Metarhizium anisopliae lamp detection primer composition and visual detection method

Abstract

The application discloses a Metarhizium rileyi LAMP detection primer composition and a visual detection method. The LAMP detection primer composition is composed of a forward inner primer MrFIP, a reverse inner primer MrFIP, a forward outer primer MrF3, a reverse outer primer MrB3, a forward loop primer MrLoopF and a reverse loop primer MrLoopB. The Metarhizium rileyi is amplified by using the primers, and the result can be determined by observing the color of the reaction solution after the detection is finished. The specific primer combination is designed according to the beta-tubin gene sequence of the Metarhizium rileyi, the LAMP technology is used for visual detection, the method has the advantages of high sensitivity, strong specificity, convenience and rapidness and the like, is helpful to rapidly identifying and distinguishing the Metarhizium rileyi, and has important significance for development and evaluation of biological control pesticide of harmful insects of the order Lepidoptera.

Description

Technical Field

[0001] This invention belongs to the field of molecular biological detection technology of pathogenic fungi, specifically involving primer composition and visualization detection method for the LAMP detection of Metarhizium anisopliae. Background Technology

[0002] Metarhizium anisopliae ( Metarhizium rileyi Also known as Leystiliae, it is a typical disexual entomopathogenic fungus that primarily infects moths of the Noctuidae family. Noctuoidea More than 60 species of Lepidoptera, mainly Lepidoptera Agricultural pest larvae, including important agricultural, forestry, and urban pests such as the cotton bollworm, beet armyworm, sugar beet armyworm, fall armyworm, and pale gray-winged armyworm, are susceptible to infection. Under natural conditions, this fungus has a high mortality rate and can cause field epidemics under suitable conditions. It can effectively control pests below the economic damage threshold, therefore, the research and development of *Metarhizium anisopliae* is receiving increasing attention. Currently, *Metarhizium anisopliae* strains pathogenic to fall armyworm, pale gray-winged armyworm, and beet armyworm have been isolated, but the pathogenicity often varies significantly among strains from different hosts and regions. To further develop the biocontrol potential of *Metarhizium anisopliae* against agricultural pests and to screen and evaluate suitable biopesticide production strains, it is necessary to further develop rapid and convenient detection and monitoring technologies for *Metarhizium anisopliae*.

[0003] Currently, commonly used methods for bacterial strain identification and detection are morphological identification and molecular biological identification (PCR). Traditional morphological identification is time-consuming and labor-intensive, has low sensitivity, is easily affected by human or environmental factors, and is limited to the isolation of pure strains. Molecular biological identification and detection has developed various methods and techniques, such as conventional PCR, nested PCR, and real-time quantitative PCR. While these methods significantly shorten identification time and improve accuracy and sensitivity compared to traditional morphology, they rely on sophisticated, high-value PCR instruments, and even expensive quantitative PCR instruments; the identification / detection process is also relatively complex, with traditional PCR requiring subsequent processing such as gel electrophoresis. For example, Chinese patent CN105349692A discloses a detection... Lactobacillus paracasei The primer pair method for strain N1115 is disclosed in Chinese patent CN114350750A, which describes a method for identifying sample strains. However, these detection instrument identification methods all require multiple procedures to determine the results.

[0004] Loop-mediated isothermal amplification (LAMP) can amplify nucleic acids under isothermal conditions in a short time. It relies on multiple primers that recognize six specific fragments of conserved DNA sequences and a strand displacement DNA polymerase to complete amplification and detection in one step. It is a simple, rapid, and accurate gene amplification method with high specificity and short processing time. This method does not rely on high-precision instruments; results can be directly determined by visual observation based on color changes. It has been used to detect various microorganisms, including *Ralstonia solanacearum* (CN106893764A), *Fusarium solani* (CN106893763A), and *Erysophora flavescens* (CN108103153B). However, there are currently no LAMP detection primers or methods specifically for *Metarhizium anisopliae*. Summary of the Invention

[0005] The purpose of this invention is to provide a primer composition and a visualization detection method for the LAMP detection of Metarhizium anisopliae. The primer composition has high specificity, and Metarhizium anisopliae can be detected with high sensitivity using the primer composition, and the detection results are visualized.

