A primer set for loop-mediated isothermal amplification detection of pine needle red spot pathogen Distichosporium septosporium and its application

Abstract

The present invention discloses a loop-mediated isothermal amplification detection primer set for the pine needle red spot pathogen, Discodium septum, and its application. The present invention designs four groups of loop-mediated isothermal amplification detection primer sets for the pine needle red spot pathogen, Discodium septum, which are primer combination T1, primer combination R1, primer combination R3, or primer combination R5. Experimental results show that the loop-mediated isothermal amplification detection primer set provided by the present invention, and the loop-mediated isothermal amplification detection method for the pine needle red spot pathogen, Discodium septum, performed using the loop-mediated isothermal amplification detection primer set, have the characteristics of strong specificity, low requirements for instruments and equipment, and easy operation. In production practice, rapid, sensitive, accurate, safe, and visual detection of the pine needle red spot pathogen can be achieved, and can be used for field surveys, early diagnosis, seedling quarantine and transportation, disease monitoring, and identification of pine needle red spot disease, providing a reliable technical and theoretical basis for early warning and prevention and control of the pine needle red spot pathogen.

Description

Technical Field

[0001] The present invention belongs to the field of plant pathogen detection, relates to the field of plant fungal disease detection, and particularly relates to a loop-mediated isothermal amplification detection primer set for the pine needle red spot pathogen, Distichosporium septoriae, and applications thereof. Background Art

[0002] Pine needle red spot is a worldwide pine needle disease that mainly harms Pinus spp. Pine ) plants, and can also infect Abies genus ( Fir tree ), Cedrus spp. ( Cedar ), Larix ( Larch ), Picea spp. ( Spruce ) and Pseudotsuga ( Pseudotsuga Currently, the disease is causing serious damage in many countries and regions around the world, causing huge losses to natural pine forests and plantations.

[0003] Based on molecular systematics and morphological characteristics, the pathogen of pine needle red spot disease is believed to be a member of the genus Colletotrichum ( Dothistroma ) are two species, namely, Psora pine ( D. pine ) and Psoralea septata ( D. septospora Through existing investigations, sampling and identification, it was determined that the pathogen of pine needle red spot disease in my country is Pseudomonas septoria.

[0004] Currently, identification of pine needle red spot disease primarily relies on disease symptoms, pathogen morphology, and molecular sequence comparison. However, the symptoms of pine needle red spot disease are highly similar to those of diseases such as brown spot disease and needle drop disease. Morphological identification relies on the production of conidiophores in the late stages of the disease, while molecular sequence comparison relies on obtaining pure cultures of the pathogen. Therefore, early detection of pine needle red spot disease is difficult and requires a high level of expertise from the identification personnel.

[0005] Loop-mediated isothermal amplification (LAMP) technology is a new nucleic acid amplification method that can quickly amplify target genes under constant temperature conditions.

[0006] In view of this, the present invention aims to provide a rapid detection method based on loop-mediated isothermal amplification (LAMP) technology for the detection of the pine needle red spot pathogen, Desmodium septoria. This method combines LAMP technology with visual detection technology, hoping to quickly detect Desmodium septoria. Summary of the Invention

[0007] In view of the technical problems in the background technology, the purpose of the present invention is to provide a loop-mediated isothermal amplification detection primer set for the pine needle red spot pathogen, Dessicaria septata, and its application.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides a loop-mediated isothermal amplification detection primer set for the pine needle red spot pathogen, the detection primer set having four primer combinations, namely primer combination T1, primer combination R1, primer combination R3, and primer combination R5, wherein:

[0010] Primer combination T1, comprising a positive inner primer FIP-1, whose nucleotide sequence is shown in SEQ ID No. 1; a negative inner primer BIP-1, whose nucleotide sequence is shown in SEQ ID No. 2; a positive outer primer F3-1, whose nucleotide sequence is shown in SEQ ID No. 3; a negative outer primer B3-1, whose nucleotide sequence is shown in SEQ ID No. 4; a positive loop primer LF-1, whose nucleotide sequence is shown in SEQ ID No. 5; and a negative loop primer LB-1, whose nucleotide sequence is shown in SEQ ID No. 6;

