Sweet potato arbuscular disease phytoplasma TaqMan fluorescent quantitative detection primer as well as kit and detection method of sweet potato arbuscular disease phytoplasma TaqMan fluorescent quantitative detection primer

Abstract

The invention discloses a sweet potato arbuscular disease phytoplasma TaqMan fluorescent quantitative detection primer, a kit and a detection method, the primer and a probe group are specially used for specific detection of sweet potato arbuscular disease phytoplasma; the invention also designs a fluorescence quantification (QPCR) kit based on the primer probe group. The detection primer group is as follows: a forward primer F as shown in SEQ ID No: 1, a reverse primer R as shown in SEQ ID No: 2, and a probe primer Probe as shown in SEQ ID No: 3; the sweet potato arbuscular disease phytoplasma can be specifically amplified within 1 hour by extracting the DNA of the sweet potato plant infected with the phytoplasma and carrying out QPCR, and the detection sensitivity is 10 copies / uL. According to the TaqMan QPCR detection technology established for the sweet potato arbuscular disease phytoplasma, the problems that an existing sweet potato arbuscular disease phytoplasma detection method is time-consuming and labor-consuming, poor in specificity and sensitivity, easy to cause pollution and the like are solved, and a technical basis is provided for early diagnosis of the sweet potato arbuscular disease phytoplasma.

Description

[Technical field]

[0001] The invention belongs to the technical field of pathogen detection, and specifically relates to a TaqMan fluorescent quantitative detection primer for sweet potato witches' broom phytoplasma and a kit and a detection method thereof. [Background technology]

[0002] Sweet potato is an asexually propagated crop that reproduces by tubers or vines. It is often infected by pathogens. After the pathogens infect sweet potatoes, they will be passed on from generation to generation, thus affecting the growth and development of sweet potatoes, causing the degradation of germplasm and species, and then affecting the quality and yield of sweet potatoes. Sweet potato witches'-broom disease is a devastating disease caused by phytoplasma (CandidatusPhytoplasma). After sweet potatoes are infected with phytoplasma, their early growth period is mainly manifested by dwarfing of plants, small leaves, yellowing lateral branches and clusters of small leaves. Initially, the leaves of the top vines become smaller, atrophied, and the leaf color is lighter. Then, the lower lateral buds of the top vines continue to sprout, the internodes shorten, and the symptoms of arbuscular branches and clusters of leaves are formed. In the middle and late stages of the disease, the tubers are small and shriveled, the potato skin becomes rough and has protrusions, and the tubers cannot be cooked. Sweet potato witches' broom caused by phytoplasmas was first reported in the southeast coast (Fujian). Currently, sweet potato phytoplasma diseases have become one of the important diseases in my country's sweet potato producing areas, especially in the southern sweet potato producing areas.

[0003] So far, China has reported 52 tentative species, 34 groups, and more than 100 subgroups of phytoplasmas, mainly caused by 11 groups of phytoplasma groups, including 16SrI, 16SrII, 16SrIII, 16SrV, 16SrVI, 16SrVII, 16SrVIII, 16SrX, 16SrXI, 16SrXII, and 16SrXIV, distributed throughout the country. The phytoplasmas reported in my country mainly belong to 11 groups, among which 16SrI members are the most, followed by 16SrII and 16SrV members. The phytoplasmas reported to cause sweet potato witches' broom disease are mostly 16SrII group. Gundersen and Lee et al. reported that the phytoplasma causing sweet potato witches' broom disease is 16SrII group. New phytoplasmas are constantly being reported, which shows that phytoplasmas are more diverse than previously thought. The inventor's research group has conducted preliminary investigations and preliminary identifications and found that the pathogen causing sweet potato witches' broom disease is phytoplasma CandidatusPhytoplasma. The 16S rRNA identification result shows that it is a member of 16SrII A group. The secY gene sequence of the sweet potato witches' broom disease phytoplasma was cloned and a secY recombinant plasmid of the sweet potato witches' broom disease phytoplasma was constructed. secY is a highly conserved segment of the phytoplasma genome and is commonly used as a target gene for phytoplasma molecular biology detection.

