Fluorescent quantitative PCR (Polymerase Chain Reaction) primer group and detection kit for detecting herpesvirus type 2 and application of fluorescent quantitative PCR primer group and detection kit

Abstract

The invention discloses a fluorescent quantitative PCR (Polymerase Chain Reaction) primer group and a detection kit for detecting type-2 agama herpesvirus and application of the fluorescent quantitative PCR primer group and the detection kit. According to the invention, a pair of specific primers IghV2-F and IghV2-R are designed for a gene conserved region of DNA-dependent DNA polymerase (pol) of iamyl herpesvirus type 2 (IghV2), and an SYBR Green dye fluorescent quantitative PCR detection method is established, so that the primer has the advantages of high sensitivity, high specificity, wide linear range, rapidness, simplicity, convenience, good repeatability and the like. The invention provides important technical support for herpesvirus infection monitoring, quarantine and protection work of rare and endangered lizard species, fills the blank of detection methods in the field, and is suitable for non-clinical medical fields such as scientific research, epidemiological investigation, quarantine monitoring, protection management and the like.

Description

Technical Field

[0001] This invention belongs to the field of wildlife pathogen detection and molecular diagnostic technology, specifically involving a fluorescent quantitative PCR primer set, detection kit, and its application for detecting iguana herpesvirus type 2. Background Technology

[0002] Herpesviridae is a family of double-stranded DNA viruses widely distributed in vertebrates. They possess the ability to establish latent infection and periodically reactivate, posing a persistent threat to host health and aquaculture. Based on host range and genomic characteristics, Herpesviridae is divided into three subfamilies: α, β, and γ. In veterinary and aquaculture fields, various herpesviruses have been proven to cause serious diseases: porcine herpesvirus type 1 (PRV-1, belonging to the alphaherpesvirus subfamily) causes pseudorabies, manifested as neurological symptoms, respiratory distress, and reproductive disorders in sows, resulting in significant economic losses to the pig industry; bovine herpesvirus type 1 (BoHV-1) causes infectious rhinotracheitis, which can lead to respiratory infections, abortion, and decreased milk production; equine herpesvirus types 1 and 4 (EHV-1 / 4) are closely associated with respiratory diseases, neurological diseases, and abortion in horses; and in aquatic animals, carp herpesvirus type 3 (CyHV-3) can cause mass mortality in koi and carp, clinically manifested as gill necrosis, renal hemorrhage, and respiratory distress.

[0003] Herpesvirus was also detected in lizards. This occurred in a variegated iguana with diffuse liver necrosis. Sauromalus varius Iguanid herpesvirus 2 (IgHV 2) was detected in green iguanas and Iguanid HV 2 was successfully isolated (Wellehan et al., 2003). Iguana iguana Iguanid HV 1, which can cause cytopathic effects, was isolated from cell cultures of the spleen, kidney, and heart (Clark and Karzon, 1972). In green monitor lizards with proliferative stomatitis... Varanus prasinus Varanid HV 1 was detected in the oral mucosa and brain of the endangered Galapagos terrestrial pink iguana (Literak et al., 2010). Conolophus marthae A novel herpesvirus, Iguanid Alphaherpesvirus 4 (IgHV4), was detected in the virus (Ainoa Nieto-Claudín et al., 2024).

[0004] DNA polymerase (pol / UL30 homology) is a core conserved gene of herpesviruses, suitable as a molecular detection target. The pol gene is essential for viral DNA replication, withstands strong evolutionary selection pressure, and exhibits significantly higher sequence conservation than easily variable genes such as envelope glycoproteins. Viruses within the same subfamily show high amino acid homology with pol, simultaneously meeting the needs for broad-spectrum detection and type identification. Databases such as NCBI provide clear and comprehensive reference sequence annotations for the herpesvirus pol gene, with well-defined conserved regions, facilitating the design of highly specific and sensitive primers. In molecular epidemiological studies of amphibian and reptile herpesviruses (such as green turtle herpesvirus and tortoise herpesvirus), pol and gB (UL27) are the most commonly used PCR targets. Broad-spectrum PCR and semi-nested PCR methods based on the conserved regions of pol have been widely validated in human and animal herpesvirus detection, proving their maturity and reliability.

