Kit for detecting FCV and FHV and application

Abstract

The invention relates to a kit for detecting FCV and FHV and application, the kit comprises: a PCR reaction liquid, the PCR reaction liquid comprises an FCV primer probe set, an FHV primer probe set, an enzyme, a stabilizer and water; the nucleic acid release liquid comprises a surfactant, an alkaline agent, a chelating agent and a splitting enhancer; wherein the FCV comprises a forward primer FCVF as shown in SEQ ID NO: 1 and a reverse primer FCVR as shown in SEQ ID NO: 2; the FHV primer probe group comprises a forward primer FHVF and a reverse primer FHVR as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and the forward primer FHVF and the reverse primer FHVR are shown in SEQ ID NO: 4 and SEQ ID NO: 5. The kit can be used for rapidly, conveniently and accurately detecting the feline cup-shaped herpes virus type I and the feline herpes virus type I, so that the kit can be applied to detection of FCV and FHV.

Description

Technical Field

[0001] The present application relates to a kit for detecting FCV and FHV and its application, belonging to the field of nucleic acid molecule detection. Background Art

[0002] Feline Calicivirus (FCV) and Feline Herpesvirus type 1 (FHV-1) are the main pathogens that cause respiratory diseases in cats and are viruses that cause upper respiratory tract infections (URIs) in cats.

[0003] In traditional detection methods, when it comes to nucleic acid extraction, most of them use magnetic bead extraction and column extraction. These processes are cumbersome and time-consuming. In the process of a large number of mechanical operations, it is easy to produce more nucleic acid fragments and aerosol pollution. At the same time, this method places high demands on detection reagents, detection equipment and detection personnel, and pet hospitals need to be tested on time and require rapid results. Therefore, the current detection methods are not adapted to the needs of terminal scenarios such as pet hospitals.

[0004] In addition, in terms of sample addition, the conventional method is to use a pipette for sample addition, but pet hospital staff are not professionally trained and are prone to using the pipette directly without adjusting the volume, which leads to inaccurate sample addition and experimental failure. Moreover, when using a pipette, the tips are mostly provided in boxes, and pet hospitals cannot take and close the tips at will to keep them clean, which can easily cause contamination.

[0005] Based on the above situation, a new method for detecting feline calicivirus and feline herpesvirus type I is urgently needed to provide technical support for clinical diagnosis and treatment. Summary of the invention

[0006] The purpose of the present application is to provide a kit and application for detecting FCV and FHV, which can quickly, conveniently and accurately detect feline calicivirus and feline herpesvirus type I.

[0007] In order to achieve the above objectives, this application provides the following technical solutions: In a first aspect, the present application provides a kit for detecting FCV and FHV, comprising: A PCR reaction solution, wherein the PCR reaction solution comprises an FCV primer probe set, an FHV primer probe set, an enzyme, a stabilizer and water; A nucleic acid releasing solution, comprising a surfactant, an alkali agent, a chelating agent and a lysis enhancer; Among them, the FCV primer probe set includes the forward primer FCVF and the reverse primer FCVR as shown in SEQ ID NO:1 and SEQ ID NO:2, and the probe FCVP; the FHV primer probe set includes the forward primer FHVF and the reverse primer FHVR as shown in SEQ ID NO:4 and SEQ ID NO:5, and the probe FHVP.

[0008] In one or more feasible embodiments, the sequence of the FCVP is as shown in SEQ ID NO:3.

[0009] In one or more feasible embodiments, the sequence of the FHVP is as shown in SEQ ID NO:6.

[0010] In one or more feasible embodiments, the PCR reaction solution also includes an internal standard primer probe group, which includes a cat housekeeping gene forward primer IF as shown in SEQ ID NO:7, a cat housekeeping gene reverse primer IR as shown in SEQ ID NO:8, and a cat housekeeping gene probe primer IP as shown in SEQ ID NO:9.

[0011] In one or more feasible embodiments, the 5' end and 3' end of the FCVP, FHVP and IP are respectively labeled with a fluorescent group and a quenching group, and the fluorescent groups labeled with the FCVP, FHVP and IP are different.

