Primer set, kit for detecting amorphophallus konjac in bean golden mosaic virus genus virus and method and application thereof

Abstract

The application relates to a primer group for detecting a bean golden yellow mosaic virus in Amorphophallus konjac, a kit and a method and application thereof, and belongs to the technical field of biology. The primer group comprises a primer pair TYLCCNV, a primer pair SyLCV and a primer pair TbLCYnV; the bean golden yellow mosaic virus is a golden yellow mosaic virus, a Yunnan tobacco leaf curl virus and a Chinese tomato yellow leaf curl virus. The primer group has the characteristics of high specificity and high sensitivity, and can quickly and efficiently detect the three viruses of TYLCCNV, SyLCV and TbLCYnV from the Amorphophallus konjac.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a primer set, reagent kit, method, and application for detecting common bean golden mosaic virus in Amorphophallus bulbifera. Background Technology

[0002] Synedrella leaf curl virus (SyLCV), Tobacco leaf curl Yunnan virus (TbLCYnV), and Tomato yellowleaf curl China virus (TYLCCNV) all belong to the genus Begomovirus. Begomovirus is the largest genus of geminiviruses and also one of the most damaging and economically important in agricultural production. When these viruses infect plants, they typically cause leaf curling, yellowing, mosaic patterns, yellow veins, thickened veins, auricles on the underside of leaves, and stunted growth.

[0003] Konjac (Amorphophallus rivieri) is a monocotyledonous perennial herbaceous plant belonging to the genus Amorphophallus in the family Araceae. Also known as konjac jelly or konjac head, it is mainly distributed in southeastern my country and the Yunnan-Guizhou Plateau. Its tubers are rich in glucomannan and it is an important economic crop widely used in food, medicine, and industry. Bulbifer (Amorphophallus bulbifer) is a special konjac germplasm resource. It can be propagated not only through tubers and seeds but also asexually through bulbils that resemble seeds and grow at the forks of petioles. Bulbifer is mainly distributed in the border region between China and Myanmar, including southern Yunnan Province and northern Myanmar. Compared to traditional cultivated varieties (such as Amorphophallus konjac and Amorphophallus vulgaris), bulbifer has many superior characteristics, such as strong disease resistance, high glucomannan content, and good yield, showing broad application prospects.

[0004] With the promotion of large-scale konjac cultivation, the planting area has been continuously expanding, providing favorable conditions for the occurrence and spread of diseases. Fungal, bacterial, and viral diseases are prevalent in major konjac producing areas both domestically and internationally. Since konjac cultivation mainly relies on asexual reproduction methods such as tubers and bulbils, viral diseases are relatively common, leading to long-term virus accumulation, seed degeneration, and increasingly serious viral damage. Yunnan, as a major konjac bulbil cultivation area in my country, has an annual planting area of ​​approximately 400,000 to 600,000 mu (approximately 26,667 to 33,333 hectares), making it one of Yunnan Province's important specialty economic crops. In recent years, due to long-term cultivation through seed tubers, the source of viruses has been continuously accumulated. Furthermore, the introduction of new bulbil konjac varieties or materials from tropical and subtropical countries such as Indonesia, Thailand, Myanmar, and Laos has led to increasingly severe konjac viral diseases in low-altitude areas of southern Yunnan. Simultaneously, new viruses are continuously damaging Yunnan bulbil konjac, with increasing annual fluctuations in the scope and severity of their damage, significantly impacting bulbil konjac production in different planting areas.

[0005] Currently, the prevention and control of Amorphophallus villosa virus disease mainly includes: using healthy seed tubers to avoid long-distance virus transmission; health-preserving cultivation, reasonable water and fertilizer management, and field planning; improving the plant's own resistance to adverse conditions; and controlling pests and diseases, selecting highly effective insecticides to kill possible virus vectors in the early stages of aphid, whitefly, and thrips outbreaks, thereby blocking cross-infection of the virus in the field.

[0006] With the increasing number of viral pathogens, the expanding incidence and severity of diseases year by year, it is of great significance to research and apply a set of accurate, simple, sensitive, and economical detection methods for important viral pathogens in Amorphophallus bulbifera for screening virus carrier rates in plants and detecting pathogens in the field. Common Bean Golden Mosaic Virus (CFMV) is a new type of viral pathogen circulating in low-altitude Amorphophallus bulbifera growing areas in Yunnan, with high detection rates in various regions. Therefore, establishing rapid and effective detection methods for this type of virus is essential for the prevention and control of viral diseases in Amorphophallus bulbifera.

[0007] Previously, methods for detecting viruses of the genus *Tylocereus undatus* (Yunnan tobacco leaf curl virus (TbLCYnV)) and *Tylocereus undatus* (Chinese tomato yellow leaf curl virus (TYLCCN)) existed, but all of them first used universal primers for the genus *Tylocereus undatus* to detect the virus, and then used specific primers to amplify the whole genome of the target virus. The process was cumbersome and time-consuming. How to achieve rapid and accurate detection of *Sylocereus undatus* leaf curl virus (SyLCVV) that infects *Amorphophallus bulbifera* is an urgent problem to be solved in this field. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide a primer set, reagent kit, method and application for detecting the genus *Amorphophallus vulgaris* virus in *Amorphophallus bulbifer*.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a primer set for detecting common bean golden mosaic virus in Amorphophallus bulbifera, including primer pair TYLCCNV, primer pair SyLCV and primer pair TbLCYnV; The sequence of the primer pair TYLCCNV is as follows: TYLCCNV-F: 5'-gtcatttccactcccgcctc-3' (SEQ ID NO. 1); TYLCCNV-R: 5'-actatatcggcgggacgctt-3' (SEQ ID NO. 2); The sequence of the primer pair SyLCV is as follows: SyLCV-F: 5'-cgacagcccatatgtgagcc-3' (SEQ ID NO.3); SyLCV-R: 5'-aagttgagacggcggcgtac-3' (SEQ ID NO.4); The sequence of the primer pair TbLCYnV is as follows: TbLCYnV-F: 5'-tatgtgagccgtgctgctgccc-3' (SEQ ID NO.5); TbLCYnV-R: 5'-tgggctgtcgaagttgagacggc-3' (SEQ ID NO. 6); The aforementioned viruses in the genus *Golden Mosaic Virus* include *Golden Sword Leaf Curved Virus*, *Yunnan Tobacco Leaf Curved Virus*, and *Chinese Tomato Yellow Leaf Curved Virus*.

[0010] A second aspect of the present invention provides a reagent or kit containing the primer set described above for detecting common bean golden mosaic virus in Amorphophallus bulbifera.

[0011] Furthermore, it also includes: PCR reaction premix, positive control, and negative control; the PCR reaction premix contains: 11.5-12.7 μL ddH2O, 25 μL 10×PCR Buffer, 10 μL 2 mmol / L dNTPs, 0.5-1.0 μL 10 μmol / L upstream primer, 0.5-1.0 μL 10 μmol / L downstream primer, 0.8-1.0 μL 1 U / μL KOD FX DNA polymerase, with a total volume of 49.5 μL; the primer pair consisting of the upstream and downstream primers is primer pair TYLCCNV, primer pair SyLCV, or primer pair TbLCYnV.

[0012] Furthermore, the PCR reaction system is as follows: 49.5 μL of PCR reaction premix; Template 0.5μL; Total volume: 50 μL; The template is the product obtained by rolling circle amplification of DNA extracted from Amorphophallus bulbifera samples, a positive control, or a negative control.

