Dpo rt-pcr primer set, detection method, kit for detecting five citrus viruses and application thereof

Abstract

The application discloses a DPO RT-PCR primer group for synchronously detecting five citrus viruses, a detection method, a kit and application of the DPO RT-PCR primer group, and sequences of the primer group are shown as SEQ ID NO. 1-SEQ ID NO. 10. The application designs specific DPO RT-PCR primers for citrus tristeza virus, citrus tatter leaf virus, citrus yellow vein clearing virus and citrus leaf mottle virus, and the detection primers can effectively remove primer dimer interference, and the five viruses can be detected from samples at the same time. The detection method is simple and efficient, and only needs to use a common PCR instrument and an electrophoresis instrument to detect, and has the characteristics of rapidness, economy, high accuracy and high sensitivity, and can be used for large-scale screening of field citrus plants and epidemic prevention detection before seedling transportation, and provides technical support for healthy seedling detection and field screening of diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant virus detection, and particularly relates to a DPORT-PCR primer group for synchronously detecting five citrus viruses, a detection method, a kit and application thereof. BACKGROUND

[0002] With the continuous expansion of citrus planting area and the introduction of new varieties, regional seedling transportation is frequent. Due to difficult supervision, a variety of diseases and pests are carried and spread through seedlings, and cause serious harm, among which viral diseases are particularly prominent.

[0003] Citrus tristeza virus (CTV) causes symptoms such as decline of citrus tree, stem sink point of branches, and small fruit; Citrus tatter leaf virus (CTLV) causes yellowing and weakness of plants, and even whole plant death; Citrus exocortis viroid (CEVd) causes dwarfing of diseased trees, few and weak new shoots, small and mostly zinc-deficient leaf, and serious flower and fruit drop; Citrus yellow vein clearing virus (CYVCV) can infect most citrus species such as lemon, lime and sweet orange, and cause different degrees of vein clearing, chlorosis and mottle symptoms, and cause serious leaf rolling on sugar orange; Citrus leaf blotch virus (CLBV) can cause abnormal grafting of trifoliate orange or grapefruit as rootstock, and the incompatibility of citrus scion and rootstock directly harms the growth of citrus trees, and further causes significant economic losses of citrus industry. The above-mentioned five citrus viruses are common RNA viruses that infect citrus, and can be transmitted by aphid vectors and scions, and the large-scale transportation of virus-carrying seedlings increases the risk of long-distance transmission of viral diseases. Therefore, it is beneficial to prevent the transmission of citrus viruses with seedlings and control the diseases from the source to establish an efficient, specific and high-sensitivity viral detection method.

[0004] Dual-primering oligonucleotide (DPO) is a new type of PCR primer design method, which has high specificity and simple primer design. It has been widely used in the detection of various pathogenic bacteria and viruses.

[0005] The information disclosed in this section of background art is only intended to increase the understanding of the overall background of the application, and should not be considered as acknowledging or implying in any form that the information constitutes prior art known to those skilled in the art. SUMMARY

[0006] The present application aims at the technical problems existing in the prior art, and provides a DPO RT-PCR primer group for synchronously detecting five citrus viruses, a detection method, a kit and application thereof.

[0007] To achieve the above-mentioned object, the technical scheme provided by the present application is as follows:

[0008] A DPO RT-PCR primer group for synchronously detecting five citrus viruses comprises two citrus tristeza virus (CTV) specific primers, two citrus tatter leaf virus (CTLV) specific primers, two citrus exocortis viroid (CEVd) specific primers, two citrus yellow vein clearing virus (CYVCV) specific primers and two citrus leaf blotch virus (CLBV) specific primers, and the specificities are as follows:

[0009] DPO-CTV-F: 5'-TGGCTCGTAGACACCIIIIICGTTCTCCGG-3', as shown in SEQ ID NO. 1;

[0010] DPO-CTV-R: 5'-CTAAGGAGAACTTCTTIIIIICACGCATACG-3', as shown in SEQ ID NO. 2;

[0011] DPO-CTLV-F: 5'-TGCTTCAACAAGCGAGGCIIIIICGGGTAGGAG-3', as shown in SEQ ID NO. 3;

