Primer pool and kit for simultaneous detection of multiple canine pathogens

Abstract

The present invention discloses a primer pool and a kit for simultaneously detecting multiple pathogens of dogs, wherein the primer pool includes primer pairs and annihilation probes for detecting canine distemper virus, canine adenovirus type II, canine parainfluenza virus, Bordetella bronchiseptica and Mycoplasma canis; the kit includes the primer pool, PCR reaction solution and buffer. The kit of the present invention is provided with a microarray qPCR detection card box, in which each sub-detection area in the microarray qPCR detection card box is only fixed with fluorescent nucleic acid of a certain pathogen, thereby being able to distinguish and detect multiple pathogens in the sample. At the same time, the present invention has targetedly optimized the primer system on the basis of the multiplex PCR rapid detection kit, and designed a new primer pool, further realizing the specific and sensitive rapid detection of multiple pathogens of dogs in clinical practice.

Description

Technical Field

[0001] The invention belongs to the technical field of pathogen nucleic acid detection, and particularly relates to a primer pool and a kit for simultaneously detecting multiple canine pathogens. Background Art

[0002] Canine distemper is a highly contagious disease of dogs caused by canine distemper virus. The virus exists in many organs and tissues such as liver, spleen, lungs, kidneys, brain and lymph nodes, and is excreted through tears, nasal fluid, saliva, urine and exhaled air. Clinically, it is characterized by biphasic fever, acute catarrhal rhinitis, conjunctivitis and subsequent bronchitis or pneumonia, gastroenteritis and neurological symptoms. Canine distemper is one of the most harmful diseases to my country's dog breeding industry, fur animal breeding industry and wildlife protection industry.

[0003] Canine parainfluenza (CPIV) is a viral infectious disease of dogs caused by canine parainfluenza virus type II, with acute respiratory inflammation as the main symptom. Canine parainfluenza virus type II is a member of the Paramyxoviridae family and the Paramyxovirus genus. It can also cause acute encephalomyelitis and intracerebral hydrops, with clinical manifestations such as hind limb paralysis and movement disorders. Canine parainfluenza virus type II can infect dogs of various ages, genders and breeds, but it is more common in young, weak and stressed dogs, and the condition is more severe, with a course of 1 to 4 weeks and a mortality rate of up to 60%. It often occurs suddenly, spreads rapidly, and is often related to contact with recently ill dogs or infected dogs. Acutely sick dogs are the main source of infection. The main natural route of infection is the respiratory tract.

[0004] Canine adenovirus (CAV) belongs to the Adenoviridae family and the mammalian adenovirus genus. It is a double-stranded, linear DNA virus. CAV is the most pathogenic virus in the mammalian adenovirus genus and has a similar genetic structure to human adenovirus. CAV can be divided into type I and type II based on its different characteristics such as hemagglutination and neutralization tests. CAV-II type can cause upper respiratory tract infections and intestinal infections. The clinical symptoms mainly cause canine contact respiratory diseases related to respiratory diseases and enteritis, but do not cause pneumonia symptoms. It can cause coughing in puppies, also known as kennel cough.

[0005] Bordetella Bronchiseptica, also known as Bordetella bronchiseptica, is an aerobic Gram-negative bacterium that can cause latent infections and acute and chronic inflammations in the respiratory tract of many mammals, such as pigs, dogs, cats, horses, cattle, rabbits, mice and other animals. Dogs of different breeds, genders and ages can be infected with this disease, but young dogs are the most sensitive and the harm is more serious. Clinically, the main symptoms are coughing and runny nose. Once the disease occurs in a group of dogs, it will quickly cause the entire litter of puppies to become ill and cough, so it is called "kennel cough."

[0006] Canis mycoplasma is a prokaryotic microorganism between bacteria and viruses. It absorbs nutrients by living on receptors on the surface of host cells and releases toxic substances, thereby damaging cells and tissues. Canis mycoplasma mainly causes canine respiratory diseases, urogenital tract diseases, anemia, arthritis and colitis. For example, dogs are susceptible to hemophilic mycoplasma, which leads to persistent fever, runny nose, loss of appetite, and in severe cases, acute clinical symptoms such as vomiting and diarrhea, and severe anemia or hemolytic anemia, and eventually death from bloody stools. Canine mycoplasma disease is often accompanied by mixed infections of mycoplasma, Escherichia coli, viruses, etc., and the condition is more severe.