[0013] Furthermore, the negative control is water; the positive control is the recombinant plasmids of TYLCCNV, SyLCV, and TbLCYnV. The recombinant plasmids of TYLCCNV, SyLCV, and TbLCYnV are prepared as follows: PCR amplification of the DNA of TYLCCNV, SyLCV, and TbLCYnV is performed using primers SEQ ID NO.1 and SEQ ID NO.2 of TYLCCNV, primers SEQ ID NO.3 and SEQ ID NO.4 of SyLCV, and primers SEQ ID NO.5 and SEQ ID NO.6 of TbLCYnV, respectively. The amplification products are then ligated with p-EASY-T to construct the recombinant plasmids T-TYLCCNV, T-SyLCV, and T-TbLCYnV.

[0014] Furthermore, the copy number of the positive control was 6.9 × 10⁻⁶ for the T-TYLCCNV recombinant plasmid. 8 The copy number of the T-SyLCV recombinant plasmid was 1.4 × 10⁻⁶ copies / μL. 9 The T-TbLCYnV recombinant plasmid contains 2.1 × 10⁻⁶ copies / μL. 8 Copy / μL.

[0015] Furthermore, for primer pair TYLCCNV, the PCR reaction program was as follows: 98℃ for 2 min; 98℃ for 10 s; 60-63℃ for 30 s; 68℃ for 3 min, repeated 28-30 times; 68℃ for 10 min; stored at 4℃. For primer pair SyLCV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 64-66℃ for 30 s; 68℃ for 3 min, repeated 28-30 times; 68℃ for 10 min; store at 4℃. For primer pair TbLCYnV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 61-63℃ for 30 s; 68℃ for 3 min, cycled 28-30 times; 68℃ for 10 min; store at 4℃.

[0016] Furthermore, the specific method for rolling circle amplification is as follows: using DNA from Amorphophallus bulbifera samples as templates; mixing 1 μL of 100 ng / μL template with 5 μL of Sample Buffer, denaturing at 95℃ for 3 min in an enzyme digester; then placing on ice for 10 min, adding 5 μL of Raction Buffer and 0.2 μL of Enzyme, reacting at 30℃ for 18 h in an enzyme digester, and terminating the reaction at 65℃ for 10 min.

[0017] A third aspect of this invention provides a method for identifying whether a virus to be tested is a virus of the genus *Amorphophallus vulgaris* found in *Amorphophallus vulgaris* for non-disease diagnosis and treatment purposes. The method uses a kit with primers for detecting *Amorphophallus vulgaris* viruses and includes the following steps: S1. Extract the nucleic acid of the virus to be tested, amplify the signal of the target nucleic acid by rolling circle amplification, and use the obtained RCA product for PCR detection; S2. Using the RCA product obtained in S1, the positive control, and the negative control as templates, respectively, prepare PCR reaction systems and perform PCR amplification reactions; the PCR reaction systems are as follows: 49.5 μL of PCR reaction premix; Template 0.5μL; Total volume: 50 μL; For primer pair TYLCCNV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 60-63℃ for 30 s; 68℃ for 3 min, repeated 28-30 times; 68℃ for 10 min; store at 4℃. For primer pair SyLCV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 64-66℃ for 30 s; 68℃ for 3 min, repeated 28-30 times; 68℃ for 10 min; store at 4℃. For primer pair TbLCYnV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 61-63℃ for 30 s; 68℃ for 3 min, cycled 28-30 times; 68℃ for 10 min; store at 4℃.

[0018] S3. Based on the reaction results, make the following judgments: The amplified PCR products were detected by agarose gel electrophoresis. If the amplified positive control PCR product had a target band of about 2700 bp, and the amplified negative control PCR product did not have a target band of about 2700 bp, and the PCR amplification product of the virus to be tested had a target band of about 2700 bp, then the virus to be tested was considered to be the target virus corresponding to the primer pair used.

[0019] A fourth aspect of this invention provides a method for identifying whether a sample to be tested contains a virus of the genus *Amorphophallus vulgaris* (bean golden mosaic virus) for non-disease diagnosis and treatment purposes. The method utilizes the aforementioned primer set for detecting *Amorphophallus vulgaris* viruses and includes the following steps: S1. Extract nucleic acid from the sample to be tested, amplify the signal of the target nucleic acid by rolling circle amplification, and use the obtained RCA product for PCR detection; S2. Using the RCA product obtained in S1, the positive control, and the negative control as templates, respectively, prepare PCR reaction systems and perform PCR amplification reactions; the PCR reaction systems are as follows: 49.5 μL of PCR reaction premix; Template 0.5μL; Total volume: 50 μL; For primer pair TYLCCNV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 60-63℃ for 30 s; 68℃ for 3 min, repeated 28-30 times; 68℃ for 10 min; store at 4℃. For primer pair SyLCV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 64-66℃ for 30 s; 68℃ for 3 min, repeated 28-30 times; 68℃ for 10 min; store at 4℃. For primer pair TbLCYnV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 61-63℃ for 30 s; 68℃ for 3 min, repeated 28-30 times; 68℃ for 10 min; store at 4℃. S3. Based on the reaction results, make the following judgments: The amplified PCR products were detected by agarose gel electrophoresis. If the amplified positive control PCR product had a target band of about 2700 bp, and the amplified negative control PCR product did not have a target band of about 2700 bp, and the PCR amplification product of the test sample had a target band of about 2700 bp, then the virus to be tested was considered to be the target virus corresponding to the primer pair used.

[0020] This invention provides a primer set for the efficient, sensitive, and specific detection of Bean Golden Mosaic Virus (BMP) viruses in Amorphophallus bulbifera using PCR technology. This invention utilizes the RCA method to amplify the target nucleic acid signal, and further performs PCR amplification and detection using virus-specific primers. It leverages the speed and sensitivity of conventional PCR to obtain the target fragment, establishing a scientific method for the large-scale, rapid, accurate, and sensitive detection of BMP viruses in Amorphophallus bulbifera and other plant samples.

[0021] The viruses of the genus *Amorphophallus* in this invention are *SyLCV*, *TbLCYnV*, and *TYLCCNV*. The primer set includes primer pairs for TYLCCNV, SyLCV, and TbLCYnV; this primer set is characterized by high specificity and sensitivity, enabling rapid and efficient detection of these three viruses from *Amorphophallus bulbifer*.

[0022] The rolling circle amplification used in this invention can amplify the target nucleic acid signal, which is beneficial for obtaining a complete genome sequence; the specific primer set is beneficial for obtaining a specific genome sequence, which greatly improves the accuracy and sensitivity of detection.

[0023] The main concept of this invention is as follows: The SyLCV, TbLCYnV, and TYLCCNV viral sequences were downloaded from GenBank. After sequence alignment using Snapgene software, the conserved region (V1 gene) of SyLCV, TYLCCNV, and TbLCYnV viruses was selected. Three pairs of back-to-back specific primers were designed for the target fragments of each region. PCR amplification was performed on the test samples or viruses. After continuous optimization of the amplification conditions, the final amplification conditions were determined.

[0024] In this invention, primer sets and PCR reagents for three different viruses are premixed to create premixed solutions for detecting the three viruses. When testing samples, the template is simply added to the premixed solution, which simplifies the process and improves efficiency.

