Loop-mediated isothermal amplification detection primer group for chestnut fruit rot pathogen pseudodiagraphis dunalii and application of loop-mediated isothermal amplification detection primer group

Abstract

The invention discloses a loop-mediated isothermal amplification detection primer group of Chinese chestnut fruit rot pathogen pseudodiagraphis dunalii and an application of the loop-mediated isothermal amplification detection primer group of chestnut fruit rot pathogen pseudodiagraphis dunalii. According to the invention, two groups of loop-mediated isothermal amplification detection primer groups, namely a primer combination E1 and a primer combination U5, of the Chinese chestnut fruit rot pathogen pseudodiagraphis taiwani are designed. Experimental results show that the two groups of loop-mediated isothermal amplification detection primer groups and the loop-mediated isothermal amplification detection method of the chestnut fruit rot pathogen pseudodiagrus schlegeli by using the loop-mediated isothermal amplification detection primer groups provided by the invention have the characteristics of strong specificity, convenience in operation and the like, and can be used for detecting the chestnut fruit rot pathogen pseudodiagrus schlegeli by using the loop-mediated isothermal amplification detection primer groups and the loop-mediated isothermal amplification detection method of the chestnut fruit rot pathogen pseudodiagrus schlegeli. In production practice, rapid, accurate, safe and visual detection of the Chinese chestnut fruit rot germs can be achieved, the method can be used for disease diagnosis and pathogenic bacterium identification after Chinese chestnut harvesting, and reliable technical and theoretical bases are provided for quality control of Chinese chestnuts in batches.

Description

Technical Field

[0001] This invention belongs to the field of plant pathogen detection, specifically the field of plant fungal disease detection, and particularly relates to a loop-mediated isothermal amplification detection primer set for *Pseudomonas dilatatus*, a pathogen causing chestnut fruit rot, and its application. Background Technology

[0002] Chestnut fruit rot is one of the most significant threats to chestnut production, drastically reducing both yield and quality. Currently, this disease occurs in all major chestnut-producing areas of my country, causing substantial economic losses.

[0003] The pathogens causing chestnut fruit rot are diverse, including *Pseudomonas dell's* (…). Gnomoniopsis daii Staphylococcus aureus ( Botryosphaeria dothidea ), Aquilaria malaccensis ( Lasiodiplodia aquilariae ), Kenyan Species of Polychaete ( Pestalotiopsis kenyana ), single-room housing ( Diaporthe eres ) and fruit anthracnose ( Colletotrichum fructicola Among them, *Pseudomonas des spp.* is a newly discovered pathogen of chestnut fruit rot in China in recent years.

[0004] Compared to other diseases, chestnut fruit rot is more insidious and may affect human health. In the early stages, the chestnut flesh shows slight discoloration and spoilage (see...). Figure 1 This disease affects the taste and texture of chestnuts; in the later stages, the chestnut flesh turns brown, becomes dry, hard, and bitter, and loses its edible value. However, identifying chestnut fruit rot through isolation and identification is currently time-consuming, labor-intensive, and requires a high level of professional expertise from practitioners and sophisticated equipment.

[0005] Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that rapidly amplifies target genes under isothermal conditions.

[0006] Therefore, this invention aims to provide a rapid detection method based on loop-mediated isothermal amplification (LAMP) technology for detecting *Pseudomonas dilatatus*, the causal agent of chestnut fruit rot. This method combines LAMP technology with visualization detection technology, and is expected to rapidly and accurately detect *Pseudomonas dilatatus*, the causal agent of chestnut fruit rot, in chestnut fruit tissue or fungal colonies. Summary of the Invention

[0007] To address the technical problems in the background art, the present invention aims to provide a loop-mediated isothermal amplification detection primer set for *Pseudomonas dilatatus*, a pathogen causing chestnut fruit rot, and its application.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides a loop-mediated isothermal amplification detection primer set for *Pseudomonas dilatatus*, a pathogen causing chestnut fruit rot. The detection primer set comprises two primer combinations: primer combination E1 and primer combination U5. The primer combination E1 includes a positive inner primer FIP-1, the nucleotide sequence of which is shown in SEQ ID No. 1; a negative inner primer BIP-1, the nucleotide sequence of which is shown in SEQ ID No. 2; a positive outer primer F3-1, the nucleotide sequence of which is shown in SEQ ID No. 3; a negative outer primer B3-1, the nucleotide sequence of which is shown in SEQ ID No. 4; a positive loop primer LF-1, the nucleotide sequence of which is shown in SEQ ID No. 5; and a negative loop primer LB-1, the nucleotide sequence of which is shown in SEQ ID No. 6. The primer combination U5 includes a positive inner primer FIP-2, the nucleotide sequence of which is shown in SEQ ID No. 7; a negative inner primer BIP-2, the nucleotide sequence of which is shown in SEQ ID No. 8; a positive outer primer F3-2, the nucleotide sequence of which is shown in SEQ ID No. 9; a negative outer primer B3-2, the nucleotide sequence of which is shown in SEQ ID No. 10; a positive loop primer LF-2, the nucleotide sequence of which is shown in SEQ ID No. 11; and a negative loop primer LB-2, the nucleotide sequence of which is shown in SEQ ID No. 12.