[0006] The technical solution adopted in this invention is as follows:

[0007] This invention provides a primer composition for LAMP detection of *Metarhizium anisopliae*, comprising a forward inner primer MrFIP, a reverse inner primer MrFIP, a forward outer primer MrF3, a reverse outer primer MrB3, a forward loop primer MrLoopF, and a reverse loop primer MrLoopB. The sequences of each primer are as follows:

[0008] MrF3: SEQ ID No. 1;

[0009] MrB3: SEQ ID No. 2;

[0010] MrFIP: SEQ ID No. 3;

[0011] MrBIP: SEQ ID No. 4;

[0012] MrLoopF: SEQ ID No. 5;

[0013] MrLoopB: SEQ ID No. 6.

[0014] Preferably, the three-pair detection primer composition is designed based on the sequence of the β-tubin specific gene of Metarhizium anisopliae.

[0015] Preferably, the molar ratio of each primer pair in the detection primer composition is (MrFIP and MrBIP):(MrF3 and MrB3):(MrLoopF ​​and MrLoopB) = 8:1:2.

[0016] This invention also provides a visual detection method for Metarhizium anisopliae, specifically including the following steps:

[0017] (1) Sample preparation: Extract genomic DNA from the sample to be tested;

[0018] (2) LAMP amplification: The above primer combination or kit is used for detection. The amplification temperature is 63-65℃ and the amplification time is 30-40min.

[0019] (3) Result interpretation: After the reaction is completed, take out the PCR reaction tube and observe the color of the reaction tube. Yellow indicates that Metarhizium anisopliae is present in the sample, while red indicates that Metarhizium anisopliae is not present in the sample.

[0020] Preferably, the sample to be tested in step (1) is the genomic DNA of the purified strain or the total DNA of the soil sample.

[0021] Preferably, the concentration of the sample to be tested is above 100 fg / μL.

[0022] Beneficial effects

[0023] (1) Visualization of detection results and visual interpretation of results: This invention utilizes WarmStart ColorimetricLAMP 2X Master Mix reagent to reflect the results after the LAMP reaction using color. The results can be directly observed by the naked eye to determine whether the sample contains Metarhizium anisopliae.

[0024] (2) High specificity: The present invention designed 6 pairs of primers for 6 specific regions of the conserved gene β-tublin of Metarhizium anisopliae, thereby ensuring the high specificity of LAMP amplification. That is, the LAMP detection method can find the corresponding target sequence for amplification from gene samples with very small differences.

[0025] (3) High sensitivity: The target DNA concentration range that can be detected by the present invention is above 100 fg / μL, which is 100 times that of ordinary PCR.

[0026] (4) This LAMP detection reaction can be completed within 30-40 minutes, which is rapid and efficient. At the same time, LAMP does not rely on high-precision, high-value PCR instruments. A temperature-controlled constant temperature water bath is sufficient for the experiment, which greatly reduces the experimental cost. Furthermore, the LAMP detection primer composition and visualization detection method prepared in this invention can detect whether Metarhizium anisopliae is present in the total DNA of soil samples, which greatly shortens the traditional isolation, screening and detection time, avoids unnecessary waste, and expands the application scope. Attached Figure Description

[0027] Figure 1 The image shows the LAMP detection results of genomic DNA extracted using different methods in Example 2. In the image, 1 represents the CTAB method, 2 represents the Omega fungal DNA extraction kit method, 3 represents the Omega soil DNA extraction kit method, 4 represents the universal genomic DNA extraction kit method, and 5 represents the negative control.

[0028] Figure 2 The images show the LAMP detection results under different temperature conditions in Example 3, where 1 represents 60℃, 2 represents 63℃, 3 represents 65℃, 4 represents 68℃, and 5 represents the negative control at 65℃.

[0029] Figure 3 The figure shows the LAMP detection results at different reaction times in Example 3. In the figure, 1 is the positive control after 30 min of reaction, 2 is the negative control after 30 min of reaction, 3 is the negative control after 40 min of reaction, and 4 is the negative control after 50 min of reaction.

[0030] Figure 4 This is a graph showing the LAMP detection results at different template concentrations in Example 4. Figure 1 It is 10 pg / μL. Figure 2 It is 1 pg / μL. Figure 3 100 fg / μL Figure 4 It is 10 fg / μL. Figure 5 The concentration was 1 fg / μL, and Figure 6 shows the negative control.