[0005] Currently, the detection of IghV2 mainly relies on traditional methods, which have obvious technical limitations: (1) Virus isolation and culture: Although it is the gold standard for etiological diagnosis, it requires specific cell lines (such as reptile-derived cell lines) and strict culture conditions, takes a long time (usually 2-4 weeks), has high technical requirements, and many herpesviruses are difficult to culture in vitro, resulting in a low detection rate. (2) Conventional PCR: Although it can detect viral nucleic acid, its sensitivity is limited (the detection limit is usually around 10). 3 -10 4 (Copies / µL) can only be used for qualitative analysis and cannot accurately assess viral load. Furthermore, it requires subsequent operations such as agarose gel electrophoresis, which increases the risk of contamination and detection time. (3) Serological methods: Due to the lack of specific antibody reagents and the latent infection characteristics of herpesvirus, antibody detection is difficult to reflect the current infection status; (4) Histopathological examination: Although characteristic pathological changes (such as intranuclear inclusions) can be observed, biopsy or autopsy samples are required. The operation is highly invasive and not suitable for live monitoring of rare and endangered species. Furthermore, it is not possible to make a diagnosis in the early stage of infection or during the incubation period.

[0006] The limitations of the aforementioned methods severely restrict early diagnosis, epidemiological surveillance, and disease control of IghV2 infection, especially for lizard populations, failing to meet the requirements for non-invasive, rapid, sensitive, and accurate detection. Furthermore, there are currently no reports on SYBR Green dye-based quantitative PCR detection methods and kits for IghV2. Therefore, there is an urgent need to establish a highly specific, sensitive, rapid, and accurate quantitative PCR detection system for IghV2 to provide crucial technical support for disease surveillance, quarantine, scientific research, and conservation management of lizard species. Summary of the Invention

[0007] To overcome the shortcomings and deficiencies of existing technologies, this invention aims to provide a primer set, detection kit, and application for the detection of iguana herpesvirus using quantitative real-time PCR. Specifically, it provides a SYBR Green dye-based quantitative real-time PCR primer set, detection kit, and detection method for iguana herpesvirus type 2 (IghV2). This primer set is designed targeting a highly conserved region of the DNA-dependent DNA polymerase (pol) gene in the IghV2 genome, exhibiting excellent specificity and sensitivity, enabling rapid, accurate, and quantitative detection of IghV2.

[0008] The first objective of this invention is to provide a primer set for detecting iguana herpesvirus type 2, the primer set comprising an upstream primer IghV2-F and a downstream primer IghV2-R, the nucleotide sequence of the upstream primer IghV2-F being shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer IghV2R being shown in SEQ ID NO.2.

[0009] A second objective of this invention is to provide a real-time quantitative PCR kit for detecting iguana herpesvirus type 2, comprising the aforementioned primer set and SYBR Green dye-based real-time PCR reagent.

[0010] Preferably, the real-time PCR kit further includes a positive control and a negative control, wherein the positive control is a recombinant plasmid containing the pol gene from the iguana herpesvirus type 2 genome, and the negative control is ddH2O.

[0011] Preferably, the concentration of the recombinant plasmid containing the pol gene from the iguana herpesvirus type 2 genome is 10. 7 The recombinant plasmid containing the pol gene from the iguana herpesvirus type 2 genome can be diluted to different concentrations as needed to construct a standard curve for quantification of iguana herpesvirus type 2 in the sample to be tested.

[0012] Preferably, the GenBank accession number of the pol gene in the iguana herpesvirus type 2 genome is NC_043063.1.

[0013] A third objective of this invention is to provide a real-time quantitative PCR method for detecting iguana herpesvirus type 2 for non-disease diagnostic purposes, comprising the following steps: S1. Extract DNA from the sample to be tested; S2. Using the aforementioned real-time PCR kit, perform real-time PCR amplification with the DNA obtained in step S1 as a template; S3. After the reaction is completed, determine whether the sample to be tested contains iguana herpesvirus type 2 based on the amplification curve. The method of determination is as follows: if the Ct value is ≤35 and the amplification curve is typical S-shaped, then the sample to be tested is determined to contain iguana herpesvirus type 2; if the Ct values ​​are all >35 or there is no Ct value, then the sample to be tested is determined not to contain iguana herpesvirus type 2.

