A primer set, reagent, kit, method for simultaneously detecting four pepper viruses and application thereof

Abstract

The application provides a primer group, reagent, kit, method and application for simultaneously detecting four pepper viruses, and belongs to the technical field of gene detection. The primer group comprises primer group 1 for detecting tomato spotted wilt virus (TSWV), primer group 2 for detecting tobacco rattle virus (TRV), primer group 3 for detecting pepper mild mottle virus (PMMoV) and primer group 4 for detecting potato virus X (PVX). The primer group provided by the application has high specificity, the primer group is used for simultaneously detecting TSWV, TRV, PMMoV and PVX, whether the sample to be detected is infected with TSWV, TRV, PMMoV or PVX virus can be determined through one-time amplification detection, and on the basis of saving detection time and reducing detection cost, the detection accuracy is improved.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology, and in particular relates to a primer set, reagent, kit, method and application for the simultaneous detection of four pepper viruses. Background Technology

[0002] chili( Capsicum annuum Chili peppers (L.) originated in the tropical and subtropical regions of South America and are one of the most widely cultivated economic crops, possessing significant economic value. With the continuous expansion of chili pepper cultivation, chili pepper diseases, especially viral diseases, are increasingly impacting chili pepper yield and quality.

[0003] Currently, over seventy viruses have been reported globally to infect chili peppers, including Cucumber mosaic virus (CMV), Tobacco mosaic virus (TMV), Pepper mild mottle virus (PMMoV), Pepper mottle virus (PepMoV), Tobacco rattle virus (TRV), Tomato spotted wilt virus (TSWV), Potato virus X (PVX), Potato virus Y (PVY), and Chilli ringspot virus (ChiRSV). Due to the large number of chili pepper viruses, co-infection with two or more viruses is common, severely weakening host resistance and leading to complex and varied symptoms after infection. Common symptoms include mosaic, yellowing, wrinkling, and malformation, resulting in a significant decline in chili pepper yield and quality.

[0004] Viruses such as PMMoV and TSWV have been detected in multiple chili-growing areas, and co-infection with TRV and PVX has been reported in other regions. However, a simultaneous detection system for co-infection of these four viruses (TSWV, TRV, PMMoV, and PVX) has not yet been established. To quickly and accurately identify the types of chili viral diseases, this study aims to provide a method for the simultaneous and rapid detection of multiple chili viruses. This system will offer a practical technical means for detecting co-infection of chili viruses and provide strong technical support for the early prevention and control of chili viral diseases. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a primer set for the simultaneous detection of four pepper viruses, which can determine whether the sample to be tested is infected with TSWV, TRV, PMMoV or PVX virus by a single amplification detection.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A primer set for the simultaneous detection of four pepper viruses, the primer set comprising primer set 1 for detecting TSWV, primer set 2 for detecting TRV, primer set 3 for detecting PMMoV and primer set 4 for detecting PVX.

[0008] The nucleotide sequences of primer set 1 are shown in SEQ ID NO.1~SEQ ID NO.2; the nucleotide sequences of primer set 2 are shown in SEQ ID NO.3~SEQ ID NO.4; the nucleotide sequences of primer set 3 are shown in SEQ ID NO.5~SEQ ID NO.6; and the nucleotide sequences of primer set 4 are shown in SEQ ID NO.7~SEQ ID NO.8.

[0009] Another object of the present invention is to provide a reagent for the simultaneous detection of four pepper viruses, the reagent comprising the primer set described above.

[0010] Another object of the present invention is to provide a kit for the simultaneous detection of four pepper viruses, the kit comprising the primer set or the reagents described above.

[0011] Another object of the present invention is to provide the application of the primer set, the reagent, or the kit in the simultaneous detection of four pepper viruses.

[0012] Another object of the present invention is to provide a method for simultaneously detecting four pepper viruses, comprising the following steps:

[0013] PCR amplification was performed using the primer set, reagent, or kit described above. After amplification, the amplification products were subjected to agarose gel electrophoresis to determine whether the sample was infected with TSWV, TRV, PMMoV, or PVX virus.