[0025] Compared with the prior art, the beneficial effects of this invention are as follows: (1) The present invention has highly conserved regions (V1 gene) of SyLCV, TYLCCNV and TbLCYnV viruses. Three sets of specific primer sequences for detecting SyLCV, TYLCCNV and TbLCYnV viruses in Amorphophallus bulbifera were designed in this region. These three sets of primers are highly specific and are not easy to amplify other viruses. Traditional detection of common bean golden mosaic virus usually involves first selecting universal primers of the genus for detection, and then performing sequencing and designing specific primers to amplify the whole genome of the virus based on the results. It usually takes about a week to obtain the whole genome sequence of the virus. However, the primer set of the present invention can directly amplify the whole genome of the virus, which can save about 2-3 days. (2) The present invention designs primers for isolates in Amorphophallus bulbifera. In the implementation case, the detection kit of the present invention was used to detect these three viruses. The detection results were consistent with the sequencing results, indicating that the detection kit of the present invention can effectively and quickly detect SyLCV, TYLCCNV and TbLCYnV viruses in Amorphophallus bulbifera.

[0026] (3) The specific primer set of the present invention can reduce non-specific amplification products. When using universal primers for amplification, a large number of non-specific bands will be amplified. Using the specific primer set for PCR amplification, the non-specific binding rate can be reduced by about 50%, and the amplification efficiency can be increased by about 80%. Attached Figure Description

[0027] Figure 1 These are the results of specific detection experiments of TYLCCNV, SyLCV, and TbLCYnV in Amorphophallus bulbifera. In A: M is DNA Marker (DL5000), lane 1 is the detection result of TYLCCNV in the Amorphophallus bulbifera sample, lane 2 is the positive control for TYLCCNV, and lane 3 is the negative control for TYLCCNV, i.e., ddH2O; B: M is DNA Marker (DL5000), lane 1 is the detection result of SyLCV in the Amorphophallus bulbifera sample, lane 2 is the positive control for SyLCV, and lane 3 is the negative control for SyLCV, i.e., ddH2O; C: M is DNA Marker (DL5000), lane 1 is the detection result of TbLCYnV in the Amorphophallus bulbifera sample, lane 2 is the positive control for TbLCYnV, and lane 3 is the negative control for TbLCYnV, i.e., ddH2O; D: M is DNA Marker (DL5000), lane 1 is the detection result of TbLCYnV in the Amorphophallus bulbifera sample, lane 2 is the positive control for TbLCYnV, and lane 3 is the negative control for TbLCYnV, i.e., ddH2O; Maker (DL5000), lane 1 is the detection result of TYLCCNV amplified by universal primer BF / BR in Amorphophallus bulbifera sample, lane 2 is the detection result of SyLCV amplified by universal primer BF / BR, and lane 3 is the detection result of TbLCYnV amplified by universal primer BF / BR. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to the embodiments.

[0029] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the field or according to the product instructions. Materials or equipment whose manufacturers are not specified are all conventional products that can be obtained by purchase.

[0030] Primer sets for detecting common bean golden mosaic virus in Amorphophallus bulbifera include primer pair TYLCCNV, primer pair SyLCV, and primer pair TbLCYnV. The sequence of the primer pair TYLCCNV is as follows: TYLCCNV-F: 5'-gtcatttccactcccgcctc-3' (SEQ ID NO. 1); TYLCCNV-R: 5'-actatatcggcgggacgctt-3' (SEQ ID NO. 2); The sequence of the primer pair SyLCV is as follows: SyLCV-F: 5'-cgacagcccatatgtgagcc-3' (SEQ ID NO.3); SyLCV-R: 5'-aagttgagacggcggcgtac-3' (SEQ ID NO.4); The sequence of the primer pair TbLCYnV is as follows: TbLCYnV-F: 5'-tatgtgagccgtgctgctgccc-3' (SEQ ID NO.5); TbLCYnV-R: 5'-tgggctgtcgaagttgagacggc-3' (SEQ ID NO. 6); The aforementioned viruses in the genus *Golden Mosaic Virus* include *Golden Sword Leaf Curved Virus*, *Yunnan Tobacco Leaf Curved Virus*, and *Chinese Tomato Yellow Leaf Curved Virus*.

[0031] Example 2 Primer sets for detecting common bean golden mosaic virus in Amorphophallus bulbifera include primer pair TYLCCNV, primer pair SyLCV, and primer pair TbLCYnV. The sequence of the primer pair TYLCCNV is as follows: TYLCCNV-F: 5'-gtcatttccactcccgcctc-3' (SEQ ID NO. 1); TYLCCNV-R: 5'-actatatcggcgggacgctt-3' (SEQ ID NO. 2); The sequence of the primer pair SyLCV is as follows: SyLCV-F: 5'-cgacagcccatatgtgagcc-3' (SEQ ID NO.3); SyLCV-R: 5'-aagttgagacggcggcgtac-3' (SEQ ID NO.4); The sequence of the primer pair TbLCYnV is as follows: TbLCYnV-F: 5'-tatgtgagccgtgctgctgccc-3' (SEQ ID NO.5); TbLCYnV-R: 5'-tgggctgtcgaagttgagacggc-3' (SEQ ID NO. 6); The aforementioned viruses in the genus *Golden Mosaic Virus* include *Golden Sword Leaf Curved Virus*, *Yunnan Tobacco Leaf Curved Virus*, and *Chinese Tomato Yellow Leaf Curved Virus*.

[0032] A reagent or kit containing the primer set as described in the claims for detecting common bean golden mosaic virus in Amorphophallus bulbifera.

[0033] The kit for detecting a common bean golden mosaic virus in Amorphophallus bulbifera includes: a PCR reaction premix, a positive control, and a negative control. The PCR reaction premix contains: 12.7 μL ddH2O, 25 μL 10×PCRBuffer, 10 μL dNTPs at 2 mmol / L, 0.5 μL of 10 μmol / L upstream primer, 0.5 μL of 10 μmol / L downstream primer, and 0.8 μL of 1 U / μL KOD FX DNA polymerase, for a total volume of 49.5 μL. The primer pair consisting of the upstream and downstream primers is TYLCCNV, SyLCV, or TbLCYnV. The negative control is ddH2O. The positive control is a recombinant plasmid of TYLCCNV, SyLCV, or TbLCYnV. The recombinant plasmids were prepared as follows: PCR amplification of the DNA of TYLCCNV, SyLCV and TbLCYnV was performed using primers SEQ ID NO.1 and SEQ ID NO.2 of TYLCCNV, primers SEQ ID NO.3 and SEQ ID NO.4 of SyLCV, and primers SEQ ID NO.5 and SEQ ID NO.6 of TbLCYnV, respectively. The amplification products were then ligated with p-EASY-T to construct the recombinant plasmids T-TYLCCNV, T-SyLCV and T-TbLCYnV.

[0034] The copy number of the positive control was 6.9 × 10⁻⁶ for the T-TYLCCNV plasmid. 8 The copy number of the T-SyLCV plasmid was 1.4 × 10⁻⁶ copies / μL. 9 The T-TbLCYnV plasmid contains 2.1 × 10⁻⁶ copies / μL. 8 Copy / μL.

[0035] The PCR reaction system is as follows: 49.5 μL of PCR reaction premix; Template 0.5μL; Total volume: 50 μL; The template is the product obtained by rolling circle amplification of DNA extracted from Amorphophallus bulbifera samples, a positive control, or a negative control.

[0036] The specific method for rolling circle amplification is as follows: using DNA from Amorphophallus bulbifera samples as template; mixing 1 μL of 100 ng / μL template with 5 μL Sample Buffer, denaturing at 95℃ for 3 min in an enzyme digester; then placing on ice for 10 min, adding 5 μL Raction Buffer and 0.2 μL Enzyme, reacting at 30℃ for 18 h in an enzyme digester, and terminating the reaction at 65℃ for 10 min.