[0014] Preferably, the reaction system for the real-time PCR amplification is 10 µL of 2×SYBR Green real-time PCR reaction premix, 0.6 µL of 10 μM upstream primer IghV2-F (as shown in SEQ ID NO.1), 0.6 µL of 10 μM downstream primer IghV2R (as shown in SEQ ID NO.2), 2 µL of DNA from the sample to be tested, and ddH2O to bring the total volume to 20 µL.

[0015] Preferably, the reaction procedure for the quantitative real-time PCR amplification is as follows: pre-denaturation at 95℃ for 30 s; followed by 40 cycles, each cycle including denaturation at 95℃ for 5 s and annealing extension at 60℃ for 30 s, with fluorescence signals collected during the annealing extension step; after amplification, melting curve analysis is performed, with the temperature increasing from 60℃ to 95℃ in increments of 0.5℃ / 5 s.

[0016] The beneficial effects of this invention are: (1) Originality and innovation: This invention establishes for the first time a SYBR Green dye-based real-time PCR detection system for iguana herpesvirus type 2, filling the technological gap in this field and providing a new technical means for pathogen monitoring of rare lizard species.

[0017] (2) High sensitivity: detection limit reaches 1.54×10 0 The limit of detection per reaction is 3.08 copies / µL, which is 100-1000 times more sensitive than conventional PCR. It can detect infections in the early stages or when the viral load is low, making early diagnosis and disease control possible.

[0018] (3) High specificity: The primers are designed for highly conserved regions of the IghV2 pol gene and have been verified to have no cross-reaction with other common reptile pathogens (including snake herpesvirus, bearded dragon adenovirus AgAdv-1, Aeromonas hydrophila, Morganella morganii, and Dermophila thunis). The specificity is 100%, avoiding false positive results.

[0019] (4) Wide linear range and accurate quantification: The standard curve is within 1.54 × 10 1 -8.7×10 7The virus exhibits good linearity within the range of copies / µL (R²=0.991), with an amplification efficiency of 96% (ideal amplification efficiency is 90-110%), enabling accurate absolute quantification of viral load and providing quantitative data support for infection severity assessment, treatment monitoring, and epidemiological studies.

[0020] (5) Fast and simple: The entire detection process, from DNA extraction to result determination, takes only about 2 hours (30 minutes for DNA extraction and 90 minutes for PCR amplification and analysis). It is easy to operate and suitable for rapid screening in grassroots laboratories and field sites.

[0021] (6) Cost-effective: As an important branch of qPCR, the SYBR Green dye method generates a fluorescent signal by non-specific binding of fluorescent dye to double-stranded DNA. There is no need to design and synthesize expensive fluorescent probes. The detection cost is about 1 / 3 to 1 / 2 of that of the TaqMan probe method. It has the characteristics of low cost, simple operation and wide applicability, and is suitable for large-scale screening and use in resource-limited areas.

[0022] (7) Wide range of applications: It is suitable for various sample types (oral swabs, cloacal swabs, whole blood, tissues, etc.), meeting different needs for live monitoring, quarantine screening and pathological research, and can be extended to different lizard species that may be infected with IghV2. Attached Figure Description

[0023] Figure 1 The results show the optimized reaction conditions for iguana herpesvirus type 2 SYBR Green dye-based quantitative PCR; a and b show the positive standard (10) under different primer concentrations (200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 650 nM), respectively. 6 Melting and amplification curves for quantitative real-time PCR were obtained using copies / µL.

[0024] Figure 2 Iguana herpesvirus type 2, 1.54 × 10⁻⁶ 7 -1.54×10 1 Amplification curve of SYBR Green dye-based quantitative PCR reaction using copies / µL of positive standards.