[0014] Preferably, the PCR amplification reaction system is as follows: ddH2O 7.4 μL, upstream primer mixture 0.4 μL, downstream primer mixture 0.4 μL, 10X PCR Buffer 1 μL, dNTPs 0.2 μL, Taq DNA polymerase 0.2 μL, and template 0.4 μL.

[0015] Preferably, the concentrations of both the upstream primer mixture and the downstream primer mixture are 0.1 × 10⁻⁶.-6 ~1.0×10 -6 μM; In the upstream primer mixture and the downstream primer mixture, the primers used to detect TSWV, TRV, PMMoV and PVX were mixed in a concentration ratio of (1~2):(1~4):(1~4):(1~4).

[0016] Preferably, the concentration of dNTPs is 0.001~0.016 mmol·L⁻¹. -1 The concentration of Taq DNA polymerase is 0.25~2 U·μL. -1 .

[0017] Preferably, the PCR amplification reaction program is as follows: 95.0℃ for 3 min; (95.0℃ for 30 s, 50.0℃ for 30 s, 72.0℃ for 1 min) for 35 cycles; 72.0℃ for 10 min, 16.0℃ for 5 s.

[0018] Preferably, the method for determining whether a sample to be tested is infected with TSWV, TRV, PMMoV, or PVX virus includes: the presence of a 201 bp specific identification band in the electrophoresis pattern indicates the presence of TSWV virus; the presence of a 311 bp specific identification band in the electrophoresis pattern indicates the presence of TRV virus; the presence of a 414 bp specific identification band in the electrophoresis pattern indicates the presence of PMMoV virus; and the presence of a 576 bp specific identification band in the electrophoresis pattern indicates the presence of PVX virus.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention provides a primer set for the simultaneous detection of four pepper viruses. A single amplification detection can clearly identify whether a sample is infected with TSWV, TRV, PMMoV, or PVX virus. This primer set has high specificity and fills the gap in the current technology for the simultaneous detection of TSWV, TRV, PMMoV, and PVX in pepper virus diseases.

[0021] The primer set of this invention can be used to simultaneously detect TSWV, TRV, PMMoV and PVX, which has a low risk of contamination. It saves detection time and reduces detection costs while improving detection accuracy. It can identify virus types more quickly, making it possible to take timely prevention and control measures. This helps to reduce the impact of viral diseases on chili pepper yield and quality and promotes the healthy development of the chili pepper industry.

[0022] This invention provides a method for simultaneously detecting multiple pepper viruses, offering a practical and feasible technical means for detecting multiple infections of pepper viruses and providing strong technical support for the early prevention and control of pepper viral diseases. Attached Figure Description

[0023] Figure 1 The results of gene sequence alignment for plasmids containing the reverse-transcribed TSWV coat protein CDS.

[0024] Figure 2 Comparison of symptoms between healthy and diseased pepper plants;

[0025] Figure 3 The results are RT-PCR detection results for four pepper viruses; where M is DL2000 Marker; 1 is TSWV (201 bp); 2 is TRV (311 bp); 3 is PMMoV (414 bp); 4 is PVX (576 bp); CK- is Negative.

[0026] Figure 4 The results show the multiplex RT-PCR detection results of four pepper viruses; where M is the DL2000 Marker; 1 is the multiplex RT-PCR of TSWV+TRV+PMMoV+PVX; 2 is the singlet PCR of TSWV (201 bp); 3 is the singlet PCR of TRV (311 bp); 4 is the singlet PCR of PMMoV (414 bp); 5 is the singlet PCR of PVX (576 bp); CK- is the Negative.

[0027] Figure 5 To optimize primer concentration; where M is the DL2000 Marker; the primer concentrations for lanes 1-10 are 0.1 × 10⁻⁶. -6 μM, 0.2×10 -6 μM, 0.3×10 -6 μM, 0.4×10 -6 μM, 0.5×10 -6 μM, 0.6×10 -6 μM, 0.7×10 -6 μM, 0.8×10 -6 μM, 0.9×10 -6 μM, 1.0×10 -6 μM; CK- is Negative;

[0028] Figure 6 For the optimization of primer concentration ratios; where M is DL 2000 DNA Maker; the TSWV:TRV:PMMoV:PVX ratios for lanes 1-9 are 1:1:1:1, 1:2:2:2, 1:4:4:4, 1:1:2:4, 2:2:4:1, 2:4:1:2, 2:2:1:4, 2:4:2:1, and 2:1:4:1, respectively; CK- is Negative.

