Specific primers and probes for detecting Fusarium graminearum strains and their applications

Abstract

The present invention belongs to the field of gene detection technology, and in particular to a specific primer, probe and application thereof for detecting a strain of Pseudograminearum Fusarium. The specific primer probe of the present invention includes an upstream primer q1490-F, a downstream primer q1490-R and a 1490-Taqman probe. The primers and probes can be used to amplify a single target fragment from the genomic DNA of Pseudograminearum Fusarium, while no amplification signal is present in other Fusarium, thereby achieving specific detection of Pseudograminearum Fusarium. Due to the addition of Taqman probes, 10 copies of sample DNA can be detected, achieving specific quantitative detection of Pseudograminearum Fusarium. At the same time, the content of Pseudograminearum Fusarium in soil with different degrees of wheat stem base rot was detected using the primers and probes of the invention, proving that the detection method has the characteristics of high specificity, high detection efficiency, low false positive rate and high sensitivity.

Description

Technical Field

[0001] The invention belongs to the technical field of gene detection, and in particular relates to a specific primer and a probe for detecting a pseudofusarium graminearum strain and an application thereof. Background Art

[0002] Fusarium pseudograminearium is a species of Fusarium. The wheat stem rot caused by it is an important soil-borne disease. The disease is widely distributed and causes serious damage in Australia, Canada, New Zealand, the United States, South Africa, the Middle East and other regions. In my country, wheat stem rot was first reported in Henan in 2011, especially in the Huanghuai wheat planting area, and is considered to be one of the most destructive diseases of wheat. Wheat stem rot was first discovered in Hebei Province in 2012. The disease is mainly concentrated in the central and southern regions of Hebei Province. The incidence rate of general plots is about 5%, and severe cases can reach 30%-50%. In addition, due to the changes in planting and farming methods such as wheat-corn rotation and straw return to the field, the occurrence of wheat stem rot has a tendency to increase year by year.

[0003] In recent years, wheat stem rot has been common in the Huanghuaihai wheat region of my country and has caused serious damage. Since the disease is a new soil-borne disease, there is a lack of disease-resistant varieties and no efficient prevention and control measures. The existing detection technologies for Pseudograminearum Fusarium are mixed detections of Pseudograminearum Fusarium and other Fusarium species, and cannot specifically predict the content of Pseudograminearum Fusarium in the soil, resulting in huge difficulties in the prediction and forecast of wheat stem rot. Therefore, there is an urgent need for detection technologies that can specifically detect the content of Pseudograminearum Fusarium in the soil to lay the foundation for scientifically formulating preventive measures and reducing the occurrence of diseases. Summary of the invention

[0004] The purpose of the present invention is to provide primers and probes for specifically detecting Fusarium graminearum.

[0005] Another object of the present invention is to provide a detection kit for Pseudomonas graminearum.

[0006] Another object of the present invention is to provide a method for detecting Pseudomonas graminearum.

[0007] The invention selects the gene Fp1490 of the pseudo-Fusarium graminearum strain, which is a gene with unknown function, and designs specific primers and probes for the pseudo-Fusarium graminearum according to the fragments of the gene.

[0008] According to a specific embodiment of the present invention, the specific primers and probes for detecting pseudofusarium graminearum include:

[0009] The nucleotide sequence of primer q1490-F is shown in SEQ ID NO.1:

[0010] 5′-TTGTCAAGCGCGTGCAA-3′;

[0011] The nucleotide sequence of primer q1490-R is shown in SEQ ID NO.2:

[0012] 5′-TGCACCAACTGCAAGAATCG-3′;

[0013] The nucleotide sequence is shown in SEQ ID NO.3 as primer 1490-Taqman:

[0014] 5′-ACCCCAAAGGCTC-3′.

[0015] According to the specific primers and probes for detecting Fusarium graminearum of a specific embodiment of the present invention, the 5' end of the probe is labeled with a fluorescent group, and the 3' end of the probe is labeled with a quenching group, wherein the fluorescent group may be FAM, HEX, TET, etc., and the quenching group may be TAMRA, BHQ (BHQ1, BHQ2), MGB, etc.

[0016] The present invention also provides the use of specific primers and probes for detecting Pseudofusorium graminearum, including the use of specific primers and probes in detecting and / or preventing Pseudofusorium graminearum, and the use of the specific primers and probes in preparing products for detecting and / or preventing Pseudofusorium graminearum.

[0017] According to a specific embodiment of the present invention, a detection kit for Pseudomonas graminearum comprises the above-mentioned specific primers and probes.

[0018] According to a specific embodiment of the detection kit of the present invention, the kit includes a DNA extraction solution, an amplification reaction solution, and a positive control solution.

[0019] Preferably, the amplification reaction solution includes 2x Fast qPCR premix and Taqman MGB probe.

[0020] According to a specific embodiment of the present invention, the method for detecting Fusarium graminearum includes the step of performing PCR amplification using the above-mentioned primers and probes.

