Novel fluorescent quantitative PCR (Polymerase Chain Reaction) detection primer, kit and method for variable chalmavirus VP gene

Abstract

The invention provides a fluorescent quantitative PCR (polymerase chain reaction) detection primer, a kit and a method for a novel variable chalmavirus VP gene, and belongs to the technical field of virus detection. A specific primer is designed and synthesized according to a conserved sequence of a VP gene of the novel variant chalmavirus, a recombinant plasmid is constructed, then the recombinant plasmid is used as a standard substance to establish a standard curve, a real-time fluorescent quantitative PCR detection system aiming at the novel variant chalmavirus is constructed, and specific detection of the novel variant chalmavirus is realized. The detection method can be used for quantitatively detecting the novel variant chalmavirus in the to-be-detected sample, and has the characteristics of high speed, high sensitivity, strong specificity and the like; the method provides a key technical support for molecular epidemiological investigation of the Sepamavirus in animal populations, is helpful for realizing early screening of infected individuals and timely blocking of virus transmission, and has important practical significance for guaranteeing population health and maintaining high mountain ecosystem stability.

Description

Technical Field

[0001] This invention belongs to the field of virus detection technology, specifically relating to a novel fluorescent quantitative PCR detection primer, kit, and method for the VP gene of variant Chapamma virus. Background Technology

[0002] Chapama viruses belong to the subfamily Chaphamaparvovirinae. They are a group of simple, non-enveloped, tiny viruses with a diameter of approximately 18-26 nanometers, whose genetic material is linear single-stranded DNA. This family of viruses includes several subfamilies, such as the Virinae subfamily, which infects vertebrates, thus having a very wide host range. The core pathogenic mechanism of chapama viruses stems from their strict dependence on host cells being in a state of active division. This leads them to specifically attack and destroy rapidly dividing tissue cells. This characteristic explains why viruses from different subfamilies cause characteristic symptoms: for example, canine parvovirus causes acute hemorrhagic enteritis in puppies by destroying intestinal epithelial cells; while human parvovirus B19 causes erythema infectivity in children or aplastic crisis in anemic patients by infecting erythroid progenitor cells in the bone marrow. Furthermore, the non-enveloped structure of chapama viruses makes them highly resistant to environmental physical and chemical factors, which further promotes the effective transmission and long-term survival of the virus, mainly through the fecal-oral route.

[0003] The genome length of the Chapama virus is estimated to be between 4 and 6 kb, exhibiting the typical icosahedral structure of viruses in this family. The virus is presumed to primarily spread through the fecal-oral route within the host population. It is excreted in the feces of infected individuals, contaminating alpine meadows, saline lands, water sources, and ridges and cliffs frequented by animals. Susceptible individuals become infected through oral ingestion during foraging, licking, or drinking. Direct contact, especially close interaction during the summer when population densities are high or when sharing saline lands, is also an important transmission chain. The pathogenicity of Chapama virus varies significantly among individuals of different ages. It is most severe in young animals, causing severe gastroenteritis symptoms, including depression, loss of appetite, watery to bloody diarrhea, and dehydration. Dehydration and electrolyte imbalances caused by diarrhea severely weaken young animals, making it difficult for them to follow the herd during necessary migrations and climbs, thus increasing their susceptibility to death from exhaustion or secondary infections, threatening the annual survival rate of young animals in specific populations. Therefore, it is necessary to develop a highly specific and stable method for detecting chapam virus to provide key technical support for molecular epidemiological investigations of chapam virus in animal populations.

[0004] Real-time quantitative PCR (qPCR) has become an important technique for the quantitative detection of pathogens due to its outstanding advantages such as high sensitivity, strong specificity, accurate quantification, and rapid efficiency. There is a need to establish a SYBR Green I real-time quantitative PCR detection method with strong specificity and good stability to fill the current technological gap in the field of rapid detection of Chapamma virus. Summary of the Invention

[0005] To address some shortcomings in existing technologies, this invention provides a novel real-time quantitative PCR detection primer, kit, and method for the VP gene of a novel variant Chapamma virus. Based on the conserved sequence of the VP gene of the novel variant Chapamma virus, this invention designs and synthesizes specific primers and constructs the recombinant plasmid pcDNA3.1HA-pvirus-VP. Then, using the recombinant plasmid as a standard, a standard curve is established to construct a real-time quantitative PCR detection system for the novel variant Chapamma virus, achieving specific detection of the novel variant Chapamma virus. The detection method can quantitatively detect the novel variant Chapamma virus in samples, featuring high speed, high sensitivity, and strong specificity. This provides key technical support for molecular epidemiological investigations of Chapamma virus in animal populations, helps to achieve early screening of infected individuals and timely blocking of virus transmission, and has significant practical implications for ensuring population health and maintaining the stability of alpine ecosystems.

