Dual fluorescent quantitative PCR (Polymerase Chain Reaction) primer and kit for gene type I and type II goose reoviruses

Abstract

The invention relates to a dual fluorescent quantitative PCR (Polymerase Chain Reaction) primer and a kit for gene type I and type II goose reoviruses. The primer comprises a first primer pair and a second primer pair, an upstream primer I-F sequence of the first primer pair is as shown in SEQ ID No: 1, and a downstream primer I-R sequence of the first primer pair is as shown in SEQ ID No: 2; the sequence of an upstream primer II-F of the second primer pair is as shown in SEQ ID No: 3, and the sequence of a downstream primer II-R of the second primer pair is as shown in SEQ ID No: 4; primers are designed aiming at the gene I type GRV and the gene II type GRV, the dual fluorescent quantitative detection kit for the gene I type GRV and the gene II type GRV is constructed, and the kit can detect virus nucleic acid with extremely low concentration, is suitable for early virus infection detection, can quantitatively analyze the existence amount of target viruses, is used for comparing virus quantity differences in different samples, and can be used for detecting early virus infection. The gene I type GRV (79.5 DEG C) and the gene II type GRV (86.5 DEG C) can be distinguished through a melting curve. The cost performance is high, and the market application prospect is wide.

Description

Technical Field

[0001] The invention belongs to the field of gene detection, and specifically relates to a dual fluorescent quantitative PCR primer and a kit for gene type I and type II goose-origin reovirus. Background Art

[0002] Goose reovirus (GRV) is mainly divided into two genotypes: genotype 1 with liver and spleen necrosis as the main symptoms and genotype 2 with liver hemorrhage and necrosis as the main symptoms. Currently, genotype 1 and genotype 2 of the disease are showing an outbreak trend in some goose-intensive breeding areas in my country.

[0003] Existing virus detection technologies mainly include serological detection methods and molecular detection methods, including enzyme-linked immunosorbent assay, neutralization test, immunofluorescence detection method, nucleic acid hybridization, etc. However, they all have various defects and shortcomings, such as relatively low sensitivity, failure to detect low concentrations of viral nucleic acid, and complex operation.

[0004] Real-time fluorescence quantitative PCR (qPCR) is an efficient and sensitive nucleic acid quantification technology that combines PCR technology and fluorescent probe technology to quantitatively detect the presence of target viruses by monitoring the intensity of the fluorescent signal in real time during the PCR process. During the PCR process, the intensity of the fluorescent signal is monitored in real time, and the fluorescent signal is proportional to the number of amplified products, thereby quantitatively detecting the presence of the target virus. The DNA binding dye method qPCR based on SYBR Green I uses all SYBR fluorescent dyes that have infiltrated double-stranded DNA amplification products to emit fluorescent signals for real-time detection, and the uniqueness of the product is judged by the melting curve peak diagram. The internal reference / external reference method can be used to quantitatively analyze unknown templates. Summary of the invention

[0005] Based on this, the present 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) to construct a dual fluorescence quantitative detection method for genotype I and genotype II GRV.

[0006] The present invention provides a dual fluorescence quantitative PCR primer for genotype I and type II goose-derived reovirus, wherein the primer comprises a first primer pair and a second primer pair.

[0007] The sequence of the upstream primer I-F of the first primer pair is shown in SEQ ID No: 1, and the sequence of the downstream primer I-R of the first primer pair is shown in SEQ ID No: 2;

[0008] The sequence of the upstream primer II-F of the second primer pair is shown in SEQ ID No:3, and the sequence of the downstream primer II-R of the second primer pair is shown in SEQ ID No:4.

[0009] The present invention also provides the use of the gene type I and type II goose-derived reovirus dual fluorescence quantitative PCR primers in preparing a reagent for detecting goose-derived reovirus.

[0010] The present invention also provides a dual fluorescence quantitative PCR kit for gene type I and type II goose-derived reovirus, and the kit comprises the primers.

[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: using the first primer pair shown in SEQ ID NO.1-SEQ ID NO.2 to PCR amplify the S2 gene of gene type I GRV, and using the second primer pair shown in SEQ ID NO.3-SEQ ID NO.4 to PCR amplify the S3 gene of gene type II GRV to obtain an amplified product, connecting the amplified product to a vector, screening positive recombinant plasmids and extracting them, so as to prepare 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 fluorescent quantitative PCR reaction reagents.

[0016] Furthermore, the fluorescent quantitative PCR reaction reagent includes TB Green Premix Ex TaqⅡ.

