Primer group, reagent, kit and method for simultaneously detecting four pepper viruses and application of primer group, reagent, kit and method

Abstract

The invention provides a primer group, a reagent, a kit and a method for simultaneously detecting four pepper viruses and application of the primer group, the reagent, the kit and the method, and belongs to the technical field of gene detection. The primer group comprises a primer group 1 for detecting tomato spotted wilf virus (TSWV), a primer group 2 for detecting tobacco embrittlement virus (TRV), a primer group 3 for detecting pepper light mottle virus (PMMoV) and a primer group 4 for detecting potato virus X (PVX). The primer group provided by the invention is high in specificity, TSWV, TRV, PMMoV and PVX are simultaneously detected by utilizing the primer group provided by the invention, whether a to-be-detected sample is infected with TSWV, TRV, PMMoV or PVX viruses or not can be determined through one-time amplification detection, and the detection accuracy is improved on the basis of saving the detection time and reducing the detection cost.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of gene detection, and particularly relates to a primer set, reagent, kit, method for simultaneously detecting four pepper viruses and application thereof. BACKGROUND

[0002] Pepper (Capsicum annuum L.) originates from the tropical and subtropical regions of South America and is one of the economic crops with the widest planting range, which has important economic value. With the continuous expansion of the pepper planting area, the influence of pepper diseases, especially pepper virus diseases, on the yield and quality of peppers is also increasing.

[0003] At present, more than seventy viruses have been reported worldwide to infect peppers, mainly 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 variety of pepper viruses, two or more viruses commonly cause complex infection, which seriously weakens the resistance of the host, causes complex and variable symptoms such as mottling, yellowing, wrinkling and deformation after infection, and causes serious decline in the yield and quality of peppers.

[0004] PMMoV and TSWV viruses have been detected in many pepper planting areas before, and there are complex infections with TRV, PVX in other regions, but there is no simultaneous detection system for the complex infection of TSWV, TRV, PMMoV and PVX. In order to quickly and accurately determine the type of pepper virus disease, a method system capable of simultaneously and rapidly detecting multiple pepper viruses is provided, which can provide a feasible technical means for detecting the complex infection of pepper viruses and provide strong technical support for the early prevention and treatment of pepper virus diseases. SUMMARY

[0005] Therefore, the present application aims to provide a primer set for simultaneously detecting four pepper viruses, which can determine whether the sample is infected with TSWV, TRV, PMMoV or PVX virus through one amplification.

[0006] To achieve the above object, the present application provides the following technical solutions.

[0007] The primer set for simultaneously detecting four pepper viruses comprises 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 sequence of the primer set 1 is shown in SEQ ID NO. 1-2; the nucleotide sequence of the primer set 2 is shown in SEQ ID NO. 3-4; the nucleotide sequence of the primer set 3 is shown in SEQ ID NO. 5-6; and the nucleotide sequence of the primer set 4 is shown in SEQ ID NO. 7-8.

[0009] Another object of the present application is to provide a reagent for simultaneously detecting four pepper viruses, which comprises the primer set.

[0010] Another object of the present application is to provide a kit for simultaneously detecting four pepper viruses, which comprises the primer set or the reagent.

[0011] Another object of the present application is to provide the primer set or the reagent or the kit for use in simultaneously detecting four pepper viruses.

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

[0013] The primer set or the reagent or the kit is used for PCR amplification, and after the amplification, the amplification product is subjected to agarose gel electrophoresis to determine whether the sample is infected with TSWV, TRV, PMMoV or PVX virus.

[0014] Preferably, the reaction system of the PCR amplification 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, template 0.4 μL.

[0015] Preferably, the concentration of the upstream primer mixture and the downstream primer mixture is 0.1x10-6 ~1.0 x 10 -6 μM; the primers for detecting TSWV, TRV, PMMoV and PVX in the upstream primer mixture and the downstream primer mixture are 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 , and the concentration of Taq DNA polymerase is 0.25-2 U·μL -1 .

[0017] Preferably, the reaction procedure of the PCR amplification 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 judging whether the sample to be tested is infected with TSWV, TRV, PMMoV or PVX virus comprises: the appearance of a 201 bp specific identification band in the electrophoretogram indicates that the sample to be tested contains TSWV virus; the appearance of a 311 bp specific identification band in the electrophoretogram indicates that the sample to be tested contains TRV virus; the appearance of a 417 bp specific identification band in the electrophoretogram indicates that the sample to be tested contains PMMoV virus; and the appearance of a 576 bp specific identification band in the electrophoretogram indicates that the sample to be tested contains PVX virus.