[0012] DPO-CTLV-R: 5'-GTATAAAGGCAGGCATGTCAIIIIICAAGACCGCG-3', as shown in SEQ ID NO. 4;

[0013] DPO-CEV-F: 5'-CGGGATCTTTCTTGAGIIIIIIGTGGTGCT-3', as shown in SEQ ID NO. 5;

[0014] DPO-CEV-R: 5'-GCTCCTGTTTCTCCGCTGGIIIIIAGTGATCC-3', as shown in SEQ ID NO. 6;

[0015] DPO-CYVCV-F: 5'-TCCATTGTCGACGAGTIIIIICTAAGCCAG-3', as shown in SEQ ID NO. 7;

[0016] DPO-CYVCV-R: 5'-GGATAGCTGCGGTAGAGAGGGTIIIIGTAGTCGAAG-3', as shown in SEQ ID NO. 8;

[0017] DPO-CLBV-F: 5'-GGATTATGTGTCTCATGTIIIIIIAGAGACGG-3', as shown in SEQ ID NO. 9;

[0018] DPO-CLBV-R: 5'-TGCAGCTTTGAGTGACIIIIICAATTCTTC-3', as shown in SEQ ID NO. 10;

[0019] The "I" in the primer sequence is inosine.

[0020] A reagent or kit comprising the primer set described above.

[0021] The method for synchronously detecting citrus tristeza virus, citrus tatter leaf virus, citrus yellowing phyllody virus, citrus leaf mottle virus and citrus yellow vein clear virus by using the primer set described above comprises the following steps:

[0022] (1) Taking the sample to be detected, extracting total RNA therefrom, and reverse transcribing the total RNA into cDNA;

[0023] (2) Using the primer set to perform PCR reaction with the cDNA of the sample to be detected as a template to obtain an amplification product;

[0024] (3) Using agarose electrophoresis to detect the PCR amplification product: if a band of 416 bp is amplified, it indicates that the sample contains citrus tristeza virus; if a band of 716 bp is amplified, it indicates that the sample contains citrus tatter leaf virus; if a band of 187 bp is amplified, it indicates that the sample contains citrus yellowing phyllody virus; if a band of 938 bp is amplified, it indicates that the sample contains citrus leaf mottle virus; and if a band of 576 bp is amplified, it indicates that the sample contains citrus yellow vein clear virus;

[0025] Preferably, the reaction system of the PCR reaction is 20 μL, and the concentration of each primer is as follows: the concentration of primer DPO-CTV-F and primer DPO-CTV-R is 0.2 μM, the concentration of primer DPO-CTLV-F and primer DPO-CTLV-R is 0.1 μM, the concentration of primer DPO-CEV-F and primer DPO-CEV-R is 0.5 μM, the concentration of primer DPO-CYVCV-F and primer DPO-CYVCV-R is 0.3 μM, and the concentration of primer DPO-CLBV-F and primer DPO-CLBV-R is 0.3 μM.

[0026] Preferably, the reaction procedure of the PCR reaction is: 50℃ for 30min, 94℃ for 5min; the 30-cycle parameters are: 94℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 2min; and finally 72℃ extension for 10min.

[0027] The application also provides application of the primer set or the kit containing the primer set in synchronous detection of citrus tristeza virus, citrus tatter leaf virus, citrus exocortis virus, citrus yellowing vein phloem virus and citrus leaf mottle virus.

[0028] Compared with the prior art, the application has the following beneficial effects:

[0029] The application can detect the five viruses from samples simultaneously by designing specific DPO RT-PCR primers for citrus tristeza virus, citrus tatter leaf virus, citrus exocortis virus, citrus yellowing vein phloem virus and citrus leaf mottle virus, and the detection method is simple and efficient, and only needs to use a common PCR instrument and an electrophoresis instrument to detect, and has the characteristics of rapidness, economy, high accuracy and high sensitivity, and can be used for large-scale screening of field citrus plants and epidemic prevention detection before seedling transportation, and provides technical support for healthy seedling detection and field screening of diseases. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Screening of the most suitable annealing temperature for single detection of citrus viruses;

[0031] Figure 2 Sensitivity detection of the five kinds of citrus viruses;

[0032] Figure 3 Screening of primer concentration combinations for multiple detection of citrus and detection electrophoretogram of different virus complex infections. DETAILED DESCRIPTION

[0033] The specific embodiments are described in detail below with reference to the accompanying drawings, but it should be understood that the protection scope of the application is not limited by the specific embodiments.

