Double fluorescent quantitative PCR primers and kit for goose type Ⅰ and type Ⅱ reovirus
By designing dual-fluorescent quantitative PCR primers and kits for genotype I and II goose reoviruses, the problem of low detection sensitivity in existing technologies has been solved, achieving high sensitivity and specificity for the detection of extremely low concentrations of viral nucleic acid, which is suitable for quantitative analysis of early viral infections.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- FOSHAN UNIVERSITY
- Filing Date
- 2024-11-06
- Publication Date
- 2026-06-23
AI Technical Summary
Existing goose reovirus detection technologies suffer from low sensitivity and complex operation, making it difficult to effectively detect low concentrations of viral nucleic acid.
Dual real-time PCR primers and kits for genotype I and II goose reovirus were designed, including specific primer pairs and positive standard plasmids. Optimized real-time PCR reaction conditions were used to achieve high sensitivity and specificity for the detection of genotype I and II GRV.
It achieves highly sensitive detection of GRV genotypes I and II, can detect extremely low concentrations of viral nucleic acid, has high specificity and good repeatability, and its sensitivity is 100 times higher than that of ordinary PCR.
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Figure CN119876484B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of gene detection, specifically relating to a dual fluorescent quantitative PCR primer and kit for goose reoviruses of genotype I and II. Background Technology
[0002] Goose reovirus (GRV) is mainly divided into two genotypes: genotype 1, characterized by liver and spleen necrosis, and genotype 2, characterized by liver hemorrhage and necrosis. Currently, both genotypes 1 and genotype 2 are showing an outbreak trend in some densely populated goose-farming areas in my country.
[0003] Existing virus detection technologies mainly include serological detection methods and molecular detection methods. These include enzyme-linked immunosorbent assays (ELISA), neutralization assays, immunofluorescence assays, and nucleic acid hybridization, but all have various defects and limitations, such as relatively low sensitivity, inability to detect low concentrations of viral nucleic acid, and complex operation.
[0004] Quantitative real-time PCR (qPCR) is a highly efficient and sensitive nucleic acid quantification technique that combines PCR and fluorescent probe technologies. It quantifies the presence of a target virus by monitoring the intensity of the fluorescence signal in real time during the PCR process. During PCR, the intensity of the fluorescence signal is directly proportional to the amount of amplified products, thus allowing for quantitative detection of the target virus. SYBR Green I-based DNA-binding dye qPCR uses SYBR fluorescent dye to emit fluorescence signals from all double-stranded DNA amplification products for real-time detection. The uniqueness of the product is determined by the melting curve peak diagram, and internal / external reference methods can be used for quantitative analysis of unknown templates. Summary of the Invention
[0005] Based on this, primers were designed for the S2 gene of type I GRV (GenBank ID: MZ546427.1) and the S3 gene of type II GRV (GenBank ID: JX145334.1) to construct a dual fluorescence quantitative detection method for type I and type II GRV.
[0006] This invention provides a dual-phase quantitative PCR primer for goose reovirus genotype I and II, comprising a first primer pair and a second primer pair.
[0007] The upstream primer I-F sequence of the first primer pair is shown in SEQ ID No: 1, and the downstream primer I-R sequence of the first primer pair is shown in SEQ ID No: 2;
[0008] The upstream primer II-F sequence of the second primer pair is shown in SEQ ID No: 3, and the downstream primer II-R sequence of the second primer pair is shown in SEQ ID No: 4.
[0009] The present invention also provides the application of the aforementioned dual fluorescent quantitative PCR primers for genotype I and II goose reovirus in the preparation of reagents for detecting goose reovirus.
[0010] The present invention also provides a dual real-time PCR kit for genotype I and II goose reovirus, the kit comprising the primers described above.
[0011] Furthermore, the kit also includes a positive standard plasmid.
[0012] Furthermore, the concentration gradient range of the positive standard plasmid is 10. 1 -10 7 copies / μL.
