PCR primer set and method for detecting bovine rotavirus subtypes

Abstract

This invention provides a PCR primer set and method for detecting bovine rotavirus subtypes, belonging to the field of virus detection technology. The invention designs primers with high specificity and sensitivity for different bovine rotavirus subtypes. The detection method is simple to operate, can greatly shorten the typing and identification time, has high accuracy, and saves costs.

Description

Technical Field

[0001] This invention relates to a PCR primer set and method for detecting bovine rotavirus subtypes, belonging to the field of virus detection technology. Background Technology

[0002] Bovine rotavirus (BRV) belongs to the family Reoviridae and the genus Rotavirus. It is a non-enveloped, segmented, double-stranded RNA virus with a diameter of 60–80 nm, comprising 11 gene segments. Rotaviruses are classified into 10 groups (A–J). Groups A, B, and C rotaviruses host humans and animals, groups D and F viruses host birds, and group E hosts pigs. Group A bovine rotavirus (BRVA) is one of the main pathogens causing diarrhea in calves. BRVA genes encode six structural proteins (VP1, VP2, VP3, VP4, VP6, VP7) and six non-structural proteins (NSP1–NSP6). VP4 and VP7 form the outer capsid of the bovine rotavirus particle. VP4 can be cleaved into two subunits, VP5 and VP8, by trypsin. BRVA genotypes are distinguished based on the genetic characteristics of VP4 and VP7, i.e., GxP[x]. To date, at least nine different G genotypes (G1, G3, G5, G6, G8, G10, G15, G21, and G24) and five different P genotypes (P[1], P[5], P

[11] , P

[14] , and P

[33] ) have been isolated. The most common BRV G and P genotypes globally are G6, G8, G10 and P[1], P[5], and P

[11] , respectively; among them, the three combinations of G6P[5], G6P[1], and G10P

[11] dominate in many parts of the world, with a combined prevalence of 40%. Bovine rotavirus often causes mixed infections with other pathogens, and there are currently no effective treatments or vaccines. Therefore, the isolation, identification, and genetic evolution analysis of BRV can clarify the genotypes of circulating strains, identify new potentially pathogenic BRV strains at an early stage, gain a deeper understanding of the genetic evolutionary relationships between different BRV strains, and obtain candidate strains for vaccine preparation, laying the foundation for the development of BRV vaccines and in-depth research on their pathogenic mechanisms. In view of this, the present invention is proposed. Summary of the Invention

[0003] To address the aforementioned technical issues and effectively differentiate and detect different genotypes of bovine rotavirus, this invention provides a primer set with high specificity and sensitivity, and its application in genotyping detection, enabling effective detection and differentiation of different subtypes.

[0004] In a first aspect, the present invention provides a set of primers for detecting bovine rotavirus subtypes, wherein the primer set comprises any combination of 1-3 of the detection primer pairs for bovine rotavirus P[5] type, P

[11] type, and P[1] type.

[0005] Secondly, the primer set provided by the present invention for PCR detection of bovine rotavirus P[5] includes an upstream primer as shown in SEQ ID NO.19 or SEQ ID NO.22 and a downstream primer as shown in SEQ ID NO.30.

[0006] Preferably, the P[5] type detection primer pair includes an upstream primer as shown in SEQ ID NO.19 and a downstream primer as shown in SEQ ID NO.30.

[0007] Thirdly, the primer pair provided by the present invention for PCR detection of bovine rotavirus P

[11] includes an upstream primer as shown in SEQ ID NO.36 and a downstream primer as shown in SEQ ID NO.54 or SEQ ID NO.56.

[0008] Preferably, the P

[11] type detection primer pair includes an upstream primer as shown in SEQ ID NO.36 and a downstream primer as shown in SEQ ID NO.54.

[0009] Fourthly, the primer pair provided by the present invention for the detection of bovine rotavirus P[1] type PCR includes an upstream primer as shown in SEQ ID NO.3 or SEQ ID NO.4 and a downstream primer as shown in SEQ ID NO.17;

[0010] Preferably, the P[1] type detection primer pair includes an upstream primer as shown in SEQ ID NO.4 and a downstream primer as shown in SEQ ID NO.17.

[0011] Furthermore, the P[1] type detection primer pair also includes an upstream primer as shown in SEQ ID NO.2 and a downstream primer as shown in SEQ ID NO.16.

[0012] Fifthly, the present invention provides a set of primers that can be used for bovine rotavirus P[5] and P

[11] typing detection, including the primer pairs for bovine rotavirus P[5] and P

[11] PCR detection.

