A double real-time fluorescent quantitative TaqMan primer probe set, kit and detection method for simultaneously detecting sweet potato feathery mottle virus and sweet potato witches' broom phytoplasma

Abstract

The application discloses a double real-time fluorescent quantitative TaqMan primer probe set, a kit and a detection method for simultaneously detecting sweet potato leaf curl virus and sweet potato witches' broom phytoplasma, which is designed according to the nucleotide sequences of SPLCV and SPWB genome sequencing in a conservative segment, and is used for specific detection of SPLCV and SPWB; and the application also designs a fluorescent quantitative kit based on the primer probe set. The primer and probe set can simultaneously detect the target of SPLCV and SPWB in one reaction, is fast and convenient, has high detection efficiency, and has high detection sensitivity of 3.3 copies / uL, which is 100 times of that of ordinary PCR. The TaqMan QPCR detection technology established by the application for SPLCV and SPWB solves the problems of time-consuming and labor-consuming, poor specificity and sensitivity, and easy pollution in the existing detection methods of SPLCV and SPWB, and provides a technical basis for early diagnosis of SPLCV and SPWB.

Description

[Technical Field]

[0001] This invention relates to the field of molecular detection technology for plant diseases, specifically a dual real-time quantitative TaqMan primer and probe set, kit, and detection method for simultaneously detecting sweet potato leaf curl virus (SPLCV) and sweet potato witches'broom phytoplasma (SPWB). [Background Technology]

[0002] Sweet potato (Ipomoea batatas) is a herbaceous plant belonging to the genus Ipomoea batatas Lam. of the family Convolvulaceae. Its scientific name is Ipomoea batatas, and it is also known as sweet potato, yam, and sweet potato. It is drought-tolerant, soil-fixing, and highly adaptable, and is widely cultivated in tropical and subtropical regions of Asia and Africa. Sweet potato is an important food and economic crop in my country, with the largest planting area in the world. my country's annual output accounts for more than 60% of the world's total output, making it a crucial crop for food security, economic value, and ecological function. Sweet potato is an asexually propagated crop, and its generations are highly susceptible to viral infections. In recent years, sweet potato diseases and pests have become severe, especially viral diseases. Sweet potato leaf curl virus (SPLCV) and sweet potato witches'broom phytoplasma (SPWB) are two major pathogens that harm sweet potato, causing symptoms such as leaf curling, yellowing, wilting, small leaves, and stunted growth, resulting in severe yield losses. Sweet potato leaf curl virus (SPV) is a single-stranded DNA virus belonging to the Geminiviridae family. It is persistently transmitted through the whitefly (Bemisiatabaci). After infection, the virus suppresses the sweet potato's gene silencing defense system, leading to leaf curling, yellowing, and malformation, resulting in a 30%-70% loss in tuber yield. SPV exhibits high recombinancy; recombinant strains in China (SPLCV-CN) have been identified, with genomic homology of only 78%-85%, posing a challenge to primer design conservation. Sweet potato wilt phytoplasma is a cell-wall-less prokaryote belonging to group 16SrII-A, transmitted by leafhoppers. Phytoplasma are mainly transmitted through grafting and leafhoppers. The phytoplasma parasitizes the phloem of plants, interfering with nutrient transport and hormone balance, leading to abnormal plant growth. Infection with phytoplasma in sweet potatoes primarily manifests as stunted growth, small leaves, yellowing, and excessive lateral branching. As the disease progresses, the leaves of the top vine gradually become smaller, wither, and pale in color. Lower lateral buds continuously sprout, internodes shorten, and typical symptoms of bushy branches and clustered leaves are formed. It induces abnormal proliferation of axillary buds, forming "broom-like" bushy branches. Accompanied by gibberellin metabolism disorder, it causes lignification of tubers, loss of quality, and a significant drop in yield, resulting in huge economic losses to the sweet potato industry.

[0003] Currently, there are no effective agents for controlling SPLCV and SPWB. Early detection and diagnosis are crucial for disease control. Most plant virus detection methods use enzyme-linked immunosorbent assay (ELISA) based on antiserum, but these methods suffer from problems such as high antiserum prices, false positives, inability to distinguish between multiple infections, and low detection sensitivity. In recent years, PCR, RCA, and qPCR-based virus detection technologies have been rapidly developing. Compared with singleton PCR, multiplex PCR offers advantages such as simultaneous detection of multiple viruses, simple operation, short detection time, and low cost, thus attracting significant attention. Preliminary investigations have shown that the co-infection rate of SPLCV and SPWB in southeastern coastal areas of my country is as high as 40%, and in severely affected areas it can reach over 80%. SPLCV weakens plant resistance, making them more susceptible to phytoplasma infection, leading to complete sweet potato crop failure. Furthermore, both SPLCV and SPWB cause leaf yellowing symptoms, which are difficult to distinguish visually in the field, requiring precise molecular identification to develop targeted control strategies.