[0021] The PCR amplification program is: 94°C for 3 min, 94°C for 5 s, 56°C for 15 s, 45 cycles, and the fluorescence signal is collected at the end of each extension.

[0022] The PCR amplification system is: 2xTaqMan Fast qPCR Master Mix 12.5 μL, q1490-F primer 0.5 μL, q1490-R primer 0.5 μL, 1490-Taqman probe 0.5 μL, template 5 μL, and DEPC water is used to make up to 25 μL.

[0023] The present invention also provides a method for predicting wheat stem base rot, the method comprising the following steps:

[0024] Take the soil to be tested and extract the DNA in the soil;

[0025] PCR detection was performed using the above primer probes;

[0026] Calculate the content of Pseudomonas graminearum. When the content of Pseudomonas graminearum in the soil is higher than 10 3 When the wheat reaches 3.5437 Wheat Stem Rot, it is judged that wheat stem rot has begun to occur.

[0027] Among them, "the beginning of wheat stem rot" means that the disease index of wheat stem rot is greater than 10.

[0028] Beneficial effects of the present invention:

[0029] The present invention can amplify a single target fragment from Pseudofusorium graminearum by using specific primers and probes, while no amplified bands are found in other Fusarium, Helicoverpa graminearum, Curvularia lunulata and Rhizoctonia graminearum, thereby achieving specific detection of Pseudofusorium graminearum.

[0030] The primers of the present invention have the characteristics of high specificity, high detection efficiency, low false positive rate, long storage time, etc. The primers can be used to quickly quantify the number of sources of Pseudomonas graminearum in soil by real-time quantitative PCR technology, which has the advantages of high throughput, rapidity and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 The standard curve of real-time fluorescence quantitative PCR reaction for detecting Fusarium graminearum is shown;

[0033] Figure 2 Shows the relationship between the wheat stem rot disease index and the changing content of Fusarium graminearum spores in the soil. DETAILED DESCRIPTION

[0034] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0035] Example 1 Design and screening of primers

[0036] The present invention selects the gene Fp1490 of the pseudo-Fusarium graminearum strain as the target gene through bioinformatics analysis, and further screens to obtain the following target sequence:

[0037] SEQ ID NO 4:

[0038]

[0039] Based on the above target sequence, the primer design software provides the following primers and probes:

[0040] q1490-rF: 5'-TCTTTGCATGATTCTT-3';

[0041] q1490-rR:5'-AAACAACAGAGTTGCCAT-3';

[0042] r1490-Taqman:FAM 5'-CCCAAAGGCTCCC-3'TamerMGB.

[0043] The primers and probes designed by the above software were tested for specificity, and the results were as follows:

[0044]

[0045]

[0046] Since the specificity of the above primers and probes is not ideal, the present invention designs specific primers and probes by itself:

[0047] q1490-F: 5'-TTGTCAAGCGCGTGCAA-3';

[0048] q1490-R: 5'-TGCACCAACTGCAAGAATCG-3';

[0049] 1490-Taqman:FAM 5'-ACCCCAAAGGCTC-3'TamerMGB.

[0050] Example 2 Specificity Detection

[0051] The genomic DNA of 13 Fusarium species, Rhizoctonia graminearum, Curvularia lunulata and Helminthosporium sp., totaling 38 strains, were extracted respectively, and the q1490-F / q1490-R primers and 1490-Taqman probe obtained in Example 1 were used for detection by fluorescence quantitative PCR.

[0052] Among them, 13 types of Fusarium were used, including Fusarium graminearum, Fusarium graminearum (5 strains), Fusarium acuminate (3 strains), Fusarium flavum (3 strains), Fusarium trilineatum (4 strains), Fusarium equisetum (3 strains), Fusarium genus (5 strains), Fusarium oxysporum (2 strains), Fusarium moniliforme (2 strains), Fusarium solani, Fusarium pubescens (2 strains), Fusarium gloeosporioides and Fusarium nigamei (4 strains).

[0053] The PCR amplification system was a 25 μL system, and the system was as follows: 2xTaqMan Fast qPCR Master Mix 12.5 μL, q1490-F primer 0.5 μL, q1490-R primer 0.5 μL, 1490-Taqman probe 0.5 μL, template 5 μL, and made up to 25 μL with DEPC water.

[0054] The PCR amplification conditions were 94°C for 3 min, 94°C for 5 s, 56°C for 15 s, and 45 cycles, and the fluorescence signal was collected at the end of each extension.

[0055] The test results are shown in Table 1:

[0056] Table 1 Specificity test results

[0057]

[0058]

[0059] The results are shown in Table 1. The primers and probes of the present invention have an amplification curve only for F. graminearum, and have no interference with other strains, and have good specificity.

[0060] Example 3 Establishing a standard curve

[0061] The genomic DNA of pseudograminearum Fusarium was used as a template, and the primers q1490-F / q1490-R screened in Example 1 were used for amplification. The amplified product was purified and cloned into the vector pMD19-T, and transformed into Escherichia coli Trans5a competent cells by a heat shock transformation method for reproduction. The single colony grown on the LB culture dish with ampicillin antibiotics was verified by PCR to obtain a recombinant plasmid containing a specific sequence. The recombinant plasmid was extracted using a plasmid extraction reagent (kit), and the extracted recombinant plasmid was digested with the endonuclease EcoRI for verification, and the correct recombinant vector verified by digestion was sequenced.