[0013] In a sixth aspect, the present invention provides a set of primers that can be used for bovine rotavirus P[5] and P[1] typing detection, including the primer pairs for bovine rotavirus P[5] and P[1] PCR detection.

[0014] In a seventh aspect, the present invention provides a set of primers that can be used for the typing detection of bovine rotavirus P[1] and P

[11] types, including the primer pairs for the PCR detection of bovine rotavirus P[1] and P

[11] types.

[0015] Eighthly, the present invention also provides a method for bovine rotavirus typing detection, the specific steps of which are to use the above-mentioned primer pair to perform multiple PCR amplifications on the virus sample in a PCR reaction system.

[0016] The PCR reaction system consisted of 12.5 μL of Premix Taq™, 0.5 μL each of the forward and reverse primers, 2 μL of Temp, and 9.5 μL of ddH2O.

[0017] The reaction program for the PCR amplification step is as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 1 min 20 s, for 35 cycles; 72℃ final extension for 10 min.

[0018] The method for bovine rotavirus typing and detection also includes a gel electrophoresis detection step.

[0019] Optionally, the specific subtype of the virus sample can be determined based on the banding results of the gel electrophoresis detection.

[0020] Furthermore, the present invention also provides a kit comprising the above-described primer pairs or primer sets.

[0021] The kit contains the primer pairs or primer sets mentioned above, and PCR reaction systems in equal numbers to the primer pairs.

[0022] The PCR reaction system contains 12.5 μL of Premix Taq™, 0.5 μL each of upstream and downstream primers, 2 μL of Temp, and 9.5 μL of ddH2O.

[0023] Furthermore, the present invention also provides the application of the above-mentioned detection primer pairs, primer sets, detection methods or kits in bovine rotavirus typing detection.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The detection primers provided by the present invention have good specificity for bovine rotavirus and can effectively and accurately identify specific P types, providing a basis for typing detection.

[0026] (2) The detection method provided by the present invention is simple to operate, greatly shortens the typing and identification time, has good repeatability, and the detection cost is much lower than that of the prior art. It can be applied on a large scale to the typing and detection of bovine rotavirus. Moreover, when multiple genotypes are infected, the accuracy of the method of the present invention is higher than that of sequencing technology. Attached Figure Description

[0027] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 The results are for the specificity verification of the inter-primer of type P[1], where M: 2000 DNA marker; 1-13: type P[1] bacterial culture; 14-29: type P[5] bacterial culture; 30-36: type P[7] bacterial culture; 37-43: type P

[11] bacterial culture; 44-47: type P

[14] bacterial culture; 48-49: type P

[29] bacterial culture; 50: type P

[33] bacterial culture;

[0029] Figure 2 The results are for the specificity verification of the inter-primer of type P[5], where M: 2000 DNA marker; 1-13: type P[1] bacterial culture; 14-29: type P[5] bacterial culture; 30-36: type P[7] bacterial culture; 37-43: type P

[11] bacterial culture; 44-47: type P

[14] bacterial culture; 48-49: type P

[29] bacterial culture; 50: type P

[33] bacterial culture;

[0030] Figure 3 The results are for the specificity verification of the inter-primer of type P

[11] , where M: 2000 DNA marker; 1-13: type P[1] bacterial culture; 14-29: type P[5] bacterial culture; 30-36: type P[7] bacterial culture; 37-43: type P

[11] bacterial culture; 44-47: type P

[14] bacterial culture; 48-49: type P

[29] bacterial culture; 50: type P

[33] bacterial culture;

[0031] Figure 4 The results are for the accuracy verification of P[1] type primers, where M: 2000 DNA marker; 1-8: BCoV, BVDV, IBRV, BRSV, BPIV3, MB, PM, MH; —: negative control; +: P[1] type positive control;

[0032] Figure 5The results are for the accuracy verification of the P[5] type primers, where M: 2000 DNA marker; 1-8: BCoV, BVDV, IBRV, BRSV, BPIV3, MB, PM, MH; —: negative control; +: P[1] type positive control;

[0033] Figure 6 The results are for the accuracy verification of the P

[11] type primers, where M: 2000 DNA marker; 1-8: BCoV, BVDV, IBRV, BRSV, BPIV3, MB, PM, MH; —: negative control; +: P[1] type positive control;

