CN117344048A - RPA-LFD primer probe group for detecting avirulence gene Avr-Pik, kit and application thereof - Google Patents
RPA-LFD primer probe group for detecting avirulence gene Avr-Pik, kit and application thereof Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention discloses an RPA-LFD primer probe group for detecting a nontoxic gene Avr-Pik, a kit and application thereof. The nucleotide sequence of the primer is as follows: forward primer 5'-GGAACTGTCGCTGTCGTCAATGCCGAAACG-3' (SEQ ID NO: 1); reverse primer: 5'-AAACAATCCAACCTGGAGGGAAGTCGCCCGA-3' (SEQ ID NO: 2); the nucleotide sequence of the probe is as follows: 5'-AAACAACGTACGTCAAGATGCTGGAACCTG-THF-TACAGCTCTTGGAAA-3' (SEQ ID NO: 3). Meanwhile, the invention also provides a kit containing the primer probe group. The invention establishes a method for rapidly detecting the avirulence gene Avr-Pik by adopting the RPA-LFD for the first time, and determines that the method can be used for detecting the avirulence gene of the rice blast bacteria in the field through specificity and sensitivity evaluation, thereby providing a new technical platform for detecting the avirulence gene Avr-Pik.
Description
Technical Field
The application relates to the technical field of non-toxic gene detection, in particular to a primer probe set for detecting a non-toxic gene Avr-Pik, a kit and application thereof.
Background
Rice blast is one of the most serious diseases affecting global rice production, and its pathogenic bacteria is a seed bag fungus rice blast (Magnaporthe oryzae). 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.
Pik locus is an important locus of rice disease resistance gene. At present, 9 alleles have been cloned, principally Pik-m, pik, pik-s, pik-p, pi1, pik-h, pi7, pik-KA and Pik-e (Xiao et al 2022). They both consist of 2 NLR protein encoding genes Pik-1, pik-2, which together activate the immune response of rice in the form of a complex, where only Pik-1 is able to recognize the avirulence gene AvrPik. The research result of the research room shows that the distribution frequency of Avr-Pik in 1293 single spore strains collected in 2018-2022 of Jiangsu province is higher and reaches more than 95%, so that the resistance level of rice varieties containing disease resistance genes Pik corresponding to the Avr-Pik is higher, but the distribution frequency of Avr-Pik fluctuates among years, and therefore, the nontoxic genes need to be monitored in real time.
Currently, in the detection of part of plant pathogenic bacteria, molecular detection methods such as qRT-PCR, lamp and PCR are widely applied, and the detection methods have better accuracy and sensitivity, but have strict technical requirements on equipment and personnel, and are not suitable for on-site identification. The Jiangsu province is a main production area of Chinese rice and is about 3300 mu of annual planting area, and the junction of subtropical zone and warm zone at the area is suitable in temperature and more in precipitation, so that rice blast generally 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 rice blast. In order to guide the scientific layout of the field disease-resistant variety and achieve the aim of disease prevention and control, real-time field monitoring of the avirulence gene Avr-Pik of the rice blast bacteria is needed, but no method capable of effectively detecting the gene in the field 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; furthermore, the accuracy of analysis of the distribution frequency of avirulence gene Avr-Pik is not high due to the limitation of the sample amount of the isolated strain.
Disclosure of Invention
In order to solve the technical problems, the invention provides an RPA-LFD primer probe group for detecting a avirulence gene Avr-Pik, a kit and application thereof. The primer probe or the kit can realize real-time monitoring of the avirulence gene Avr-Pik of the rice blast bacteria, and can effectively monitor the avirulence gene Avr-Pik 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 invention is as follows:
in a first aspect, the present invention provides a primer and probe combination for detecting avirulence gene Avr-Pik, wherein the nucleotide sequence of the primer is as follows: forward primer
5'-GGAACTGTCGCTGTCGTCAATGCCGAAACG-3' (SEQ ID NO: 1); reverse primer: 5'-AAACAATCCAACCTGGAGGGAAGTCGCCCGA-3' (SEQ ID NO: 2); the nucleotide sequence of the probe is as follows: 5' -AAACAACGTACGTCAAGATGCTGGAACCTG
-THF-TACAGCTCTTGGAAA-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 invention also provides an RPA-LFD method for detecting avirulence gene Avr-Pik, which comprises the following steps:
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 invention 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-Pik; 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-Pik.
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, deionized water were added to a 0.2mL TwistAmp reaction cell 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 invention also provides an RPA-LFD kit for detecting avirulence gene Avr-Pik, 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 invention 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-Pik.
