Primer and kit for rapidly detecting canine respiratory tract pathogen and preparation method

Abstract

The invention discloses primers and a kit for rapidly detecting canine respiratory tract pathogens and a preparation method, and belongs to the technical field of biology. The kit comprises five groups of specific primers for rapidly detecting the canine respiratory pathogens, can rapidly and accurately detect the five respiratory pathogens of canine parainfluenza virus, canine distemper virus, canine adenovirus type II, canine crown virus and canine bordetella within 15 minutes, and is low in detection cost and low in detection environment requirement. The kit can be applied to clinical rapid diagnosis and on-site screening to timely diagnose infection, so that veterinarians can rapidly take effective treatment measures; the technical problems of short time consumption, high accuracy, low cost and simple operation cannot be simultaneously considered in the detection process in the prior art are solved.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology and relates to a primer, a kit and a preparation method for rapid detection of canine respiratory pathogens. Background Art

[0002] Dogs are loyal companions to humans, providing emotional comfort and companionship. Good physical condition helps dogs maintain good mood and behavior, and prolongs their lifespan. Canine respiratory diseases are common throughout the year, and in severe cases, they may develop into fatal pneumonia. Biological factors are the main causes of canine respiratory diseases: 1) Canine distemper virus (CDV) is an acute, highly contagious viral disease of dogs worldwide. It is easy to be mixed or secondary infected with bacteria and other viruses, with an incidence rate of up to 80%. 2) Canine adenovirus type II (CVA) can cause infectious laryngotracheitis and pneumonia symptoms in dogs. 3) Canine parainfluenza virus (CPIV) is the main respiratory infectious disease of dogs infected with canine parainfluenza virus; clinical manifestations include fever, cough, runny nose and other symptoms. 4) Bordetella bronchiseptica (Bb) is a gram-negative bacterium. In the late stage of the disease, dogs have difficulty breathing, sit in a dog-like posture, and have a poor appetite. 5) Canine coronavirus (CCV) can cause frequent vomiting, diarrhea, depression, anorexia and other symptoms in dogs. This disease can occur all year round; the virus is often mixed with canine parvovirus, rotavirus and other gastrointestinal diseases.

[0003] Timely diagnosis of infection helps veterinarians to quickly adopt effective treatment measures. The clinical manifestations of the above five canine respiratory infectious diseases are very similar and difficult to distinguish.

[0004] The mainstream methods for detecting canine respiratory tract in the market are qPCR and colloidal gold immunochromatography. The qPCR method has high detection sensitivity, but requires professional instruments and reagents, the equipment cost is high, the sample pre-treatment steps are complicated, there are certain requirements for the operator, and the detection time is relatively long, taking more than 90 minutes, which is not suitable for clinical rapid diagnosis and on-site screening. The colloidal gold immunochromatography method is simple and fast to operate, but the accuracy is low and it is easy to cause misjudgment.

[0005] The LAMP constant temperature amplification method has high sensitivity and can detect trace amounts of nucleic acid. The reaction process is simple and does not require professional instruments. It can be used for simple on-site or home testing. The reaction time is short and results are usually available within 30 minutes, making it suitable for rapid on-site testing.

[0006] However, the LAMP isothermal amplification method has high requirements for primer design; and the detection speed of the LAMP isothermal amplification method still needs to be improved.

[0007] Therefore, there is a need for a rapid, accurate and low-cost detection method suitable for rapid clinical diagnosis and on-site screening of pathogens. Summary of the invention

[0008] In order to solve the above problems, a primer, a kit and a preparation method for rapid detection of canine respiratory pathogens are provided. The kit of this application contains 5 sets of specific primers for rapid detection of canine respiratory pathogens, which can quickly and accurately detect five respiratory pathogens, namely canine parainfluenza virus, canine distemper virus, canine adenovirus type II, canine coronavirus and canine Bordetella within 15 minutes, and the detection cost is low and the detection environment requirements are low. It can be used for clinical rapid diagnosis and on-site screening to promptly diagnose infection, so that veterinarians can quickly adopt effective treatment measures; it solves the technical problem that the detection process in the prior art cannot take into account short time, high accuracy, low cost and simple operation at the same time.

