Use of first gene fragment and second gene fragment, primer set and probe, kit and construction method thereof

Abstract

The present application belongs to the field of biotechnology, and discloses the use of preparing a primer set and a probe for rapidly identifying feline parvovirus and canine parvovirus by using a first gene fragment of feline parvovirus and a second gene fragment of canine parvovirus. In addition, the present application also discloses a primer set and a probe for rapidly identifying feline parvovirus and canine parvovirus. Due to the high similarity between the first gene fragment and the second gene fragment, the antagonistic effect between the primers and the probe in the present application is small, and effective screening of feline parvovirus and canine parvovirus can still be achieved. In addition, the present application also discloses a kit for rapidly identifying feline parvovirus and canine parvovirus and a method for constructing the above kit.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to uses of a first gene fragment and a second gene fragment, a primer set and a probe, a kit and a construction method thereof. Background Art

[0002] Feline parvovirus (FPV), also known as feline panleukopenia virus, commonly known as feline plague virus, can cause fever, vomiting, hemorrhagic enteritis and leukopenia in cats, with a high mortality rate. Canine parvovirus (CPV) can cause gastroenteritis and myocarditis, with a rapid onset and high mortality rate. With the drift of viral antigens, the virus can also infect cats, bears, minks and other animals. Feline parvovirus (FPV) and canine parvovirus (CPV) are two closely related parvoviruses. Both belong to the Parvoviridae family, the Protoparvovirus genus, and the Protoparvovirus 1 species of carnivores. They are single-stranded DNA viruses. In recent years, cats have been infected with canine parvovirus disease from time to time. Since the clinical symptoms of feline parvovirus infection in cats are similar to those of canine parvovirus infection, the possibility of canine parvovirus infection is often ignored in the diagnosis of cats with acute viral hemorrhagic enteritis, resulting in a low cure rate. When animals are co-infected with feline parvovirus and canine parvovirus, it is difficult to make a differential diagnosis based on clinical symptoms, and laboratory testing is necessary to confirm the diagnosis.

[0003] Isolation of feline parvovirus and canine parvovirus by cell culture technology is the gold standard method for diagnosis. Although it is highly specific, it is time-consuming, labor-intensive and expensive, and requires skilled professionals, so its application range is narrow. At present, a variety of immunological methods have been developed for direct identification, including enzyme-linked immunosorbent assay (ELISA), latex agglutination test (LAT), hemagglutination test (HA), fluorescent antibody test (FAT), etc. Most of these laboratory methods are used to detect specific antigens or antibodies. Data show that the homology between FPV and CPV is more than 98%. CPV and FPV can produce cross-immunity and serological cross-reactions, so immunological methods have the problems of low sensitivity and poor specificity, and cannot distinguish between feline parvovirus and canine parvovirus. PCR technology can be used to amplify pathogens that cannot be cultured. Since PCR detection is a specific primer sequence designed to completely match the pathogen sequence, it is highly specific and can detect the target gene in a short time.

[0004] Evolutionary analysis data show that CPV and FPV sequences have high homology, and the VP2 gene has only 0.5% difference. Some reports believe that they may have evolved from the same ancestor to adapt to different hosts. CPV can infect cats, but FPV has not been reported to infect dogs so far; and many studies have reported mixed infection of cats with CPV and FPV. The existing technologies for differential diagnosis of feline parvovirus and canine parvovirus are as follows:

[0005] DNA sequencing and hemagglutination inhibition test using monoclonal antibodies can be used to identify FPV and CPV. The former requires the extraction of viral DNA for PCR amplification and generally needs to be sent to a company for sequencing, which is time-consuming and labor-intensive. The latter requires multiple monoclonal antibodies and is not suitable for general use.

[0006] Decaro et al. designed a pair of primers and probes based on the single nucleotide polymorphism site G3752A and established a TaqMan MGB real time PCR method that can identify and detect FPV and CPV DNA (Decaro et al., 2008). However, the conservation of the sequence greatly affects the test results. Recently, China reported that the HB3 strain (GenBank No. KT162043) had a mutation at the 3752 base site, which made it impossible to identify the single nucleic acid mutation.