[0025] Figure 3 Amplification curves (a) and melting curves (b) for the specific detection of iguana herpesvirus type 2 by SYBR Green dye-based quantitative PCR are shown. Among them, 1 is the amplification curve of IghV2, and 2-7 are the amplification curves and melting curves of snake herpesvirus, bearded dragon adenovirus AgAdv-1, Aeromonas hydrophila, Morganella morganii, Dermophilic bacteria of turtles and negative control, respectively.

[0026] Figure 4 The results of sensitivity detection of iguana herpesvirus type 2 using SYBR Green dye-based quantitative PCR are shown in the figure. The figure displays different concentrations of positive standards for iguana herpesvirus type 2 (1.54 × 10⁻⁶). 7 -1.54×10 0 Amplification curves of SYBR Green dye-based quantitative PCR reaction (copies / µL) were obtained.

[0027] Figure 5 To use positive standards at different concentrations (1.54 × 10⁻⁶) 7 -1.54×10 0 A standard curve was constructed using SYBR Green dye-based quantitative PCR reaction results with copies / µL as templates. Detailed Implementation

[0028] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0029] Example 1: Primer design, synthesis and reaction system optimization 1.1 Target gene selection and primer design The DNA-dependent DNA polymerase (pol) gene of IgHV2 (Iguanid herpesvirus 2) (GenBank accession number: NC_043063.1) was selected as the detection target. The pol gene is one of the core genes of the Herpesviridae family, encoding a DNA polymerase necessary for viral DNA replication. It is highly conserved among different herpesviruses, while also exhibiting sufficient sequence differences between species, making it suitable as a target for species-specific detection.

[0030] Design primers using the following steps: (1) Download the complete DNA-dependent DNA polymerase (pol) gene sequence (NC_043063.1) of IghV2 and the remaining annotated pol gene sequences of the virus from the NCBI GenBank database; (2) Multiple sequence alignment was performed using MEGA 7.0 software to determine the conserved and variable regions of the pol gene; (3) Primers were designed in the conservative region using Primer Premier 5.0 and Oligo 7.0 software. The design principles were: primer length 18-25 bp, GC content 40-60%, Tm value 58-62℃, to avoid hairpin structure, primer dimer and mismatch. (4) Use the NCBI BLAST tool for specificity analysis to ensure that the primers only match the IghV2 sequence and have no significant homology with other species (including the host crocodile lizard) and other pathogens; (5) The following primer pairs were finally determined: Upstream primer IghV2-F: 5'-CTGGGCTACCAGAGAGAATCTA-3' (SEQ ID NO.1, 22 bp); Downstream primer IghV2-R: 5'-ACTGAGTCGGTGTCTCCATA-3' (SEQ ID NO.2, 20 bp).

[0031] The amplification product of this primer pair is 105 bp in length and is located in a conserved region of the pol gene (gene sequence position: 403-507 bp). The physicochemical parameters of the primers are shown in Table 1.

[0032] Table 1 Physicochemical parameters of primers Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., purified by HPLC, dissolved in nuclease-free water, and prepared into a 100 μM stock solution, which was stored at -20℃ for later use.

[0033] 1.2 Construction of positive standards To establish a standard curve and perform quantitative detection, positive standards containing the target gene fragment need to be constructed. The specific steps are as follows: (1) Gene fragment design: A 780 bp fragment containing the primer binding site and its upstream and downstream sequences in the IghV2 pol gene was selected as the standard template. The nucleotide sequence of this fragment is shown in SEQ ID NO.3.

[0034] (2) Gene synthesis: The above 780 bp fragment was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and directly cloned into the SmaI restriction site (multiple cloning site) of the pUC57 vector to obtain the recombinant plasmid pUC57-IghV2-pol.

[0035] (3) Plasmid transformation and screening: The recombinant plasmid was transformed into Escherichia coli Top10 competent cells, plated on LB solid medium containing 100 μg / mL ampicillin, and incubated overnight at 37°C. Single colonies were picked for colony PCR identification, and positive clones were sent for sequencing verification.

