CN117165711A - RPA-LFD primer probe group for detecting avirulence gene Avr-Piz-t, kit and application thereof - Google Patents
RPA-LFD primer probe group for detecting avirulence gene Avr-Piz-t, kit and application thereof Download PDFInfo
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Abstract
The application discloses an RPA-LFD primer probe group for detecting a nontoxic gene Avr-Piz-t, a kit and application thereof. The nucleotide sequence of the primer is as follows: forward primer 5'-TTCTCAACCATCATCACCGTGTGCCTCTTC-3' (SEQ ID NO: 1); reverse primer: 5'-CCAACGGTGCCGGAGGAGAGAACATCAGTG-3' (SEQ ID NO: 2); the nucleotide sequence of the probe is as follows: 5'-FAM-GCTTCGTACAATGCAATCATCATCTCCTGT-THF-CAATGGCAGACACTG-3' (SEQ ID NO: 3). Meanwhile, the application also provides a kit containing the primer probe group. The application establishes a method for rapidly detecting the avirulence gene Avr-Piz-t by adopting the RPA-LFD for the first time, can be used for detecting the avirulence gene of the rice blast bacteria by specificity and sensitivity evaluation, and provides a new technical platform for detecting the avirulence gene Avr-Piz-t. Meanwhile, by combining a polyethylene glycol-sodium hydroxide (PEG-NaOH) nucleic acid extraction technology, the rapid and accurate detection of pathological tissues in the field is realized.
Description
Technical Field
The application relates to the technical field of non-toxic gene detection, in particular to a primer probe group for detecting a non-toxic gene Avr-Piz-t, a kit and application thereof.
Background
Rice blast fungus (Magnaporthe oryzae) is a seed bag fungus, and the rice blast caused by the seed bag fungus is one of the most serious diseases affecting global rice production. At present, the cultivation of resistant varieties is the most economical, effective and environment-friendly measure for preventing and treating rice blast. Up to now, more than 100 major Resistance (Resistance) genes have been found in cultivated and wild rice, of which more than 30 have been cloned. The vast majority of disease-resistant genes are proteins of the type that encode NLR (nucleic acid-binding-rich repeat), and the "gene-to-gene" hypothesis is met between these disease-resistant genes and Avirulence genes (Avirulence genes), i.e., the specific interaction between the disease-resistant genes and the Avirulence genes induces the host to produce a disease-resistant response. Therefore, the distribution frequency of the nontoxic genes can be used as an important index of the same effectiveness as the corresponding disease resistance genes. In conclusion, the monitoring of the distribution of the main Avr genes has important significance for guiding scientific layout and breeding of rice disease-resistant varieties.
To date, 12 avirulence genes have been cloned. The avirulence gene Avr-Piz-t of the rice blast fungus is obtained by identification in 2009, and the Avr-Piz-t regulates the pathogenicity of the rice blast fungus and the disease resistance of the rice through target rice transcription factors APIP5, E3 ubiquitin ligase APIP6 and APIP10, potassium ion channel protein OsAKT1 and trypsin inhibitor APIP 4. The research result of the research room shows that the distribution frequency of Avr-Piz-t in 1183 single spore strains collected in 2017-2021 of Jiangsu province is higher and reaches more than 80%, so that the resistance level of rice varieties containing disease-resistant genes Piz-t corresponding to the Avr-Piz-t is higher, but the distribution frequency of Avr-Piz-t fluctuates among years, so that the non-toxic genes need to be monitored in real time.
Currently, molecular detection methods such as PCR (Polymerase chain reaction), qRT-PCR (qReal-Time PCR) and Lamp (Loop mediated isothermal amplification) have been applied to detection of part of plant pathogenic bacteria, and these detection methods have better accuracy and sensitivity, but have strict technical requirements on equipment and personnel, and are not suitable for field identification. Recombinase (Recombinase polymerase amplification, RPA) was first proposed by Twist DX in 2006. The principle is that under the constant temperature condition, the T4Uvs X protein is combined with a primer to form a nucleoprotein filament with the assistance of T4Uvs Y. The resulting complex searches for homologous sequences in double stranded DNA. The recombinase enables the primer to determine the homologous sequence of the DNA double stranded template and to melt the template DNA with the aid of the single stranded DNA binding protein SSB, and then form a new complementary DNA strand in the presence of the DNA aggregate.