[0007] In her dissertation titled "Establishment and Preliminary Application of Molecular Identification Methods for Canine Parvoviruses of Different Antigenic Types and Feline Parvoviruses", Sun Yaru from the Chinese Academy of Agricultural Sciences downloaded all the complete genome sequences of FPV and CPV from GenBank, used MEGA7 for sequence comparison analysis, and found a stable SNP A4408C site, and a conserved fragment near the site. A pair of primers F1 / R1 was designed to amplify a 361bp conservative fragment containing the mutation site and construct a standard plasmid; a pair of primers F2 / R2 was designed to establish a qPCR-HRM method for identifying FPV and CPV, but this method requires the use of expensive PCR instruments and analysis software, and the qPCR-HRM reaction amplification procedure takes about 2 hours, which is time-consuming and not suitable for use in pet hospitals. In addition, through sequence analysis, it was found that the specific primers in this method cannot distinguish FPV and CPV;

[0008] Amplification Refractory Mutation System (ARMS) technology was first established by Newton et al. to detect known mutations. The basic principle of ARMS-QPCR, an identification detection method developed on this basis, is that the 3' end base of the primer is not complementary to the template base, and cannot be extended by DNA polymerase, while the primer complementary to the template base can be extended normally by DNA polymerase. Combined with fluorescent probe detection, real-time fluorescent identification detection of SNP sites can be performed. At present, this method has been used to detect point mutations in many diseases and has also begun to be used in pathogen identification detection. The base type during primer design has a great influence on PCR amplification. The mismatch of different base types introduced at the 3' end of the primer has inconsistent amplification efficiency. The amplification efficiency of the 3' end of the primer is the lowest when G changes to C, T changes to G, and C changes to A, which greatly hinders amplification. This can be used to provide a reference for the specificity of primer design when designing primers. Studies have shown that when the base binding between the primer and the template is CC, GA, AG, and AA, the binding rate is the lowest and the amplification reaction is most significantly hindered.

[0009] TaqMan probe is a dual-labeled, self-quenched hydrolysis probe, with the 5' and 3' ends labeled with a fluorescent group and a quenching group, respectively. When the probe structure is complete, the two are close to each other, and the signal of the fluorescent group can be quenched. During the PCR amplification process, if the TaqMan probe and the target sequence are completely matched, the probe can be bound to the DNA template. At this time, when the Taq enzyme extends to the probe position, its exonuclease activity will cut the hydrolysis probe, release the fluorescent group, and enhance the fluorescence signal. Moreover, as the number of amplified products increases, the fluorescent signal becomes stronger and stronger, so that the change process of the fluorescent signal can be monitored in real time by the instrument. For biallelic SNPs, two corresponding probes can be designed respectively. Only when the probe and the template are completely matched, the probe can be hydrolyzed to produce a strong fluorescent signal during the amplification process. If there is a mismatch between the probe and the target sequence, its fluorescence intensity will be weakened, so that the SNP site can be detected by fluorescence intensity. The number of nucleotide mismatches that the probe detection can tolerate is 6, and the position in the middle of the probe is more sensitive than the two ends of the probe, and the Tm value decreases faster. When a nucleotide mismatch occurs in the probe, it has little effect on the amplification efficiency, and the main impact is the change in the Tm value. When the mismatch increases to a certain number, the amplification curve will directly appear negative.

