Primer probe combination for detecting cat pathogen nucleic acid and application thereof

Abstract

The invention relates to the related technical field of cat pathogen nucleic acid detection, and particularly discloses a primer probe combination for detecting cat pathogen nucleic acid and application of the primer probe combination. The primer probe combination comprises a first primer probe combination and / or a second primer probe combination; the first primer probe combination comprises an FPV primer probe group for detecting feline parvovirus, an FHV-1 primer probe group for detecting feline herpes virus type I and a TOXO primer probe group for detecting feline toxoplasma gondii; the second primer probe combination comprises an FCV primer probe set for detecting feline calicivirus, an FCoV primer probe set for detecting feline coronavirus and an FeLV primer probe set for detecting feline leukemia virus. The primer and the probe designed in the invention have strong specificity so as to realize simultaneous detection of three cat digestive tract pathogens, and / or simultaneous detection of three cat respiratory tract pathogens, and / or simultaneous detection of three cat digestive tract pathogens and three cat respiratory tract pathogens.

Description

Technical Field

[0001] The present application relates to the technical field related to cat pathogen nucleic acid detection, and specifically to a primer-probe combination for detecting cat pathogen nucleic acid and its use. Background Art

[0002] Common and frequent feline diseases are mainly caused by six pathogens: feline parvovirus (FPV), feline herpesvirus type-1 (FHV-1), toxoplasmosis (TOXO), feline calicivirus (FCV), feline coronavirus (FCoV), and feline leukemia virus (FeLV). FPV, FHV-1, and TOXO can cause digestive tract diseases in cats, while FCV, FCoV, and FeLV can cause respiratory diseases in cats.

[0003] FPV can cause feline panleukopenia (FP), an acute, highly contagious disease in cats. Symptoms include biphasic fever, vomiting, diarrhea, dehydration, circulatory impairment, severe leukopenia, and hemorrhagic enteritis. FPV primarily infects kittens under one year old, with a mortality rate of 50-60% for cats under one year old and as high as 80-90% for kittens under five months old. In pregnant females, the virus can be transmitted vertically through the placenta, leading to embryonic resorption, fetal mummification, miscarriage, or stillbirth.

[0004] FHV-1 is the most common cause of conjunctivitis and keratitis in domestic cats, and feline rhinotracheitis caused by FHV-1 is a highly contagious respiratory disease with a mortality rate of up to 100% if no treatment is taken, especially in kittens.

[0005] Cats are the definitive host of TOXO. While cats may be asymptomatic after infection, some may develop central nervous system disorders, encephalitis, pneumonia, and eye symptoms. Initial infection in pregnant cats may result in miscarriage, stillbirth, and neurological disorders in newborns. TOXO can also infect humans.

[0006] FCV is highly contagious and can cause acute oral problems and upper respiratory tract diseases. It is one of the most common pathogens of infectious upper respiratory tract diseases in cats and even has the risk of inducing stomatitis.

[0007] Feline leukemia is a malignant tumor infectious disease caused by FeLV, which suppresses the cat's immune system. It is one of the most common causes of cancer in cats, accounting for more than 30% of all cancers in cats.

[0008] Feline enteric coronavirus disease is an enteric-transmitted disease of cats caused by FCoV, which belongs to the Coronaviridae family and the Coronavirus genus. It may cause dehydration and mainly causes enteritis in kittens aged (42-84) days.

[0009] At present, there is no detection method in the prior art for simultaneously detecting three cat digestive tract pathogens, namely, feline parvovirus, feline herpesvirus type 1, and feline Toxoplasma gondii, and / or simultaneously detecting three cat respiratory tract pathogens, namely, feline calicivirus, feline coronavirus, and feline leukemia virus, and / or simultaneously detecting six cat pathogens, namely, feline parvovirus, feline herpesvirus type 1, feline Toxoplasma gondii, feline calicivirus, feline coronavirus, and feline leukemia virus. Summary of the Invention

[0010] In view of this, the present application provides a primer probe combination for detecting cat pathogen nucleic acid and its use.

