Fluorescent quantitative PCR (Polymerase Chain Reaction) detection primer, kit and method for VP (Virus Pvovirus) gene of Candida parvovirus

Abstract

The invention discloses a fluorescent quantitative PCR (Polymerase Chain Reaction) detection primer, a kit and a method for a VP (Virus Pvovirus) gene of a civilus parvovirus, and belongs to the technical field of virus detection. The primer provided by the invention has specificity in detection of the poop parvovirus, based on the primer, the invention provides a kit and a detection method for detecting the poop parvovirus VP gene, and experiments prove that the primer and the method provided by the invention can be used for detecting the poop parvovirus PuBOV, and can be used for detecting the poop parvovirus VP gene. The kit has the advantages of good stability, strong specificity, good repeatability and accurate quantification, the lowest detection concentration of PuBOV is 1 * 10 < 1 > copies / [mu] L and reaches 1000 times that of common PCR, and the kit has good detection effect and application value.

Description

Technical Field

[0001] The invention relates to primers, a kit and a method for real-time fluorescence quantitative PCR detection of the snow leopard parvovirus VP gene, and belongs to the technical field of virus detection. Background Art

[0002] Parvoviruses belong to the Parvoviridae family and are small, non-enveloped viruses with icosahedral symmetry and a diameter of approximately 18-26 nanometers. Their genomes consist of single-stranded DNA, approximately 4-6 kilobase pairs (kb) in length. Depending on the virus species, the genome can be either positive-stranded or negative-stranded. The Parvoviridae family encompasses multiple genera, each infecting a diverse host population, encompassing a wide range of mammals and humans. Parvoviruses are transmitted in various ways, primarily through the fecal-oral route, where the virus is excreted in the host's feces, contaminating the environment, and other animals can become infected after ingesting contaminated material. The virus can also be transmitted through direct contact and airborne transmission, and some viruses, such as B19, can be vertically transmitted to the fetus across the placenta. Parvoviruses are highly pathogenic to young animals and humans. Infection in young animals often results in severe gastrointestinal symptoms, such as vomiting, diarrhea, and bloody stools, as well as respiratory symptoms, such as coughing and difficulty breathing. For example, canine parvovirus can cause acute hemorrhagic enteritis in puppies, with a high mortality rate. Symptoms of human infection with parvoviruses, such as B19, vary depending on age and immune status. Children and adolescents may experience mild respiratory symptoms or a rash, while adults are usually asymptomatic or have mild symptoms. However, in immunocompromised individuals, infection can cause severe illness.

[0003] Snow leopards (Panthera uncia) are endangered species primarily found in Tibet, Qinghai, Xinjiang, Gansu, Sichuan, and Inner Mongolia in my country. As a feline species, snow leopards are susceptible to parvovirus-related diseases. Researchers have identified a new parvovirus from wild snow leopard fecal samples using viral metagenomics. Bioinformatics analysis of high-throughput sequencing yielded the full genome sequence of this snow leopard parvovirus (GenBank No. OQ627713). Comparison with the NCBI genome revealed that the newly detected virus shares only 77.67% nucleotide identity with the known feline parvovirus (GenBank No. MT633128). The VP gene shares 78.17% nucleotide identity with the known feline parvovirus (GenBank No. MT633128). Phylogenetic analysis suggests that this virus may be a new species of the genus Bocaparvovirus. However, at present, the research on snow leopard parvovirus is still in its initial stage, and a sensitive and effective detection system has not yet been established, making it impossible to effectively detect snow leopard parvovirus.