[0010] Prior art 1: Chinese patent application CN202111628075.0 discloses a PCR detection kit for cat and / or dog pathogens, as well as a detection method and application. The kit of this scheme includes primers and probes for detecting canine distemper virus, canine influenza A virus, canine parainfluenza virus, canine parvovirus, canine coronavirus, canine rotavirus, canine babesia, canine ascaris, canine ehrlichia, canine Brucella, rabies virus, Borrelia burgdorferi, internal reference gene ACTB, feline herpesvirus, feline calicivirus, feline parvovirus, feline coronavirus, feline immunodeficiency virus, feline leukemia virus, feline hemomycoplasma, feline Mycoplasma, feline Chlamydia, Giardia, Toxoplasma, Bartonella and internal reference gene GAPDH. The collected cat and dog DNA and RNA are added to the kit, and a real-time fluorescence PCR instrument is used to perform a PCR reaction. FAM, HEX, ROX and CY5 fluorescence signals are collected in each cycle to analyze the relevant pathogens. The specificity and sensitivity are higher than those of traditional detection methods.

[0011] Further observation of the protocol's instructions shows that, although the protocol is capable of detecting multiple viruses simultaneously, there are differences in the primers and probes used in the test process, i.e., each virus uses its own highly specific fragments for primer design and amplification.

[0012] Prior art 2: Chinese patent application CN202310047212.4 discloses a method, primer composition and kit for rapid detection of cat and dog pathogens to solve technical problems such as low detection accuracy and high cost of existing pet pathogen detection methods, or complex and time-consuming detection process, and very high cost of equipment.

[0013] The method includes:

[0014] 1. Collect samples of pathogens to be tested;

[0015] 2. The sample is purified by nucleic acid or directly lysed by lysis buffer;

[0016] 3. Amplification reaction; 3.1. Determine a specific primer set for each pathogen to be detected and design an amplification system;

[0017] 3. Add the sample treated in step 2 and the corresponding primer set to the amplification system for amplification reaction;

[0018] 4. Visual detection: observe whether the color of the reaction solution changes after the amplification reaction. If there is a color change, it means the corresponding pathogen is infected. If there is no color change, it means the corresponding pathogen is not infected.

[0019] Further observation of the scheme shows that although the scheme can detect multiple viruses including feline parvovirus and canine parvovirus at the same time, the scheme also designs different amplification systems for specific fragments of different viruses and integrates multiple amplification systems to achieve simultaneous detection of multiple viruses;

[0020] In summary, although there are kits in the prior art that can detect multiple viruses at the same time, the above kits are all designed with corresponding amplification primers based on the specific fragments of multiple viruses, and because the specific fragments of multiple viruses are quite different, during the detection process, multiple sets of primers and probes will inevitably produce antagonism, thereby causing deviations in the detection results.

[0021] The problem to be solved by the present application is: how to design primers and probes based on fragments with high similarity between FPV and CPV, and to be able to distinguish FPV and CPV during the detection process. Summary of the invention

[0022] The purpose of the present application is to provide a primer set and a probe for rapid identification of feline parvovirus and canine parvovirus. Due to the high similarity between the first gene fragment and the second gene fragment referenced in the design process of the primer set and the probe, the antagonism between the primers and the probe in the present application is small, and effective screening of feline parvovirus and canine parvovirus can still be achieved.

[0023] To achieve the above-mentioned purpose, the present application discloses the use of a primer set and a probe for rapidly distinguishing feline parvovirus from canine parvovirus by using a first gene segment of feline parvovirus and a second gene segment of canine parvovirus;

[0024] The nucleotide sequence of the first gene fragment is shown in SEQ ID NO.1;

[0025] The nucleotide sequence of the second gene fragment is shown as SEQ ID NO.2.

[0026] In addition, the present application also discloses a primer set and a probe for rapid identification of feline parvovirus and canine parvovirus, wherein the primer set comprises a first primer set and a second primer set;

[0027] The first primer set is designed by the first gene fragment of feline parvovirus;

[0028] The second primer set is designed by the second gene segment of canine parvovirus;

[0029] The first primer set includes a first upstream primer and a first downstream primer;

[0030] The second primer set includes a second upstream primer and a second downstream primer;

[0031] At least one set of nucleotide sequences of the first upstream primer and the second upstream primer or the first downstream primer and the second downstream primer are not completely identical;

[0032] The probes include a first probe corresponding to a specific segment in the first gene segment and a second probe corresponding to a specific segment in the second gene segment.