Compared with the prior art, the application can carry out the avirulence gene Avr-Pik on the rice blast bacteria in the field without separating pathogenic bacteria in a laboratoryThe method is convenient and quick for real-time monitoring. Meanwhile, the RPA-LFD primer probe group and the kit for detecting the avirulence gene Avr-Pik can detect the sensitivity of the avirulence gene Avr-Pik to 10 fg. Mu.L -1 And has high specificity. Therefore, the detection method of the RPA-LFD of the avirulence gene Avr-Pik can guide the scientific layout of the field disease-resistant variety 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-Pik gene, wherein, 1 st to 12 th are 12 rice blast bacteria isolated from Jiangsu, 13 th to 15 th are other 3 plant pathogenic bacteria, and 16 th is negative control.
FIG. 2 is the sensitivity detection result of the RPA-LFD detection method of Avr-Pik gene.
FIG. 3 is the results of the inoculation experiment of the RPA-LFD method for detecting Avr-Pik gene.
Detailed Description
The present application is further described in detail below with reference to the attached drawings and examples, and is specifically described as follows: 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-Pik (AB 916598), a forward primer 5'-GGAACTGTCGCTGTCGTCAATGCCGAAACG-3' (SEQ ID NO: 1) was designed; reverse primer: 5'-AAACAATCCAACCTGGAGGGAAGTCGCCCGA-3' (SEQ ID NO: 2); the nucleotide sequence of the probe is as follows: 5'-AAACAACGTACGTCAAGATGCTGGAACCTG-THF-TACAGCTCTTGGAAA-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-Pik 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, DNA2.0. Mu.L, starter 3. Mu.L, deionized water were added to a 0.2mL reaction cell 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 have a strip in the detection area, so that the sample does not contain the gene Avr-Pik.
As shown in FIG. 1, in 12 rice blast pathogens, 11 strains containing avirulence gene Avr-Pik can be detected by using the test strip, and in addition, 1 strain can not detect Avr-Pik, which indicates that the detection rate of Avr-Pik in the 12 rice blast pathogens is 91.67%; the Avr-Pik 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 RPA primers and probes in this study were very specific.
Example 2
DNA concentration of the Avr-Pik Gene-positive strain was determined to be 100 ng/. Mu.L using a 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 、10fg·μL -1 Two brown bands appear on the test strip of the gene Avr-pik, and a 25 mu L reaction system contains 1 fg. Mu.L -1 The paper strip of the Avr-Pik 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 10 fg.mu.L -1 。
The genomic DNA of the Avr-Pik gene positive strain with the same concentration is used as an amplification template, and a PCR amplification reaction is carried out by using a conventional PCR method (Avr-Pik-F: 5'-ATGCGTGTTACCAC-3' (SEQ ID NO: 4); avr-Pik-R:5'-TTAAAAGCCGGGCC-3' (SEQ ID NO: 5)), and the sensitivity of the two methods is compared; the experiment was repeated 3 times to determine the sensitivity of the PCR method to detect the genomic DNA of the Avr-Pik 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 10fg.mu.L -1 The target band can still be seen, which shows that the sensitivity of the RPA lateral flow chromatography test strip detection method is 10 of that of the PCR method 5 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 needed, so that the operation procedure is simple and convenient, and the method is more beneficial to popularization and application in production.
Example 3
To evaluate the applicability of the RPA-LFD test method for Avr-Pik gene, the present application examined diseased rice tissue. 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-Pik, 11 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 Avr-Pik gene in the affected tissue, and the PEG-NaOH rapid extraction method is fully applicable.
The present embodiment is merely illustrative of the present application and is not intended to be limiting, and those skilled in the art, after having read the present specification, may make modifications to the present embodiment without creative contribution as required, but is protected by patent laws within the scope of the claims of the present application.
Claims (7)
1. The primer and probe combination for detecting avirulence gene Avr-Pik is characterized in that the nucleotide sequence of the primer is as follows: forward primer
5'-GGAACTGTCGCTGTCGTCAATGCCGAAACG-3' (SEQ ID NO: 1); reverse primer: 5'-AAACAATCCAACCTGGAGGGAAGTCGCCCGA-3' (SEQ ID NO: 2); the nucleotide sequence of the probe is as follows: 5' -AAACAACGTACGTCAAGATGCTGGAACCTG
-THF-TACAGCTCTTGGAAA-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-Pik, comprising the steps of:
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-Pik; 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-Pik.
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, DNA2.0. Mu.L, starter 3. Mu.L, deionized water were added to a 0.2mL reaction cell 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.
5. An RPA-LFD kit for detecting avirulence gene Avr-Pik, 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-Pik.
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