Primer probe set, kit for detecting capripoxvirus based on constant temperature insulation fluorescence PCR platform and application
By designing specific primer and probe sets and using freeze-drying technology, combined with a temperature-controlled isolated fluorescent PCR platform, the problem of low detection rate of existing quantitative fluorescent PCR methods in detecting goat type C Kubvirus has been solved, achieving high sensitivity and high accuracy in on-site detection, which is suitable for rapid diagnosis in goat farming.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SOUTHWEST UNIVERSITY FOR NATIONALITIES
- Filing Date
- 2025-11-05
- Publication Date
- 2026-07-03
AI Technical Summary
Existing quantitative real-time PCR methods for detecting goat type C Kubvirus have problems such as low detection rate, complicated operation, and unsuitability for rapid on-site detection, especially in the case of gene mutations, they are difficult to detect accurately.
Design specific primer and probe sets, use a temperature-controlled isolated fluorescent PCR platform, and use lyophilization technology to preserve reaction solutions and controls to simplify the operation process and make it suitable for on-site testing.
It achieves high sensitivity and high accuracy detection of goat type C Kubuvir, simplifies the operation process, reduces storage requirements, and facilitates transportation and field application.
Smart Images
Figure CN122326809A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a primer and probe set, kit, and application for detecting goat type C Kubuvir based on a isothermal isolated fluorescent PCR platform. Background Technology
[0002] Goat Coovirus type C (CKoV) was first detected in the feces of Korean black goats by Lee MH et al. in 2012. In 2019, ABI KM et al. first discovered goat Coovirus type C in the feces of goats with diarrhea, proving the presence of the virus in China and confirming that goat Coovirus type C is an important pathogen causing diarrhea in goats. The main clinical symptom of goat Coovirus type C infection is diarrhea, with a high mortality rate, causing huge economic losses to the goat farming industry.
[0003] Many pathogens can cause diarrhea in goats, including circovirus, enterovirus, Bopivirus, Bocaparvovirus, astrovirus, Escherichia coli, Salmonella, Cryptosporidium, and coccidia. Their symptoms are similar and difficult to distinguish, making early differential diagnosis crucial. Pathogen detection provides direct evidence of infection and is the most reliable diagnostic method. Quantitative real-time PCR (qPCR) is the most commonly used pathogen detection method and has become the mainstream approach for animal disease diagnosis. However, the use of conventional qPCR methods for rapid on-site detection is limited by the need for large equipment, complex operation, and long reaction time.
[0004] Meanwhile, due to regional differences, the goat C-type Kubvirus gene has undergone significant mutations, especially the 3D gene, which is often used as a target gene. The mutations in the 3D gene affect the detection rate of PCR methods that use it as a target gene, making it difficult to detect accurately.
[0005] Therefore, establishing a detection method that is accurate, fast, efficient, and suitable for field use is of great significance for improving the accuracy and stability of detection. Summary of the Invention
[0006] To address the aforementioned problems, the present invention aims to provide a method for detecting goat Kubvirus type C and its applications. The primer and probe set, reagent kit, and detection method provided by the present invention, combined with a temperature-controlled isolated fluorescent PCR platform, can achieve highly sensitive and accurate detection of goat Kubvirus type C.
[0007] To achieve the above objectives, the present invention provides the following technical solution:
[0008] A primer and probe set for detecting goat type C Kub virus based on a temperature-controlled isolated fluorescent PCR platform includes upstream and downstream primers for goat type C virus and a fluorescent probe.
[0009] The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the fluorescent probe is shown in SEQ ID NO: 3.
[0010] The fluorescent probe nucleotide sequence has a FAM tag attached to its 5' end and a BHQ1 fluorescence quencher attached to its 3' end.
[0011] The application of the above primer and probe set in the preparation of a goat type C Kub virus detection kit.
[0012] A kit for detecting goat type C Kubvirus based on a isothermal isolated fluorescent PCR platform, comprising the aforementioned primer and probe set.
