A SYBR Green I fluorescent quantitative PCR detection primer for detecting LSDV and application thereof

Abstract

The application belongs to the technical field of virus detection, and particularly relates to a SYBR Green I fluorescent quantitative PCR detection primer for detecting LSDV and application. The primer pair for detecting bovine nodular dermatopathy virus by SYBR Green I fluorescent quantitative PCR comprises an upstream primer LSDV-F, the nucleotide sequence of which is shown as SEQ ID NO. 1, and a downstream primer LSDV-R, the nucleotide sequence of which is shown as SEQ ID NO. 2. The application is based on the GTPV, SPPV and LSDV whole genome sequences disclosed in GenBank, and a pair of specific amplification primers are designed after whole genome alignment, and a fluorescent quantitative PCR detection method capable of specifically detecting LSDV is successfully established, thereby providing technical support for distinguishing capripoxvirus infection and infection monitoring in the clinic.

Description

Technical Field

[0001] This invention belongs to the field of virus detection technology, and in particular relates to a SYBR Green I fluorescent quantitative PCR detection primer for detecting LSDV and its application. Background Technology

[0002] Bovine lumpy skin disease (LSD) is an acute, contagious disease caused by bovine lumpyskin disease virus (LSDV). Infected cattle typically exhibit persistent high fever (40-41°C), emaciation, increased discharge from the eyes, nose, and mouth, widespread nodules on the skin, mucous membranes, and internal organs, and swollen lymph nodes.

[0003] Studies have reported a morbidity rate of 5%–45% and a mortality rate of 10% for LSD (Low Spur Cattle Disease). Although most affected cattle recover to a sub-healthy state, they may still experience mastitis, pneumonia, and skin nodules. This disease severely impacts the development of the cattle industry and international trade, causing significant economic losses. Currently, LSD is listed as a notifiable bovine disease by the World Organisation for Animal Health (OIE).

[0004] LSDV belongs to the Poxviridae family ( Poxviridae ), Chordopoxvirus subfamily ( Chordopoxvirinae ChPV), Goatpoxvirus (ChPV) Capripoxvirus A member of CaPV, and related to goatpox virus (CaPV). Goatpox virus GTPV) and sheep pox virus ( Sheeppox virus These three viruses (SPPV, SHPV, SHPV) belong to the same genus. They exhibit cross-protection, and serological tests cannot distinguish between them.

[0005] In clinical practice, sheep pox vaccine is often used to immunize cattle. Therefore, it is particularly important to establish a highly sensitive, specific, and reproducible quantitative real-time PCR detection method for LSDV that can distinguish it from GTPV in naturally infected cattle or cattle carrying the virus after immunization. Summary of the Invention

[0006] Therefore, in order to establish a specific real-time PCR detection method for LSDV that can be distinguished from GTPV and SPPV, this invention is based on the whole genome sequences of GTPV, SPPV and LSDV published in GenBank (search date: May 30, 2024, 116 LSDV sequences, 22 SPPV sequences, 10 GTPV sequences). After whole genome alignment, a pair of specific amplification primers were designed, and a real-time PCR detection method that can specifically detect LSDV was successfully established, providing technical support for the clinical differentiation of goatpoxvirus infections and infection monitoring.

[0007] This invention provides a SYBR Green I fluorescent quantitative PCR primer pair for detecting bovine nodular dermatosis virus, the primer pair comprising: The upstream primer LSDV-F has the nucleotide sequence shown in SEQ ID NO.1; The downstream primer LSDV-R has the nucleotide sequence shown in SEQ ID NO.2.

[0008] The present invention provides a detection reagent comprising primer pairs.

[0009] Furthermore, the reagents also include SYBR Green I real-time PCR reaction premix, positive control, and negative control.

[0010] This invention provides a method for detecting bovine nodular dermatitis virus using primer pairs for non-diagnostic purposes, comprising the following steps: (1) Extract DNA from the sample to be tested; (2) Using the extracted DNA as a template, SYBR Green I real-time PCR amplification was performed using primer pairs; (3) Analyze the amplification curve and melting curve to determine the detection results.

[0011] Further, the reaction system for the SYBR Green I real-time PCR in step (2) is as follows: 2 × ChamQ Universal SYBR qPCR Master Mix 10 μL; 0.8 μL of 10 µM upstream primer LSDV-F; 0.6 μL of 10 µM downstream primer LSDV-R; 1 μL of DNA template; Add ddH2O to bring the total to 20 μL.