[0004] Real-time fluorescence quantitative PCR detection methods have been widely used in the quantitative detection of pathogenic pathogens due to their advantages such as high sensitivity, strong specificity, accurate quantification, and rapid detection. The present invention aims to establish an effective real-time fluorescence quantitative PCR detection method for the VP gene of snow leopard parvovirus, which can fill the technical gap in snow leopard parvovirus detection methods, and is expected to carry out more extensive molecular biological detection of snow leopard parvovirus, timely blocking the spread of the virus, which is of great significance for protecting the health of snow leopards and maintaining the stability of the species. Summary of the Invention

[0005] The purpose of the present invention is to address some problems existing in the prior art and provide a fluorescent quantitative PCR detection primer, a kit and a method for snow leopard parvovirus VP gene to achieve rapid, effective, specific and highly sensitive quantitative detection of the virus.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention first provides a fluorescent quantitative PCR detection primer for the VP gene of snow leopard parvovirus, the primer sequences of which are shown in SEQ ID No: 1 and SEQ ID No: 2:

[0008] SEQ ID No: 1 (upstream primer PuBOV-VP-F):

[0009] 5'-AGAAACTGCCGGACGCGGAA-3',

[0010] SEQ ID No: 2 (downstream primer PuBOV-VP-R):

[0011] 5′-TGCCCTGGTTTTGACGGGCA-3′.

[0012] Furthermore, the target fragment amplified by the primers has a size of 104 bp;

[0013] Furthermore, the snow leopard parvovirus includes PuBOV (Panthera uncia bocaparvovirus), whose gene sequence is accession number No. OQ627713 in GenBank.

[0014] Furthermore, the snow leopard parvovirus VP gene is located at positions 3118-5256 of GenBank sequence number No. OQ627713.

[0015] The present invention also provides a snow leopard parvovirus fluorescence quantitative PCR detection kit, which comprises the primers.

[0016] Furthermore, the kit also includes a recombinant plasmid standard and a real-time fluorescence quantitative PCR reaction reagent, and the real-time fluorescence quantitative PCR reaction reagent includes SYBR Green Mix.

[0017] Further preferably, the method for preparing the recombinant plasmid standard is as follows: primers are designed according to the VP gene sequence of the snow leopard parvovirus, the DNA fragment of the snow leopard parvovirus is amplified, and after connecting to the vector, it is transformed into Escherichia coli competent cells, and positive clones are picked to extract the plasmid to obtain the recombinant plasmid standard.

[0018] More preferably, the primer sequences used in the preparation of the recombinant plasmid standard are as shown in SEQ ID No: 3 and SEQ ID No: 4.

[0019] SEQ ID No: 3 and SEQ ID No: 4 are as follows:

[0020] Upstream primer Pu-F: 5'-ATGGCACCAACTAACAGGCGTC-3' (SEQ ID No: 3)

[0021] Downstream primer Pu-R: 5'-TTACAGAACTTTATTGATTCCG-3' (SEQ ID No: 4)

[0022] The present invention also provides a fluorescent quantitative PCR detection method for snow leopard parvovirus, which comprises:

[0023] (1) Using recombinant plasmid standards of different concentrations as templates, fluorescent quantitative PCR was performed using primers shown in SEQ ID No: 1 and SEQ ID No: 2, and a fluorescent quantitative standard curve was established based on the concentration and Ct value of the recombinant plasmid standards;

[0024] (2) Extract the DNA of the sample to be tested, and use the primers shown in SEQ ID No: 1 and SEQ ID No: 2 to perform fluorescent quantitative PCR reaction. The corresponding Ct value of the sample to be tested is obtained and substituted into the linear regression equation of the standard curve to achieve quantitative detection of snow leopard parvovirus.

[0025] Furthermore, in the fluorescent quantitative PCR reaction, the final concentration of the primers in the reaction system is 1-2 μM. Preferably, the final concentration of the primers in the reaction system is 1.75 μM.

[0026] Furthermore, the annealing temperature of the fluorescent quantitative PCR reaction is 54-58°C, preferably, the annealing temperature is 57°C.