[0009] The specific technical solutions of this application are as follows: A primer for rapid detection of canine respiratory pathogens, the primer comprising one or more of the following five groups, each group comprising inner primers FIP / BIP, outer primers F3 / B3 and loop primers LF / LB, the specific sequences are as follows: Canine parainfluenza virus group primers: FIP is shown in SEQ ID No. 1, BIP is shown in SEQ ID No. 2, F3 is shown in SEQ ID No. 3, B3 is shown in SEQ ID No. 4, LF is shown in SEQ ID No. 5, and LB is shown in SEQ ID No. 6; Canine distemper virus group primers: FIP is shown as SEQ ID No. 7, BIP is shown as SEQ ID No. 8, F3 is shown as SEQ ID No. 9, B3 is shown as SEQ ID No. 10, and LB is shown as SEQ ID No. 11; Canine adenovirus type II group primers: FIP is shown in SEQ ID No. 12, BIP is shown in SEQ ID No. 13, F3 is shown in SEQ ID No. 14, B3 is shown in SEQ ID No. 15, LF is shown in SEQ ID No. 16, and LB is shown in SEQ ID No. 17; Canine coronavirus group primers: FIP is shown in SEQ ID No. 18, BIP is shown in SEQ ID No. 19, F3 is shown in SEQ ID No. 20, B3 is shown in SEQ ID No. 21, and LF is shown in SEQ ID No. 22; Primers for the Bordetella canis group: FIP is shown as SEQ ID No. 23, BIP is shown as SEQ ID No. 24, F3 is shown as SEQ ID No. 25, B3 is shown as SEQ ID No. 26, LB is shown as SEQ ID No. 27, and LF is shown as SEQ ID No. 28.

[0010] The present application also provides a kit for rapid detection of canine respiratory pathogens containing the above primers.

[0011] Optionally, the kit includes a canine parainfluenza virus detection tube, a canine distemper virus detection tube, a canine adenovirus type II detection tube, a canine coronavirus detection tube, a canine Bordetella detection tube, and a negative colorimetric tube; The canine parainfluenza virus detection tube includes canine parainfluenza virus group primers, the canine distemper virus detection tube includes canine distemper virus group primers, the canine adenovirus type II detection tube includes canine adenovirus type II group primers, the canine coronavirus detection tube includes canine coronavirus group primers, and the canine Bordetella detection tube includes canine Bordetella group primers.

[0012] Optionally, each detection tube also includes a nucleic acid direct amplification release agent, liquid paraffin, and a lyophilizer.

[0013] The present application also provides a method for preparing the above-mentioned kit for rapid detection of canine respiratory pathogens, which includes the step of using a lyophilizer to prepare a lyophilized ball from a LAMP reaction solution containing primers.

[0014] Optionally, the LAMP reaction solution also includes Bst polymerase, dNTP mixed solution, magnesium ions, and phenol red.

[0015] Optionally, the nucleic acid direct amplification release agent includes guanidine hydrochloride, Tris, adsorption resin XAD-7, and polysorbate.

[0016] Furthermore, the present application also provides the use of the above-mentioned primers for rapid detection of canine respiratory pathogens in the preparation of products for detecting canine respiratory pathogens.

[0017] The present application also provides the use of the above-mentioned kit for rapid detection of canine respiratory pathogens in rapid on-site screening of canine respiratory pathogens and in clinical rapid diagnosis of canine respiratory pathogens.

[0018] The beneficial effects of this application include but are not limited to: 1. The test kit of this application can quickly and accurately detect five respiratory pathogens, namely canine parainfluenza virus, canine distemper virus, canine adenovirus type II, canine coronavirus and canine Bordetella canis, within 15 minutes. Canine parainfluenza virus can even be detected within 11.25 minutes. This is much shorter than the positive detection time of more than one hour in the current existing technology, and does not require the use of professional and expensive instruments and equipment, so the detection cost is low; the detection environment requirements are low. It can be used for rapid clinical diagnosis and on-site screening (including homes and other places) to confirm infection, allowing veterinarians to quickly adopt effective treatment measures; it solves the technical problem that the detection process in the existing technology cannot take into account short time, high accuracy, low cost and simple operation at the same time.