[0036] (4) Large-scale plasmid extraction: The positive clones with correct sequencing were inoculated into 5 mL LB liquid medium (containing 100 μg / mL ampicillin) and cultured at 37℃ with shaking for 12-16 hours. Plasmids were extracted using a plasmid mini-prep kit (DP103, Tiangen Biotech Co., Ltd.).

[0037] (5) Plasmid concentration determination and copy number calculation: The plasmid concentration was determined to be 57.2 ng / µL using a NanoDrop 2000 spectrophotometer, and the A260 / A280 ratio was 1.88, indicating good plasmid purity. The full-length plasmid was 3370 bp (2590 bp of pUC57 vector + 780 bp of insert fragment). The plasmid copy number was calculated using the following formula: Copy number (copies / µL) = [concentration (ng / µL) × 6.02 × 10] 23 The plasmid concentration was calculated to be 1.54 × 10⁻⁶ / [plasmid length (bp) × 660]. 10 copies / µL.

[0038] (6) Plasmid preservation: Dilute the plasmid with nuclease-free water to a concentration of 1.54 × 10⁻⁶. 8 Aliquots / µL, aliquot and store at -80°C for later use. Perform 10-fold serial dilutions before use to prepare a 1.54×10⁻⁶ oz. 7 -1.54×10 1 positive standards in copies / µL series.

[0039] 1.3 Optimization of the reaction system To obtain the best amplification efficiency and specificity, the primer concentration in the real-time PCR reaction system was optimized.

[0040] 1.3.1 Primer Concentration Optimization In a 20 µL quantitative PCR reaction system, add 10 µL of SYBR Green qPCR Mix, and add forward primers (IghV2-F) and reverse primers (IghV2-R) with final concentrations of 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, and 650 nM, respectively. 6A positive standard of 2 µL (copies / µL) was used as a template, and ddH2O was added to bring the total volume to 20 µL. The amplification effect was tested at primer concentrations of 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, and 650 nM. The reaction program was as follows: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s; 60℃ annealing extension for 30 s; 40 cycles. FAM fluorescence signal was collected at the 60℃ step. After amplification, melting curve analysis was performed, with the temperature increasing from 60℃ to 95℃ in increments of 0.5℃ / 5 s.

[0041] The results show that ( Figure 1 When the primer concentration is 300 nM, the Ct value is the lowest (Ct=18.66), the amplification efficiency is the highest, and there is no non-specific amplification or primer dimer formation. When the primer concentration is too low (200 nM), the Ct value is too high, and the sensitivity decreases. When the primer concentration is too high (≥500 nM), primer dimer formation is likely to occur, leading to increased background fluorescence. Therefore, the optimal primer concentration was determined to be 300 nM.

[0042] 1.3.2 Optimal Reaction System Based on the above optimization results, the optimal reaction system (total volume of 20 µL) is determined as shown in Table 2.

[0043] Table 2. qPCR reaction system for detecting IghV2 1.3.3 Optimal Reaction Procedure Based on the above optimization results, the determined real-time PCR reaction procedure is shown in Table 3.

[0044] Table 3. qPCR reaction procedure for detecting IghV2 Using this optimized reaction system and procedure, at 1.54 × 10 7 -1.54×10 1 The positive standards in copies / µL series were used as templates for detection. The amplification results are shown in the figure. Figure 2 The amplification curves showed a typical S-shape, with Ct values ​​differing between dilutions by approximately 3.3 cycles, consistent with the theoretical value for a 10-fold dilution.

[0045] Instruments and reagents: All quantitative PCR experiments were performed on a Roche LightCycler 480 II real-time PCR instrument. SYBR Green qPCR Mix (SYBR Green quantitative PCR reaction premix) was purchased from Takara (product number: RR820A), and nuclease-free water was purchased from Thermo Fisher Scientific.