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

[0020] The present application provides a primer set for simultaneously detecting four kinds of pepper viruses, which can determine whether the sample to be tested is infected with TSWV, TRV, PMMoV or PVX virus through one-time amplification detection, and has high specificity, thereby making up for the vacancy of simultaneous detection of TSWV, TRV, PMMoV and PVX in the current pepper virus disease.

[0021] The primer set of the present application is used for simultaneous detection of TSWV, TRV, PMMoV and PVX, has a small risk of contamination, saves detection time and reduces detection cost, improves detection accuracy, can more quickly identify the types of viruses, provides the possibility of timely prevention and control measures, and helps to reduce the impact of viral diseases on the yield and quality of peppers and promote the healthy development of the pepper industry.

[0022] The present application provides a method for simultaneously detecting multiple pepper viruses, which provides a feasible technical means for detecting the complex infection of pepper viruses and provides strong technical support for the early prevention and control of pepper virus diseases. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1The result of plasmid gene sequence alignment containing reverse transcription TSWV coat protein CDS;

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

[0025] Figure 3 RT-PCR detection results of four pepper viruses; M is DL2000 Marker; 1 is TSWV (201bp); 2 is TRV (311bp); 3 is PMMoV (417bp); 4 is PVX (576bp); CK- is Negative;

[0026] Figure 4 Multiple RT-PCR detection results of four pepper viruses; M is DL2000 Marker; 1 is multiple RT-PCR of TSWV+TRV+PMMoV+PVX; 2 is single PCR of TSWV (201bp); 3 is single PCR of TRV (311bp); 4 is single PCR of PMMoV (417bp); 5 is single PCR of PVX (576bp); CK- is Negative;

[0027] Figure 5 Optimization of primer concentration; M is DL2000 Marker; lanes 1-10 correspond to primer concentrations of 0.1×10 -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 Optimization of primer concentration ratio; M is DL2000 DNA Marker; lanes 1-9 correspond to TSWV:TRV:PMMoV:PVX of 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, 2:1:4:1, respectively; CK- is Negative;

[0029] Figure 7Optimization of annealing temperature; wherein, 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 Optimization of dNTPs concentration; wherein, M is DL2000 Maker; lanes 1-9 correspond to dNTPs concentration of 0.001 mmol·L -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- is Negative;

[0031] Figure 9 Optimization of Taq DNA polymerase concentration; wherein, M is DL2000 Maker; lanes 1-9 correspond to enzyme concentration of 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 ; CK- is Negative;

[0032] Figure 10 Sensitivity test of multiplex RT-PCR; wherein, M is DL2000 Maker; lanes 1-7 correspond to virus concentration of 10 ng, 1 ng, 1×10 -1 ng, 1×10 -2 ng, 1×10 -3 ng, 1×10 -4 ng, 1×10 -5 ng; CK- is Negative;

[0033] Figure 11The detection results of field pepper samples; wherein, M is a DL 2000 Maker; lanes 1-15 are the detection results of 15 abnormal pepper leaf samples; CK- is the detection result of 1 normal pepper leaf sample. DETAILED DESCRIPTION

[0034] The application provides a primer group for simultaneously detecting four pepper viruses, the primer group comprising 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 nucleotide sequence of the primer group 1 is shown as SEQ ID NO. 1-SEQ ID NO. 2; the nucleotide sequence of the primer group 2 is shown as SEQ ID NO. 3-SEQ ID NO. 4; the nucleotide sequence of the primer group 3 is shown as SEQ ID NO. 5-SEQ ID NO. 6; and the nucleotide sequence of the primer group 4 is shown as SEQ ID NO. 7-SEQ ID NO. 8.

[0035] The application designs the primer for detecting TSWV, TRV, PMMoV and PVX virus according to the gene number HQ260978.1 of TSWV, the gene number NC_003811.1 of TRV, the gene number LC817996.1 of PMMoV and the gene number M95516.1 of PVX.

[0036] The application further provides a reagent for simultaneously detecting four pepper viruses, wherein the reagent comprises the primer group.

[0037] The application further provides a kit for simultaneously detecting four pepper viruses, wherein the kit comprises the primer group or the reagent.

[0038] The application further provides application of the primer group or the reagent or the kit in simultaneously detecting four pepper viruses.