[0004] Accurate detection of pathogens is a prerequisite for effective disease prevention and control, and is conducive to cutting off the spread of diseases at the source. Since phytoplasmas are polymorphic, cell wall-free prokaryotes that parasitize the phloem of plants and are difficult to culture artificially, traditional pathogen identification methods are not suitable for phytoplasmas, which brings certain difficulties to the prevention and control of phytoplasmas. At present, the main methods for detecting sweet potato witches' broom phytoplasmas include grafting, electron microscopy, and 16SrRNA nested PCR. However, these methods cannot meet the needs of modern production due to their low sensitivity, long cycle, high false positive rate, cumbersome operation, time-consuming and labor-intensive operation, etc. Therefore, it is very important to establish a set of rapid, accurate and efficient sweet potato witches' broom phytoplasma detection technology to provide technical support for preventing sweet potato witches' broom and improving the quality of virus-free seedlings.

[0005] In summary, TaqMan QPCR detection technology has been widely used in the detection of animal and plant pathogens because of its sensitivity, specificity and rapidity. However, the application of this technology in the detection of sweet potato witches' broom phytoplasma has not been reported. Therefore, the establishment of TaqMan QPCR detection technology for sweet potato witches' broom phytoplasma has broad application prospects in the rapid diagnosis of sweet potato pathogens. [Summary of the invention]

[0006] The technical problem to be solved by the present invention is to provide a TaqMan fluorescent quantitative detection primer for sweet potato witches' broom disease phytoplasma and a kit and a detection method thereof. The primer has the advantages of rapidity, sensitivity, qualitative and quantitative detection, and can be used for the detection of sweet potato witches' broom disease phytoplasma in field sweet potato seedlings, virus-free seedlings, etc., thereby ensuring the healthy production of sweet potatoes.

[0007] The present invention is achieved in that:

[0008] A TaqMan fluorescent quantitative detection primer for sweet potato witches' broom phytoplasma, wherein the TaqMan detection primer comprises the following primer set:

[0009] Forward primer F: 5'-GGAATGAACAAGGTTCTATTGGTAAG-3', as shown in SEQ ID No: 1;

[0010] Reverse primer R: 5'-GCAATATCAAACATTCTAGAGCGAA-3', as shown in SEQ ID No: 2;

[0011] Probe primer: 5'-AGCTTTAGGACAAGGATTATCCTCTGT-3', as shown in S EQ ID No: 3;

[0012] The 5' end of the probe also contains a fluorescent group, and the 3' end also contains a quenching group;

[0013] The fluorescent group includes FAM; the quenching group includes BHQ.

[0014] Furthermore, a kit for specifically detecting sweet potato witches' broom phytoplasma comprises the primer probe set and QPCR detection reagent.

[0015] Furthermore, the QPCR detection kit comprises: IIProbe qPCR SuperMixUDG, 50×ROX Reference Dye II, positive standard, negative control and Nuclease-free Water.

[0016] Furthermore, the positive standard contains a recombinant plasmid of the target fragment of the secY gene of the sweet potato witches' broom phytoplasma amplified by an upstream primer with a nucleotide sequence as shown in SEQ ID No: 1 and a downstream primer with a nucleotide sequence as shown in SEQ ID No: 2; and the negative control is Nuclease-free Water.

[0017] Further, a TaqMan QPCR method for qualitatively detecting sweet potato witches' broom phytoplasma comprises the following steps:

[0018] (1) DNA extraction: Process the sample and extract the sample DNA;

[0019] (2) Mix the sample DNA with the IIProbe qPCR SuperMix UDG, 50×ROX Reference Dye II, forward primer, reverse primer, probe primer set and Nuclease-free Water were used to prepare QPCR reaction system. The detection of fluorescent signal indicated the presence of sweet potato witches' broom phytoplasma.

[0020] Further, a TaqMan QPCR method for quantitatively detecting sweet potato witches' broom phytoplasma comprises the following steps:

[0021] (1) The positive sample in the kit was diluted 10 times in a gradient manner as a positive template and then mixed with the positive sample in the kit. IIProbe qPCR SuperMix UDG, 50×ROX Reference Dye II, forward primer, reverse primer, probe primer set and Nuclease-free Water were used to prepare QPCR reaction system, and QPCR reaction was performed to establish the standard curve of fluorescence quantitative detection;

[0022] (2) Using the sample DNA to be tested as a template, IIProbe qPCRSuperMix UDG, 50×ROX Reference Dye II, forward primer, reverse primer, probe primer set and Nuclease-free Water were used to prepare the QPCR reaction system, and the QPCR reaction was performed to quantify the phytoplasma copy number in the sample to be tested according to the standard curve established in (1).