[0013] It also includes reaction buffer, real-time PCR reaction solution, positive control and negative control.
[0014] Further, the reaction buffer comprises the following components: 500mM KCl, pH 8.3, 100mM Tris-HCl, 15mM MgCl2, with the balance being ddH2O.
[0015] Furthermore, the real-time PCR reaction solution includes Premix Ex Taq™ enzyme, reverse transcriptase, dNTPs, and the primer and probe set described above.
[0016] The fluorescence quantitative PCR reaction solution, positive control, and negative control are in a lyophilized state.
[0017] A method for detecting goat type C Kubvirus includes the following steps:
[0018] (1) Preparation of RNA template: Take fecal samples, extract total RNA from the samples to prepare RNA template;
[0019] (2) The above primer and probe set or the above kit is used to carry out the reaction in a constant temperature isolated fluorescent PCR instrument.
[0020] (3) Test result interpretation: "+" indicates positive, "-" indicates negative.
[0021] Compared with the prior art, the present invention has the following technical effects:
[0022] This invention, through specific primer and probe design and use with a temperature-controlled isolated fluorescent PCR platform, achieves highly sensitive and accurate detection of goat type C Kubuvir, demonstrating the best positive detection rate for clinical samples compared to existing quantitative fluorescent PCR methods. Furthermore, the fluorescent quantitative PCR reaction solution and both positive and negative controls used in this invention are prepared by lyophilization, reducing the storage requirements of the kit and allowing it to be stored at 4°C for one year, facilitating transportation and storage.
[0023] This invention provides a detection method based on a temperature-controlled isolated fluorescent PCR platform. It features high sensitivity and specificity, solving the technical problems of low detection rate and inability to rapidly and accurately detect various goat type C Kubuvirus variants in existing technologies. Moreover, it is simple to operate, fast and efficient (requiring only 42 minutes), and is handheld, allowing for rapid on-site detection. It can be used for molecular epidemiological surveys of goat type C Kubuvirus, which is of great significance for ensuring the healthy development of the goat farming industry.
[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the embodiments do not limit the present invention in any way. For those skilled in the art, any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention. Attached Figure Description
[0025] Figure 1 It's a POCKIT TM A structural diagram of the instrument.
[0026] Figure 2 These are the results of the sensitivity test for positive samples, where 1: 3.99 × 10⁻⁶. 2 copies / μL; 2: 3.99×10 1 copies / μL; 3: 3.99×10 0 copies / μL; 4: 3.99×10 -1 copies / μL. Detailed Implementation
[0027] The present invention will be further described in detail below with reference to the embodiments, but the present invention is not limited thereto.
[0028] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.
[0029] Example 1: A kit for detecting goat type C Kubvirus based on a temperature-controlled isolated fluorescent PCR platform
[0030] The kit of the present invention comprises the following components:
[0031] (1) Reaction buffer: 3 ml
[0032] It consists of 500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM MgCl2 and ddH2O (balance).
[0033] (2) Lyophilized tubes of real-time PCR reaction solution (50 tubes):
[0034] The real-time PCR reaction solution includes 1 μL of Premix Ex Taq™ enzyme per tube (5 U / μL) -1 ), reverse transcriptase 1.25 μL / tube (20 U·μL⁻¹), forward and reverse primers 3.5 μL / tube (10 μmol·μL¹) and probe 0.25 μL / tube (10 μmol·μL¹) for detection. -1 ), dNTPs 4μL / tube (2.5mM); add the above mixture to a 0.5ml PCR tube and cool to -50℃, then freeze-dry at 10pa pressure for 24h. After forming a dry powder, close the tube to obtain the lyophilized tube of the real-time PCR reaction solution.
[0035] Among them, three upstream and downstream primers and probes were used to detect goat type C Kubuvirus, and the length of the goat type C Kubuvirus 3D gene was 143bp.