[0029] Figure 7For the optimization of annealing temperature; where M is DL2000 Maker; 1 is Tm = 50℃; 2 is Tm = 51℃; 3 is Tm = 52℃; 4 is Tm = 53℃; 5 is Tm = 54℃; 6 is Tm = 55℃; 7 is Tm = 56℃; 8 is Tm = 57℃; 9 is Tm = 58℃; 10 is Tm = 59℃; CK- is Negative;

[0030] Figure 8 To optimize the concentration of dNTPs; where M is the DL 2000 Maker; the dNTP concentrations for lanes 1-9 are 0.001 mmol·L⁻¹. -1 0.002 mmol·L -1 0.004 mmol·L -1 0.006 mmol·L -1 0.008 mmol·L -1 0.010 mmol·L -1 0.012 mmol·L -1 0.014 mmol·L -1 0.016 mmol·L -1 CK- stands for Negative;

[0031] Figure 9 To optimize the concentration of Taq DNA polymerase; where M is DL 2000 Maker; the enzyme concentrations for lanes 1-9 are 0.25 U·μL. -1 0.5 U·μL -1 1 U·μL -1 1.5 U·μL -1 2 U·μL -1 2.5 U·μL -1 3U·μL -1 3.5 U·μL -1 4 U·μL -1 CK- stands for Negative;

[0032] Figure 10 This is a sensitivity test for multiplex RT-PCR; where M is the DL 2000 Maker; the viral concentrations corresponding to lanes 1-7 are 10 ng, 1 ng, and 1×10⁻⁶, respectively. -1 ng, 1×10 -2 ng, 1×10 -3 ng, 1×10 -4 ng, 1×10 -5 ng; CK- stands for Negative;

[0033] Figure 11 The results are for field chili pepper samples; M represents the DL 2000 Maker; lanes 1-15 represent the results for leaf samples from 15 chili pepper plants with abnormal growth; CK- represents the results for leaf samples from 1 chili pepper plant with normal growth. Detailed Implementation

[0034] This invention provides a primer set for the simultaneous detection of four pepper viruses. The primer set includes primer set 1 for detecting Tomato spotted wilt virus (TSWV), primer set 2 for detecting Tobacco rattle virus (TRV), primer set 3 for detecting Pepper mild mottle virus (PMMoV), and primer set 4 for detecting Potato virus X (PVX). The nucleotide sequences of primer set 1 are shown in SEQ ID NO.1 to SEQ ID NO.2; the nucleotide sequences of primer set 2 are shown in SEQ ID NO.3 to SEQ ID NO.4; the nucleotide sequences of primer set 3 are shown in SEQ ID NO.5 to SEQ ID NO.6; and the nucleotide sequences of primer set 4 are shown in SEQ ID NO.7 to SEQ ID NO.8.

[0035] This invention designs primers for detecting TSWV, TRV, PMMoV and PVX viruses based on the TSWV gene number HQ260978.1, TRV gene number NC_003811.1, PMMoV gene number LC817996.1 and PVX gene number M95516.1.

[0036] The present invention also provides a reagent for simultaneously detecting four pepper viruses, the reagent comprising the primer set described above.

[0037] The present invention also provides a kit for the simultaneous detection of four pepper viruses, the kit comprising the primer set or the reagents described herein.

[0038] The present invention also provides the application of the primer set, reagent, or kit described herein in the simultaneous detection of four pepper viruses.

[0039] This invention also provides a method for simultaneously detecting four pepper viruses, comprising the following steps: performing PCR amplification using the primer set, the reagent, or the kit described herein; after amplification, performing agarose gel electrophoresis on the amplification products to determine whether the sample to be tested is infected with TSWV, TRV, PMMoV, or PVX virus.

