A lamp primer composition for detecting c. gattii and a method for detecting the same

Abstract

The application discloses a LAMP primer composition for detecting Altemaria alternate and a detection method thereof. The primer composition for detecting Altemaria alternate is composed of a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3, a loop primer LF and LB. The LAMP reaction procedure is: 63 DEG C isothermal condition amplification for 60 min. The Altemaria alternate existing in the sample to be detected can be detected rapidly, conveniently, efficiently, with high specificity and high sensitivity. The Altemaria alternate does not need complex instruments, can better meet the on-site detection of the Altemaria alternate, is suitable for the inspection and quarantine of entry and exit plants and plants, disease investigation, rapid diagnosis and monitoring and the like, has important significance for preventing the Altemaria alternate from being transmitted into China, and meanwhile, the establishment of the system provides technical guidance and theoretical basis for the detection of other pathogenic bacteria.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a rapid detection method for perennial heterobasidiomycetes using LAMP primer composition. Background Technology

[0002] Perennial heterobasidion fungi (Heterobasidion annosum) belong to the phylum Basidiomycota, class Agaricomycetes, order Russulales, family Bondarzewiaceae, and genus Heterobasidion. They primarily infect pine trees, causing basal rot of the coniferous trunks and resulting in significant economic losses. Currently, there are few detection techniques for perennial heterobasidion fungi. Traditional detection methods mainly rely on plate isolation, identifying perennial heterobasidion fungi based on their morphology. This method is time-consuming and labor-intensive, requiring extensive and specialized experience in fungal identification, and it is difficult to distinguish perennial heterobasidion fungi from closely related species based on morphological characteristics, thus failing to meet the needs of forestry production. With the development of molecular biology, especially the widespread use of PCR technology, more and more molecular biology techniques are being applied to the detection of perennial heterobasidion fungi. However, these detection technologies often need to be carried out in laboratories with specialized instruments, reagents, and strict environmental conditions. The instruments and reagents are expensive, the operation requirements are high, the process is complex, and the detection time is still long. They cannot meet the needs of rapid detection and are not suitable for use and promotion in grassroots environments.

[0003] Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technique developed in recent years. Due to its simple operation, rapid speed, high specificity, and low cost, it has become a viable alternative to conventional PCR. It involves designing four specific primers targeting six regions of a target gene. Under the action of Bst large-fragment polymerase, a self-circulating chain displacement reaction is initiated. Within 60 minutes at 60–65°C, a large amount of target DNA is synthesized, accompanied by the production of a white magnesium pyrophosphate precipitate as a byproduct. Adding chromogenic dyes such as SYBR Green and hydroxynaphthol blue (HNB) to the reaction system allows for visual observation of color changes to determine the amplification. Because LAMP amplification relies on recognizing six independent regions of the target sequence, the reaction has high specificity. Furthermore, the nucleic acid amplification process is carried out under isothermal conditions; a simple water bath or equipment with a stable heat source is sufficient, significantly reducing detection costs. Currently, this technique is widely used for the rapid detection of fungi, bacteria, viruses, and oomycetes, but its application in the detection of perennial heterobasidiomycetes has not been reported domestically or internationally.

[0004] This invention uses the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as the target sequence for detection, designs a specific LAMP primer composition for perennial heterobasidiomycetes, and establishes a rapid LAMP detection method for perennial heterobasidiomycetes based on this. Summary of the Invention

[0005] The purpose of this invention is to address the problems of long detection cycles, time-consuming, labor-intensive, cumbersome, and poor specificity of existing biological detection methods for perennial heterobasidiomycetes, as well as the problem that PCR detection technology requires thermal cycling instruments and cannot quickly detect perennial heterobasidiomycetes. This invention provides a LAMP detection primer composition and molecular detection method for rapid detection of perennial heterobasidiomycetes. This method has a short detection cycle, high accuracy, high sensitivity, and allows for visual observation of the detection results.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] The LAMP primer composition for detecting perennial heterobasidiomycetes comprises the forward inner primer FIP as shown in SEQ ID NO.1, the reverse inner primer BIP as shown in SEQ ID NO.2, the forward outer primer F3 as shown in SEQ ID NO.3, the reverse outer primer B3 as shown in SEQ ID NO.4, the forward loop primer LF as shown in SEQ ID NO.5, and the reverse loop primer LB as shown in SEQ ID NO.6.