[0006] To achieve the above-mentioned technical objectives, the present invention employs the following technical means:

[0007] This invention first provides novel fluorescent quantitative PCR primers for detecting the VP gene of variant Chapamma virus, wherein the primers include an upstream primer PsNV-VP-qF and a downstream primer PsNV-VP-qR;

[0008] The nucleotide sequence of the upstream primer PsNV-VP-qF is shown in SEQ ID No:1: 5'-CACGCTAGGTGCTCAAGACA-3';

[0009] The nucleotide sequence of the downstream primer PsNV-VP-qR is shown in SEQ ID No:2: 5'-TGTGGGAAGAACGAGTCGTG-3'.

[0010] Preferably, the novel variant of the Chapama virus is Pseudois nayaur parvovirus, with accession number GeneBase:C_AA104993; the VP gene is located at 2732-4153 bp of the novel variant of the Chapama virus.

[0011] The present invention also provides a novel real-time quantitative PCR detection kit for variant chapam virus, the kit comprising the above-mentioned real-time quantitative PCR detection primers.

[0012] Preferably, the kit further comprises recombinant plasmid standards and real-time quantitative PCR reaction reagents;

[0013] The recombinant plasmid standard contains a DNA fragment of a novel variant of Chapamma virus;

[0014] The real-time quantitative PCR reaction reagents include SYBR Green Mix.

[0015] Preferably, the method for preparing the recombinant plasmid standard includes:

[0016] Primers were designed based on the VP gene of the novel variant Chapamma virus as a template. Using the cDNA of the novel Chapamma virus as a template, the DNA fragment of the VP gene of the novel variant Bocavirus was amplified by PCR. After ligation into a vector, the DNA fragment was transformed into competent E. coli cells. Positive clones were picked and plasmids were extracted to obtain recombinant plasmid standards.

[0017] Preferably, the primer sequences include the upstream primer PsNV-VP-F and the downstream primer PsNV-VP-R;

[0018] Upstream primer (PsNV-VP-F): 5'-ATGGCTGACTCAGTCTCGTT-3' (SEQ ID No:3);

[0019] Downstream primer (PsNV-VP-R): 5'-TTAGGTATGCATACGGGATA-3' (SEQ ID No:4).

[0020] The present invention also provides a novel real-time quantitative PCR detection method for variant Chapamma virus, the method being implemented based on the above-mentioned real-time quantitative PCR detection primers or the above-mentioned real-time quantitative PCR detection kit;

[0021] The method includes:

[0022] (1) Using recombinant plasmid standards of different concentrations as templates, real-time quantitative PCR was performed using primers shown in SEQ ID No:1 and SEQ ID No:2. A quantitative PCR standard curve was established based on the copy number concentration and Ct value of the recombinant plasmid standards.

[0023] (2) Extract total nucleic acid from the sample to be tested and reverse transcribe it into cDNA. Use the primers shown in SEQ ID No:1 and SEQ ID No:2 to perform real-time fluorescence quantitative PCR reaction. The corresponding Ct value of the sample to be tested is then substituted into the linear regression equation of the standard curve to achieve quantitative detection of the novel variant Chapam virus.

[0024] Preferably, the real-time quantitative PCR system in steps (1) and (2) includes: 1 μL 2×SYBR GreenⅠMix, 0.2 μL upstream primer PsNV-VP-qF, 0.2 μL downstream primer PsNV-VP-qR, 5 μL DNA template, and 3.6 μL ddH2O;

[0025] In the real-time quantitative PCR system, the final concentrations of PsNV-VP-qF and PsNV-VP-qR in the reaction system are 0.05-0.20 μM.

[0026] Preferably, the final concentrations of PsNV-VP-qF and PsNV-VP-qR in the reaction system are 0.20 μM.