[0007] The clinical symptoms of the above diseases are similar after infection, and mixed infection is easy to occur, which greatly interferes with clinical diagnosis. In addition, the current single canine pathogen detection is time-consuming and cannot achieve rapid and accurate detection and identification of multiple pathogens. Therefore, it is necessary to establish a rapid early diagnosis method that can simultaneously achieve a large number of different pathogens to save diagnosis time so that effective prevention and control measures can be taken as soon as possible. Summary of the invention

[0008] The present invention aims to solve the technical problems that the existing technology for detecting canine pathogens is mainly single detection, which is time-consuming and cannot simultaneously realize rapid and accurate detection and identification of multiple pathogens. The present invention aims to provide a primer pool and a kit that can simultaneously realize rapid diagnosis of multiple canine pathogens and detect multiple canine pathogens.

[0009] The primer pool and kit for simultaneously detecting multiple canine pathogens of the present invention include the following primer pairs and annihilation probes:

[0010] The CDV-F forward primer, CDV-R reverse primer and CDV-P annihilation probe for detecting canine distemper virus have nucleotide sequences shown in SEQ ID NOs: 1 to 3 in the sequence table;

[0011] The CAV-1-F forward primer, CAV-1-R reverse primer and CAV-1-P annihilation probe for detecting canine adenovirus have nucleotide sequences shown in SEQ ID NOs: 4 to 6 in the sequence table;

[0012] The CPIV-F forward primer, CPIV-R reverse primer and CPIV-P annihilation probe for detecting canine parainfluenza virus type II have nucleotide sequences shown in SEQ ID NOs: 7 to 9 in the sequence table.

[0013] Bb-F forward primer, Bb-R reverse primer and Bb-P annihilation probe for detecting Bordetella bronchiseptica, the nucleotide sequences of which are shown in SEQ ID NOs: 10 to 12 in the sequence table;

[0014] The nucleotide sequences of the MC-F forward primer, MC-R reverse primer and MC-P annihilation probe for detecting Mycoplasma canis are shown in SEQ ID NOs: 13 to 15 in the sequence table.

[0015] Preferably, it comprises the primer pool, PCR reaction solution and buffer as described in claim 1.

[0016] Preferably, the primer pool is a primer pool freeze-dried powder; the PCR reaction solution is a PCR reaction solution freeze-dried powder; and the buffer solution is a Tris buffer solution with a pH of 8.0.

[0017] Preferably, the PCR reaction solution freeze-dried powder comprises a heat-resistant DNA polymerase, a reverse transcriptase, an RNase inhibitor, a divalent magnesium ion and deoxyribonucleotide triphosphates.

[0018] Preferably, the PCR reaction solution freeze-dried powder and the primer pool freeze-dried powder are configured with the buffer to form a PCR reaction solution with the following concentration:

[0019] The concentration of the thermostable DNA polymerase is 0.01 to 1 unit / μL, preferably 0.01 to 0.1 unit / μL;

[0020] Reverse transcriptase 100-400 U / μL, preferably 100-150 U / μL;

[0021] RNase inhibitor 30-50U / μL, preferably 35-45U / μL;

[0022] The concentration of divalent magnesium ions is 1 to 4 mmol / L, preferably 2.5 to 3.5 mmol / L;

[0023] The concentration of deoxyribonucleotide triphosphate is 10 to 500 μmol / L, preferably 100 to 200 μmol / L;

[0024] The concentrations of each forward primer, each reverse primer and each annihilation probe are 10 to 500 nmol / L, preferably 100 to 200 nmol / L.

[0025] Preferably, the PCR reaction solution freeze-dried powder and the primer pool freeze-dried powder are configured with the buffer to form a PCR reaction solution with the following concentration:

[0026] The concentration of thermostable DNA polymerase was 0.05 units / μL;

[0027] The concentration of reverse transcriptase was 125 U / μL;

[0028] The concentration of RNase inhibitor was 40 U / μL;

[0029] The concentration of divalent magnesium ions is 3mmol / L;

[0030] The concentration of deoxyribonucleotide triphosphates was 200 μmol / L;

[0031] The concentrations of each forward primer, each reverse primer and each annihilation probe were 200 nmol / L, respectively.

[0032] Preferably, the kit further comprises PCR quality control products, and the PCR quality control products comprise positive quality control products and negative quality control products.

[0033] Preferably, the positive quality control product is a plasmid or nucleic acid template standard, and the negative quality control product is sterilized diethyl pyrocarbonate treated water.

[0034] Preferably, the nucleotide sequence of the plasmid or nucleic acid template standard is shown in SEQ ID NO: 16-20 in the sequence listing.

