A primer, probe and kit for detecting ustiago sphaerogena based on recombinase polymerase amplification method

Abstract

The application discloses a primer, a probe and a kit for detecting Ustilaginoidea virens based on a recombinase polymerase amplification method, the primer comprises a forward primer F-primer shown in SEQ ID NO. 1 and a reverse primer R-primer shown in SEQ ID NO. 2; and the sequence of the probe Probe is shown in SEQ ID NO. 3. Compared with a traditional detection technology for identifying Ustilaginoidea virens based on morphological characteristics and using ordinary PCR, the application has higher accuracy, sensitivity and effectiveness, is convenient to operate, has good practicability, provides a new technical platform for detection of Ustilaginoidea virens, and can be used for high-sensitivity and rapid detection of rice smut caused by Ustilaginoidea virens.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to primers, probes, and kits for detecting rice false smut based on a recombinase polymerase amplification method. Background Technology

[0002] Rice false smut is a significant fungal disease affecting rice production, caused by *Pseudomonas aeruginosa*. This pathogen primarily infects the panicle of rice plants during the booting stage. The mycelium then colonizes the floral organs, absorbing nutrients from the panicle and eventually transforming into rice false smut balls covered with dark green, powdery, thick-walled spores. The formation of these rice false smut balls not only hinders grain filling but also affects the sterility of other spikelets, resulting in severe yield losses. Furthermore, these rice false smut balls produce toxic fungal toxins, threatening human and animal health. Currently, there are no highly effective control measures for rice false smut. Strengthening quarantine and preventing the spread of the pathogen are among the most effective measures for controlling this disease. Therefore, rapid detection methods should be established to provide technical support for the detection of *Pseudomonas aeruginosa* in the field and for the prediction and forecasting of rice false smut, thereby reducing losses caused by the disease.

[0003] Currently, there are few detection technologies for rice false smut caused by *Strombus oryzae*. Traditional detection methods mainly rely on the morphology of the fungus for identification. The difficulty in isolating the fungus and the long culture period cause significant inconvenience for detection. With the development of molecular biology, especially the widespread use of PCR technology, more and more molecular biology techniques are being applied to the detection of *Strombus oryzae*, including PCR and LAMP techniques. Although these techniques have greatly improved in specificity and sensitivity, the detection time is still relatively long, and they rely on sophisticated temperature cycling devices, making the detection process complex and unable to meet the needs of rapid field detection.

[0004] Recombinase polymerase amplification (RPA) is a novel nucleic acid amplification technique that has become a viable alternative to PCR due to its simplicity, speed, high specificity, and low equipment requirements. The RPA reaction primarily relies on three enzymes: recombinase, DNA polymerase, and single-stranded binding protein (SSB). The complex of these three enzymes is active at room temperature. The protein-DNA complex formed by the recombinase and primers searches for homologous sequences in double-stranded DNA. Once the primers locate a homologous sequence, a strand exchange reaction occurs, initiating DNA synthesis under the action of DNA polymerase, resulting in exponential amplification of the target region on the template. The replaced single-stranded DNA strand binds to the SSB, preventing further substitution. In this system, a synthetic event is initiated by two opposing primers. The entire process is very rapid, yielding detectable amplification products within as little as ten minutes. In RPA assays based on colloidal gold lateral flow chromatography strips, a Twist Amp nfo probe is required. During RPA amplification, the Twist Amp nfo probe binds to the RPA product. The nfo, acting as a DNA repair enzyme, recognizes internal empty base sites (tetrahydrofuran, THF) and cleaves the probe, allowing amplification to continue. RPA technology is convenient, time-efficient, and exhibits high specificity and sensitivity, making it ideal for field testing. In just a few years, this technology has rapidly developed and is now widely used in disease diagnosis, pathogen detection, and transgenic detection; however, reports on its use in the detection of plant pathogens are scarce.