[0037] For primer pair TYLCCNV, the PCR reaction program was as follows: 98℃ for 2 min; 98℃ for 10 s; 60℃ for 30 s; 68℃ for 3 min, repeated 28 times; 68℃ for 10 min; stored at 4℃. For primer pair SyLCV, the PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s; 64℃ for 30 s; 68℃ for 3 min, repeated 28 times; 68℃ for 10 min; stored at 4℃. For primer pair TbLCYnV, the PCR reaction program was as follows: 98℃ for 2 min; 98℃ for 10 s; 61℃ for 30 s; 68℃ for 3 min, repeated 28 times; 68℃ for 10 min; stored at 4℃.

[0038] A method for identifying whether a virus to be tested belongs to the genus *Amorphophallus vulgaris* in Amorphophallus bulbifera for non-disease diagnosis and treatment purposes, using the primer set described above for detecting *Amorphophallus vulgaris* viruses in Amorphophallus bulbifera, includes the following steps: S1. Extract the nucleic acid of the virus to be tested, amplify the signal of the target nucleic acid by rolling circle amplification, and use the obtained RCA product for PCR detection; S2. Using the RCA product obtained in S1, the positive control, and the negative control as templates, respectively, prepare PCR reaction systems and perform PCR amplification reactions; the PCR reaction systems are as follows: 49.5 μL of PCR reaction premix; Template 0.5μL; Total volume: 50 μL; For primer pair TYLCCNV, the PCR reaction program was as follows: 98℃ for 2 min; 98℃ for 10 s; 60℃ for 30 s; 68℃ for 3 min, repeated 28 times; 68℃ for 10 min; stored at 4℃. For primer pair SyLCV, the PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s; 64℃ for 30 s; 68℃ for 3 min, repeated 28 times; 68℃ for 10 min; stored at 4℃. For primer pair TbLCYnV, the PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s; 61℃ for 30 s; 68℃ for 3 min, repeated 28 times; 68℃ for 10 min; stored at 4℃. S3. Based on the reaction results, make the following judgments: The amplified PCR products were detected by agarose gel electrophoresis. If the amplified positive control PCR product had a target band of about 2700 bp, and the amplified negative control PCR product did not have a target band of about 2700 bp, and the PCR amplification product of the virus to be tested had a target band of about 2700 bp, then the virus to be tested was considered to be the target virus corresponding to the primer pair used.

[0039] A method for identifying whether a sample contains a bean golden mosaic virus (BMP) in Amorphophallus bulbifera for non-disease diagnosis and treatment purposes, using the aforementioned primer set for detecting BMP in Amorphophallus bulbifera, includes the following steps: S1. Extract nucleic acid from the sample to be tested, amplify the signal of the target nucleic acid by rolling circle amplification, and use the obtained RCA product for PCR detection; S2. Using the RCA product obtained in S1, the positive control, and the negative control as templates, respectively, prepare PCR reaction systems and perform PCR amplification reactions; the PCR reaction systems are as follows: 49.5 μL of PCR reaction premix; Template 0.5μL; Total volume: 50 μL; For primer pair TYLCCNV, the PCR reaction program was as follows: 98℃ for 2 min; 98℃ for 10 s; 60℃ for 30 s; 68℃ for 3 min, repeated 28 times; 68℃ for 10 min; stored at 4℃. For primer pair SyLCV, the PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s; 64℃ for 30 s; 68℃ for 3 min, repeated 28 times; 68℃ for 10 min; stored at 4℃. For primer pair TbLCYnV, the PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s; 61℃ for 30 s; 68℃ for 3 min, repeated 28 times; 68℃ for 10 min; stored at 4℃. S3. Based on the reaction results, make the following judgments: The amplified PCR products were detected by agarose gel electrophoresis. If the amplified positive control PCR product had a target band of about 2700 bp, and the amplified negative control PCR product did not have a target band of about 2700 bp, and the PCR amplification product of the test sample had a target band of about 2700 bp, then the virus to be tested was considered to be the target virus corresponding to the primer pair used.

[0040] Example 3 Primer sets for detecting common bean golden mosaic virus in Amorphophallus bulbifera include primer pair TYLCCNV, primer pair SyLCV, and primer pair TbLCYnV. The sequence of the primer pair TYLCCNV is as follows: TYLCCNV-F: 5'-gtcatttccactcccgcctc-3' (SEQ ID NO. 1); TYLCCNV-R: 5'-actatatcggcgggacgctt-3' (SEQ ID NO. 2); The sequence of the primer pair SyLCV is as follows: SyLCV-F: 5'-cgacagcccatatgtgagcc-3' (SEQ ID NO.3); SyLCV-R: 5'-aagttgagacggcggcgtac-3' (SEQ ID NO.4); The sequence of the primer pair TbLCYnV is as follows: TbLCYnV-F: 5'-tatgtgagccgtgctgctgccc-3' (SEQ ID NO.5); TbLCYnV-R: 5'-tgggctgtcgaagttgagacggc-3' (SEQ ID NO. 6); The aforementioned viruses in the genus *Golden Mosaic Virus* include *Golden Sword Leaf Curved Virus*, *Yunnan Tobacco Leaf Curved Virus*, and *Chinese Tomato Yellow Leaf Curved Virus*.

[0041] A reagent or kit containing the primer set as described in the claims for detecting common bean golden mosaic virus in Amorphophallus bulbifera.

[0042] The kit for detecting a common bean golden mosaic virus in Amorphophallus bulbifera includes: a PCR reaction premix, a positive control, and a negative control. The PCR reaction premix contains: 11.5 μL ddH2O, 25 μL 10×PCRBuffer, 10 μL dNTPs at 2 mmol / L, 1.0 μL upstream primer at 10 μmol / L, 1.0 μL downstream primer at 10 μmol / L, and 1.0 μL KOD FX DNA polymerase at 1 U / μL, for a total volume of 49.5 μL. The primer pair consisting of the upstream and downstream primers is TYLCCNV, SyLCV, or TbLCYnV. The negative control is ddH2O. The positive control is a recombinant plasmid of TYLCCNV, SyLCV, or TbLCYnV. The recombinant plasmids were prepared as follows: PCR amplification of the DNA of TYLCCNV, SyLCV and TbLCYnV was performed using primers SEQ ID NO.1 and SEQ ID NO.2 of TYLCCNV, primers SEQ ID NO.3 and SEQ ID NO.4 of SyLCV, and primers SEQ ID NO.5 and SEQ ID NO.6 of TbLCYnV, respectively. The amplification products were then ligated with p-EASY-T to construct the recombinant plasmids T-TYLCCNV, T-SyLCV and T-TbLCYnV.

[0043] The copy number of the positive control was 6.9 × 10⁻⁶ for the T-TYLCCNV plasmid. 8 The copy number of the T-SyLCV plasmid was 1.4 × 10⁻⁶ copies / μL. 9 The T-TbLCYnV plasmid contains 2.1 × 10⁻⁶ copies / μL. 8 Copy / μL.

[0044] The PCR reaction system is as follows: 49.5 μL of PCR reaction premix; Template 0.5μL; Total volume: 50 μL; The template is the product obtained by rolling circle amplification of DNA extracted from Amorphophallus bulbifera samples, a positive control, or a negative control.