[0017] Furthermore, the PCR reaction program of the kit is: ① 95°C for 3 min; ② 95°C for 3 s, ③ 64°C for 30 s, ②-③ 40 cycles, ⑤ melting curve program according to the default settings of the device AriaMx Real-Time PCR G8830A.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1) Strong specificity: The cDNA of genotype I GRV and genotype II GRV were used as positive controls, and the cDNA / DNA of avian influenza viruses (AIV), duck tembusu virus (DTMUV), goose astrovirus (GoAstV), goose parvovirus (GPV), goose circovirus (GoCV), and fowl aviadenovirus (FAdV) were used as templates. At the same time, negative controls were set up and amplification was performed using the optimized system to verify the specificity of the method.

[0020] 2) High sensitivity: using 10 7 ~10 1 The 18T-Ⅰ-GRV and 18T-Ⅱ-GRV plasmid standards with 100 copies / μL were used as templates and ddH2O was negative. The optimized reaction conditions were used for qPCR amplification to determine the minimum copy number detected by the single-plex and double-plex fluorescence quantitative methods, and the sensitivity of the method was verified by using ordinary PCR as a reference. The minimum detection values ​​of genotype Ⅰ and Ⅱ GRV were 6.466×101 copies / μL and 4.979×101 copies / μL, respectively, which were 100 times more sensitive than ordinary PCR.

[0021] 3) Good repeatability: Select a gradient of 10 7 , 10 5 , 10 3 The 18T-Ⅰ-GRV and 18T-Ⅱ-GRV standard positive plasmids were repeated three times at three different time points with the optimized reaction system, and a negative control was set. The coefficient of variation (CV) was less than 0.51%, and the repeatability was good. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the result of identification of genotype Ⅰ and Ⅱ GRV plasmids;

[0023] Figure 2 is the Tm value of the real-time fluorescence PCR amplification product, where A is the melting curve of genotype I GRV, B is the melting curve of genotype II GRV, and C is the double melting curve;

[0024] Figure 3 The results of the standard curve of genotype I GRV; A is the melting curve of type I, and B is the standard curve;

[0025] Figure 4 The results of the standard curve of genotype II GRV; A is the melting curve of type II, and B is the standard curve;

[0026] Figure 5 This is the specific result of single-plex fluorescence quantitative PCR for genotype I GRV;

[0027] Figure 6 This is the specific result of single-plex fluorescence quantitative PCR for genotype II GRV;

[0028] Figure 7 It is the specific result of dual fluorescence quantitative PCR for genotype Ⅰ and Ⅱ GRV;

[0029] Figure 8 This is the sensitivity test result of genotype I GRV;

[0030] Fig. 9 This is the result of sensitivity test for genotype II GRV;

[0031] Fig.10 These are the results of sensitivity testing for genotype Ⅰ and Ⅱ GRV. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0033] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making any creative work shall fall within the scope of protection of the present invention.

[0034] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; the experimental methods are all conventional experimental methods in the art unless otherwise specified.

[0035] Example 1, primer design

[0036] The S2 gene of genotype I GRV (GenBank ID: MZ546427.1) and the S3 gene of genotype II GRV (GenBank ID: JX145334.1) were aligned by MEGA11, and their relatively conservative regions were selected. Multiple pairs of primers were designed using Oligo7. After excluding primer dimers and comparing Tm values, the two most suitable pairs of specific primers were screened out: Ⅰ-F / Ⅰ-R and Ⅱ-F / Ⅱ-R (Table 1). The target fragment sizes were 87 bp and 139 bp, 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 target fragments

[0041] Using the cDNA of genotype Ⅰ and Ⅱ GRV as templates, primers Ⅰ-F / Ⅰ-R and Ⅱ-F / Ⅱ-R were used for amplification respectively. The conventional 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: ① 94°C for 10 min; ② 94°C for 30 s, ③ 55°C for 30 s, ④ 72°C for 10 s, ②-④ for 30 cycles; ⑤ 72°C for 10 min, stored at 12°C.

[0045] After the PCR program was completed, the amplified product was subjected to 3% agarose gel electrophoresis and the corresponding band was cut out and recovered using the Novezan FastPure Gel DNA Extraction Mini Kit.

[0046] (2) Connection of target fragments

[0047] The PCR purified product obtained according to the instruction manual of Tarkara's pMD18-T Vector was connected to the vector, as shown in Table 3:

[0048] Table 3 Ligation reaction system

[0049] Components Volume (μL) PCR purification product 4 pMD18-T Vector 1 Solution I 5 total 10

[0050] After mixing, place in a metal bath at 16°C for 4 hours to obtain a connection product.