[0013] Furthermore, the positive standard plasmid is prepared by the following method: PCR amplification of the S2 gene of type I GRV using the first primer pair shown in SEQ ID NO.1-SEQ ID NO.2, and PCR amplification of the S3 gene of type II GRV using the second primer pair shown in SEQ ID NO.3-SEQ ID NO.4, respectively, to obtain amplification products. The amplification products are ligated into a vector, and positive recombinant plasmids are screened and extracted to obtain the positive standard plasmid; wherein the GenBank ID of the S2 gene is MZ546427.1, and the GenBank ID of the S3 gene is JX145334.1.
[0014] Furthermore, the PCR amplification system reagents include TaKaRa Premix Taq.
[0015] Furthermore, the kit also includes real-time PCR reaction reagents.
[0016] Furthermore, the real-time PCR reagent includes TB Green Premix Ex Taq II.
[0017] Furthermore, the PCR reaction program of the kit is as follows: ① 95℃ for 3 min; ② 95℃ for 3 s; ③ 64℃ for 30 s; ②-③ for 40 cycles; ⑤ The melting curve program is set to the default settings of the AriaMx Real-Time PCR G8830A device.
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] 1) High specificity: Using cDNA of genotype I GRV and genotype II GRV as positive controls, and cDNA / DNA of avian influenza viruses (AIV), duck tembusu virus (DTMUV), goose astrovirus (GoAstV), goose parvovirus (GPV), goose circovirus (GoCV), and avian adenovirus (FAdV) as templates, while setting up negative controls, the optimized system was used for amplification to verify the specificity of the method.
[0020] 2) High sensitivity: utilizing 10 7 ~10 1 Using 18T-Ⅰ-GRV and 18T-Ⅱ-GRV plasmid standards at a concentration of [copy / μL] as templates and ddH2O negative, qPCR amplification was performed under optimized reaction conditions. The minimum copy number detected by singlet and doublet quantitative PCR methods was determined, and conventional PCR was used as a reference to verify the sensitivity of the method. The minimum detection values for genotype I and II GRV were 6.466 × 10¹ copies / μL and 4.979 × 10¹ copies / μL, respectively, which is 100 times more sensitive than conventional PCR.
[0021] 3) Good repeatability: Gradient of 10 was selected. 7 10 5 10 3 The 18T-Ⅰ-GRV and 18T-Ⅱ-GRV standard positive plasmids were replicated three times at three different time points using an optimized reaction system, with a negative control included. The coefficient of variation (CV) was less than 0.51% for all three replicates, indicating good reproducibility. Attached Figure Description
[0022] Figure 1 Figure showing the identification results of GRV plasmids of genotypes I and II;
[0023] Figure 2 Tm values of real-time fluorescence PCR amplification products, where A is the melting curve of type I GRV, B is the melting curve of type II GRV, and C is the double melting curve;
[0024] Figure 3 The results are the standard curves for genotype I GRV; where A is the melting curve for genotype I and B is the standard curve.
[0025] Figure 4 The results are the standard curves for genotype II GRV; where A is the melting curve for type II and B is the standard curve.
[0026] Figure 5 This is a specific result of singlet quantitative PCR for type I GRV.
[0027] Figure 6 This is a specific result of singlet quantitative PCR for type II GRV.
[0028] Figure 7 This is a specific result of dual real-time PCR for GRV types I and II.
[0029] Figure 8 This is the result of sensitivity testing for type I GRV;
[0030] Figure 9 This is the result of sensitivity testing for type II GRV.
[0031] Figure 10 The results are for sensitivity testing of GRV types I and II. Detailed Implementation
[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.
[0033] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0034] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all test methods are conventional test methods in this field.
[0035] Example 1: Primer Design
[0036] After MEGA11 alignment of the S2 gene of genotype I GRV (GenBank ID: MZ546427.1) and the S3 gene of genotype II GRV (GenBank ID: JX145334.1), relatively conserved regions were selected. Multiple primer pairs were designed using Oligo7. After eliminating primer dimers and comparing Tm values, the two most suitable specific primer pairs were selected: I-F / I-R and II-F / II-R (Table 1). The target fragment sizes were 87bp and 139bp, respectively. The primers were synthesized by Guangzhou Tianyi Huiyuan Co., Ltd.