The Jiangsu province is a main production area of Chinese rice, the annual planting area reaches 220 ten thousand hectares, the temperature in the area is proper, the precipitation is more, the rice blast commonly occurs in the Jiangsu rice planting area, and the planting of disease-resistant varieties is the most economic and effective way for preventing and controlling the rice blast. In order to guide the scientific layout of field disease-resistant varieties and achieve the aim of disease prevention and control, real-time monitoring of avirulence genes Avr-Piz-t of rice blast bacteria is needed, but no detection method capable of effectively detecting the avirulence genes in fields exists at present. Secondly, the existing detection methods all need to separate pathogenic bacteria in a laboratory, extract genome DNA, and amplify by designing specific primers, and the sensitivity can reach ng level, but the method is complicated, has the defects of high technical threshold and low sensitivity, and the distribution frequency analysis accuracy of the avirulence gene Avr-Piz-t is not high due to the limitation of the sample amount of the separated strain.
Disclosure of Invention
In order to solve the technical problems, the application provides an RPA-LFD primer probe group for detecting a nontoxic gene Avr-Piz-t, a kit and application thereof. The primer probe or the kit can realize real-time monitoring of the avirulence gene Avr-Piz-t of the rice blast bacteria, and can effectively monitor the avirulence gene Avr-Piz-t of the rice blast bacteria in the field without separating pathogenic bacteria in a laboratory.
In order to achieve the above purpose, the technical scheme provided by the application is as follows:
in a first aspect, the present application provides a primer and probe combination for detecting avirulence gene Avr-Piz-t, wherein the nucleotide sequence of the primer is as follows: forward primer
5'-TTCTCAACCATCATCACCGTGTGCCTCTTC-3' (SEQ ID NO: 1); reverse primer: 5'-CCAACGGTGCCGGAGGAGAGAACATCAGTG-3' (SEQ ID NO: 2); the nucleotide sequence of the probe is as follows: 5'-GCTTCGTACAATGCAATCATCATCTCCTGT-THF-CAATGGCAGACACTG-3' (SEQ ID NO: 3).
Further, FAM is added to the 5 'end of the probe, and C3-spacer is added to the 3' end of the probe.
In a second aspect, the present application also provides an RPA-LFD method for detecting avirulence gene Avr-Piz-t, comprising the steps of:
1) Extracting DNA of a sample to be detected;
2) Using DNA as a template, adopting the primer and probe combination of the first aspect of the application to carry out RPA amplification;
3) Detecting an amplification product by using a lateral flow chromatography test strip; when the test strip has two brown strips, one strip is positioned in the quality control area, and the other strip is positioned in the detection area, the result is positive, which indicates that the sample contains a nontoxic gene Avr-Piz-t; when the test strip has only a brown band in the quality control area and no band in the detection area, the result is negative, which indicates that the sample does not contain the avirulence gene Avr-Piz-t.
Further, the RPA amplification in step 2): buffer 25. Mu.L, 10. Mu.M upstream primer 2.1pL, 10. Mu.M downstream primer 2.1. Mu.L, probe 0.6. Mu.L, DNA2.0uL, starter 3. Mu.L inside the PCR tube cap and deionized water to 47. Mu.L were added to a 0.2mL TwistAmp reaction unit tube containing lyophilized enzyme powder; and (3) fully and uniformly mixing the RPA amplification system, centrifuging for 10s at 5.000xg, placing the mixture on a metal bath at 39 ℃ for reaction for 20min, incubating for 4 min, uniformly mixing the reaction tube again, centrifuging for 3-5s, and placing the mixture in a water bath kettle at 39 ℃ for continuous reaction for 16min.
In a third aspect, the application also provides an RPA-LFD kit for detecting avirulence gene Avr-Piz-t, which at least comprises more than 1 dose of any one of the primer and probe combination reagent.
Further, the RPA-LFD kit also comprises: standard positive DNA, buffer, sterile double distilled water, reaction driving solution, product dilution, and lateral flow chromatography reagents.
In a fourth aspect, the application also provides an application of the primer and probe combination according to the first aspect or the kit according to the third aspect in detection of avirulence gene Avr-Piz-t.
Compared with the prior art, the method can realize real-time monitoring of the avirulence gene Avr-Piz-t of the rice blast bacteria, does not need to separate pathogenic bacteria in a laboratory, can effectively monitor the avirulence gene Avr-Piz-t of the rice blast bacteria in the field, and is convenient and quick. Meanwhile, the RPA-LFD primer probe group and the kit for detecting the avirulence gene Avr-Piz-t provided by the application can detect the sensitivity of the avirulence gene Avr-Piz-t to 100 fg. Mu.L -1 And has specificity. Therefore, the detection method of the RPA-LFD of the avirulence gene Avr-Piz-t can guide the scientific layout of the disease-resistant varieties in the field so as to achieve the aim of disease prevention and control.
Drawings
FIG. 1 shows the result of specific detection of the RPA-LFD method of Avr-Piz-t gene, wherein, the 1 st to the 12 th rice blast bacteria isolated from Jiangsu are 12 rice blast bacteria, the 13 th to the 15 th rice blast bacteria are other 3 plant pathogenic bacteria, and the 16 th rice blast bacteria are negative controls.