[0023] Furthermore, the QPCR reaction system 20 μL contains IIProbe qPCR SuperMix UDG 10μL, 10μmol / L upstream primer and downstream primer 0.2μL each, 10μmol / L probe primer 0.2μL, 50×ROXReference Dye II 0.4μL template DNA 2μL, and the rest is Nuclease-free Water.

[0024] Furthermore, the QPCR reaction procedure is: 25°C for 3 min, 95°C for 5 min, 95°C for 10 sec, annealing and extension at 60°C for 30 sec, collecting fluorescence signals, and 40 cycles.

[0025] The present invention has the following advantages:

[0026] The present invention provides a primer and probe set for specific detection of sweet potato witches' broom phytoplasma, and the primer and probe set can specifically, sensitively and quickly detect sweet potato witches' broom phytoplasma. The present invention also designs a QPCR kit based on the primer and probe set, and the detection result of the kit has good specificity, the amplification curve of sweet potato witches' broom phytoplasma is good, and other sweet potato pathogens do not show specific amplification curves, and the sensitivity is high, and the detection sensitivity is 10 copies / μL, which is 10 times that of ordinary PCR; the detection is fast, and the detection can be completed within 1 hour, and false positives are not easy to occur. The problems of time-consuming and labor-intensive, poor specificity and sensitivity of the existing detection method are solved, and it can be used for the detection of sweet potato witches' broom phytoplasma in field sweet potato seedlings, virus-free seedlings, etc., so as to ensure the healthy production of sweet potatoes.

Brief Description of the Drawings

[0027] The present invention will be further described below in conjunction with embodiments with reference to the accompanying drawings.

[0028] Figure 1 The standard curve amplification diagram of the sweet potato witches' broom phytoplasma TaqMan QPCR detection method in Example 1 of the present invention; the curves A in the figure are 1×10 8 copies / μL to 1×10 1 A is the amplification curve of the positive standard diluted with copies / μL, and B is the standard curve graph.

[0029] Figure 2 This is a specific amplification diagram of the sweet potato witches' broom phytoplasma TaqMan QPCR detection method in Example 2 of the present invention.

[0030] Figure 3 The sensitivity detection diagram of sweet potato witches' broom phytoplasma in Example 3 of the present invention, wherein A is a sensitivity curve diagram of the TaqMan QPCR detection method, and B is a sensitivity electrophoresis diagram of the PCR detection method. The sample concentrations of lanes 1-11 are 1:10 respectively. 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:ddH2O、11:ddH2O. [Specific implementation method]

[0031] The following will be combined with the attached Figure 1-3 The technical scheme of the present invention is clearly and completely described in detail with specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0032] Example 1. TaqMan QPCR detection method standard curve

[0033] The invention provides a sweet potato witches' broom phytoplasma TaqMan QPCR detection primer and probe set, comprising a forward primer F as shown in SEQ ID No: 1; a reverse primer R as shown in SEQ ID No: 2; and a probe primer Probe as shown in SEQ ID No: 3.

[0034] Test samples: sweet potato witches' broom phytoplasma genomic DNA, sweet potato feathery mottle virus (SPMFV), sweet potato virus G (SPVG), sweet potato latent virus (SPLV) cDNA; sweet potato leaf curl virus (SPLCV), sweet potato blight, and sweet potato black rot DNA were all provided by the Crop Research Institute of Fujian Academy of Agricultural Sciences.

[0035] The specific steps are as follows: According to the secY gene sequence in the phytoplasma 16SrII A subgroup genome published by GenBank, the 16SrII A subgroup secY gene sequence was designed and amplified, and the secY sequence was obtained by amplification to design TaqMan QPCR detection primers and probe sets, upstream primer F (SEQ ID No: 1): 5'-GGAATGAACAAGGTTCTATTGGTAAG-3'; downstream primer R (SEQ ID No: 2): 5'-GCAATATCAAACATTCTAGAGCGAA-3'; probe primer Probe (SEQ ID No: 3): 5'-AGCTTTAGGACAAGGATTATCCTCTGT-3', the 5' end of the probe primer Probe was modified by FAM; the 3' end was modified by BHQ, and the primers and probes were synthesized by Sangon Biotechnology Co., Ltd. and purified by HPLC.

[0036] DNA extraction: DNA was extracted according to the instructions of the plant total DNA extraction kit and stored in a -20°C refrigerator.