[0004] Due to the high recombination rate of SPLCV, primer design faces challenges of conservation. Current literature on SPLCV detection includes CN107475460A and CN105132588A, both involving methods for detecting only one type of SPLCV. These methods suffer from low detection efficiency, low sensitivity, cumbersome procedures, and susceptibility to aerosol contamination with the LAMP method. SPWB cannot be cultured in vitro, lacks a standard genomic database, and relies on molecular detection. While its 16S rRNA gene is conserved, subgroup variations such as 16SrII-B and II-D exist in the field, limiting detection. Literature on arbuscular phytoplasma detection mainly includes CN113667769A and CN119193875A, involving PCR and qPCR detection of 16S rRNA. These methods also suffer from limitations such as detecting only one pathogen, low sensitivity (10 copies), and poor specificity (inability to distinguish subspecies). Therefore, developing a dual-system approach for simultaneous detection and quantification of pathogens faces challenges such as conservatism in primer and probe design and cross-interference in pathways. Currently, there are no reported dual real-time quantitative PCR methods for simultaneously detecting SPLCV and SPWB. The SecA gene of phytoplasma is a core molecular tool for studying its phylogeny, rapid detection, and pathogenic mechanisms. Its high resolution compensates for the shortcomings of 16S rRNA, and it has higher variability than traditional markers, better distinguishing phytoplasmas from closely related strains that may belong to different species. It has become one of the standards for modern phytoplasma classification and disease diagnosis. SecA can serve as a control target in the dynamic regulation of host-host interactions. Our research group previously obtained the SecA gene sequence of sweet potato arbuscular mycorrhizal phytoplasma through phytoplasma genome sequencing. Comparative analysis revealed that the SecA gene is highly conserved in sweet potato arbuscular mycorrhizal phytoplasma. Therefore, developing TaqMan specific primers and probe sets, as well as detection methods, for simultaneous real-time quantitative PCR of SPLCV and SPWB using the conserved sequence of sweet potato leaf curl virus and the SecA gene sequence of sweet potato arbuscular mycorrhizal phytoplasma shows great promise.

[0005] Therefore, how to provide TaqMan specific primers and probe sets, as well as detection methods, for real-time quantitative PCR of SPLCV and SPWB with high detection sensitivity is a technical problem that urgently needs to be solved by those skilled in the art. [Summary of the Invention]

[0006] Based on this, the present invention provides a primer and probe set, detection method, kit and application for simultaneous detection of SPLCV and SPWB, which can simultaneously detect the two major pathogens of sweet potato, SPLCV and SPWB, and has the advantages of high detection efficiency and high sensitivity.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A TaqMan fluorescent quantitative detection primer and probe set for simultaneously detecting sweet potato leaf curl virus and sweet potato wisteria phytoplasma includes the following 6 sets of primers:

[0009] The forward primer for sweet potato leaf curl virus, F1: 5'-ACGTTTGTCTGTGTCTCGGA-3', is shown in SEQ ID No:1;

[0010] The reverse primer for sweet potato leaf curl virus, R1, is 5'-GGCCACATTGTCATCCATCC-3', as shown in SEQ ID No:2;

[0011] Sweet potato leaf curl virus probe primer Probe1: 5'-ACCCAAGCGATGGGTAAGCCCA-3', as shown in SEQ ID No:3;

[0012] The probe also contains a fluorescent group at its 5' end and a quenching group at its 3' end;

[0013] The forward primer for sweet potato hop phytoplasm F2: 5'-TGGCTGGAAGAGGTACAGAT-3', as shown in SEQ ID No:4;

[0014] The reverse primer R2 for sweet potato hop phytoplasm is 5'-TCGCACTTCATGCCTTTCTG-3', as shown in SEQ ID No:5;

[0015] The sweet potato hop phytoplasma probe primer Probe2: 5'-CCGCCTAACTCAGCTACACCTTCTGC-3', as shown in SEQ ID No: 6;

[0016] The probe also contains a fluorescent group at its 5' end and a quenching group at its 3' end.