[0046] Example 2: Specificity Detection To verify the specificity of the method established in Example 1, six common reptile pathogens were selected for cross-reactivity testing. These six common reptile pathogens include: 1. IghV2 (Iguana herpesvirus type 2): Positive control, recombinant plasmid pUC57-IghV2-pol, concentration 10 6 copies / µL; 2. Opheodrys herpesvirus 1: DNA extracted from oral swabs of infected snakes (corn snakes); 3. Bearded dragon adenovirus (Agamid Adenovirus 1, AgAdv-1): DNA extracted from the feces of diseased iguanas; 4. Aeromonas hydrophila: Laboratory-preserved strains, genomic DNA extracted; 5. Morganella morganii: Laboratory-preserved strains from which genomic DNA was extracted; 6. *Austwickia chelonae*: A fungus isolated from skin lesions of diseased turtles; genomic DNA was extracted from it. 7. Negative control: Nuclease-free water.

[0047] Use the DNA or plasmid of the above pathogens as a template (2 µL of each template, DNA concentration of approximately 50 ng / µL or 10 ng / µL). 6 (copies / µL) were used for real-time PCR amplification and detection according to the reaction system optimized in Example 1. Three technical replicates were set for each sample.

[0048] Result: As Figure 3 As shown in a, only the IghV2 positive control showed a typical S-shaped amplification curve with a Ct value of 21.87±0.15 (n=3). A Ct value ≤35 and a typical S-shaped amplification curve indicate a positive result. The other five pathogens and the negative control showed no amplification signal, with Ct values ​​>35 or no Ct value, indicating a negative result. Melting curve ( Figure 3 Analysis b) showed that the IghV2 positive control exhibited a single melting peak with a Tm value of 80.3℃, while other samples showed no melting peak.

[0049] The results demonstrate that the primer set IghV2-F / IghV2-R is highly specific to IghV2 (100% specificity) and has no cross-reactivity with other common reptile pathogens (including Opheodrys herpesvirus 1, which is also a herpesvirus), thus avoiding false positive results.

[0050] Example 3: Sensitivity Measurement To determine the detection limit (sensitivity) of the method established in Example 1, the positive standard pUC57-IghV2-pol (initial concentration 1.54 × 10⁻⁶) was used. 10 The concentration gradient was obtained by serially diluting the sample (copies / µL) 10-fold to obtain the following concentration gradient: 1.54 × 10⁻⁶. 7 1.54×10 6 1.54×10 5 1.54×10 4 1.54×10 3 1.54×10 2 1.54×10 1 1.54×10 0 1.54×10 -1 copies / µL.

[0051] Each concentration gradient was performed in triplicate, and the reaction system optimized in Example 1 was used for quantitative real-time PCR amplification and detection. A positive result was defined as a Ct value ≤ 35, an amplification curve exhibiting a typical S-shape, and a melting curve showing a single peak.

[0052] Result: As Figure 4 As shown, the concentration is ≥1.54×10 0 All dilution gradients of copies / µL were stably detected, and the average Ct values ​​and coefficients of variation for each concentration are shown in Table 4.

[0053] Table 4. Mean Ct values ​​and coefficients of variation for positive standards at different concentrations When the concentration is 1.54 × 10 0 At a concentration of 1.54 × 10⁻⁶ copies / µL, three repeated assays all yielded positive results with a Ct value ≤ 35 (Ct = 33.90 ± 0.25), the amplification curve showed a typical S-shape, and the melting curve showed a single peak (Tm = 80.3℃); when the concentration was reduced to 1.54 × 10⁻⁶, positive results were obtained. -1When the concentration was set to copies / µL, no amplification signal was observed, indicating that the concentration was below the stable detection limit. Therefore, the detection limit (sensitivity) of this method was determined to be 1.54 × 10⁻⁶. 0 The limit of detection for each reaction is 3.08 copies / µL (2 µL template × 1.54 copies / µL).

[0054] This sensitivity is higher than that of conventional PCR methods (the detection limit is typically 10). 3 -10 4 The number of copies / µL increased by approximately 100-1000 times, reaching the single-copy level, enabling detection in the early stages of infection or when the viral load is low.

[0055] Example 4: Standard Curve Establishment and Linearity Range Verification To achieve accurate quantification of viral load, a standard curve needs to be established. Different concentrations of positive standards (1.54 × 10⁻⁶) from Example 3 were used. 7 -1.54×10 1 Using copies / µL (7 concentration gradients in total) as templates, and with 3 technical replicates for each concentration, the reaction system established in Example 1 was used for real-time PCR amplification.

