LAMP primer set, kit and detection method for detecting pinus armandii gasteria

Abstract

The application discloses a LAMP primer group, a kit and a detection method for detecting Pinus armandii rust fungus, and belongs to the technical field of genetic engineering. The application takes the specific nucleotide sequence of the large subunit ribosomal RNA of the Pinus armandii rust fungus as a target gene to design and screen a primer group which is specific and high in sensitivity, and can be used for LAMP detection of the Pinus armandii rust fungus. The LAMP primer group provided by the application has the advantages of high universality, high specificity, high sensitivity and good accuracy, and the detection limit can reach 4.5x10 ‑4 ng / μL, which is 100 times higher than the sensitivity of the LAMP detection of different species in the same genus in the prior art, the detection method can rapidly detect the Pinus armandii rust fungus from spore piles and host plant materials, and is suitable for the detection of Pinus armandii seedling materials in forest fields and customs quarantine ports.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a LAMP primer set, kit, and detection method for detecting Pinus armandii rust. Background Technology

[0002] Pine blister rust is a significant quarantine disease of forest trees worldwide, listed in my country's "List of Quarantine Pests for Imported Plants" and "National List of Quarantine Pests in Forestry." It primarily affects five-needle pines such as Chinese white pine and Korean pine, causing necrosis of branches and trunks, ultimately leading to death and severe economic and ecological losses. The pathogen causing pine blister rust is *Cronartium ribicola*, belonging to the genus *Cronartium* within the order Pucciniales. In recent years, a new species of *Cronartium* rust affecting Chinese white pine has been discovered: *Cronartium armandii*. This new species is widely distributed in Shaanxi, Sichuan, and Yunnan provinces of my country, primarily affecting the branches and trunks of Chinese white pine, causing trunk rust. Studies have shown that *Crassostrea huashanensis* shares similar spore morphology, host species, and disease symptoms with *Crassostrea gigas* and *Cronartium orientale*, making taxonomic confusion highly likely. Currently, these closely related species can only be distinguished through conventional PCR reactions and phylogenetic analysis.

[0003] To effectively reduce the risk of the spread of rust in Huashan pine, accurate identification and diagnosis in the early stages of disease occurrence are crucial for rapid and accurate detection of the pathogen. Previous studies have used methods such as observing the morphological characteristics of each spore stage and conventional PCR reactions for detecting column rust fungi. However, morphological identification of closely related rust species often requires professionals with a solid foundation in fungal taxonomy, while conventional PCR techniques are cumbersome, taking 6-8 hours from sampling to obtaining results, are time-consuming and labor-intensive, have low sensitivity, and must be performed in a laboratory. Loop-mediated isothermal amplification (LAMP) is an alternative to PCR, utilizing 4 to 6 oligonucleotide primers and strand displacement DNA polymerase (Bst DNA polymerase) for amplification at a constant temperature. It offers advantages such as high versatility, high specificity, high sensitivity, and good accuracy, while reducing the requirements for experimental equipment, testing environment, and technical personnel, making it more suitable for rapid molecular detection in the field. In 2023, Kozhar et al. used comparative genomics to screen specific primers for *Cronartium ribicola* and established a rapid molecular detection system for this fungus using LAMP, with a detection sensitivity of 40 pg / ul. However, there is currently no rapid molecular detection and diagnostic technology for *Cronartium ribicola*, which can cause the same disease in *Pinus armandii*. Summary of the Invention

[0004] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a LAMP primer set, reagent kit, and detection method for detecting *Pinus armandii* rust, thereby resolving the current lack of rapid molecular detection and diagnostic technologies for *Pinus armandii* rust.

[0005] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A LAMP primer set for detecting *Pinus armandii* rust fungus is provided, comprising the following primer sequences:

[0006] Forward outer primer F3: 5'-GTTGTGTTATAGCTCATGACTT-3' (SEQ ID NO.1);

[0007] Reverse outer primer B3: 5'-CAGATCTCATTTACATTCACTTTCA-3' (SEQ ID NO.2);

[0008] Forward inner primer FIP:

[0009] 5'-GTGAGAAGATCCCCAAAGGCACTTAGGATTGAGGAACACAGT-3' (SEQ ID NO. 3);

[0010] Reverse inner primer BIP:

[0011] 5'-TGGTGTAATAGCTTTAAATGACCCCTTATGCATAAGGGTTTCAACA-3' (SEQ ID NO. 4).

[0012] This invention provides a kit for detecting *Pinus armandii* rust fungus, comprising the aforementioned LAMP primer set.