[0011] Primer combination R1, including a positive inner primer FIP-6, whose nucleotide sequence is shown in SEQ ID No. 7, a negative inner primer BIP-6, whose nucleotide sequence is shown in SEQ ID No. 8; a positive outer primer F3-6, whose nucleotide sequence is shown in SEQ ID No. 9; a negative outer primer B3-6, whose nucleotide sequence is shown in SEQ ID No. 10; a positive loop primer LF-6, whose nucleotide sequence is shown in SEQ ID No. 11; and a negative loop primer LB-6, whose nucleotide sequence is shown in SEQ ID No. 12;

[0012] Primer combination R3, including a positive inner primer FIP-8, whose nucleotide sequence is shown in SEQ ID No. 13; a negative inner primer BIP-8, whose nucleotide sequence is shown in SEQ ID No. 14; a positive outer primer F3-8, whose nucleotide sequence is shown in SEQ ID No. 15; a negative outer primer B3-8, whose nucleotide sequence is shown in SEQ ID No. 16; a positive loop primer LF-8, whose nucleotide sequence is shown in SEQ ID No. 17; and a negative loop primer LB-8, whose nucleotide sequence is shown in SEQ ID No. 18;

[0013] Primer combination R5 includes a forward inner primer FIP-10, whose nucleotide sequence is shown in SEQ ID No. 19; a negative inner primer BIP-10, whose nucleotide sequence is shown in SEQ ID No. 20; a forward outer primer F3-10, whose nucleotide sequence is shown in SEQ ID No. 21; a negative outer primer B3-10, whose nucleotide sequence is shown in SEQ ID No. 22; a forward loop primer LF-10, whose nucleotide sequence is shown in SEQ ID No. 23; and a negative loop primer LB-10, whose nucleotide sequence is shown in SEQ ID No. 24.

[0014] The second aspect of the present invention provides a loop-mediated isothermal amplification (LAMP) detection reagent for the pine needle red spot pathogen, Condylosporium septosporium, comprising any one primer combination of the above-mentioned loop-mediated isothermal amplification detection primer set.

[0015] The third aspect of the present invention provides a loop-mediated isothermal amplification (LAMP) detection kit for the pine needle red spot pathogen, Condylosporium septosporium, comprising any one primer combination of the above-mentioned loop-mediated isothermal amplification detection primer set.

[0016] A fourth aspect of the present invention provides a method for detecting the pine needle red spot pathogen, Desporium septoria, by loop-mediated isothermal amplification (LAMP) using any one primer combination in the above-mentioned loop-mediated isothermal amplification (LAMP) detection primer set, comprising the following steps:

[0017] S1. Extract DNA from pine needle red spot disease tissue or pure culture of the pathogen;

[0018] S2. Performing a LAMP amplification reaction using DNA as a template using any one primer combination in the loop-mediated isothermal amplification detection primer set;

[0019] S3. Perform a SYBR Green I colorimetric reaction and determine whether the pine needle tissue sample or pure culture of the pathogen contains the pine needle red spot pathogen, Dessosporium septosporium, based on the color.

[0020] Preferably, in step S2, the reaction system of the LAMP amplification reaction is 25 μL, including the following components: 10 × IsothermalAmp Buffer 2.5 μL, 100 mM MgSO4 1.5 μL, 25 mM dNTPs 1.4 μL, 10 μM positive inner primer FIP 4 μL, 10 μM negative inner primer BIP 4 μL, 10 μM positive outer primer F3 0.5 μL, 10 μM negative outer primer B3 0.5 μL, 10 μM forward loop primer LF 2 μL, 10 μM negative loop primer LB 2 μL, 0.5 M Betaine 1 μL, 8U / μL Bst II Pro DNA Polymerase Large Fragment 1 μL, DNA template 1 μL, and sterile water 2 μL.

[0021] Preferably, in step S2, the reaction conditions of the LAMP amplification reaction are: 65° C., 50 min.

[0022] Preferably, in step S3, the step of performing SYBR Green 1 color development reaction is: after the LAMP amplification reaction is completed, 2.5 μL of SYBR Green 1 reagent is added to the reaction system, mixed, and the color change is observed.

[0023] Preferably, the judgment principle is: if it shows fluorescent yellow-green color, it is judged that the pine needle red spot disease needle tissue of the sample to be tested or the pure culture of the pathogen contains spore spores of pine needle red spot pathogen; if it shows orange-red color, it is judged that the pine needle red spot disease needle tissue of the sample to be tested or the pure culture of the pathogen does not contain spore spores of pine needle red spot pathogen.