[0027] Preferably, the reaction program for real-time quantitative PCR in steps (1) and (2) is as follows: pre-denaturation 95℃ 30s; cycling reaction: denaturation 95℃ 10s, annealing 50~60℃ 30s, 40 cycles; melting curve: extension 95℃ 15s, 60℃ 60s.

[0028] Preferably, the annealing temperature is 50°C.

[0029] The present invention also provides the application of the above-mentioned real-time quantitative PCR detection primers, or the above-mentioned real-time quantitative PCR detection kits, or the above-mentioned real-time quantitative PCR detection methods in the detection of novel variant chapam virus for non-therapeutic and diagnostic purposes.

[0030] Preferably, the application includes:

[0031] (1) Detection of chapamma infection; and / or

[0032] (2) Epidemiological investigation and monitoring of Parma virus infection status in animal populations; and / or

[0033] (3) Accurate quantitative analysis of viral load provides a basis for assessing the infection process and formulating prevention and control strategies.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] This invention, based on the conserved sequence of the VP gene of Chapamma virus, provides a novel primer, kit, and method for the detection of the VP gene of variant Chapamma virus using real-time quantitative PCR, filling a technological gap in the current field of rapid detection of Chapamma virus. The application of this method will provide crucial technical support for molecular epidemiological investigations of Chapamma virus in animal populations, facilitating early screening of infected individuals and timely interruption of virus transmission. This has significant practical implications for protecting population health and maintaining the stability of alpine ecosystems.

[0036] The method described in this invention has the following technical advantages:

[0037] (1) High specificity: The detection system established based on the real-time fluorescence quantitative PCR detection primers for the VP gene of Chapamma virus described in this invention has no cross-reaction with Sendai virus, porcine coronavirus, etc., showing high specificity and being able to accurately identify the target virus.

[0038] (2) High sensitivity: The detection method based on the primers has a detection limit of 1×10¹ copies / μL and a sensitivity of 100 times that of conventional PCR methods, which can effectively detect latent or early infected individuals with low viral load.

[0039] (3) Good repeatability and simple operation: The coefficients of variation of the method in intra-group and inter-group repeated experiments were less than 0.91% and 1.40%, respectively, showing excellent stability. The detection process does not require agarose gel electrophoresis, and the results are automatically interpreted by the system software, which significantly simplifies the operation process and shortens the detection time. The detection technology established by this invention provides key technical support for the formulation of effective wildlife virus prevention and control strategies, and is of great significance for reducing the risk of zoonotic disease transmission and ensuring public health and ecological security. Attached Figure Description

[0040] Figure 1 This is a graph showing the optimized annealing temperature for quantitative real-time PCR of novel Chapama virus (PsNV); the lanes from left to right are: 1: 2000 DNA ladder Marker; 2: 50℃; 3: 52℃; 4: 54℃; 5: 56℃; 6: 58℃; 7: 60℃; 8: negative control.

[0041] Figure 2 This is a diagram showing the optimized primer concentrations for quantitative real-time PCR of novel Chapama virus (PsNV); the lanes from left to right are: 1: 2000 DNA ladder marker; 2: 0.05 μM; 3: 0.10 μM; 4: 0.15 μM; 5: 0.20 μM; 6: negative control.

[0042] Figure 3This is a standard curve for quantitative real-time PCR of novel chapam virus (PsNV).

[0043] Figure 4 This is a comparison chart of the specificity verification of the novel chapam virus (PsNV) by real-time quantitative PCR; each amplification curve corresponds to: 1: recombinant plasmid pcDNA3.1HA-pvirus-VP standard; 2: vesicular stomatitis virus (VSV); 3: Sendai virus (SeV); 4: enterovirus 71 (EV71); 5: coxsackievirus B3 (CBV3); 6: blank control (ddH2O).

[0044] Figure 5 This is a sensitivity validation graph for real-time quantitative PCR of novel chapamma virus (PsNV); curves 1-8 in the graph correspond to 5.01×10⁻⁸. 8 Eight gradients of ~5.01×10¹ copies / μL, with 9 serving as a blank control for ddH2O.