[0062] The correctly sequenced recombinant vector was digested by enzymes, and the product was subjected to agarose gel electrophoresis and then the corresponding fragments were recovered by cutting the gel. The fragments were recovered using the Gel Extension Kit of Bioengineering. The DNA concentration of the linearized recombinant plasmid was determined using a spectrophotometer, and the copy number of the recombinant plasmid DNA was calculated according to the formula.

[0063] The linearized plasmid vector was diluted to 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 1×10 1 copy / μL diluent.

[0064] The above-mentioned DNA with gradient dilution of copy number was used as template, q1490-F / q1490-R as primer, 1490-Taqman as probe, and TaqMan fluorescent probe method were used to perform real-time fluorescence quantitative PCR reaction. A standard curve of plasmid copy number and Ct value was established.

[0065] The results are as follows Figure 1 As shown, the standard curve established is y = -3.4445x + 37.505 (R 2 =0.999), the amplification efficiency was 95.13%, wherein x represents the logarithm of the copy number, and y represents the Ct value; the primers of the present invention were amplified in 1×10 1 ~1×10 6 The linear relationship is good within the copy number range, and the number of Pseudomonas graminearum in the sample can be accurately detected.

[0066] In addition, when the recombinant plasmid has 10 copies, the Ct value can be measured to be 33.77. Therefore, the primers of the present invention can detect that the minimum copy number of Pseudomonas graminearum is 10, and the primers of the present invention have a high sensitivity to Pseudomonas graminearum.

[0067] Example 4 Application of specific primers and probes in detecting Fusarium graminearum

[0068] The spore suspension of Pseudomonas graminearum strain 2035 was shaken to a concentration of 10 6 / mL, the spore suspension was diluted in a gradient of 10 5 , 10 4 , 10 3 , 10 2 , 10 1 The concentration of CFU / g soil was mixed into the sterilized and dried soil matrix to ensure that the amount of fungal spores in each part of the soil matrix was consistent. 600g of soil was mixed for each, and the sterilized soil without bacteria was used as a blank control. The mixed fungal soil was divided into two parts, one for extracting DNA from the soil, and the other for cultivating wheat seedlings for disease level detection; wheat seeds were planted in seedling box soil containing different amounts of bacteria, and cultured under greenhouse conditions at 25℃ during the day and 18℃ at night. After 30 days, the incidence of wheat treated with different concentrations of pathogens was investigated. At the same time, 300g of fungal soil was retained in each gradient to extract DNA after drying, and the number of pathogens was detected by qPCR.

[0069] The fitting relationship between the number of pathogens and the severity of wheat stem rot disease was analyzed.

[0070] like Figure 2 As shown in Figure 2, the disease index of wheat stem rot increases with the increase of spore content in the soil. 3 CFU / g, wheat begins to become ill, so according to the experimental results, the spore concentration in the soil is 10 3 CFU / g is the critical value for the occurrence of the disease and provides a predictive forecast for the occurrence of wheat stem rot.

[0071] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. Specific primers and probes for detecting pseudofusarium graminearum, characterized in that: include Primer q1490-F having a nucleotide sequence as shown in SEQ ID NO.1; Primer q1490-R having a nucleotide sequence as shown in SEQ ID NO.2; The nucleotide sequence of probe 1490-Taqman is shown as SEQ ID NO.

3.

2. The specific primers and probe for detecting Pseudomonas graminearum according to claim 1, characterized in that: The 5' end of the probe is labeled with a fluorescent group, and the 3' end of the probe is labeled with a quenching group.

3. Use of the specific primers and probes according to claim 1 in detecting Fusarium graminearum.

4. Use of the specific primers and probes according to claim 1 in the preparation of products for detecting Pseudomonas graminearum.

5. A detection kit for Pseudomonas graminearum, characterized in that: The kit comprises the specific primers and probes according to claim 1.

6. The detection kit according to claim 5, characterized in that The detection kit comprises a DNA extraction solution, an amplification reaction solution and a positive control solution.

7. A method for detecting Pseudomonas graminearum, characterized in that: The method comprises the step of performing PCR amplification using the specific primers and probes described in claim 1.

8. The detection method according to claim 7, characterized in that: The PCR amplification program was: 94°C for 3 min, 94°C for 5 s, 56°C for 15 s, 45 cycles, and the fluorescence signal was collected at the end of each extension.

9. The detection method according to claim 7, characterized in that: The PCR amplification system was as follows: 12.5 μL of 2xTaqMan FastqPCR Master Mix, 0.5 μL of q1490-F primer, 0.5 μL of q1490-R primer, 0.5 μL of 1490-Taqman probe, 5 μL of template, and the volume was made up to 25 μL with DEPC water.

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