[0040] In the method for simultaneously detecting four pepper viruses of the present invention, the PCR amplification reaction system comprises: 7.4 μL ddH₂O, 0.4 μL upstream primer mixture, 0.4 μL downstream primer mixture, 1 μL 10X PCR Buffer, 0.2 μL dNTPs, 0.2 μL Taq DNA polymerase, and 0.4 μL template; the preferred concentrations of both the upstream and downstream primer mixtures are 0.1 × 10⁻⁶. -6 ~1.0×10 -6 μM; in the upstream and downstream primer mixtures, the primers used for detecting TSWV, TRV, PMMoV, and PVX are preferably mixed in a concentration ratio of (1~2):(1~4):(1~4):(1~4), more preferably 2:2:1:4; the concentration of dNTPs is preferably 0.001~0.016 mmol·L. -1 More preferably, it is 0.004~0.014 mmol·L⁻¹. -1 Further preferred is 0.006 mmol·L -1 The preferred concentration of Taq DNA polymerase is 0.25–2 U / μL. -1 More preferably, 1~1.5 U·μL -1 .

[0041] In the method for simultaneous detection of four pepper viruses of the present invention, the PCR amplification reaction program is as follows: 95.0℃ for 3 min; (95.0℃ for 30 s, 50.0℃ for 30 s, 72.0℃ for 1 min) for 35 cycles; 72.0℃ for 10 min, 16.0℃ for 5 s.

[0042] In the method for simultaneously detecting four types of pepper viruses of the present invention, the method for determining whether the sample to be tested is infected with TSWV, TRV, PMMoV or PVX virus includes: the presence of a 201 bp specific identification band in the electrophoresis pattern indicates the presence of TSWV virus; the presence of a 311 bp specific identification band in the electrophoresis pattern indicates the presence of TRV virus; the presence of a 414 bp specific identification band in the electrophoresis pattern indicates the presence of PMMoV virus; and the presence of a 576 bp specific identification band in the electrophoresis pattern indicates the presence of PVX virus.

[0043] This invention achieves a synergistic improvement in amplification efficiency, detection sensitivity, and economy by systematically optimizing reaction components and amplification parameters. It can determine whether a sample is infected with TSWV, TRV, PMMoV, or PVX viruses with a single amplification, saving detection costs and improving detection efficiency.

[0044] Using the detection method provided by this invention, even with a template amount as low as 1×10⁻⁶, -3 Even at ng, all target bands can be stably amplified without non-specific amplification products. This led to the optimization of a sensitive, rapid, and economical detection system, significantly improving detection efficiency. The method provided by this invention for detecting field pepper samples offers crucial technical support for the early diagnosis and integrated control of pepper viral diseases. The widespread application of this invention is expected to further promote the improvement of pepper viral disease control systems and contribute to the sustainable development of the pepper industry.

[0045] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0046] In a specific embodiment of the present invention, the infectious clones of TSWV, TRV, PMMoV and PVX viruses were provided by the Plant Antiviral Immunology Laboratory of the College of Plant Protection, Anhui Agricultural University, and the diseased leaves of infected plants in the field were collected from Fuyang area, Anhui Province.

[0047] The reverse transcription kit was provided by Hifair AdvanceFast 1st Strand cDNASynthesis Kit from Yisheng Company; the FreeZol Reagent total RNA extraction kit, Phanta Max Super-Fidelity DNA polymerase, and ClonExpress II One Step Cloning Kit were purchased from Nanjing Novizan Biotechnology Co., Ltd.; the Taq Plus DNA polymerase was purchased from Sangon Biotech (Shanghai) Co., Ltd.; primer synthesis and DNA sequencing services were performed by Beijing Qingke Biotechnology Co., Ltd.; and the plasmid miniprep kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0048] Example 1

[0049] A primer set for the simultaneous detection of four pepper viruses.

[0050] Primers for detecting TSWV, TRV, PMMoV and PVX viruses were designed based on the TSWV gene number HQ260978.1, TRV gene number NC_003811.1, PMMoV gene number LC817996.1 and PVX gene number M95516.1. The primer sequences are shown in Table 1.