[0011] In the first aspect, the present application provides a primer probe combination for detecting cat pathogen nucleic acids, the primer probe combination comprising a first primer probe combination and / or a second primer probe combination; the first primer probe combination comprises an FPV primer probe group for detecting feline parvovirus, an FHV-1 primer probe group for detecting feline herpesvirus type I, and a TOXO primer probe group for detecting feline Toxoplasma gondii; the second primer probe combination comprises an FCV primer probe group for detecting feline calicivirus, an FCoV primer probe group for detecting feline coronavirus, and a FeLV primer probe group for detecting feline leukemia virus.

[0012] In some optional embodiments, the primer probe combination includes the first primer probe combination, and the primer probe combination simultaneously detects feline parvovirus, feline herpesvirus type I, and feline Toxoplasma gondii.

[0013] In some optional embodiments, the primer probe combination includes the second primer probe combination, and the primer probe combination simultaneously detects feline calicivirus, feline coronavirus and feline leukemia virus.

[0014] In some optional embodiments, the primer probe combination includes the first primer probe combination and the second primer probe combination, and the primer probe combination simultaneously detects feline parvovirus, feline herpesvirus type 1, feline Toxoplasma gondii, feline calicivirus, feline coronavirus and feline leukemia virus.

[0015] In some optional embodiments, the FPV primer probe set includes an FPV upstream primer, an FPV downstream primer and an FPV probe;

[0016] The FPV upstream primer is selected from the nucleotide sequence shown in SEQ ID NO.1; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO.1, and has the same or similar functions;

[0017] The FPV downstream primer is selected from the nucleotide sequence shown in SEQ ID NO. 2; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO. 2, and has the same or similar functions;

[0018] The FPV probe is selected from the nucleotide sequence shown in SEQ ID NO.3; and / or the nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO.3, and has the same or similar functions.

[0019] In some specific embodiments, the FPV upstream primer is selected from the nucleotide sequence shown in SEQ ID NO.1.

[0020] In some specific embodiments, the FPV downstream primer is selected from the nucleotide sequence shown in SEQ ID NO.2.

[0021] In some specific embodiments, the FPV probe is selected from the nucleotide sequence shown in SEQ ID NO.3.

[0022] In some optional embodiments, the FHV-1 primer probe set includes a FHV-1 upstream primer, a FHV-1 downstream primer and a FHV-1 probe;

[0023] The FHV-1 upstream primer is selected from the nucleotide sequence shown in SEQ ID NO.4; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO.4, and has the same or similar functions;

[0024] The FHV-1 downstream primer is selected from the nucleotide sequence shown in SEQ ID NO.5; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO.5, and has the same or similar functions;

[0025] The FHV-1 probe is selected from the nucleotide sequence shown in SEQ ID NO.6; and / or the nucleotide sequence shown in SEQ ID NO.6 by substitution, and / or deletion, and / or addition of 1-3 bases, and has the same or similar functions.

[0026] In some specific embodiments, the FHV-1 upstream primer is selected from the nucleotide sequence shown in SEQ ID NO.4.

[0027] In some specific embodiments, the FHV-1 downstream primer is selected from the nucleotide sequence shown in SEQ ID NO.5.

[0028] In some specific embodiments, the FHV-1 probe is selected from the nucleotide sequence shown in SEQ ID NO.6.

[0029] In some optional embodiments, the TOXO primer probe set includes a TOXO upstream primer, a TOXO downstream primer and a TOXO probe;

[0030] The TOXO upstream primer is selected from the nucleotide sequence shown in SEQ ID NO. 7; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO. 7, and has the same or similar functions;

[0031] The TOXO downstream primer is selected from the nucleotide sequence shown in SEQ ID NO. 8; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO. 8, and has the same or similar functions;

[0032] The TOXO probe is selected from the nucleotide sequence shown in SEQ ID NO.9; and / or the nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO.9, and has the same or similar functions.

[0033] In some specific embodiments, the TOXO upstream primer is selected from the nucleotide sequence shown in SEQ ID NO.7.

[0034] In some specific embodiments, the TOXO downstream primer is selected from the nucleotide sequence shown in SEQ ID NO.8.

[0035] In some specific embodiments, the TOXO probe is selected from the nucleotide sequence shown in SEQ ID NO.9.