[0045] Figure 6 This is a graph showing the sensitivity of routine PCR testing for the novel chapam virus (PsNV). Detailed Implementation

[0046] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. It should be noted that the following embodiments are merely typical implementations to illustrate the technical solution of the present invention, and their content does not constitute any limitation on the scope of protection of the present invention. The present invention has been described through specific embodiments, and those skilled in the art should understand that appropriate adjustments or modifications can be made to the described implementation schemes without departing from the principles of the present invention. Any equivalent substitutions, improvements, or changes made within the scope defined by the claims of the present invention should be considered to fall within the scope of protection of the present invention.

[0047] Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are conventional commercially available reagents or reagents prepared according to conventional methods.

[0048] Example 1: Design of novel primers for detecting variant Chapamma virus and construction of recombinant plasmid standard pcDNA3.1HA-pvirus-VP

[0049] The nucleotide sequence of the VP gene of the novel variant Chapamma virus (GeneBase: C_AA104993), located at positions 2732-4153 (1422 bp in length), was compared with that of bat Chapamma virus (GeneBank: OQ420634), sheep Chapamma virus (GeneBank: MW344047), mouse Chapamma virus (GeneBank: PV605140), and pangolin Chapamma virus (GeneBank: ON024117) using the MUSCLE function of MEGA software. Based on the MUSCLE analysis, specific primers PsNV-VP-qF and PsNV-VP-qR were designed to target the differentially expressed region of the novel Chapamma virus (positions 437-765 bp of the VP fragment).

[0050] Upstream primer PsNV-VP-qF: 5'-CACGCTAGGTGCTCAAGACA-3' (SEQ ID No:1);

[0051] Downstream primer PsNV-VP-qR: 5'-TGTGGGAAGAACGAGTCGTG-3' (SEQ ID No:2);

[0052] The amplification product was 151 bp in length, and the primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0053] The VP gene of the novel mutant Chapamma virus was compared with the viral sequences of bat Chapamma virus (GeneBank: OQ420634), sheep Chapamma virus (GeneBank: MW344047), mouse Chapamma virus (GeneBank: PV605140), and pangolin Chapamma virus (GeneBank: ON024117) using MEGA software. Based on PCR primer design principles, specific amplification primers were designed for the VP gene region to amplify the DNA fragment of the novel mutant Chapamma virus VP. The specific amplification primers are as follows:

[0054] Upstream primer (PsNV-VP-F): 5'-ATGGCTGACTCAGTCTCGTT-3' (SEQ ID No:3);

[0055] Downstream primer (PsNV-VP-R): 5'-TTAGGTATGCATACGGGATA-3' (SEQ ID No:4);

[0056] The target fragment size was 1422 bp; primers were synthesized by Shanghai Bioengineering Co., Ltd.

[0057] Fresh feces were collected from wild blue sheep or other blue sheep individuals confirmed to be infected with the novel variant Chapama virus in areas such as the Qinghai-Tibet Plateau. Immediately after collection, the feces were placed in ice packs for temporary storage and transported to the laboratory via a complete cold chain. Upon arrival at the laboratory, the samples were thoroughly resuspended in DPBS buffer, centrifuged, and the supernatant was collected. Nucleic acid was then extracted using a viral nucleic acid extraction kit (TaKaRa), and cDNA was obtained after reverse transcription. The cDNA was then used as a template for PCR amplification to obtain a 1422bp DNA fragment of the novel variant Chapama virus VP.

[0058] The collection and processing methods for fresh feces were based on the study "Viral metagenomics reveals parvovirus dark matter of herbivorous wildlife from the Qinghai Province-Xizang Autonomous Region Plateau" (DOI: 10.1186 / s12866-025-04281-0). After collection, metagenomic sequencing of the target samples revealed an unknown viral sequence. Full-length genome acquisition and evolutionary analysis confirmed it belonged to a novel variant of Chapama virus. The verification method for whether fresh feces were infected with the novel variant of Chapama virus was as follows: using the novel variant of Chapama virus as a template, qPCR detection was performed using specific primers PsNV-VP-qF and PsNV-VP-qR. Positive samples were confirmed, yielding fecal samples verified as infected with the novel variant of Chapama virus.

[0059] The PCR amplification reaction system described above was as follows: 1 μL cDNA template, 25 μL TaKaRa Taq, 0.8 μL PsNV-VP-F, 0.8 μL PsNV-VP-qR, and ddH2O added to a final volume of 50 μL.