[0036] SEQ ID NO: 1 Upstream primer sequence: 5'-CAAACTAACTGGAGATGAAA-3';
[0037] SEQ ID NO: 2 Downstream primer sequence: 5'-AGTGTTGATGATGGAAGTA-3';
[0038] SEQ ID NO: 3 Probe sequence: 5'-TGCCGCCAATCATCTCATACG-3' (5' with FAM label added, 3' with BHQ1 fluorescence quencher group added).
[0039] (3) Positive control lyophilized tube (1 tube):
[0040] 100 μL of an RNA sample containing a fragment of goat type C Kubuvirus RNA was loaded into a PCR tube, cooled to -50°C, and then freeze-dried at 10 Pa for 24 h to form a dry powder. The tube was then closed to prepare the sample. The sequence of the goat type C Kubuvirus RNA is as follows (SEQ ID NO: 4).
[0041] SEQ ID NO: 4
[0042]
[0043] (4) Negative control lyophilized tube (1 tube):
[0044] 100 μL of a sample containing goat type C Kubu virus RNA fragment was loaded into a PCR tube, cooled to -50°C, and then freeze-dried at 10 Pa for 24 h to form a dry powder. The tube was then closed to prepare the sample. The sequence of goat type C Kubu virus RNA is shown in SEQ ID NO: 4.
[0045] This kit should be stored at 4°C and transported with ice packs.
[0046] Example 2: Method of using the goat C-type Kubvirus Detection Kit of the present invention
[0047] The method includes the following steps:
[0048] (1) RNA template: Take 400 μL of fecal sample supernatant and extract total RNA from the sample supernatant using the Trizol method to prepare RNA template;
[0049] (2) Preparation of fluorescent PCR reaction system: The fluorescent PCR reaction system was prepared on ice;
[0050] Add 100 μL of reaction buffer to the positive control lyophilized tube and mix to prepare the positive control. Then add 50 μL of reaction buffer and 5 μL of positive control to the real-time PCR reaction solution lyophilized tube to prepare the positive control reaction system.
[0051] Add 100 μL of reaction buffer to a lyophilized tube of negative control to prepare negative control. Then add 50 μL of reaction buffer and 5 μL of negative control to a lyophilized tube of real-time PCR reaction solution to prepare negative control reaction system.
[0052] Add 50 μL of reaction buffer and 5 μL of RNA from the detection sample to a lyophilized tube containing the real-time PCR reaction solution to prepare the RNA reaction system for the detection sample.
[0053] Then, transfer 50 μL of the mixture from each reaction system into a temperature-controlled, isolated fluorescent PCR reaction tube. Avoid light exposure during sample addition. Briefly centrifuge the prepared fluorescent quantitative PCR reaction tube at high speed for 5 seconds, avoiding the formation of air bubbles.
[0054] (3) Amplification detection: Place the PCR reaction tubes inside a temperature-controlled, insulated fluorescent PCR instrument (POCKIT). TM )(like Figure 1 (As shown), press the run button to start the reaction;
[0055] (4) Result interpretation: "+" indicates positive and "-" indicates negative.
[0056] Note: Test results may show a "?", which indicates a suspected result, possibly due to invalid data, weak signal, or air bubbles during sample addition. If a "?" appears, you should retest to confirm the result.
[0057] Example 3: Preparation of Standards and Determination of Sensitivity
[0058] Take 400 μL of fecal sample supernatant, extract total RNA from the sample supernatant using the Trizol method, and then reverse transcribe the total RNA into cDNA according to the instructions of the TaKaRa reverse transcription kit.
[0059] The cDNA prepared above was amplified by PCR using primers for goat type C Kubuvirus, with the corresponding pathogen cDNA as a positive control and no template as a negative control.
[0060] The reaction system consisted of: 2.0 μL cDNA, 10 μL 2×Taq PCR Master Mix, 1.0 μL each of upstream and downstream primers, 6 μL ddH2O, for a total volume of 20 μL.
[0061] The PCR amplification conditions were: 95℃ for 3 min, 95℃ for 15 s, 58.7℃ for 15 s, 72℃ for 20 s, 35 cycles, 72℃ for 5 min.