[0012] Furthermore, the amplification program for SYBR Green I real-time quantitative PCR in step (2) is as follows: Pre-denaturation at 95℃ for 30 seconds; The process involved denaturation at 95℃ for 10 seconds, annealing at 42-50℃ for 30 seconds, and extension at 72℃ for 30 seconds, for a total of 40 cycles. Then, a dissolution curve analysis was performed.

[0013] Furthermore, the criteria for determining the dissolution curve are as follows: When the temperature of the melting curve is 74.5±0.5, it can be determined as LSDV specific amplification; if the temperature of the melting curve is not 74.5±0.5, it can be determined as primer non-specific amplification.

[0014] Furthermore, the primer pair is used to distinguish between bovine nodular dermatosis virus and goatpox virus.

[0015] This invention provides the application of primer pairs or reagents in the preparation of bovine nodular dermatitis virus detection products.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention analyzes the conserved regions of the whole genome sequences of LSDV, GTPV, and SPPV published in the GenBank database, designs specific primers, and establishes a qPCR detection method based on SYBR Green I. After system optimization, the optimal amplification system is as follows: 10 μL of 2 × ChamQ Universal SYBR qPCR Master Mix, 0.8 μL of Primer 1 (10 µM), 0.6 μL of Primer 2 (10 µM), 1 μL of Template DNA, and 7.6 μL of dH2O. The amplification program is 95 ℃ for 30 s, 95 ℃ for 10 s, 49.5 ℃ for 30 s, and 72 ℃ for 30 s, for a total of 40 cycles. Analysis of the melting curve using the machine's default program showed a melting temperature of 74.5 ± 0.5 ℃, exhibiting good amplification efficiency (E=96.5%) and a detection limit of 10. 2 The sample has a low copy number (μL), good repeatability, and can be distinguished from GTPV. Serial dilutions of the same nucleic acid sample revealed that its detection sensitivity is 10 times higher than that of the national standard detection primers. In conclusion, this detection method can provide technical support for the clinical detection of LSDV. Attached Figure Description

[0017] Figure 1 The result is the LSDV amplification result in Example 2, where M: DL 2000 Marker, 1: LSDV.

[0018] Figure 2 The results of temperature optimization in Example 3 are shown, where: A is the amplification curve; B is the melting curve.

[0019] Figure 3 The results of primer concentration optimization in Example 3 are as follows: A, primer concentration optimization results for test wells; B, primer concentration optimization melting curve for test wells; C, primer concentration optimization results for repeat wells; D, primer concentration optimization melting curve for repeat wells.

[0020] Figure 4 The amplification results for different copy numbers in Example 4 are shown below: A, amplification curve; B, melting curve; 1-7 are respectively 10 8 10 7 10 6 10 5 10 4 10 3 10 2 Copies / μL of plasmid standard.

[0021] Figure 5 This is the LSDV standard curve diagram in Example 4.

[0022] Figure 6 The results of sensitivity verification in Example 5 are shown, where: A, amplification curve; B, melting curve; 1, 10 3 copies / μL; 2, 10 2 copies / μL.

[0023] Figure 7 The results of the specificity test in Example 6 are as follows: A, amplification curve; B, melting curve; 1, positive control of the system; 2, LSDV; 3, goatpox virus; 4, negative control of the system.

[0024] Figure 8 The results of comparison with the national standard primers in Example 8 are shown below: A, LSDV national standard primer PCR detection results; B, LSDV qPCR amplification curve; C, LSDV qPCR melting curve; 1, negative control of the system; 2, nucleic acid 10 -3 Dilution, 3, nucleic acid 10 -4 Dilution, 4, nucleic acid 10 -5 Dilution, 5, nucleic acid 10 -6 Dilution, 6, positive control of the system. Detailed Implementation