[0060] The PCR reaction procedure is shown in Table 1:

[0061] Table 1. PCR amplification reaction procedure

[0062]

[0063] After the PCR reaction was completed, the amplification products were electrophoresed in a 1% agarose gel. The electrophoresis results were observed under a UV gel imaging system and the correct target fragment (1422bp) was cut out. The DNA fragment was then recovered using a DNA gel recovery kit (Kangwei Century Biotechnology Co., Ltd.).

[0064] The purified DNA fragment was ligated into the linearized pcDNA3.1HA vector (Nanjing Novizan Biotechnology Co., Ltd.). After mixing the ligation system, it was incubated at 16°C for 4 hours to obtain the ligation product. The ligation system consisted of 1 μL of pcDNA3.1-HA vector, 4 μL of Solution 1, and 5 μL of DNA fragment.

[0065] The ligation product was transformed into DH5α *E. coli* competent cells and cultured overnight at 37°C. Positive single colonies were then selected and enriched in LB broth containing ampicillin (1 μg / mL). Following this, a bacterial PCR reaction was performed (pre-denaturation 95°C for 5 min, denaturation 95°C for 30 s, annealing 50°C for 30 s, extension 72°C for 1 min 30 s, 30 cycles, extension 72°C for 10 min, storage 16°C for 2 min). Suitable bacterial cultures were sequenced (Sangon Biotech). Bacterial cultures with sequencing results identical to the target gene fragment were cultured overnight at 37°C. Plasmids were extracted using an endotoxin-free plasmid extraction kit (Kangwei Century Biotechnology Co., Ltd.) to obtain recombinant plasmid standards. The obtained recombinant plasmid OD260 / OD280 ratio was between 1.8 and 2.0, which can be used for subsequent standard curve establishment.

[0066] The concentration of the extracted plasmid was determined, and the DNA copy number was calculated using the following formula:

[0067] Copy number (copies / μL) = [concentration (ng / μL) × 6.022 × 10] 14 [DNA length (bp) × 660]

[0068] The extracted plasmid concentration was 374.6 ng / μL, and the copy number calculated using the above formula was 5.01 × 10⁻⁶. 10 copies / μL.

[0069] In this embodiment, the VP gene sequence of a novel variant of Chapam virus (GeneBase:C_AA104993) can also be directly synthesized, then ligated with the pcDNA3.1HA vector and transformed into DH5α Escherichia coli competent cells, and the plasmid can be extracted to obtain the recombinant plasmid standard pcDNA3.1HA-pvirus-VP.

[0070] Example 2: Optimization and Establishment of a SYBR Green I Real-Time Quantitative PCR Detection Method for Novel Variant Chapamma Virus

[0071] Using the recombinant plasmid standard pcDNA3.1HA-pvirus-VP obtained in Example 1 as a template, conventional PCR methods were employed. Six temperature gradients (50℃, 52℃, 54℃, 56℃, 58℃, 60℃) and four primer final concentration gradients (0.05μM, 0.1μM, 0.15μM, 0.2μM) were set within the range of 50-60℃ to optimize the primer annealing temperature and final primer concentration during PCR amplification. The optimization results are shown below. Figure 1 and Figure 2 As shown.

[0072] from Figure 1 and Figure 2 It can be seen that PsNV-VP-qF and PsNV-VP-qR are optimal when the final concentration in the reaction system is 0.2 μM and the annealing temperature is 50 °C.

[0073] The optimized reaction system is as follows:

[0074]

[0075] The optimized PCR amplification reaction procedure is as follows:

[0076]

[0077] Note: Different types of instruments require different melting curve acquisition programs. You can use the instrument's default melting curve acquisition program.

[0078] Example 3: Establishment of a standard curve for the SYBR Green I real-time quantitative PCR detection method for a novel variant of Chapamma virus

[0079] Based on the length (6819 bp) of the recombinant plasmid standard pcDNA3.1HA-pvirus-VP in Example 1 and the formula in Example 1, the copy number concentration was calculated to be 5.01 × 10⁻⁶. 10 copies / μL.