[0005] This invention analyzes the differences in genomic sequences between *Strombus oryzae* and other fungi through sequence alignment, designs primers and probes specific to *Strombus oryzae*, and establishes a rapid detection method for recombinase polymerase of rice false smut caused by *Strombus oryzae*. Summary of the Invention

[0006] To address the problems of existing biological detection methods for rice false smut, such as long detection cycles, poor specificity, low sensitivity, and difficulty in field application, the present invention aims to provide primers, probes, and kits for detecting rice false smut caused by rice false smut based on recombinase polymerase amplification.

[0007] To achieve the aforementioned objectives, this invention first provides a primer pair and probe for detecting rice false smut caused by *Strombus oryzae* based on a recombinase polymerase amplification method. This primer pair utilizes bioinformatics tools and comparative genomics methods to identify sequence-specific genes on the *Strombus oryzae* genome as detection targets. First, the nucleic acid sequences of 8322 genes on the *Strombus oryzae* genome are compared with those of fifteen other sequenced fungi using a BLAST method. Magnaporthe oryzae , Rhizoctonia solani ,Fusarium fujikuroi , Metarhizium anisopliae , Fusarium graminearum , Fusarium culmorum , Fusarium oxysporum , Fusarium chlamydosporum , Fusarium solani , Sclerotinia sclerotiorum , Colletotrichum scovillei , Colletotrichum higginsianu m、 Phomopsis longicolla , Colletotrichum gloeosporioides , Macrophomina phaseolina All gene sequences of *Strombus haematobium* were analyzed, identifying 168 genes specific to the *Strombus haematobium* genome. A BLAST comparison with the NCBI NR database was then performed, and one specific gene, UV8b_00275, was selected as the detection target. RPA primers and probes were designed, and their specificity was verified experimentally. These specific primers and probes are also the sequences claimed in this patent. The probe includes the forward primer F-primer as shown in SEQ ID NO.1 and the reverse primer R-primer as shown in SEQ ID NO.2; the sequence of the probe is shown in SEQ ID NO.3.

[0008] The primers and probes involved in this invention are derived from the rice false smut gene UV8b_00275 (XM_043137773.1), and the rice false smut genome sequence is obtained from the NCBI website. This gene and the corresponding primer and probe sequences actually exist on the rice false smut genome.

[0009] The application of the above primer pairs and probes in detecting rice false smut caused by rice false smut fungus.

[0010] The application of the above primer pairs and probes in the preparation of a kit for detecting rice false smut caused by *Strombus oryzae*.

[0011] A kit for detecting rice false smut caused by *Strombus oryzae* based on a recombinase polymerase amplification method, the kit containing the aforementioned primer pairs and probes.

[0012] The kit also contains reagents commonly used in recombinase polymerase amplification techniques.

[0013] The volumes of each reagent in the above kit are: 40.9 μL A Buffer, 2 μL 10 μmol·L⁻¹ -1 Forward and reverse primers, 0.6 μL 10 μmol·L -1 Probe, 2 μL DNA template, 2.5 μL B Buffer.

[0014] The above-mentioned primer and probe combinations and kits are used in the detection of rice false smut caused by *Strombus oryzae*.

[0015] A method for detecting rice false smut caused by *Strombus oryzae* based on recombinase polymerase amplification involves extracting DNA from the target microorganism, using the DNA as a template, and performing recombinase polymerase amplification using the aforementioned primer pairs and probes. The product is then passed through colloidal gold lateral flow chromatography strips, and the presence or absence of *Strombus oryzae* is determined based on the color development of the control and detection lines. The reaction procedure for recombinase polymerase amplification is: amplification at 40℃ for 20 min.

[0016] The specific steps of this method are as follows: extract the DNA of the microorganism to be tested, take 2 μL of DNA solution as the reaction template, add 48 μL of the detection solution in the kit for RPA, the RPA reaction program is: reaction amplification at 40℃ for 20 min, then take 10 μL of product and add it to the end of the colloidal gold test strip, vertically immerse the test strip in 190 μL of HybriDetect Assay Buffer, and read the result after 3~5 min.