[0045] The specific method for rolling circle amplification is as follows: using DNA from Amorphophallus bulbifera samples as template; mixing 1 μL of 100 ng / μL template with 5 μL Sample Buffer, denaturing at 95℃ for 3 min in an enzyme digester; then placing on ice for 10 min, adding 5 μL Raction Buffer and 0.2 μL Enzyme, reacting at 30℃ for 18 h in an enzyme digester, and terminating the reaction at 65℃ for 10 min.

[0046] For primer pair TYLCCNV, the PCR reaction program was as follows: 98℃ for 2 min; 98℃ for 10 s; 62℃ for 30 s; 68℃ for 3 min, repeated 30 times; 68℃ for 10 min; stored at 4℃. For primer pair SyLCV, the PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s; 65℃ for 30 s; 68℃ for 3 min, repeated 30 times; 68℃ for 10 min; stored at 4℃. For primer pair TbLCYnV, the PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s; 62℃ for 30 s; 68℃ for 3 min, repeated 30 times; 68℃ for 10 min; stored at 4℃. A method for identifying whether a virus to be tested belongs to the genus *Amorphophallus vulgaris* in Amorphophallus bulbifera for non-disease diagnosis and treatment purposes, using the primer set described above for detecting *Amorphophallus vulgaris* viruses in Amorphophallus bulbifera, includes the following steps: S1. Extract the nucleic acid of the virus to be tested, amplify the signal of the target nucleic acid by rolling circle amplification, and use the obtained RCA product for PCR detection; S2. Using the RCA product obtained in S1, the positive control, and the negative control as templates, respectively, prepare PCR reaction systems and perform PCR amplification reactions; the PCR reaction systems are as follows: 49.5 μL of PCR reaction premix; Template 0.5μL; Total volume: 50 μL; For primer pair TYLCCNV, the PCR reaction program was as follows: 98℃ for 2 min; 98℃ for 10 s; 62℃ for 30 s; 68℃ for 3 min, repeated 30 times; 68℃ for 10 min; stored at 4℃. For primer pair SyLCV, the PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s; 65℃ for 30 s; 68℃ for 3 min, repeated 30 times; 68℃ for 10 min; stored at 4℃. For primer pair TbLCYnV, the PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s; 62℃ for 30 s; 68℃ for 3 min, repeated 30 times; 68℃ for 10 min; stored at 4℃. S3. Based on the reaction results, make the following judgments: The amplified PCR products were detected by agarose gel electrophoresis. If the amplified positive control PCR product had a target band of about 2700 bp, and the amplified negative control PCR product did not have a target band of about 2700 bp, and the PCR amplification product of the virus to be tested had a target band of about 2700 bp, then the virus to be tested was considered to be the target virus corresponding to the primer pair used.

[0047] A method for identifying whether a sample contains a bean golden mosaic virus (BMP) in Amorphophallus bulbifera for non-disease diagnosis and treatment purposes, using the aforementioned primer set for detecting BMP in Amorphophallus bulbifera, includes the following steps: S1. Extract nucleic acid from the sample to be tested, amplify the signal of the target nucleic acid by rolling circle amplification, and use the obtained RCA product for PCR detection; S2. Using the RCA product obtained in S1, the positive control, and the negative control as templates, respectively, prepare PCR reaction systems and perform PCR amplification reactions; the PCR reaction systems are as follows: 49.5 μL of PCR reaction premix; Template 0.5μL; Total volume: 50 μL; For primer pair TYLCCNV, the PCR reaction program was as follows: 98℃ for 2 min; 98℃ for 10 s; 62℃ for 30 s; 68℃ for 3 min, repeated 30 times; 68℃ for 10 min; stored at 4℃. For primer pair SyLCV, the PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s; 65℃ for 30 s; 68℃ for 3 min, repeated 30 times; 68℃ for 10 min; stored at 4℃. For primer pair TbLCYnV, the PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s; 62℃ for 30 s; 68℃ for 3 min, repeated 30 times; 68℃ for 10 min; stored at 4℃. S3. Based on the reaction results, make the following judgments: The amplified PCR products were detected by agarose gel electrophoresis. If the amplified positive control PCR product had a target band of about 2700 bp, and the amplified negative control PCR product did not have a target band of about 2700 bp, and the PCR amplification product of the test sample had a target band of about 2700 bp, then the virus to be tested was considered to be the target virus corresponding to the primer pair used.

[0048] Example 4 Primer sets for detecting common bean golden mosaic virus in Amorphophallus bulbifera include primer pair TYLCCNV, primer pair SyLCV, and primer pair TbLCYnV. The sequence of the primer pair TYLCCNV is as follows: TYLCCNV-F: 5'-gtcatttccactcccgcctc-3' (SEQ ID NO. 1); TYLCCNV-R: 5'-actatatcggcgggacgctt-3' (SEQ ID NO. 2); The sequence of the primer pair SyLCV is as follows: SyLCV-F: 5'-cgacagcccatatgtgagcc-3' (SEQ ID NO.3); SyLCV-R: 5'-aagttgagacggcggcgtac-3' (SEQ ID NO.4); The sequence of the primer pair TbLCYnV is as follows: TbLCYnV-F: 5'-tatgtgagccgtgctgctgccc-3' (SEQ ID NO.5); TbLCYnV-R: 5'-tgggctgtcgaagttgagacggc-3' (SEQ ID NO. 6);; The aforementioned viruses in the genus *Golden Mosaic Virus* include *Golden Sword Leaf Curved Virus*, *Yunnan Tobacco Leaf Curved Virus*, and *Chinese Tomato Yellow Leaf Curved Virus*.

[0049] A reagent or kit containing the primer set as described in the claims for detecting common bean golden mosaic virus in Amorphophallus bulbifera.

[0050] The kit for detecting a common bean golden mosaic virus in Amorphophallus bulbifera further includes: a PCR reaction premix, a positive control, and a negative control; the PCR reaction premix contains: 12.2 μL ddH2O, 25 μL 10×PCRBuffer, 10 μL dNTPs at 2 mmol / L, 0.7 μL of 10 μmol / L upstream primer, 0.7 μL of 10 μmol / L downstream primer, and 0.9 μL of 1 U / μL KOD FX DNA polymerase, for a total volume of 49.5 μL; the primer pair consisting of the upstream and downstream primers is primer pair TYLCCNV, primer pair SyLCV, or primer pair TbLCYnV; the negative control is ddH2O; the positive control is a recombinant plasmid of TYLCCNV, SyLCV, and TbLCYnV. The recombinant plasmids were prepared as follows: PCR amplification of the DNA of TYLCCNV, SyLCV and TbLCYnV was performed using primers SEQ ID NO.1 and SEQ ID NO.2 of TYLCCNV, primers SEQ ID NO.3 and SEQ ID NO.4 of SyLCV, and primers SEQ ID NO.5 and SEQ ID NO.6 of TbLCYnV, respectively. The amplification products were then ligated with p-EASY-T to construct the recombinant plasmids T-TYLCCNV, T-SyLCV and T-TbLCYnV.

[0051] The copy number of the positive control was 6.9 × 10⁻⁶ for the T-TYLCCNV plasmid. 8 The copy number of the T-SyLCV plasmid was 1.4 × 10⁻⁶ copies / μL. 9 The T-TbLCYnV plasmid contains 2.1 × 10⁻⁶ copies / μL. 8 Copy / μL.