[0012] In one or more feasible embodiments, the fluorescent group can be any one of HEX, FAM, TET, CF532, JOE, TAMRA, ROX, CY3, CY5, Texas Red, NED, Alexa Flour or VIC.

[0013] In one or more feasible embodiments, the quenching group may be any one of MGB, TAMRA, BHQ1, BHQ2, BHQ3 or QSY4.

[0014] In one or more feasible embodiments, the enzyme includes a DNA polymerase, a thermosensitive reverse transcriptase, and an anti-pollution enzyme.

[0015] In one or more feasible embodiments, the stabilizer is selected from any one or more of Tween 20, glycerol, BSA, ammonium sulfate and DTT.

[0016] In one or more feasible embodiments, the PCR reaction solution includes 10-200 mM Tris-HCL, 10-100 mM KCl, 1-6 mM MgCL2, 0.2-0.6 mM dNTPs, 0.1-0.8 µm FCVF / FCVR / FCVP / FHVF / FHVR / FHVP, 0.05-0.4 µm IF / IR / IP, 0.1%-1% Tween 20, 1-5% glycerol, 0.05-1% BSA, 1-20 mM ammonium sulfate, 1-5 mM DTT, 0.01-0.1% Proclin 300, 0.5U-4U DNA polymerase, 20-100U thermosensitive reverse transcriptase, UDG enzyme and DEPC water.

[0017] In one or more feasible embodiments, the surfactant is selected from sodium dodecylbenzene sulfonate, CHAPS or a combination thereof.

[0018] In one or more feasible embodiments, the alkaline agent is sodium hydroxide.

[0019] In one or more feasible embodiments, the chelating agent is disodium ethylenediaminetetraacetate.

[0020] In one or more feasible embodiments, the lysis enhancer includes sodium tripolyphosphate and aminotriacetic acid.

[0021] In one or more feasible embodiments, the nucleic acid releasing solution includes 1-100 mM potassium chloride, 0.1-1% aminotriacetic acid, 0.01%-2% 3-[3-(cholanidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.05-1M sodium hydroxide, 0.05%-2% sodium tripolyphosphate, 0.01-1% sodium dodecylbenzenesulfonate and 1-50 mM disodium ethylenediaminetetraacetate.

[0022] In one or more feasible embodiments, the kit is used for rapid fluorescent PCR reaction.

[0023] In one or more feasible embodiments, the kit further comprises a disposable sample loading ring having a sampling end and a hand-held end, and a radial dimension of the disposable sample loading ring gradually decreases from the hand-held section to the sampling end.

[0024] In a second aspect, the present application provides use of the kit according to the first aspect in detecting FCV and FHV.

[0025] In one or more feasible embodiments, the application includes preparing a rapid PCR nucleic acid amplification reaction tube.

[0026] In one or more possible embodiments, the application includes using a disposable sample loading loop for loading the sample, wherein the disposable sample loading loop has a sampling end and a hand-held end.

[0027] In one or more feasible embodiments, the radial dimension of the disposable sample adding ring gradually decreases from the handheld section to the sampling end.

[0028] Through the implementation methods of this application, the beneficial effects of this application are: In the kit of the present application, the nucleic acid release solution used does not require magnetic bead extraction, high temperature lysis, column centrifugation and other complex operations. The designed primer probe set has few dimers and is used in combination with a thermosensitive reverse transcriptase and a double-antibody DNA polymerase, which plays an important role in the stability of the reaction solution. At the same time, a stabilizer is configured in the PCR reaction solution to improve the room temperature stability and 2~8°C stability of the fully premixed reaction solution, saving solution preparation time and reducing losses caused by incorrect storage and operation.