[0027] The present invention also provides applications of the primers, the kit, or the method in detecting snow leopard parvovirus, including: application in detecting snow leopard parvovirus infection, conducting epidemiological surveys on snow leopard parvovirus infection, accurately detecting the copy number of snow leopard parvovirus, and understanding the infection process of snow leopard parvovirus.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The present invention provides a real-time fluorescence quantitative PCR detection primer for the VP gene of snow leopard parvovirus. The detection primer of the present invention has strong specificity. The real-time fluorescence quantitative PCR detection system established with the primer for the newly identified new strain of snow leopard parvovirus PuBOV has strong specificity and has no cross-reaction with other viruses (such as Sendai virus (SeV), CVA6, CVB3, etc.).

[0030] (2) The present invention further provides a detection method based on the real-time fluorescence quantitative PCR detection primers for snow leopard parvovirus. The detection method provided by the present invention has high sensitivity and can detect the virus at 1×10 8 copies / μL to 1×10 1 There is a good linear relationship in the range of copies / μL, and the lowest detection template concentration is 1×10 1 copies / μL, which is 1000 times that of ordinary PCR and can efficiently detect snow leopards infected with or carrying PuBOV.

[0031] (3) The detection method of the present invention is fast and efficient, and the test results can be displayed directly through the software without the need for presentation through methods such as agarose gel electrophoresis, which saves detection time and simplifies the experimental steps. It can detect PuBOV efficiently and quickly, which is conducive to carrying out more extensive molecular biological detection of snow leopard parvovirus and timely blocking the spread of the virus, which is of great significance to protecting the health of snow leopards and maintaining the stability of the species. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a graph showing the annealing temperature optimization results for the SYBR Green I fluorescent quantitative PCR detection method for the VP gene of the new snow leopard parvovirus (PuBOV) strain in Example 2. From left to right in the figure are: M: DNA ladder marker; 1: 56°C; 2: 56.5°C; 3: 57°C; 4: 57.5°C; 5: 58°C; 6: H2O.

[0033] Figure 2This figure shows the primer concentration optimization results for the SYBR Green I fluorescent quantitative PCR detection method for the VP gene of the new strain of snow leopard parvovirus (PuBOV) in Example 2. From left to right in the figure are: M: DNA ladder marker; 1: final primer concentration 1 μM; 2: final primer concentration 1.25 μM; 3: final primer concentration 1.5 μM; 4: final primer concentration 1.75 μM; 5: final primer concentration 2 μM; 6: H2O.

[0034] Figure 3 The standard curve of the SYBR Green I fluorescence quantitative PCR detection method of the VP gene of the new strain of snow leopard parvovirus (PuBOV) in Example 3 is shown; the logarithm of the copy number concentration of the recombinant plasmid standard is the abscissa, the Ct value is the ordinate, the linear equation is y=-3.541x+38.95, and the correlation coefficient (R 2 ) is 0.9989.

[0035] Figure 4 The sensitivity validation results of the fluorescent quantitative PCR detection method for the VP gene of the new strain of snow leopard parvovirus (PuBOV) in Example 4 are as follows: 1:1×10 8 copies / μL; 2: 1×10 7 copies / μL; 3: 1×10 6 copies / μL; 4: 1×10 5 copies / μL; 5: 1×10 4 copies / μL; 6: 1×10 3 copies / μL; 7: 1×10 2 copies / μL; 8: 1×10 1 copies / μL; 9:H2O.

[0036] Figure 5 The sensitivity validation gel images of the conventional quantitative PCR detection method for the VP gene of the new strain of snow leopard parvovirus (PuBOV) in Example 5 are shown in the figure. From left to right in the figure, they are: 1:1×10 8 copies / μL; 2: 1×10 7 copies / μL; 3: 1×10 6 copies / μL; 4: 1×10 5 copies / μL; 5: 1×10 4 copies / μL; 6: 1×10 3 copies / μL; 7: 1×10 2 copies / μL; 8: 1×10 1copies / μL; 9:H2O.