[0052] The PCR reaction system is as follows: 49.5 μL of PCR reaction premix; Template 0.5μL; Total volume: 50 μL; The template is the product obtained by rolling circle amplification of DNA extracted from Amorphophallus bulbifera samples, a positive control, or a negative control.

[0053] The specific method for rolling circle amplification is as follows: using DNA from Amorphophallus bulbifera samples as template; mixing 1 μL of 100 ng / μL template with 5 μL Sample Buffer, denaturing at 95℃ for 3 min in an enzyme digester; then placing on ice for 10 min, adding 5 μL Raction Buffer and 0.2 μL Enzyme, reacting at 30℃ for 18 h in an enzyme digester, and terminating the reaction at 65℃ for 10 min.

[0054] For primer pair TYLCCNV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 63℃ for 30 s; 68℃ for 3 min, repeated 28-30 times; 68℃ for 10 min; store at 4℃. For primer pair SyLCV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 66℃ for 30 s; 68℃ for 3 min, repeat 28-30 times; 68℃ for 10 min; store at 4℃. For primer pair TbLCYnV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 63℃ for 30 s; 68℃ for 3 min, cycled 28-30 times; 68℃ for 10 min; store at 4℃.

[0055] A method for identifying whether a virus to be tested belongs to the genus *Amorphophallus vulgaris* in Amorphophallus bulbifera for non-disease diagnosis and treatment purposes, using the primer set described above for detecting *Amorphophallus vulgaris* viruses in Amorphophallus bulbifera, includes the following steps: S1. Extract the nucleic acid of the virus to be tested, amplify the signal of the target nucleic acid by rolling circle amplification, and use the obtained RCA product for PCR detection; S2. Using the RCA product obtained in S1, the positive control, and the negative control as templates, respectively, prepare PCR reaction systems and perform PCR amplification reactions; the PCR reaction systems are as follows: 49.5 μL of PCR reaction premix; Template 0.5μL; Total volume: 50 μL; For primer pair TYLCCNV, the PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s; 63℃ for 30 s, 68℃ for 3 min, repeated 29 times; 68℃ for 10 min; stored at 4℃. For primer pair SyLCV, the PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s; 66℃ for 30 s; 68℃ for 3 min, repeated 29 times; 68℃ for 10 min; stored at 4℃. For primer pair TbLCYnV, the PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s; 63℃ for 30 s; 68℃ for 3 min, repeated 29 times; 68℃ for 10 min; stored at 4℃. S3. Based on the reaction results, make the following judgments: The amplified PCR products were detected by agarose gel electrophoresis. If the amplified positive control PCR product had a target band of about 2700 bp, and the amplified negative control PCR product did not have a target band of about 2700 bp, and the PCR amplification product of the virus to be tested had a target band of about 2700 bp, then the virus to be tested was considered to be the target virus corresponding to the primer pair used.

[0056] A method for identifying whether a sample contains a bean golden mosaic virus (BMP) in Amorphophallus bulbifera for non-disease diagnosis and treatment purposes, using the aforementioned primer set for detecting BMP in Amorphophallus bulbifera, includes the following steps: S1. Extract nucleic acid from the sample to be tested, amplify the signal of the target nucleic acid by rolling circle amplification, and use the obtained RCA product for PCR detection; S2. Using the RCA product obtained in S1, the positive control, and the negative control as templates, respectively, prepare PCR reaction systems and perform PCR amplification reactions; the PCR reaction systems are as follows: 49.5 μL of PCR reaction premix; Template 0.5μL; Total volume: 50 μL; For primer pair TYLCCNV, the PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s; 63℃ for 30 s, 68℃ for 3 min, repeated 29 times; 68℃ for 10 min; stored at 4℃. For primer pair SyLCV, the PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s; 66℃ for 30 s; 68℃ for 3 min, repeated 29 times; 68℃ for 10 min; stored at 4℃. For primer pair TbLCYnV, the PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s; 63℃ for 30 s; 68℃ for 3 min, repeated 29 times; 68℃ for 10 min; stored at 4℃. S3. Based on the reaction results, make the following judgments: The amplified PCR products were detected by agarose gel electrophoresis. If the amplified positive control PCR product had a target band of about 2700 bp, and the amplified negative control PCR product did not have a target band of about 2700 bp, and the PCR amplification product of the test sample had a target band of about 2700 bp, then the virus to be tested was considered to be the target virus corresponding to the primer pair used.

[0057] Application Example 1: Detection of SyLCV, TYLCCNV and TbLCYnV viruses in leaf samples of Amorphophallus bulbifera plants collected in the field.

[0058] The specific steps are as follows: (1) Total DNA was extracted from 12 leaf samples of Amorphophallus bulbifera collected in the field. The extracted DNA was dissolved in ddH2O. The concentration and quality of the DNA samples were detected by an ultra-micro spectrophotometer. After passing the test, the samples were stored at -20℃ for later use.

[0059] (2) Rolling circle amplification (RCA) system (total volume 11.2 μL): Mix 1 μL of DNA template with 5 μL of Sample Buffer at a concentration of 100 ng / μL evenly, denature at 95 °C for 3 min in an enzyme digester; then place on ice for 10 min, add 5 μL of Raction Buffer and 0.2 μL of Enzyme, react at 30 °C for 18 h in an enzyme digester, and terminate the reaction at 65 °C for 10 min.

[0060] (3) PCR amplification system (total volume 50 μL): ddH2O 12.7 μL, 10×PCR Buffer 25 μL, 2 mmol / L dNTPs 10 μL, 10 μmol / L upstream and downstream primers 0.5 μL each, RCA product template 0.5 μL, 1 U / μL KOD FX DNA polymerase 0.8 μL. Add the above reagents to the PCR tube and perform DNA amplification in the PCR instrument.

[0061] For primer pair TYLCCNV, the PCR reaction program was as follows: 98℃ for 2 min; 98℃ for 10 s; 60℃ for 30 s; 68℃ for 3 min, repeated 28 times; 68℃ for 10 min; stored at 4℃. For primer pair SyLCV, the PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s; 64℃ for 30 s; 68℃ for 3 min, repeated 28 times; 68℃ for 10 min; stored at 4℃. For primer pair TbLCYnV, the PCR reaction program was as follows: 98℃ for 2 min; 98℃ for 10 s; 61℃ for 30 s; 68℃ for 3 min, repeated 28 times; 68℃ for 10 min; stored at 4℃.

[0062] (4) The test results showed that the amplified PCR products were detected by agarose gel electrophoresis. The amplified positive control PCR product had a target band of about 2700bp, while the amplified negative control PCR product did not have a target band of about 2700bp. At the same time, the PCR amplification products of TYLCCNV, SyLCV and TbLCYnV had target bands of about 2700bp. The test results were verified by the sequencing results, proving that the test results of the present invention are true and reliable.

[0063] Application Example 2: Detection of SyLCV, TYLCCNV and TbLCYnV viruses in seed tuber samples of Amorphophallus bulbifer collected in the field.

[0064] The specific steps are as follows: (1) Total DNA was extracted from 10 samples of Amorphophallus bulbifera seed tubers collected in the field. The extracted DNA was dissolved in ddH2O. The concentration and quality of the DNA samples were detected by an ultra-micro spectrophotometer. After passing the test, the samples were stored at -20℃ for later use.

[0065] (2) Rolling circle amplification (RCA) system (total volume 11.2 μL): Mix 1 μL of DNA template with 5 μL Sample Buffer at a concentration of 100 ng / μL and denature at 95 °C for 3 min in an enzyme digester; then place on ice for 10 min, add 5 μL of Raction Buffer and 0.2 μL of Enzyme, react at 30 °C for 18 h in an enzyme digester, and terminate the reaction at 65 °C for 10 min.