[0033] Preferably, the nucleotide sequences of the first upstream primer and the second upstream primer are not completely the same, and the nucleotide sequences of the first downstream primer and the second downstream primer are the same.

[0034] Preferably, the nucleotide sequence of the first upstream primer is as shown in SEQ ID NO.3;

[0035] The nucleotide sequence of the second upstream primer is shown in SEQ ID NO.4;

[0036] The nucleotide sequence of the first downstream primer is shown in SEQ ID NO.5;

[0037] The nucleotide sequence of the first probe is shown in SEQ ID NO.6;

[0038] The nucleotide sequence of the second probe is shown in SEQ ID NO.7.

[0039] In addition, the present application also discloses a kit for rapid identification of feline parvovirus and canine parvovirus, comprising the primer set and probe for rapid identification of feline parvovirus and canine parvovirus as described above;

[0040] The primer set and the probe are added to the kit in the form of a reaction solution, and the reaction solution also includes 5×MDX140 Mix and sterile water.

[0041] Preferably, the reaction solution specifically includes 4 µL of 5×MDX140 Mix, 1 µL of the first upstream primer, 1 µL of the second upstream primer, 2 µL of the first downstream primer, 0.5 µL of the first probe, 0.5 µL of the second probe, and 10 µL of sterile water.

[0042] Preferably, the kit is composed of a reaction solution, a positive control substance, and a negative control substance. The positive control substance is a dilution of a mixed plasmid of a first plasmid containing a first gene fragment and a second plasmid containing a second gene fragment. The dilution is prepared by diluting the mixed plasmid prepared by the first plasmid and the second plasmid to 10 6 copies / μL, and the mass ratio of the first plasmid to the second plasmid was 1:1;

[0043] The negative control substance is ddH2O.

[0044] Preferably, the kit specifically includes 10 tubes of reaction solution at 19 μL / tube, 1 tube of positive control substance at 50 μL / tube, and 1 tube of negative control substance at 50 μL / tube.

[0045] In addition, the present application also discloses a method for constructing the above-mentioned kit for rapid identification of feline parvovirus and canine parvovirus, comprising the following steps:

[0046] Step 1: Screening to obtain the first gene fragment and the second gene fragment;

[0047] Step 2: designing a first upstream primer, a second upstream primer, and a first downstream primer according to the same nucleotide sequence parts in the first gene fragment and the second gene fragment;

[0048] Step 3: designing a first probe based on the specific fragment in the first gene fragment, and designing a second probe based on the specific fragment in the second gene fragment;

[0049] Step 4: Prepare a reaction solution with the first upstream primer, the second upstream primer, the first downstream primer, the first probe, and the second probe, and mix them with a positive control substance and a negative control substance to obtain a kit for rapid identification of feline parvovirus and canine parvovirus.

[0050] The beneficial effects of the present application are as follows: on the one hand, the primer set and probe of the present application can be used for rapid differential diagnosis of feline parvovirus and canine parvovirus, and the qPCR direct amplification detection method is used to greatly shorten the time required for detection, and the sample is directly added, and only one-step fluorescence observation is required without sequencing, so that the diagnosis of feline parvovirus and canine parvovirus can be quickly differentiated;

[0051] On the other hand, the qPCR method of the present application uses targets with large differences between feline parvovirus and canine parvovirus as specific targets, and adopts the method of combining the amplification refractory mutation system (ARMS) technology with Taqman bi-allelic SNP to detect feline parvovirus and canine parvovirus, which has high specificity;

[0052] And because of the high similarity between the first gene fragment and the second gene fragment referenced in the design process of the primer set and the probe, the antagonism between the primers and probes in the present application is relatively small, and effective screening of feline parvovirus and canine parvovirus can still be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is the fluorescence curve of FAM channel (feline parvovirus);