[0037] Table 1 Specific primer sequences
[0038]
[0039] Example 2: Preparation of plasmid standards
[0040] (1) PCR amplification and purification of the target fragment
[0041] Using cDNA from genotype I and genotype II GRV as templates, amplification was performed using primers I-F / I-R and II-F / II-R, respectively. The standard PCR reaction system is shown in Table 2.
[0042] Table 2 Conventional PCR reaction system
[0043] Sample Name Volume (μL) TaKaRa Premix Taq 12.5 Upstream primer (10 μM) 1 Downstream primer (10 μM) 1 template 2 <![CDATA[ddH2O]]> 8.5 Total volume 25
[0044] The reaction procedure was as follows: ① 94℃ for 10 min; ② 94℃ for 30 s; ③ 55℃ for 30 s; ④ 72℃ for 10 s; ②-④ for 30 cycles; ⑤ 72℃ for 10 min; and then stored at 12℃.
[0045] After the PCR program was completed, the amplification products were subjected to 3% agarose gel electrophoresis, and the corresponding bands were cut off and recovered using the Novizan FastPure Gel DNAExtraction Mini Kit.
[0046] (2) Connection of target segments
[0047] The PCR purified product obtained according to the Tarkara pMD18-T Vector instruction manual was used for vector ligation, as shown in Table 3:
[0048] Table 3 Connection Reaction System
[0049] Components Volume (μL) PCR purified products 4 pMD18-T Vector 1 Solution I 5 total 10
[0050] After mixing, the mixture was placed in a metal bath at 16°C for 4 hours to obtain the ligation product.
[0051] (3) Screening and identification of recombinant plasmids
[0052] The ligation product obtained in step (2) was added to 50 μL of LDH5α chemocompetent cells, incubated on ice for 30 min, heat-shocked at 42℃ for 45 s, incubated on ice for 5 min, and then 1 mL of antibiotic-free LB liquid medium was added. The cells were then cultured in a shaker at 37℃ for 1 h. After centrifugation and discarding 700 μL of supernatant, 100 μL of the resuspended bacterial solution was spread onto LB agar plates containing ampicillin (100 μg / mL) and incubated upside down at 37℃ for 12–16 h.
[0053] Single colonies with good growth were selected and placed in 500 μL of LB broth containing ampicillin. After incubation at 37°C with shaking for 3 h, bacterial colonies were identified by PCR, as shown in Table 4.
[0054] Table 4 PCR reaction system
[0055] Components Volume (μL) TaKaRa Premix Taq 5.0 <![CDATA[ddH2O]]> 3.0 upstream primer 0.5 Downstream primer 0.5 bacterial solution 1.0 total 10.0
[0056] The reaction procedure was as follows: ① 94℃ for 10 min; ② 94℃ for 30 s; ③ 55℃ for 30 s; ④ 72℃ for 10 s; repeat steps ②-④ for 30 cycles; ⑤ 72℃ for 10 min; store at 12℃. The selected positive bacterial cultures were sent to Sangon Biotech Co., Ltd. for sequencing.
[0057] (4) Extraction of positive plasmids
[0058] The bacterial culture with correct sequencing results from step (3) was expanded, and plasmid extraction was performed using the Novizan FastPure Plasmid Mini Kit. Figure 1 )
[0059] The plasmid concentration was measured using an ultra-micro fluorescence spectrophotometer, and the gene copy number (copy / μL) was calculated to be 6.02 × 10⁻⁶. 23 × plasmid concentration (ng / μL) × 10 -9 The number of gene copies is calculated by using the formula: / [plasmid size (bp) × 660].
[0060] Example 3: Optimization of the Real-Time PCR Reaction System
[0061] According to TB Premix Ex Taq TM II. The system and procedure in the instruction manual were optimized for the annealing temperature (55℃, 60℃, 65℃) and primer concentration (0.4μM / L, 0.6μM / L, 0.8μM / L) of the qPCR reaction system. The optimal reaction conditions and system for the single fluorescence quantitative detection method and the dual fluorescence quantitative detection method for GRV type I and type II were determined, as shown in Tables 5 and 6.