[0009] A second aspect of the present invention provides a loop-mediated isothermal amplification (LAMP) detection reagent for *Pseudomonas dilatatus*, a pathogen causing fruit rot in chestnuts, comprising any one of the primer combinations in the above-mentioned LAMP detection primer set.

[0010] A third aspect of the present invention provides a loop-mediated isothermal amplification (LAMP) detection kit for *Pseudomonas dilatatus*, a pathogen causing fruit rot in chestnuts, comprising any one of the primer combinations in the above-mentioned LAMP detection primer set.

[0011] A fourth aspect of the present invention provides a method for detecting *Pseudomonas dilatatus*, a chestnut fruit rot pathogen, using any combination of primers from the above-described loop-mediated isothermal amplification (LAMP) detection primer set, comprising the following steps: S1. Extract DNA from chestnut fruit tissue or pure culture of pathogenic bacteria in the sample to be tested; S2. Using DNA as a template, perform LAMP amplification reaction using any combination of primers from the loop-mediated isothermal amplification detection primer set; S3. Perform the SYBR Green I colorimetric reaction and determine whether the chestnut fruit sample or the pure culture of the pathogen contains the chestnut fruit rot pathogen *Pseudomonas dell'sus*.

[0012] Preferably, the determination principle is as follows: if the color is fluorescent yellow-green, it is determined that the chestnut fruit tissue or pure culture of pathogen in the test sample contains *Pseudomonas dell's husk*, the pathogen causing chestnut fruit rot; if the color is orange-red, it is determined that the chestnut fruit tissue or pure culture of pathogen in the test sample does not contain *Pseudomonas dell's husk*, the pathogen causing chestnut fruit rot.

[0013] The fifth aspect of the present invention provides the application of the above-described loop-mediated isothermal amplification detection reagent or loop-mediated isothermal amplification detection kit in the detection of *Pseudomonas dilatatus*, the pathogen of chestnut fruit rot.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention designs two sets of loop-mediated isothermal amplification detection primers for the detection of *Pseudomonas dilatatus*, a pathogen of chestnut fruit rot. In the loop-mediated isothermal amplification detection of *Pseudomonas dilatatus*, a pathogen of chestnut fruit rot, using any of the above-mentioned loop-mediated isothermal amplification detection primers, the LAMP reaction can be completed in a constant temperature condition of 60 °C for 60 min, which is efficient and rapid.

[0015] (2) The present invention utilizes loop-mediated isothermal amplification detection primers to detect the loop-mediated isothermal amplification of the chestnut fruit rot 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.

[0016] (3) The loop-mediated isothermal amplification detection of *Pseudomonas dilatatus*, a pathogen of chestnut fruit rot, using the detection primers of this invention has high specificity: it specifically amplifies only *Pseudomonas dilatatus*, while not amplifying other endophytic or saprophytic fungi of chestnuts, such as *Staphylococcus aureus*. Botryosphaeria dothidea ), Aquilaria malaccensis ( Lasiodiplodia aquilariae ), Kenyan Species of Polychaete ( Pestalotiopsis kenyana ), single-room housing ( Diaporthe eres ) and fruit anthracnose ( Colletotrichum fructicola No response was observed. (4) The results obtained by the loop-mediated isothermal amplification detection of chestnut fruit rot pathogen Pseudomonas dilatatus using the loop-mediated isothermal amplification detection primers of the present invention are easy to interpret. The results can be interpreted by observing the color of the product with the naked eye.

[0017] (5) The loop-mediated isothermal amplification detection of chestnut fruit rot pathogen Pseudomonas dilatatus using the loop-mediated isothermal amplification detection primers of the present invention has good safety and does not use highly toxic reagents (such as EB) during the detection process.

