Primer pair, detection reagent and RPA-LFD kit for visually, ultrasensitively and rapidly detecting puccinia zeae and application

Abstract

The invention belongs to the technical field of molecular biological detection, and particularly relates to a primer pair, a detection reagent and an RPA-LFD kit for visually, ultrasensitively and rapidly detecting puccinia zeae and application. The sequences of an upstream primer and a downstream primer of the RPA primer pair are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2. The RPA primer pair can be used for visually detecting the puccinia zeae through RPA amplification or RPA-LFD by naked eyes, the sensitivity is ultrahigh (0.01 pg / mu L), and the specificity is strong; and no specific band exists on puccinia polystachys, bipolaris maydis, corn ear rot pathogen and curvularia maydis leaf spot pathogen.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, specifically relating to a primer pair, detection reagent, RPA-LFD kit, and application for the visualization, ultrasensitive, and rapid detection of corn stalk rust fungus. Background Technology

[0002] Maize is one of the world's most important food crops, playing an increasingly diverse role in global agricultural and food systems and food security. However, maize production is threatened by various diseases, among which southern rust and common rust are two significant ones. Southern rust is caused by a fungus called *Cephalotaxus fortunei*. Puccinia polysora An airborne disease of maize caused by *Underw.* . Caused by *Underw.* rust fungus (*Underw.* ). P. sorghi Common rust of maize (Schw.) is widely distributed in tropical, subtropical, temperate, and highland environments. After infection with the rust fungus, maize plants have an incubation period of approximately 14 days, making early symptoms difficult to detect. Current diagnostic techniques mainly rely on the identification of typical symptoms in the later stages of infection and microscopic observation of urediniospores. This traditional method has significant limitations: firstly, it cannot provide early warning in the initial stages of infection; secondly, the morphological characteristics of southern maize rust and common rust are easily confused, leading to misdiagnosis based solely on morphological identification. Therefore, developing rapid and accurate molecular detection technologies for early rust detection is of great significance for the comprehensive prevention and control of maize rust and for promoting safe production in the maize industry.

[0003] Common rust of maize, caused by *Russula sessiliflora*, is the most widespread disease in my country. Its morphological characteristics and field symptoms are remarkably similar to those of *Russula multifiliis*, the pathogen of southern maize rust, making it easily confused and difficult to distinguish among field technicians. Therefore, there is an urgent need to establish a visual, ultrasensitive, and rapid detection system. Previous studies have established detection methods for southern and common maize rust using conventional PCR technology. These methods can differentiate between *Russula multifiliis* and *Russula sessiliflora*, but they are time-consuming and require sophisticated instruments such as PCR machines and gel imaging systems, making them unsuitable for field sample testing (e.g., Xing et al., 2017). Subsequent studies used recombinase polymerase amplification (RPA) to differentiate between *Russula multiflora* and *Russula maize*. However, this method is time-consuming and requires multiple instruments such as real-time quantitative PCR, gel imaging systems, and electrophoresis apparatus. Among these, real-time quantitative PCR is an expensive and sophisticated instrument with cumbersome operation, making it very difficult to promote at the grassroots level and impossible to perform on-site detection of field samples. In addition, the detection sensitivity of this method is very limited, with the RPA detection sensitivity for *Russula maize* DNA being only 9.9 pg / μL (e.g., Wu et al., 2024).

[0004] At present, the research on molecular detection technology of corn smut is relatively less in China, and the existing detection methods still have many shortcomings such as long detection period, low sensitivity, expensive detection equipment, inconvenient operation, and inability to detect visually, and many other shortcomings, and the development of visual, ultra-sensitive, rapid and accurate early molecular detection technology has become an urgent need for current rust prevention and control research. Lateral flow dipstick (LFD) is an endpoint detection technology for visual observation of amplification products, and the products of RPA amplification form a direct visual detection result at the detection line position through antigen-antibody combination. At present, there is no report on the application of RPA-LFD technology in rapid detection of corn smut. SUMMARY

[0005] Based on this, the purpose of the present application is to provide a RPA primer pair and application for detecting corn smut (Puccinia sorghi) Puccinia sorghi The primer pair can be used for specific RPA amplification of the DNA of corn smut, and has high specificity, and a RPA-LFD visual ultra-sensitive rapid detection method for corn smut can be constructed based on the primer pair, and has high accuracy.