[0035] Preferably, the kit further comprises a microarray qPCR detection cartridge, wherein each sub-detection area in the microarray qPCR detection cartridge is respectively fixed with a fluorescent nucleic acid hybridized with a corresponding annihilation probe.

[0036] The positive and progressive effects of the present invention are:

[0037] The kit of the present invention is provided with a microarray qPCR detection card box, and each sub-detection area in the microarray qPCR detection card box is only fixed with the fluorescent nucleic acid of a certain pathogen, so that multiple pathogens in the sample can be detected and differentiated. At the same time, the present invention has made targeted optimization of the primer system on the basis of the multiplex PCR rapid detection kit, and designed a new primer pool, which further realizes the clinical specific and sensitive rapid detection of multiple canine pathogens. Compared with the existing single canine pathogen detection, the present invention can realize the simultaneous detection of five canine pathogens, including canine distemper virus, canine parainfluenza virus, canine adenovirus type II, Bordetella bronchiseptica, and Mycoplasma canis, through a single PCR amplification reaction system, and has strong specificity and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a CDV amplification curve diagram in Example 3 of the present invention;

[0039] Figure 2 is a CAV-1 amplification curve diagram in Example 3 of the present invention;

[0040] Figure 3 is a CPIV amplification curve diagram in Example 3 of the present invention;

[0041] Figure 4It is a Bb amplification curve diagram in Example 3 of the present invention;

[0042] Figure 5 is the MC amplification curve diagram in Example 3 of the present invention;

[0043] Figure 6 This is the amplification curve of the negative control product in Example 3 of the present invention. DETAILED DESCRIPTION

[0044] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention 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 invention.

[0045] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials and reagents used are all commercially available unless otherwise specified. The present invention provides a primer pool and a kit for simultaneously detecting multiple canine pathogens. The primer pool and the kit will be further described in conjunction with specific examples. The specific contents are as follows:

[0046] Example 1 Design of primer pool

[0047] Using bioinformatics knowledge and related bioinformatics software, the genome sequences of CDV, CAV-1, CPIV, Bb, and MC were compared and analyzed based on the NCBI (nt and bacterial virus) database. The sequences of these genes were compared with other microorganisms (including environmental and related microorganisms, such as Escherichia coli, canine parvovirus, etc.) using the public BLAST software in Genbank, and the sequence segments with specificity ≥ 98% were selected. Then, the corresponding upstream and downstream primers and probes were designed in these specific sequences in combination with the software Primer 5.0 and oligo7.

[0048] The design of primer probes follows three basic principles: first, the sequences of the primer and template must be closely complementary; second, the formation of stable dimers or hairpin structures between primers should be avoided; and third, the primer should not induce DNA polymerization at non-target sites in the template (i.e., mismatch).

[0049] A new primer pool was designed through the above process. The specific sequences of the primers and probes included in the primer pool are shown in Table 1 below:

[0050] Table 1 Primer and annihilation probe sequences

[0051]

[0052] It should be noted that the primer pool for simultaneously detecting multiple canine pathogens of the present invention includes but is not limited to the primers and annihilation probes in Table 1.

[0053] Example 2 Sensitivity test of the kit

[0054] As an example, the kit for simultaneously detecting multiple pathogens of dogs of the present invention comprises the primer pool freeze-dried powder of the primer pool described in Example 1, the PCR reaction solution freeze-dried powder and the Tris buffer of pH=8.0, wherein the PCR reaction solution freeze-dried powder comprises a heat-resistant DNA polymerase, a reverse transcriptase, an RNase inhibitor, a divalent magnesium ion and a triphosphate deoxyribonucleotide. The specific component concentrations of the reagent components of the kit for simultaneously detecting multiple pathogens of dogs of the present invention are: 0.05 units / μL of heat-resistant DNA polymerase, 125U / μL of reverse transcriptase, 40U / μL of RNase inhibitor, 3mmol / L of divalent magnesium ions, 200μmol / L of triphosphate deoxyribonucleotide, and 200nmol / L of the forward primer, reverse primer and annihilation probe in Table 1, respectively.

[0055] The kit of the present invention also includes a microarray qPCR detection cassette (flash microarray-qPCR reagent cassette, i.e., containing the multiple ligation probe microarray described in CN2019107011550), and each sub-detection area in the microarray qPCR detection cassette is respectively fixed with a fluorescent nucleic acid hybridized with a corresponding annihilation probe. The kit of the present invention also includes a PCR quality control product, which uses a plasmid or a nucleic acid template standard as a positive quality control product, and uses sterilized diethyl pyrocarbonate treated water as a negative quality control product.