[0013] This invention also provides a method for detecting rust fungus in Pinus armandii, comprising the following steps:

[0014] (1) Extract DNA from the sample to be tested;

[0015] (2) LAMP amplification was performed using LAMP primer sets;

[0016] (3) Add a reaction indicator to the amplification product obtained in step (2) and determine whether it contains Pinus armandii rust by color change and fluorescence intensity;

[0017] The primer sequences for the LAMP primer set are as follows:

[0018] Forward outer primer F3: 5'-GTTGTGTTATAGCTCATGACTT-3' (SEQ ID NO.1);

[0019] Reverse outer primer B3: 5'-CAGATCTCATTTACATTCACTTTCA-3' (SEQ ID NO.2);

[0020] Forward inner primer FIP:

[0021] 5'-GTGAGAAGATCCCCAAAGGCACTTAGGATTGAGGAACACAGT-3'

[0022] (SEQ ID NO.3);

[0023] Reverse inner primer BIP:

[0024] 5'-TGGTGTAATAGCTTTAAATGACCCCTTATGCATAAGGGTTTCAACA-3' (SEQ ID NO. 4).

[0025] Furthermore, the LAMP amplification reaction system was 25 μL, including: 0.4-0.6 μL of forward inner primer FIP, 0.4-0.6 μL of reverse inner primer BIP, 0.4-0.6 μL of forward outer primer F3, 0.4-0.6 μL of reverse outer primer B3, 3-4 μL of dNTPs, 1-3 μL of MgSO4, 4-6 μL of betaine, 2-3 μL of 1×Bst Reaction, 0.5-1.5 μL of Bst DNA polymerase, and 0.5-1.5 μL of the DNA solution to be tested, which was then brought to a final volume of 25 μL with ddH2O.

[0026] Furthermore, the concentrations of the forward inner primer FIP were 1.5-1.7 μM, the reverse inner primer BIP were 1.5-1.7 μM, the forward outer primer F3 was 0.1-0.3 μM, the reverse outer primer B3 was 0.1-0.3 μM, the dNTPs were 8-12 mM, the MgSO4 was 80-120 mM, the betaine was 0.5-1.5 mM, and the Bst DNA polymerase was 7-9 U / μL.

[0027] Furthermore, the LAMP amplification reaction system was 25 μL, including: 0.5 μL of 1.6 μM forward inner primer FIP, 0.5 μL of 1.6 μM reverse inner primer BIP, 0.5 μL of 0.2 μM forward outer primer F3, 0.5 μL of 0.2 μM reverse outer primer B3, 3.5 μL of 10 mM dNTPs, 1.5 μL of 100 mM MgSO4, 5 μL of 1 mM betaine, 2.5 μL of 1×Bst Reaction buffer, 1 μL of 8 U / μL Bst DNA polymerase, and 1 μL of template DNA, with ddH2O added to bring the total to 25 μL.

[0028] Furthermore, the LAMP amplification temperature was 50-65℃ and the time was 30-75 min.

[0029] Furthermore, the reaction indicator is the dye SYBR Green I. After adding SYBR Green I to the amplification product obtained in step (2), when the color of the amplification product is yellow-green, it indicates that the sample to be tested is positive, and when the color of the amplification product is orange, it indicates that the sample to be tested is negative.

[0030] The present invention has the following beneficial effects:

[0031] (1) Filling a gap: *Pinus armandii* rust and *Rhizoctonia solani* rust are morphologically similar and cause the same disease symptoms, making accurate identification and differentiation impossible using traditional taxonomic methods. Furthermore, there is currently no LAMP detection method specifically for *Pinus armandii* rust. This study designed primers using the specific nucleotide sequence of the large subunit ribosomal RNA of *Pinus armandii* rust as the target gene, establishing a specific LAMP detection system for *Pinus armandii* rust. This system can specifically amplify *Pinus armandii* rust, providing important practical value and significance for early warning of *Pinus armandii* rust disease.

[0032] (2) High specificity: This invention uses a specific fragment of the large subunit ribosomal RNA of Pinus armandii as the target gene to design primers, and through primer screening, it can accurately distinguish other closely related species and non-quarantine pathogenic fungi that can cause rust disease.

[0033] (3) High sensitivity: Traditional morphological detection methods are time-consuming and the results are unstable, while the specific primer set and efficient detection system of Pinus armandii screened in this invention can achieve a sensitivity of up to 450 fg / ul.