[0031] Figure 5 The image shows the detection results in Example 5. In the image, 1 is Metarhizium anisopliae, 2 is Metarhizium anisopliae (soil sample), 3 is Metarhizium scutellarioides, 4 is Metarhizium scabra, 5 is Beauveria bassiana, 6 is Colloidal anthracnose, 7 is Alternaria alternata, 8 is Fusarium tumefaciens, 9 is Fusarium solani, 10 is Staphylococcus aureus, 11 is negative control, and 12 is negative control (soil sample). Detailed Implementation

[0032] WarmStart Colorimetric LAMP 2X Master Mix is ​​being sought after by BioLabs.

[0033] Omega Fungal DNA Extraction Kit D3390-001 was purchased from Omega.

[0034] Omega Soil DNA Kit D5625 was purchased from Omega.

[0035] The Universal Genomic DNA Extraction Kit 26240 was purchased from Shanghai Shangbao Biotechnology Co., Ltd.

[0036] Example 1

[0037] The design and screening of LAMP-specific primer combinations for Metarhizium anisopliae are as follows:

[0038] (1) The closely related species of Metarhizium anisopliae were identified on NCBI, and ITS and β-tublin were selected as specific primer target genes. In this series, the ITS (Genbank number: KX641194.1) and β-tublin (Genbank number: MK054129.1) gene sequences of Metarhizium anisopliae were used as the basic sequences for primer design.

[0039] (2) Based on the conserved intraspecific and interspecific variation regions of ITS and β-tublin sequences, LAMP primer combinations for specific amplification of Metarhizium anisopliae were designed using Primer Explorer (http: / / primerexplorer.jp / e / v5_manual / index.html) software and sent to Sangon Biotech (Shanghai) Co., Ltd. for primer synthesis. The primer combinations and sequences used in this embodiment are shown in Table 1.

[0040] (3) Prepare 100 μM storage solutions of the synthesized primers with ddH2O and store them at -20 ℃ for later use;

[0041] Table 1 Primer Combination Screening Table

[0042]

[0043] (4) Using the genomic DNA of Metarhizium anisopliae strain DT2011N7 as a template, primer pairs (MrFIP and MrBIP), (MrF3 and MrB3) and (MrLoopF ​​and MrLoopB) were prepared as 10× primer mix at concentrations of 16 μM, 2 μM and 4 μM. LAMP reaction was performed using WarmStart Colorimetric LAMP 2X Master Mix, and combinations that did not change color after the reaction were eliminated.

[0044] (5) Using the genomic DNA of *Metarhizium anisopliae* strain DT2011N7 as a positive template, primer mixtures were prepared using primer combinations screened in step (4). Genomic DNA of *Metarhizium anisopliae*, a closely related species of *Metarhizium anisopliae*, was used as a negative control. LAMP reactions were performed using WarmStart Colorimetric LAMP 2X Master Mix. Combinations that could distinguish between *Metarhizium anisopliae* and *Metarhizium anisopliae* were selected as candidate specific primer combinations for the visual detection of *Metarhizium anisopliae*. After screening, it was found that only primer combination 7 could specifically distinguish between *Metarhizium anisopliae* and *Metarhizium anisopliae*. Detailed primer information is shown in Table 2.

[0045] Table 2. Primers for LAMP detection based on the β-tubulin gene.

[0046]

[0047] Example 2

[0048] The specific methods for extracting genomic DNA from the tested strains are as follows:

[0049] (1) The mycelium of Metarhizium anisopliae was picked up with a needle and inoculated onto a SMAY medium plate for activation culture;

[0050] (2) Inoculate the activated cultured strain onto the corresponding culture medium, culture at 25℃ for 5-7 days, scrape off the mycelium and collect it into EP tubes for later use;

[0051] (3) The template DNA of the test strain was extracted using the CTAB method, the fungal DNA extraction kit, the soil DNA kit and the universal genomic DNA extraction kit, respectively, and the concentration was adjusted to 1-100 ng / μL.

[0052] (4) Perform the LAMP reaction according to the WarmStart Colorimetric LAMP 2X Master Mix product instructions, with ddH2O as the negative control.

[0053] The results show that ( Figure 1Compared with the negative control, all four methods of genomic DNA extracted could be detected by LAMP as Metarhizium anisopliae, indicating that the genomic DNA extracted by different methods and kits did not affect the subsequent experimental results.