[0054] Figure 2It is the fluorescence curve of HEX channel (canine parvovirus);

[0055] Figure 3 This is a graph showing specificity test results. DETAILED DESCRIPTION

[0056] The present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. In the description of the present invention, it should be noted that, if no specific conditions are specified in the embodiments, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0057] Before presenting the examples, the information of the experimental consumables such as the seeds, reagents, instruments, etc. used in the examples is described as necessary, and the specific information is shown in Table 1:

[0058] Table 1

[0059] Experimental consumables Supplier Information Experimental consumables Supplier Information Weijiawu (canine distemper, canine adenovirus type 2, canine parainfluenza, canine parvovirus disease) quadruple inactivated vaccine Zoetis (Shanghai) Enterprise Management Co., Ltd. Felocell vaccine (feline panleukopenia, feline calicivirus, herpesvirus) Zoetis (Shanghai) Enterprise Management Co., Ltd. Air-dried feces direct amplification qPCR master mix (MDX140) Beijing Median Biotechnology Co., Ltd. Viral DNA / RNA Extraction Kit Produced by Guangzhou Yueyang Biotechnology Co., Ltd.

[0060] Example 1

[0061] 1.1 Design of primers and probes

[0062] All the whole genome sequences of feline parvovirus and canine parvovirus were downloaded from GenBank, and the whole genome sequences were compared and analyzed by MAFFTversion 7 and MGA7 to find the gene fragments with stable base differences between the two viruses. Probe sequences were designed respectively, and 6 SNP sites with stable base differences between feline parvovirus and canine parvovirus were found, namely A2754G, A2794C, T3214C, T3682C, A4206G, and C4218G. Multiple primer probe sequences were designed according to the gene fragments where the differential SNP sites were located. The primer probes and amplified fragments are shown in Table 2-6 below:

[0063] Table 2: Primer sets and probes designed based on A2754G and A2794C sites

[0064]

[0065] Table 3: Primer sets and probes designed based on A2794C site

[0066]

[0067] Table 4: Primer sets and probes designed based on T3214C ​​site

[0068]

[0069] Table 5: Primer sets and probes designed based on T3682C site

[0070]

[0071] Table 6: Primer sets and probes designed based on A4206G and C4218G sites

[0072]

[0073] Example 2

[0074] Establishment of a single qPCR detection method for feline parvovirus and canine parvovirus

[0075] 2.1 Experimental Materials

[0076] Weijiawu (canine distemper, canine adenovirus type 2, canine parainfluenza, canine parvovirus disease) quadruple inactivated vaccine and Myovirus-3 vaccine (feline distemper, feline calicivirus, herpesvirus) were purchased from Zoetis (Shanghai) Enterprise Management Co., Ltd.; air-dried fecal direct amplification qPCR premix (MDX140) was purchased from Beijing Median Biotechnology Co., Ltd.; viral DNA / RNA extraction kits were produced by our company.

[0077] 2.2 Experimental methods

[0078] 2.2.1 Nucleic acid extraction: Take 200 μl of each of the Vanguard vaccine and the Milbevac vaccine liquid, and use the viral DNA / RNA extraction kit to extract nucleic acid for later use.

[0079] 2.2.2 Establishment of single qPCR detection methods for feline parvovirus and canine parvovirus

[0080] In order to evaluate the specificity of the SNP site probes designed in the first part, 10 sets of primer probes were preliminarily tested, and the following materials were used to establish the basic system of the qPCR method: air-dried fecal direct amplification qPCR premix, MDX140 (Beijing Maidian Biotechnology Co., Ltd.), 5μL, 1μL of upstream and downstream primers, 10μM, 0.5μL of probes, 1μL of Weijiawu vaccine and Miaosanduo vaccine nucleic acid template, and positive samples of feline parvovirus and canine parvovirus, respectively, and deionized water was added to 20μL. Reaction procedure: 95℃ pre-denaturation for 3 min; 95℃10 s, 60℃30 s (fluorescence signal collection), detection using a fluorescence quantitative PCR instrument, using FAM and HEX channels to collect fluorescence signals, and preliminarily established a single qPCR detection system for feline parvovirus and canine parvovirus.