[0024] A fifth aspect of the present invention provides the use of the above-mentioned loop-mediated isothermal amplification detection reagent or loop-mediated isothermal amplification detection kit in the detection of pine needle red spot fungus, Spore s ...

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention designs four sets of loop-mediated isothermal amplification (LAMP) detection primers for the detection of spore-forming spores of the pine needle red spot pathogen. In the loop-mediated isothermal amplification (LAMP) detection of spore-forming spores of the pine needle red spot pathogen using the LAMP detection primers, the LAMP reaction can be completed at a constant temperature of 65°C for 50 minutes, and the detection is efficient and rapid.

[0027] (2) The present invention uses loop-mediated isothermal amplification detection primers to perform loop-mediated isothermal amplification detection of the pine needle red spot pathogen, which has low requirements for instruments and equipment. It does not require conventional detection and identification instruments such as PCR instruments, electrophoresis instruments, gel imaging systems, and microscopes. The detection can be completed using a water bath.

[0028] (3) The loop-mediated isothermal amplification detection of the pine needle red spot pathogen, the pine needle red spot pathogen, by using the loop-mediated isothermal amplification detection primers has strong specificity: it only specifically amplifies the pine needle red spot pathogen, the pine needle red spot pathogen, the pine needle red spot pathogen, the pine cone spore ...

[0029] (4) The results obtained by the loop-mediated isothermal amplification detection of the pine needle red spot pathogen, Dictyophora septoria, using the loop-mediated isothermal amplification detection primers of the present invention are easy to interpret, and the results can be interpreted by observing the color of the product with the naked eye.

[0030] (5) The loop-mediated isothermal amplification detection of the pine needle red spot pathogen, Dictyophora septoria, using the loop-mediated isothermal amplification detection primers of the present invention is safe, and no highly toxic reagents (such as EB) are used in the detection process.

[0031] In summary, the loop-mediated isothermal amplification detection primer set provided by the present invention, and the loop-mediated isothermal amplification detection method for pine needle red spot fungus, Sporangiophora septata, performed using the loop-mediated isothermal amplification detection primer set, have the characteristics of strong specificity, low requirements for instruments and equipment, and easy operation. In production practice, they can achieve rapid, sensitive, accurate, safe, and visual detection of pine needle red spot fungus, and can be used for field surveys, early diagnosis, seedling quarantine and transportation, disease monitoring, and identification of pine needle red spot disease, providing a reliable technical and theoretical basis for early warning and prevention and control of pine needle red spot fungus. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0033] Figure 1 The figure shows the results of screening available primer combinations (where Ds is the pine needle red spot pathogen, CK is the sterile water negative control, and T1-R5 are the alternative primer combinations listed in Table 1);

[0034] Figure 2Figure 2 is the LAMP amplification specificity detection result of four primer combinations (wherein, Ds1 is the diseased pine needle sample, Ds2 is the pure culture colony sample of Distichosporium septosporum, Ca is Distichosporium pine needles, Cc is Cladosporium cladosporioides, Lc is Leptosporium coniferum, Lp is Leptosporium pine needles, Sp is Scutellaria pinaster, and Ss is Scedosporium polysporum; T1, R1, R3, and R5 are the primer sets listed in Table 1). DETAILED DESCRIPTION

[0035] The present invention will be further illustrated and described below in conjunction with the embodiments, but the embodiments described are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the present invention and embodiments, all other inventions and embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0036] Example 1

[0037] 1. Experimental Materials

[0038] In this embodiment, the target strain used for detection was isolated from the needles infected with pine needle red spot disease, which was the spore-forming ... Collectrichumaenigma ), Cladosporium cladosporioides ( Cladosporiumcladosporioides ), coniferous speckled shell ( Lophodermium conigenum )、Pine needle speckle shell( Lophodermium pinastri ), Conchocystis spp. ( Sphaeropsis sapinea ) and Scedosporium polysporum ( Sydowia polyspora ), all purchased from China Forestry Culture Collection Center.