[0034] Figure 7 The results were used to verify the limit of detection of the P[1] type primers, where M: 2000 DNA marker; 1-9: 10 9 copies / µL~10 1 copies / µL plasmid;

[0035] Figure 8 The results were used to verify the limit of detection of the P[5] type primers, where M: 2000 DNA marker; 1-9: 10 9 copies / µL~10 1 copies / µL plasmid;

[0036] Figure 9 The results of the validation of the lowest detection limit of the P

[11] type primers, where M: 2000 DNA marker; 1-9: 10 9 copies / µL~10 1 copies / µL plasmid;

[0037] Figure 10 The results are for P[1] type Tm verification, where M: 2000 DNA marker; 1-8: 46℃, 48℃, 50℃, 52℃, 54℃, 56℃, 58℃, 60℃;

[0038] Figure 11 The results are for the verification of P[5] type Tm, where M: 2000 DNA marker; 1-8: 46℃, 48℃, 50℃, 52℃, 54℃, 56℃, 58℃, 60℃;

[0039] Figure 12 The results are for P

[11] type Tm verification, where M: 2000 DNA marker; 1-8: 46℃, 48℃, 50℃, 52℃, 54℃, 56℃, 58℃, 60℃. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0041] The main reagents used in the following examples and their purchase information are shown in Table 1:

[0042] Table 1. Reagents and their manufacturers

[0043]

[0044] Example 1 Primer Screening

[0045] In this embodiment, the bovine rotavirus VP4 gene sequence was compared. Multiple primer pairs were designed based on all differential sites in different subtypes for preliminary screening. Based on bovine rotavirus P[1] types (GenBank: OK574452, OK539645, MW687596, MT240628, MN807286, MK638873, MF940712, LC727728, JN79018) downloaded from NCBI, the primer pairs were also used for preliminary screening. 7. JF693040, FJ969816, DQ838596, AB119636); P[5] type (GenBank: U53923, OQ807043, OP169139, ON 504858, OM212042, OL988947, MN937516, MN478801, M22306, LC727979, LC133539, KX212870, KC89 5831, KC815661, JF693062, D16351); P[7] type (GenBank: MF940657, MF940654, KF500177, JX971572 , HM988970, EU873009, D16341); P

[11] type (GenBank: ON711387, ON012977, MZ913004, MK250427, M9 Positive plasmids were synthesized from the VP4 gene sequences of P

[14] type (GenBank: MN067458, GU984756, EF554140, AB158430); P

[29] type (GenBank: AB486010, AB454420); and P

[33] type (GenBank: AB513836), and used as templates for primer screening. Multiple primer pairs were designed as shown in Table 2 below, and PCR amplification was performed on the corresponding strains.

[0046] Table 2. Primer pair sequence listing

[0047]

[0048] Note: The degenerate bases are R: A / G; Y: C / T; M: A / C; K: G / T; S: G / C; W: A / T; H: A / T / C; B: G / T / C; V: G / A / C; D: G / A / T; N: A / T / C / G.

[0049] The primers in Table 2 above were used to perform PCR amplification of the corresponding bovine rotavirus P[1], P[5] and P

[11] positive plasmids. The PCR reaction system used was Premix Taq. ™ The initial volume was 12.5 μL, containing 0.5 μL each of forward and reverse primers, 2 μL of Temp, and ddH2O to a final volume of 25 μL. The reaction program was as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 2 min, repeated 35 times; final extension at 72℃ for 10 min. After amplification, 1% agarose gel electrophoresis was performed, and the results were observed and recorded. The results are shown in Table 3.

[0050] Table 3 Primer amplification screening results

[0051]

[0052] Based on the results recorded in Table 3, a secondary screening was conducted using primer type specificity assays to amplify the corresponding bovine rotavirus types in Table 3. The PCR reaction system used was as follows: 12.5 μL of Premix Taq™, 0.5 μL each of forward and reverse primers, 2 μL of Temp, and ddH2O to a final volume of 25 μL. The reaction program was as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 2 min, repeated 35 times; final extension at 72℃ for 10 min. After amplification, 1% agarose gel electrophoresis was performed to verify the primer type specificity. The results are shown in Table 4. Figures 1-3 .