[0037] Figure 6 The results of specificity verification of the SYBR Green I fluorescent quantitative PCR detection method for the VP gene of the new strain of snow leopard parvovirus (PuBOV) in Example 6 are shown in FIG. 1 is a recombinant plasmid standard; 2-5 are in order from top to bottom, namely 2: H2O; 3: CVA6 virus; 4: CVB3; 5: Sendai virus. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to better understand the technical solutions of the present invention, preferred embodiments of the present invention are described in detail below. However, the following embodiments do not limit the scope of protection of the present invention.

[0039] In the embodiments of the present invention, conventional experimental methods are used unless otherwise specified. The processes involved in the embodiments are all understandable and easily implementable by those skilled in the art based on the product instructions or basic knowledge in the field. The reagents, materials, etc. involved are all conventional methods and conventional commercially available reagents unless otherwise specified.

[0040] Example 1: Preparation of VP gene standard of new strain of snow leopard parvovirus (PuBOV)

[0041] The complete genome sequences of other feline parvoviruses were downloaded from the GenBank database (access link: https: / / www.ncbi.nlm.nih.gov / nuccore / ?term=Feline+bocaparvovirus+3) and compared with the sequence of PuBOV (GenBank No. OQ627713). Sequences were aligned using Mega software (v6.19.9). Specific primers were designed based on the conserved region of the VP gene according to the principles of real-time fluorescence quantitative PCR primer design:

[0042] Pu-F: 5'-ATGGCACCAACTAACAGGCGTC-3' (SEQ ID No: 3)

[0043] Pu-R: 5'-TTACAGAACTTTATTGATTCCG-3' (SEQ ID No: 4)

[0044] The amplified target fragment was 2,139 bp in size, namely the VP gene sequence.

[0045] The primers were synthesized by Shanghai Bioengineering Co., Ltd. The nucleic acid was extracted using a viral nucleic acid extraction kit (TaKaRa) and PCR amplification was performed to obtain the target gene fragment.

[0046] The nucleic acid was extracted from snow leopard feces, which were collected by the inventors in a mountainous area of Heilongjiang Province in northeast China using disposable sterile containers, then transported to the laboratory for storage on dry ice, and tested for samples containing PuBOV virus. The samples were the same as those in the article "Viral metagenomic analysis reveals diverse viruses and a novel bocaparvovirus in the enteric virome of snow leopard (Panthera uncia) [J]. Microelectronics Journal, 2024, 10(8).". Snow leopard feces can also be provided by zoos.

[0047] Approximately 1 g of fecal sample was resuspended in 2 mL of phosphate-buffered saline (PBS), vigorously vortexed for 5 minutes, and centrifuged at 15,000 g for 10 minutes. To remove bacterial and eukaryotic cell-sized particles, the supernatant was collected and filtered through a 0.45 μm filter (Merck Millipore, MA, USA). The virus-enriched filtrate was collected and treated with a cocktail of various nucleases (DNase: TurboDNase, Ambion, USA; Baseline-ZERO, Epicentre, USA; Benzonase, Novagen, Germany; RNase: Promega, USA) at 37°C for 60 minutes to digest unprotected nucleic acids. Viral nucleic acids were then extracted using a kit.

[0048] The PCR amplification reaction system and reaction conditions are as follows:

[0049] PCR amplification reaction system:

[0050] Reagents Reaction volume (μL) DNA template 5 Pu-F 1 Pu-R 1 TakaRa rTaq enzyme 25 <![CDATA[ddH2O]]> 18

[0051] PCR amplification reaction conditions:

[0052]

[0053] After the PCR reaction, the product was electrophoresed on a 1% agarose gel. The electrophoresis results were observed under a UV gel imager, and the specific band corresponding to the target gene fragment size (2,139 bp) was excised and recovered using a DNA gel recovery kit (purchased from Kangwei Century Biotechnology Co., Ltd.). The recovered and purified target gene fragment was ligated with the pMD18-T vector (purchased from Nanjing Novezan Biotechnology Co., Ltd.) using the following ligation system:

[0054]

[0055]

[0056] After mixing the reagents, incubate at 16°C for 4 hours. The ligation product was transformed into DH5α Escherichia coli competent cells, and the positive clone bacterial solution was selected for sequencing (Sangon Biotechnology). The bacterial solution with the sequencing result exactly the same as the target gene fragment sequence was selected and cultured overnight at 37°C. The plasmid was extracted using an endotoxin-free plasmid extraction kit (purchased from Kangwei Century Biotechnology Co., Ltd.) to obtain the recombinant plasmid standard.