[0029] That is, the test kit provided by this application has strong specificity, wide coverage of cat cup and herpes detection, and higher sensitivity than the group standard: 20 copies / test. At the same time, the use of the test kit of this application for rapid PCR reaction is not only easy to operate, but also has an anti-pollution system and sample loop error prevention facilities. More importantly, the application is time-saving, with a total of 45 minutes; low cost, high throughput, and multiple application scenarios. It not only meets the use of hospitals, but can also be used for catteries, pet entry and exit detection, etc.

[0030] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present application in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of PCR detection of feline calicivirus shown in one of the embodiments of the present application; Figure 2 A schematic diagram of PCR detection of feline herpesvirus type I shown in one of the embodiments of the present application; Figure 3 This is a schematic diagram of the structure of a disposable sample adding ring shown in one of the embodiments of the present application. DETAILED DESCRIPTION

[0032] The following describes the embodiments of the present application through specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the contents disclosed in this specification. The present application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that the following embodiments and features in the embodiments can be combined with each other without conflict.

[0033] It should be noted that the illustrations provided in the following embodiments are only used to illustrate the basic concept of the present application in a schematic manner, and therefore the illustrations only show components related to the present application rather than being drawn according to the number, shape and size of components in actual implementation. In actual implementation, the type, quantity and proportion of each component may be changed arbitrarily, and the component layout may also be more complicated.

[0034] Some exemplary embodiments of the present application are described for the purpose of explanation, and it should be understood that the present application may be implemented in other ways not specifically shown in the drawings.

[0035] The present application provides a kit for detecting FCV and FHV, which is mainly used for rapid PCR reaction, and comprises: A PCR reaction solution, wherein the PCR reaction solution comprises an FCV primer probe set, an FHV primer probe set, an enzyme, a stabilizer and water; A nucleic acid releasing solution, comprising a surfactant, an alkali agent, a chelating agent and a lysis enhancer; Among them, the FCV primer probe set includes the forward primer FCVF and the reverse primer FCVR as shown in SEQ ID NO:1 and SEQ ID NO:2, and the probe FCVP; the FHV primer probe set includes the forward primer FHVF and the reverse primer FHVR as shown in SEQ ID NO:4 and SEQ ID NO:5, and the probe FHVP.

[0036] In at least one embodiment of the present application, the sequence of the FCVP is shown as SEQ ID NO:3, and the sequence of the FHVP is shown as SEQ ID NO:6.

[0037] As an example but not a limitation, the PCR reaction solution also includes an internal standard primer probe group, which includes a cat housekeeping gene forward primer IF as shown in SEQ ID NO:7, a cat housekeeping gene reverse primer IR as shown in SEQ ID NO:8, and a cat housekeeping gene probe primer IP as shown in SEQ ID NO:9.

[0038] The applicant found through comparison that the use of the above three sets of primer probe sets reduced the dimer at the 3' end, effectively prevented the generation of non-specificity, and played an important role in the stability of the system.

[0039] It is understood by those skilled in the art that the 5' end and 3' end of FCVP, FHVP and IP are respectively labeled with a fluorescent group and a quenching group, and the fluorescent groups labeled by the FCVP, FHVP and IP are different. For example, the fluorescent group can be any one of HEX, FAM, TET, CF532, JOE, TAMRA, ROX, CY3, CY5, Texas Red, NED, Alexa Flour or VIC. The quenching group can be any one of MGB, TAMRA, BHQ1, BHQ2, BHQ3 or QSY4.

[0040] In one embodiment of the present application, the 5' end of the FCVP probe is labeled with a FAM fluorescent group and the 3' end is labeled with an MGB quencher group, the 5' end of the FHVP probe is labeled with a VIC fluorescent group and the 3' end is labeled with a BHQ1 quencher group, and the 5' end of the IP primer is labeled with a roX fluorescent group and the 3' end is labeled with a BHQ2 quencher gene.

[0041] As an example but not a limitation, the enzymes in the PCR reaction solution include DNA polymerase, thermosensitive reverse transcriptase and anti-contamination enzyme. The added stabilizer is selected from any one or more of Tween 20, glycerol, BSA, ammonium sulfate and DTT.