[0080] The specific steps are as follows:

[0081] First, dilute the recombinant plasmid standard to 10. 8 copies / μL, then serially diluted 10-fold to obtain a set from 10 8 ~10 1Standards were then used in SYBR GreenI real-time quantitative PCR, with different gradients of standards as templates. The real-time quantitative PCR system and reaction procedure were the same as those optimized in Example 2. Based on the real-time quantitative PCR data of novel Chapamma virus (PsNV), a standard curve was plotted with Ct value on the ordinate and the copy number logarithm of recombinant plasmid on the abscissa. Figure 3 ).

[0082] Linear regression analysis yielded the equation y = -3.357x + 34.31, with a correlation coefficient R² = 0.9987, confirming a good linear relationship between the two.

[0083] Example 4: Specificity validation of the SYBR GreenI real-time quantitative PCR method for detecting the VP gene of novel chapamma virus (PsNV).

[0084] This embodiment uses cDNA from five viruses (feline parvovirus (FPV), porcine deltacoronavirus (PDCoV), Sendai virus (SeV), vesicular stomatitis virus (VSV), and enterovirus 71 (EV71) – all preserved in our laboratory and publicly known materials – and the recombinant plasmid standard constructed in Example 1 as templates. Nuclease-free water was used as a blank control. SYBR Green I real-time quantitative PCR was performed according to the reaction system and procedure of Example 2. The results are as follows: Figure 4 As shown.

[0085] from Figure 4 As can be seen, except for the recombinant plasmid standard which showed a specific amplification curve, no significant amplification was observed in the other control viruses and blank control, indicating that this method has good detection specificity for the novel Chapam virus.

[0086] Example 5: Sensitivity validation of SYBR GreenI real-time quantitative PCR method for detecting novel chapamma virus (PsNV) VP gene

[0087] The recombinant plasmid standard pcDNA3.1HA-pvirus-VP was serially diluted 10-fold to obtain concentrations of 5.01×10¹ to 5.01×10¹. 8 A series of gradient standards were prepared at copy number / μL. Using each copy number standard as a template and ddH2O as a blank control, real-time quantitative PCR and conventional PCR were performed. The results are shown below. Figure 5 and Figure 6 As shown.

[0088] Combination Figure 5 and Figure 6It can be seen that the detection limit of conventional PCR is 5.01 × 10³ copies / μL, while real-time quantitative PCR can still effectively detect it at a level as low as 5.01 × 10¹ copies / μL. The results indicate that the detection sensitivity of the real-time quantitative PCR method established in this study is approximately 100 times that of conventional PCR.

[0089] Example 6: Repeatability test of SYBR GreenI real-time quantitative PCR method for detecting novel chapamma virus (PsNV) VP gene

[0090] In Example 1, five gradients (10) were selected. 3 ~10 8 The copies / μL) were used as templates for intra-group and inter-group repeated experiments, with each experiment performed 3 times, and the standard deviation (SD) and coefficient of variation (CV) were measured.

[0091] The average value of Ct (Ct̅) is calculated as (Ct1 + Ct2 + Ct3) / 3;

[0092] Standard deviation of detection (SD) = ,X i , (X) i (where Ct is the value and u is the average value).

[0093] Coefficient of variation (CV) = (SD / Ct̅) × 100%.

[0094] The test results are shown in Table 2.

[0095] Table 2. Repeatability test results

[0096]

[0097] As shown in Table 2, the SYBR Green I real-time quantitative PCR detection method for the novel chapamma virus (PsNV) VP gene had the highest intra- and inter-group repeatability coefficients of variation of 0.91% and 1.40%, respectively, indicating that the method exhibited good stability and repeatability at different repeatability levels.

[0098] In summary, this invention successfully establishes a real-time quantitative PCR detection method for the VP gene of novel Chapama virus (PsNV). This method possesses advantages such as high specificity, high sensitivity, good repeatability, and excellent stability, providing a reliable technical means for the rapid identification and in-depth study of Chapama virus.

[0099] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. 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 shall fall within the protection scope of the present invention.

Claims

1. A novel primer for real-time PCR detection of the VP gene of variant Chapamma virus, characterized in that, The real-time quantitative PCR detection primers include the upstream primer PsNV-VP-qF and the downstream primer PsNV-VP-qR; The nucleotide sequence of the upstream primer PsNV-VP-qF is shown in SEQ ID No:1: 5'-CACGCTAGGTGCTCAAGACA-3'; The nucleotide sequence of the downstream primer PsNV-VP-qR is shown in SEQ ID No:2: 5'-TGTGGGAAGAACGAGTCGTG-3'.