[0019] 2. The canine respiratory pathogen primers of this application are highly sensitive and can be used for different quality control templates (10 3 After amplification of 1000 copies / ml, all of them could be successfully detected; 3. The canine respiratory pathogen primers of the present application have high specificity. After using the canine respiratory pathogen primers to amplify different types of plasmid templates, only the sites (primers) corresponding to the primers and templates are successfully amplified.

[0020] The kit of the present application can ultimately determine a positive result in different amplification instruments, although the performance and reaction conditions (such as temperature control accuracy, heating and cooling rates, etc.) of the instruments may differ, indicating that the primers of the present application can well distinguish target sequences from non-target sequences; it can also be shown that the primers have stable amplification efficiency and good adaptability. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings: Figure 1 This is a diagram of the sample processing process before amplification for this application; Figure 2 Amplification process diagram for this application; Figure 3 Validation results for primer sensitivity; Figure 4 Arrangement of the rapid detection of five canine respiratory pathogens in the test kit; Figure 5 The results of FireMini-8P, BioRad PCR instrument, and ABI 7500 amplification reaction tubes are shown; Figure 6 It is the amplification diagram of FireMini-8P, Bio-Rad PCR instrument, and ABI 7500; Figure 7 for Figure 6The enlarged picture of the FireMini-8P result; Figure 8 for Figure 6 ABI 7500 results are shown in the enlarged view. DETAILED DESCRIPTION

[0022] The present application is described in detail below in conjunction with the examples, but the present application is not limited to these examples. Unless otherwise specified, the raw materials, reagents, and equipment in the examples of the present application are purchased through commercial channels.

[0023] The present application designs five sets of nucleic acid sequences for specific detection of pathogens for five combinations of respiratory pathogens, namely canine parainfluenza virus, canine distemper virus, canine adenovirus type II, canine coronavirus and canine Bordetella canis.

[0024] Among them, FIP / BIP are inner primers, F3 / B3 are outer primers, and LF / LB are loop primers.

[0025] This application prepares a kit for detecting 5 canine respiratory pathogens. The kit includes freeze-dried balls (primers, enzymes, buffers and other reagents) required for the LAMP reaction system, as well as nucleic acid release agents for nucleic acid extraction and liquid paraffin for sealing. The specific operation is as follows: 1) Use a swab to thoroughly wipe under the eyelid to collect an eye swab sample; use the same swab to moderately wipe oral and nasal secretions to collect a nasopharyngeal swab sample. After the swab sample is collected, the swab head should be quickly broken and placed in the nucleic acid release agent, shaken for 10 seconds to fully mix, and placed at room temperature for 5 minutes to allow the pathogens on the swab head to fully dissolve in the nucleic acid release agent.

[0026] 2) Prepare the reagents according to the following table: Table 1 Reagent preparation

[0027] 3) Place the eight-tube strip into the FireMini-8P constant temperature nucleic acid amplifier and start the nucleic acid amplification reaction.

[0028] Result interpretation: During the experiment, you can check the indicator light color to read the positive result in advance (red indicator light). After the experiment, the green indicator light is interpreted as a negative result, and the red indicator light is interpreted as a positive result. You can also view detailed information from the software and print the test report.

[0029] Example 1 Primer design and screening According to the genes of each pathogen in GenBank, specific primers were designed and screened on the PrimerExplorer V5 (http: / / primerexplorer.jp / e / ) website. The standard products of each pathogen are plasmids synthesized by biological companies. The test primers are shown in Table 2: Table 2 Primer sequences for canine respiratory tract design and testing

[0030] Different primers have different binding efficiencies with templates, which are reflected in the difference in TTR values ​​in the FireMini-8P constant temperature amplification instrument. For example, for the same concentration of template, three different primers will present different TTR values. The faster the color changes to yellow, the easier it is for this primer to bind to the template, and the smaller the final TTR value, the better the design, otherwise it is bad, see the results in Table 3.