[0008] The application of the primer composition described in this invention in the detection of perennial heterobasidiomycetes, wherein the detection is for non-disease diagnosis purposes.

[0009] The application of the primer composition described in this invention in the preparation of a LAMP detection kit for perennial heterobasidiomycetes.

[0010] A LAMP kit for detecting perennial heterobasidiomycetes, containing the primer composition described in this invention.

[0011] As a preferred embodiment of the present invention, the LAMP kit for detecting perennial heterobasidiomycetes further comprises 10×ThermoPol Buffer, MgSO4, dNTPs, betaine, Bst DNA polymerase, and hydroxynaphthol blue.

[0012] The kit described in this invention is used for the detection of perennial heterobasidiomycetes, and the detection is for non-disease diagnosis purposes.

[0013] A method for detecting perennial heterobasidiomycetes using LAMP is characterized by extracting DNA from the microorganism to be tested, using the DNA solution as a reaction template, performing a LAMP reaction with the primer composition, and then observing the color change of the amplification product. If the color is sky blue, the test result is positive and perennial heterobasidiomycetes are present; if the color is purple, the test result is negative and perennial heterobasidiomycetes are not present.

[0014] As a preferred embodiment of the present invention, the LAMP reaction system comprises: 2.5 μL 10×ThermoPol Buffer, 8 mmol·L⁻¹ -1 MgSO4, 1.2 mmol·L -1 dNTPs, 1.6 μmol·L⁻¹ of internal primers FIP and BIP -1 0.4 μmol·L⁻¹ of outer primers F3 and B3. -1 0.8 μmol·L⁻¹ of loop primers LF and LB -1 0.8 μmol·L -1 Betaine, 8 U·μL -1 Bst DNApolymerase, 180mmol·L -1 Hydroxynaphthol blue, 2 μL template DNA, total system volume 26 μL.

[0015] As a preferred embodiment of the present invention, the LAMP reaction procedure is as follows: reaction amplification at 63°C for 60 min.

[0016] The method for detecting perennial heterobasidiomycetes of the present invention uses extracted DNA as a template and performs a LAMP reaction using the aforementioned LAMP primer composition; Hydroxylnaphthol blue (HNB) is a type of metal ion indicator. HNB is Mg 2+ The titrant's color changes with the solution's pH, therefore, the color of Mg in the LAMP reaction system can be monitored. 2+ The concentration change and solution pH affect the color indicator. Before the reaction, HNB is added to the reaction solution; the reaction system turns purple. During the reaction, Mg... 2+ It combines with the byproducts of the LAMP reaction to produce a large amount of precipitate, and Mg in the solution 2+ As the concentration decreases, the pH changes, causing the color of HNB to change from purple to sky blue. Therefore, the presence or absence of perennial heterobasidiomycetes can be determined by observing the color change of the reaction system after the reaction: sky blue indicates a positive result, indicating the presence of perennial heterobasidiomycetes; purple indicates a negative result, indicating the absence of perennial heterobasidiomycetes.

[0017] Compared with the prior art, the advantages and positive effects of this invention are as follows:

[0018] (1) Good practicality. Gel electrophoresis of products from conventional PCR reactions can easily cause product diffusion, which is a major source of laboratory contamination; moreover, ethidium bromide (EB) is highly toxic and can accumulate and cause cancer; long-term observation under ultraviolet light can also cause certain harm to laboratory personnel. In contrast, LAMP reactions can be carried out in a constant temperature water bath. After the reaction, the presence of the target strain can be determined by observing the change in the color of the solution, thus improving its application value in the field.

[0019] (2) Isothermal amplification. Unlike PCR, which requires thermal cycling, LAMP eliminates the dependence on thermal cycling equipment. As long as there is a stable heat source, the LAMP reaction can occur, greatly expanding the scope of LAMP applications. The reason why LAMP can react under a constant heat source is that betaine is added to the LAMP reaction solution, which keeps the double-stranded DNA in a dynamic equilibrium of melting, and amplification is achieved under the action of Bst DNA polymerase.