[0081] 2.3 Experimental Results

[0082] Ten systems were tested. System 1 and system 2 can identify feline parvovirus and canine parvovirus vaccine nucleic acids and clinical samples. System 9 and system 10 can identify vaccine strain nucleic acids but cannot identify clinical samples. System 3, system 4, system 5, system 6, system 7, and system 8 cannot identify vaccine strains and clinical samples. The specific system construction and test results are shown in Table 7:

[0083] Table 7: Results of nucleic acid tests on 10 feline parvovirus and canine parvovirus systems and vaccines

[0084] qPCR system Primer probe Ct vaccine Vanguard Vaccine (Ct) Feline Parvovirus Sample Canine Parvovirus Sample System 1 FPV-F1, FC-R1, FPV-T1 16.21 — 26.01 — System 2 CPV-F1, FC-R1, CPV-T1 — 10.57 — 23.21 System 3 FC-F2, FC-R1, FPV-T2 16.56 12.45 26.37 24.08 System 4 FC-F2, FC-R1, CPV-T2 17.84 11.63 28.62 24.24 System 5 FC-F3, FC-R3, FPV-T3 17.65 10.61 26.87 24.69 System 6 FC-F3, FC-R3, CPV-T3 17.43 11.33 26.83 24.17 System 7 FC-F3, FC-R3, FPV-T4 16.65 10.55 27.66 25.28 System 8 FC-F4, FC-R4, CPV-T4 17.36 11.27 26.82 24.29 System 9 FC-F5, FC-R5, FPV-T5 16.86 _ 26.86 24.35 System 10 FC-F5, FC-R5, CPV-T5 _ 10.66 27.36 25.38

[0085] Example 3

[0086] Establishment of a dual fluorescence PCR detection method for feline parvovirus and canine parvovirus

[0087] Based on the preliminary test results of the primer probes in Example 2, the primer probe sequences of System 1 and System 2 that can identify both vaccine strains and clinical samples were selected to establish the third part of the dual fluorescence PCR detection method for feline parvovirus and canine parvovirus.

[0088] 3.1 Synthesis of positive plasmid

[0089] The positive plasmids of feline parvovirus and canine parvovirus in the positive control were delivered to Anhui General Co., Ltd. for synthesis. The specific sequence and vector information are shown in Table 8-9:

[0090] Table 8: Sequence and vector information of FPV positive plasmid construction

[0091]

[0092] Table 9: Sequence and vector information of CPV positive plasmid construction

[0093]

[0094] 3.2 Preparation of dual fluorescence PCR detection system for feline parvovirus and canine parvovirus

[0095] The PCR reaction solution composition formula and amplification parameters of the kits for feline parvovirus and canine parvovirus are shown in Table 10-11:

[0096] Table 10: PCR reaction solution formula for feline parvovirus and canine parvovirus

[0097]

[0098] Table 11: Instrument PCR amplification parameters

[0099]

[0100] 3.3 Sensitivity test of dual fluorescence PCR detection system for feline parvovirus and canine parvovirus

[0101] Dilute the positive plasmids of the two pathogens to 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL, 10 0 The sensitivity test was carried out with 6 concentration gradients of 10 copies / μL.

[0102] refer to Figure 1-2 , from left to right are 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL, 10 0 The amplification results of 100 copies / μL show that the detection sensitivity of the dual fluorescence PCR detection kit of the present application for feline parvovirus and canine parvovirus can reach 10 copies / μL, indicating that the dual fluorescence PCR detection kit and detection method of the present application have high sensitivity for the differential diagnosis of feline parvovirus and canine parvovirus.