2. The real-time fluorescence quantitative PCR detection primers according to claim 1, characterized in that, The novel variant of parvovirus is Pseudois nayaur parvovirus, with accession number GeneBase:C_AA104993; the VP gene is located at 2732-4153bp of the novel variant of parvovirus.

3. A novel real-time quantitative PCR detection kit for variant chapam virus, characterized in that, The real-time quantitative PCR detection kit contains the real-time quantitative PCR detection primers as described in claim 1.

4. The real-time fluorescence quantitative PCR detection kit according to claim 3, characterized in that, The real-time quantitative PCR detection kit also includes recombinant plasmid standards and real-time quantitative PCR reaction reagents; The recombinant plasmid standard contains a DNA fragment of a novel variant of the Chapamma virus; The real-time quantitative PCR reaction reagents include SYBR Green Mix.

5. The real-time fluorescence quantitative PCR detection kit according to claim 4, characterized in that, The preparation method of the recombinant plasmid standard includes: Primers were designed based on the VP gene of the novel variant Chapamma virus as a template. Using the cDNA of the novel Chapamma virus as a template, the DNA fragment of the VP gene of the novel variant Bocavirus was amplified by PCR. After ligation into a vector, the DNA fragment was transformed into competent E. coli cells. Positive clones were picked and plasmids were extracted to obtain recombinant plasmid standards.

6. The real-time fluorescence quantitative PCR detection kit according to claim 5, characterized in that, The primer sequences include the upstream primer PsNV-VP-F and the downstream primer PsNV-VP-R; Upstream primer PsNV-VP-F: 5'-ATGGCTGACTCAGTCTCGTT-3' (SEQ ID No:3); Downstream primer PsNV-VP-R: 5'-TTAGGTATGCATACGGGATA-3' (SEQ ID No:4).

7. A novel real-time quantitative PCR detection method for variant chapam virus, characterized in that, The method is implemented based on the real-time fluorescence quantitative PCR detection primers according to claim 1, or the real-time fluorescence quantitative PCR detection kit according to any one of claims 3-6; The method includes: (1) Using recombinant plasmid standards of different concentrations as templates, real-time quantitative PCR was performed using primers shown in SEQ ID No:1 and SEQ ID No:

2. A quantitative PCR standard curve was established based on the copy number concentration and Ct value of the recombinant plasmid standards. (2) Extract total nucleic acid from the sample to be tested and reverse transcribe it into cDNA. Use the primers shown in SEQ ID No:1 and SEQ ID No:2 to perform real-time fluorescence quantitative PCR reaction. The corresponding Ct value of the sample to be tested is then substituted into the linear regression equation of the standard curve to achieve quantitative detection of the novel variant Chapam virus.

8. The real-time fluorescence quantitative PCR detection method according to claim 7, characterized in that, The real-time quantitative PCR system for steps (1) and (2) includes: 1 μL 2×SYBR GreenⅠMix, 0.2 μL upstream primer PsNV-VP-qF, 0.2 μL downstream primer PsNV-VP-qR, 5 μL DNA template, and 3.6 μL ddH2O; In the real-time quantitative PCR system, the final concentrations of PsNV-VP-qF and PsNV-VP-qR in the reaction system are 0.05-0.20 μM; The reaction procedures for real-time quantitative PCR in steps (1) and (2) are as follows: pre-denaturation 95℃ 30s; cycling reaction: denaturation 95℃ 10s, annealing 50~60℃ 30s, 40 cycles; melting curve: extension 95℃ 15s, 60℃ 60s.

9. The application of the real-time quantitative PCR detection primers according to claim 1 or 2, or the real-time quantitative PCR detection kit according to any one of claims 3-6, or the real-time quantitative PCR detection method according to any one of claims 7-8 in the detection of novel variant chapam virus for non-therapeutic and diagnostic purposes.

10. The application according to claim 9, characterized in that, The applications include: (1) Detection of chapamma infection; and / or (2) Epidemiological investigation and monitoring of Parma virus infection status in animal populations; and / or (3) Accurate quantitative analysis of viral load provides a basis for assessing the infection process and formulating prevention and control strategies.

Images