[0025] The following examples use the following materials and instruments: 1. Test materials ChamQ Universal SYBR qPCR Master Mix (purchased from Nanjing Novizan Biotechnology Co., Ltd., Cat: Q711); pMD TM 18-T Vector Cloning Kit (purchased from Takara Bio Inc., Cat: 6011); DH5α competent cells (purchased from Tiangen Biotech (Beijing) Co., Ltd., Cat: CB101); 2×Es Taq Master Mix (purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd., Cat: CW0690M); LSDV nucleic acid was extracted from skin samples of clinical bovine sarcoidosis (samples were stored in inactivated sampling tubes). All nucleic acid tests were performed in a biosafety cabinet in the BSL-2 laboratory of Hebei Agricultural University. No experiments were conducted on live virus isolation or virus proliferation. DL2000 DNA Marker (purchased from Takara Bio Inc., Cat: 3427Q); YeaRed nucleic acid dye (10000× DMSO solution) (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., Cat: 10203ES76); Conventional agarose gel DNA recovery kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., Cat: DP209); Goat pox live vaccine (attenuated goat pox virus strain, CVCC AV41) (purchased from Jilin Zhengye Biological Products Co., Ltd., veterinary drug production license number: 070224003); Viral DNA extraction kit (purchased from Beijing Solarbio Technology Co., Ltd., Cat: D2400); Plasmid miniprep kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., Cat: DP103).

[0026] 2. Test instruments PCR instrument; Real-time PCR instrument (Bio Rad CFX96); Nucleic acid electrophoresis apparatus.

[0027] Example 1 Primer design and synthesis Based on the whole genome sequences of GTPV, SPPV, and LSDV already published in GenBank (search date: May 30, 2024, 116 LSDV sequences, 22 SPPV sequences, 10 GTPV sequences, accession numbers for each sequence are detailed in Tables 1-3). Table 1. GenBank numbers of sequences involved in LSDV

[0028] Table 2. GenBank numbers of sequences involved in SPPV

[0029] Table 3 GenBank numbers of the sequences involved in GTPV

[0030] After whole-genome alignment, differences were found among the three strains at the LSDV-F position, indicating that this position is a differential site among the three strains. However, the LSDV-R position at the 3' end can bind to all three strains and cannot distinguish them. At the same time, sequence analysis of LSDV-F / LSDV-R revealed that the sequence lengths of LSDV, GTPV, and SPPV strains are different, suggesting different melting temperatures after amplification. Therefore, a pair of specific amplification primers was designed based on the region corresponding to LSDV-F / LSDV-R. The primer sequences (SEQ ID NO.1: LSDV-F; SEQ ID NO.2: LSDV-R) are shown in Table 4.

[0031] Table 4 LSDV amplification primer information

[0032] Example 2 Preparation of plasmid standards Using LSDV positive nucleic acid as a template, the target gene was amplified using the primer pairs in Table 1. The amplification system is shown in Table 5, and the amplification program is shown in Table 6. Table 5 Amplification System

[0033] Table 6 Amplification Procedure

[0034] After amplification, the results were analyzed by agarose gel electrophoresis, and the target gene was recovered using a standard agarose gel DNA recovery kit. The recovered target gene was then compared with pMD. TM The 18-T Vector Cloning Kit was used for overnight ligation at 16 ℃, followed by transformation to DH5α. Clone samples were then picked and sent to Sanger sequencing at Sangon Biotech (Shanghai) Co., Ltd. Experimental results showed that ( Figure 1 The LSDV-F / LSDV-R primers successfully amplified the target gene, which was verified by NCBI Blast after Sanger sequencing. The sequence was 100% consistent with the expected sequence.

[0035] The sequence obtained from sequencing is as follows, SEQ ID NO.3: Tatactccttcatcactcttagaaatatttcgtattagtaatttttctttgcttttttatatttaaaattattgtttatttttccctgtctttaaaccaatttacttcataagaacaattaaacgtatttataaagttttttgtatttatacacaacaactcatagtttgtgccaatgacagagtttatagtctc; For correctly sequenced plasmid samples, plasmids were extracted using a plasmid miniprep kit according to the instructions. The extracted plasmid concentration was then measured using Nanodrop One, and the plasmid copy number was calculated. The copy number calculation formula is as follows: DNA copy number (copies / μL) = (DNA concentration (ng / μL) × 6.022 × 10⁻⁶) 23 ) / (plasmid length (bp) × 1×10 9 × 660); The plasmid concentration was 682.321 ng / μL, A260 / A280 = 1.974, and its copy number was 21,542,383,4397.98 copies / μL. It was diluted to 10... 10 Copies / μL are available for later use.