[0066] (3) PCR amplification system (total volume 50 μL): ddH2O 12 μL, 10×PCR Buffer 25 μL, 2 mmol / L dNTPs 10 μL, 10 μmol / L upstream and downstream primers 0.8 μL each, RCA product template 0.5 μL, 1 U / μL KOD FX DNA polymerase 0.9 μL. Add the above reagents to the PCR tube and perform DNA amplification in the PCR instrument.

[0067] For primer pair TYLCCNV, the PCR reaction program was as follows: 98℃ for 2 min; 98℃ for 10 s; 62℃ for 30 s; 68℃ for 3 min, repeated 30 times; 68℃ for 10 min; stored at 4℃. For primer pair SyLCV, the PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s; 65℃ for 30 s; 68℃ for 3 min, repeated 30 times; 68℃ for 10 min; stored at 4℃. For primer pair TbLCYnV, the PCR reaction program was as follows: 98℃ for 2 min; 98℃ for 10 s; 62℃ for 30 s; 68℃ for 3 min, repeated 30 times; 68℃ for 10 min; stored at 4℃.

[0068] (4) The test results showed that the amplified PCR products were detected by agarose gel electrophoresis. The amplified positive control PCR product had a target band of about 2700bp, while the amplified negative control PCR product did not have a target band of about 2700bp. At the same time, the PCR amplification products of TYLCCNV, SyLCV and TbLCYnV had target bands of about 2700bp. The test results were verified by the sequencing results, proving that the test results of the present invention are true and reliable.

[0069] Application Example 3: Detection of SyLCV, TYLCCNV and TbLCYnV viruses in leaf surface samples of Amorphophallus bulbifera collected in the field.

[0070] The specific steps are as follows: (1) Total DNA was extracted from 15 leaf fruit samples of Amorphophallus bulbifera collected in the field. The extracted DNA was dissolved in ddH2O. The concentration and quality of the DNA samples were detected by an ultra-micro spectrophotometer. After passing the test, the samples were stored at -20℃ for later use.

[0071] (2) Rolling circle amplification (RCA) system (total volume 11.2 μL): Mix 1 μL of DNA template with 5 μL of Sample Buffer at a concentration of 100 ng / μL evenly, denature at 95 °C for 3 min in an enzyme digester; then place on ice for 10 min, add 5 μL of Raction Buffer and 0.2 μL of Enzyme, react at 30 °C for 18 h in an enzyme digester, and terminate the reaction at 65 °C for 10 min.

[0072] (3) PCR amplification system (total volume 50 μL): ddH2O 11.5 μL, 10×PCR Buffer 25 μL, 2 mmol / L dNTPs 10 μL, 1.0 μL each of 10 μmol / L upstream and downstream primers, 0.5 μL RCA product template, and 1.0 μL 1 U / μL KOD FX DNA polymerase. Add the above reagents to the PCR tube and perform DNA amplification in the PCR instrument.

[0073] For primer pair TYLCCNV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 63℃ for 30 s; 68℃ for 3 min, repeated 28-30 times; 68℃ for 10 min; store at 4℃. For primer pair SyLCV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 66℃ for 30 s; 68℃ for 3 min, repeat 28-30 times; 68℃ for 10 min; store at 4℃. For primer pair TbLCYnV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 63℃ for 30 s; 68℃ for 3 min, cycled 28-30 times; 68℃ for 10 min; store at 4℃.

[0074] (4) The test results showed that the amplified PCR products were detected by agarose gel electrophoresis. The amplified positive control PCR product had a target band of about 2700bp, while the amplified negative control PCR product did not have a target band of about 2700bp. At the same time, the PCR amplification products of TYLCCNV, SyLCV and TbLCYnV had target bands of about 2700bp. The test results were verified by the sequencing results, proving that the test results of the present invention are true and reliable.

[0075] Specificity test of the reagent kit of this invention: Nucleic acid was extracted from the leaves of Amorphophallus bulbifera plants. The samples were then tested using specific primers for TYLCCNV, SyLCV, and TbLCYnV, as well as universal primers BF (5′-accggatggccgcgattttt-3′; SEQ ID NO.7) and BR (5′-aatattatacggatggccgc-3′; SEQ ID NO.8). The specificity of the primer sets for the detection of TYLCCNV, SyLCV, and TbLCYnV was verified. The specific implementation method was the same as in Application Case 1.

[0076] See results Figure 1 The specific detection results of TYLCCNV, SyLCV and TbLCYnV in Amorphophallus bulbifera are shown in the electrophoresis results. Figure 1 As shown in the figure, TYLCCNV ( ) amplified using the specific primers of the kit of this invention Figure 1 A), SyLCV ( Figure 1 B) and TbLCYnV ( Figure 1 C) The results showed excellent specificity with no extraneous bands; TYLCCNV, SyLCV, and TbLCYnV were amplified using universal primers BF / BR. Figure 1 D) Numerous extraneous bands. This result indicates that the primer pairs in the kit of this invention have good specificity, and the experimental results can be directly used for subsequent experiments.

[0077] The results showed that regardless of changes in annealing temperature, primer concentration, enzyme concentration, or other conditions, SyLCV, TYLCCNV, and TbLCYnV could be detected in the leaves, seed tubers, and foliar fruits of Amorphophallus bulbifera collected from the field under the same experimental conditions. No false positives were observed in the negative controls, demonstrating high specificity and reliability. This invention employs rolling circle replication (RCA) to amplify the target nucleic acid signal and then uses virus-specific primers for PCR detection. This method enables rapid, efficient, and specific acquisition of the target fragment, establishing a scientific method for the large-scale, rapid, accurate, and sensitive detection of TYLCCNV, SyLCV, and TbLCYnV in Amorphophallus bulbifera leaf, seed tuber, and foliar fruit samples.

[0078] The above examples demonstrate the design of specific primer sets for the detection of three viruses—TYLCCNV, SyLCV, and TbLCYnV—from three batches of samples collected from the field: leaves, seed tubers, and leaf fruits of Amorphophallus bulbifera plants. These primer sets and kits exhibit high specificity, targeting the conserved regions (V1 gene) of TYLCCNV, SyLCV, and TbLCYnV, thus avoiding non-specific binding. This is the first time that specific primers for TYLCCNV, SyLCV, and TbLCYnV viruses have been used for the detection of these viral pathogens in Amorphophallus bulbifera, enabling rapid, specific, and efficient detection of TYLCCNV, SyLCV, and TbLCYnV in different parts of the plant.

[0079] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A primer set for detecting *Amorphophallus quinquefolius* viruses in *Amorphophallus bulbifer*, characterized in that... This includes primer pair TYLCCNV, primer pair SyLCV, and primer pair TbLCYnV; The sequence of the primer pair TYLCCNV is as follows: TYLCCNV-F: 5'-gtcatttccactcccgcctc-3' (SEQ ID NO. 1); TYLCCNV-R: 5'-actatatcggcgggacgctt-3' (SEQ ID NO. 2); The sequence of the primer pair SyLCV is as follows: SyLCV-F: 5'-cgacagcccatatgtgagcc-3' (SEQ ID NO.3); SyLCV-R: 5'-aagttgagacggcggcgtac-3' (SEQ ID NO.4); The sequence of the primer pair TbLCYnV is as follows: TbLCYnV-F: 5'-tatgtgagccgtgctgctgccc-3' (SEQ ID NO. 5); TbLCYnV-R: 5'-tgggctgtcgaagttgagacggc-3' (SEQ ID NO. 6); The aforementioned viruses in the genus *Golden Mosaic Virus* include *Golden Sword Leaf Curved Virus*, *Yunnan Tobacco Leaf Curved Virus*, and *Chinese Tomato Yellow Leaf Curved Virus*.