[0062] The PCR products were identified by 3% agarose gel electrophoresis, the target fragment was recovered using a gel extraction kit, and cloned into the pMD19-T vector (Takara Bio Inc.). The vector was then transformed into E. coli DH5α competent cells, and positive clones were screened and inoculated into LB liquid medium containing ampicillin. The cells were cultured at 37°C for 8 hours, and the recombinant plasmid was extracted using the Plas-mid Mini kit plasmid extraction kit and sent to Sangon Biotech Inc. for sequencing.
[0063] The correctly sequenced positive plasmid was used as a positive standard for goat type C Kubvirus. Its concentration was determined using a nucleic acid protein analyzer, and the nucleic acid concentration was calculated using the following formula: Positive standard concentration. (copies / μL) = (6.022 × 10⁻⁶) 23 (copies / mol) × (61.5 ng μL) × 10 -3 / (9.27×10 5 g / mol) = 3.99 × 10 10 copies / μL.
[0064] The positive standard was serially diluted 10-fold (10 -2 ~10 -12), for 10 2 10 1 10 0 and 10 -1 Four dilutions of the sample were tested, and each reaction tube was placed in a POCKIT. TM To open the reaction well, press the "Run" button. The reaction will end after 42 minutes. A positive result will be indicated by "+", and a negative result by "-".
[0065] The results are as follows Figure 2 As shown, 10 2 10 1 10 0 The result is "+" 10 -1 A result of "-" indicates that the limit of detection for this kit is 39.9 copies / μL.
[0066] Example 4: Specificity and stability testing
[0067] The kit developed in this invention was used to detect prepared positive standards and seven related pathogens: Torovirus, Enterovirus, Bopivirus, Bocaparvovirus, Astrovirus, Escherichia coli, and Salmonella. DEPCH2O was used as a negative control to evaluate the kit's specificity. The results showed positive detection of the positive standards and negative detection of the seven related pathogens and DEPC H2O, demonstrating the high specificity of this method.
[0068] The stability of the developed kit was evaluated by testing positive standards of goat type C Kubvirus at seven dilutions three times. The results showed that the three tests were consistent, indicating that the kit has good stability.
[0069] Example 5: Clinical application of the kit of the present invention and comparison with conventional real-time PCR methods
[0070] The nucleic acids extracted from 28 goat type C Kubuvir-positive samples were detected using the kit of this invention and the fluorescence quantitative PCR method reported in existing literature. The concordance rates of the five PCR methods were compared. The 28 goat type C Kubuvir-positive samples were isolated from cells in our laboratory and their positivity was confirmed.
[0071] The isothermal isolated fluorescence PCR instrument used in this experiment was purchased from Ruiji Marine Biotechnology Co., Ltd. (product number: POCKIT™); the quantitative fluorescence PCR instrument was purchased from Thermo Fisher Scientific (Guangzhou) Biotechnology Co., Ltd. (product number: QuantStudio 1Plus).
[0072] The existing literature reports the following:
[0073] Abikhamo et al. (Abikhamo, Yu Zhonghua, Jing Zhizhong, et al. Establishment and application of real-time fluorescence quantitative PCR method for detecting goat ridge virus [J]. Journal of Animal Husbandry and Veterinary Medicine, 2021, 52 (05): 1349-1358).
[0074] MELEGARI I et al. (MELEGARI I, DI PROFIO F, SARCHESE V, et al. First molecular evidence of kobuviruses in goats in Italy [J]. Arch Virol, 2016, 161(11): 3245-3248);
[0075] REUTER G et al. (REUTER G, BOLDIZSAR A, PANKOVICS P. Complete nucleotide and amino acid sequences and genetic organization of porcine kobuvirus, a member of a new species in the genus Kobuvirus, family Picornaviridae [J]. ArchVirol, 2009, 154(1): 101-108.);
[0076] Lee MH et al. (Lee MH, Jeoung HY, Lim JA, et al. Kobuvirus blackgoats[J]. Virus genes, 2012, 45(1): 186-189).