[0103] 3.4 Specificity test of dual fluorescence PCR detection system for feline parvovirus and canine parvovirus

[0104] In order to test the specificity of the kit of this application, other pathogenic nucleic acids were tested respectively, and the test results were as follows Figure 3 As shown, only the nucleic acid of feline parvovirus and canine parvovirus samples showed normal amplification, while the negative control (ddH2O) and positive samples such as feline coronavirus, canine coronavirus, and Tritrichomonas fetus did not show amplification. The above results show that the fluorescent PCR detection kit of the present application can specifically amplify feline parvovirus and canine parvovirus without cross-reacting with other pathogenic nucleic acids. This shows that the method and kit of the present application have good specificity and no false negatives occur.

[0105] 3.5 Repeatability test of dual fluorescence PCR detection system for feline parvovirus and canine parvovirus

[0106] The prepared reagents were frozen and thawed five times at 0 day, 4 degrees for 7 days, and -20 degrees for 30 days. Compared with the reagents at 0 day, the amplification sensitivity was basically the same. The test results are shown in Table 12, and the coefficient of variation was less than 2%.

[0107] Table 12: Repeatability test of dual fluorescence PCR detection system for feline parvovirus and canine parvovirus

[0108] Time / pathogen detection 0 days / Ct 3 days / Ct 7 days / Ct 15 days / Ct 30 days / Ct Coefficient of variation FPV (FAM) 32.07 32.05 33.15 32.03 32.24 1.32% CPV (HEX) 31.11 31.19 30.78 31.08 30.82 0.53%

[0109] 3.6 Clinical sample testing

[0110] In order to verify that the primers, probes, and reagents described in this application can be used normally under actual conditions, the primers, probes, and reagents of this application are used to detect actual samples collected from pet hospitals. The double fluorescence PCR detection reagent of this application was used to detect feline parvovirus and canine parvovirus in 10 cat fecal swabs and 10 dog fecal swabs collected from Guangzhou Barbie Hall Animal Hospital, South China Agricultural University Animal Hospital, Lanbo Pet Hospital, etc. The fecal swab samples were directly amplified and nucleic acid was extracted before comparison testing. The negative compliance rate was 100%, and the positive compliance rate was about 90%. Some weakly positive (Ct>35) samples were not detected by direct amplification, but direct amplification does not require nucleic acid extraction, which can save about 30 minutes of extraction time. The test can be completed in less than 1 hour from sampling to the end of the test. The specific test results are shown in Table 13:

[0111] Table 13: Clinical sample test results

[0112]

[0113] In summary, the dual fluorescence PCR detection system for feline parvovirus and canine parvovirus of the present application has the advantages of rapid, real-time, sensitive and accurate identification of feline parvovirus and canine parvovirus, and provides a rapid and accurate method for clinical accurate identification and detection of pathogens of feline and canine diarrhea.

[0114] 3.7. A kit for rapid identification of feline parvovirus and canine parvovirus

[0115] A kit for rapid identification of feline parvovirus and canine parvovirus, comprising an FPV / CPV reaction solution, a positive control substance, and a negative control substance. The FPV / CPV reaction solution is prepared according to the third formula table and packaged in 19 μl / tube. The positive control substance is a mixed plasmid containing a positive plasmid containing a gene fragment region of feline parvovirus and canine parvovirus, which is diluted to 10 6 copies / μL, and the mass ratio of the first plasmid to the second plasmid is 1:1, the negative control is dd H2O, and the main components and contents of the kit are shown in Table 14:

[0116] Table 14: Main components and contents of the kit

[0117] Serial number Element content 1 FPV / CPV reaction solution 10 tubes (19 μL / tube) 2 FPV / CPV positive control 1 tube (50 μL / tube) 3 Negative Control 1 tube (50 μL / tube) 4 manual 1 serving

[0118] The procedure in the kit is as follows:

[0119] Sample processing and PCR amplification

[0120] Use cotton swabs to collect dog and cat fecal swab samples, put them into EP tubes filled with physiological saline, stir and elute, centrifuge at 12000r for 2min, take 1 microliter of supernatant and add it to FPV / CPV reaction solution, mark and place it in the corresponding position of the fluorescent PCR sample slot, record the sequence, set the amplification parameters according to Table 15, and perform PCR amplification. The PCR amplification parameters are shown in Table 15:

[0121] Table 15: PCR amplification parameters

[0122]

[0123] Result judgment:

[0124] Reasonably adjust the threshold line. Different instruments can be adjusted according to the instrument noise conditions.