[0017] Furthermore, the fluorescent group includes TAMRA or FAM; the quenching group includes BHQ.

[0018] Furthermore, the application of the TaqMan fluorescent quantitative detection primer and probe set for simultaneously detecting sweet potato leaf curl virus and sweet potato wisteria phytoplasma in the preparation of gene products for identifying sweet potato leaf curl virus and sweet potato wisteria phytoplasma.

[0019] Furthermore, a kit for simultaneously detecting sweet potato leaf curl virus and sweet potato wisteria phytoplasma includes the aforementioned primer and probe set.

[0020] Furthermore, the kit also includes Probe qPCR Mix, 50×ROX Reference Dye, and Nuclease-free Water.

[0021] Furthermore, a detection method for simultaneously detecting sweet potato leaf curl virus and sweet potato wilting phytoplasma involves using the aforementioned kit to perform TaqMan qPCR amplification of the pathogens to be detected. The presence of a fluorescent signal during the amplification process indicates the presence of both sweet potato leaf curl virus and sweet potato wilting phytoplasma.

[0022] Furthermore, the composition of the TaqMan qPCR reaction system is as follows:

[0023] The total volume of the reaction system was 25 μL, containing 12.5 μL of Probe qPCR Mix, 0.25 μL of ROXReference Dye II, 2 μL of 10 ng / μL template, 0.2–0.5 μL of sweet potato leaf curl virus forward primers, 0.2–0.5 μL of sweet potato leaf curl virus reverse primers, 0.3–0.5 μL of sweet potato leaf curl virus probe primers; 0.2–0.5 μL of sweet potato wilt phytoplasma forward primers, 0.2–0.5 μL of sweet potato wilt phytoplasma reverse primers, 0.3–0.5 μL of sweet potato wilt phytoplasma probe primers, and the remaining components were Nuclease-free water.

[0024] The TaqMan qPCR reaction procedure is as follows: preheat at 25℃ for 2 min, predenature at 95℃ for 2 min; denature at 95℃ for 10 s, anneal and extend at 58-64℃ for 25 s, collect signal, and repeat 40 times.

[0025] Furthermore, the initial concentration of all forward primers, reverse primers, and probe primers in the TaqMan qPCR reaction system before being added to the reaction system is 10 μmol / L, and the concentrations of forward primers, reverse primers, and probe primers in each pair of primers in the reaction system are equal.

[0026] Furthermore, in the TaqMan qPCR reaction system, the final concentrations of the forward and reverse primers for sweet potato leaf curl virus were both 0.08-0.2 μmol / L; the final concentrations of the sweet potato leaf curl virus probe primers were both 0.12-0.2 μmol / L; the final concentrations of the forward and reverse primers for sweet potato hops phytoplasma were both 0.08-0.2 μmol / L; and the final concentrations of the sweet potato hops phytoplasma probe primers were both 0.12-0.2 μmol / L.

[0027] Furthermore, when a single red S-shaped amplification curve appears and the Ct value is ≤35, it indicates that the sample contains sweet potato leaf curl virus; when a single blue S-shaped amplification curve appears and the Ct value is ≤35, it indicates that the sample contains sweet potato wisteria phytoplasma; when both a single red and a single blue S-shaped amplification curve appear and the Ct value is ≤35, it indicates that the sample contains both sweet potato leaf curl virus and sweet potato wisteria phytoplasma; when no S-shaped amplification curve appears or the Ct value of the amplification curve is >35, it indicates that the sample does not contain the detected pathogen.

[0028] The present invention has the following advantages:

[0029] This invention targets the nucleotide sequences of SPLCV (single-stranded DNA virus) and SPWB (cell wall-less prokaryote) genomes, designing specific TaqMan detection primer sets and probe primers for their conserved regions. Using these two primer sets and probe primer sets, TaqMan qPCR reactions are simultaneously performed on SPLCV and SPWB in sweet potato tissues, establishing a novel TaqMan qPCR reaction system and procedure. Amplification curves for SPLCV and SPWB were obtained, achieving the goal of simultaneous detection of SPLCV and SPWB in a single TaqMan qPCR reaction tube. Furthermore, the primer sets demonstrate high specificity, showing good amplification curves for sweet potato leaf curl virus and sweet potato wilfordii phytoplasma, while no non-specific amplification curves were observed for other sweet potato pathogens. The detection sensitivity is high, at 3.3 copies / µL, 100 times that of conventional PCR; the detection is rapid, completed within 1 hour, with a low false positive rate. [Attached Image Description]

[0030] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0031] Figure 1 This is an amplification diagram of the concentration of TaqMan primer set and probe primer combination for simultaneous detection of sweet potato leaf curl virus and sweet potato wisteria phytoplasma in Example 1 of the present invention.

