A one-step RT-PCR detection kit for detecting porcine gattai virus infection and application thereof

Abstract

The application provides a one-step RT-PCR detection kit for detecting porcine getah virus infection and application thereof, and belongs to the technical field of virus detection. The application provides a one-step RT-PCR detection kit for detecting porcine getah virus (GETV) infection and application thereof, wherein the kit contains a pair of specific primers for detecting the getah virus. The kit has good detection result specificity, and has no cross reaction with main members of pig diseases and common pig, cattle and sheep pathogens; the kit has high sensitivity, and the sensitivity for detecting plasmid standard products and GETV cell toxins is 5.9 copies and 0.3 TCID 50 respectively; the kit is rapid, sensitive, simple, low in cost, reverse transcription and PCR are performed in one reaction tube, and the pollution opportunity is reduced.

Description

Technical Field

[0001] This invention belongs to the field of virus detection technology, specifically relating to a one-step RT-PCR detection kit for detecting porcine Geytavirus infection and its application. Background Technology

[0002] Getah virus (GETV) has a clear taxonomic classification, belonging to the genus Alphavirus of the family Bleoviridae, group Semliki Forest Virus Complex. It exhibits typical characteristics of mosquito-borne vector viruses and can circulate among vertebrate hosts. Genomically, GETV possesses a non-segmented, single-stranded positive-sense RNA of approximately 12 kb in length. Its genome has clearly defined functional regions, including two open reading frames (ORFs). The 5' ORF encodes four non-structural proteins (NSP1-NSP4), while the 3' ORF encodes five structural proteins (E1, E2, E3, C, and 6K). These proteins play crucial roles in viral replication, assembly, and host cell recognition. Importantly, the NSP3 gene, a non-structural protein, is essential for the synthesis of negative-sense RNA and subgenomic RNA. This protein domain possesses ADP-nucleotide 1' phosphatase and RNA-binding activity, potentially related to inducing apoptosis in infected cells. Similar to the NSP2 gene, a portion of the NSP3 gene is also located on the nuclear envelope, suggesting its association with viral pathogenicity. This unique gene structure has become a core target for the design of specific primers and probes in GETV molecular biology detection, providing an important basis for the development of precision detection technology. Regarding its host range, GETV shows a continuously expanding trend. Currently, GETV antibodies have been detected in a variety of animals, including horses, cattle, goats, dogs, foxes, rabbits, kangaroos, chickens, and some wild birds, indicating that these animals may all be hosts for GETV. More concerningly, neutralizing antibodies against GETV have also been detected in human serum samples, suggesting a potential risk of transmission to humans and the development of disease. Currently, laboratory detection methods for GETV mainly fall into four categories: virus isolation and identification, immunoelectron microscopy observation, serological diagnosis, and viral nucleic acid detection. Each method has its own characteristics and limitations in application. Given the limitations of existing detection technologies and the serious impact of GETV on economically important animals, establishing a rapid, specific, sensitive, and qualitative GETV detection method has become an urgent task. Summary of the Invention

[0003] This invention provides a one-step RT-PCR detection kit for detecting porcine Gettavirus infection and its application. The one-step detection of GETV has the characteristics of being specific, sensitive, rapid, simple, and low-cost.

[0004] This invention provides a set of primer pairs for detecting porcine Getta virus, including nucleotide sequences such as GETV-F shown in SEQ ID No. 1 and GETV-R shown in SEQ ID No. 2.

[0005] The present invention also provides a detection kit for porcine getta virus containing the above-mentioned primer pairs.

[0006] In a preferred embodiment of the present invention, the detection kit is an RT-PCR detection kit.

[0007] The present invention also provides a one-step RT-PCR detection kit for porcine getta virus containing the above-mentioned primer pairs.

[0008] In a preferred embodiment of the present invention, the invention further includes One Step Reaction Mix, TransScript® OneStep Enzyme Mix, positive standard, positive control, negative control, and enzyme-free water.

[0009] In a preferred embodiment of the present invention, the positive standard is obtained by ligating the fragment obtained by amplification using the above-mentioned primer pair into a cloning vector.

[0010] In a preferred embodiment of the present invention, the positive control is an RNA extract of an inactivated porcine Geyta virus cell culture or a synthetically produced plasmid containing the porcine Geyta virus NSP3 gene.