[0018] (6) This invention is based on specific genes TEF1 and TUB2Two primer combinations were designed to effectively avoid the risk of missed detection caused by a single gene mutation, and further improve the reliability of the test results.

[0019] In summary, the loop-mediated isothermal amplification (LMA) primer set and the LMA detection method for *Pseudomonas dilatatus*, the chestnut fruit rot pathogen, provided by this invention have the characteristics of high specificity, low requirements for instruments and equipment, and convenient operation. In production practice, it can realize rapid, accurate, safe, and visual detection of chestnut fruit rot, and can be used for post-harvest disease diagnosis and pathogen identification of chestnuts, providing a reliable technical and theoretical basis for batch quality control of chestnuts. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Images showing early symptoms of chestnut fruit rot fungus; Figure 2 The image shows the LAMP amplification specificity detection results of primer combination E1 (where GD is a pure culture colony sample of *Pseudomonas dell'sria*, FRUIT is a chestnut fruit sample with fruit rot, and BD is *Botrytis cinerea*). Botryosphaeria dothidea Pure culture colony sample, LA is agaric hairy dispora ( Lasiodiplodia aquilariae Pure culture colony samples, PK was Kenyan platycodon ( Pestalotiopsis kenyana Pure culture colony samples, DE is a single-celled shell ( Diaporthe eres Pure culture colony samples, CF is fruit-borne anthracnose ( Colletotrichum fructicola (Pure cultured bacterial colony samples); Figure 3 The image shows the LAMP amplification specificity detection results of primer combination U5 (where GD is a pure culture colony sample of *Pseudomonas dell'sria*, FRUIT is a chestnut fruit sample with fruit rot, and BD is *Botrytis cinerea*). Botryosphaeria dothidea Pure culture colony sample, LA is agaric hairy dispora ( Lasiodiplodia aquilariae Pure culture colony samples, PK was Kenyan platycodon ( Pestalotiopsis kenyana Pure culture colony samples, DE is a single-celled shell ( Diaporthe eres Pure culture colony samples, CF is fruit-borne anthracnose ( Colletotrichum fructicola Pure culture colony samples). Detailed Implementation

[0022] The present invention will be further described and illustrated below with reference to embodiments. However, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the present invention and the embodiments, all other inventions and embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1 1. Experimental Materials In this embodiment, the target strain used for detection was isolated from chestnut fruit rot diseased fruit, specifically *Pseudobularia davidii*. Gnomoniopsis daii Five negative control strains were identified, namely *Staphylococcus aureus* (…). Botryosphaeria dothidea ), Aquilaria malaccensis ( Lasiodiplodia aquilariae ), Kenyan Species of Polychaete ( Pestalotiopsis kenyana ), single-room housing ( Diaporthe eres ) and fruit anthracnose ( Colletotrichum fructicola All of them were purchased from the China Forestry Microbial Culture Collection Center (CFCC).

[0024] 2. Test methods 2.1 Primer combination design According to Dai's proposed sun-gauge shell ( Gnomoniopsis daii ) TEF1 and TUB2 To ensure gene sequence specificity, two primer sets were designed using an online primer design website (http: / / primerexplorer.jp / lampv5e / index.html) for loop-mediated isothermal amplification (LAMP). Primer set E1 was designed to target specific gene sequences. TEF1 Gene design, primer set U5 targeting TUB2 The gene design involves a total of 12 primers, as shown in Table 1 below.

[0025] Table 1

[0026] 2.2 Specificity test (1) DNA (FRUIT) was extracted from the chestnut fruit rot tissue of the test sample, and the shell of *Pseudoraska dauricus* was extracted. Gnomoniopsis daii , GD), Staphylococcus aureus ( Botryosphaeria dothidea , BD), Agarwood hairy two-spore ( Lasiodiplodia aquilariae , LA), Kenyan Species of Polytrichum candida ( Pestalotiopsis kenyana ,PK), single-seat shell ( Diaporthe eres DE) and fruit anthracnose ( Colletotrichum fructicola DNA from pure cultures (CF) was used to detect the specificity of primer combinations E1 and U5; (2) Using the extracted DNA as a template, LAMP amplification reactions were performed using primer combinations E1 and U5 listed in Table 1. The LAMP amplification reaction system was 25 μL, consisting of: 10 × IsothermalAmp Buffer 2.5 μL, 100mM MgSO4 2 μL, 25 mM dNTPs 3.5 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 positive loop primer LF 2 μL, 10 μM negative loop primer LB 2 μL, 8 U / μL Bst II Pro DNA Polymerase Large Fragment 1 μL, DNA template 1 μL, and sterile water 2 μL. The reaction mixture was thoroughly mixed in a PCR tube and incubated at 60 °C for 60 min. After the LAMP amplification reaction, the tube was opened, and 1 μL of SYBR Green I was added to the reaction solution. The mixture was then mixed, and the SYBR Green I colorimetric reaction was performed. The color of the solution was observed; a fluorescent yellow-green color indicated that the primer set could detect *Pseudomonas dell'sria*, the causal agent of chestnut fruit rot; an orange-red color indicated that the primer set could not detect *Pseudomonas septum*. Results are shown below. Figure 2 and Figure 3 .