[0039] 2. Test methods

[0040] 2.1 Design and screening of primer combinations

[0041] According to the spore spore ( Dothistromaseptospora ) tef1 and rpb2 To determine the specificity of gene sequences, 10 primer combinations were designed using the loop-mediated isothermal amplification (LAMP) assay primer online design website (http: / / primerexplorer.jp / lampv5e / index.html), containing a total of 54 primers, as shown in Table 1.

[0042] Table 1

[0043]

[0044] The alternative primer combinations listed in Table 1 were used for LAMP amplification reactions. The LAMP amplification reaction system was 25 μL and consisted of: 10 × IsothermalAmp Buffer 2.5 μL, MgSO4 (100 mM) 1.5 μL, dNTPs (25 mM) 1.4 μL, positive inner primer FIP (10 μM) 4 μL, negative inner primer BIP (10 μM) 4 μL, positive outer primer F3 (10 μM) 0.5 μL, negative outer primer B3 (10 μM) 0.5 μL, positive loop primer LF (10 μM) 2 μL, negative loop primer LB (10 μM) 2 μL, Betaine (0.5 M) 1 μL, Bst II Pro DNA Polymerase Large Fragment (8 U / μL) 1 μL, and a DNA template of 1 μL. 1 μL, 2 μL sterile water. The reaction system was mixed in a PCR tube and placed in a constant temperature condition of 65 ℃ for 50 min. After the reaction was completed, 0.5 μL of SYBR Green I was added for color development. The color of the solution was then observed. If it showed fluorescent yellow-green, it indicated that the primer combination could detect the spore spore spore; if it showed orange-red, it indicated that the primer combination could not detect the spore spore spore spore. The results are shown in Figure 1 .

[0045] Figure 1 The results showed that only four primer combinations, T1, R1, R3 and R5, showed fluorescent yellow-green color. Therefore, it can be concluded that the detection method for the red spot fungus, Dessicaria septifolia, established based on the above four primer combinations can detect Dessicaria septifolia.

[0046] 3. Specificity test

[0047] DNA was extracted from the infected needle tissue of the pine needle red spot sample (Ds1), and the spore spore spores were extracted from the spore spores of the spore spores ( Dothistromaseptospora , Ds2), and the six control strains were Pseudomonas aeruginosa ( Collectrichumanigma , Ca), Cladosporium cladosporioides ( Cladosporiumcladosporioides , Cc), coniferous speckle shell ( Lophodermium conigenum , Lc), pine needle speckle shell ( Lophodermium pinastri , Lp), Conchoceras pinicola ( Sphaeropsis sapinea , Ss) and Scedosporium polysporum ( Sydowia polyspora , Sp) pure culture DNA was used to detect the specificity of the four primer combinations screened above, namely primer combinations T1, R1, R3, and R5.

[0048] The extracted DNA was used as a template for LAMP amplification using primer combination T1, primer combination R1, primer combination R3, or primer combination R5. The LAMP amplification reaction system was 25 μL and consisted of the following: 10 × IsothermalAmp Buffer 2.5 μL, MgSO4 (100 mM) 1.5 μL, dNTPs (25 mM) 1.4 μL, positive inner primer FIP (10 μM) 4 μL, negative inner primer BIP (10 μM) 4 μL, positive outer primer F3 (10 μM) 0.5 μL, negative outer primer B3 (10 μM) 0.5 μL, positive loop primer LF (10 μM) 2 μL, negative loop primer LB (10 μM) 2 μL, Betaine (0.5 M) 1 μL, and Bst II Pro DNA Polymerase Large Fragment (8 U / μL). 1 μL, 1 μL of the above-extracted DNA template, and 2 μL of sterile water. LAMP amplification reaction was carried out at 65°C for 50 min. After the LAMP amplification reaction, 0.5 μL of SYBR Green I was added to the tube and the SYBR Green I color reaction was performed. The color of the solution was observed. If it showed fluorescent yellow-green, it indicated that the primer combination could detect the spore spore; if it showed orange-red, the primer combination could not detect the spore spore. Results are shown in the figure. Figure 2 .

[0049] Depend on Figure 2 The results showed that the four primer combinations T1, R1, R3 and R5 could only detect the needles of pine needle red spot disease and pure culture of Dessicaria septoria, which indicated that the four primer combinations T1, R1, R3 and R5 had high specificity.