[0011] This invention also provides a detection method for the above-mentioned one-step RT-PCR detection kit for non-disease diagnosis and treatment purposes, including extracting RNA from the sample to be tested when performing quantitative detection of porcine Geyta virus, constructing a one-step RT-PCR reaction system using RNA and serially diluted positive standards as templates to perform RT-PCR reaction, constructing a standard curve based on the Ct value and concentration of the positive standards, and substituting the Ct value of the RNA of the sample to be tested into the standard curve to obtain the content of porcine Geyta virus in the sample to be tested.

[0012] In a preferred embodiment of the present invention, the RT-PCR reaction system, in 20 μL, comprises: 10 μL One Step Reaction Mix, 0.4 μL TransScript® One Step Enzyme Mix, 1 μL each of 10 μmol / L upstream and downstream primers, 3 μL viral RNA, and the remainder ddH2O.

[0013] In a preferred embodiment of the present invention, the RT-PCR reaction program includes: 45℃ for 20 min, 94℃ for 3 min; denaturation at 94℃ for 30 sec, annealing at 57.0℃ for 30 sec, extension at 72.0℃ for 45 sec, 35 cycles; and extension at 72℃ for 10 min.

[0014] Beneficial Effects: This invention provides a one-step RT-PCR detection kit for detecting porcine Gettavirus (GETV) infection and its application. The kit contains a pair of specific primers for detecting Gettavirus. The kit exhibits high specificity, showing no cross-reactivity with major swine disease members such as classical swine fever virus (CSFV), porcine epidemic diarrhea (PED), highly pathogenic porcine reproductive and respiratory syndrome (PRRS), and bovine viral diarrhea virus (BVDV), as well as common swine, bovine, and ovine pathogens. It also demonstrates high sensitivity, with sensitivities of 5.9 copies for plasmid standards and 0.3 TCID for GETV cytotoxicity. 50 The test is rapid, sensitive, simple, and inexpensive. Reverse transcription and PCR are performed in one reaction tube, reducing the chance of contamination. It can fill the gap in the current lack of commercially available GETV detection kits in China and can be widely used in clinical sample detection, disease diagnosis, and molecular epidemiological surveys of GETV. It plays an important role in ensuring the diagnosis and monitoring of Geta disease in my country, more effectively preventing and controlling Geta disease, protecting livestock health, and promoting industrial development.

[0015] Biological Preservation Information The porcine Getah virus strain GETV-BJ-2022 is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, on January 14, 2026, with accession number CCTCCNO:V202606. Attached Figure Description

[0016] Figure 1 The figure shows the results of the sensitivity test of the RT-PCR detection method for Gettavirus. Figure 2This is a graph showing the results of a specific detection test using the RT-PCR method for Gettavirus. In the graph, positive indicates the amplification result of GETV; negative indicates the negative control; 1-19 are the main representative strains of swine, cattle, and sheep (including classical swine fever virus C strain, classical swine fever virus Thiveral strain, bovine viral diarrhea virus type 1, bovine viral diarrhea virus type 2, highly pathogenic porcine reproductive and respiratory syndrome live vaccine (TJM-F92 strain), porcine reproductive and respiratory syndrome live vaccine (CH-1R strain), pseudorabies live vaccine (Bartha-K61 strain), goatpox live vaccine (AV41 strain), peste des petits ruminants live vaccine (Clone9 strain), bovine parainfluenza virus type 3, porcine circovirus type 2, porcine transmissible gastroenteritis SCJY-1 strain, porcine epidemic diarrhea SCSZ-1 strain, SIV-H1N1 LN strain, SIV-H3N2 HLJ strain, mink-canine distemper live vaccine, porcine transmissible gastroenteritis, porcine epidemic diarrhea, and porcine rotavirus (G5 type) triple live vaccine, etc.). Figure 3 This is a sensitivity test of the RT-PCR method for detecting Getta virus. In the figure: 1, 2, 3, 4, 5, 6, 7, and 8 represent serial dilutions of 10-fold (10... 1 ~10 8 3.16×10 after) 4 TCID 50 up to 0.003 TCID 50 Amplification results of the GETV strain; Figure 4 Image showing the results of a Getta virus RT-PCR detection kit applied to clinical samples; Figure 5 Figure showing the results of the storage conditions test for the Getta virus RT-PCR detection kit; Figure 6 The figure shows the repeatability test results of the GETV RT-PCR detection method. M in the figure represents the DL2000 marker; negative represents the negative control. Detailed Implementation

[0017] This invention provides a set of primer pairs for detecting porcine Getta virus, including nucleotide sequences such as GETV-F shown in SEQ ID No. 1 and GETV-R shown in SEQ ID No. 2.