[0036] In some optional embodiments, the FCV primer probe set includes an FCV upstream primer, an FCV downstream primer and an FCV probe;

[0037] The FCV upstream primer is selected from the nucleotide sequence shown in SEQ ID NO. 10; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO. 10, and has the same or similar functions;

[0038] The FCV downstream primer is selected from the nucleotide sequence shown in SEQ ID NO. 11; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO. 11, and has the same or similar functions;

[0039] The FCV probe is selected from the nucleotide sequence shown in SEQ ID NO.12; and / or the nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO.12, and has the same or similar functions.

[0040] In some specific embodiments, the FCV upstream primer is selected from the nucleotide sequence shown in SEQ ID NO.10.

[0041] In some specific embodiments, the FCV downstream primer is selected from the nucleotide sequence shown in SEQ ID NO.11.

[0042] In some specific embodiments, the FCV probe is selected from the nucleotide sequence shown in SEQ ID NO.12.

[0043] In some optional embodiments, the FCoV primer probe set includes an FCoV upstream primer, an FCoV downstream primer and an FCoV probe;

[0044] The FCoV upstream primer has a nucleotide sequence as shown in SEQ ID NO.13; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence as shown in SEQ ID NO.13, and has the same or similar functions;

[0045] The FCoV downstream primer has a nucleotide sequence as shown in SEQ ID NO. 14; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence as shown in SEQ ID NO. 14, and has the same or similar functions;

[0046] The FCoV probe has a nucleotide sequence as shown in SEQ ID NO.15; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence as shown in SEQ ID NO.15, and has the same or similar functions.

[0047] In some specific embodiments, the FCoV upstream primer has a nucleotide sequence as shown in SEQ ID NO.13.

[0048] In some specific embodiments, the FCoV downstream primer has a nucleotide sequence as shown in SEQ ID NO.14.

[0049] In some specific embodiments, the FCoV probe has a nucleotide sequence as shown in SEQ ID NO.15.

[0050] In some optional embodiments, the FeLV primer probe set includes a FeLV upstream primer, a FeLV downstream primer, and a FeLV probe;

[0051] The FeLV upstream primer is selected from the nucleotide sequence shown in SEQ ID NO. 16; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO. 16, and has the same or similar functions;

[0052] The FeLV downstream primer is selected from the nucleotide sequence shown in SEQ ID NO. 17; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO. 17, and has the same or similar functions;

[0053] The FeLV probe is selected from the nucleotide sequence shown in SEQ ID NO.18; and / or the nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO.18, and has the same or similar functions.

[0054] In some specific embodiments, the FeLV upstream primer is selected from the nucleotide sequence shown in SEQ ID NO.16.

[0055] In some specific embodiments, the FeLV downstream primer is selected from the nucleotide sequence shown in SEQ ID NO.17.

[0056] In some specific embodiments, the FeLV probe is selected from the nucleotide sequence shown in SEQ ID NO.18.

[0057] In some optional embodiments, the 5' ends of the FPV probe, the FHV-1 probe, the TOXO probe, the FCV probe, the FCoV probe, and the FeLV probe are respectively labeled with a fluorescent group, and the 3' ends are respectively labeled with a quenching group.

[0058] In some optional embodiments, the fluorescent group includes at least one of a FAM fluorescent group, a VIC fluorescent group, a Texas Red fluorescent group, a CY5 fluorescent group, a FAM fluorescent group, and a ROX fluorescent group.

[0059] In some optional embodiments, the quencher group includes at least one of a BHQ1 quencher group, a BHQ2 quencher group, a MGB quencher group, and a TAMRA quencher group.

[0060] In some optional embodiments, the fluorescent group of the FPV probe, the fluorescent group of the FHV-1 probe, and the fluorescent group of the TOXO probe are selected from different fluorescent groups.

[0061] In some optional embodiments, the fluorescent group labeled with the FPV probe is selected from the FAM fluorescent group;

[0062] And / or, the fluorescent group labeled with the FHV-1 probe is selected from the VIC fluorescent group;

[0063] And / or, the fluorescent group labeled with the TOXO probe is selected from the Texas Red fluorescent group.

[0064] In some optional embodiments, the fluorescent group of the FCV probe, the fluorescent group of the FCV probe, and the fluorescent group of the FeLV probe are selected from different fluorescent groups.