[0039] The application further provides a method for simultaneously detecting four pepper viruses, comprising the following steps: performing PCR amplification by using the primer group or the reagent or the kit; and after the amplification is completed, performing agarose gel electrophoresis on the amplification product to determine whether the sample to be detected is infected with TSWV, TRV, PMMoV or PVX virus.

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

[0041] In the method for simultaneously detecting the four pepper viruses, the reaction procedure of the PCR amplification is as follows: 95.0 ℃ for 3 min; 95.0 ℃ for 30 s, 50.0 ℃ for 30 s, and 72.0 ℃ for 1 min for 35 cycles; 72.0 ℃ for 10 min, and 16.0 ℃ for 5 s.

[0042] In the method for simultaneously detecting the four pepper viruses, the method for judging whether the sample to be detected is infected with TSWV, TRV, PMMoV or PVX virus comprises: the appearance of a 201 bp specific identification band in an electrophoretogram indicates that the sample contains TSWV virus; the appearance of a 311 bp specific identification band in an electrophoretogram indicates that the sample contains TRV virus; the appearance of a 417 bp specific identification band in an electrophoretogram indicates that the sample contains PMMoV virus; and the appearance of a 576 bp specific identification band in an electrophoretogram indicates that the sample contains PVX virus.

[0043] The present application realizes the synergistic promotion of amplification efficiency, detection sensitivity and economy by optimizing the reaction components and amplification parameters, and can determine whether the sample to be detected is infected with TSWV, TRV, PMMoV or PVX virus through one amplification, thereby saving the detection cost and improving the detection efficiency.

[0044] The detection method provided by the present application can be used to detect a template amount as low as 1×10 -3All target bands can be stably amplified and no non-specific amplification products are generated at 30 cycles, and finally a sensitive, rapid and economical detection system is optimized, which significantly improves the detection efficiency. The method provided by the application for detecting field pepper samples provides key technical support for early diagnosis and comprehensive prevention and control of pepper virus diseases. The popularization and application of the application are expected to further promote the perfection of the pepper virus disease prevention and control system and help the sustainable development of the pepper industry.

[0045] The technical solutions provided by the application will be described in detail below in combination with embodiments, but they should not be understood as limiting the scope of protection of the application.

[0046] In specific embodiments of the application, the TSWV, TRV, PMMoV and PVX virus infectious clones are provided by the Plant Virus Immunity Laboratory of the Plant Protection College of Anhui Agricultural University, and the diseased plant leaves collected from the Fuyang area of Anhui Province.

[0047] The reverse transcription kit is Hifair AdvanceFast 1st Strand cDNA Synthesis Kit provided by Yixing Company; the FreeZol Reagent total RNA extraction kit, Phanta Max Super-Fidelity DNA polymerase and ClonExpress II One Step Cloning Kit are purchased from Nanjing Nuowezan Biotechnology Co., Ltd.; TaqPlus DNA polymerase is purchased from Shengong Biotechnology (Shanghai) Co., Ltd.; primer synthesis and DNA sequencing services are completed by Beijing Qikexin Biotechnology Co., Ltd.; the plasmid extraction kit is purchased from Tiangeng Biochemical Technology (Beijing) Co., Ltd.

[0048] Example 1

[0049] A primer set for simultaneously detecting four kinds of pepper viruses.

[0050] According to the TSWV gene number HQ260978.1, the TRV gene number NC_003811.1, the PMMoV gene number LC817996.1 and the PVX gene number M95516.1, primers for detecting TSWV, TRV, PMMoV and PVX viruses are designed, and the primer sequences are shown in Table 1.

[0051] Table 1 Specific sequences of primers for amplifying four virus target gene fragments by multiplex RT-PCR

[0052]

[0053] Example 2

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

[0055] According to the instructions of the FreeZol Reagent total RNA extraction kit, total RNA was extracted from virus-infected pepper leaves, and the total RNA concentration was determined using an ultramicro spectrophotometer (Yisheng). cDNA was synthesized according to the instructions of the HifairAdvanceFast 1st Strand cDNA Synthesis Kit provided by Yisheng Company, and the synthesized cDNA was immediately subjected to subsequent PCR amplification.