[0050] The present invention is not limited to the above-mentioned specific implementation methods. Various changes made by ordinary technicians in this field based on the above-mentioned conception without creative work are all within the scope of protection of this invention.

Claims

1. A primer set for loop-mediated isothermal amplification detection of the pine needle red spot pathogen, Dictyophora septoria, characterized in that: The detection primer set includes four primer combinations, namely primer combination T1, primer combination R1, primer combination R3, and primer combination R5, wherein: Primer combination T1, comprising a positive inner primer FIP-1, whose nucleotide sequence is shown in SEQ ID No. 1; a negative inner primer BIP-1, whose nucleotide sequence is shown in SEQ ID No. 2; a positive outer primer F3-1, whose nucleotide sequence is shown in SEQ ID No. 3; a negative outer primer B3-1, whose nucleotide sequence is shown in SEQ ID No. 4; a positive loop primer LF-1, whose nucleotide sequence is shown in SEQ ID No. 5; and a negative loop primer LB-1, whose nucleotide sequence is shown in SEQ ID No. 6; Primer combination R1, comprising a positive inner primer FIP-6, whose nucleotide sequence is shown in SEQ ID No. 7, a negative inner primer BIP-6, whose nucleotide sequence is shown in SEQ ID No. 8; a positive outer primer F3-6, whose nucleotide sequence is shown in SEQ ID No. 9; a negative outer primer B3-6, whose nucleotide sequence is shown in SEQ ID No. 10; a positive loop primer LF-6, whose nucleotide sequence is shown in SEQ ID No. 11; and a negative loop primer LB-6, whose nucleotide sequence is shown in SEQ ID No. 12; Primer combination R3, comprising a positive inner primer FIP-8, whose nucleotide sequence is shown in SEQ ID No. 13; a negative inner primer BIP-8, whose nucleotide sequence is shown in SEQ ID No. 14; a positive outer primer F3-8, whose nucleotide sequence is shown in SEQ ID No. 15; a negative outer primer B3-8, whose nucleotide sequence is shown in SEQ ID No. 16; a positive loop primer LF-8, whose nucleotide sequence is shown in SEQ ID No. 17; and a negative loop primer LB-8, whose nucleotide sequence is shown in SEQ ID No. 18; Primer combination R5 includes a forward inner primer FIP-10, whose nucleotide sequence is shown in SEQ ID No. 19; a negative inner primer BIP-10, whose nucleotide sequence is shown in SEQ ID No. 20; a forward outer primer F3-10, whose nucleotide sequence is shown in SEQ ID No. 21; a negative outer primer B3-10, whose nucleotide sequence is shown in SEQ ID No. 22; a forward loop primer LF-10, whose nucleotide sequence is shown in SEQ ID No. 23; and a negative loop primer LB-10, whose nucleotide sequence is shown in SEQ ID No.

24.

2. A loop-mediated isothermal amplification detection reagent for the pine needle red spot pathogen, characterized in that: A primer combination comprising any one of the loop-mediated isothermal amplification detection primer sets according to claim 1.

3. A LAMP detection kit for pine needle red spot fungus, characterized in that: A primer combination comprising any one of the loop-mediated isothermal amplification detection primer sets according to claim 1.

4. A method for detecting the pine needle red spot pathogen Diabrotica septoria by loop-mediated isothermal amplification using any one of the primer combinations in the loop-mediated isothermal amplification detection primer set according to claim 1, characterized in that: The following steps are involved: S1. Extract DNA from pine needle red spot disease tissue or pure culture of the pathogen; S2. Performing a LAMP amplification reaction using DNA as a template using any one primer combination in the loop-mediated isothermal amplification detection primer set; S3. Perform a SYBR Green I colorimetric reaction and determine whether the pine needle tissue sample or pure culture of the pathogen contains the pine needle red spot pathogen, Dessosporium septosporium, based on the color.

5. The loop-mediated isothermal amplification detection method according to claim 4, characterized in that The judgment principle is: if it shows fluorescent yellow-green color, it is judged that the pine needle red spot disease needle tissue of the sample to be tested or the pure culture of the pathogen contains spore spores of pine needle red spot pathogen; if it shows orange-red color, it is judged that the pine needle red spot disease needle tissue of the sample to be tested or the pure culture of the pathogen does not contain spore spores of pine needle red spot pathogen.

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