[0034] (4) Good practicality: This invention can directly extract DNA from the diseased parts and spore masses of Pinus armandii for rapid molecular detection, which is more suitable for rapid detection of rust fungi in the wild forest farm, customs site and other environments; secondly, the ordinary PCR reaction requires gel electrophoresis of the product, which requires ethidium bromide (EB) staining and observation under ultraviolet light to judge the result, which prolongs the detection time, while the LAMP reaction can be carried out in a constant temperature water bath, with a short detection cycle. After the reaction is completed, the result can be judged directly by the naked eye under natural light by the color change of SYBR Green I. Therefore, it is more suitable for rapid detection of imported Pinus armandii seedlings in the wild and at customs quarantine ports, and has important application value. Attached Figure Description

[0035] Figure 1 Image showing the initial screening results of 5 primer sets for LAMP (3 replicates);

[0036] Figure 2 Figure showing the optimization results of the reaction temperature for the detection method of rust fungus in Pinus armandii;

[0037] Figure 3 Figure showing the optimized reaction time results for the detection method of rust fungus in Pinus armandii;

[0038] Figure 4 The figure shows the experimental results for the specificity of LAMP primer set primer 1;

[0039] Figure 5The figure shows the sensitivity test results of the detection method for rust fungus in Pinus armandii.

[0040] Figure 6 This is a sample test result of *Pinus armandii* rust fungus. Detailed Implementation

[0041] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0042] Example 1: Design and Screening of LAMP Primers

[0043] (1) The specific nucleotide sequence (SEQ ID NO.5) of the large subunit ribosomal RNA of Pinus armandii was selected as the target sequence for detecting Pinus armandii. Five sets of LAMP primers were designed online using https: / / www.neb.cn. The five sets of primers are primer 1, primer 2, primer 3, primer 4 and primer 5. The sequences of the five sets of primers are shown in Table 1.

[0044] (2) The designed primer sets 1, 2, 3, 4 and 5 were subjected to LAMP amplification experiments. The specific steps are as follows:

[0045] ① Genomic DNA of Pinus armandii rust fungus extracted by the slide method was used as a template;

[0046] ② The detection reaction system includes: 0.5 μL of 1.6 μM forward inner primer FIP, 0.5 μL of 1.6 μM reverse inner primer BIP, 0.5 μL of 0.2 μM forward outer primer F3, 0.5 μL of 0.2 μM reverse outer primer B3, 3.5 μL of 10 mM dNTPs, 1.5 μL of 100 mM MgSO4, 5 μL of 1 mM betaine, 2.5 μL of 1×Bst Reaction buffer, 1 μL of 8 U / μL Bst DNA polymerase, and 1 μL of template DNA, with ddH2O added to a final volume of 25 μL. The reaction includes a non-template control (NTC) to eliminate false positives.

[0047] ③ After the reaction system is prepared, add 1 μL of 100×SYBR Green I fluorescent dye working solution to the inner wall of the PCR tube cap, tighten the PCR tube cap, and carry out the LAMP amplification reaction. The reaction conditions are: 65℃ for 45 min; 85℃ for 10 min for denaturation, and then terminate the reaction.

[0048] ④ After the LAMP amplification reaction is complete, without opening the PCR tube, centrifuge or gently shake it to mix the SYBR Green I fluorescent dye on the inner wall of the PCR tube cap with the LAMP amplification product. Invert the tube to mix thoroughly and observe the color change of the mixture with the naked eye. Under natural light, if the mixture turns green, it indicates that the SYBR Green I dye has bound to the double-stranded DNA, which is a positive reaction, indicating that the sample contains *Pinus armandii* rust. If the mixture turns orange, it is a negative reaction, indicating that the sample does not contain *Pinus armandii* rust.

[0049] (3) The obtained LAMP amplification products were subjected to agarose gel electrophoresis to detect whether the sample contained the DNA of Pinus armandii. If a ladder-shaped electrophoresis band appeared, it was a positive reaction, indicating that the sample contained Pinus armandii. If no ladder-shaped electrophoresis band appeared, it was a negative reaction, indicating that the sample did not contain Pinus armandii.

[0050] The results are as follows Figure 1 As shown, by Figure 1 It was found that the reaction tube of primer set 1 was green (positive), and the gel electrophoresis results showed a ladder-shaped electrophoretic band. The other four primer sets failed to achieve successful amplification, and NTC was negative. Primer set 1 was able to successfully amplify the target gene. Therefore, primer set 1 was selected as the LAMP primer set for *Pinus armandii* rust.

[0051] Table 1. Nucleotide sequences of 5 LAMP primers

[0052]

[0053]

[0054] Example 2: Optimal reaction temperature and reaction time for the detection method of rust fungus in Pinus armandii.