[0020] (3) High accuracy: Traditional methods for detecting perennial heterobasidiomycetes rely solely on morphological characteristics for identification. However, the growth of perennial heterobasidiomycetes is influenced by environmental factors, leading to unstable morphology and the influence of similar species, making accurate identification difficult. This invention, based on the genome sequence of perennial heterobasidiomycetes, uses Blast software to compare the genome sequence of perennial heterobasidiomycetes with that of other heterobasidiomycetes. The GAPDH gene sequence of perennial heterobasidiomycetes was selected to design specific LAMP primers. This gene is not used as a target in conventional PCR for primer designing identification primers for perennial heterobasidiomycetes. The LAMP reaction specifically recognizes six independent regions on the target sequence using four primers (FIP, BIP, F3, B3), demonstrating high specificity (Table 1). Furthermore, the forward loop primer LF and the reverse loop primer LB improve the reaction rate. Together with the other four primers, this invention enables rapid detection of perennial heterobasidiomycetes while ensuring reaction accuracy. The LAMP reaction uses four primers to specifically recognize six independent regions on the target sequence, which is more specific and sensitive than the two independent regions recognized by PCR primers.

[0021] (4) High sensitivity: The LAMP detection method for perennial heterobasidiomycetes established in this invention has very high sensitivity, reaching 100 pg DNA, indicating that the detection method is sufficient to accurately and rapidly detect perennial heterobasidiomycetes even at low DNA concentrations.

[0022] To better understand and implement this invention, the following detailed description is provided in conjunction with the accompanying drawings. Attached Figure Description

[0023] Figure 1 The nucleic acid sequence and primer positions of the GAPDH gene in perennial heterobasidobacteria are shown.

[0024] Figure 2 Figure 3 To validate the specificity of LAMP primers for detecting perennial heterobasidiomycetes:

[0025] The primers designed in this invention were used to perform LAMP amplification on DNA samples from different perennial heterobasidiomycete strains and other fungal strains. After amplification at a constant temperature of 63℃ for 60 min in a water bath, the color change of the reaction solution was observed. The results showed that all different perennial heterobasidiomycete strains exhibited a sky-blue positive reaction. Figure 2 The solutions in the reaction tubes of other tested bacterial strains all showed a purple negative reaction. Figure 3 ).

[0026] Figure 3 Sensitivity of LAMP detection for perennial heterobasidiomycetes:

[0027] LAMP amplification of genomic DNA at different concentrations; amplification results of 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, and 10 fg DNA in 26 μL reaction systems ( Figure 4 ). Detailed Implementation

[0028] To make the objectives and technical solutions of this invention clearer, specific examples are used for further explanation, but these examples are not the only ones.

[0029] Example 1: Detection of perennial heterobasidiomycetes using LAMP

[0030] 1. LAMP primer composition for detecting perennial heterobasidiomycetes: forward inner primer FIP as shown in SEQ ID NO.1, reverse inner primer BIP as shown in SEQ ID NO.2, forward outer primer F3 as shown in SEQ ID NO.3, reverse outer primer B3 as shown in SEQ ID NO.4, forward loop primer LF as shown in SEQ ID NO.5, and reverse loop primer LB as shown in SEQ ID NO.6.

[0031] 2. Detection Method: Extract DNA from the microorganism to be tested. Use the DNA solution as a reaction template and add it to the detection solution for LAMP reaction. The LAMP reaction system for detecting perennial heterobasidiomycetes is: 2.5 μL 10×ThermoPol Buffer, 8 mmol·L⁻¹ -1 MgSO4, 1.2 mmol·L -1 dNTPs, 1.6 μmol·L⁻¹ of internal primers FIP and BIP -1 0.4 μmol·L⁻¹ of outer primers F3 and B3. -10.8 μmol·L⁻¹ of loop primers LF and LB -1 0.8 μmol·L -1 Betaine, 8 U·μL -1 Bst DNApolymerase, 180mmol·L -1 Hydroxynaphthol blue, 2 μL template DNA, total system volume 26 μL; the LAMP reaction procedure is as follows: amplify at 63℃ for 60 min, then observe the color change of the amplification product. If it is sky blue, the test result is positive and perennial heterobasidiomycetes are present. If it is purple, the test result is negative and perennial heterobasidiomycetes are not present.