[0031] The purchased canine parainfluenza virus, canine distemper virus, canine adenovirus type II, canine coronavirus virus, and canine Bordetella caninum quality control products were diluted to 10 using a nucleic acid releaser. 5 100 copies / ml were used as amplification templates, with 3 parallels each. Taking the primers for detecting canine parainfluenza virus as an example, it can be seen from the TTR value in Table 3 that after using the No.2 canine parainfluenza primer in Table 2, the average TTR value is less than that of No.1 canine parainfluenza and No.3 canine parainfluenza, and the time when the instrument is judged as positive in real time is also the fastest for the No.2 canine parainfluenza primer, that is, the amplification sensitivity is the highest, so the No.2 canine parainfluenza primer is selected as the amplification primer combination of this kit. The detection of canine distemper virus, canine adenovirus type II, canine coronavirus, and canine Bordetella canis showed the same results. The primers of the present invention did not produce cross reactions, so the sensitivity was higher and the specificity was better. However, when other primer combinations in Table 2 were used, the amplification sensitivity was very poor, and the detection of canine respiratory pathogens could not be achieved. Therefore, the subsequent experiments selected five combinations of No.2 canine parainfluenza, No.4 canine distemper, No.1 canine gland, No.2 canine crown, and No.1 canine wave for specificity and sensitivity detection. The primer information is shown in Table 3.

[0032] Table 3 Screening results of different primer combinations

[0033] In addition, according to the data in the above table, the time from the start of amplification to the positive judgment of primers No.2 Canine Parapneumonia, No.4 Canine Distemper, No.1 Canine Glandular, No.2 Canine Coronary, and No.1 Canine Wave is very short, all within 15 minutes, and No.2 Canine Parapneumonia can even be judged as positive within 11.25 minutes.

[0034] Regarding the calculation of judgment time: According to the TTR value of the instrument, the amplification time can be calculated. The specific formula is TTR / 2+3.5=amplification completion time (minutes). The principle of the TTR value judged as positive by the instrument can refer to patent CN 118726078 B.

[0035] The positive detection time is much shorter than the one-hour time taken by the existing technology, and it does not require the use of professional and expensive instruments and equipment, so the detection cost is low; the detection environment requirements are low. It can be used for rapid clinical diagnosis and on-site screening to confirm infection, so that veterinarians can quickly adopt effective treatment measures; it solves the technical problem that the detection process in the existing technology cannot take into account short time, high accuracy, low cost and simple operation at the same time.

[0036] Table 4 Primers finally used for canine respiratory pathogens

[0037] Example 2 Primer sensitivity detection Purchase canine parainfluenza virus, canine distemper virus, canine adenovirus type II, canine coronavirus virus, and canine Bordetella canis quality control products, and dilute the quality control products to 10% using a nucleic acid release agent. 3 Copies / ml were used as amplification template and amplified using FireMini-8P amplification instrument for 30 minutes to obtain the amplification results (including TTR values) and Figure 3 Primer sensitivity validation results.

[0038] Table 5 Amplification results

[0039] According to Table 5 and Figure 3 The amplification results showed that the canine respiratory pathogen primers were used to amplify different quality control templates (10 3 After further dilution of the control sample, canine adenovirus type II can be detected to 10 2 However, no other sites could be detected, so the minimum detection limit of this primer combination, i.e., the sensitivity, was 10 3 At this time, the time for positive determination can still be within 20 minutes; even the positive detection time for canine adenovirus type II is only 12.91 minutes.

[0040] Figure 3 When the TTR value of the sample to be tested is within the reference range of 0-60 (excluding the two values ​​of 0 and 60) and the amplification curve has an obvious slope, the test result is positive; when the TTR value of the sample to be tested is outside the reference range, or the TTR value is within the reference range but there is no amplification curve, the test result is negative.