[0018] This invention targets a highly conserved fragment of the NSP3 gene in the non-structural protein of GETV and designs a pair of specific primers (GETV-F / GETV-R).

[0019] GETV-F (SEQ ID No. 1): 5'-GCACCGTCATACAGGGTCCGCCGCG-3'; GETV-R (SEQ ID No. 2): 5'-CGCGGTCATCGCGTACCGACAT-3'.

[0020] The present invention also provides a detection kit for porcine getta virus containing the above-mentioned primer pairs.

[0021] The detection kit described in this invention is an RT-PCR detection kit, which contains reagents for RT-PCR detection.

[0022] The present invention also provides a one-step RT-PCR detection kit for porcine getta virus containing the above-mentioned primer pairs.

[0023] The kit of the present invention also includes One Step Reaction Mix (2×), TransScript® OneStep Enzyme Mix, positive standard, positive control, negative control and enzyme-free water, wherein the positive standard is obtained by ligating the fragment obtained by amplification using the above primer pairs into a cloning vector.In one embodiment of the present invention, the nucleotide sequence of the recovered product obtained by amplification is shown in SEQ ID No. 3. The product shown in SEQ ID No. 3 was ligated into the pEASY®-T1 Cloning Vector (Beijing TransGen Biotech Co., Ltd., Cat#: CT101-01), transformed into Trans-T1 competent cells, and positive clones were obtained by ampicillin resistance screening, PCR identification and sequencing. Plasmids were extracted according to the plasmid miniprep kit as positive standards.

[0024] The positive control described in this invention is an RNA extract from an inactivated porcine Gettavirus cell culture or a synthetically produced plasmid containing the porcine Gettavirus NSP3 gene. The negative control described in this invention is a normal cell control such as VERO or BHK-21 / BSR cells or an RNA extract from anticoagulated blood of GETV-negative animals.

[0025] This invention also provides a detection method for the above-mentioned one-step RT-PCR detection kit for non-disease diagnosis and treatment purposes, including extracting RNA from the sample to be tested when performing quantitative detection of porcine Geyta virus, constructing a one-step RT-PCR reaction system using RNA and serially diluted positive standards as templates to perform RT-PCR reaction, constructing a standard curve based on the Ct value and concentration of the positive standards, and substituting the Ct value of the RNA of the sample to be tested into the standard curve to obtain the content of porcine Geyta virus in the sample to be tested.

[0026] The RT-PCR reaction system described in this invention, in 20 μL volume, comprises: 10 μL One Step Reaction Mix, 0.4 μL TransScript® One Step Enzyme Mix, 1 μL each of 10 μmol / L upstream and downstream primers, 3 μL viral RNA, and the remainder ddH2O.

[0027] The RT-PCR reaction procedure described in this invention includes: 45℃ for 20 min, 94℃ for 3 min; denaturation at 94℃ for 30 sec, annealing at 57.0℃ for 30 sec, extension at 72.0℃ for 45 sec, 35 cycles; and extension at 72℃ for 10 min. When using the method described in this invention for detection, the established method achieves a sensitivity of 5.9 copies / μL, demonstrating good sensitivity. Using serially diluted 10-fold doses of the GETV strain with determined viral content as templates, the established Getta virus RT-PCR detection method achieves a sensitivity of 0.3 TCID⁻¹. 50 The sensitivity is good; the amplification results are clear and accurate for different template concentrations, indicating that the established RT-PCR detection method has good stability.

[0028] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a one-step RT-PCR detection kit for detecting porcine Geytavirus infection and its application, should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1: Design of specific primers targeting the conserved region of the NSP3 gene in the GETV non-structural protein The reference sequence of the GETV strain was downloaded from GenBank. Based on the sequence alignment results, a pair of specific primers (GETV-F / GETV-R) were designed targeting the highly conserved fragment of the NSP3 gene in the non-structural protein of GETV.

[0030] Establishment and optimization of RT-PCR detection method for Getta virus T Following the instructions of the Body Fluid Virus DNA / RNA Mini-Preparation Kit (Axygen, catalog number AP-MN-BF-VNA-250), the GETV genome was extracted as a template. The template concentration was fixed, and the optimal primer concentration, probe concentration, and reaction conditions were determined by comprehensively considering factors such as primer concentration and annealing temperature.