[0006] In order to achieve the above purpose, the present application can adopt the following technical solutions: The present application provides a RPA primer pair for detecting corn smut, and the sequences of the upstream primer and the downstream primer of the RPA primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively.

[0007] The present application provides a detection reagent for detecting corn smut, which comprises the above-mentioned RPA primer pair.

[0008] Preferably, the above-mentioned detection reagent further comprises a probe, and the sequence of the probe is shown in SEQ ID NO: 3.

[0009] More preferably, in the above-mentioned detection reagent, the 5' end of the probe is labeled with an antigenic marker, the 3' end is modified with a blocking gene, and a base substitute dSpcacer is provided between the 31st and 32nd bases of the probe.

[0010] More preferably, in the above-mentioned detection reagent, the antigenic marker is 5-FAM, the blocking gene is C3-spacer, and the base substitute dSpcacer is tetrahydrofuran.

[0011] The present application further provides a RPA-LFD kit for detecting corn smut, which comprises the above-mentioned RPA primer pair or the above-mentioned detection reagent.

[0012] In still another aspect of the present application, the RPA primer pair or the detection reagent or the RPA-LFD kit is used for detecting Puccinia sorghi.

[0013] In still another aspect of the present application, a method for detecting Puccinia sorghi is provided, which comprises: (1) extracting DNA of a sample to be detected as template DNA; (2) using the RPA primer pair or the detection reagent to perform RPA amplification to obtain an amplification product; (3) using a nucleic acid test strip to detect the amplification product, and then determining whether Puccinia sorghi exists.

[0014] Preferably, in the method, the temperature for RPA amplification is 27-42°C; and / or the time for RPA amplification is ≥5 min.

[0015] More preferably, in the method, the temperature for RPA amplification is 37-42°C; and / or the time for RPA amplification is 15 min.

[0016] The present application has the following advantages: the RPA primer pair provided by the present application can be used for detecting Puccinia sorghi through RPA amplification or RPA-LFD, and has high sensitivity (0.01 pg / μL) and strong specificity; no specific bands are generated for Puccinia polysora, Helminthosporium maydis, Gibberella zeae and Cercospora zeae-maydis. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Specific detection of Puccinia sorghi RPA primer (RPs1-F / RPs1-R); wherein, M: DL 2000 Marker; 1: Puccinia sorghi; 2: Puccinia polysora; 3: Gibberella zeae; 4: Cercospora zeae-maydis; 5: Helminthosporium maydis; 6: sterile water; Figure 2 Specific detection of Puccinia sorghi RPA primer (RPs2-F / RPs2-R); wherein, M: DL 2000 Marker; 1: Puccinia sorghi; 2: Puccinia polysora; 3: Gibberella zeae; 4: Cercospora zeae-maydis; 5: Helminthosporium maydis; 6: sterile water; Figure 3 Specific detection of Puccinia sorghi RPA primer (RPs3-F / RPs4-R); wherein, M: DL 2000 Marker; 1: Puccinia sorghi; 2: Puccinia polysora; 3: Gibberella zeae; 4: Cercospora zeae-maydis; 5: Helminthosporium maydis; 6: sterile water; Figure 4 Optimization of Puccinia sorghi RPA-LFD detection time; Figure 5 Optimization of the detection temperature for Puccinia sorghi RPA-LFD Figure 6 Specific detection of Puccinia sorghi RPA-LFD; wherein, 1: Puccinia sorghi; 2: Puccinia polysora; 3: Cochliobolus heterostrophus; 4: Cochliobolus sativus; 5: Gibberella zeae; Figure 7 Sensitivity detection of Puccinia sorghi RPA-LFD detection system; wherein, 1-7: Puccinia sorghi template DNA concentration is 1000 pg / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 0.1 pg / μL, 0.01 pg / μL and 0.001 pg / μL, respectively; Figure 8 Field sample detection of Puccinia sorghi RPA-LFD detection system; wherein, CK1: Puccinia polysora; CK2: Puccinia sorghi; 3-7: Lanzhou samples in Gansu; 8-9: Shangqiu samples; 10: Xinxiang samples. DETAILED DESCRIPTION

[0018] The embodiments are provided to better illustrate the present application, but are not intended to limit the present application to only the embodiments. Therefore, the skilled in the art can make non-essential improvements and adjustments to the embodiments according to the above description, which still belong to the protection scope of the present application.

[0019] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless there is a clear different meaning in the context, the singular form includes the plural form. As used herein, it should be understood that terms such as "include", "have", "contain", etc. are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present application are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations can exist or can be added. As used herein, " / " can be interpreted as "and" or "or" depending on the circumstances.