[0041] Figure 3 In the figure, the vertical axis is the starting device that starts heating. After reaching the set temperature, the light-emitting bodies on the circuit board light up in turn every other cycle, and the values ​​collected after lighting up every 30 seconds. The horizontal axis is the light-emitting body collecting light signals every 30 seconds, which represents one round. The experimental time is 30 minutes, which means that 6 light-emitting bodies collect 60 rounds of light signals.

[0042] Example 3 Specificity detection of primers Select 10 canine parainfluenza virus, canine distemper virus, canine adenovirus type II, canine coronavirus, and canine Bordetella canis quality control products 5 The copies / ml were used as amplification templates, and each template was replicated twice to obtain the following amplification results, as shown in Table 6.

[0043] Table 6 Primer specificity verification

[0044] From the amplification results in the above table, after using canine respiratory pathogen primers to amplify different types of plasmid templates, only the sites (primers) corresponding to the primers and templates were amplified successfully, and the results showed that the TTR value was within the normal range; for other quality control templates, the TTR results showed N / A (not detected), indicating that the specificity of this amplification system is good.

[0045] Example 4 Preparation of nucleic acid direct amplification release agent and reaction system The LAMP reaction solution is formulated as follows: Based on a 50 μl system, it includes Bst polymerase 8U, betaine 100 mM, dNTP mixture 2.5 mM, magnesium ion 6 mM, phenol red 50 µM, 10×primer, buffer mix; 10×primer contains 16μM FIP / BIP, 2μM F3 / B3, and 4μM LF / LB.

[0046] Buffer Mix contains 1% Tween-20, 100 mM (NH4)2SO4, 500 mM KCl, 200 mMTris-HCl; Make up to 50 μl with sterile water.

[0047] Nucleic acid direct amplification release agent: obtained by preparing a solution of 1M guanidine hydrochloride, 10 mM Tris, adsorption resin XAD-7 (1 g / L), and 0.1% polysorbate 20.

[0048] Example 5 Determining the instrument amplification threshold (1) Sample preparation: Prepare standard samples (or positive control samples) with the lowest detection limit. Use the same sample pretreatment steps as the actual test samples to process these standard samples.

[0049] (2) LAMP isothermal amplification reaction: Add the standard sample to the LAMP reaction system according to the instrument operating instructions. The configuration method is as described in Example 4, and the amplification reaction is performed. Collect instrument data to monitor the reaction results.

[0050] (3) Determine the detection threshold: Repeat the test on standard samples of different concentrations to obtain the signal value at each concentration. According to the distribution of signal values, determine the signal value corresponding to the lowest detection concentration as the detection threshold.

[0051] (4) Verify the detection threshold: Use real positive and negative samples for verification to ensure that positive and negative results can be accurately distinguished under this detection threshold.

[0052] Table 7 Signal value detection

[0053] Based on the data collected by the above instruments, the collected data (final value - initial value) / initial value is used as the threshold. The threshold line can clearly distinguish the reaction curves of positive and negative samples. The threshold line of this method is set to 1.1. After entering this value into the instrument, the negative and positive results of the reagent can be determined.

[0054] Determining a uniform amplification threshold helps to obtain comparable experimental results under different experimental conditions (such as different laboratories, different operators, different batches of reagents and instruments). As long as all experiments are performed according to the same threshold setting principle, the impact of differences in human judgment and fluctuations in experimental conditions on the results can be reduced.

[0055] Example 6 Establishment of a kit for rapid detection of five canine respiratory pathogens In combination with the system in Specific Example 4, freeze drying was performed according to the following procedure: prepare LAMP freeze-dried reaction solution, the formula is as follows: Bst polymerase 8U, betaine 1µL, dNTP mixture 2.5 mM, magnesium ion 6 mM, phenol red 50 µM, sterile water to 50 µL, FIP 1.6 µM, BIP 1.6 µM, F3 0.2 µM, B3 0.2 µM, LF 0.4 µM, LB 0.4 µM, lyophilization protectant 20 µL. Prepare the reagent according to the above formula, fill it into the bead counting machine, the bead counting machine drips into liquid nitrogen at 50 µL / drop, quickly transfer the pre-frozen microspheres to the pre-frozen vials, half-cap them and transfer them to the pre-frozen vacuum freeze dryer, vacuum freeze-dry according to the procedure of this laboratory, and after obtaining the freeze-dried beads, follow the Figure 4The “Assignment of Rapid Detection of Five Canine Respiratory Pathogens in the Kit” was dispensed into 0.2 ml transparent PCR tubes.