[0065] In some optional embodiments, the fluorescent group labeled with the FCV probe is selected from the FAM fluorescent group;

[0066] and / or, the fluorescent group labeled with the FCV probe is selected from a VIC fluorescent group;

[0067] And / or, the fluorescent group labeled with the FeLV probe is selected from the Texas Red fluorescent group.

[0068] In a second aspect, the present application provides a PCR amplification system, which includes the primer-probe combination described in the first aspect.

[0069] In some optional embodiments, the amplification program of the PCR amplification system is: reverse transcription: 50°C, 10 min, 1 cycle; pre-denaturation: 95°C, 30 s, 1 cycle; PCR: 95°C, 10 s, 58°C, 30 s, 40 cycles.

[0070] In a third aspect, the present application provides the use of any of the following items in the preparation of a product for detecting cat pathogen nucleic acids:

[0071] (I) the primer-probe combination described in the first aspect;

[0072] (II) The PCR amplification system described in the second aspect.

[0073] In a fourth aspect, the present application provides a kit for detecting nucleic acids of cat pathogens, wherein the kit comprises the primer-probe combination described in the first aspect, and / or the PCR amplification system described in the second aspect.

[0074] In some optional embodiments, the test results of the kit are determined as follows:

[0075] If the sample test result Ct value is ≤35 and there is a significant exponential growth, it is judged as positive;

[0076] If the sample test result Ct value is greater than 38 or no Ct value is found, the line is straight or slightly slanted, and there is no obvious exponential growth, it is considered negative;

[0077] If the test result of the sample is 35<Ct value≤38, the sample is tested again; if the repeated test result of the sample is 35<Ct value≤38 and there is a significant exponential growth, it is judged as positive.

[0078] This application has the following beneficial effects:

[0079] The present application designs specific primers and probes for the highly conserved regions in the VP2 gene of feline parvovirus, the U27 gene of feline herpesvirus type I, the Rep529 gene of feline Toxoplasma gondii, the VP1 gene of feline calicivirus, the ORF1 gene of feline coronavirus, and the Pol protein gene of feline leukemia virus. The primers and probes have strong specificity, so as to achieve the simultaneous detection of three cat digestive tract pathogens, namely, feline parvovirus, feline herpesvirus type I, and feline Toxoplasma gondii, and / or the simultaneous detection of three cat respiratory pathogens, namely, feline calicivirus, feline coronavirus, and feline leukemia virus, and / or the simultaneous detection of six cat pathogens, namely, feline parvovirus, feline herpesvirus type I, feline Toxoplasma gondii, feline calicivirus, feline coronavirus, and feline leukemia virus. DETAILED DESCRIPTION

[0080] The present application discloses a primer-probe combination for detecting cat pathogen nucleic acid and its use. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and they are all considered to be included in this application. The methods and applications of this application have been described through preferred embodiments. Relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of this application to implement and apply the technology of this application.

[0081] General Definitions and Terminology:

[0082] The term “and / or” used in this application should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0083] As used herein, “comprise” or “comprising” means including stated elements, integers or steps, but not excluding any other elements, integers or steps.

[0084] In order to make the purpose, technical solutions and advantages of this application clearer, the implementation scheme of this application will be further described in detail with reference to examples below.

[0085] Unless otherwise specified, the experimental methods involved in this application are conventional methods in the art using default parameters, steps, etc., unless otherwise specified; the experimental materials used are commercially available products unless otherwise specified. If specific techniques or conditions are not specified in the examples, they are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the corresponding product instructions. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0086] Unless otherwise specified, all experimental reagents were of analytical grade, and the experimental water should meet the requirements of Grade 1 water in GB / T 6682.

[0087] Example 1, specific primers and probes:

[0088] The present application designs specific primers and probes for three common pathogens that cause common digestive tract diseases in cats (feline parvovirus, feline herpesvirus type I, feline toxoplasma) and three pathogens of respiratory diseases (feline calicivirus, feline coronavirus, feline leukemia virus). Specifically, specific primers and probes are designed for the highly conserved regions in the VP2 gene of feline parvovirus (FPV), the U27 gene of feline herpesvirus type-1 (FHV-1), the Rep529 gene of toxoplasmosis (TOXO), the VP1 gene of feline calicivirus (FCV), the ORF1 gene of feline coronavirus (FCoV), and the Pol protein gene of feline leukemia virus (FeLV) (as shown in Table 1).