[0125] Test kit validity determination

[0126] Negative control: no Ct value, the line shape is straight or slightly inclined, and there is no obvious exponential growth.

[0127] Positive control: Ct values ​​of FAM (parvovirus) and HEX (canine parvovirus) channels were ≤30, with obvious exponential growth and a typical S-shaped curve.

[0128] Sample result determination

[0129] Positive: The Ct value of the detection channel is ≤38, and the curve has an obvious exponential growth curve.

[0130] Suspicious: If the detection channel is 38<Ct value≤45, it is recommended to repeat the test. If the repeated test result is still 38<Ct value≤45 and there is an obvious amplification curve, it is positive, otherwise it is negative.

[0131] Negative: The sample test result has no Ct value and no obvious amplification curve.

Claims

1. A primer set and probe for rapid identification of feline parvovirus and canine parvovirus, characterized in that: The primer set includes a first primer set and a second primer set; The first primer set is designed by the first gene fragment of feline parvovirus, and the nucleotide sequence of the first gene fragment is shown in SEQ ID NO.1; The second primer set is designed by the second gene fragment of canine parvovirus, and the nucleotide sequence of the second gene fragment is shown in SEQ ID NO.2; The first primer set includes a first upstream primer and a first downstream primer; The second primer set includes a second upstream primer and a second downstream primer; The nucleotide sequence in the first downstream primer is completely identical to that in the second downstream primer; The nucleotide sequence of the first upstream primer is shown in SEQ ID NO.3; The nucleotide sequence of the second upstream primer is shown in SEQ ID NO.4; The nucleotide sequence of the first downstream primer is shown in SEQ ID NO.5; The probes include a first probe corresponding to a specific segment in the first gene segment and a second probe corresponding to a specific segment in the second gene segment; The nucleotide sequence of the first probe is shown in SEQ ID NO.6; The nucleotide sequence of the second probe is shown in SEQ ID NO.

7.

2. A kit for rapid identification of feline parvovirus and canine parvovirus, characterized in that: Containing the primer set and probe for rapid identification of feline parvovirus and canine parvovirus as described in claim 1; The primer set and the probe are added to the kit in the form of a reaction solution, and the reaction solution also includes 5×MDX140Mix and sterile water.

3. The kit for rapid identification of feline parvovirus and canine parvovirus according to claim 2, characterized in that: The reaction solution specifically includes 4 µL of 5×MDX140 Mix, 1 µL of the first upstream primer, 1 µL of the second upstream primer, 2 µL of the first downstream primer, 0.5 µL of the first probe, 0.5 µL of the second probe, and 10 µL of sterile water.

4. The kit for rapid identification of feline parvovirus and canine parvovirus according to claim 2, characterized in that: The kit is composed of a reaction solution, a positive control substance, and a negative control substance. The positive control substance is a dilution of a mixed plasmid of a first plasmid containing a first gene fragment and a second plasmid containing a second gene fragment. The dilution is prepared by diluting the mixed plasmid prepared by the first plasmid and the second plasmid to 10 6 copies / μL, and the mass ratio of the first plasmid to the second plasmid was 1:1; The negative control substance is ddH2O.

5. The kit for rapid identification of feline parvovirus and canine parvovirus according to claim 4, characterized in that: The kit specifically includes 10 tubes of reaction solution at 19 μL / tube, 1 tube of positive control substance at 50 μL / tube, and 1 tube of negative control substance at 50 μL / tube.

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