[0056] Example 7 Comparison of the detection effects of kits for 5 canine respiratory pathogens on different instruments Dilute the canine parainfluenza virus, canine distemper virus, canine adenovirus type II, canine coronavirus, and canine Bordetella canis quality control products to 10 using a nucleic acid releaser. 3 Copies / ml were used as amplification template and amplified for 30 minutes using FireMini-8P, Bio-Rad PCR instrument and ABI 7500 respectively. Figure 5 This is the result of amplifying the above samples using three instruments: FireMini-8P, Bio-Rad PCR Instrument, and ABI 7500. The results of the three instruments on whether the samples are positive are consistent.

[0057] From this point of view 1) the primers of the present application are highly specific. Although the performance and reaction conditions (such as temperature control accuracy, heating and cooling rates, etc.) of the instruments may vary in different amplification instruments, positive results can be determined in the end, which shows that the primers can well distinguish target sequences from non-target sequences.

[0058] It can also be shown that the primer has stable amplification efficiency and good adaptability.

[0059] Furthermore, the principle of this application is based on the addition of phenol red, which has a pH indicator function, to the reagent. Since a large amount of H+ will be generated during the reaction, causing the pH of the reaction system to drop, the reaction solution will appear red before amplification. If there is a target sequence in the reaction after the reaction, the reaction solution will appear yellow. Therefore, on the FireMini-8P that collects data based on light changes, the instrument can collect data on light changes to determine negative and positive in real time, but Bio-Rad PCR and ABI 7500 cannot collect data on light changes (color changes), such as Figure 6 As shown; however, as long as the amplification temperature of Bio-Rad PCR and ABI 7500 is kept at 65°C, the amplification results can also be observed by the naked eye.

[0060] From this point of view 2) the use of the FireMini-8P thermal cycler is more convenient and can be read through data, which is more efficient; compared to interpreting the results with the naked eye, it will not cause subjectivity or misjudgment.

[0061] Example 8 Testing of real samples of the kit for rapid detection of 5 canine respiratory pathogens Swab samples were collected from 30 dogs in pet shops. One swab sample from the same dog included the eyes, nose, and mouth. The procedure was as follows: 1) Use a swab to thoroughly wipe under the eyelid to collect an eye swab sample; use the same swab to moderately smear the dog's oral and nasal secretions to collect a nasopharyngeal swab sample. After the swab sample is collected, the swab head should be quickly broken and placed in the nucleic acid release agent, shaken for 10 seconds to fully mix, and placed at room temperature for 5 minutes to allow the pathogens on the swab head to fully dissolve in the nucleic acid release agent.

[0062] 2) Prepare the reagents according to the following table: the amplification sites are canine parainfluenza virus, canine distemper virus, canine adenovirus type II, canine coronavirus virus, and canine Bordetella canis.

[0063] Table 8 Reagent preparation

[0064] 3) Open the cover of FireMini-8P constant temperature nucleic acid amplification instrument, first put the negative control sample into the instrument "N" position, then put the test sample into the test position (make sure it is consistent with the test hole position entered in the experiment), and close the cover.

[0065] Insert the Bluetooth module into the computer (tablet, etc.) according to the instrument manual, open the instrument software to connect the device, and enter the information. Click "Start Detection", and the status indicator light will flash in colorful breathing, indicating that the nucleic acid amplification reaction has started. After the reaction is completed, click "End Detection" → "Detection Record" and select the experimental results to print. The following Table 9 shows the reaction results.