[0051] Table 1 Primer-specific sequences for multiplex RT-PCR amplification of target gene fragments from four viruses.

[0052]

[0053] Example 2

[0054] A method for simultaneously detecting four pepper viruses, comprising the following steps:

[0055] The FreeZol Reagent Total RNA Extraction Kit was used to extract total RNA from infected pepper leaves. The total RNA concentration was measured using a micro spectrophotometer (Yisheng). cDNA was synthesized according to the instructions of the Hifair Advance Fast 1st Strand cDNA Synthesis Kit provided by Yisheng Company. The synthesized cDNA was immediately subjected to subsequent PCR amplification.

[0056] PCR amplification was performed using the primer set from Example 1; the upstream primers for TSWV, TRV, PMMoV, and PVX viruses from Example 1 were mixed at a concentration ratio of 2:2:1:4 to obtain an upstream primer mixture with a primer concentration of 0.5 × 10⁻⁶. -6 μM; The downstream primers of TSWV, TRV, PMMoV and PVX viruses in Example 1 were mixed at a concentration ratio of 2:2:1:4 to obtain a downstream primer mixture with a primer concentration of 0.5 × 10 μM. -6 μM;

[0057] The reaction mixture consisted of: 7.4 μL ddH₂O, 0.4 μL upstream primer mixture, 0.4 μL downstream primer mixture, 1 μL 10X PCR Buffer, and 0.2 μL dNTPs (0.006 mmol·L⁻¹). -1 ), Taq DNA polymerase 0.2 μL (1.5 U·μl) -1 ), template 0.4 μL;

[0058] The reaction program was as follows: 95.0℃ for 3 min; (95.0℃ for 30 s, 50.0℃ for 30 s, 72.0℃ for 1 min) for 35 cycles; 72.0℃ for 10 min, 16.0℃ for 5 s;

[0059] After amplification, the amplification products were subjected to agarose gel electrophoresis to determine whether the sample was infected with TSWV, TRV, PMMoV, or PVX viruses: a 201 bp specific identification band indicated the presence of TSWV virus; a 311 bp specific identification band indicated the presence of TRV virus; a 414 bp specific identification band indicated the presence of PMMoV virus; and a 576 bp specific identification band indicated the presence of PVX virus.

[0060] Example 3

[0061] Results of singleton PCR and multiplex RT-PCR detection.

[0062] 1. Plasmid construction

[0063] According to the method in Example 2, total RNA was extracted from pepper leaves infected with TSWV, TRV, PMMoV and PVX viruses respectively, and cDNA was synthesized.

[0064] Following the instructions of the ClonExpress II One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd., TSWV coat protein sequence CDS plasmid recombination was performed. The recombinant plasmid was transformed into *E. coli* DH5α competent cells, heat-shocked, and then plated on LB solid medium containing the corresponding antibody. After the emergence of visible single colonies, positive clones were screened using the Taq Plus DNA polymerase from Sangon Biotech (Shanghai) Co., Ltd. Single colonies containing the target plasmid were amplified and screened. Plasmids were extracted according to the instructions of the plasmid miniprep kit from Tiangen Biotech (Beijing) Co., Ltd., and then sent to Beijing Qingke Biotechnology Co., Ltd. for gene sequencing. Gene sequence alignment was performed using SnapGene 6.0.2 software. The results are as follows: Figure 1 As shown.

[0065] Using the same method, plasmids containing TRV, PMMoV, and PVX were constructed respectively.

[0066] 2. Friction inoculation

[0067] Take 0.1 g of tobacco leaves infected with TSWV, TRV, PMMoV and PVX viruses respectively, add 200 μL of 1×PBS solution, grind into a homogenate, sprinkle carborundum evenly on the chili pepper leaves, then take the homogenate and spread it on the leaves sprinkled with carborundum, rub 2-3 times, and change gloves every time a different type of virus is inoculated.

[0068] like Figure 2As shown, pepper plants infected with the four viruses all exhibited typical viral disease symptoms, including varying degrees of leaf wrinkling and mosaic symptoms. Notably, plants infected with TRV and PMMoV also showed a significant dwarfing phenotype. These results indicate that these four viruses can significantly affect the growth and development of pepper plants, with TRV and PMMoV exhibiting particularly prominent pathogenic effects.