[0054] Example 3: Establishment of the LAMP Visual Detection Method

[0055] Using the genomic DNA of *Metarhizium anisopliae* extracted in Example 2 as a template, LAMP amplification was performed using the three primer pairs in Table 2 of Example 1 to determine the optimal reaction conditions. The specific steps are as follows:

[0056] I. Using the recommended reaction system of WarmStart Colorimetric LAMP 2X Master Mix, LAMP assays were performed on the genomic DNA of *Metarhizium anisopliae* under different temperature conditions to determine the optimal reaction temperature. The specific steps included:

[0057] (1) Preparation of primer mixture: Take 16 µL, 16 µL, 2 µL, 2 µL, 4 µL, and 4 µL of primers MrFIP, MrBIP, MrF3, MrB3, MrLoopF, and MrLoopB (100 µM storage solution), respectively, add 56 µL of sterile deionized water, mix well and set aside; the primer mixture contains the above 6 specific primers (SEQ ID No. 1-6), wherein MrFIP / MrBIP:MrF3 / MrB3:MrLoopF / MrLoopB=8:1:2;

[0058] (2) Template DNA preparation: Genomic DNA of the tested Metarhizium anisopliae was extracted according to the method in Example 2;

[0059] (3) According to the recommended system of WarmStart Colorimetric LAMP 2X Master Mix, add 2.5 μL of primer mixture, 1 μL of template DNA, 12.5 μL of WarmStart Colorimetric LAMP 2X Master Mix reaction solution, and ddH2O to make up to 25 μL;

[0060] (4) The above reaction system was placed in a constant temperature water bath at 60℃, 63℃, 65℃ and 68℃ and incubated for 30 min respectively;

[0061] (5) Result interpretation: After the reaction is completed, take out the reaction tube and observe the color change of the reaction solution to interpret the result.

[0062] The results are as follows Figure 2As shown, compared to the negative control, the liquid in the reaction tubes was yellow at 63℃, 65℃, and 68℃, indicating that the LAMP reaction could occur at all three temperatures, thus detecting *Metarhizium anisopliae*. The yellow color was more intense in the reaction tubes at 63℃ and 65℃, while the liquid in the reaction tube at 68℃ only turned slightly yellow. The liquid in the reaction tube at 60℃ was red, indicating that the LAMP reaction could not occur at this temperature, and it was impossible to detect the presence of *Metarhizium anisopliae* in the sample. This shows that the LAMP reaction can occur normally between 63-68℃, with the highest reaction efficiency between 63-65℃.

[0063] II. The specific steps for determining the optimal reaction time under the recommended reaction system of WarmStart Colorimetric LAMP 2X Master Mix are as follows:

[0064] (1) The preparation of primer mixture and template DNA and the reaction system are the same as described in steps (1)-(3) of (I);

[0065] (2) Set the LAMP reaction program to run for 30, 40 and 50 min respectively to observe the results of the negative blank control. At the same time, set a positive control containing Metarhizium anisopliae DNA template at 30 min.

[0066] (3) Result interpretation: After the reaction is completed, take out the reaction tube and observe the color change of the reaction solution to interpret the result. Yellow indicates that the sample contains Metarhizium anisopliae, and red indicates that the sample does not contain Metarhizium anisopliae.

[0067] The results show that ( Figure 3 At 30 min, the liquid in the positive control tube turned yellow, indicating that the LAMP reaction was successful and *Metarhizium anisopliae* was detected. After 30 and 40 min, the negative control tube was red; after 50 min, it turned slightly yellow, indicating that a small amount of non-specific amplification between primers may occur with prolonged reaction time. Therefore, the LAMP reaction time should be controlled between 30 and 40 min.

[0068] Example 4: Sensitivity Detection of the LAMP Visual Detection Method

[0069] The genomic DNA of Metarhizium anisopliae was quantified, and after adjusting the concentration, it was diluted 10-fold to obtain test genomic DNA samples with concentrations of 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, and 1 fg / μL, respectively. These samples were then used as template DNA for LAMP detection, with a negative blank control set up. The LAMP reaction was carried out according to the system and optimal reaction temperature and time in Example 3.

[0070] The results showed that ( Figure 4 The liquids in reaction tubes 1-3 are yellow, indicating that *Metarhizium anisopliae* can be detected by LAMP at concentrations of 10 pg / μL, 1 pg / μL, and 100 fg / μL. The liquids in reaction tubes 4-5 remain red, similar to the negative control sample tube 6, indicating that *Metarhizium anisopliae* cannot be detected by LAMP at sample DNA concentrations of 10 fg / μL and 1 fg / μL. This demonstrates that the detection sensitivity of this method can be as low as 100 fg / μL.