[0051] (3) Screening and identification of recombinant plasmids

[0052] The ligation product obtained in step (2) was added to 50 μL of DH5α chemically competent cells, placed on ice for 30 min, heat-shocked at 42°C for 45 s, placed on ice for 5 min, and 1 mL of LB liquid medium without resistance was added, and cultured in a constant temperature shaker at 37°C for 1 h. After centrifugation and discarding 700 μL of supernatant, 100 μL of the resuspended bacterial solution was spread on an LB agar plate containing ampicillin (100 μg / mL), and cultured inverted at 37°C for 12 to 16 h.

[0053] A single colony with good growth was selected and placed in 500 μL of LB liquid culture medium containing ampicillin antibiotics, and cultured in a constant temperature shaker at 37°C for 3 hours before performing PCR identification of the bacterial solution, 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 liquid 1.0 total 10.0

[0056] The reaction procedure was: ① 94°C for 10 min; ② 94°C for 30 s, ③ 55°C for 30 s, ④ 72°C for 10 s, ②-④ for 30 cycles; ⑤ 72°C for 10 min, stored at 12°C. The screened positive bacterial solution was sent to Shenggong Biotechnology Co., Ltd. for sequencing.

[0057] (4) Extraction of positive plasmid

[0058] The bacterial solution with the correct sequencing result in step (3) was expanded and cultured, and the plasmid was extracted using the Novezan FastPure PlasmidMini Kit ( Figure 1 )

[0059] The plasmid concentration was measured using an ultra-micro fluorescence spectrophotometer and the gene copy number (copy / μL) = 6.02 × 10 23 ×Plasmid concentration (ng / μL)×10 -9 / [plasmid size (bp) × 660], and calculate its gene copy number.

[0060] Example 3: Optimization of Fluorescence Quantitative PCR Reaction System

[0061] According to TB Premix Ex Taq TM II instruction manual, the annealing temperature (55°C, 60°C, 65°C) and primer concentration (0.4μM / L, 0.6μM / L, 0.8μM / L) of the qPCR reaction system were optimized, and the optimal reaction conditions and systems for the single fluorescence quantitative detection method and double fluorescence quantitative detection method of genotype I and type II GRV were determined, see Tables 5 and 6.

[0062] Table 5 Single-plex 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: ①95℃ for 30s; ②95℃ for 5s, ③60℃ for 30s, ②-③40 cycles, ⑤Melting curve program according to the machine default settings.

[0067] Example 4: Melting curve analysis

[0068] The Tm value was determined based on the melting curve of the single-plex fluorescence quantitative PCR detection method for genotype I and genotype II GRV. The Tm value of genotype I GRV was 79.5°C, and the Tm value of genotype II GRV was 86.5°C. The mixed plasmids were simultaneously subjected to fluorescence quantitative PCR, and the Tm value of the melting curve was consistent with that of the single-plex fluorescence quantitative PCR method ( Figure 2 ).

[0069] Example 5: Specificity test

[0070] In order to verify the specificity of the single fluorescence quantitative detection method and the dual fluorescence quantitative detection method for genotype Ⅰ and Ⅱ GRV, specific detection was performed on genotype Ⅰ and Ⅱ GRV and six common pathogens (AIV, DTMUV, GoAstV, GPV, GoCV, FAdV). The results showed that only genotype Ⅰ and Ⅱ GRV were detected positive, and no specific amplification was found in the control group, indicating that the method has good specificity ( Figure 5 , Figure 6 , Figure 7 ).

[0071] Example 6, sensitivity test

[0072] In order to verify the sensitivity of single fluorescence quantitative detection method and double fluorescence quantitative detection method for genotype I and II GRV, 10 7 ~10 1 The sensitivity of the method was tested by amplifying 18T-Ⅰ-GRV and 18T-Ⅱ-GRV plasmid standards with a concentration of 18T-Ⅰ-GRV and 18T-Ⅱ-GRV copies / μL. The results showed that the minimum detection values ​​of genotype Ⅰ and genotype Ⅱ GRV were 6.466×10 1 copies / μL, 4.979×10 1 copies / μL, 100 times more sensitive than conventional PCR ( Figure 8 , Fig. 9 , Fig.10 ).

[0073] Example 7, establishment of standard curve

[0074] The 18T-Ⅰ-GRV and 18T-Ⅱ-GRV standard positive plasmids were diluted to 10 in a 10-fold gradient. 8 ~10 2 copies / μL, five concentration gradients (10 8 , 10 7 , 10 6 , 10 5 , 10 4) was used as a template to construct the standard curve of the single fluorescence quantitative detection method for genotype I and II GRV, and the results were repeated three times, and a negative control was set up at the same time. The results showed that the standard curve equation of genotype I GRV was y=-3.128X+38.21, the correlation coefficient was 0.997, and the amplification efficiency was 104.54 ( Figure 3 The standard curve equation of genotype II GRV is y=-3.265X+38.58, the correlation coefficient is 0.999, and the amplification efficiency is 102.41( Figure 4 ).