[0032] Figure 2 This is a standard curve amplification diagram of a dual real-time quantitative TaqMan detection method for simultaneously detecting sweet potato leaf curl virus and sweet potato wisteria phytohedrosis, as described in Example 2 of this invention; curve A in the diagram represents sweet potato leaf curl virus at 1×10⁻⁶. 8 copies / μL to 1×10 1 Amplification curves of positive standards diluted to copies / μL, B is the standard curve of sweet potato leaf curl virus; curve C is the amplification curve of sweet potato arbuscular phytoplasma at 1×10⁻⁶ copies / μL. 8 copies / μL to 1×10 1Amplification curves of positive standards diluted to copies / μL, where D is the standard curve of Mycoplasma sweet potatoensis.

[0033] Figure 3 This is a specific amplification curve of the dual real-time quantitative PCR method for simultaneously detecting sweet potato leaf curl virus and sweet potato wisteria phytoparasite in Example 3 of the present invention. In the figure, 1 is the amplification curve of a positive sample of sweet potato leaf curl virus, 2 are the amplification curves of positive sweet potato wisteria phytoparasite, and 3-9 are the amplification curves of SPBV, SPCV, sweet potato blast, sweet potato black rot, SPMFV, SPVG, SPLV and Nuclease-free Water, respectively.

[0034] Figure 4 This is an amplification graph showing the sensitivity of the dual real-time quantitative PCR method for simultaneously detecting sweet potato leaf curl virus and sweet potato wisteria phytoplasma in Example 4 of this invention. In the graph, A represents the sensitivity curve of the TaqMan qPCR method (1:10). 8 2:10 7 3:10 6 4:10 5 5:10 4 6:10 3 7:10 2 8:10 1 9:10 0 10-12: Healthy sweet potato samples, Nuclease-free Water, Nuclease-free Water; B is an electrophoresis image showing the sensitivity of the singlet PCR detection method for sweet potato leaf curl virus, 1:10. 8 2:10 7 3:10 6 4:10 5 5:10 4 6:10 3 7:10 2 8:10 1 9:10 0 10: Nuclease-free Water; C represents the sensitivity of the singlet PCR detection method for sweet potato arbuscular phytoplasma. Electrophoresis diagram: Sample concentrations in lanes 1-10 are 1:10. 8 2:10 7 3:10 6 4:10 5 5:10 4 6:10 3 7:10 2 8:10 1 9:10 0 10: Nuclease-free Water.

Detailed Implementation Methods

[0035] The following will be combined with the appendix Figure 1-4 The technical solution of the present invention will be clearly and completely described in detail with specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0036] Example 1: Detection of concentrations using dual real-time quantitative TaqMan qPCR primer set and probe primers for sweet potato leaf curl virus and sweet potato wisteria phytoparasite:

[0037] Test samples: sweet potato leaf curl virus DNA, sweet potato witches' broom disease phytoplasma DNA.

[0038] The specific steps are as follows: Based on the sequencing sequences of sweet potato leaf curl virus and sweet potato wilting phytoplasma obtained by the research group, and after comparison with the sequences of sweet potato leaf curl virus and sweet potato wilting phytoplasma published in GenBank, primers were designed for conserved regions. The TaqMan qPCR detection primer and probe set was designed as follows:

[0039] The forward primer for sweet potato leaf curl virus, F1: 5'-ACGTTTGTCTGTGTCTCGGA-3', is shown in SEQ ID No:1;

[0040] The reverse primer for sweet potato leaf curl virus, R1, is 5'-GGCCACATTGTCATCCATCC-3', as shown in SEQ ID No:2;

[0041] Sweet potato leaf curl virus probe primer Probe1: 5'-ACCCAAGCGATGGGTAAGCCCA-3', as shown in SEQ ID No:3;

[0042] The probe also contains a fluorescent group TAMRA at its 5' end and a quenching group BHQ at its 3' end.