[0031] The optimized reaction system consisted of 20 μL: 10 μL One Step Reaction Mix (2×), 0.4 μL TransScriptOne Step Enzyme Mix, 1.0 μL upstream primer (10 μM), 1.0 μL downstream primer (10 μM), 3 μL template, and 4.6 μL enzyme-free water. The final reaction conditions were: reverse transcription at 55℃ for 30 min; pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 1 min, annealing at 55℃ for 1 min, extension at 72℃ for 1 min, for a total of 35 cycles; and extension at 72℃ for 10 min.

[0032] The RT-PCR products were recovered and purified using a large volume agarose gel DNA recovery kit (TIANGEN, catalog number DP210-02). The recovered products were ligated into the pEASY®-T1 Cloning Vector (Beijing TransGen Biotech Co., Ltd., Cat#: CT101-01), transformed into Trans-T1 competent cells, and positive clones were obtained by ampicillin resistance screening, PCR identification and sequencing. Plasmids were extracted as positive standards according to the plasmid miniprep kit (TIANGEN Biotech, catalog number DP103).

[0033] According to the formula N = (plasmid concentration ng / μL × 10) -9 ×6.02×10 23 Calculate the copy number of the recombinant plasmid by dividing the base number by 660 (bp × 660). The recombinant plasmid was initially calculated from 5.9 × 10⁻⁶. 10 copies / μL up to 5.9×10 -1 Copies / μL were serially diluted 10 times to serve as positive standards.

[0034] Example 2: Specificity results of the Gettavirus Quantitative RT-PCR Detection Kit 1. Sample processing (1) Samples: GETV (GETV-BJ-2022 strain, CCTCC NO: V202606), classical swine fever virus C strain (published in the article: Zhang Zhihui. Establishment and proteomics analysis of porcine testicular cell line carrying high titer classical swine fever virus C strain. Acta Virologica Sinica. 2021), classical swine fever virus Thiverval strain (published in the article: Fan Yunfeng, Zhao Qizu. Effect of the 3' uncoding region insertion fragment of classical swine fever virus Thiverval strain on virus rescue. Chinese Journal of Preventive Veterinary Medicine. Vol. 31, No. 6, June 2009), bovine viral diarrhea virus type 1 (Oregon C24V, published in the article: L COGGINS et al. Attenuation of virus diarrhea virus (strain Oregon C24V) for vaccine purposes[J]. Cornell Vet, 1961 Oct.), bovine viral diarrhea virus type 2 (890 strain, published in the article: Ridpath JF et al. Thegenomic sequence of a virulent bovine viral diarrhea virus (BVDV) from thetype 2 genotype: detection of a large genomic insertion in a noncytopathicBVDV[J]. Journal of Virology, 1995), highly pathogenic porcine reproductive and respiratory syndrome live vaccine (TJM-F92 strain, the production-specific strain has been used in the research and development of multiple combination vaccines, all of which were approved by Huawit (Jiangsu) Biopharmaceutical Co., Ltd. as the main research unit), porcine reproductive and respiratory syndrome live vaccine (CH-1R strain, which obtained the national Class II new veterinary drug registration certificate on April 23, 2007, certificate number (2007) New Veterinary Drug Certificate No. 15), pseudorabies live vaccine (Bartha-K61 strain, GenBank accession number: JF797217.1), goat pox live vaccine (AV41 strain, Harbin Pharmaceutical Group Biological Vaccine Co., Ltd., trade name: Doubiying, approval number: Veterinary Drug Production Certificate No. 080074003), small ruminant plague live vaccine (Clone9 strain, published in the article: Wang Yu, Gao Yueyi, Gao Jinyuan, et al. Construction of infectious cDNA clone of small ruminant plague virus and virus rescue).Journal of Animal Husbandry and Veterinary Medicine, 2023, 54 (7): 2956-2963), Bovine Parainfluenza Virus Type 3 NMG strain (published in the Chinese Journal of Pathogenic Biology published by Inner Mongolia University for Nationalities / Inner Mongolia Animal Disease Prevention and Control Center: Isolation, Identification and Whole Genome Sequence Analysis of a Bovine Parainfluenza Virus Type 3 Strain, Vol. 19, No. 5, 2024), Porcine Circovirus Type 2 (LN strain, published in the article: Menali, Wang Ganggang, Li Tianli, Wang Yadan. Preliminary Isolation and Whole Genome Sequencing of Porcine Circovirus Type 2 LN Strain. Animal Husbandry and Feed Science, 2015), Porcine Transmissible Gastroenteritis SCJY-1 strain (published in the literature: Safety and Immunogenicity Evaluation of Porcine Transmissible Gastroenteritis and Porcine Epidemic Diarrhea Bivalent Live Vaccine (SCJY-1 strain + SCSZ-1 strain). Chinese Journal of Biological Products, 2018), Porcine Epidemic Diarrhea SCSZ-1 strain, SIV-H1N1 LN strain (Lu Wei, Zhang Xiuhua, Wang Xiudong, Wu Hua. Isolation, identification and genetic analysis of H1N1 subtype swine influenza virus isolates. Acta Virologica Sinica, 2010, Vol. 26, No. 5, pp. 396–401), SIV-H3N2 HLJ strain (published in the literature: Whole genome sequence analysis of swine influenza H3N2 HLJ strain. Chinese Journal of Biological Products, 2018), mink-canine distemper live vaccine (GenBank accession number: FJ592174), porcine transmissible gastroenteritis, porcine epidemic diarrhea, and porcine rotavirus (G5 type) trivalent live vaccine (New Veterinary Drug Registration: (2014) New Veterinary Drug Certificate No. 54), etc.