[0020] In a first aspect, the embodiments of the present application provide a RPA primer pair for detecting Puccinia sorghi, the sequences of the upstream primer and the downstream primer of the RPA primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0021] It should be noted that the RPA primer pair (SEQ ID NO: 1 and SEQ ID NO: 2) in the application is for the research of molecular detection of the ITS region of Puccinia sorghi, and a plurality of RPA primer pairs of Puccinia sorghi are designed, and the primer pair with stable amplification and high efficiency is screened out. The specific fragment size of the amplification primer pair of Puccinia sorghi is 172 bp, and there is no specific band for Puccinia polysora, Cercospora zeae-maydis, Cercospora zeae-maydis and Cercospora zeae-maydis. It is suitable for RPA amplification detection and RPA-LFD amplification detection.

[0022] In a second aspect, the embodiments of the application provide a detection reagent for detecting Puccinia sorghi, which comprises the RPA primer pair described above.

[0023] It should be noted that the RPA primer pair in the application can be combined with conventional auxiliary reagents to form a detection reagent for detecting Pratylenchus coffeae; the conventional auxiliary reagents are known in the art, such as RPA freeze-dried powder tube, A buffer, positive control primer mix, positive control DNA template and B buffer in the RPA amplification reaction system; in some specific embodiments, the above-mentioned detection reagent can comprise: ddH2O 9.1 μL, A buffer 29.4 μL, B buffer 2.5 μL, 10 μM of upstream primer (SEQ ID NO: 1) and downstream primer (SEQ ID NO: 2) each 2 μL, and DNA template of Puccinia sorghi 5 μL.

[0024] In some specific examples, the above-mentioned detection reagent further comprises a probe, and the sequence of the probe is shown as SEQ ID NO: 3.

[0025] In some specific examples, in the above-mentioned detection reagent, the 5 'end of the probe is labeled with an antigenic marker, the 3 'end is blocked with a blocking gene, and a base substitute dSpcacer is arranged between the 31st and 32nd bases of the probe.

[0026] In some specific examples, in the above-mentioned detection reagent, the antigenic marker is 5-FAM, the blocking gene is C3-spacer, and the base substitute dSpcacer is tetrahydrofuran.

[0027] In a third aspect, the embodiments of the application provide an RPA-LFD kit for detecting Puccinia sorghi, which comprises the RPA primer pair described above or the detection reagent described above.

[0028] It should be noted that the composition of the RPA-LFD kit is in the form known in the art, such as a detection reagent kit will be provided with a kit instruction, such as there will be reagent bottles containing various detection reagents, such as there will be a number of small cells for placing detection reagent bottles, etc. In addition, the RPA primer pair or detection reagent in the present application can also be combined with the test strip for the detection of Puccinia sorghi, combined with the high efficiency and convenience of the test strip, it will bring greater convenience to the detection of corn common rust.

[0029] In a fourth aspect, the embodiments of the present application provide an application of the above-mentioned RPA primer pair or the above-mentioned detection reagent or the above-mentioned RPA-LFD kit in the detection of Puccinia sorghi.

[0030] In a fifth aspect, the embodiments of the present application provide a method for detecting Puccinia sorghi, the method comprising: (1) extracting DNA of a sample to be detected as template DNA; (2) using the above-mentioned RPA primer pair or the above-mentioned detection reagent for RPA amplification to obtain an amplification product; (3) using a nucleic acid test strip to detect the amplification product, and then judging whether Puccinia sorghi exists.

[0031] It should be noted that RPA amplification is a new type of isothermal amplification method relying on recombinase, strand displacement DNA polymerase and single-strand binding protein. This method only needs a pair of 30 bp-35 bp specific primers, and can complete the detection at 37℃-42℃ isothermal reaction for 10-20 min. RPA amplification has the advantages of high sensitivity, short reaction time and low reaction temperature. It can quickly perform DNA specific amplification without the need for precise instruments, providing technical support for on-site rapid detection and popularization and application at the grassroots level. At present, there is no report on the application of RPA-LFD technology in rapid detection of Puccinia sorghi. The above-mentioned amplification primer pair has strong specificity for RPA-LFD amplification, making the detection of Puccinia sorghi highly sensitive and accurate. In addition, the RPA amplification product is generally analyzed qualitatively by using a test strip. In addition, the RPA amplification based on the amplification primer pair of the present application has the characteristics of short time consumption, simple operation, high sensitivity, simple reaction and strong specificity, which can well meet the detection requirements at the grassroots level and provide reference for rapid detection of other types. It has important significance for field monitoring, early diagnosis and disease control.