[0057] In this embodiment, the VP gene sequence in PuBOV (GenBank No. OQ627713) can also be directly commissioned for synthesis, ligated with the pMD18-T vector, transformed into DH5α Escherichia coli competent cells, and the plasmid is extracted to obtain a recombinant plasmid standard.

[0058] The concentration of the extracted recombinant plasmid was measured and the DNA copy number was calculated using the following formula: copy number (copies / μL) = Atschoff constant (6.02×10 23 )×plasmid concentration (500ng / μL)×10 -9 / fragment size (bp) × 660 ≈ 2.13 × 10 11 copies / μL.

[0059] Example 2: SYBR Green I fluorescence quantitative PCR detection method and condition optimization of Snow Leopard Parvovirus (PuBOV) VP gene

[0060] As described in Example 1, the complete genome sequences of other feline parvoviruses (GenBank No. MT633128, PP541575, ON595886, and KM017744) and the sequence of PuBOV (GenBank No. OQ627713) were downloaded from the GenBank database, and sequence alignment was performed using Mega software (v6.19.9). Specific primers were designed using partial gene differential region sequences (or hypervariable region sequences) of VP according to the principles of real-time fluorescence quantitative PCR primer design:

[0061] PuBOV-VP-F: 5'-AGAAACTGCCGGACGCGGAA-3' (SEQ ID No: 1)

[0062] PuBOV-VP-R: 5'-TGCCCTGGGTTTTGACGGGCA-3' (SEQ ID No: 2)

[0063] The amplified fragment was 104 bp, which was the partial gene difference region of the VP.

[0064] The primers were also synthesized by Shanghai Bioengineering Co., Ltd. Using the recombinant plasmid standard obtained in Example 1 as a template, 5 gradient annealing temperatures (56°C, 56.5°C, 57°C, 57.5°C, 58°C) and 5 gradient primer final concentrations (1 μM, 1.25 μM, 1.5 μM, 1.75 μM, 2 μM) were set for reaction optimization:

[0065] (1) Optimization of primer annealing temperature

[0066] First, a conventional PCR method was used with a total reaction volume of 10 μL. 1 μL of the recombinant plasmid standard was used as a template, 0.2 μL of each upstream and downstream primer (PuBOV-VP-F / PuBOV-VP-R), and 5 μL of TakaRa rTaq enzyme were added to 10 μL with ddH2O. The reaction conditions were 95°C pre-denaturation for 5 minutes, 95°C denaturation for 30 seconds, and annealing for 30 seconds. Five annealing temperature gradients were set, from left to right: 56°C, 56.5°C, 57°C, 57.5°C, and 58°C for 1 minute. The above three steps were cycled 30 times, and then extended at 72°C for another 10 minutes. The reaction products were detected by electrophoresis, and the results were as follows: Figure 1 As shown, Figure 1 It can be seen that the band specificity is strongest when the annealing temperature is 57°C.

[0067] (2) Optimization of final primer concentration

[0068] Conventional PCR was still used, with a total reaction volume of 10 μL. 1 μL of the recombinant plasmid standard was used as the template. The final concentrations of the upstream and downstream primers were 1 μM, 1.25 μM, 1.5 μM, 1.75 μM, and 2 μM, respectively. 5 μL of TakaRa rTaq enzyme was added to 10 μL with ddH2O. The reaction conditions were pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for 1 minute. The above three steps were cycled 30 times, and then extended at 72°C for another 10 minutes. The reaction products were detected by electrophoresis, and the results were as follows: Figure 2 As shown, Figure 2 As can be seen from the figure, the band specificity is strongest when the final primer concentration is 1.75 μM.