[0053] Table 4. Results of primer type specificity screening

[0054]

[0055] Based on the results recorded in Table 4, a primer species-specificity verification test was further conducted. Primer pairs with good inter-type specificity in Table 4 were screened three times to verify their primer species-specificity. The viruses used were: bovine coronavirus (BCoV), bovine viral diarrhea virus (BVDV), bovine infectious rhinotracheitis virus (IBRV), bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus type 3 (BPIV3), bovine mycoplasma (MB), Pasteurella multocida (PM), and hemolytic Mansonia solani (MH). The PCR reaction system used was: 12.5 μL of Premix Taq™, 0.5 μL each of forward and reverse primers, 2 μL of Temp, and ddH2O to make up to 25 μL. The reaction program was: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 2 min, for 35 cycles; and 72℃ final extension for 10 min. After amplification, 1% agarose gel electrophoresis was performed to verify the primer specificity. The results are shown in Table 5. Figures 4-6 .

[0056] Table 5. Results of Species-Specific Screening of Primers

[0057]

[0058] Based on the results recorded in Table 5, the sensitivity of the primer pairs selected in the table was verified. The concentration of positive plasmids was determined using a spectrophotometer, according to the formula: DNA copy number = plasmid concentration (ng / µL) × 10⁻⁶. -9 Calculate the plasmid copy number by (660 × number of standard plasmid bases). Dilute the positive plasmid with the standard (1 × 10⁻⁶). 9 copies / µL ~1×10 1 (copies / µL) PCR was performed on standards of different diluted concentrations to determine the limit of detection for each genotyping technique. The reaction system, reaction procedure, and detection method were the same as above. The results are shown in Table 6. Figures 7-9 .

[0059] Table 6. Results of primer limit of detection screening (four-stage screening)

[0060]

[0061] According to Table 6, Figure 7-9 According to the results recorded, the detection limit of primer pair 4.17 in type P[1] is 10. 2 copies / µL; the detection limit for primer pairs 2.16 and 3.17 is 10 copies / µL. 4 The limit of detection for primer pair 19.30 in type P[5] is 10 copies / µL. 2The limit of detection for primer pair 22.30 is 10 copies / µL. 3 The detection limit of primer pair 36.54 in type P

[11] is 10 copies / µL; the detection limit of primer pair 36.56 is 10 copies / µL. 2 copies / µL.

[0062] The gel electrophoresis results showed that the primer pairs presented in Table 7 could detect the corresponding positive plasmids, and no cross-reaction occurred between different types. The accuracy was good and the sensitivity was higher, indicating that the primers screened in Table 7 were the best and could be used for bovine rotavirus typing.

[0063] Table 7 Primer screening results

[0064]

[0065] Example 2: Optimal Tm Value Screening

[0066] Annealing temperatures were selected from 46℃ to 60℃, with lower Tm values ​​chosen based on CT values. The reaction mixture consisted of 12.5 μL of Premix Taq™, 0.5 μL each of forward and reverse primers, 2 μL of Temp, and ddH2O to a final volume of 25 μL. The reaction program was as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 1 min 20 s, for 35 cycles; and a final extension at 72℃ for 10 min. After PCR amplification, 1% agarose gel electrophoresis was performed, and the results are shown below. Figures 10-12 As shown.

[0067] The results showed that P[1] type had better amplification effect at 46~52℃, P[5] type had better amplification effect at 46~50℃, and P

[11] type had better amplification effect at 46~58℃. To avoid non-specific amplification caused by excessively low temperature, 50℃ was finally selected as the optimal Tm value.

[0068] Example 3: Comparison of this method with sequencing methods

[0069] The samples used in this embodiment were five anal swabs collected from a cattle farm in Xinjiang. After shaking and mixing to extract nucleic acid, and identification as BRV infection by RT-qPCR, the positive nucleic acids were reverse transcribed. Specifically:

[0070] (1) Clinical samples, shake to mix, centrifuge at 12000r / min for 5min to extract nucleic acid.

[0071] (2)Species identification of nucleic acids was carried out by RT-qPCR method. The primers used are shown in Table 6. Reaction system: Premix 5 μL, Primer F (10 μM) 0.5 μL, Primer R (10 μM) 0.5 μL, Probe 0.25 μL, RNA 2 μL, ddH2O 16.75 μL. Reaction procedure: 55 °C for 15 min, 94 °C for 2 min, (94 °C for 10 s, 60 °C for 30 s) for 40 cycles.

[0072] Table 8 RT-qPCR Primers and Probes

[0073]

[0074] Result determination: Samples with Ct value ≤ 35 in the FAM channel and specific amplification curves are positive samples; samples with no signal value in the FAM channel and no specific amplification curves are negative samples; samples with 35 < Ct value ≤ 40 in the FAM channel and specific amplification curves need to be retested once. If there is still a Ct value, it is judged as positive; if there is no Ct value, it is judged as negative.