[0069] 3. Singleton PCR amplification

[0070] Following the method described in Example 2, total RNA was extracted from each pepper leaf, and cDNA was synthesized and then amplified by PCR. The results are as follows: Figure 3 As shown.

[0071] Using target virus cDNA synthesized from total RNA extracted from leaves of previously infected pepper plants as a template, PCR amplification was performed using specific primers. Figure 3 As shown, lanes 1-4 exhibited single, bright amplified bands at DNA molecular weight standards of 201, 311, 414, and 576 bp, respectively. The size of each band perfectly matched the expected design, and the band edges were clear without tailing. Notably, the negative control sample did not show any non-specific amplification products in the corresponding regions, indicating that this PCR system has good specificity.

[0072] 4. Detect and analyze mixed infection samples and single pathogenic samples separately.

[0073] The mixed infection sample was prepared by mixing plasmids containing the inserted TSWV, TRV, PMMoV, and PVX coat protein (CP) sequences in a concentration ratio of 1:1:1:1. Amplification was performed using the method described in Example 2, and the results are as follows: Figure 4 As shown.

[0074] like Figure 4 As shown, lane 1 of the mixed sample detection showed clear single amplification bands at all four target sites, with migration rates perfectly matching the theoretical amplification lengths of each virus (201 bp for TSWV, 311 bp for TRV, 414 bp for PMMoV, and 576 bp for PVX). In the detection of single pathogenic samples, lanes 2-5 showed specific bands at their respective target sites, and no non-target amplification products were observed. The negative control group showed no amplification signal in the entire electrophoresis pattern, fully demonstrating that the multiplex detection system of this invention has high specificity, no cross-reactivity between primer pairs, and high sensitivity, effectively distinguishing between mixed and single infections.

[0075] Example 4

[0076] Optimization of multiplex RT-PCR reaction system.

[0077] 1. Optimization of primer concentration and primer concentration ratio

[0078] Since multiplex RT-PCR specifically amplifies multiple target fragments simultaneously in the same reaction system, primer concentration and primer competition can affect the performance of multiplex RT-PCR. Therefore, it is necessary to optimize the primer concentration and primer ratio.

[0079] The difference from Example 2 is that the primers were mixed in a concentration ratio of 2:2:1:4, and the primer concentration was adjusted to 0.1 × 10⁻⁶. -6 μM, 0.2×10 -6 μM, 0.3×10 -6 μM, 0.4×10 -6 μM, 0.5×10 -6 μM, 0.6×10 -6 μM, 0.7×10 -6 μM, 0.8×10 -6 μM, 0.9×10 -6 μM, 1.0×10 -6 μM.

[0080] A mixed infection sample (concentration 10 ng / μL) containing plasmids with inserted TSWV, TRV, PMMoV, and PVX sequences in a concentration ratio of 1:1:1:1 was used as a template, and the method described in Example 2 was employed for detection. The results are as follows: Figure 5 As shown, the primer concentration range is from 0.1 × 10⁻⁶. -6 Up to 1.0×10 -6 μM, within this primer concentration range, can amplify 4 bands.

[0081] The concentration of the mixed primers was set at 0.5 × 10⁻⁶. -6 μM, the concentration ratio of each primer was set according to Table 2. A mixed infection sample (concentration 10 ng / μL) was prepared by mixing plasmids containing CP sequences of TSWV, TRV, PMMoV, and PVX in a concentration ratio of 1:1:1:1 as a template, and detection was performed using the method of Example 2. Results are as follows: Figure 6 As shown in Table 2, all the concentration ratios listed can amplify specific bands, but the bands are clearest and most distinct when the primer concentration ratio is TSWV:TRV:PMMoV:PVX=2:2:1:4.

[0082] Table 2 Primer Concentration Ratio Reference Table

[0083]

[0084] 2. Optimization of annealing temperature

[0085] To establish a stable multiplex amplification system, suitable amplification conditions are required. The DNA template is activated by Taq DNA polymerase, and the annealing temperature plays an important role in the annealing process, affecting the specificity of PCR.