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

[0057] The reaction system was 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 (0.006 mmol·L -1 ), Taq DNA polymerase 0.2 μL (1.5 U·μl -1 ), template 0.4 μL;

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

[0059] After amplification, the amplified product is subjected to agarose gel electrophoresis to determine whether the sample is infected with TSWV, TRV, PMMoV or PVX virus: if a 201bp specific identification band appears in the electrophoresis pattern, it contains TSWV virus; if a 311bp specific identification band appears in the electrophoresis pattern, it contains TRV virus; if a 417bp specific identification band appears in the electrophoresis pattern, it contains PMMoV virus; if a 576bp specific identification band appears in the electrophoresis pattern, it contains PVX virus.

[0060] Example 3

[0061] Results of singleplex PCR and multiplex RT-PCR.

[0062] 1. Plasmid Construction

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

[0064] According to the ClonExpress II One Step Cloning Kit manual of Nanjing Nvwa Biological Technology Co., Ltd., the TSWV coat protein sequence CDS plasmid was recombined, the recombined plasmid was transformed into E. coli DH5a competent cells, and then coated on LB solid medium with corresponding antibodies. After waiting for the visible single colonies to grow, the TaqPlus DNA polymerase manual of Shengong Bioengineering (Shanghai) Co., Ltd. was used for positive clone screening, and the single colony containing the target plasmid was screened and expanded. According to the plasmid extraction kit manual of Tiangen Biochemical Technology (Beijing) Co., Ltd., the plasmid was extracted and sent to Beijing Qikang Biological Technology Co., Ltd. for gene sequencing. The gene sequence alignment was performed by SnapGene 6.0.2 software, and the results are shown in Figure 1

[0065] The same method was used to construct plasmids containing TRV, PMMoV and PVX, respectively.

[0066] 2. Rubbing inoculation

[0067] 0.1 g of N. benthamiana leaves infected with TSWV, TRV, PMMoV and PVX viruses, respectively, was added to 200 μL of 1×PBS solution, ground into homogenate, and evenly spread on the pepper leaves with sandpaper. Then the homogenate was sucked and smeared on the leaves with sandpaper, and rubbed 2-3 times. Gloves were changed for each virus type inoculated.

[0068] As shown in Figure 2 , the pepper plants infected with the four viruses all showed typical virus disease symptoms, including different degrees of leaf curling and mosaic symptoms. Notably, the plants infected with TRV and PMMoV also showed a significant dwarfing phenotype. The above results showed that the four viruses could have a significant impact on the growth and development of pepper plants, and the pathogenic effects of TRV and PMMoV were particularly prominent.

[0069] 3. Single-plex PCR amplification

[0070] The total RNA of each pepper leaf was extracted and cDNA was synthesized, and then subjected to PCR amplification according to the method of Example 2. The results are shown in Figure 3

[0071] The target virus cDNA synthesized from the total RNA extracted from the previously infected pepper plant leaves was used as a template for PCR amplification with specific primers. As shown in Figure 3 ​​As shown in Figure 2, lanes 1-4 showed single and bright amplification bands at the positions of 201, 311, 417 and 576 bp of the DNA molecular weight marker, respectively, and the sizes of each band were completely consistent with the expected design, and the band edges were clear without tailing phenomenon. Notably, the negative control sample did not have non-specific amplification products in the corresponding region, indicating that the PCR system had good specificity.

[0072] 4. Detection and analysis of mixed infection samples and single pathogenicity samples, respectively.

[0073] The mixed infection sample was mixed by plasmids inserted with TSWV, TRV, PMMoV and PVX coat protein (CP) sequences at a concentration ratio of 1:1:1:1. The amplification was carried out by the method in Example 2, and the results are shown in Figure 3. Figure 4

[0074] As shown in Figure 3, the mixed sample detection lane (lane 1) showed clear single amplification bands at the four target sites, and the migration rate was completely matched with the theoretical amplification length of each virus (201 bp for TSWV, 311 bp for TRV, 417 bp for PMMoV, and 576 bp for PVX). In the single pathogenicity sample detection, lanes 2-5 showed specific bands at the corresponding target sites, and no non-target amplification products were present. The negative control group had no amplification signal in the entire electropherogram, fully proving that the multiplex detection system of the application has strong specificity, no cross-reaction between primer pairs, high sensitivity, and can effectively distinguish mixed infection from single infection. Figure 4

[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 simultaneously and specifically amplifies multiple target fragments in the same reaction system, primer concentration and primer competition can affect the effect of multiplex RT-PCR. Therefore, the primer concentration and primer concentration ratio were optimized.