[0034] Example 1 primer design

[0035] According to the whole genome sequences of citrus tristeza virus, citrus tatter leaf virus, citrus exocortis virus, citrus yellowing vein phloem virus and citrus leaf mottle virus published by NCBI, the DPO RT-PCR detection primers for synchronous detection of the five viruses are designed.

[0036] All pathogenic genomics sequences of citrus tristeza virus (accession number: AF001623, AF260651, AY170468, FJ525436, JQ965169, etc.), citrus tatter leaf virus (accession number: AY646511, EU553489, MH108985, KC588948, KY706358, etc.), citrus exocortis virus (accession number: DQ44444, M30870, S67437, M30868, etc.), citrus yellow shoot phloem virus (accession number: KT124646, KX156742, MK415924, MF563877, etc.), citrus leaf mottle virus (accession number: EU857539, MT863785, MG572236, MN495980, etc.) were subjected to alignment analysis, and the conserved sequences among different pathogenic types of the same virus and the gene sequences with large differences between different viruses (excluding the conserved sequences between different viruses) were selected for primer design. The specificity of the primers was improved by avoiding the different sequences between the gene sequences of different pathogenic types of the same virus and the conserved sequences between different virus gene sequences. At the same time, the 3 segments of the primer sequence were modified by increasing the hypoxanthine, further reducing the formation of dimers, so as to achieve the purposes of high specificity, high sensitivity and reducing the interference of dimers of the primers.

[0037] All the finally designed primers have no obvious homology with other sequences in the nucleic acid sequence database, and the sizes of the amplified target bands are different, which can be easily separated and distinguished by agarose gel electrophoresis. The primer sequences are as follows:

[0038] DPO-CTV-F: 5'-TGGCTCGTAGACACCIIIIICGTTCTCCGG-3', as shown in SEQ ID NO. 1;

[0039] DPO-CTV-R: 5'-CTAAGGAGAACTTCTTIIIIICACGCATACG-3', as shown in SEQ ID NO. 2;

[0040] DPO-CTLV-F: 5'-TGCTTCAACAAGCGAGGCIIIIICGGGTAGGAG-3', as shown in SEQ ID NO. 3;

[0041] DPO-CTLV-R: 5'-GTATAAAGGCAGGCATGTCAIIIIICAAGACCGCG-3', as shown in SEQ ID NO. 4;

[0042] DPO-CEV-F: 5'-CGGGATCTTTCTTGAGIIIIIIGTGGTGCT-3', as shown in SEQ ID NO. 5;

[0043] DPO-CEV-R: 5'-GCTCCTGTTTCTCCGCTGGIIIIIAGTGATCC-3', as shown in SEQ ID NO. 6;

[0044] DPO-CYVCV-F: 5'-TCCATTGTCGACGAGTIIIIICTAAGCCAG-3', as shown in SEQ ID NO. 7;

[0045] DPO-CYVCV-R: 5'-GGATAGCTGCGGTAGAGAGGGTIIIIGTAGTCGAAG-3', as shown in SEQ ID NO. 8;

[0046] DPO-CLBV-F: 5'-GGATTATGTGTCTCATGTIIIIIIAGAGACGG-3', as shown in SEQ ID NO. 9;

[0047] DPO-CLBV-R: 5'-TGCAGCTTTGAGTGACIIIIICAATTCTTC-3', as shown in SEQ ID NO. 10;

[0048] The "I" in the primer sequence is hypoxanthine;

[0049] The PCR amplification products are detected by agarose electrophoresis: if a band of 416 bp is amplified, it indicates that the sample contains citrus tristeza virus; if a band of 716 bp is amplified, it indicates that the sample contains citrus tatter leaf virus; if a band of 187 bp is amplified, it indicates that the sample contains citrus exocortis virus; if a band of 938 bp is amplified, it indicates that the sample contains citrus yellowing vein phloem virus; and if a band of 576 bp is amplified, it indicates that the sample contains citrus leaf mottle virus.