[0056] The method for detecting the sensitivity of the kit for simultaneously detecting multiple canine pathogens of the present invention comprises the following steps:

[0057] Step S1, sample preparation. Five plasmid or nucleic acid template standards were placed in physiological saline. The five plasmid or nucleic acid template standards were produced by a commissioned supplier (Shanghai Sangon Biotechnology Co., Ltd.), and the sequences are shown in Table 2. Five target standards (concentration of 8×10 8 copy / mL), and then diluted with physiological saline to a concentration of 1.5×10 5 copy / mL, 1.5×10 4 copy / mL and 1.5×10 3 The three test sample concentrations were 10.0 copy / mL.

[0058] Table 2 Sequences of plasmid or nucleic acid template standards

[0059]

[0060] Step S2, detection kit sensitivity. Take 100 μL of the three samples diluted in step S1 respectively, and then add them to the sample injection port of the microarray qPCR detection card box in the kit of the present invention respectively (or use the CN201810022723X surface probe quantitative PCR method, or CN2020102299920 multiple quantitative PCR detection system based on universal probe chip is also possible). The disposable microarray qPCR detection card box contains the reagent components in the kit for simultaneous detection of multiple canine pathogens of the present invention, and the specific component concentrations are: the concentration of heat-resistant DNA polymerase is 0.05 units / μL, reverse transcriptase 125U / μL, RNase inhibitor 40U / μL, divalent magnesium ion concentration is 3mmol / L, triphosphate deoxyribonucleotide concentration is 200μmol / L, and the forward primer, reverse primer and annihilation probe in Table 1 are 200nmol / L respectively; the buffer in the microarray qPCR detection card box is a Tris buffer with a pH of 8.0.

[0061] The amplification detection experiment uses the FDx-1000 nucleic acid amplification detection analyzer (National Medical Device Registration No. 20223221095) of Shanliang Company as the corresponding fluorescent real-time quantitative PCR instrument (patent number: CN111621551B), and selects the preset rapid automatic detection program as 45 cycles. After detecting the model of the microarray qPCR detection cartridge, the device automatically runs the selected program and performs fully automatic amplification detection. Among them, the denaturation temperature of the PCR amplification detection is 92 degrees for 30 seconds, the renaturation temperature is 65 degrees for 45 seconds, and the amplification temperature is 72 degrees for 45 seconds. The FDx-1000 system automatically measures the Ct value of each detection target of the amplification reaction through the amplification curve: if Ct≤38, the sample is positive; if 38<Ct<40, the sample is a sample at the edge of sensitivity and needs to be resampled for detection; if Ct≥40, the sample is negative. At the same time, sterilized diethyl pyrocarbonate treated water is used as a negative quality control product as a control.

[0062] The test results are shown in Table 3. Through this experiment, it can be observed that the amplification system can stably detect virus samples with lower concentrations, even if the sample concentration is as low as 1.5×10 3 copy / mL can still be detected.

[0063] Table 3 Detection sensitivity results of 5 canine pathogen standards using the kit of the present invention

[0064]

[0065] Example 3 Specificity detection of the kit

[0066] The specific detection method of the kit for simultaneously detecting multiple canine pathogens of the present invention is as follows: the test samples are pet samples (inactivated cat clinical samples) with positive clinical infections of 7 kinds of CDV, CAV-1, CPIV, Bb, MC, Toxoplasma, and Trichomonas. The above 7 kinds of clinical positive samples and negative quality control products (sterilized diethyl pyrocarbonate treated water) are detected and analyzed using the kit of the present invention under the detection method of Example 2. The detection results are shown in Table 4.

[0067] Table 4 Specific detection results of 7 clinical positive samples and negative quality control products using the kit of the present invention

[0068]

[0069] Depend on Figures 1 to 6 As shown in Table 4, the negative-positive coincidence rate of the positive-negative samples used in Example 3 of the present invention is 100%, which shows that the specific detection experiment is effective. Among them, CDV, CAV-1, CPIV, Bb, and MC are all detected on the corresponding targets, and there is no amplification in the clinical positive samples of Toxoplasma gondii, the clinical positive samples of Trichomonas, and the negative quality control products containing other viruses, indicating that the kit of the present invention has good specificity.

[0070] The present invention is described in detail above in conjunction with the embodiments of the accompanying drawings. A person skilled in the art can make various variations of the present invention according to the above description. Therefore, some details in the embodiments should not be construed as limiting the present invention, and the present invention shall be protected by the scope defined by the attached claims.