FIG. 2 is the sensitivity detection result of the RPA-LFD method of Avr-Piz-t gene.
FIG. 3 is the results of the inoculation experiment of the RPA-LFD method for detecting Avr-Piz-t gene.
Detailed Description
The application is described in further detail below with reference to the drawings and examples, in which: the following examples, in which no specific conditions are noted, are conducted under conventional conditions or conditions recommended by the manufacturer, and the raw materials used in the following examples are commercially available from ordinary sources except for the specific descriptions.
Example 1
Based on the sequence of the gene Avr-Piz-t (XP_ 003721287.1), the forward primer 5'-TTCTCAACCATCATCACCGTGTGCCTCTTC-3' (SEQ ID NO: 1) and the reverse primer were designed: 5'-CCAACGGTGCCGGAGGAGAGAACATCAGTG-3' (SEQ ID NO: 2), probes: 5'-GCTTCGTACAATGCAATCATCATCTCCTGT-THF-CAATGGCAGACACTG-3' (SEQ ID NO: 3) the probe has FAM added at its 5 'end and C3-spacer at its 3' end.
In order to verify the specificity of the RPA lateral flow chromatography test strip detection method, 12 rice blast bacteria and 3 other plant pathogenic bacteria separated from Jiangsu are used as experimental materials, and the details are shown in Table 1.
TABLE 1 plant pathogenic bacteria experimental materials
Extracting genome DNA as a template, and adopting a primer combination of Avr-Piz-t genes to perform RPA reaction in the following reaction system: buffer 25. Mu.L, 10. Mu.M upstream primer 2.1pL, 10. Mu.M downstream primer 2.1. Mu.L, probe 0.6. Mu.L, DNA2.0uL, starter 3. Mu.L inside the PCR tube cap and deionized water to 47. Mu.L were added to a 0.2mL TwistAmp reaction unit tube containing lyophilized enzyme powder; and (3) fully and uniformly mixing the RPA amplification system, centrifuging for 10s at 5.000xg, placing the mixture on a metal bath at 39 ℃ for reaction for 20min, incubating for 4 min, uniformly mixing the reaction tube again, centrifuging for 3-5s, and placing the mixture in a water bath kettle at 39 ℃ for continuous reaction for 16min. Meanwhile, negative control: procedure sample detection, 2.0. Mu.L of template DNA was changed to 2.0. Mu.L of sterilized ddH 2 O。
The result of the RPA lateral flow chromatography test strip detection method shows that two brown strips appear on the test strip of Jiangsu isolate, one strip is positioned in the quality control area, and the other strips of pathogenic fungi are positive if the result is positive, and only one brown strip appears on the quality control area, and the result is negative if the test strip does not appear on the detection area, so that the sample does not contain the gene Avr-Piz-t.
As shown in FIG. 1, 8 strains of Pyricularia oryzae containing avirulence gene Avr-Piz-t can be detected by using the test strip, and the other 4 strains of Pyricularia oryzae can not detect Avr-Piz-t, which shows that the detection rate of Avr-Piz-t in the 12 strains of Pyricularia oryzae is 66.67%; the Avr-Piz-t could not be detected by the other 3 pathogens and clear water (negative control), and the same results were obtained by PCR, which indicated that the RPA primer and probe specificity in this study was very high.
Example 2
DNA concentration of the positive strain for Avr-Piz-t Gene was determined to be 100 ng/. Mu.L using Nanodro 2000 micro-spectrophotometer -1 Which are diluted to 100 ng/. Mu.L in turn -1 、10ng·μL -1 、1ng·μL -1 、100pg·μL -1 、10pg·μL -1 、1pg·μL -1 、100fg·μL -1 、10fg·μL -1 And 1 fg. Mu.L -1 The RPA and PCR assays were performed on DNA at different concentrations, according to the primers, reaction system and reaction conditions used in example 1.
As a result, as shown in FIG. 2, each of the 25. Mu.L reaction systems contained 100 ng. Mu.L -1 、10ng·μL -1 、1ng·μL -1 、100pg·μL -1 、10pg·μL -1 、1pg·μL -1 、100fg·μL -1 Two brown bands appear on the Avr-pizz gene test strip, and a 25 mu L reaction system contains 1 fg. Mu.L -1 The paper strip of the Avr-Piz-t gene of (2) presents a brown band and has negative reaction; the color development result shows that the sensitivity of the RPA side-flow chromatography test strip reaches 100 fg.mu.L -1 。
The genomic DNA of the Avr-Piz-t gene positive strain with the same concentration was used as an amplification template, and a PCR amplification reaction was performed by a conventional PCR method (Avr-Piz-t-F: 5'-AATCCCGTCACTTTCATTCTCC-3' (SEQ ID NO: 4); avr-Piz-t-R:5'-GTCGCAAGCCTCGTACTACCTTT-3' (SEQ ID NO: 5)), and the sensitivities of the two methods were compared; the experiment was repeated 3 times to determine the sensitivity of the PCR method to detect the genomic DNA of the Avr-Piz-t gene positive strain. As shown in FIG. 2, the concentration of genomic DNA was 100 ng/. Mu.L -1 、10ng·μL -1 、1ng·μL -1 When the PCR detection is carried out, the specificity band can be detected, the specificity amplification is proved to occur, and the detection result is judged to be positive.