[0050] Example 2 Construction of plasmids

[0051] Using the specific primers designed in Example 1, the target fragments were amplified from the collected samples of citrus tristeza virus, citrus tatter leaf virus, citrus yellow vein clearing virus, citrus leaf mottle virus, respectively, and then the amplified fragments were recovered and purified by using a gel recovery kit, and then ligated with pMDT-20T vector, and then transformed into E. coli DH5a competent cells for culture, and colony PCR was performed to verify the correct colonies, and the bacterial liquid was sent to Shanghai Sangon Biological Engineering Co., Ltd. for sequencing. The plasmids of the correct colonies were extracted according to the sequencing results, and the recombinant plasmids pCEV, pCTV, pCLBV, pCTLV and pCYVCV were obtained. Then the concentration of the plasmid DNA was determined by using a nucleic acid protein analyzer to determine the concentration of the sample DNA, and the concentrations of the plasmids were 70.7 ng / μL, 47.4 ng / μL, 50.8 ng / μL, respectively. According to the formula copies / μL = {6.02×10 23 (copies / mol)×DNA concentration(ng / μL)×10 -9} / {base number(bp)×660(ng / mol)}(Wilhelm et al., 2003), the copy numbers of the plasmids were calculated as 1.39×10 10 copies, 1.19×10 10 copies, 1.23×10 10 copies, 1.12×10 10 copies, and 1.60×10 10 copies, respectively. The five plasmids were diluted, and the dilution gradients are shown in Table 1.

[0052] Table 1 Concentration gradient dilution of five citrus virus plasmids and calculation of copy number

[0053]

[0054] Example 3 Effect of different annealing temperatures on single-plex PCR detection results

[0055] The cloned recombinant plasmids were diluted 10 2 times as templates for PCR amplification. The PCR reaction system was 20 μL: Premix Ex TaqTM 10 μL, final concentration of upstream and downstream primers 0.50 μmol / L, plasmid DNA template 1 μL, and ddH2O 20 μL. The PCR reaction parameters were: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 48-62℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; finally 72℃ extension for 10 min, cooling to 12℃ to end the reaction.

[0056] Other conditions remain unchanged, screening annealing temperature, set 8 treatments: 48℃, 50℃, 52℃, 54℃, 56℃, 58℃, 60℃, 62.0℃.

[0057] PCR product electrophoresis detection: 6 μL PCR product and 1 μL 6x loading buffer were mixed, and 1.2% agarose gel (Goldview nucleic acid dye was added) electrophoresis was carried out at room temperature, the electrophoresis condition was voltage 120V, time 30min, and the electrophoresis result was observed in the gel imaging system after electrophoresis.

[0058] The experimental results are shown in Figure 1 , wherein M is DL2000 plus marker (Genstar); lanes 1-8 correspond to low to high annealing temperature 48℃, 50℃, 52℃, 54℃, 56℃, 58℃, 60℃, 62.0℃ respectively; from top to bottom: A figure is CEVd; B figure is CTV; C figure is CLBV; D figure is CTLV; E figure is CYVCV. It can be seen from Figure 1 , the specific primers for detecting five citrus viruses in the application can detect a single band with a size consistent with the theoretical value at all annealing temperatures above, and there is no primer dimer interference, and subsequent experiments verify that the annealing temperature is selected as 56℃.

[0059] Example 4 detection sensitivity

[0060] On the basis of example 3, other conditions remain unchanged, the sensitivity of each primer is verified, and the TA cloned plasmid template is diluted (Table 1) in turn, and 7 treatments are set: 10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 . CEV can still detect the target gene band when diluted to 10 -3 , CTV and CYVCV can detect the target gene band when diluted to 10 -4 , CLBV and CTLV can still detect the target gene band when diluted to 10 -5 . The results show that CEV, CTV, CLBV, CTLV and CYVCV single PCR have high detection sensitivity, and the copies of the detection threshold are 1.39E+06, 1.19E+06, 1.23E+05, 1.12E+05, 1.60E+06 respectively.