[0032] In some specific examples, in the above-mentioned method, the temperature of RPA amplification is 27℃-42℃; and / or the time of RPA amplification is ≥5 min.

[0033] In some specific examples, in the above-mentioned method, the temperature of RPA amplification is 37℃-42℃; and / or the time of RPA amplification is 15 min.

[0034] It should be noted that, in the present application, the time of RPA amplification can be preferably 37-42℃, such as 37℃, 38℃, 39℃, 40℃, 41℃ or 42℃, etc.; in addition, the time of RPA amplification can be preferably 15 min; when detection is carried out under this condition, higher detection accuracy can be obtained in the shortest time.

[0035] In order to better understand the present application, the content of the present application will be further illustrated below in combination with specific examples, but the content of the present application is not limited only to the following examples.

[0036] In the following examples, the RPA kit DNA constant temperature rapid amplification kit (basic type) and DNA constant temperature rapid amplification kit (colloidal gold test strip type) used are purchased from Amp Future (Changzhou) Biotechnology Co., Ltd. The recombinant enzyme, strand displacement DNA polymerase and single-stranded binding protein are in the form of freeze-dried enzyme powder in the RPA reaction tube, which is dissolved with A buffer / AD buffer when used, and B buffer is added to start the reaction. The entire RPA reaction is carried out in the RPA reaction tube.

[0037] Example 1 The present application provides the design of RPA primers for Puccinia sorghi and the specific detection conditions.

[0038] (1) Primer and probe design The ITS nucleotide sequence of Puccinia sorghi was analyzed, and 3 pairs of primers and probes suitable for RPA reaction were designed, as shown in Table 1 below. After screening of the primers and probes, the first pair of primers and probes was selected by the present application.

[0039] Table 1 Primers for screening of Puccinia sorghi RPA detection .

[0040] (2) Preparation of Puccinia sorghi DNA template The DNA of Puccinia sorghi is extracted by CTAB method. The specific operation is as follows: collect the appropriate mycelium in a sterile 2 mL centrifuge tube in a clean bench, and add 2 sterile steel balls and 800 μL of CTAB solution preheated in a 65°C water bath; then quickly place it in a multi-sample tissue grinder and grind at 50HZ for 300S; after grinding, place the 2 mL centrifuge tube in a 65°C water bath for 40 min, and mix every 15 min; after water bath, centrifuge at 12000 rpm for 10 min at 4°C; after centrifugation, take 800 μL of supernatant into a new 2 mL centrifuge tube, and add 800 μL of extraction solution I-phenol: chloroform: isopropyl alcohol (volume ratio 25:24:1) (same volume as supernatant), mix well, and centrifuge at 12000 rpm for 10 min at 4°C; take 800 μL of supernatant into a new 2 mL centrifuge tube, add 800 μL of extraction solution II-chloroform: isopropyl alcohol (volume ratio 24:1), mix well, and centrifuge at 12000 rpm for 10 min at 4°C; after centrifugation, take 650 μL of supernatant into a new 1.5 mL centrifuge tube, add 650 μL of pre-cooled isopropyl alcohol, mix well, and freeze at-20°C for 30 min; after freezing, centrifuge at 12000 rpm for 10 min at 4°C; discard the supernatant, wash the precipitate with 500 μL of 70% ethanol solution, centrifuge at 12000 rpm for 5 min at 4°C, discard the supernatant, remove the residual ethanol solution with a pipette, and dry in a clean bench for 15 minutes (the drying time can be appropriately prolonged according to the actual situation); finally, add 50 μL of sterile deionized water to the centrifuge tube and shake to mix the precipitate, measure the DNA concentration with a UV spectrophotometer, and store in a-20°C refrigerator for standby.

[0041] (3) RPA reaction Using the DNA of Puccinia sorghi as a template, RPA amplification is carried out with the corresponding primers; the RPA reaction system is prepared according to the RPA constant temperature rapid amplification kit (basic type) instruction; 50 μL reaction system: A Buffer 29.4 μL, upstream primer (10 μmol / L) 2 μL, downstream primer (10 μmol / L) 2 μL, DNA template 5 μL, add ddH2O 9.1 μL, mix well with a pipette, then add 2.5 μL B Buffer and mix well; after the reaction is completed, the RPA product is added to 50 μL Tris saturated phenol / chloroform / isopropyl alcohol (volume ratio 25:24:1) DNA extraction solution, mix well, centrifuge at 12000 rpm for 5 min, take 5 μL of supernatant and mix with 1 μL of 6×Loadingbuffer, and then perform agarose gel electrophoresis detection.