[0089] Table 1. Specific primers and probes:

[0090]

[0091] Example 2, kit for detecting cat pathogen nucleic acid:

[0092] In this embodiment, the kit can be a kit for detecting nucleic acids of pathogens of cat digestive tract diseases, a kit for detecting nucleic acids of pathogens of cat respiratory tract diseases, or a kit for detecting nucleic acids of pathogens of cat digestive tract diseases and cat respiratory tract diseases simultaneously.

[0093] Among them, the kit for detecting nucleic acids of pathogens of cat digestive tract diseases uses fluorescent RT-PCR technology and one-tube three-channel amplification. It uses a fully automatic fluorescent quantitative PCR amplifier to monitor and output the signal intensity of the corresponding channels during the PCR process in real time, thereby realizing qualitative analysis of nucleic acid detection of pathogens of common cat digestive tract diseases.

[0094] The kit for detecting nucleic acids of pathogens of feline respiratory diseases utilizes fluorescent RT-PCR technology and three-channel amplification in one tube. A fully automatic fluorescent quantitative PCR amplifier is used to monitor and output the signal intensity of the corresponding channels in real time during the PCR process, thereby enabling qualitative analysis of nucleic acid detection of common respiratory disease pathogens in cats.

[0095] The kit can simultaneously detect nucleic acids of pathogens of cat digestive tract diseases and cat respiratory diseases. It uses fluorescent RT-PCR technology and three-channel amplification in two tubes. When the reaction system contains the target genome template, the PCR reaction can proceed and release fluorescent signals. The fully automatic fluorescent quantitative PCR amplifier monitors and outputs the signal intensity of the corresponding channel in real time during the PCR process, realizing qualitative analysis of the nucleic acid detection of pathogens of common cat digestive tract diseases and cat respiratory diseases.

[0096] Table 2. Main components of the kit:

[0097] Main components Dosage for 50 qPCR reactions Reaction solution A1 1 tube (500 μL) Reaction solution B1 1 tube (500 μL) Reaction solution A2 1 tube (500 μL) Reaction solution B2 1 tube (500 μL) Mixed enzyme solution 1 tube (100 μL) Negative control 1 tube (100 μL) Positive control 1 tube (100 μL)

[0098] The mixed enzyme solution includes tag enzyme and reverse transcriptase mixed in a volume ratio of 1:1.

[0099] Negative control: RNase-free water.

[0100] Positive control: a positive plasmid containing the target fragment.

[0101] Table 3. qPCR reaction system:

[0102]

[0103]

[0104] The kit in this application is suitable for automatic fluorescence quantitative PCR instruments such as ABI7000, ABI7300, ABI7500, ABI7900, MX3000P, MX3005P, iCycleriQ4, iCycleriQ5, MJ-chromo4, Light cycler R480 and SLAN.

[0105] The method for detecting cat pathogen nucleic acid with the kit of this application specifically comprises the following steps:

[0106] Step 1: Sample collection and nucleic acid extraction:

[0107] Swab sample: Take a cotton swab and insert it deep into the nose and eyelids and scrape it back and forth 3-5 times, or pick up eye and nasal secretions; take another cotton swab and insert it deep into the anus and rotate it 2-3 times to pick up feces; immerse both swabs together in a sterile enzyme-free centrifuge tube containing physiological saline or PBS buffer (1.0 ml) for later use.

[0108] Tissue samples: Weigh a small amount of sample (approximately 1 g) from three random locations of each tissue. Cut and mix with surgical scissors, then grind 0.05 g in a grinder. Add 1.5 ml of normal saline and continue grinding. After homogenization, transfer to a 1.5 ml sterile centrifuge tube and centrifuge at 8000 rpm for 2 minutes. Transfer 200 μl of the supernatant to a 1.5 ml sterile enzyme-free centrifuge tube for testing.

[0109] Liquid samples: Use a sterile syringe to draw a liquid sample and place it in a 1.5ml sterile enzyme-free centrifuge tube for testing.