[0079] The difference from Example 2 is that the primers were mixed at a concentration ratio of 2:2:1:4, and the primer concentration was adjusted to 0.1×10 -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] The mixed infection sample (concentration 10 ng / μL) containing plasmids inserted with the CP sequences of TSWV, TRV, PMMoV, and PVX at a concentration ratio of 1:1:1:1 was used as a template and the method of Example 2 was used for detection. Figure 5 As shown, the interval of primer concentration was selected from 0.1×10 -6 to 1.0×10 -6 μM, 4 bands can be amplified within this primer concentration range.

[0081] The concentration of the mixed primers was set to 0.5×10 -6 μM, the concentration ratio of each primer was set according to Table 2, and the mixed infection sample (concentration of 10 ng / μL) was used as the template (concentration of 10 ng / μL) to mix the plasmids inserted with the CP sequence of TSWV, TRV, PMMoV and PVX at a concentration ratio of 1:1:1:1. The results were shown in Example 2. Figure 6 As shown in Table 2, all the concentration ratios listed in Table 2 can amplify specific bands, but the bands are most clear when the primer concentration ratio is TSWV:TRV:PMMoV:PVX=2:2:1:4.

[0082] Table 2 Primer concentration ratio reference table

[0083] Trial No. TSWV:TRV:PMMoV:PVX 1 1:1:1:1 2 1:2:2:2 3 1:4:4:4 4 1:1:2:4 5 2:2:4:1 6 2:4:1:2 7 2:2:1:4 8 2:4:2:1 9 2:1:4:1

[0084] 2 Optimization of annealing temperature

[0085] In order to establish a stable multiplex amplification system, appropriate amplification conditions are required. The DNA template is under the action of TaqDNA polymerase, and the annealing temperature plays an important role in the annealing process, affecting the specificity of PCR.

[0086] A mixed infection sample containing plasmids inserted with the CP sequences of TSWV, TRV, PMMoV, and PVX at a concentration ratio of 1:1:1:1 was used as a template (concentration of 10 ng / μL). Amplification was performed using the method of Example 2, except that the annealing temperatures in the amplification program were adjusted to 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, and 59°C, respectively, according to the annealing temperatures of the primers. The results are shown in FIG. Figure 7 As shown in the figure, the four bands can be amplified when the annealing temperature is between 50 and 59 °C. Among them, the amplified bands are the clearest and most distinct when the annealing temperature is 55 °C.

[0087] 3. Optimization of dNTPs and Taq DNA polymerase concentrations

[0088] A mixed infection sample containing plasmids inserted with the CP sequences of TSWV, TRV, PMMoV, and PVX at a concentration ratio of 1:1:1:1 was used as a template (concentration of 10 ng / μL). Amplification was performed using the method of Example 2, except that the dNTPs concentrations were adjusted to 0.001 mmol·L -1 , 0.002mmol·L -1 , 0.004mmol·L -1 , 0.006mmol·L -1 , 0.008mmol·L -1 , 0.010mmol·L -1 , 0.012mmol·L -1 , 0.014mmol·L -1 , 0.016mmol·L -1 The results are as follows Figure 8 As shown, when the dNTPs concentration is between 0.001 and 0.016 mmol·L -1 However, from the perspective of effect and economy, when the dNTPs concentration is 0.006mmol·L -1 The strip is best.

[0089] A mixed infection sample containing plasmids inserted with the CP sequences of TSWV, TRV, PMMoV, and PVX at a concentration ratio of 1:1:1:1 was used as a template (concentration of 10 ng / μL). Amplification was performed using the method of Example 2, except that the concentration of Taq DNA polymerase was adjusted to 0.25 U·μL. -1 , 0.5U·μL -1 , 1U·μL -1 , 1.5U·μL -1 , 2U·μL -1 , 2.5U·μL -1 , 3U·μL -1 , 3.5U·μL -1 , 4U·μL -1 The results are as follows Figure 9 As shown, the concentration of Taq DNA polymerase is 0.25~2U·μL -1 The range can amplify stable and clear bands, where the concentration of Taq DNA polymerase is 1.5U·μL -1 The band is clearest when the enzyme concentration is > 2U·μL -1 When the enzyme concentration increases, the tailing phenomenon increases, resulting in the inability to distinguish between bands.

[0090] Example 5

[0091] Sensitivity of reaction system

[0092] The virus concentration in the plant body naturally infected by the virus is much lower than that in the plant body specially inoculated in the laboratory, so the detection system is required to have sufficient sensitivity.