[0036] Example 3 Optimization of SYBR Green I qPCR reaction system and conditions 1. Optimization of qPCR amplification temperature With 10 6 Using copies / μL plasmid as a template, the amplification temperature of the system was optimized by selecting eight temperatures: 50.0, 49.5, 48.7, 47.1, 45.1, 43.6, 42.5, and 42.0℃. The reaction system is shown in Table 7, and the amplification program is shown in Table 8. The amplification curves are shown below. Figure 2 As shown in (A), the dissolution curve is as follows: Figure 2 As shown in (B), by analyzing the Cq value (Table 9), it was found that the Cq value was the lowest when the annealing temperature was 49.5 ℃. Therefore, the optimal amplification temperature of this primer was determined to be 49.5 ℃.

[0037] Table 7 Amplification System

[0038] Table 8 Amplification Procedure

[0039] Table 9. Cq values ​​corresponding to different annealing temperatures

[0040] 2. Optimization of qPCR amplification system (primer concentration) Based on the optimal amplification temperature determined in step 1, upstream primer concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 μM and downstream primer concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 μM were selected, and an orthogonal design was performed (the amplification system and primer orthogonal design are shown in Tables 10 and 11, respectively). Amplification was carried out using the optimal amplification program (Table 12). The optimal amplification system was determined by analyzing the mean values. The experimental results show that ( Figure 3 (See Table 13). When the final concentration of the upstream primer was 0.4 μM and the final concentration of the downstream primer was 0.3 μM, the amplification efficiency of the template was good, and the Cq value was the lowest. In summary, the optimal annealing temperature was determined to be 49.5 ℃, and the determined final primer concentrations were 0.4 μM for the upstream primer and 0.3 μM for the downstream primer.

[0041] Table 10 Amplification System

[0042] Primer concentrations were tested using the checkerboard method. The amplification program was based on the optimized amplification program in Table 3. The sample addition methods for primers of different concentrations are shown in Table 11. The stock concentration of the primers is 10 μM. The dilution method is to add the primers according to the dilution column in Table 11. After mixing, add 1 μL to obtain the final concentration of the system.

[0043] Table 11 Comparison of Primer Additions at Different Concentrations

[0044] Note: single underscore The volume shown by the line is the primer volume. Double underscore Linear volumes represent the volume of the diluent (dH2O), and all volume units are in μL; bold italic text represents the volume added to the reaction system.

[0045] Table 12 Amplification Procedure

[0046] Table 13 Amplification with different primer concentrations (10) 6 Cq value corresponding to copies / μL plasmid

[0047] Example 4 Standard curve Based on the optimal amplification conditions determined in Example 3, 10 were selected respectively. 8 10 7 106 10 5 10 4 10 3 10 2 Amplification was performed using copies / μL plasmid standards as templates. After selecting an appropriate threshold, the relationship between different concentrations of plasmid standards and Cq values ​​was statistically analyzed. At the same time, a regression equation was established and the E value was calculated (E=10^(-1 / slope)-1).

[0048] Amplification results as follows Figure 4 As shown, the exported data is shown in Table 14. The standard curve is shown below. Figure 5 As shown. Experimental results show that the standard curve formula of this primer is: Y = -3.410X + 39.85, R2=0.9984, E=96.5%, indicating good linear correlation and good amplification efficiency.

[0049] Table 14. Data Exported from Standard Curve Establishment

[0050] Example 5 Sensitivity test Based on the optimal amplification conditions determined in Example 3, 10 were selected respectively. 3 10 2 10 1 10 0 Amplification was performed using copies / μL of plasmid standard as templates. Melting curves and amplification curves were analyzed to determine the detection limit of this method.

[0051] Amplification results as follows Figure 6 As shown. Analysis of the amplification and melting curves revealed that the lower limit of amplification for this reaction system was 10. 2 copies / μL.

[0052] Example 6 Specificity test Based on the optimal amplification conditions determined in Example 3, goatpox live vaccine was used as a sample, and goatpox virus nucleic acid was extracted using a viral DNA extraction kit (the extraction method was performed according to the instructions of the viral DNA extraction kit). Amplification was performed using goatpox virus nucleic acid and LSDV nucleic acid, respectively, to determine the specificity of the detection method.

[0053] Amplification results as follows Figure 7 As shown in the figure. The experimental results indicate that this amplification system can successfully distinguish between LSDV and goatpox, demonstrating its good specificity.