[0061] The results are shown in Figure 2Figure 1 shows the electrophoresis results of the sensitivity detection of the multiplex PCR, wherein M is DL2000 plus marker (Genstar); lanes 1-8 correspond to dilution factors of 10 from low to high 0 -1 -2 -3 -4 -5 -6 8 is a negative control; Figure A is an electrophoresis chart of the sensitivity detection of CEVd; Figure B is an electrophoresis chart of the sensitivity detection of CTV; Figure C is an electrophoresis chart of the sensitivity detection of CLBV; Figure D is an electrophoresis chart of the sensitivity detection of CTLV; and Figure E is an electrophoresis chart of the sensitivity detection of CYVCV.

[0062] Example 5: Influence of different primer concentrations on the detection results of the multiplex PCR

[0063] On the basis of Examples 3 and 4, the primer concentrations were optimized to establish a multiplex PCR system for simultaneously detecting five citrus viruses. Other conditions were unchanged, 16 primer concentration combinations were set, the combination conditions are shown in Table 2, and the plasmid templates with dilution factors of 10 -2 were selected to perform multiplex PCR verification, and the results are shown in Figure 3 Figure A, except that combinations 5 and 11 failed to detect the five viruses, the other primer combinations can detect five bands with sizes consistent with the theoretical values.

[0064] Table 2: Screening of the optimal primer combinations for simultaneously detecting multiple citrus viruses

[0065]

[0066]

[0067] Example 6: Specificity and stability test of the multiplex PCR

[0068] On the basis of Example 5, primer combination 2 in Table 2 was selected to perform multiplex PCR verification on the mixed plasmids of one, two, three, four, and five viruses. The electrophoresis results are shown in Figure 3 Figure B, M is DL2000 plus marker (Genstar); lanes 1-5 are single virus templates: CEVd, CTV, CLBV, CTLV, and CYVCV; lanes 6-15 are complex templates of random combinations of two, three, four, and five viruses; and lane 16 is a negative control. The results show that the multiplex detection system of the present application can stably detect the target viruses in one, two, three, four, and five viruses.

[0069] ​​​​​​The foregoing description of specific exemplary embodiments of the application will be better understood when read in conjunction with the accompanying drawings. It should be understood that the description is illustrative but not limiting of the present application. While specific embodiments have been illustrated and described, it is recognized that numerous modifications and changes can well be made by those skilled in the art. It is intended to embrace all such modifications and changes and, accordingly, the scope of the application should be judged in terms of the claims and their equivalents. SEQUENCE LISTING <110> Guangxi Academy of Agricultural Sciences <120> DPO RT-PCR primer set for simultaneous detection of five citrus viruses, detection method and kit <130> NKY <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (16)..(20) <223> i is hypoxanthine <400> 1 tggctcgtag acaccIIIII cgttctccgg 30 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (17)..(21) <223> i is hypoxanthine <400> 2 ctaaggagaa cttcttIIII Icacgcatac g 31 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (19)..(23) <223> i is hypoxanthine <400> 3 tgcttcaaca agcgaggcII IIIcgggtag gag 33 <210> 4 <211> 35 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (21)..(25) <223> i is hypoxanthine <400> 4 gtataaaggc aggcatgtca IIIIIcaaga ccgcg 35 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (17)..(22) <223> i is hypoxanthine <400> 5 cgggatcttt cttgagIIII IIgtggtgct 30 <210> 6 <211> 32 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (20)..(24) <223> i is hypoxanthine <400> 6 gctcctgttt ctccgctggI IIIIagtgat cc 32 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (17)..(21) <223> i is hypoxanthine <400> 7 tccattgtcg acgagtIIII Ictaagccag 30 <210> 8 <211> 36 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (23)..(26) <223> i is hypoxanthine <400> 8 ggatagctgc ggtagagagg gtIIIIgtag tcgaag 36 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (19)..(24) <223> i is hypoxanthine <400> 9 ggattatgtg tctcatgtII IIIIagagac gg 32 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (17)..(21) <223> i is hypoxanthine <400> 10 tgcagctttg agtgacIIII Icaattcttc 30