[0056] The logarithm of the number of copies per microliter of starting template (log 10 Using (copies / µL) as the x-axis and Ct as the y-axis, perform linear regression analysis and plot the standard curve.

[0057] Result: As Figure 5 As shown, at 1.54×10 1 -1.54×10 7 Within the concentration range of copies / µL, the standard curve exhibits a good linear relationship. The equation of the standard curve is: y = -3.4164x + 39.176, where y is the Ct value and x is logarithm. 10 (copies / µL).

[0058] The quality parameters of the standard curve are shown in Table 5.

[0059] Table 5 Quality parameters of the standard curve The amplification efficiency (E) is calculated using the formula: E = (10 -1 / 斜率 -1) × 100% = (10 -1 / (-3.4164) -1) ×100% = 96%.

[0060] The above results show that the established standard curve has excellent linearity (R²=0.991) over a wide concentration range of 7 orders of magnitude, and the amplification efficiency is close to the theoretical value of 100% (actually 96%, within the ideal range of 90-110%). It can be used for accurate absolute quantification of IghV2 viral DNA copy number in the sample to be tested.

[0061] In practical applications, one or more samples can be collected from lizards, such as oral swabs, cloacal swabs, whole blood, serum, plasma, tissue homogenate, or organ tissue.

Claims

1. A primer set for detecting iguana herpesvirus type 2, characterized in that, The primer set includes an upstream primer IghV2-F and a downstream primer IghV2-R. The nucleotide sequence of the upstream primer IghV2-F is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer IghV2R is shown in SEQ ID NO.

2.

2. A real-time PCR kit for detecting iguana herpesvirus type 2, characterized in that, It contains the primer set as described in claim 1 and the SYBR Green dye-based real-time PCR reagent.

3. The reagent kit according to claim 2, characterized in that, It also includes a positive control and a negative control, wherein the positive control is a recombinant plasmid containing the pol gene from the iguana herpesvirus type 2 genome, and the negative control is ddH2O.

4. The reagent kit according to claim 3, characterized in that, The concentration of the recombinant plasmid containing the pol gene from the iguana herpesvirus type 2 genome is 10. 7 Copies / µL or more.

5. The reagent kit according to claim 3, characterized in that, The GenBank accession number for the pol gene in the iguana herpesvirus type 2 genome is NC_043063.

1.

6. A real-time quantitative PCR method for detecting iguana herpesvirus type 2 for non-disease diagnostic purposes, characterized in that, Includes the following steps: S1. Extract DNA from the sample to be tested; S2. Using the aforementioned real-time PCR kit, perform real-time PCR amplification with the DNA obtained in step S1 as a template; S3. After the reaction is completed, determine whether the sample to be tested contains iguana herpesvirus type 2 based on the amplification curve. The method of determination is as follows: if the Ct value is ≤35 and the amplification curve is typical S-shaped, then the sample to be tested is determined to contain iguana herpesvirus type 2; if the Ct values ​​are all >35 or there is no Ct value, then the sample to be tested is determined not to contain iguana herpesvirus type 2.

7. The method according to claim 6, characterized in that, The reaction system for the quantitative real-time PCR amplification was 10 µL of 2×SYBR Green quantitative real-time PCR reaction premix, 0.6 µL of 10 μM upstream primer IghV2-F (as shown in SEQ ID NO.1), 0.6 µL of 10 μM downstream primer IghV2R (as shown in SEQ ID NO.2), 2 µL of DNA from the sample to be tested, and ddH2O to bring the total volume to 20 µL.

8. The method according to claim 6, characterized in that, The reaction procedure for the quantitative real-time PCR amplification is as follows: pre-denaturation at 95℃ for 30 s; followed by 40 cycles, each cycle including denaturation at 95℃ for 5 s and annealing extension at 60℃ for 30 s, with fluorescence signals collected during the annealing extension step; after amplification, melting curve analysis is performed, with the temperature increasing from 60℃ to 95℃ in increments of 0.5℃ / 5 s.

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