[0017] The method for detecting rice blast fungus caused by *Strombus oryzae* of this invention uses extracted DNA as a template and performs an RPA reaction using the aforementioned RPA primers and probe. Since the probe contains a FAM group at its 5' end, a blocking group at its 3' end, and a THF site in the middle, when the probe binds to the amplification product, the nfo enzyme recognizes the THF site and cleaves the blocking group on the probe, thus allowing product amplification to continue. The reverse primer contains a biotin group at its 5' end. When both the FAM and biotin groups are present in the product, the product will cause both the detection line and the control line to show color simultaneously when passed through a colloidal gold lateral flow chromatography test strip; otherwise, only the control line will show color. Therefore, the presence or absence of *Strombus oryzae* is determined by the color development of the colloidal gold lateral flow chromatography test strip after the reaction.

[0018] This invention, through extensive research, identified the genes targeted by the RPA method for detecting rice false smut fungus and designed primer pairs and probes accordingly, ultimately achieving an effective detection method. Therefore, this invention has the following advantages:

[0019] (1) Convenient operation: The RPA method for detecting rice false smut provided by this invention overcomes the problems of long cycle, time-consuming, labor-intensive, cumbersome and poor specificity of existing biological detection methods for rice false smut, as well as the problem that PCR detection technology requires thermal cycling instruments and cannot quickly detect rice false smut. The detection method of this invention can accurately, quickly and efficiently detect rice false smut in just 20 minutes under isothermal conditions of 40℃. It has simple requirements for the experimental site, does not require complex instruments, and can better meet the needs of on-site detection of rice false smut.

[0020] (2) High accuracy: Traditional methods for detecting rice false smut rely solely on morphological characteristics for identification. However, the growth of rice false smut is affected by factors such as temperature, resulting in unstable morphology and slow growth, making accurate identification difficult. Traditional detection primers are mostly based on ITS sequences, while the primers in this patent application are designed based on the genome sequence of rice false smut. Using comparative genomics, the Blast software is employed to compare the genome sequence of rice false smut with that of other fungi. A segment of a conserved gene unique to rice false smut was selected, and specific RPA primers and probes were designed. RPA primers are longer than ordinary primers, thus exhibiting higher specificity and the ability to distinguish rice false smut from other fungi.

[0021] (3) High sensitivity: The RPA detection method for rice false smut established in this invention has high sensitivity, reaching 10 pg DNA, indicating that the detection method is sufficient to accurately and quickly detect rice false smut even at low DNA concentrations. Attached Figure Description

[0022] Figure 1 The results show the specificity validation of the RPA primers and probes for *Strombus oryzae*. Uv1-3: *Strombus oryzae*; Mo: *Strombus oryzae*; Rs: *Sheath blight*; Ff: *Fusarium oxysporum*; NC: negative control.

[0023] Figure 2 The results validate the sensitivity of the RPA detection method for rice false smut. From left to right, the figures represent different concentrations of rice false smut genome; NC: negative control. Detailed Implementation

[0024] To make the objectives and technical solutions of this invention clearer, specific examples are used for further explanation, but the invention is not limited to these examples.

[0025] Based on bioinformatics tools and comparative genomics methods, the inventors identified sequence-specific genes in the genome of *Strombus oryzae* as detection targets. First, the nucleic acid sequences of 8322 genes in the *Strombus oryzae* genome were compared with those of fifteen other sequenced fungi using BLAST. Magnaporthe oryzae , Rhizoctonia solani , Fusarium fujikuroi , Metarhizium anisopliae , Fusarium graminearum , Fusarium culmorum , Fusarium oxysporum , Fusarium chlamydosporum , Fusarium solani , Sclerotinia sclerotiorum , Colletotrichum scovillei , Colletotrichum higginsianu m、 Phomopsis longicolla , Colletotrichum gloeosporioides ,Macrophomina phaseolina All gene sequences of *Strombus haematobium* were analyzed, identifying 168 genes specific to *Strombus haematobium*. Then, by comparing with the NCBI NR database Blast, one specific gene, UV8b_00275, was selected as the detection target. RPA primers and probes were designed, and their specificity was verified experimentally. The RPA primers include the forward primer F-primer shown in SEQ ID NO.1 and the reverse primer R-primer shown in SEQ ID NO.2; the sequence of the probe is shown in SEQ ID NO.3.

