system was the same as step 5, and the reaction program was as shown in Table 9.

[0087] Table 9 Reaction program for annealing temperature optimization

[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 shown in Table 10, and 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 as 64°C. Figure 2

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

[0091] 8. Establishment of standard curve

[0092] ​Three synthesized standard plasmids with known copy numbers were serially diluted 10-fold. RT-qPCR was performed using the kit prepared in this invention on a SLAN-96S instrument, with three replicates for each plasmid concentration. A standard curve was plotted using Graphpad Prism 8 software, with the CT value of the amplification curve on the ordinate and the logarithm of the template copy number on the x-axis. The primer pool standard curve is shown below. Figures 3-4 As shown. The slopes of the standard curves for amplifying FIPV N, FIPVⅠ-S, and FIPVⅡ-S plasmids using the primer pool were -3.104, -3.386, and -3.284, respectively. Their amplification efficiencies were calculated to be 109.97%, 97.39%, and 101.61%, respectively. R 2 All values ​​were greater than 0.99, indicating that the primer pool had high amplification efficiency.

[0093] 9. Sensitivity Experiment

[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. Using the mixed plasmid as a template, 12 replicates were performed for each concentration to determine the detection limit of the kit prepared in this invention for each serotype of FIPV. The detection limit for that genotype or lineage strain was defined as a detection rate of 95% or higher in the lowest detection replicate experiment. The detection results of plasmids at each concentration are shown in Table 10, indicating that the detection limit of the kit prepared in this invention can reach 0.5 copies / μl for FIPV N and FIPV II-S genes, and 5 copies / μl for FIPV I-S gene, demonstrating the high sensitivity of the kit. The amplification curves of FIPV N and FIPV II-S plasmids at a concentration of 0.5 copies / μl and FIPV I-S plasmid at a concentration of 5 copies / μl are attached. Figure 5 .

[0095] Table 10 Sensitivity Experiment Results

[0096]

[0097] 10. Repeatability Experiments

[0098] As attached Figure 6 - Appendix 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. Use of a kit in the preparation of a product for the serotyping of feline infectious peritonitis virus; the kit comprising a primer probe combination; the primer probe combination comprising FIPV N gene specific primer probe, FIPV I serotype S gene specific primer probe and 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 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 FIPV I 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 FIPV II 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; the primer probe combination is mixed to form a primer pool; 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; 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; the concentration of the upstream primer of the FIPV II 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.

2. Use according to claim 1, characterized in that, the 5' end of the probe 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 probe of the FIPV I serotype S gene specific primer probe is modified with a VIC group, and the 3' end is modified with MGB; the 5' end of the probe of the FIPV II serotype S gene specific primer probe is modified with a FAM group, and the 3' end is modified with MGB.

Images