[0055] (1) Eight temperature gradients were set for LAMP amplification: 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, and 80℃. Amplification was performed at each temperature for 60 min. Each reaction group included a non-template control (NTC) to eliminate false positives. After amplification, color development was performed, and the obtained LAMP products were detected by agarose gel electrophoresis. The results are shown below. Figure 2 As shown.

[0056] (2) Five different amplification times were set for LAMP amplification: 15 min, 30 min, 45 min, 60 min, and 75 min. Amplification was performed at 65℃ for each time. Each reaction included a non-template control (NTC) to eliminate false positives. After amplification, color development was performed, and the obtained LAMP products were detected by agarose gel electrophoresis. The results are as follows: Figure 3 As shown.

[0057] Depend on Figure 2 and Figure 3 It is known that the LAMP primer set primer 1 of this invention can successfully amplify the target gene at 50-65℃ for 30-75 min, and all NTCs are negative. By visually observing the color brightness of the mixture in the reaction tube and the brightness of the gel electrophoresis bands, the optimal reaction temperature and reaction time for the detection method of Pinus armandii rust fungus are 65℃ and 45 min, respectively.

[0058] Example 3: Specificity experiment of LAMP primer set primer 1

[0059] Using *C. ribicola*, *C. orientale*, *C. flaccidum*, *C. quercuum*, *Chrysomyxa qilianensis*, *Melampsora laricis-epitea*, and *Penicillium wortmannii* as controls, LAMP amplification experiments were performed using the detection reaction system and method described in Example 1. The reaction conditions were 65℃ for 45 min. Each reaction group included a non-template control (NTC) to eliminate false positives. After amplification, color development was performed, and the obtained LAMP products were detected by agarose gel electrophoresis. The results are as follows: Figure 4 As shown.

[0060] Depend on Figure 4 It can be seen that only the reaction tube containing the DNA of *Pinus armandii* was green (positive) and had a ladder-shaped electrophoretic band, while the rest of the amplification reactions were orange and had no ladder-shaped electrophoretic band. The NTC solution was negative. This indicates that the LAMP primer set primer 1 and the established LAMP detection method have high specificity for the detection of *Pinus armandii*.

[0061] Example 4: Sensitivity Experiment of the Detection Method for Rust Fungus in Pinus armandii

[0062] The concentration of extracted genomic DNA from *Pinus armandii* rust fungus was determined using a NanoDrop 8000 ultraviolet spectrophotometer. The genomic DNA was diluted to different concentration gradients using ddH2O: 450 ng / μL, 45 ng / μL, 4.50 ng / μL, 450 pg / μL, 45 pg / μL, 4.50 pg / μL, 450 fg / μL, 45 fg / μL, 4.5 fg / μL, 450 ag / μL, and 45 ag / μL. 1.0 μL of each dilution was used as a template, and LAMP amplification was performed using the detection reaction system and method described in Example 1. The reaction conditions were: amplification at 65℃ for 45 min. Each reaction group included a non-template control (NTC) to eliminate false positives. After amplification, color development was performed, and the obtained LAMP products were detected by agarose gel electrophoresis. The results... Figure 5 As shown.

[0063] Depend on Figure 5 It can be seen that when the concentration of *Pinus armandii* DNA in the reaction tube is diluted to 450 fg / μL, the reaction tube is green (positive) and has a ladder-shaped electrophoretic band. The other amplification products are all orange and have no ladder-shaped electrophoretic band. NTC is negative. The lowest detection limit of LAMP primer set primer 1 and the established LAMP detection method for the detection of *Pinus armandii* is 450 fg / μL.

[0064] Test case

[0065] Bark samples from both diseased and healthy Pinus armandii trees were collected from the forest floor. Plant tissue DNA was extracted using the CTAB method. The obtained genomic DNA was used as a template for LAMP amplification experiments using the detection reaction system and method described in Example 1. The reaction conditions were: amplification at 65℃ for 45 min. Each reaction group included a non-template control (NTC) to eliminate false positives. After amplification, color development was performed, and the obtained LAMP products were detected by agarose gel electrophoresis. The results are as follows: Figure 6 As shown.

[0066] Depend on Figure 6 It can be seen that the reaction tubes of diseased Pinus armandii bark samples were positive (green) and showed a ladder-shaped electrophoretic band; the reaction tubes of healthy Pinus armandii bark samples were negative (orange) and showed no ladder-shaped electrophoretic band, and NTC was negative in both cases. This indicates that the LAMP primer set of the present invention can rapidly detect Pinus armandii column rust fungus.