[0075] Example 8, Repeatability Test

[0076] In order to verify the repeatability of single fluorescence quantitative detection method and double fluorescence quantitative detection method for genotype I and II GRV, a gradient of 10 7 , 10 5 , 10 3 The 18T-Ⅰ-GRV and 18T-Ⅱ-GRV standard positive plasmids were repeated three times at three different time points, and the Tm value and Ct value were recorded to calculate the coefficient of variation and the standard deviation within and between groups. The results showed that the coefficient of variation within the single qPCR detection method for genotype I GRV was 0.22% to 0.49%, and the coefficient of variation between groups was 0.43% to 0.58%; the coefficient of variation within the single qPCR detection method for genotype II GRV was 0.24% to 0.36%, and the coefficient of variation between groups was 0.32% to 0.41%); the coefficient of variation within the double qPCR detection method for genotype I and type II GRV was 0.27% to 0.34%, and the coefficient of variation between groups was 0.16% to 0.51% (see Tables 7 to 9).

[0077] Table 7 Results of repeatability test of genotype I GRV

[0078]

[0079] Table 8 Results of repeatability test of genotype II GRV

[0080]

[0081] Table 9 Results of repeatability test of genotype Ⅰ and Ⅱ GRV

[0082]

[0083] Comparative experiment

[0084] The sensitivity was compared using the common PCR method, and 10 7 ~10 1The 18T-Ⅰ-GRV and 18T-Ⅱ-GRV plasmid standards with a concentration of 18T-Ⅰ-GRV and 18T-Ⅱ-GRV were amplified using the amplification system and reaction procedure in Table 2. The results showed that conventional PCR could only amplify to 10 3 copies / μL, while binding Figure 8-10 The minimum detection values ​​of genotype I and II GRV in this application were 6.466×10 1 copies / μL, 4.979×10 1 The dual fluorescence quantitative PCR of the present invention is 100 times more sensitive than the common PCR.

[0085] In summary, the present 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). The primers can be used to construct single fluorescence detection of genotype I and genotype II GRV, and can also be used to construct dual fluorescence detection. Both can detect extremely low concentrations of viral nucleic acid, which is suitable for early viral infection detection. The amount of target virus can be quantitatively analyzed for comparing the difference in the number of viruses in different samples, wherein the dual fluorescence detection can distinguish genotype I GRV (79.5°C) and genotype II GRV (86.5°C) through the melting curve. It is cost-effective and has a wide market application prospect.

[0086] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned 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 above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Double fluorescent quantitative PCR primers for genotype I and II goose-derived reovirus, characterized in that: The primers include a first primer pair and a second primer pair, The sequence of the upstream primer I-F of the first primer pair is shown in SEQ ID No: 1, and the sequence of the downstream primer I-R of the first primer pair is shown in SEQ ID No: 2; The sequence of the upstream primer II-F of the second primer pair is shown in SEQ ID No:3, and the sequence of the downstream primer II-R of the second primer pair is shown in SEQ ID No:

4.

2. Use of the dual fluorescent quantitative PCR primers for genotype I and type II goose-derived reovirus as claimed in claim 1 in the preparation of a reagent for detecting goose-derived reovirus.

3. A dual fluorescence quantitative PCR kit for genotype I and II goose-derived reovirus, characterized in that: Comprising the primer as claimed in claim 1.

4. The kit according to claim 3, characterized in that: A positive standard plasmid is also included.

5. The 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 kit according to claim 4, characterized in that: The positive standard plasmid is prepared by the following method: using the first primer pair shown in SEQ ID NO.1-SEQ ID NO.2 to PCR amplify the S2 gene of gene type I GRV, and using the second primer pair shown in SEQ ID NO.3-SEQ ID NO.4 to PCR amplify the S3 gene of gene type II GRV to obtain an amplified product, connecting the amplified product to a vector, screening positive recombinant plasmids and extracting them, thereby preparing 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.

7. The kit according to claim 6, characterized in that: The PCR amplification system reagents include TaKaRa Premix Taq.

8. The kit according to claim 4, characterized in that: The kit also includes fluorescent quantitative PCR reaction reagents.

9. The kit according to claim 8, characterized in that: The fluorescent quantitative PCR reaction reagent includes TBGreen Premix Ex TaqⅡ.

10. The kit according to claim 9, characterized in that The PCR reaction program of the kit is: ① 95°C for 3 min; ② 95°C for 3 s, ③ 64°C for 30 s, ②-③ 40 cycles, ⑤ melting curve program according to the default settings of the device AriaMxReal-Time PCRG8830A.

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