[0110] Samples should be tested promptly after collection, or stored at -20±5℃ for testing. For long-term storage, they should be kept below -70℃.

[0111] Sample nucleic acid extraction: Use a nucleic acid extraction kit and follow the instructions to extract the DNA or RNA template from the sample. If the DNA or RNA template cannot be tested immediately, it is recommended to store it below -70°C.

[0112] Step 2: Reagent preparation:

[0113] 20 min before the experiment, the kit and corresponding reagents of this application were taken out from the -20°C refrigerator to equilibrate to room temperature (15-25°C), and the reagents were completely melted. The tubes were centrifuged for 15 s to allow all the liquid to settle to the bottom.

[0114] Calculate the number of reactions (n) required for the experiment; where n = number of negative controls (1 reaction) + number of positive controls (1 reaction) + error margin + number of samples.

[0115] Calculate the required amounts of various reagents for the experiment based on the K1 well reaction system formula (as shown in Table 4) and the K2 well reaction system formula (as shown in Table 5). Important: Each sample must be tested simultaneously in wells K1 and K2, and the reagents for wells K1 and K2 must be prepared and packaged separately.

[0116] Table 4, formula of K1 well reaction system:

[0117]

[0118] Table 5. Formula of K2 well reaction system:

[0119]

[0120] Since the pathogens detected by K1 well and K2 well are different, the K1 well reaction system does not require the mixed enzyme solution component, while the K2 well reaction system does require the mixed enzyme solution component.

[0121] Add the reagents of the K1 well reaction system to a sterile enzyme-free centrifuge tube and mix well. Centrifuge at 2000 rpm for 10 seconds. Dispense the prepared K1 well reagent into sterile enzyme-free PCR reaction tubes at a volume of 20.0 μl / tube. Close the PCR reaction tube caps tightly and mark them as K1. Add the reagents of the K2 well reaction system to another sterile enzyme-free centrifuge tube and mix well. Centrifuge at 2000 rpm for 10 seconds. Dispense the prepared K2 well reagent into sterile enzyme-free PCR reaction tubes at a volume of 20.0 μl / tube. Close the PCR reaction tube caps tightly and mark them as K2.

[0122] If there is any remaining reagent in well K1 or well K2, the remaining reagent can be frozen and stored at -20°C.

[0123] Step 3: Add sample:

[0124] Open the caps of the PCR reaction tubes labeled K1 and add a positive control (10 μl) and a negative control (10 μl) to two of them, respectively. Add a sample template (10 μl) to the remaining PCR reaction tubes. Open the caps of the PCR reaction tubes labeled K2 and add a positive control (10 μl) and a negative control (10 μl) to two of them, respectively. Add a sample template (10 μl) to the remaining PCR reaction tubes. Record the order of sample addition.

[0125] Cover the PCR reaction tube, shake the PCR reaction solution in the PCR reaction tube to mix, and centrifuge at 2000 rpm for 10 seconds.

[0126] Step 4: PCR reaction:

[0127] Turn on the instrument to warm up and check its performance.

[0128] Place the PCR reaction tubes in the corresponding positions of the sample slots in the multi-channel fluorescence quantitative qPCR amplification instrument and record the placement order.

[0129] Set the nucleic acid amplification parameters of the multi-channel fluorescence quantitative qPCR amplifier according to Table 6 and perform PCR amplification.

[0130] Table 6. Nucleic acid amplification parameters of multi-channel fluorescence quantitative qPCR amplifier:

[0131]

[0132] Note: For ABI series fluorescence quantitative PCR instruments, do not select ROX correction and select None as the quenching group.

[0133] Step 5: Result analysis and judgment:

[0134] Result analysis condition setting: The principle of threshold setting is to reasonably adjust the threshold line. Different instruments can be adjusted according to the instrument noise conditions.

[0135] Test kit validity determination:

[0136] Positive control: The Ct values of FAM, VIC, and Texas Red channels in wells K1 and K2 were all ≤30, indicating a significant exponential increase.

[0137] Negative control: The Ct values of the FAM, VIC, and Texas Red channels in wells K1 and K2 were all greater than 38 or had no Ct value, and the line shape was a straight line or a slightly slanted line, with no obvious exponential growth.