[0075] (3)Samples with positive species identification results were reverse transcribed to obtain cDNA. Reaction procedure: 37 °C for 20 min, 85 °C for 15 s. Reaction system: 5×Buffer 2 μL, RT Enzyme Mix 0.5 μL, Oligo dT Primer 0.5 μL, Random 6 mers 2 μL, RNA 5 μL.

[0076] (4)The cDNA was typed by using the detection method in Example 1 of the present invention. At the same time, the VP4 gene of positive samples was cloned, sent to General Biology (Anhui) Co., Ltd. for sequencing, and the detection results of the method of the present invention were compared with the sequencing results. The results are shown in Table 9.

[0077] Table 9 Comparison Results between the Present Method and Sequencing Method

[0078]

[0079] The results showed that the detection results obtained by the present invention were consistent with the typing results of sequencing analysis, indicating that the method has high accuracy and is suitable for daily detection applications.

[0080] Example 4 Cost Accounting Comparison

[0081] Taking the number of samples tested in the epidemiological survey in Xinjiang as an example, assuming 10 samples need to be tested to complete one typing identification, the calculated unit price for sequencing is 124 yuan, while the unit price for the PCR method of this invention is 48 yuan (the calculation basis is: nucleic acid extraction cost 3 yuan / sample, PCR cost 0.6 yuan / sample; implementing the typing detection of this invention requires simultaneous testing of 3 types, therefore the unit price cost of this invention = 3 + 0.6 * 3). The sequencing cost is 1240 yuan, while the cost of the PCR method of this invention is 48 yuan. Compared with sequencing, the method of this invention can reduce the typing cost by as much as 25 times. Therefore, the method of this invention has the advantages of low cost, high efficiency, and accuracy, and can be widely used for routine typing detection of bovine rotavirus.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer set for detecting bovine rotavirus subtypes, characterized in that, The primer set includes any combination of 1-3 of the detection primer pairs for bovine rotavirus P[5], P[11], and P[1] types, wherein the detection primer pair for P[5] type includes an upstream primer as shown in SEQ ID NO.19 or SEQ ID NO.22 and a downstream primer as shown in SEQ ID NO.

30.

2. The genotyping primer set according to claim 1, characterized in that, The P[5] type detection primer pair includes an upstream primer as shown in SEQ ID NO.19 and a downstream primer as shown in SEQ ID NO.

30.

3. The genotyping primer set according to claim 1, characterized in that, The P[11] type detection primer pair includes an upstream primer as shown in SEQ ID NO.36 and a downstream primer as shown in SEQ ID NO.54 or SEQ ID NO.56; Preferably, the P[11] type detection primer pair includes an upstream primer as shown in SEQ ID NO.36 and a downstream primer as shown in SEQ ID NO.

54.

4. The detection primer set according to claim 1, characterized in that, The primer set further includes a bovine rotavirus P[1] type detection primer pair; Preferably, the P[1] type detection primer pair includes an upstream primer as shown in SEQ ID NO.3 or SEQ ID NO.4 and a downstream primer as shown in SEQ ID NO.17; More preferably, the P[1] type detection primer pair includes an upstream primer as shown in SEQ ID NO.4 and a downstream primer as shown in SEQ ID NO.

17.

5. The detection primer set according to claim 4, characterized in that, The P[1] type detection primer pair includes an upstream primer as shown in SEQ ID NO.2 and a downstream primer as shown in SEQ ID NO.

16.

6. A method for detecting bovine rotavirus subtypes, characterized in that, Using the primer set described in any one of claims 1-5, PCR amplification of samples containing bovine rotavirus strains was performed in a PCR reaction system.

7. The method according to claim 6, characterized in that, The PCR amplification reaction system is as follows: 12.5 μL Premix Taq™, 0.5 μL each of upstream and downstream primers, 2 μL Temp, and ddH2O to make up to 25 μL of reaction system.

8. The detection method according to claim 7, characterized in that, The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 1 min 30 s, for 35 cycles; 72℃ final extension for 10 min.

9. A kit for typing and detecting bovine rotavirus, characterized in that, The kit contains a primer set according to any one of claims 1-5, and a PCR reaction system in equal numbers to the primer pairs.

10. The use of the detection primer set, detection method or kit according to any one of claims 1-9 in the detection of bovine rotavirus subtypes.

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