[0037] Preparation of positive recombinant plasmid standard, using primers secYf / secY-r, positive sweet potato witches' broom sample DNA as template, healthy sweet potato sample DNA negative control, ddH2O as blank control, PCR amplification. PCR product was detected by agarose gel electrophoresis, the gel with the target band was cut out, and the target band was purified using an agarose gel recovery kit. The purified product was connected to Zero Cloning Kit, and transformed into DH5a competent cells, screened and extracted the plasmids of positive clones, and sent them to Shanghai Biotech Co., Ltd. for sequencing. The concentration of the correctly sequenced plasmid was measured using an ultra-micro nucleic acid analyzer and stored at -20°C for later use. The copy number of the recombinant plasmid was calculated using the Shirima method (Shirima RR et al, 2017). The recombinant plasmid copy number (copies / uL) = (60.2×10 23 × plasmid concentration) / (10 9 × the number of base pairs of the recombinant plasmid × 660 dalton / bp). The final calculated copy number of the recombinant plasmid was 1.71 × 10 10 copies / uL.

[0038] The positive plasmid standard was used as the initial template and diluted 1.71×10 8 copies / uL to 1.71×10 1copies / uL, using 8 gradient concentrations of positive plasmid standards as templates, healthy sweet potato samples and Nuclease-free Water as controls, QPCR was performed, the reaction system II Probe qPCR SuperMix UDG 10μL, 10μmol / L upstream primer and downstream primer 0.2μL each, 10μmol / L probe primer 0.2μL, 50×ROX Reference Dye II 0.4μL, template DNA 2μL, and the rest is Nuclease-free Water. The reaction program is: 25℃3min, 95℃5min, 95℃10sec, annealing and extension at 60℃30sec, collect fluorescence signals, 40 cycles.

[0039] The obtained kinetic amplification curve and PCR amplification cycle threshold (Ct value) were used to draw a standard curve with the Ct value as the ordinate and the plasmid copy number of 10 as the base and the vertical axis as the abscissa.

[0040] The standard curve showed that the Ct value was linearly related to the logarithm of the copy number. The standard curve equation was y = -0.35617x + 37.953, the amplification efficiency was 94.2%, and the related dilution R 2 is 0.9994 (e.g. Figure 1 B).

[0041] Example 2: Specificity verification of TaqMan QPCR detection method

[0042] The recombinant plasmid of sweet potato witches' broom phytoplasma and positive sample DNA were used as positive controls, healthy sweet potato samples and Nuclease-free Water were used as negative controls, and SPMFV, SPVG, SPLV cDNA, SPLCV, sweet potato blight and sweet potato black rot DNA were used as templates for specific detection using the QPCR reaction system.

[0043] Specific reference Figure 2 , among which 1-2: positive plasmid; 3-5 positive DNA, 6-13: SPMFV, SPVG, SPLV, SPLCV, sweet potato blight, sweet potato black rot, healthy sweet potato, Nuclease-free Water. The results showed that only the recombinant plasmid of sweet potato witches' broom phytoplasma and the positive sample DNA showed amplification curves, while the healthy samples, SPMFV, SPVG, SPLV, SPLCV, sweet potato blight, and sweet potato black rot had no expansion curves, indicating that the detection method has good specificity for the detection of sweet potato witches' broom phytoplasma, and other sweet potato genomes and pathogens did not produce nonspecific interference.

[0044] Example 3: Sensitivity verification of TaqMan QPCR detection method

[0045] The positive standard was made into 1×10 8 copies / uL to 1×10 0 The established TaqMan QPCR detection method for border disease virus was used as a template after 10-fold serial dilution of copies / uL, and compared with the common PCR detection method.

[0046] The results showed that the sensitivity of the established method could reach 10 copies / uL, which is 10 times that of ordinary PCR, and the sensitivity is good (such as Figure 3 shown).

[0047] Example 4: Repeatability test of TaqMan QPCR detection method

[0048] Select 1.71×10 5 copies / uL, 1.71×10 4 copies / uL, 1.71×10 3 The QPCR test was performed with 1000 copies / uL and three concentrations of positive plasmid standards as templates. Each concentration was repeated 3 times, and the intra-group repeatability test was performed to calculate the coefficient of variation of the Ct value. The repeatability of the established QPCR detection method was analyzed.

[0049] The results are shown in Table 1. The coefficients of variation of different templates were all less than 1%, indicating that the established QPCR detection method has good repeatability.