Claims

1. A primer pool for simultaneously detecting multiple canine pathogens, characterized in that The primer pool includes the following primer pairs and annihilation probes: The CDV-F forward primer, CDV-R reverse primer and CDV-P annihilation probe for detecting canine distemper virus have nucleotide sequences shown in SEQ ID NOs: 1 to 3 in the sequence table; The CAV-1-F forward primer, CAV-1-R reverse primer and CAV-1-P annihilation probe for detecting canine adenovirus type II have nucleotide sequences shown in SEQ ID NOs: 4 to 6 in the sequence table; The CPIV-F forward primer, CPIV-R reverse primer and CPIV-P annihilation probe for detecting canine parainfluenza virus have nucleotide sequences shown in SEQ ID NOs: 7 to 9 in the sequence table. Bb-F forward primer, Bb-R reverse primer and Bb-P annihilation probe for detecting Bordetella bronchiseptica, the nucleotide sequences of which are shown in SEQ ID NOs: 10 to 12 in the sequence table; The nucleotide sequences of the MC-F forward primer, MC-R reverse primer and MC-P annihilation probe for detecting Mycoplasma canis are shown in SEQ ID NOs: 13 to 15 in the sequence table.

2. A kit for simultaneously detecting multiple pathogens in dogs, characterized in that: It comprises the primer pool, PCR reaction solution and buffer solution as described in claim 1.

3. The kit according to claim 2, characterized in that The primer pool is freeze-dried powder of the primer pool; the PCR reaction solution is freeze-dried powder of the PCR reaction solution; and the buffer solution is Tris buffer solution with a pH of 8.

0.

4. The kit according to claim 3, characterized in that The PCR reaction solution freeze-dried powder comprises a heat-resistant DNA polymerase, a reverse transcriptase, an RNase inhibitor, a divalent magnesium ion and deoxyribonucleotide triphosphate.

5. The kit according to claim 4, characterized in that The PCR reaction solution freeze-dried powder and the primer pool freeze-dried powder are configured with the buffer to form a PCR reaction solution with the following concentrations: The concentration of the thermostable DNA polymerase is 0.01 to 1 unit / μL; Reverse transcriptase 100-400 U / μL; RNase inhibitor 30-50U / μL; The concentration of divalent magnesium ions is 1 to 4 mmol / L; The concentration of deoxyribonucleotide triphosphates is 10 to 500 μmol / L; The concentrations of each forward primer, each reverse primer and each annihilation probe are 10 to 500 nmol / L, respectively.

6. The kit according to claim 5, characterized in that The PCR reaction solution freeze-dried powder and the primer pool freeze-dried powder are configured with the buffer to form a PCR reaction solution with the following concentrations: The concentration of the thermostable DNA polymerase is 0.01 to 0.1 units / μL; Reverse transcriptase 100-150 U / μL; RNase inhibitor 35-45U / μL; The concentration of divalent magnesium ions is 2.5-3.5 mmol / L; The concentration of deoxyribonucleotide triphosphates is 100-200 μmol / L; The concentrations of each forward primer, each reverse primer and each annihilation probe are 100-200 nmol / L, respectively.

7. The kit according to claim 6, characterized in that The PCR reaction solution freeze-dried powder and the primer pool freeze-dried powder are configured with the buffer to form a PCR reaction solution with the following concentrations: The concentration of thermostable DNA polymerase was 0.05 units / μL; The concentration of reverse transcriptase was 125 U / μL; The concentration of RNase inhibitor was 40 U / μL; The concentration of divalent magnesium ions is 3mmol / L; The concentration of deoxyribonucleotide triphosphates was 200 μmol / L; The concentrations of each forward primer, each reverse primer and each annihilation probe were 200 nmol / L, respectively.

8. The kit according to claim 2, characterized in that The kit also includes PCR quality control products, and the PCR quality control products include positive quality control products and negative quality control products.

9. The kit according to claim 8, characterized in that The positive quality control product is a plasmid or nucleic acid template standard product, and the negative quality control product is sterilized diethyl pyrocarbonate treated water.

10. The kit according to claim 9, characterized in that The nucleotide sequences of the plasmid or nucleic acid template standard are shown in SEQ ID NOs: 16 to 20 in the sequence table.

11. The kit according to claim 2, characterized in that The kit further comprises a microarray qPCR detection card box, and each sub-detection area in the microarray qPCR detection card box is respectively fixed with a fluorescent nucleic acid hybridized with a corresponding annihilation probe.

Images