[0086] A mixed infection sample (concentration 10 ng / μL) containing plasmids with inserted TSWV, TRV, PMMoV, and PVX CP sequences in a concentration ratio of 1:1:1:1 was used as a template. Amplification was performed using the method described in Example 2, with the difference being that the annealing temperatures in the amplification program were adjusted to 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, and 59℃ according to the annealing temperatures of each primer. The results are as follows: Figure 7 As shown, four bands can be amplified in the annealing temperature range of 50~59℃, with the most distinct and clear bands amplified at an annealing temperature of 55℃.

[0087] 3. Optimization of dNTP concentration and Taq DNA polymerase concentration

[0088] Using a mixed infection sample (concentration 10 ng / μL) prepared by mixing plasmids containing CP sequences of TSWV, TRV, PMMoV, and PVX in a concentration ratio of 1:1:1:1, amplification was performed according to the method in Example 2, except that the concentration of dNTPs was adjusted to 0.001 mmol·L⁻¹. -1 0.002 mmol·L -1 0.004 mmol·L -1 0.006 mmol·L -1 0.008 mmol·L -1 0.010 mmol·L -1 0.012 mmol·L -1 0.014 mmol·L -1 0.016 mmol·L -1 The result is as follows Figure 8 As shown, when the concentration of dNTPs is between 0.001 and 0.016 mmol·L⁻¹ -1 Stable and clear bands can be amplified at all times, but from the perspective of effectiveness and economy, when the dNTP concentration is 0.006 mmol·L⁻¹, it is less effective. -1 The optimal time for striping is when the strips are in place.

[0089] Using a mixed infection sample (concentration 10 ng / μL) prepared by mixing plasmids containing CP sequences of TSWV, TRV, PMMoV, and PVX in a concentration ratio of 1:1:1:1, amplification was performed according to the method in Example 2, the difference being that the concentration of Taq DNA polymerase was adjusted to 0.25 U·μL. -1 0.5 U·μL -1 1 U·μL -1 1.5 U·μL -1 2 U·μL -1 2.5 U·μL -1 3 U·μL -1 3.5 U·μL -1 4 U·μL -1 The result is as follows Figure 9 As shown, the concentration of Taq DNA polymerase is between 0.25 and 2 U / μL. -1 The range can amplify stable and clear bands, with a Taq DNA polymerase concentration of 1.5 U·μL. -1 The bands are clearest when the enzyme concentration is >2 U·μL. -1 At this time, the tailing phenomenon intensifies with increasing enzyme concentration, making it impossible to distinguish between bands.

[0090] Example 5

[0091] Sensitivity of the reaction system.

[0092] The concentration of virus in plants naturally infected by viruses is often much lower than the concentration of virus in plants specially inoculated in the laboratory. This requires the detection system to have sufficient sensitivity.

[0093] Using a mixed infection sample prepared by mixing plasmids containing CP sequences of TSWV, TRV, PMMoV, and PVX in a concentration ratio of 1:1:1:1 as a template, the viral concentration in the template was adjusted to 10 ng, 1 ng, and 1×10⁻⁶, respectively. -1 ng, 1×10 -2 ng, 1×10 -3 ng, 1×10 -4 ng, 1×10 -5 ng was detected using the method described in Example 2.

[0094] The results are as follows Figure 10 As shown, the levels were detected in the range of 10 ng to 10 ng. -3 Clear bands of four viruses can be amplified simultaneously within the ng interval.

[0095] In summary, this invention fills the technological gap in the simultaneous detection of multiple infections of TSWV, TRV, PMMoV, and PVX. This system complements existing methods for detecting viruses such as CMV and TMV. Aiming to establish an efficient and accurate multiplex RT-PCR detection system, this invention constructs a one-step multiplex RT-PCR system for the simultaneous detection of four viruses: TSWV, TRV, PMMoV, and PVX. Through systematic optimization of reaction components and amplification parameters, this invention achieves a synergistic improvement in amplification efficiency, detection sensitivity, and cost-effectiveness.