[0043] The forward primer for sweet potato hop phytoplasm F2: 5'-TGGCTGGAAGAGGTACAGAT-3', as shown in SEQ ID No:4;

[0044] The reverse primer R2 for sweet potato hop phytoplasm is 5'-TCGCACTTCATGCCTTTCTG-3', as shown in SEQ ID No:5;

[0045] The sweet potato hop phytoplasma probe primer Probe2: 5'-CCGCCTAACTCAGCTACACCTTCTGC-3', as shown in SEQ ID No: 6;

[0046] The probe also contains a fluorescent group FAM at its 5' end and a quenching group BHQ at its 3' end.

[0047] Primers and probes were synthesized by Sangon Biotech Co., Ltd., and the probes and primers were purified by HPLC.

[0048] DNA extraction was performed using the CTAB method. Sweet potato tissue was flash-frozen in liquid nitrogen, ground into a fine powder, and 100 mg of powder was added to 600 μL of CTAB lysis buffer preheated to 65 °C. The mixture was incubated in a 65 °C water bath for 60 min, vortexing every 10 min. After cooling, chloroform / isoamyl alcohol at a volume ratio of 24:1 was added, and the mixture was gently shaken and centrifuged at 12000 rpm for 10 min. 300 μL of the supernatant was transferred to a 1.5 mL centrifuge tube, and 600 μL of anhydrous ethanol was added. The mixture was incubated at -20 °C for 30 min, centrifuged at 12000 rpm for 5 min, and the supernatant was discarded. The mixture was washed with 500 μL of 75% ethanol, repeated twice, and the supernatant was discarded. After drying in a clean bench, 100 μL of preheated sterile water was added to dissolve the DNA, and 10 μL of 50 μg / mL RNase A was added. The DNA was stored at -20 °C.

[0049] Using the primer pairs for sweet potato leaf curl virus (SPV) and sweet potato wilting disease (SWD) as described above, and with DNA from positive SPV and SWD samples as templates, and DNA from healthy sweet potato samples as negative controls and Nuclease-free Water as controls, TaqMan qPCR was performed. The total volume of the reaction system was 25 μL, containing 12.5 μL of Probe qPCR Mix, 0.25 μL of LOX Reference Dye II, 2 μL of 10 ng / μL template, 0.2–0.5 μL of SPV forward primers, 0.2–0.5 μL of SPV reverse primers, and 0.3–0.5 μL of SPV probe primers; 0.2–0.5 μL of SWD forward primers, 0.2–0.5 μL of SWD reverse primers, and 0.3–0.5 μL of SWD probe primers; and the remainder being Nuclease-free Water. The reaction procedure was as follows: preheating at 25℃ for 2 min, pre-denaturation at 95℃ for 2 min, denaturation at 95℃ for 10 s, annealing and extension at 58-64℃ for 25 s, signal collection, and repeating 40 times.

[0050] See Figure 1The primer concentration combinations shown in the figure are as follows: Curve 1: 0.2 μL of sweet potato leaf curl virus forward primer, 0.2 μL of sweet potato leaf curl virus reverse primer, and 0.3 μL of sweet potato leaf curl virus probe primer; 0.2 μL of sweet potato wilt phytoplasma forward primer, 0.2 μL of sweet potato wilt phytoplasma reverse primer, and 0.3 μL of sweet potato wilt phytoplasma probe primer;

[0051] Curve 2: 0.3 μL of sweet potato leaf curl virus forward primer, 0.3 μL of sweet potato leaf curl virus reverse primer, 0.4 μL of sweet potato leaf curl virus probe primer; 0.3 μL of sweet potato wilt phytoplasma forward primer, 0.3 μL of sweet potato wilt phytoplasma reverse primer, 0.4 μL of sweet potato wilt phytoplasma probe primer;

[0052] Curve 3: 0.4 μL of sweet potato leaf curl virus forward primer, 0.4 μL of sweet potato leaf curl virus reverse primer, 0.5 μL of sweet potato leaf curl virus probe primer; 0.4 μL of sweet potato wilt phytoplasma forward primer, 0.4 μL of sweet potato wilt phytoplasma reverse primer, 0.5 μL of sweet potato wilt phytoplasma probe primer;

[0053] Curve 4: 0.5 μL of sweet potato leaf curl virus forward primer, 0.5 μL of sweet potato leaf curl virus reverse primer, 0.5 μL of sweet potato leaf curl virus probe primer; 0.5 μL of sweet potato wilt phytoplasma forward primer, 0.5 μL of sweet potato wilt phytoplasma reverse primer, 0.5 μL of sweet potato wilt phytoplasma probe primer.