[0138] Sample result determination:

[0139] If the sample test result Ct value is ≤35 and there is a significant exponential growth, it is judged as positive;

[0140] If the sample test result Ct value is greater than 38 or no Ct value is found, the line is straight or slightly slanted, and there is no obvious exponential growth, it is considered negative;

[0141] If the test result of the sample is 35<Ct value≤38, the sample is tested again; if the repeated test result of the sample is 35<Ct value≤38 and there is a significant exponential growth, it is judged as positive.

[0142] Table 7. Interpretation of sample test results:

[0143]

[0144]

[0145] Example 3: Detection of nucleic acid samples with known background based on the kit of this application:

[0146] Based on the detection of nucleic acid samples with known background by the kit of this application (the nucleic acid comes from clinically tested disease materials or commercial vaccines, and has been qualitatively detected using the corresponding pathogen fluorescence PCR detection kit), the test results are shown in Table 8.

[0147] Table 8. Test results of nucleic acid samples with known background:

[0148]

[0149] As can be seen from Table 8, the primers and probes designed in this application have good specificity, can specifically amplify specific sample nucleic acids, and have no non-specific reactions.

[0150] The above describes in detail the primer-probe combination for detecting feline pathogen nucleic acids and its uses provided by this application. This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above examples is only intended to help understand the method and core concept of this application. It should be noted that those skilled in the art can make various improvements and modifications to this application without departing from the principles of this application, and such improvements and modifications also fall within the scope of protection of the claims of this application.

Claims

1. A primer-probe combination for detecting cat pathogen nucleic acid, characterized in that: The primer probe combination includes a first primer probe combination for detecting cat digestive tract pathogens and / or a second primer probe combination for detecting cat respiratory tract pathogens; the first primer probe combination includes an FPV primer probe group for detecting feline parvovirus, an FHV-1 primer probe group for detecting feline herpesvirus type I, and a TOXO primer probe group for detecting cat Toxoplasma gondii; the second primer probe combination includes an FCV primer probe group for detecting feline calicivirus, an FCoV primer probe group for detecting feline coronavirus, and a FeLV primer probe group for detecting feline leukemia virus; The FPV primer probe group includes an FPV upstream primer, an FPV downstream primer and an FPV probe; The FPV upstream primer is selected from the nucleotide sequence shown in SEQ ID NO.1; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO.1, and has the same or similar functions; The FPV downstream primer is selected from the nucleotide sequence shown in SEQ ID NO. 2; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO. 2, and has the same or similar functions; The FPV probe is selected from the nucleotide sequence shown in SEQ ID NO. 3; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO. 3, and has the same or similar functions; The FHV-1 primer probe group includes an FHV-1 upstream primer, an FHV-1 downstream primer and an FHV-1 probe; The FHV-1 upstream primer is selected from the nucleotide sequence shown in SEQ ID NO.4; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO.4, and has the same or similar functions; The FHV-1 downstream primer is selected from the nucleotide sequence shown in SEQ ID NO.5; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO.5, and has the same or similar functions; The FHV-1 probe is selected from the nucleotide sequence shown in SEQ ID NO.6; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO.6, and has the same or similar functions; The TOXO primer probe set includes a TOXO upstream primer, a TOXO downstream primer and a TOXO probe; The TOXO upstream primer is selected from the nucleotide sequence shown in SEQ ID NO. 7; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO. 7, and has the same or similar functions; The TOXO downstream primer is selected from the nucleotide sequence shown in SEQ ID NO. 8; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO. 8, and has the same or similar functions; The TOXO probe is selected from the nucleotide sequence shown in SEQ ID NO. 9; and / or a nucleotide sequence obtained by substituting, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO. 9, and has the same or similar functions; The FCV primer probe set includes an FCV upstream primer, an FCV downstream primer and an FCV probe; The FCV upstream primer is selected from the nucleotide sequence shown in SEQ ID NO. 10; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO. 10, and has the same or similar functions; The FCV downstream primer is selected from the nucleotide sequence shown in SEQ ID NO. 11; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO. 11, and has the same or similar functions; The FCV probe is selected from the nucleotide sequence shown in SEQ ID NO. 12; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO. 12, and has the same or similar functions; The FCoV primer-probe group includes an FCoV upstream primer, an FCoV downstream primer and an FCoV probe; The FCoV upstream primer has a nucleotide sequence as shown in SEQ ID NO.13; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence as shown in SEQ ID NO.13, and has the same or similar functions; The FCoV downstream primer has a nucleotide sequence as shown in SEQ ID NO. 14; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence as shown in SEQ ID NO. 14, and has the same or similar functions; The FCoV probe has a nucleotide sequence as shown in SEQ ID NO. 15; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence as shown in SEQ ID NO. 15, and has the same or similar functions; The FeLV primer-probe set includes a FeLV upstream primer, a FeLV downstream primer and a FeLV probe; The FeLV upstream primer is selected from the nucleotide sequence shown in SEQ ID NO. 16; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO. 16, and has the same or similar functions; The FeLV downstream primer is selected from the nucleotide sequence shown in SEQ ID NO. 17; and / or a nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO. 17, and has the same or similar functions; The FeLV probe is selected from the nucleotide sequence shown in SEQ ID NO.18; and / or the nucleotide sequence obtained by replacing, and / or deleting, and / or adding 1-3 bases to the nucleotide sequence shown in SEQ ID NO.18, and has the same or similar functions.