[0050] Table 1

[0051]

[0052] In summary, the present invention provides a primer and probe set for specific detection of sweet potato witches' broom phytoplasma, and the primer and probe set can specifically, sensitively and rapidly detect sweet potato witches' broom phytoplasma. The present invention also designs a QPCR kit based on the primer and probe set, and the detection result of the kit has good specificity, the amplification curve of sweet potato witches' broom phytoplasma is good, and other sweet potato pathogens do not show specific amplification curves, and the sensitivity is high, and the detection sensitivity is 10 copies / μL, which is 10 times that of ordinary PCR; the detection is fast, and the detection can be completed within 1 hour, and false positives are not easy to occur. The problems of time-consuming and labor-intensive, poor specificity and sensitivity of the existing detection methods are solved, and it can be used for the detection of sweet potato witches' broom phytoplasma in field sweet potato seedlings, virus-free seedlings, etc., thereby ensuring the healthy production of sweet potatoes.

[0053] Although the specific implementation modes of the present invention are described above, those skilled in the art should understand that the specific implementation modes described are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A TaqMan fluorescent quantitative detection primer for sweet potato witches' broom phytoplasma, characterized in that: The TaqMan detection primers include the following primer set: Forward primer F: 5'-GGAATGAACAAGGTTCTATTGGTAAG-3', as shown in SEQ ID No: 1; Reverse primer R: 5'-GCAATATCAAACATTCTAGAGCGAA-3', as shown in SEQ ID No: 2; Probe primer: 5'-AGCTTTAGGACAAGGATTATCCTCTGT-3', as shown in SEQ ID No: 3; The 5' end of the probe also contains a fluorescent group, and the 3' end also contains a quenching group; The fluorescent group includes FAM; the quenching group includes BHQ.

2. A kit for specifically detecting sweet potato witches' broom phytoplasma, characterized in that: It comprises the primer probe set and QPCR detection reagent according to claim 1.

3. A kit for specifically detecting sweet potato witches' broom phytoplasma according to claim 2, characterized in that: The QPCR detection kit comprises: IIProbe qPCR SuperMix UDG, 50×ROX ReferenceDye II, positive standard, negative control and Nuclease-free Water.

4. A kit for specifically detecting sweet potato witches' broom phytoplasma according to claim 3, characterized in that: The positive standard contains a recombinant plasmid of the target fragment of the sweet potato witches' broom phytoplasma secY gene amplified by an upstream primer with a nucleotide sequence as shown in SEQ ID No: 1 and a downstream primer with a nucleotide sequence as shown in SEQ ID No: 2; the negative control is Nuclease-free Water.

5. A TaqMan QPCR method for qualitative detection of sweet potato witches' broom phytoplasma, characterized in that: The following steps are involved: (1) DNA extraction: Process the sample and extract the sample DNA; (2) mixing the sample DNA with the kit described in claim 3 IIProbe qPCR SuperMix UDG, 50×ROX Reference Dye II, forward primer, reverse primer, probe primer set and Nuclease-free Water were used to prepare the QPCR reaction system. The detection of fluorescent signal indicated the presence of sweet potato witches' broom phytoplasma.

6. A TaqMan QPCR method for quantitative detection of sweet potato witches' broom phytoplasma, characterized in that: The following steps are involved: (1) The positive sample in the kit of claim 3 or 4 is diluted 10 times in a gradient manner as a positive template and then mixed with the positive sample in the kit IIProbe qPCR SuperMix UDG, 50×ROX Reference Dye II, forward primer, reverse primer, probe primer set and Nuclease-free Water were used to prepare QPCR reaction system, and QPCR reaction was performed to establish the standard curve of fluorescence quantitative detection; (2) Using the sample DNA to be tested as a template, IIProbe qPCR SuperMixUDG, 50×ROX Reference Dye II, forward primer, reverse primer, probe primer set and Nuclease-free Water were used to prepare the QPCR reaction system, and the QPCR reaction was performed to quantify the phytoplasma copy number in the sample to be tested according to the standard curve established in (1).

7. A TaqMan QPCR method for quantitatively detecting sweet potato witches' broom phytoplasma according to claim 6, characterized in that: QPCR reaction system 20μL, including IIProbe qPCR SuperMix UDG 10μL, 10μmol / L upstream primer and downstream primer 0.2μL each, 10μmol / L probe primer 0.2μL, 50×ROX Reference Dye II 0.4μL template DNA 2μL, and the rest is Nuclease-free Water.

8. The TaqMan QPCR method for quantitatively detecting sweet potato witches' broom phytoplasma according to claim 6, characterized in that: The QPCR reaction program is: 25°C for 3 min, 95°C for 5 min, 95°C for 10 sec, annealing and extension at 60°C for 30 sec, collecting fluorescence signals, and 40 cycles.

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