[0054] Standard curves showed that 0.2–0.5 μL of sweet potato leaf curl virus forward primers, 0.2–0.5 μL of sweet potato leaf curl virus reverse primers, and 0.3–0.5 μL of sweet potato leaf curl virus probe primers; 0.2–0.5 μL of sweet potato wilt phytoplasma forward primers, 0.2–0.5 μL of sweet potato wilt phytoplasma reverse primers, and 0.3–0.5 μL of sweet potato wilt phytoplasma probe primers could all amplify the corresponding amplification curves. Among them, 0.5 μL of sweet potato leaf curl virus forward and reverse primers, 0.5 μL of sweet potato leaf curl virus probe primers, 0.5 μL of sweet potato wilt phytoplasma forward and reverse primers, and 0.5 μL of sweet potato wilt phytoplasma probe primers produced the best amplification curves.

[0055] Example 2: Standard Curve

[0056] Test samples: sweet potato leaf curl virus DNA, sweet potato witches' broom phytoplasma DNA, sweet potato baculovirus (SPBV) DNA, sweet potato cauliflower mosaic virus (SPCV) DNA, sweet potato feather mottle virus (SPMFV), sweet potato G virus (SPVG), sweet potato latent virus (SPLV) cDNA; sweet potato blast and sweet potato black rot DNA were provided by the Crop Research Institute of Fujian Academy of Agricultural Sciences.

[0057] The primer and probe set is the same as that shown in Example 1.

[0058] The specific steps are as follows: Preparation of positive recombinant plasmid standards: Using primers for sweet potato leaf curl virus and sweet potato wilting disease phytoparasite, DNA from positive sweet potato leaf curl virus and sweet potato wilting disease samples was used as templates, with DNA from healthy sweet potato samples as negative controls and Nuclease-free Water as a blank control for PCR amplification. The PCR products were detected by agarose gel electrophoresis. The gel containing the target band was excised and purified using an agarose gel extraction kit. The purified product was ligated into a Zero Cloning Kit and transformed into DH5α competent cells. Plasmids from positive clones were screened and extracted, and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The concentration of correctly sequenced plasmids was determined using an ultra-micro nucleic acid analyzer and stored at -20℃ for later use. The copy number in the recombinant plasmid was calculated using Shirima's method (Shirima RR et al., 2017): Recombinant plasmid copy number (copies / uL) = (6.022 × 10⁻⁶). 23 × plasmid concentration (ng / μL) / (10 9 (×recombinant plasmid base pairs × 660 dalton / bp). The final calculated copy number of the sweet potato leaf curl virus recombinant plasmid and the sweet potato arbuscular phytoplasma recombinant plasmid were 3.3 × 10⁻⁶. 10 copies / uL.

[0059] Using the positive plasmid standard as the initial template, it was diluted 10-fold to a concentration of 3.3 × 10⁻⁶. 8 copies / uL up to 3.3×10 0Using nine gradient concentrations of positive plasmid standards as templates, and healthy sweet potato samples and Nuclease-free Water as controls, TaqMan qPCR was performed. The total volume of the reaction system was 25 μL, containing 12.5 μL of Probe qPCR Mix, 0.25 μL of LOX Reference Dye II, 2 μL of 10 ng / μL template, 0.5 μL of sweet potato leaf curl virus forward primer, 0.5 μL of sweet potato leaf curl virus reverse primer, 0.5 μL of sweet potato leaf curl virus probe primer; 0.5 μL of sweet potato hopsporidis forward primer, 0.5 μL of sweet potato hopsporidis reverse primer, 0.5 μL of sweet potato hopsporidis probe primer, and the remaining components were Nuclease-free Water. The reaction procedure was as follows: preheating at 25℃ for 2 min, pre-denaturation at 95℃ for 2 min, denaturation at 95℃ for 10 s, annealing and extension at 58-64℃ for 25 s, signal collection, and repeating 40 times.

[0060] The obtained kinetic amplification curves and PCR amplification cycle thresholds (Ct values) were plotted as a standard curve with Ct values ​​on the ordinate and the base plasmid copy number (10) on the abscissa. (See details below.) Figure 2 As shown.

[0061] The standard curve showed a linear relationship between the Ct value and the logarithm of the copy number. The standard curve equation for sweet potato leaf curl virus was y = -3.416x + 33.96, with an amplification efficiency of 98.3% and a relevant dilution Rt. 2 It is 0.997 (e.g.) Figure 2 (As shown in B). The standard curve equation for sweet potato leaf curl virus is y = -3.381x + 34.48, with an amplification efficiency of 98.6% and a relevant dilution R0. 2 It is 0.998 (e.g.) Figure 2 (as shown in D).