[0026] Example 1: Detection of rice false smut fungus using RPA method

[0027] RPA primers and probes for detecting rice false smut: forward primer F-primer as shown in SEQ ID NO.1, reverse inner primer R-primer as shown in SEQ ID NO.2, and probe as shown in SEQ ID NO.3.

[0028] The total volume of the RPA kit for detecting rice false smut is 50 μL, and the volumes of each reagent are: 40.9 μL A Buffer, 2 μL 10 μmol·L⁻¹ -1 Forward and reverse primers, 0.6 μL 10 μmol·L -1 Probe, 2 μL DNA template, 2.5 μLB Buffer.

[0029] RPA detection method: Extract DNA from the microorganism to be tested, take 2 μL of DNA solution as the reaction template, add 48 μL of the detection solution in the kit for RPA. The RPA reaction program is as follows: react and amplify at 40℃ for 20 min, then take 10 μL of the product and add it to the end of the colloidal gold test strip. Immerse the test strip vertically into 190 μL of HybriDetect Assay Buffer, and read the result after 3~5 min.

[0030] To verify the specificity of the RPA method, *Strombus oryzae*, *Strombus oryzae*, *Sheath blight*, and *Bakanaenia bakanae* were used as test materials. The detection results are shown below. Figure 1 As can be seen, using RPA-specific primers and probes for *Strombus oryzae* to detect different pathogens using RPA, the results showed that only the reaction products of *Strombus oryzae* showed color on both the control and test lines after passing through the colloidal gold lateral flow chromatography test strip, while other fungi only showed color on the control line and not the test line. In other words, the RPA detection results showed that the reaction products of three *Strombus oryzae* strains were positive, while those of other fungi were negative, indicating that the RPA method can specifically detect *Strombus oryzae*.

[0031] Example 2: Sensitivity test of RPA reaction of rice false smut fungus

[0032] To determine the sensitivity of the RPA detection method, rice false smut DNA extracted using a genome extraction kit was serially diluted 10-fold using a Qubit 4.0 real-time fluorescence instrument to determine its concentration. The DNA concentration range was set from 10 ng to 10 fg. 2 μL of each diluted DNA solution was used as a template and added to 48 μL of the kit solution for the RPA reaction. The reaction program was: 40℃ for 40 min. The results are as follows: Figure 2 As shown, the color of the test line on the test strip gradually fades as the amount of DNA template decreases. The test line remains visible even when the DNA concentration is 10 pg, indicating that the method of this invention has very high sensitivity.

Claims

1. A primer pair and probe combination for detecting rice false smut caused by Sphacelotheca reilliana based on a recombinase polymerase amplification method, characterized by comprising a primer pair of SEQ ID NOs: 1 and 2 and a probe of SEQ ID NO:

3. The primer pair comprises a forward primer F-primer as shown in SEQ ID NO. 1 and a reverse primer R-primer as shown in SEQ ID NO. 2; and the probe Probe has a sequence as shown in SEQ ID NO.

3.

2. The primer pair and probe combination of claim 1 are used in the detection of rice sheath blight caused by Rhizoctonia solani.

3. The primer pair and probe combination of claim 1 are used in the preparation of a kit for the detection of rice sheath blight caused by Rhizoctonia solani.

4. A kit for detecting rice false smut caused by *Strombus oryzae* based on a recombinase polymerase amplification method, characterized in that... The kit comprises the primer pair and probe combination of claim 1.

5. The kit of claim 4 is used in the detection of rice sheath blight caused by Rhizoctonia solani.

6. A method for detecting rice false smut caused by *Strombus oryzae* based on recombinase polymerase amplification, characterized in that: DNA of the microorganism to be detected is extracted, and recombination enzyme polymerase amplification is performed using the primer pair and probe combination of claim 1 with the DNA of the microorganism to be detected as a template, and the amplification product is detected.

7. The method of claim 6, wherein: The reaction product is detected by a colloidal gold lateral flow chromatographic test strip; if both the quality control line and the detection line show color, the sample is positive, and if only the quality control line shows color and the detection line does not show color, the sample is negative.

8. The method according to claim 6 or 7, characterized in that: The reaction procedure of the recombination enzyme polymerase amplification is 40 °C for 20 min.

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