The result shows that the concentration of PCR detection is 1ng mu L -1 Whereas the RPA detection concentration was 100 fg. Mu.L -1 The target strip can still be seen, which shows that the sensitivity of the RPA side-stream chromatography test strip detection method is 10 higher than that of the PCR method 4 Multiple times. Meanwhile, the PCR detection process needs 2.5 hours, the RPA detection time only needs 30 minutes, and expensive instruments such as a PCR instrument and the like are not neededThe device has simple and convenient operation program and is more beneficial to popularization and application in production.
Example 3
In order to evaluate the practicability of the RPA-LFD detection method of Avr-Piz-t gene, the application detects the pathogenic rice tissues. The 1 st to 12 th strains in Table 1 were inoculated with the leaves and ears of Lijiang New-cluster black grain variety LTH, and all strains were capable of causing typical diseases to rice leaves and ears (see FIG. 3).
And extracting genome DNA of the pathological tissues by adopting a PEG-NaOH rapid extraction method, and taking the genome DNA as a template for RPA-LFD and PCR detection. In the RPA-LFD test of the gene Avr-Piz-t, 8 positive bands were detected, which was identical to the PCR test result (FIG. 3). These results again demonstrate that the RPA-LFD method can accurately detect the presence of the Avr-Piz-t gene in the tissue of origin.
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.
Claims (7)
1. The primer and probe combination for detecting avirulence gene Avr-Piz-t is characterized in that the nucleotide sequence of the primer is as follows: forward primer
5'-TTCTCAACCATCATCACCGTGTGCCTCTTC-3' (SEQ ID NO: 1); reverse primer: 5'-CCAACGGTGCCGGAGGAGAGAACATCAGTG-3' (SEQ ID NO: 2); the nucleotide sequence of the probe is as follows:
5’-GCTTCGTACAATGCAATCATCATCTCCTGT-THF-CAATGGCAGACACTG-3’(SEQ ID NO:3)。
2. the primer and probe combination of claim 1, wherein the probe has FAM added at the 5 'end and C3-spacer added at the 3' end.
3. An RPA-LFD method for detecting avirulence gene Avr-Piz-t, which is characterized by comprising the following steps:
1) Extracting DNA of a sample to be detected;
2) Performing RPA amplification using the primer and probe combination of claim 2 using DNA as a template;
3) Detecting an amplification product by using a lateral flow chromatography test strip; when the test strip has two brown strips, one strip is positioned in the quality control area, and the other strip is positioned in the detection area, the result is positive, which indicates that the sample contains a nontoxic gene Avr-Piz-t; when the test strip has only a brown band in the quality control area and no band in the detection area, the result is negative, which indicates that the sample does not contain the avirulence gene Avr-Piz-t.
4. A method according to claim 3, wherein in step 2) RPA amplification: buffer 25. Mu.L, 10. Mu.M upstream primer 2.1pL, 10. Mu.M downstream primer 2.1. Mu.L, probe 0.6. Mu.L, DNA2.0uL, starter 3. Mu.L and deionized water were added to the tube of the unit containing lyophilized enzyme powder; and (3) fully and uniformly mixing the RPA amplification system, centrifuging for 10s at 5.000xg, placing the mixture on a metal bath at 39 ℃ for reaction for 20min, incubating for 4 min, uniformly mixing the reaction tube again, centrifuging for 3-5s, and placing the mixture in a water bath kettle at 39 ℃ for continuous reaction for 16min.
5. An RPA-LFD kit for detecting avirulence gene Avr-Piz-t, comprising at least 1 dose of the primer and probe combination reagent of any one of claims 1-2.
6. The RPA-LFD kit of claim 5, further comprising: standard positive DNA, buffer, sterile double distilled water, reaction driving solution, product dilution, and lateral flow chromatography reagents.
7. Use of a primer and probe combination according to any one of claims 1-2 or a kit according to any one of claims 5-6 for detecting avirulence gene Avr-Piz-t.
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| CN117757980A (en) * | 2023-12-29 | 2024-03-26 | 江苏省农业科学院 | Primer and probe combination for rapidly and accurately detecting rice blast germ and application thereof |
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