[0062] Example 3: Specificity Verification

[0063] Using positive samples of sweet potato leaf curl virus and sweet potato wisteria phytoplasma as positive controls, and healthy sweet potato samples and Nuclease-free Water as negative controls, SPBV, SPCV, sweet potato blast, and sweet potato black rot DNA, as well as SPMFV, SPVG, and SPLV cDNA as templates, the samples were specifically detected using a qPCR reaction system.

[0064] For details, please refer to [link / reference]. Figure 3Among them, 1-2 are positive samples of sweet potato leaf curl virus and sweet potato wilfordii; 3-9 are amplification curves of SPBV, SPCV, sweet potato blast, sweet potato black rot, SPMFV, SPVG, SPLV, and Nuclease-free Water, respectively. The results show that only the DNA of the positive samples of sweet potato leaf curl virus and sweet potato wilfordii showed amplification curves, while SPBV, SPCV, sweet potato blast, sweet potato black rot, SPMFV, SPVG, SPLV, and Nuclease-free Water did not show amplification curves. This indicates that the detection method has good specificity for the detection of sweet potato leaf curl virus and sweet potato wilfordii, and no non-specific interference was caused by other sweet potato genomes and pathogens.

[0065] Example 4: Sensitivity Verification

[0066] The positive standard was tested at 3.3 × 10⁻⁶. 8 The samples were serially diluted 10-fold (copies / µL) as templates and detected using the established TaqMan qPCR method for borderline viruses. The results were compared with conventional PCR methods.

[0067] The results show that the established method has a sensitivity of 3.3 × 10⁻⁶. 0 The copies / uL ratio is 100 times that of conventional PCR, with good sensitivity (e.g., ...). Figure 4 (As shown).

[0068] Example 5: Repeatability Test

[0069] Select 3.3×10 5 copies / uL, 3.3×10 4 copies / uL, 3.3×10 3 Using positive plasmid standards at three concentrations (copies / µL) as templates, TaqMan qPCR was performed. Each concentration was repeated three times, and intra-group repeatability tests were conducted. The coefficient of variation (Ct) was calculated. The reproducibility of the established TaqMan qPCR detection method was analyzed.

[0070] The results are shown in Table 1. The coefficient of variation for different templates was less than 1%, indicating that the established TaqMan qPCR detection method has good reproducibility.

[0071] Table 1

[0072]

[0073] In summary, this invention provides a dual real-time quantitative qPCR primer and probe set capable of simultaneously detecting sweet potato leaf curl virus (SPV) and sweet potato wilfordii. This primer and probe set provides specific, sensitive, and rapid detection of both SPV and wilfordii. Based on this primer and probe set, a TaqMan qPCR kit was designed. This kit exhibits high specificity, with good amplification curves for SPV and wilfordii, while no non-specific amplification curves were observed for other sweet potato pathogens. It also demonstrates high sensitivity (3.3 copies / µL), 100 times that of conventional PCR, and rapid detection within 1 hour with a low false positive rate. This invention solves the problems of existing detection methods, such as time-consuming and labor-intensive methods, poor specificity and sensitivity, and the inability to simultaneously detect SPV and wilfordii. It can be used for the detection of sweet potato wilfordii phytopathogens in field sweet potato seedlings and virus-free seedlings, thereby ensuring healthy sweet potato production.

[0074] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A TaqMan fluorescent quantitative detection primer and probe set for simultaneously detecting sweet potato leaf curl virus and sweet potato wisteria phytoprophyte, characterized in that: The primers include the following 6 groups: sweet potato feathery mottle virus forward primer F1: 5'-ACGTTTGTCTGTGTCTCGGA-3', as shown in SEQ ID No: 1; sweet potato feathery mottle virus reverse primer R1: 5'-GGCCACATTGTCATCCATCC-3', as shown in SEQ ID No: 2; sweet potato feathery mottle virus probe Probe1: 5'-ACCCAAGCGATGGGTAAGCCCA-3', as shown in SEQ ID No: 3; The 5' end of the probe further comprises a fluorescent group, and the 3' end further comprises a quenching group; sweet potato witches' broom phytoplasma forward primer F2: 5'-TGGCTGGAAGAGGTACAGAT-3', as shown in SEQ ID No: 4; sweet potato witches' broom phytoplasma reverse primer R2: 5'-TCGCACTTCATGCCTTTCTG-3', as shown in SEQ ID No: 5; sweet potato witches' broom phytoplasma probe Probe2: 5'-CCGCCTAACTCAGCTACACCTTCTGC-3', as shown in SEQ ID No: 6; The 5' end of the probe further comprises a fluorescent group, and the 3' end further comprises a quenching group.