[0035] (2) Sample processing: use the original virus solution directly; centrifuge the above solution at 3000r / min 4℃ for 10min, and take the supernatant for later use.

[0036] 2. Take 200 μL of virus sample and extract viral genome as template according to the instructions of the Body Fluid Virus DNA / RNA Mini-Preparation Kit (Axygen, catalog number AP-MN-BF-VNA-250). Detection was performed using the established Getta virus RT-PCR method. Except for GETV, no specific amplification bands were detected in other samples. Figure 2 The established detection method has good specificity.

[0037] Example 3: Determination of the lowest detection limit of the Getta virus RT-PCR detection kit (1) Dilute the positive standard 10 times according to the calculated copy number. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -810 -9 10 -10 and 10 -11 As a template for amplification, the established RT-PCR detection method for gettavirus was used for detection. The sensitivity of the established method can reach 5.9 copies / μL (see results). Figure 1 ).

[0038] Nucleic acid was extracted from GETV (GETV-BJ-2022 strain) after a 10-fold serial dilution to determine viral load, and used as a template. The established RT-PCR method for detecting gettavirus was then used, and the sensitivity of the established method reached 0.3 TCID⁻¹. 50 (See results) Figure 3 ), with good sensitivity.

[0039] Example 4: Repeatability testing of the Getta virus RT-PCR detection kit With 1×10 -9 Up to 10 -7 Diluted positive standards were used as templates, and each concentration of samples was tested in duplicate. The reproducibility of the established RT-PCR detection method was analyzed. Results are as follows: Figure 6 As shown, the amplification results for different template concentrations were clear and accurate, indicating that the established RT-PCR detection method has good stability.

[0040] Example 5: Application of the Gettavirus RT-PCR Detection Kit in Clinical Sample Detection 1. Sample processing (1) 126 clinical samples: GETV (GETV-BJ-2022 strain), highly pathogenic porcine reproductive and respiratory syndrome live vaccine (JXAI-R strain, published in the article: Tian Kegong, et al. Isolation, identification and molecular epidemiological analysis of highly pathogenic porcine reproductive and respiratory syndrome virus [J]. Chinese Journal of Preventive Veterinary Medicine, 2007, 29 (5): 323-327.), Highly pathogenic porcine reproductive and respiratory syndrome live vaccine (TJM-F92 strain, the production-specific strain has been used in the research and development of multiple combination vaccines, all of which were approved by Huawit (Jiangsu) Biopharmaceutical Co., Ltd. as the main research unit), Porcine reproductive and respiratory syndrome live vaccine (CH-1R strain, National Class II New Veterinary Drug Registration Certificate, Certificate No. (2007) New Veterinary Drug Certificate No. 15), Porcine reproductive and respiratory syndrome live vaccine (PC strain, published in Chinese Patent CN113151195A), Highly pathogenic porcine reproductive and respiratory syndrome heat-resistant protective live vaccine (JXAI-R strain suspension culture, published in Chinese Patent CN102727903A), Porcine pseudorabies live vaccine (HB-98 strain, Chinese New Veterinary Drug Certificate No. 07 [Z]. 2006.); Commercially available vaccines include: live vaccine for porcine pseudorabies (Bartha-K61 strain, cell-derived), live vaccine for classical swine fever (cell-derived), freeze-dried vaccine for classical swine fever (rabbit-derived), live vaccine for classical swine fever (rabbit-derived), live vaccine for peste des petits ruminants (PPR) and goatpox (capital pox), live vaccine for goatpox (AV41 strain), live vaccine for PPR (Clone9 strain), live vaccine for fowlpox (quail-derived attenuated strain), live vaccine for infectious bursal disease in chickens, live vaccine for infectious bronchitis (H120 strain), live vaccine for Newcastle disease in chickens (La Sota strain), live vaccine for duck viral hepatitis (type 3), recombinant fowlpox virus genetically engineered vaccine for infectious laryngotracheitis in chickens, live vaccine for mink and canine distemper, and live vaccine for porcine transmissible gastroenteritis, porcine epidemic diarrhea, and porcine rotavirus (G5 type). Serum and other materials: PAN fetal bovine serum, horse serum, piglet serum, newborn calf serum, Vero cell suspension, MDBK cell suspension, BHK-21 cell suspension, BSR cell suspension, goat kidney cell suspension, pancreatic enzymes, etc.