[0071] Example 5: Specificity of the LAMP visualization detection method and its application in soil samples.

[0072] The specificity of the LAMP detection method was tested using genomic DNA templates of *Metarhizium anisopliae*, *Metarhizium scutellarioides*, *Metarhizium anisopliae*, *Beauveria bassiana*, *Colletotrichum gloeosporioides*, *Alternaria alternata*, *Fusarium oxysporum*, *Fusarium solani*, and *Staphylococcus aureus* (Table 3), with ddH2O as a negative control template. The reaction system and conditions were the same as in Example 3, and the concentration of the genomic DNA template was 1 ng / μL.

[0073] Total DNA was extracted from soil samples irrigated with Metarhizium anisopliae spore solution and soil samples without Metarhizium anisopliae, using a soil DNA extraction kit as templates. The kit was then used to detect Metarhizium anisopliae in soil samples. The reaction system and optimal reaction conditions were the same as in Example 3, with a total soil DNA concentration of 1 ng / μL.

[0074] The results are as follows Figure 5 As shown, compared with the negative blank control 11, PCR tube 1 is yellow, indicating that the sample to be tested contains Metarhizium anisopliae. PCR tubes 3-10 are red, indicating that these reaction tubes do not contain Metarhizium anisopliae. This demonstrates that the primer composition and visualization detection method designed in this invention can specifically detect Metarhizium anisopliae.

[0075] The results are as follows Figure 5 As shown, compared with the negative blank control (soil sample), the reaction solution in tube 2 is yellow, indicating that the LAMP detection primer composition and the visualization detection method can accurately detect whether or not Metarhizium repens is present in the DNA of soil samples. Therefore, it can be directly used to detect whether or not Metarhizium repens is present in soil samples.

[0076] The test strains used in this embodiment were all sent to the company for sequencing in advance. The LAMP visualization detection results were consistent with the sequencing results, which can accurately and quickly detect Metarhizium anisopliae from a variety of test strains, indicating that the method has high specificity.

[0077] Table 3. Strains used in the LAMP analysis

[0078]

[0079] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the present invention. The scope of protection of the present invention should be determined by the contents of the claims. Equivalent substitutions made by those skilled in the art without inventive effort all fall within the scope of protection of the present invention.

Claims

1. A primer composition for LAMP detection of Metarhizium rileyi, characterized in that: It consists of a forward inner primer MrFIP, a reverse inner primer MrFIP, a forward outer primer MrF3, a reverse outer primer MrB3, a forward loop primer MrLoopF, and a reverse loop primer MrLoopB. The primer sequences are as follows: MrF3: SEQ ID No. 1; MrB3: SEQ ID No. 2; MrFIP: SEQ ID No. 3; MrBIP: SEQ ID No. 4; MrLoopF: SEQ ID No. 5; MrLoopB: SEQ ID No. 6; The detection primer composition was designed based on the sequence of the β-tubin gene, a specific gene of Metarhizium rileyi.

2. The LAMP detection primer composition for Metarhizium rileyi according to claim 1, characterized in that: The molar ratio of each primer pair in the detection primer composition is (MrFIP and MrBIP):(MrF3 and MrB3):(MrLoopF ​​and MrLoopB) = 8:1:

2.

3. A visual detection method for Metarhizium rileyi, characterized in that: Includes the following steps: (1) Sample preparation: Extract genomic DNA from the sample to be tested; (2) LAMP amplification: LAMP detection is performed using the primer composition according to any one of claims 1-2; (3) Result interpretation: After the reaction is completed, take out the PCR reaction tube and observe the color of the reaction tube. Yellow indicates that Metarhizium rileyi is present in the sample, while red indicates that Metarhizium rileyi is not present in the sample.

4. A visual detection method for Metarhizium rileyi according to claim 3, characterized in that: The sample to be tested in step (1) is the genomic DNA of the purified strain or the total DNA of the soil sample.

5. A visual detection method for Metarhizium rileyi according to claim 4, characterized in that: The concentration of the sample to be tested in step (1) is above 100 fg / μL.

6. A visual detection method for Metarhizium rileyi according to claim 3, characterized in that: The amplification temperature during LAMP detection in step (2) is 63-65℃, and the amplification time is 30-40min.

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