[0069] The final reaction system and reaction conditions are as follows:

[0070] Reaction system:

[0071] Reagents Reaction volume (μL) DNA template 1 PuBOV-VP-F (1.75 μM) 0.2 PuBOV-VP-R (1.75 μM) 0.2 SYBR Green Mix 5 ddH2O 3.6

[0072] Reaction conditions:

[0073]

[0074] Example 3: Establishment of a standard curve for SYBR Green I fluorescence quantitative PCR detection of the VP gene of a new strain of snow leopard parvovirus (PuBOV)

[0075] According to the recombinant plasmid copy number obtained in Example 1, the sample concentration was first diluted to 1×10 8 Then, a set of recombinant plasmid standards with different concentrations (1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 Using recombinant plasmid standards of different concentrations as templates, the reaction system and conditions were the same as those optimized in Example 2, and finally the Ct values corresponding to different samples (i.e., recombinant plasmid standards of different concentrations) were obtained.

[0076] The logarithm of the copy number concentration of the recombinant plasmid standard was used as the abscissa and the Ct value was used as the ordinate to obtain the linear regression equation and establish a standard curve.

[0077] The results are as follows Figure 3 As shown, when the concentration of the standard is 1×10 1 ~1×10 8 When the Ct value was 0.01, the logarithm of the recombinant plasmid copy number concentration had a good linear relationship, and the linear equation was y = -3.541x + 38.95. The correlation coefficient (R 2 ) is 0.9989.

[0078] Example 4: Sensitivity Verification of SYBR Green I Fluorescence Quantitative PCR Detection Method for VP Gene of New Snow Leopard Parvovirus (PuBOV)

[0079] A set of recombinant plasmid standards (1×10 1 ~1×10 8 Copies / μL) were used as templates, and nuclease-free water was used as blank control. Conventional PCR and SYBR GreenⅠ fluorescence quantitative PCR were performed respectively.

[0080] The total reaction volume of conventional PCR was 10 μL, with 1 μL of DNA template, 1.75 μM final concentrations of upstream and downstream primers (PuBOV-VP-F / PuBOV-VP-R), 5 μL of TakaRa rTaq enzyme, and ddH2O added to 10 μL. The reaction conditions were pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 1 min. The above three steps were cycled 30 times, and then extended at 72°C for 10 min.

[0081] Fluorescence quantitative PCR adopted the reaction system and reaction conditions optimized in Example 2.

[0082] The experimental results are as follows Figure 4 and Figure 5 As shown in the figure, in a 10 μL reaction system, the detection limit of conventional PCR is 10 4 copies / μL, while the detection limit of real-time fluorescence quantitative PCR is 10 1 copies / μL, which improves the detection sensitivity by a thousand times compared with the former.

[0083] Example 5: Specificity Verification of SYBR Green I Fluorescence Quantitative PCR Detection Method for the VP Gene of the New Snow Leopard Parvovirus (PuBOV)

[0084] Sendai virus, CVA6, and CVB3 viruses (all preserved in this laboratory and publicly known materials) and the recombinant plasmid standard obtained in Example 1 were used as templates, and nuclease-free water was used as a blank control. SYBR Green I fluorescence quantitative PCR detection was performed according to the reaction system and conditions in Example 2.

[0085] The results are as follows Figure 6 As shown in the figure, 1 is a recombinant plasmid standard; 2-5 are H2O; CVA6 virus; CVB3; Sendai virus; it can be seen from the figure that except for the recombinant plasmid standard, other samples did not obtain typical amplification curves. The results show that the SYBR GreenⅠ fluorescent quantitative PCR detection method for the new strain of snow leopard parvovirus established by the present invention can specifically detect snow leopard parvovirus, but cannot perform amplification reactions on Sendai virus, Coxsackievirus A6 (CVA6), and Coxsackievirus B3 (CVB3).