Claims

1. A DPO RT-PCR primer set for simultaneous detection of five citrus viruses, characterized in that, Includes the following primers: Two citrus decline virus-specific primers: DPO-CTV-F: 5'-TGGCTCGTAGACACCIIIIICGTTCTCCGG-3', as shown in SEQ ID NO.1; DPO-CTV-R:5'-CTAAGGAGAACTTCTTIIIIICACGCATACG-3', as shown in SEQ ID NO.2; Two citrus leaf fragment virus-specific primers: DPO-CTLV-F: 5'-TGCTTCAACAAGCGAGGCIIIICCGGGTAGGAG-3', as shown in SEQ ID NO.3; DPO-CTLV-R: 5'-GTATAAAGGCAGGCATGTCAIIIIICAAGACCGCG-3', as shown in SEQ ID NO.4; Two specific primers for citrus peel cracking virus: DPO-CEV-F:5'-CGGGATCTTTCTTGAGIIIIIIGTGGTGCT-3', as shown in SEQ ID NO.5; DPO-CEV-R: 5'- GCTCCTGTTTCTCCGCTGGIIIIIAGTGATCC-3', as shown in SEQ ID NO.6; Two citrus yellowing vein virus-specific primers: DPO-CYVCV-F: 5'-TCCATTGTCGACGAGTIIIIICTAAGCCAG-3', as shown in SEQ ID NO.7; DPO-CYVCV-R: 5'-GGATAGCTGCGGTAGAGAGGGTIIIIGTAGTCGAAG-3', as shown in SEQ ID NO.8; Two specific primers for citrus leaf spot virus are as follows: DPO-CLBV-F: 5'-GGATTATGTGTCTCATGTIIIIIIAGAGACGG-3', as shown in SEQ ID NO.9; DPO-CLBV-R: 5'-TGCAGCTTTGAGTGACIIIIICAATTCTTC-3', as shown in SEQ ID NO.10; Among them, I represents hypoxanthine.

2. A detection reagent or kit, characterized in that, The detection reagent or kit contains the DPO RT-PCR primer set as described in claim 1.

3. A method for simultaneously detecting citrus senescence virus, citrus leaf fragmentation virus, citrus peel cracking virus, citrus yellowing and vein clearing virus, and citrus leaf spot virus, characterized in that, Includes the following steps: (1) Take the sample to be tested, extract its total RNA, and reverse transcribe it into cDNA; (2) Using the DPO RT-PCR primer set described in claim 1, a PCR reaction is performed with cDNA of the sample to be tested as a template to obtain the amplification product; (3) Detect PCR amplification products using agarose electrophoresis: If a band of 416 bp is amplified, it indicates that the sample contains citrus degeneration virus; if a band of 716 bp is amplified, it indicates that the sample contains citrus leaf fragmentation virus; if a band of 187 bp is amplified, it indicates that the sample contains citrus peel cracking virus; if a band of 938 bp is amplified, it indicates that the sample contains citrus yellowing vein virus; if a band of 576 bp is amplified, it indicates that the sample contains citrus leaf mottle virus.

4. The method according to claim 3, characterized in that, The PCR reaction system consisted of 20 µL, and the concentrations of each primer were as follows: DPO-CTV-F and DPO-CTV-R were both 0.2 µM, DPO-CTLV-F and DPO-CTLV-R were both 0.1 µM, DPO-CEV-F and DPO-CEV-R were both 0.5 µM, DPO-CYVCV-F and DPO-CYVCV-R were both 0.3 µM, and DPO-CLBV-F and DPO-CLBV-R were both 0.3 µM.

5. The method according to claim 4, characterized in that, The PCR reaction procedure was as follows: 50℃ for 30 min, 94℃ for 5 min; the parameters for 30 cycles were: denaturation at 94℃ for 30 s, annealing at 56℃ for 30 s, extension at 72℃ for 2 min; and a final extension at 72℃ for 10 min.

6. The application of the DPO RT-PCR primer set according to claim 1 in the simultaneous detection of citrus degeneration virus, citrus leaf fragmentation virus, citrus peel cracking virus, citrus yellowing vein virus, and citrus leaf mottle virus.

7. The application of the detection reagent or kit according to claim 2 in the simultaneous detection of citrus degeneration virus, citrus leaf fragmentation virus, citrus peel cracking virus, citrus yellowing vein virus, and citrus leaf mottle virus.

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