[0042] (4) Primer specificity detection The RPA amplification product obtained in the above step was mixed with 5 μL of loading buffer, and then subjected to 1% agarose gel electrophoresis to detect the amplification result and observe the characteristics of the amplification band.

[0043] The detection results of the primer pair (RPs1-F and RPs1-R) are shown in Figure 1 The results show that the primer pair can amplify the specific band of P. zeae, and no band is amplified for P. polysora, C. zeae, D. zeae and C. zeina, indicating strong specificity, and the target fragment amplified by P. zeae is 172 bp. The detection results of the primer pair (RPs2-F and RPs2-R) are shown in Figure 2 The results show that the primer pair cannot amplify P. zeae, P. polysora, C. zeae, D. zeae and C. zeina, indicating that the specificity is not strong. The detection results of the primer pair (RPs3-F and RPs3-R) are shown in Figure 3 The results show that the primer pair can not only amplify the non-specific target band (two bands) of P. zeae, but also amplify P. polysora and D. zeae, and cannot amplify C. zeina and C. zeae, indicating that the specificity is not strong. It can be known that only the primer pair (RPs1-F and RPs1-R) has excellent specificity for P. zeae, and the primer pair (RPs1-F and RPs1-R) is selected for amplification detection in the present application.

[0044] Example 2 The present application provides a kind of P. zeae RPA-LFD detection system of establishment, optimization and specific detection condition.

[0045] (1) RPA-LFD detection system construction P. zeae ( P. sorghi ), P. polysora ( P. polysora ), C. zeae ( Bipolaris maydis ), D. zeae ( Fusarium gminearum ) and C. zeina ( Curvularia lunata(Table 2) Different fungal DNAs were extracted as templates according to the method in Example 1, and RPA amplification was performed using the designed primers. The RPA reaction system was prepared according to the DNA isothermal rapid amplification kit (colloidal gold test strip type). The 50 μL reaction system consisted of: 29.4 μL AD Buffer, 2 μL upstream primer (10 μmol / L), 2 μL downstream primer (10 μmol / L), 0.6 μL probe (the 5' end of the probe was labeled with an antigen marker, the 3' end was modified with a blocking gene, and a base substitute dSpcacer was set between the 31st and 32nd bases of the probe; the antigen marker was 5-FAM, the blocking gene was C3-spacer, and the base substitute dSpcacer was tetrahydrofuran), 5 μL DNA template, 8.5 μL ddH2O was added, and after mixing with a pipette tip, 2.5 μL B Buffer was added. The reaction was carried out at 37℃ for 15 min. After the reaction, 10 μL of the amplification product was mixed with 90 Mix μL of sterile water thoroughly, and then add 80 μL of the mixture into the sample well of the test strip.

[0046] Table 2 Information on fungi used in the test .

[0047] (2) Optimization of reaction time for detection of corn stalk rust fungus Under the condition of reaction temperature of 37℃, the reaction time is 5-25 min, and a gradient is set every 5 min for a total of 5 gradients. The detection is carried out according to the conditions of (1) above.

[0048] Test results as follows Figure 4 As shown, the results indicate that a positive band can be detected after 5 minutes of reaction, but the band color is relatively light. The color becomes darker after 15 minutes. In order to improve the detection efficiency of corn rust fungus, this invention can select 15 minutes as the optimal reaction time.

[0049] (3) Optimization of reaction temperature for detecting corn stalk rust fungus The reaction temperatures were set to 22℃, 27℃, 32℃, 37℃ and 42℃ respectively, for a total of 5 reaction temperatures, and the tests were carried out according to the conditions in (1) above.

[0050] Test results as follows Figure 5 As shown, the results indicate that, except for 22℃ where no bands were detected, obvious detection bands appeared at 27℃, 32℃, 37℃, and 42℃. Therefore, in field applications, the appropriate reaction temperature can be selected according to the actual situation, with 32℃ and 37℃ being preferred.