[0086] Example 6: Verification of Repeatability and Stability of SYBR Green I Fluorescence Quantitative PCR Detection Method for VP Gene of New Snow Leopard Parvovirus (PuBOV)

[0087] Select 1×10 from Example 3 4 ~1×10 8Recombinant plasmid standards, containing five different copies / μL, were used as templates for three replicates each between and within each group. The mean Ct value, standard deviation (SD), and coefficient of variation (CV) were determined. The results are shown in Table 1. The maximum inter-group coefficient of variation for the SYBR Green I fluorescence quantitative PCR assay for the novel snow leopard parvovirus (PuBOV) was 2.82%, and the maximum intra-group coefficient of variation was 1.05%, demonstrating good reproducibility and stability.

[0088] in,

[0089] Mean Ct Value u=total Ct value / number of repetitions,

[0090] X i is the Ct value each time, u is the average value,

[0091] Coefficient of variation (CV) = (standard deviation / mean) x 100%.

[0092] Table 1. Repeatability test results of SYBR Green I fluorescence quantitative PCR detection method for VP gene of snow leopard parvovirus (PuBOV)

[0093]

[0094] In summary, the present invention has established a fluorescent quantitative PCR detection method for the VP gene of a new strain of snow leopard parvovirus (PuBOV). This method has high specificity, strong sensitivity, good repeatability and stability, and provides reliable technical support for the research and identification of snow leopard parvovirus.

[0095] The embodiments described are preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention are within the scope of protection of the present invention.

Claims

1. Fluorescence quantitative PCR detection primers for snow leopard parvovirus VP gene, characterized in that: The primer sequences are shown as SEQ ID No: 1 and SEQ ID No:

2.

2. The primer according to claim 1, characterized in that The snow leopard parvovirus includes PuBOV.

3. A snow leopard parvovirus fluorescent quantitative PCR detection kit, characterized in that: The kit comprises the primer according to claim 1 or 2.

4. The kit according to claim 3, wherein The kit further comprises a recombinant plasmid standard and a fluorescent quantitative PCR reaction reagent, wherein the fluorescent quantitative PCR reaction reagent comprises SYBR Green Mix.

5. The kit according to claim 4, characterized in that The preparation method of the recombinant plasmid standard is as follows: Primers were designed based on the VP gene of snow leopard parvovirus to amplify the DNA fragment of snow leopard parvovirus. After ligation with the vector, the fragment was transformed into competent Escherichia coli cells. Positive clones were picked to extract the plasmid and obtain the recombinant plasmid standard.

6. The kit according to claim 5, characterized in that The primer sequences are shown in SEQ ID No: 3 and SEQ ID No:

4.

7. A fluorescent quantitative PCR detection method for snow leopard parvovirus, characterized in that: The method comprises: (1) Using recombinant plasmid standards of different concentrations as templates, using the primers described in claim 1 or 2, perform fluorescence quantitative PCR, and establish a fluorescence quantitative standard curve based on the concentration and Ct value of the recombinant plasmid standards; (2) Extract the DNA of the sample to be tested, use the primers described in claim 1 or 2 to perform fluorescent quantitative PCR reaction, and obtain the corresponding Ct value of the sample to be tested, and then substituting it into the linear regression equation of the standard curve to achieve quantitative detection of snow leopard parvovirus.

8. The method according to claim 7, characterized in that In the fluorescent quantitative PCR reaction, the final concentration of the primers in the reaction system is 1-2 μM. Preferably, the final concentration of the primers in the reaction system is 1.75 μM.

9. The method according to claim 7, characterized in that The annealing temperature of the fluorescent quantitative PCR reaction is 54-58°C, preferably, the annealing temperature is 57°C.

10. Use of the primer according to claim 1 or 2, or the kit according to any one of claims 3 to 6, or the method according to any one of claims 7 to 9 in detecting snow leopard parvovirus.

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