[0054] In the single amplification system, it is impossible to determine whether the amplified curve is GTPV. As long as the melting curve temperature is 74.5±0.5, it can be determined as LSDV specific amplification. If the melting curve temperature is not 74.5±0.5, it is determined as primer non-specific amplification.

[0055] Example 7 Repeatability test Based on the optimal amplification conditions determined above, 10 were selected respectively. 7 10 6 10 5 Amplification was performed using copies / μL of plasmid standard as templates, and CV was calculated. The experimental results (Table 15) show that the coefficient of variation for each group is less than 3%, indicating good reproducibility.

[0056] Table 15 Results of intra-group and inter-group repeatability tests

[0057] Example 8 Referring to the national testing standard document GB / T 39602-2020, standard PCR detection primers were synthesized. After determining the nucleic acid concentration using Nanodrop One, the nucleic acid samples were serially diluted 10-fold, and the following two methods were used for detection: (1) National standard PCR method: LSDV national standard primers (SEQ ID NO.4: F: 5′-CCTCCTTTTAAGCTACTTTTTCTTA-3′, SEQ ID NO.5: R: 5′-GATACATGTAGGAACATTGTTACCTA-3′) were used for routine PCR amplification, and the products were analyzed by agarose gel electrophoresis; (2) Method of the present invention: SYBR Green I real-time quantitative PCR amplification was performed using LSDV qPCR detection primers. The amplification systems and conditions for the two methods are shown in Tables 16 and 17, respectively, and the sensitivity of the two detection methods was compared.

[0058] The test results are as follows Figure 8 As shown. Experimental results indicate that the concentration of LSDV-positive nucleic acid samples was 137.311 μg / μL (A260 / A280=1.816), and 10 μg / μL could be detected using the LSDV national standard detection primers. -3 Nucleic acid diluted 10 times -4 The diluted nucleic acid bands were faint, leading to a suspected diagnosis, while LSDV qPCR could detect 10... -4 The nucleic acid was diluted several times, indicating that the detection sensitivity of the LSDV qPCR method is 10 times that of the LSDV national standard detection primers.

[0059] Table 16 Amplification System

[0060] Table 17 Amplification Procedure

[0061] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A SYBR Green I fluorescent quantitative PCR primer pair for detecting bovine nodular dermatosis virus, characterized in that, The primer pair includes: The upstream primer LSDV-F has the nucleotide sequence shown in SEQ ID NO.1; The downstream primer LSDV-R has the nucleotide sequence shown in SEQ ID NO.

2.

2. A detection reagent, characterized in that, The reagent comprises the primer pair as described in claim 1.

3. The reagent according to claim 2, characterized in that, The reagents also include SYBR Green I real-time PCR reaction premix, positive control, and negative control.

4. A method for detecting bovine nodular dermatosis virus using the primer pair of claim 1 for non-diagnostic purposes, characterized in that, Includes the following steps: (1) Extract DNA from the sample to be tested; (2) Using the extracted DNA as a template, perform SYBR Green I real-time PCR amplification using the primer pair described in claim 1; (3) Analyze the amplification curve and melting curve to determine the detection results.

5. The method according to claim 4, characterized in that, The reaction system for SYBR Green I real-time PCR in step (2) is as follows: 2 × ChamQ Universal SYBR qPCR Master Mix 10 μL; 0.8 μL of 10 µM upstream primer LSDV-F; 0.6 μL of 10 µM downstream primer LSDV-R; 1 μL of DNA template; Add ddH2O to bring the total to 20 μL.

6. The method according to claim 5, characterized in that, The amplification program for SYBR Green I real-time PCR in step (2) is as follows: Pre-denaturation at 95℃ for 30 seconds; The process involved denaturation at 95℃ for 10 seconds, annealing at 42-50℃ for 30 seconds, and extension at 72℃ for 30 seconds, for a total of 40 cycles. Then, a dissolution curve analysis was performed.

7. The method according to claim 6, characterized in that, The criteria for determining the dissolution curve are as follows: When the temperature of the melting curve is 74.5±0.5, it can be determined as LSDV specific amplification; if the temperature of the melting curve is not 74.5±0.5, it can be determined as primer non-specific amplification.

8. The method according to claim 4, characterized in that, The primer pair is used to distinguish between bovine nodular dermatosis virus and goatpox virus.

9. The use of the primer pair of claim 1 or the reagent of claim 2 in the preparation of a bovine nodular dermatosis virus detection kit.

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