[0093] The mixed infection sample mixed in the ratio of 1:1:1:1 in concentration with the plasmid inserted with the CP sequence of TSWV, TRV, PMMoV and PVX is taken as the template, the virus concentration in the template is adjusted to 10 ng, 1 ng, 1x10 -1 - 2 -3 -4 -5 ng, and the method in Example 2 is adopted for detection.

[0094] The results are shown in Table 2. Figure 10 As shown in Table 2, clear bands of the four viruses can be amplified simultaneously in the range of 10 ng to 10 -3 ng.

[0095] According to the above results, the present application fills the technical blank of synchronous detection of TSWV, TRV, PMMoV and PVX complex infection. The system is complementary to the existing CMV, TMV and other virus detection methods. The present application aims to establish an efficient and accurate multiplex RT-PCR detection system, and a one-step multiplex RT-PCR system for simultaneously detecting TSWV, TRV, PMMoV and PVX and other four viruses is constructed. The present application realizes the synergistic improvement of amplification efficiency, detection sensitivity and economy by optimizing the reaction components and amplification parameters.

[0096] By adopting the method of the present application, all target bands can be stably amplified when the template amount is as low as 1x10 -3 ng, and there is no non-specific amplification product. The present application optimizes a sensitive, rapid and economical detection system, and significantly improves the detection efficiency. The field pepper samples are detected by the method of the present application, which provides key technical support for early diagnosis and comprehensive prevention and control of pepper virus diseases.

[0097] Example 6

[0098] Detection results of field pepper samples

[0099] According to the method of Example 2, 15 abnormal growth pepper leaf samples and 1 normal growth pepper leaf sample in Fuyang area of Anhui Province are detected, and the results are shown in Table 3. Figure 11 ​​​​As shown, the detection finds that 12 of 15 samples have virus infection, and all are composite infection, and the composite infection rate is as high as 80%, wherein samples 1, 3 and 4 are not detected to have bands, samples 2 and 5 are detected to have PMMoV and PVX simultaneously, samples 6, 7, 8, 9, 10, 13, 14 are detected to have TSWV, TRV and PVX simultaneously, samples 11, 12 and 15 are detected to have TSWV and TRV simultaneously, and the result of field detection proves that the present application can stably, reliably, clearly and effectively detect the infection type of viruses in samples.

[0100] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A primer set for 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 sequence of the primer set 1 is shown as SEQ ID NO.1 to SEQ ID NO.2; the nucleotide sequence of the primer set 2 is shown as SEQ ID NO.3 to SEQ ID NO.4; the nucleotide sequence of the primer set 3 is shown as SEQ ID NO.5 to SEQ ID NO.6; and the nucleotide sequence of the primer set 4 is shown as SEQ ID NO.7 to SEQ ID NO.

8.

2. A reagent for simultaneously detecting four types of pepper viruses, characterized in that: The reagents include the primer set according to claim 1.

3. A kit for simultaneously detecting four pepper viruses, characterized in that: The kit comprises the primer set according to claim 1 or the reagent according to claim 2.

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

5. A method for simultaneously detecting four pepper viruses, characterized in that: The steps include: PCR amplification is performed using the primer set of claim 1, the reagent of claim 2, or the kit of claim 3; after the amplification, the amplified product is subjected to agarose gel electrophoresis to determine whether the sample to be tested is infected with TSWV, TRV, PMMoV or PVX virus.

6. The method according to claim 5, characterized in that 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.

7. The method according to claim 6, characterized in that The concentrations of the upstream primer mix and the downstream primer mix were both 0.1×10 -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 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 is 0.001 to 0.016 mmol·L -1 The concentration of Taq DNA polymerase is 0.25~2U·μL -1 .

9. The method according to claim 5, characterized in that The reaction procedure of the PCR amplification was as follows: 95.0°C for 3 min; 35 cycles of (95.0°C for 30 s, 50.0°C for 30 s, 72.0°C for 1 min); 72.0°C for 10 min, 16.0°C for 5 s.

10. The method according to claim 5, characterized in that The method for determining whether the sample to be tested is infected with TSWV, TRV, PMMoV or PVX virus includes: if a 201bp specific identification band appears on the electrophoresis spectrum, it is infected with TSWV virus; if a 311bp specific identification band appears on the electrophoresis spectrum, it is infected with TRV virus; If a 417bp specific identification band appears in the electrophoresis spectrum, the virus is infected with PMMoV virus; if a 576bp specific identification band appears in the electrophoresis spectrum, the virus is infected with PVX virus.

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