[0067] The specific nucleotide sequence of the large subunit ribosomal RNA of *Pinus sinensis* rust fungus of this invention is as follows:

[0068] (SEQ ID NO.5).

[0069] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A LAMP primer set for detecting *Pinus armandii* rust fungus, characterized in that, Includes the following primer sequences: Forward outer primer F3: 5'-GTTGTGTTATAGCTCATGACTT-3' (SEQ ID NO.1); Reverse outer primer B3: 5'-CAGATCTCATTTACATTCACTTTCA-3' (SEQ ID NO.2); Forward inner primer FIP: 5'-GTGAGAAGATCCCCAAAGGCACTTAGGATTGAGGAACACAGT-3' (SEQ ID NO. 3); Reverse inner primer BIP: 5'-TGGTGTAATAGCTTTAAATGACCCCTTATGCATAAGGGTTTCAACA-3' (SEQ ID NO. 4).

2. A kit for detecting *Pinus armandii* rust fungus, characterized in that, Includes the LAMP primer set as described in claim 1.

3. A method for detecting *Pinus armandii* rust fungus, characterized in that, Includes the following steps: (1) Extract DNA from the sample to be tested; (2) LAMP amplification was performed using LAMP primer sets; (3) Add a reaction indicator to the amplification product obtained in step (2) and determine whether it contains Pinus armandii rust by color change and fluorescence intensity; The primer sequences for the LAMP primer set are as follows: Forward outer primer F3: 5'-GTTGTGTTATAGCTCATGACTT-3' (SEQ ID NO.1); Reverse outer primer B3: 5'-CAGATCTCATTTACATTCACTTTCA-3' (SEQ ID NO.2); Forward inner primer FIP: 5'-GTGAGAAGATCCCCAAAGGCACTTAGGATTGAGGAACACAGT-3' (SEQ ID NO. 3); Reverse inner primer BIP: 5'-TGGTGTAATAGCTTTAAATGACCCCTTATGCATAAGGGTTTCAACA-3' (SEQ ID NO. 4).

4. The method for detecting *Pinus armandii* rust fungus according to claim 3, characterized in that, The LAMP amplification reaction system is 25 μL, comprising: 0.4-0.6 μL of forward inner primer FIP, 0.4-0.6 μL of reverse inner primer BIP, 0.4-0.6 μL of forward outer primer F3, 0.4-0.6 μL of reverse outer primer B3, 3-4 μL of dNTPs, 1-3 μL of MgSO4, 4-6 μL of betaine, 2-3 μL of 1×BstReaction, 0.5-1.5 μL of Bst DNA polymerase, and 0.5-1.5 μL of the DNA solution to be tested, and then brought to 25 μL with ddH2O.

5. The method for detecting *Pinus armandii* rust fungus according to claim 4, characterized in that, The concentrations of the forward inner primer FIP and the reverse inner primer BIP are 1.5-1.7 μM, the concentrations of the forward outer primer F3 and the reverse outer primer B3 are 0.1-0.3 μM, the concentrations of the dNTPs are 8-12 mM, the concentrations of the MgSO4 are 80-120 mM, the concentrations of the betaine are 0.5-1.5 mM, and the concentration of the Bst DNA polymerase is 7-9 U / μL.

6. The method for detecting *Pinus armandii* rust fungus according to claim 4 or 5, characterized in that, The LAMP amplification reaction system consisted of 25 μL, including: 0.5 μL of 1.6 μM forward inner primer FIP, 0.5 μL of 1.6 μM reverse inner primer BIP, 0.5 μL of 0.2 μM forward outer primer F3, 0.5 μL of 0.2 μM reverse outer primer B3, 3.5 μL of 10 mM dNTPs, 1.5 μL of 100 mM MgSO4, 5 μL of 1 mM betaine, 2.5 μL of 1×Bst Reaction buffer, 1 μL of 8 U / μL Bst DNA polymerase, and 1 μL of template DNA, with ddH2O added to bring the total volume to 25 μL.

7. The method for detecting *Pinus armandii* rust fungus according to claim 3, characterized in that, The LAMP amplification was performed at a temperature of 50-65℃ for 30-75 minutes.

8. The method for detecting *Pinus armandii* rust fungus according to claim 3, characterized in that, The reaction indicator is the dye SYBR Green I. After adding SYBR Green I to the amplification product obtained in step (2), when the color of the amplification product is yellow-green, it indicates that the sample to be tested is positive, and when the color of the amplification product is orange, it indicates that the sample to be tested is negative.

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