[0027] Depend on Figure 2 The results showed that primer combination E1 could only detect chestnut fruit rot diseased fruits and pure cultures of *Pseudomonas dell'sria* shells, but could not detect other endophytic fungi or pathogens, indicating that primer combination E1 has high specificity.

[0028] Depend on Figure 3 The results showed that primer combination U5 could only detect chestnut fruit rot diseased fruits and pure cultures of *Pseudomonas dell's palustris* shells, but could not detect other endophytic fungi or pathogens, indicating that primer combination U5 has high specificity.

[0029] This invention is not limited to the specific embodiments described above. Any modifications made by those skilled in the art based on the above concept without creative effort are within the scope of protection of this invention.

Claims

1. A set of loop-mediated isothermal amplification primers for detecting *Pseudomonas dilatatus*, a pathogen causing chestnut fruit rot, characterized in that... The loop-mediated isothermal amplification detection primer set has two primer combinations: primer combination E1 and primer combination U5. The primer combination E1 includes a positive inner primer FIP-1, the nucleotide sequence of which is shown in SEQ ID No. 1; a negative inner primer BIP-1, the nucleotide sequence of which is shown in SEQ ID No. 2; a positive outer primer F3-1, the nucleotide sequence of which is shown in SEQ ID No. 3; a negative outer primer B3-1, the nucleotide sequence of which is shown in SEQ ID No. 4; a positive loop primer LF-1, the nucleotide sequence of which is shown in SEQ ID No. 5; and a negative loop primer LB-1, the nucleotide sequence of which is shown in SEQ ID No.

6. The primer combination U5 includes a positive inner primer FIP-2, the nucleotide sequence of which is shown in SEQ ID No. 7; a negative inner primer BIP-2, the nucleotide sequence of which is shown in SEQ ID No. 8; a positive outer primer F3-2, the nucleotide sequence of which is shown in SEQ ID No. 9; a negative outer primer B3-2, the nucleotide sequence of which is shown in SEQ ID No. 10; a positive loop primer LF-2, the nucleotide sequence of which is shown in SEQ ID No. 11; and a negative loop primer LB-2, the nucleotide sequence of which is shown in SEQ ID No.

12.

2. A loop-mediated isothermal amplification detection reagent for *Pseudomonas dilatatus*, a pathogen causing chestnut fruit rot, characterized in that... It includes any one of the primer combinations in the loop-mediated isothermal amplification detection primer set described in claim 1.

3. A loop-mediated isothermal amplification detection kit for *Pseudomonas dilatatus*, a pathogen causing chestnut fruit rot, characterized in that... It includes any one of the primer combinations in the loop-mediated isothermal amplification detection primer set described in claim 1.

4. A method for detecting *Pseudomonas dilatatus*, the causal agent of chestnut fruit rot, using any combination of primers from the loop-mediated isothermal amplification detection primer set described in claim 1, characterized in that... Includes the following steps: S1. Extract DNA from chestnut fruit tissue or pure culture of pathogenic bacteria in the sample to be tested; S2. Using DNA as a template, perform LAMP amplification reaction using any combination of primers from the loop-mediated isothermal amplification detection primer set; S3. Perform the SYBR Green I colorimetric reaction and determine whether the chestnut fruit sample or the pure culture of the pathogen contains the chestnut fruit rot pathogen *Pseudomonas dell'sus*.

5. The loop-mediated isothermal amplification detection method according to claim 4, characterized in that, The judgment principle is as follows: if the color is fluorescent yellow-green, it is determined that the chestnut fruit sample or the pure culture of the pathogen contains the chestnut fruit rot pathogen *Pseudomonas dell's husk*; if the color is orange-red, it is determined that the chestnut fruit sample or the pure culture of the pathogen does not contain the chestnut fruit rot pathogen *Pseudomonas dell's husk*.

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