[0032] 3. To verify the specificity of the LAMP method, three different perennial heterobasidiomycete strains and 20 other fungal strains were used as test materials (Table 2). The LAMP results showed that only the reaction tube solution using perennial heterobasidiomycetes as a template exhibited a sky-blue positive reaction. Figure 2 Other tested fungal strains and the negative control all showed a purple negative reaction. Figure 3 ).

[0033] Table 1

[0034]

[0035]

[0036] Table 2

[0037]

[0038]

[0039] a BJFC: Herbarium of the Institute of Microbiology, Beijing Forestry University

[0040] b JSAFC: Jiangsu Vocational College of Agriculture and Forestry Specimen Museum

[0041] Example 2: Sensitivity of LAMP detection in perennial heterobasidiomycetes

[0042] To determine the sensitivity of the LAMP detection method, the concentration of extracted DNA from perennial heterobasidiomycetes was measured using a spectrophotometer and diluted sequentially in 10-fold increments to achieve a mass concentration of 10 ng / μL. -1 1 ng·μL -1 100 pg·μL -1 10 pg·μL -1 1 pg·μL -1 100 fg·μL -1 and 10 fg·μL-1 Two μL of each sample was used as template for LAMP reaction. The reaction program was 63℃ for 60 min. HNB colorimetric results indicated that when the DNA concentration of perennial heterobasidiomycetes reached 100 pg·μL... -1 The solution in the reaction tube will turn sky blue. Figure 4 ).

[0043] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A LAMP primer composition for detecting perennial heterobasidiomycetes, characterized in that, The primer composition consists of the following primers: the forward inner primer FIP as shown in SEQ ID No. 1, the reverse inner primer BIP as shown in SEQ ID No. 2, the forward outer primer F3 as shown in SEQ ID No. 3, the reverse outer primer B3 as shown in SEQ ID No. 4, the forward loop primer LF as shown in SEQ ID No. 5, and the reverse loop primer LB as shown in SEQ ID No.

6.

2. The use of the LAMP primer composition according to claim 1 in the detection of perennial heterobasidiomycetes.

3. The use of the LAMP primer composition of claim 1 in the preparation of a kit for detecting perennial heterobasidiomycetes.

4. A LAMP kit for detecting perennial heterobasidiomycetes, characterized in that, This kit contains the LAMP primer composition of claim 1.

5. The LAMP kit for detecting perennial heterobasidiomycetes according to claim 4, characterized in that, The kit also contains ThermoPol Buffer, MgSO4, dNTPs, and betaine. Bst DNA polymerase, hydroxynaphthol blue.

6. The use of the LAMP kit according to claim 4 or 5 in the detection of perennial heterobasidiomycetes.

7. A LAMP detection method for perennial heterobasidiomycetes, characterized in that... DNA was extracted from the microorganism to be tested. The DNA solution was used as a reaction template, and a LAMP reaction was performed using the primer composition described in claim 1. The color change of the amplification product was then observed. A sky-blue color indicated a positive result and the presence of perennial heterobasidiomycetes, while a purple color indicated a negative result and the absence of perennial heterobasidiomycetes. The LAMP reaction system consisted of 2.5 μL of 10×ThermoPol Buffer and 8 mmol·L⁻¹. -1 MgSO4, 1.2 mmol·L -1 dNTPs, 1.6 μmol·L⁻¹ of internal primers FIP and BIP -1 0.4 μmol·L⁻¹ of outer primers F3 and B3. -1 0.8 μmol·L⁻¹ of loop primers LF and LB -1 0.8 μmol·L -1 Betaine, 8 U·μL -1 Bst DNA polymerase, 180 mmol·L⁻¹ -1 Hydroxynaphthol blue, 2 μL template DNA, total system volume 26 μL; the LAMP reaction procedure was: amplification at 63℃ for 60 min.

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