2. The primer-probe combination according to claim 1, characterized in that The 5' ends of the FPV probe, the FHV-1 probe, the TOXO probe, the FCV probe, the FCoV probe, and the FeLV probe are respectively labeled with a fluorescent group, and the 3' ends are respectively labeled with a quenching group; Optionally, the fluorescent group includes at least one of a FAM fluorescent group, a VIC fluorescent group, a Texas Red fluorescent group, a CY5 fluorescent group, a FAM fluorescent group, and a ROX fluorescent group; Optionally, the quencher group includes at least one of a BHQ1 quencher group, a BHQ2 quencher group, a MGB quencher group, and a TAMRA quencher group.

3. The primer-probe combination according to claim 2, characterized in that The fluorescent group of the FPV probe, the fluorescent group of the FHV-1 probe, and the fluorescent group of the TOXO probe are selected from different fluorescent groups; Optionally, the fluorescent group labeled with the FPV probe is selected from the FAM fluorescent group; And / or, the fluorescent group labeled with the FHV-1 probe is selected from the VIC fluorescent group; And / or, the fluorescent group labeled with the TOXO probe is selected from the Texas Red fluorescent group.

4. The primer-probe combination according to claim 2, characterized in that The fluorescent group of the FCV probe, the fluorescent group of the FCV probe, and the fluorescent group of the FeLV probe are selected from different fluorescent groups; Optionally, the fluorescent group labeled with the FCV probe is selected from the FAM fluorescent group; and / or, the fluorescent group labeled with the FCV probe is selected from a VIC fluorescent group; And / or, the fluorescent group labeled with the FeLV probe is selected from the Texas Red fluorescent group.

5. A PCR amplification system, characterized in that: The PCR amplification system comprises the primer-probe combination according to any one of claims 1 to 4.

6. The PCR amplification system according to claim 5, characterized in that The amplification program of the PCR amplification system is as follows: reverse transcription: 50°C, 10 min, 1 cycle; pre-denaturation: 95°C, 30 s, 1 cycle; PCR: 95°C, 10 s, 40 cycles.

7. Use of any of the following in the preparation of a product for detecting nucleic acid of a feline pathogen: (I) a primer-probe combination according to any one of claims 1 to 4; (II) The PCR amplification system according to any one of claims 5 to 6.

8. A kit for detecting cat pathogen nucleic acid, characterized in that: The kit comprises the primer-probe combination according to any one of claims 1 to 4, and / or the PCR amplification system according to any one of claims 5 to 6.

9. The kit according to claim 8, characterized in that The test results of the kit are determined as follows: If the sample test result Ct value is ≤35 and there is a significant exponential growth, it is judged as positive; If the sample test result Ct value is greater than 38 or no Ct value is found, the line is straight or slightly slanted, and there is no obvious exponential growth, it is considered negative; If the test result of the sample is 35<Ct value≤38, the sample is tested again; if the repeated test result of the sample is 35<Ct value≤38 and there is a significant exponential growth, it is judged as positive.

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