[0042] In at least one embodiment of the present application, the PCR reaction solution includes 10-200 mM Tris-HCL, 10-100 mM KCl, 1-6 mM MgCL2, 0.2-0.6 mM dNTPs, 0.1-0.8 µm FCVF / FCVR / FCVP / FHVF / FHVR / FHVP, 0.05-0.4 µm IF / IR / IP, 0.1%-1% Tween 20, 1-5% glycerol, 0.05-1% BSA, 1-20 mM ammonium sulfate, 1-5 mM DTT, 0.01-0.1% Proclin 300, 0.5U-4U DNA polymerase, 20-100U thermosensitive reverse transcriptase, UDG enzyme and DEPC water.

[0043] As an example but not a limitation, the surfactant is selected from sodium dodecylbenzene sulfonate, CHAPS or a combination thereof. For example, the alkali agent is sodium hydroxide, the chelating agent is disodium ethylenediaminetetraacetic acid, and the cleavage enhancer includes sodium tripolyphosphate and aminotriacetic acid.

[0044] In at least one embodiment of the present application, the nucleic acid releasing solution includes 1-100 mM potassium chloride, 0.1-1% aminotriacetic acid, 0.01%-2% CHAPS, 0.05-1M sodium hydroxide, 0.05%-2% sodium tripolyphosphate, 0.01-1% sodium dodecylbenzene sulfonate and 1-50 mM disodium ethylenediaminetetraacetic acid.

[0045] Most products on the market store the enzyme solution and reaction solution separately, or provide the reaction solution in a freeze-dried form. This solves the transportation and storage problems on the one hand, and reduces the dimer reaction that renders the reagent ineffective on the other. However, an additional mixing or re-dissolution step is required for terminal use, which increases the number of operation steps. For pet hospital staff, professional PCR training has not been received, which invisibly increases the possibility of contamination and operational errors.

[0046] The above-mentioned PCR reaction solution is a fully premixed reagent, which can be stable for 3 days at room temperature and 7 months at 2-8°C, saving time in liquid preparation and reducing losses caused by incorrect storage and operation, meeting the needs of end customers.

[0047] The applicant found that the above-mentioned kit can be widely used in the detection of FCV and FHV. Specifically, the sample to be tested is added to the nucleic acid release solution of the above-mentioned kit, and then the sample to be tested in the nucleic acid release solution is transferred to the PCR reaction solution using a sampling tool, and then a rapid PCR reaction is performed. Finally, the positive and negative results are judged according to the Ct value: the internal standard channel, that is, the ROX channel, must have an obvious amplification curve. If the FAM channel has an obvious amplification curve, it is judged as cat cup positive; if the VIC channel has an obvious amplification curve, it is judged as cat herpes positive.

[0048] In terms of sample addition, pipettes are usually used for sample addition. However, pet hospital staff are not professionally trained and may use pipettes directly without adjusting the volume, resulting in inaccurate sample addition and experimental failure. In addition, when using pipettes, the tips are mostly provided in boxes. Pet hospitals rarely take and close the tips to keep them clean, which is prone to contamination.

[0049] In at least one embodiment of the present application, the sampling tool is a disposable sampling ring.

[0050] The present application will be further described in detail below in conjunction with specific embodiments.

[0051] PCR reaction solution The plasmids constructed with the conservative sequences of feline calix and feline herpes genes were quantified using Qubit and diluted to 2000 copies / μL. 2 μL of FCV / FHV plasmid was added to the reaction solution. The reaction solution conditions were shown in Table 1. In addition, 0.1 mM dNTPs, 2.5 U DNA polymerase, 20 U thermosensitive reverse transcriptase, and 1 U thermosensitive UNG enzyme were added. The concentrations of FCVF / FCVR / FCVP were 250 nM, FHVF / FHVR / FHVP were 250 nM, and IF / IR / IP were 150 nM. DEPC water was used to make up the final volume to 18 μL.