[0096] Using the method of this invention, the template amount can be as low as 1×10 -3 Even at ng, all target bands can be stably amplified without non-specific amplification products. This invention ultimately optimizes a sensitive, rapid, and economical detection system, significantly improving detection efficiency. Using the method of this invention to detect field pepper samples provides key technical support for the early diagnosis and integrated prevention and control of pepper viral diseases.

[0097] Example 6

[0098] Results of field chili pepper sample testing.

[0099] Following the method in Example 2, leaf samples from 15 abnormally growing chili pepper plants and 1 normally growing chili pepper plant in Fuyang, Anhui Province were tested. The results are shown in the attached figure. Figure 11 As shown, the test revealed that 12 out of 15 samples were infected with the virus, and all of them were co-infected, with a co-infection rate as high as 80%. Among them, no bands were detected in samples 1, 3, and 4; PMMoV and PVX were detected in samples 2 and 5; TSWV, TRV, and PVX were detected in samples 6, 7, 8, 9, 10, 13, and 14; and TSWV and TRV were detected in samples 11, 12, and 15. The results of field testing prove that the present invention can stably, reliably, clearly, and effectively detect the type of virus infection in samples.

[0100] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer set for the simultaneous detection of four pepper viruses, characterized in that, The primer set includes primer set 1 for detecting TSWV, primer set 2 for detecting TRV, primer set 3 for detecting PMMoV, and primer set 4 for detecting PVX. The nucleotide sequences of primer set 1 are shown in SEQ ID NO.1~SEQ ID NO.2; the nucleotide sequences of primer set 2 are shown in SEQ ID NO.3~SEQ ID NO.4; the nucleotide sequences of primer set 3 are shown in SEQ ID NO.5~SEQ ID NO.6; and the nucleotide sequences of primer set 4 are shown in SEQ ID NO.7~SEQ ID NO.

8.

2. A reagent for simultaneously detecting four pepper viruses, characterized in that, The reagent comprises the primer set as described in claim 1.

3. A kit for simultaneously detecting four pepper viruses, characterized in that, The kit includes the primer set of claim 1 or the reagent of claim 2.

4. The application of the primer set of claim 1, the reagent of claim 2, or the kit of claim 3 in the simultaneous detection of four pepper viruses.

5. A method for simultaneously detecting four pepper viruses, characterized in that, Includes the following steps: PCR amplification was performed using the primer set described in claim 1, the reagent described in claim 2, or the kit described in claim 3. After amplification, the amplification products were subjected to agarose gel electrophoresis to determine whether the sample to be tested was infected with TSWV, TRV, PMMoV, or PVX virus. Methods for determining whether a sample is infected with TSWV, TRV, PMMoV, or PVX viruses include: the presence of a 201 bp specific identification band in the electrophoresis pattern indicates TSWV infection; a 311 bp specific identification band indicates TRV infection; a 414 bp specific identification band indicates PMMoV infection; and a 576 bp specific identification band indicates PVX infection.

6. The method according to claim 5, characterized in that, The PCR amplification reaction system consisted of: 7.4 μL ddH2O, 0.4 μL upstream primer mixture, 0.4 μL downstream primer mixture, 1 μL 10X PCR Buffer, 0.2 μL dNTPs, 0.2 μL Taq DNA polymerase, and 0.4 μL template.

7. The method according to claim 6, characterized in that, In both the upstream and downstream primer mixtures, the primers used to detect TSWV, TRV, PMMoV, and PVX were mixed at a concentration ratio of (1~2):(1~4):(1~4):(1~4).

8. The method according to claim 6, characterized in that, The concentration of dNTPs ranged from 0.001 to 0.016 mmol·L⁻¹ -1 The concentration of Taq DNA polymerase is 0.25~2 U·μL. -1 .

9. The method according to claim 5, characterized in that, The PCR amplification reaction program was as follows: 95.0℃ for 3 min; 95.0℃ for 30 s, 50.0℃ for 30 s, 72.0℃ for 1 min, 35 cycles; 72.0℃ for 10 min, 16.0℃ for 5 s.

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