[0062] Table 5 Single-color fluorescence quantitative reaction system
[0063] Sample Name Volume (μL) TB Green Premix Ex TaqⅡ 12.5 I-F / II-F (10μM) 1 I-R / II-R (10μM) 1 template 2.0 <![CDATA[ddH2O]]> 8.5 Total volume 25
[0064] Table 6 Dual fluorescence quantitative reaction system
[0065]
[0066] Reaction program: ① 95℃ for 30s; ② 95℃ for 5s; ③ 60℃ for 30s; ②-③ 40 cycles; ⑤ Melting curve program is set to machine default settings.
[0067] Example 4: Analysis of Melting Curve Results
[0068] The melting point (Tm) was determined based on the melting curves of singlet quantitative PCR for GRV types I and II. The Tm value for GRV type I was 79.5℃, and the Tm value for GRV type II was 86.5℃. When the mixed plasmid was simultaneously subjected to quantitative PCR, the Tm values of the melting curves were consistent with those of the singlet quantitative PCR method. Figure 2 ).
[0069] Example 5, Specificity Test
[0070] To verify the specificity of single-fluorescence quantitative detection methods and dual-fluorescence quantitative detection methods for GRV types I and II, specific detection was performed on GRV types I and II and six common pathogens (AIV, DTMUV, GoAstV, GPV, GoCV, and FAdV). The results showed that only GRV types I and II were detected, and no specific amplification was observed in the control group, indicating that this method has good specificity. Figure 5 , Figure 6 , Figure 7 ).
[0071] Example 6, Sensitivity Test
[0072] To verify the sensitivity of single-fluorescence quantitative detection methods and dual-fluorescence quantitative detection methods for genotype I and II GRV, 10 [units of measurement] were selected based on the optimized reaction conditions. 7 ~10 1 Amplification was performed using 18T-Ⅰ-GRV and 18T-Ⅱ-GRV plasmid standards at a concentration of 1 copy / μL to test the sensitivity of this method. The results showed that the lowest detection limit for genotype I and genotype II GRV was 6.466 × 10⁻⁶. 1 Copy / μL, 4.979×10 1 copies / μL, 100 times more sensitive than conventional PCR ( Figure 8 , Figure 9 , Figure 10 ).
[0073] Example 7: Establishment of the Standard Curve
[0074] The 18T-Ⅰ-GRV and 18T-Ⅱ-GRV standard positive plasmids were diluted 10-fold in a 10-fold serial sequence to 100 μL. 8 ~10 2 Copy / μL, select five concentration gradients (10 8 10 7 10 6 10 5 10 4Using [a specific template], standard curves for single-color quantitative PCR detection of GRV types I and II were constructed, with three replicates and a negative control included. The results showed that the standard curve equation for GRV type I was y = -3.128X + 38.21, with a correlation coefficient of 0.997 and an amplification efficiency of 104.54 ([a specific value]). Figure 3 The standard curve equation for genotype II GRV is y = -3.265X + 38.58, with a correlation coefficient of 0.999 and an amplification efficiency of 102.41. Figure 4 ).
[0075] Example 8, Repeatability Test
[0076] To verify the repeatability of the single-fluorescence quantitative detection method and the dual-fluorescence quantitative detection method for genotype I and II GRV, a gradient of 10 was selected. 7 10 5 10 3 The 18T-Ⅰ-GRV and 18T-Ⅱ-GRV standard positive plasmids were replicated three times at three different time points. Tm and Ct values were recorded, and the coefficient of variation and within-group and between-group standard deviations were calculated. The results showed that the within-group coefficient of variation for the single-pair qPCR detection method for type I GRV was 0.22%–0.49%, and the between-group coefficient of variation was 0.43%–0.58%; the within-group coefficient of variation for the single-pair qPCR detection method for type II GRV was 0.24%–0.36%, and the between-group coefficient of variation was 0.32%–0.41%; and the within-group coefficient of variation for the dual-pair qPCR detection method for type I and type II GRV was 0.27%–0.34%, and the between-group coefficient of variation was 0.16%–0.51% (see Tables 7–9).