[0052] Table 1 Serial number Tris-HCl pH 8.5 KCl <![CDATA[MgCl2]]> 1 100 mM 50 mM 3 mM 2 150 mM 50 mM 3 mM 3 200 mM 50 mM 3 mM 4 150 mM 100 mM 3 mM 5 150 mM 200 mM 3 mM 6 150 mM 50 mM 4 mM 7 150 mM 50 mM 5 mM The fluorescence amplification curves of the PCR reaction solution systems of FCV and FHV are shown in Figure 1 and Figure 2 As shown in the figure, it can be seen that the PCR reaction solution system of group 7 has the best application effect in FCV and FHV detection.

[0053] Clinical sample testing Since feline calicivirus is an RNA virus with a very high mutation rate, clinical samples were used to verify whether the selected primer probes can well identify real samples. The primers in the control group refer to the group standard T / CVMA 38-2020 issued by the Chinese Veterinary Association.

[0054] Samples: Eye, mouth and nose swabs were collected from 20 cats with respiratory symptoms from the pet hospital, with 2 swabs collected for each sample.

[0055] The experimental group used a one-step release method, and 20 eye, mouth and nose swabs were directly inserted into 1 mL of release solution, the swabs were broken, the cotton head was left in the release solution, the lid was screwed on, and it was shaken up and down. After leaving it at room temperature for 3 minutes, 2 μL was added to the reaction solution and tested on the machine. The release solution components are: 100 mM potassium chloride, 0.5% aminotriacetic acid, 0.05% 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt, 0.1 M sodium hydroxide, 0.1% sodium tripolyphosphate, 0.05% sodium dodecylbenzenesulfonate and 5 mM disodium ethylenediaminetetraacetic acid solution.

[0056] The control group used the magnetic bead method. First, 20 swab samples were soaked in 1 ml of saline for 5 minutes, and then 200 μL was taken for extraction. After elution with 200 μL of DEPC water, 2 μL was added to the reaction solution and tested on the machine. The results are shown in Table 2.

[0057] Table 2 Sample No. Experimental group - cup Control group - cup 1 Undetect Undetect 2 33.8 36.1 3 25.8 27.6 4 34.3 36.5 5 35.2 36.83 6 Undetect Undetect 7 27.9 26.3 8 35.5 Undetect 9 31.2 30.4 10 30.1 36.5 11 Undetect Undetect 12 34.0 35.2 13 Undetect Undetect 14 Undetect Undetect 15 27.1 34.2 16 22.9 27.9 17 Undetect Undetect 18 34.9 38.8 19 Undetect Undetect 20 Undetect Undetect As shown in Table 2, the positive rates of the primers in the experimental group and the control group are basically the same, and the Ct values ​​are also at the front. On the one hand, it shows that the primer set selected by the experimental group can well identify cat cup primers, and on the other hand, it also shows that the sensitivity of the one-step release method is higher.

[0058] Rapid PCR reaction procedure Select cat cup clinical positive samples with Ct of about 30 as cup program screening samples, select FCV / FHV plasmid 40 copies / μL 2μL as feline herpes program screening samples, use the program in Table 3 for amplification verification, the number of cycles is 40, and each program is repeated 10 times.

[0059] Table 3

[0060] The cup-shaped results are shown in Table 4. It can be seen from Table 4 that the best result was obtained using the conditions of Program 1, Programs 2 and 4 were basically the same, and Programs 3 and 5 were slightly worse, indicating that the reverse transcription process was better when it was more than 3 minutes.

[0061] The herpes results are shown in Table 5. It can be seen from Table 5 that except for the slightly delayed Ct of Program 3, the other groups are basically the same, indicating that the extension time controlled at more than 15s can meet our needs. Combining the experimental results of the cup and herpes groups, the relatively short program 2 is selected to meet the market demand without affecting the judgment of the experimental results.