[0066] Table 9 TTR values ​​of real dog samples

[0067] From the results in the table above, we can see that among the 30 dogs, 21 (70%) were completely healthy, 3 (10%) were infected with canine Bordetella, 1 (3.3%) were infected with canine adenovirus type II, 4 (13.3%) were infected with canine parainfluenza virus, 1 (3.3%) were infected with canine coronavirus, and 0 (0%) were infected with canine distemper virus. Among the 9 dogs with respiratory diseases, 4 dogs had severe sneezing and tearing, and the remaining 5 had no obvious symptoms. The 30 dog samples were retested using qPCR, and the test results (table) were 100% consistent with the kit for rapid detection of 5 canine respiratory pathogens, proving that the accuracy of this kit is consistent with qPCR.

[0068] The above is only the embodiment of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the technical ideas and principles of the present application should be included in the protection scope of the present application.

Claims

1. A primer for rapid detection of canine respiratory pathogens, characterized in that: The primers include one or more of the following five groups, each group includes inner primers FIP / BIP, outer primers F3 / B3 and loop primers LF / LB, and the specific sequences are as follows: Canine parainfluenza virus group primers: FIP is shown as SEQ ID No. 1, BIP is shown as SEQ ID No. 2, F3 is shown as SEQ ID No. 3, B3 is shown as SEQ ID No. 4, LF is shown as SEQ ID No. 5, and LB is shown as SEQ ID No. 6; Canine distemper virus group primers: FIP is shown in SEQ ID No. 7, BIP is shown in SEQ ID No. 8, F3 is shown in SEQ ID No. 9, B3 is shown in SEQ ID No. 10, and LB is shown in SEQ ID No. 11; Canine adenovirus type II group primers: FIP is shown as SEQ ID No. 12, BIP is shown as SEQ ID No. 13, F3 is shown as SEQ ID No. 14, B3 is shown as SEQ ID No. 15, LF is shown as SEQ ID No. 16, and LB is shown as SEQ ID No. 17; Canine coronavirus group primers: FIP is shown in SEQ ID No. 18, BIP is shown in SEQ ID No. 19, F3 is shown in SEQ ID No. 20, B3 is shown in SEQ ID No. 21, and LF is shown in SEQ ID No. 22; Primers for the Bordetella canis group: FIP is shown as SEQ ID No. 23, BIP is shown as SEQ ID No. 24, F3 is shown as SEQ ID No. 25, B3 is shown as SEQ ID No. 26, LB is shown as SEQ ID No. 27, and LF is shown as SEQ ID No.

28.

2. A kit for rapid detection of canine respiratory pathogens containing the primers as claimed in claim 1.

3. The kit according to claim 2, characterized in that The test kit comprises a canine parainfluenza virus detection tube, a canine distemper virus detection tube, a canine adenovirus type II detection tube, a canine coronavirus detection tube, a canine Bordetella detection tube and a negative colorimetric tube; The canine parainfluenza virus detection tube includes canine parainfluenza virus group primers, the canine distemper virus detection tube includes canine distemper virus group primers, the canine adenovirus type II detection tube includes canine adenovirus type II group primers, the canine coronavirus detection tube includes canine coronavirus group primers, and the canine Bordetella detection tube includes canine Bordetella group primers.

4. The kit according to claim 3, wherein each detection tube further comprises a nucleic acid direct amplification release agent, liquid paraffin, and a lyophilizer.

5. The kit according to claim 4, characterized in that The nucleic acid direct expansion release agent comprises guanidine hydrochloride, Tris, adsorption resin XAD-7 and polysorbate.

6. The method for preparing the kit according to any one of claims 2 to 5, characterized in that: The method comprises the step of using a lyophilizer to prepare a lyophilized ball from a LAMP reaction solution containing primers.

7. The preparation method according to claim 6, characterized in that: The LAMP reaction solution also includes Bst polymerase, dNTP mixed solution, magnesium ions and phenol red.

8. Use of the primers as claimed in claim 1 in preparing products for detecting canine respiratory pathogens.

9. Use of the kit as described in any one of claims 2 to 5 in rapid on-site screening of canine respiratory pathogens.

10. Use of the kit as described in any one of claims 2 to 5 in the clinical rapid diagnosis of canine respiratory pathogens.

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