[0077] Table 7 Results of repeatability tests for genotype I GRV
[0078]
[0079] Table 8 Results of repeatability tests for type II GRV
[0080]
[0081] Table 9. Repeatability test results for GRV types I and II.
[0082]
[0083] Comparative experiment
[0084] Sensitivity was compared using standard PCR methods, with 10... 7 ~10 1The 18T-Ⅰ-GRV and 18T-Ⅱ-GRV plasmid standards were amplified using the amplification system and reaction procedure shown in Table 2. The results showed that conventional PCR could only amplify to 10 copies / μL. 3 Copy / μL, while binding Figure 8-10 The lowest detection values for genotype I and genotype II GRV in this application are 6.466 × 10⁻⁶. 1 Copy / μL, 4.979×10 1 With a concentration of 1 copy / μL, the dual-fluorescence quantitative PCR of this invention is 100 times more sensitive than conventional PCR.
[0085] In summary, this invention designs primers for the S2 gene of genotype I GRV (GenBank ID: MZ546427.1) and the S3 gene of genotype II GRV (GenBank ID: JX145334.1). These primers can be used to construct singleton and dual-fluorescence detection systems for both genotype I and genotype II GRV, both capable of detecting extremely low concentrations of viral nucleic acid. They are suitable for early viral infection detection and can quantitatively analyze the presence of the target virus to compare viral quantity differences in different samples. The dual-fluorescence detection system can distinguish between genotype I GRV (79.5℃) and genotype II GRV (86.5℃) using melting curves. It offers high cost-effectiveness and has broad market application prospects.
[0086] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0087] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.
Claims
1. Dual fluorescent quantitative PCR primers for goose reovirus genotypes I and II, characterized in that, The primers consist of a first primer pair and a second primer pair; The upstream primer I-F sequence of the first primer pair is shown in SEQ ID No: 1, and the downstream primer I-R sequence of the first primer pair is shown in SEQ ID No: 2; The upstream primer II-F sequence of the second primer pair is shown in SEQ ID No: 3, and the downstream primer II-R sequence of the second primer pair is shown in SEQ ID No:
4.
2. The application of the dual fluorescent quantitative PCR primers for genotype I and II goose reovirus as described in claim 1 in the preparation of reagents for detecting goose reovirus.
3. A dual real-time PCR kit for goose reovirus genotypes I and II, characterized in that, Includes the primers as described in claim 1.
4. The reagent kit according to claim 3, characterized in that: It also includes positive standard plasmids.
5. The reagent kit according to claim 4, characterized in that, The concentration gradient range of the positive standard plasmid is 10. 1 -10 7 copies / μL.
6. The reagent kit according to claim 4, characterized in that: The positive standard plasmid is prepared by the following method: the S2 gene of type I GRV is amplified by PCR using the first primer pair shown in SEQ ID NO: 1-SEQ ID NO: 2, and the S3 gene of type II GRV is amplified by PCR using the second primer pair shown in SEQ ID NO: 3-SEQ ID NO: 4, respectively. The amplification products are obtained, the amplification products are ligated into a vector, positive recombinant plasmids are screened and extracted, thus the positive standard plasmid is prepared.
7. The reagent kit according to claim 6, characterized in that: The PCR amplification reagents include TaKaRa PremixTaq.
8. The reagent kit according to claim 4, characterized in that: The kit also includes reagents for quantitative real-time PCR.
9. The reagent kit according to claim 8, characterized in that: The reagents for the quantitative real-time PCR reaction include TBGreen Premix Ex Taq II.
10. The reagent kit according to claim 9, characterized in that, The PCR reaction program of the kit is as follows: ① 95℃ for 3 min; ② 95℃ for 3 s; ③ 64℃ for 30 s; ②-③ for 40 cycles; ④ The melting curve program is set to the default settings of the AriaMx Real-Time PCR G8830A device.