[0062] Table 4 Serial number Procedure 1 Procedure 2 Procedure 3 Procedure 4 Procedure 5 1 30.26 31.36 31.34 31.47 32.13 2 30.27 32.13 32 31.50 34.62 3 31.11 32.03 31.08 30.66 32.90 4 31.13 31.8 34.28 31.30 32.00 5 30.02 31.2 32.61 32.17 33.33 6 31.22 31.04 32.17 31.90 33.14 7 31.44 31.9 33.38 32.61 34.82 8 31.21 31.09 32.21 32.01 32.87 9 30.25 32.17 34.30 32.48 33.27 10 31.25 31.15 32.17 31.11 32.10 Mean 30.81 31.58 32.55 31.72 33.11 CV 1.76% 1.46% 3.41% 1.96% 2.95% Table 5 Serial number Procedure 1 Procedure 2 Procedure 3 Procedure 4 Procedure 5 1 34.14 33.82 35.72 34.59 35.41 2 33.53 33.49 34.7 35.24 35.97 3 33.54 34.92 35.7 34.45 34.46 4 34.01 34.46 34.68 35.15 34.85 5 33.52 34.01 35.62 34.14 34.19 6 33.47 34.87 36.03 34.56 34.35 7 34.08 33.49 34.8 34.32 33.91 8 33.84 34.53 34.79 35.29 35.35 9 34.43 34.08 35.12 34.06 34.50 10 33.84 34.8 36.46 35.2 34.12 Mean 33.84 34.247 35.362 34.7 34.711 CV 0.96% 1.59% 1.78% 1.38% 1.92% Sensitivity verification of disposable sample loop

[0063] The FCV / FHV plasmid was diluted to 10 copies / μL as a sensitivity reference, and the experimental group used a disposable sample loop for sample loading. The disposable sample loop structure is as follows Figure 3 It has a sampling end and a hand-held end. The radial size of the disposable sampling ring gradually decreases from the hand-held section to the sampling end, and the sampling end has an annular structure with a through hole in the middle to facilitate the passage of liquid.

[0064] The control group was a 2 μL sample pipetted with a pipette. This was repeated 20 times. The results are shown in Table 6. All the results were positive, indicating that the sensitivity of the sample loop was comparable to that of the pipette.

[0065] Table 6

[0066] Stabilizer

[0067] The PCR reaction solution additives were prepared as shown in Table 7, and the fully premixed reaction solution was stored at 2~8℃ and 25℃ respectively. The sample was a moderately strong positive clinical sample, which was lysed by a one-step release solution, and the test was repeated 3 times to take the average value. The control group was without additives, and the enzyme solution and the reaction solution were stored separately at -20℃. When testing, the reaction solution was mixed and used according to 17.4 μL+0.6 μL enzyme solution. The results are shown in Table 8. Formulas 3 and 4 are relatively stable, but Formula 4 initially showed a Ct delay compared with the control group.

[0068] Table 7 Serial number glycerin Tween20 BSA Ammonium sulfate DTT Proclin300 1 0% 0% 0% 0mM 0mM 0% 2 2% 0.05% 0.05% 10mM 1mM 0.05% 3 4% 0.1% 0.1% 20mM 5mM 0.05% 4 8% 0.5% 0.5% 50mM 10mM 0.05% Table 8

[0069] Based on the above embodiments, in terms of the kit, the nucleic acid release solution provided in the present application does not require magnetic bead extraction, high-temperature lysis, column centrifugation and other complex operations, and can release viral nucleic acids very well. The whole process takes only 5 minutes or even less, is easy to operate, does not require professional training, and reduces the contamination of nucleic acid fragments.

[0070] In addition, according to the embodiments of the present application, three groups of primer probes are preferably selected in the present application, and primer probes for monitoring the cat housekeeper gene are added, which can well monitor the sampling and amplification process; and through different combinations, combinations with fewer dimers are screened out, and the use of thermosensitive reverse transcriptase and double-antibody DNA polymerase plays an important role in the stability of the reaction solution.

[0071] At the same time, this application optimizes the additives of the reaction solution, so that the room temperature stability and 2~8℃ stability of the fully premixed reaction solution are improved, which saves the time of liquid preparation and reduces the losses caused by incorrect storage and operation, meeting the needs of end customers. The use of the UNG enzyme system can effectively eliminate the pollution of products in the environment, which is more suitable for the use scenarios of small pet hospitals.