[0041] (2) Sample processing: use the original virus solution directly; centrifuge the above solution at 3000r / min 4℃ for 10min, and take the supernatant for later use.

[0042] 2. Take 200 μL of virus sample and extract viral genome as template according to the instructions of the Body Fluid Virus DNA / RNA Mini-Preparation Kit (Axygen, catalog number AP-MN-BF-VNA-250). Use the established Getta virus RT-PCR detection method to detect 126 vaccine products and biological raw materials. A total of 21 positive samples were detected (positive rate of 17%), which fully demonstrates that the established detection method is rapid, accurate and specific (see results). Figure 4 ).

[0043] Example 6: Storage Condition Test of Geita virus RT-PCR Detection Kit The kit contents, including One Step Reaction Mix (2×), TransScript One Step Enzyme Mix, positive standard, positive control, negative control, and enzyme-free water, were stored at -20°C for 1 month, 3 months, 6 months, 9 months, and 12 months, respectively, under the same amplification conditions. The amplification products were analyzed by electrophoresis with 1.0% agarose gel to determine the storage conditions. The results showed that this kit can be stored at -20°C for at least 9 months, and bright bands were amplified in all samples (see results below). Figure 5 ).

[0044] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A set of primer pairs for detecting porcine Geyta virus, characterized in that, This includes nucleotide sequences such as GETV-F shown in SEQ ID No. 1 and GETV-R shown in SEQ ID No.

2.

2. A detection kit for porcine getta virus comprising the primer pair of claim 1.

3. The detection kit according to claim 2, characterized in that, The detection kit is an RT-PCR detection kit.

4. A one-step RT-PCR detection kit for porcine getta virus comprising the primer pair of claim 1.

5. The one-step RT-PCR detection kit according to claim 4, characterized in that, It also includes One Step Reaction Mix, TransScript® One Step Enzyme Mix, positive standard, positive control, negative control, and enzyme-free water.

6. The one-step RT-PCR detection kit according to claim 5, characterized in that, The positive standard is obtained by ligating the fragment obtained by amplification using the primer pair described in claim 1 into a cloning vector.

7. The one-step RT-PCR detection kit according to claim 5, characterized in that, The positive control is an RNA extract from an inactivated porcine Geyta virus cell culture or a synthetically produced plasmid containing the porcine Geyta virus NSP3 gene.

8. The one-step RT-PCR detection kit according to any one of claims 4 to 7, for detection methods not for disease diagnosis and treatment, is characterized in that, This includes extracting RNA from the sample to be tested during quantitative detection of porcine Geita virus, constructing a one-step RT-PCR reaction system using RNA and serially diluted positive standards as templates, performing RT-PCR, constructing a standard curve based on the Ct value and concentration of the positive standards, and substituting the Ct value of the RNA from the sample to be tested into the standard curve to obtain the content of porcine Geita virus in the sample.

9. The detection method according to claim 8, characterized in that, The RT-PCR reaction system, in 20 μL volume, contains: 10 μL One Step Reaction Mix, 0.4 μL TransScript® One Step Enzyme Mix, 1 μL each of 10 μmol / L upstream and downstream primers, 3 μL viral RNA, and the remainder ddH2O.

10. The detection method according to claim 8, characterized in that, The RT-PCR reaction procedure includes: 45℃ for 20 min, 94℃ for 3 min; 94℃ denaturation for 30 sec, 57.0℃ annealing for 30 sec, 72.0℃ extension for 45 sec, 35 cycles; 72℃ extension for 10 min.

Images