2. A reagent or kit containing the primer set of claim 1 for detecting common bean golden mosaic virus in Amorphophallus bulbifera.

3. The kit for detecting *Amorphophallus glaucus* virus in *Amorphophallus bulbifer* according to claim 2, characterized in that, Also includes: PCR reaction premix, positive control, and negative control; the PCR reaction premix contains: 11.5-12.7 μL ddH2O, 25 μL 10×PCR Buffer, 10 μL dNTPs at 2 mmol / L, 0.5-1.0 μL upstream primer at 10 μmol / L, 0.5-1.0 μL downstream primer at 10 μmol / L, and 0.8-1.0 μL KOD FX DNA polymerase at 1 U / μL, for a total volume of 49.5 μL; the primer pair consisting of the upstream and downstream primers is primer pair TYLCCNV, primer pair SyLCV, or primer pair TbLCYnV.

4. The kit for detecting *Amorphophallus glaucus* virus in *Amorphophallus bulbifer* according to claim 3, characterized in that, The PCR reaction system is as follows: 49.5 μL of PCR reaction premix; Template 0.5μL; Total volume: 50 μL; The template is the product obtained by rolling circle amplification of DNA extracted from Amorphophallus bulbifera samples, a positive control, or a negative control.

5. The kit for detecting *Amorphophallus glaucus* virus in *Amorphophallus bulbifer* according to claim 3, characterized in that, The negative control is water; the positive control is a recombinant plasmid of TYLCCNV, SyLCV, and TbLCYnV. The recombinant plasmids TYLCCNV, SyLCV, and TbLCYnV were prepared as follows: PCR amplification of the DNA of TYLCCNV, SyLCV, and TbLCYnV was performed using primers SEQ ID NO.1 and SEQ ID NO.2 of TYLCCNV, primers SEQ ID NO.3 and SEQ ID NO.4 of SyLCV, and primers SEQ ID NO.5 and SEQ ID NO.6 of TbLCYnV, respectively. The amplification products were then ligated with p-EASY-T to construct the recombinant plasmids T-TYLCCNV, T-SyLCV, and T-TbLCYnV.

6. The kit for detecting *Amorphophallus glaucus* virus in *Amorphophallus bulbifer* according to claim 5, characterized in that, The copy number of the positive control was 6.9 × 10⁻⁶ for the T-TYLCCNV recombinant plasmid. 8 The copy number of the T-SyLCV recombinant plasmid was 1.4 × 10⁻⁶ copies / μL. 9 The T-TbLCYnV recombinant plasmid contains 2.1 × 10⁻⁶ copies / μL. 8 Copy / μL.

7. The kit for detecting *Amorphophallus glaucus* virus in *Amorphophallus bulbifer* according to claim 3, characterized in that: For primer pair TYLCCNV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 60-63℃ for 30 s; 68℃ for 3 min, repeated 28-30 times; 68℃ for 10 min; store at 4℃. For primer pair SyLCV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 64-66℃ for 30 s; 68℃ for 3 min, repeat 28-30 times; 68℃ for 10 min; store at 4℃. For primer pair TbLCYnV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 61-63℃ for 30 s; 68℃ for 3 min, cycled 28-30 times; 68℃ for 10 min; store at 4℃.

8. The kit for detecting a common bean golden mosaic virus in Amorphophallus bulbifera according to claim 4, characterized in that: The specific method for rolling circle amplification is as follows: using DNA from Amorphophallus bulbifera samples as template; mixing 1 μL of 100 ng / μL template with 5 μL of sample buffer, denaturing at 95℃ for 3 min in an enzyme digester; then placing on ice for 10 min, adding 5 μL of reaction buffer and 0.2 μL of enzyme, reacting at 30℃ for 18 h in an enzyme digester, and terminating the reaction at 65℃ for 10 min.

9. A method for identifying whether a virus to be tested belongs to the genus *Amorphophallus vulgaris* in Amorphophallus bulbifera for non-disease diagnosis and treatment purposes, comprising a kit for detecting *Amorphophallus vulgaris* viruses according to any one of claims 3-8, characterized in that... Includes the following steps: S1. Extract the nucleic acid of the virus to be tested, amplify the signal of the target nucleic acid by rolling circle amplification, and use the obtained RCA product for PCR detection; S2. Using the RCA product obtained in S1, the positive control, and the negative control as templates, respectively, prepare PCR reaction systems and perform PCR amplification reactions; the PCR reaction systems are as follows: 49.5 μL of PCR reaction premix; Template 0.5μL; Total volume: 50 μL; For primer pair TYLCCNV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 60-63℃ for 30 s; 68℃ for 3 min, repeated 28-30 times; 68℃ for 10 min; store at 4℃. For primer pair SyLCV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 64-66℃ for 30 s; 68℃ for 3 min, repeat 28-30 times; 68℃ for 10 min; store at 4℃. For primer pair TbLCYnV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 61-63℃ for 30 s; 68℃ for 3 min, repeated 28-30 times; 68℃ for 10 min; store at 4℃. S3. Based on the reaction results, make the following judgments: The amplified PCR products were detected by agarose gel electrophoresis. If the amplified positive control PCR product had a target band of about 2700 bp, and the amplified negative control PCR product did not have a target band of about 2700 bp, and the PCR amplification product of the virus to be tested had a target band of about 2700 bp, then the virus to be tested was considered to be the target virus corresponding to the primer pair used.

10. A method for identifying whether a sample contains a virus of the genus *Amorphophallus* in *Amorphophallus konjac* for non-disease diagnosis and treatment purposes, comprising a kit for detecting *Amorphophallus konjac* viruses according to any one of claims 3-8, characterized in that... Includes the following steps: S1. Extract nucleic acid from the sample to be tested, amplify the signal of the target nucleic acid by rolling circle amplification, and use the obtained RCA product for PCR detection; S2. Using the RCA product obtained in S1, the positive control, and the negative control as templates, respectively, prepare PCR reaction systems and perform PCR amplification reactions; the PCR reaction systems are as follows: 49.5 μL of PCR reaction premix; Template 0.5μL; Total volume: 50 μL; For primer pair TYLCCNV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 60-63℃ for 30 s; 68℃ for 3 min, repeated 28-30 times; 68℃ for 10 min; store at 4℃. For primer pair SyLCV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 64-66℃ for 30 s; 68℃ for 3 min, repeat 28-30 times; 68℃ for 10 min; store at 4℃. For primer pair TbLCYnV, the PCR reaction program is as follows: 98℃ for 2 min; 98℃ for 10 s; 61-63℃ for 30 s; 68℃ for 3 min, repeated 28-30 times; 68℃ for 10 min; store at 4℃. S3. Based on the reaction results, make the following judgments: The amplified PCR products were detected by agarose gel electrophoresis. If the amplified positive control PCR product had a target band of about 2700 bp, and the amplified negative control PCR product did not have a target band of about 2700 bp, and the PCR amplification product of the test sample had a target band of about 2700 bp, then the virus to be tested was considered to be the target virus corresponding to the primer pair used.

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