2. The TaqMan fluorescent quantitative detection primer and probe set for simultaneously detecting sweet potato feathery mottle virus and sweet potato witches' broom phytoplasma according to claim 1, characterized in that: The fluorescent group comprises TAMRA or FAM; and the quenching group comprises BHQ.

3. Use of the TaqMan fluorescent quantitative detection primer and probe set for simultaneously detecting sweet potato feathery mottle virus and sweet potato witches' broom phytoplasma according to claim 1 or 2 in the preparation of a sweet potato feathery mottle virus and sweet potato witches' broom phytoplasma gene product.

4. A kit for simultaneous detection of Ipomoea leaf crinkle virus and Ipomoea bushy stunt phytoplasma, characterized by: The primer and probe set according to claim 1 or 2.

5. The kit for simultaneous detection of Ipomoea leaf crinkle virus and Ipomoea bushy top phytoplasma according to claim 4, characterized in that: The kit further comprises Probe qPCR Mix, 50x ROX Reference Dye, and Nuclease-free Water.

6. A method for detecting sweet potato feil leaf virus and sweet potato little leaf phytoplasma simultaneously, characterized in that: The kit according to claim 4 is used for TaqMan QPCR amplification of the pathogen to be detected, and the presence of sweet potato feathery mottle virus and sweet potato witches' broom phytoplasma is indicated by the detection of fluorescent signals during the amplification process.

7. The method according to claim 6, wherein the method is for simultaneous detection of Ipomoea leaf crinkle virus and Ipomoea bushy top phytoplasma. The composition of the TaqMan QPCR reaction system is as follows: The total volume of the reaction system is 25 μL, which contains 12.5 μL of Probe qPCR Mix, 0.25 μL of ROX Reference Dye II, 2 μL of 10 ng / μL of template, 0.2-0.5 μL of sweet potato feathery mottle virus forward primer, 0.2-0.5 μL of sweet potato feathery mottle virus reverse primer, 0.3-0.5 μL of sweet potato feathery mottle virus probe; 0.2-0.5 μL of sweet potato witches' broom phytoplasma forward primer, 0.2-0.5 μL of sweet potato witches' broom phytoplasma reverse primer, 0.3-0.5 μL of sweet potato witches' broom phytoplasma probe, and the remaining components are Nuclease-free Water; The TaqMan QPCR reaction program is as follows: preheating at 25 DEG C for 2 min, pre-denaturation at 95 DEG C for 2 min; denaturation at 95 DEG C for 10 s, annealing and extension at 58-64 DEG C for 25 s, signal collection, and cycling for 40 times.

8. The method according to claim 7, wherein the method is for simultaneous detection of Ipomoea leaf crinkle virus and Ipomoea bushy top phytoplasma. The initial concentration of all forward primers, reverse primers and probes in the TaqMan QPCR reaction system before being added into the reaction system is 10 mol / L.

9. The method according to claim 8, wherein the method is for simultaneous detection of Ipomoea leaf crinkle virus and Ipomoea bushy top phytoplasma. In the TaqMan QPCR reaction system, the final concentration of the sweet potato leaf roll virus forward primer and reverse primer is 0.08-0.2 mol / L; the final concentration of the sweet potato leaf roll virus probe is 0.12-0.2 mol / L; the final concentration of the sweet potato phylloid forward primer and reverse primer is 0.08-0.2 mol / L; and the final concentration of the sweet potato phylloid probe is 0.12-0.2 mol / L.

10. The method according to claim 6, wherein the method is for simultaneous detection of sweet potato feathery mottle virus and sweet potato little leaf phytoplasma. When one red S-shaped amplification curve appears and the Ct value is less than 35, it indicates that the detection sample contains the sweet potato leaf roll virus; when one blue S-shaped amplification curve appears and the Ct value is less than 35, it indicates that the detection sample contains the sweet potato phylloid; when one red and one blue S-shaped amplification curve appear and the Ct value is less than 35, it indicates that the detection sample contains the sweet potato leaf roll virus and the sweet potato phylloid; and when no S-shaped amplification curve appears or the Ct value of the amplification curve is greater than 35, it indicates that the detection sample does not contain the detected pathogen.

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