[0072] In terms of operation, this application uses a disposable sample loading loop to simplify the loading process, thereby reducing the possibility of incorrect operation and reducing contamination. It also combines nucleic acid release liquid, rapid PCR program and disposable sample loading loop, which is easy to operate and takes about 45 minutes, meeting the needs of terminal hospitals.

[0073] In summary: The kit provided in this application has strong specificity, wide coverage of cat cup and herpes detection, and higher sensitivity than the group standard: 20 copies / test. At the same time, the use of the kit of this application for rapid PCR reaction is not only easy to operate, but also has an anti-pollution system and sample loop error prevention facilities. More importantly, the application is time-saving, with a total of 45 minutes; low cost, high throughput, and multiple application scenarios. It not only meets the use of hospitals, but can also be used for catteries, pet entry and exit detection, etc.

[0074] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0075] The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be construed as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent application shall be subject to the attached claims.

Claims

1. A kit for detecting FCV and FHV, characterized in that, include: A PCR reaction solution, wherein the PCR reaction solution comprises an FCV primer probe set, an FHV primer probe set, an enzyme, a stabilizer and water; A nucleic acid releasing solution, comprising a surfactant, an alkali agent, a chelating agent and a lysis enhancer; Among them, the FCV primer probe set includes the forward primer FCVF and the reverse primer FCVR as shown in SEQ ID NO:1 and SEQ ID NO:2, and the probe FCVP; the FHV primer probe set includes the forward primer FHVF and the reverse primer FHVR as shown in SEQ ID NO:4 and SEQ ID NO:5, and the probe FHVP.

2. The kit according to claim 1, characterized in that: The sequence of the FCVP is shown in SEQ ID NO: 3; and / or, The sequence of the FHVP is shown in SEQ ID NO:

6.

3. The kit according to claim 1 or 2, characterized in that The PCR reaction solution also includes an internal standard primer probe group, which includes a cat housekeeping gene forward primer IF as shown in SEQ ID NO:7, a cat housekeeping gene reverse primer IR as shown in SEQ ID NO:8, and a cat housekeeping gene probe primer IP as shown in SEQ ID NO:

9.

4. The kit according to claim 3, characterized in that The 5' end and 3' end of the FCVP, FHVP and IP are respectively labeled with a fluorescent group and a quenching group, and the fluorescent groups labeled with the FCVP, FHVP and IP are different.

5. The kit according to claim 1 or 2, characterized in that The enzymes include DNA polymerase, thermosensitive reverse transcriptase and anti-pollution enzyme; and / or, The stabilizer is selected from any one or more of Tween 20, glycerol, BSA, ammonium sulfate and DTT.

6. The kit according to claim 1, characterized in that The surfactant is selected from sodium dodecylbenzene sulfonate, CHAPS or a combination thereof; and / or, The alkaline agent is sodium hydroxide; and / or, The chelating agent is disodium ethylenediaminetetraacetate; and / or, The lysis enhancer includes sodium tripolyphosphate and aminotriacetic acid.

7. The kit according to claim 1 or 6, characterized in that The nucleic acid release solution includes 1-100 mM potassium chloride, 0.1-1% aminotriacetic acid, 0.01%-2% CHAPS, 0.05-1M sodium hydroxide, 0.05%-2% sodium tripolyphosphate, 0.01-1% sodium dodecylbenzene sulfonate and 1-50 mM disodium ethylenediaminetetraacetate.

8. The kit according to claim 1, wherein The kit is used for rapid fluorescent PCR reaction; the kit also includes a disposable sample loading ring, the disposable sample loading ring has a sampling end and a hand-held end, and the radial size of the disposable sample loading ring gradually decreases from the hand-held section to the sampling end.

9. Use of the kit according to any one of claims 1 to 8 in detecting FCV and FHV.

10. The use according to claim 9, characterized in that The application includes preparing a rapid PCR nucleic acid amplification reaction tube.

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