[0077] The results showed that among the 28 positive samples of goat type C Kubvirus, the kit of this invention detected 27 positive samples, with a concordance rate of 96.42%.
[0078] The quantitative real-time PCR method reported by Abikhamo et al. detected 19 positive samples for total RNA, with a concordance rate of 67.85%.
[0079] The quantitative real-time PCR method reported by MELEGARI I et al. detected 9 positive samples for total RNA, with a concordance rate of 32.14%.
[0080] REUTER G et al. reported that the real-time quantitative PCR method detected 25 positive samples for total RNA, with a concordance rate of 89.28%.
[0081] The quantitative real-time PCR method reported by Lee MH et al. detected 0 positive samples for total RNA, with a concordance rate of 0%.
[0082] In addition, when the primer and probe set of the present invention was used on a conventional real-time PCR instrument to detect the above 28 samples, only 12 positive samples were found, with a concordance rate of 42.86%. Moreover, the amplification efficiency and detection sensitivity were lower than those when used on a temperature-controlled isolated real-time PCR instrument.
[0083] Sequencing of PCR products that were not detected by quantitative real-time PCR but were detected positive by the kit confirmed that they were specific sequences of the goat type C Kubvirus 3D gene.
[0084] It is evident that the kit of this invention has a better detection effect on goat type C Kubuvirus than the existing fluorescent quantitative PCR method. At the same time, it is simpler to operate and more portable than the existing PCR method, making it more suitable for on-site detection and diagnosis. It provides a powerful and convenient tool for the diagnosis and epidemiological investigation of goat type C Kubuvirus.
[0085] In summary, the kit of this invention overcomes the problem of low detection rate in existing quantitative PCR methods and can detect goat type C Kubvirus with high efficiency. This kit, combined with POCKIT... TM The kit is easy to use, portable, and suitable for clinical applications. Furthermore, the reagents in this invention are lyophilized, reducing storage requirements and facilitating transportation and clinical use.
Claims
1. A primer and probe set for detecting goat type C Kubvirus based on a isothermal isolated fluorescent PCR platform, characterized in that, It includes upstream and downstream primers and a fluorescent probe for goat type C virus. The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the fluorescent probe is shown in SEQ ID NO:
3.
2. The primer-probe set according to claim 1, characterized in that, The fluorescent probe nucleotide sequence has a FAM tag attached to the 5' end and a BHQ1 fluorescence quencher group attached to the 3' end.
3. The application of the primer and probe set according to claim 1 or 2 in the preparation of a goat type C Kub virus detection kit.
4. A kit for detecting goat type C Kubuvir based on a isothermal isolated fluorescent PCR platform, characterized in that, It includes the primer-probe set as described in claim 1 or 2.
5. The reagent kit according to claim 4, characterized in that, It also includes reaction buffer, real-time PCR reaction solution, positive control and negative control.
6. The reagent kit according to claim 5, characterized in that, The reaction buffer comprises the following components: 500 mM MCl, pH 8.3, 100 mM Tris-HCl, 15 mM MgCl2, with the balance being ddH2O.
7. The reagent kit according to claim 5, characterized in that, The real-time PCR reaction solution includes Premix ExTaq™ enzyme, reverse transcriptase, dNTPs, and the primer and probe set as described in claim 1 or 2.
8. The kit according to claim 5 or 7, characterized in that, The fluorescence quantitative PCR reaction solution, positive control, and negative control were in a lyophilized state.
9. A method for detecting goat type C Kubvirus, characterized in that, Includes the following steps: (1) Preparation of RNA template: Take fecal samples, extract total RNA from the samples to prepare RNA template; (2) The reaction is carried out in a constant temperature isolated fluorescence PCR instrument using the primer and probe set as described in claim 1 or 2, or the kit as described in claim 4 or 5. (3) Result determination: "+" indicates positive and "-" indicates negative.