[0051] (4) Specificity testing of the detection system The DNA of corn smut, corn multiple stem rust, corn head rot, corn small spot and corn curved spore leaf spot was detected by using the corn smut RPA-LFD detection system (the reaction time of RPA reaction was 15 min, and the reaction temperature was 37 ℃) established above, and the specificity of the detection system was evaluated.

[0052] The detection results are shown in Figure 6 The results show that only the test strip of corn smut appears obvious detection line (T) at the same time as the quality control line (C), and the detection lines of other control samples do not appear obvious bands, which indicates that the RPA primer designed in the application has specificity for corn smut.

[0053] Example 3 The sensitivity detection experiment (RPA & LFD) of the corn smut RPA-LFD detection system (the reaction time of RPA reaction was 15 min, and the reaction temperature was 37 ℃) is provided in the embodiments of the application, and the specific conditions are as follows: the DNA extraction method in Example 1 is used to extract the DNA of corn smut, and the DNA template of corn smut is diluted by 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 and 10 7 times respectively, and the sensitivity of the corn smut RPA-LFD detection system established in Example 2 is tested.

[0054] The results are shown in Figure 7 The results show that when the concentration of corn smut template DNA is 1000 pg / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 0.1 pg / μL and 0.01 pg / μL, obvious positive bands can be detected, and when the concentration of pathogen template DNA is 0.001 pg / μL, positive bands cannot be detected, which indicates that the sensitivity of the corn smut RPA-LFD detection system established in the application can reach 0.01 pg / μL.

[0055] Example 4 The detection experiment (RPA & LFD) of the corn smut RPA-LFD detection system constructed above on field samples is provided in the embodiments of the application, and the specific conditions are as follows: the RPA-LFD detection system (the reaction time of RPA reaction was 15 min, and the reaction temperature was 37 ℃) constructed in Example 2 is used to detect 8 corn rust samples collected in the field.

[0056] The detection results are shown in Figure 8As shown, the results show that corn rust samples collected from Lanzhou and Xinxiang, Henan, 6 corn rust samples were detected, and 2 corn rust samples collected from Shangqiu, Henan, were not detected. The detection results are consistent with the conventional PCR detection results. The enzyme used in the conventional PCR detection is 2x San Taq PCR Mix premix, and the method is carried out according to the instruction manual, which is purchased from Shanghai Sangon Biological Engineering Co., Ltd.

[0057] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the purpose and scope of the present application, and they should be covered by the scope of the claims of the present application.

Claims

1. Used for detecting corn stalk rust fungus ( Puccinia sorghi The RPA primer pair of ( ) is characterized in that, The upstream primer and the downstream primer of the RPA primer pair are respectively as shown in SEQ ID NO: 1 and SEQ ID NO:

2.

2. A detection reagent for detecting Puccinia graminis f. sp. gram inis, characterized by, The RPA primer pair of claim 1.

3. The detection reagent according to claim 2, characterized in that, The detection reagent further comprises a probe, and a sequence of the probe is as shown in SEQ ID NO:

3.

4. The detection reagent according to claim 2 or 3, characterized in that, The 5' end of the probe is labeled with an antigenic marker, the 3' end is blocked by a gene modification, and a base substitute dSpcacer is arranged between the 31st and 32nd bases of the probe.

5. The detection reagent according to claim 4, characterized in that, The antigenic marker is 5-FAM, the blocking gene is C3-spacer, and the base substitute dSpcacer is tetrahydrofuran.

6. A RPA-LFD kit for detecting Puccinia sorghi, characterized by, The RPA primer pair of claim 1 or the detection reagent of any one of claims 2 to 4.

7. The RPA primer pair of claim 1 or the detection reagent of any one of claims 2 to 5 or the RPA-LFD kit of claim 6 in the detection of Puccinia sorghi.

8. A method of detecting Puccinia graminis, the method comprising contacting a sample with the antibody of claim 1 and detecting the presence of the antibody bound to the sample. Comprising: (1) extracting DNA of a sample to be detected as template DNA; (2) using the RPA primer pair in claim 1 or the detection reagent of any one of claims 2 to 5 to perform RPA amplification to obtain an amplification product; (3) using a nucleic acid test strip to detect the amplification product, and then judging whether Puccinia sorghi exists.

9. The method of claim 8, wherein, the temperature of RPA amplification is 27-42℃; and / or the time of RPA amplification is ≥5 min.

10. The method of claim 8 or 9, wherein, the temperature of RPA amplification is 37-42℃; and / or the time of RPA amplification is 15 min.

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