A lamp primer composition for identifying leucophaeocybe portentosa and application thereof

Abstract

The application discloses a LAMP primer composition for identifying Lactarius volemus and application thereof, and the LAMP primer composition comprises a forward inner primer FIP shown in SEQ ID No. 7, a reverse inner primer BIP shown in SEQ ID No. 8, a forward outer primer F3 shown in SEQ ID No. 9, a reverse outer primer B3 shown in SEQ ID No. 10, a reverse loop primer LF shown in SEQ ID No. 11 and a reverse loop primer LB shown in SEQ ID No. 12. The LAMP primer composition disclosed by the application can be used for rapidly and efficiently identifying Lactarius volemus, and the identification method has the advantages of high specificity, high accuracy, good sensitivity, simple operation, and the like, and the result can be observed by naked eyes; when the DNA reaches 100 pg per microliter ‑1 , the Lactarius volemus can be identified, and the LAMP primer composition has high practicability.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a LAMP primer composition for identifying *Agaricus fulvidraco* and its application. This case is a divisional application of application number 202310176610.6, entitled "A LAMP Primer Composition and Its Application". Background Technology

[0002] *Lepiota lilacea*, belonging to the phylum Basidiomycota, class Agaricomycetes, order Agaricales, family Agaricomyceae, and genus *Lepiota*, has a small fruiting body with a scaly cap that is pale purple. The stipe is small and has a ring, with free gills. The basidiospores are colorless, smooth, and ellipsoid to oblong. This species is mainly distributed in Australia, North America, South America, Europe, and Asia. In my country, it is mainly found in Hainan, Guizhou, Jilin, and Heilongjiang provinces. It is a potentially poisonous mushroom. Because its appearance is similar to edible mushrooms such as matsutake, hazelnut, and shiitake, it is difficult for non-professionals to distinguish, easily leading to accidental ingestion and poisoning. Therefore, establishing a rapid and accurate identification method is of great significance for the identification and tracing of the source of *Lepiota lilacea* poisoning.

[0003] Currently, there are few detection technologies for *Amanita lilacinus*. Traditional detection methods mainly rely on the morphology of *Amanita lilacinus* for identification. This method requires extensive and professional experience in mushroom identification, and it is difficult to distinguish *Amanita lilacinus* from closely related species based on morphological characteristics alone. PCR technology, on the other hand, often requires laboratories with specialized instruments, reagents, and strict environmental conditions. The instruments and reagents are expensive, the operation is demanding, the process is complex, and the detection time is still relatively long, failing to meet the needs for rapid detection and making it unsuitable for widespread use in grassroots environments.

[0004] *Lepiota brunneoincarnata*, belonging to the phylum Basidiomycota, class Agaricomycetes, order Agaricales, family Agaricomyceae, and genus *Lepiota*, has a small, pale pinkish-flesh-colored fruiting body with a cap diameter of 2-6 cm. The flesh is thin and white, and the gills are free. The white to off-white cap has brown to dark brown scales arranged in nearly concentric rings. It lacks a volva and has an annulus. Despite its unassuming appearance, *Lepiota brunneoincarnata* is a highly poisonous mushroom, containing amatoxins and phallotoxins. Because of its resemblance to edible mushrooms such as pine mushrooms, hazel mushrooms, and shiitake mushrooms, it is difficult for non-professionals to distinguish, easily leading to accidental ingestion and poisoning. Like the highly toxic Amanita phalloides, it can cause acute liver damage. From 2019 to 2021, it caused at least 35 poisoning incidents in my country, resulting in 85 poisonings and 8 deaths, truly earning the title of "small but mighty mushroom king." *Amanita fulva* is widely distributed in Northeast, North, and Northwest my country, and in recent years has also been found in Central, East, and Southwest China. Therefore, establishing a rapid and accurate identification method is of great significance for the identification and tracing of the toxin source in *Amanita fulva* poisoning.

[0005] Currently, there are many methods for detecting *Amanita fulva*, such as chromatographic assays, enzyme-linked immunosorbent assays (ELISA), immunochromatography, and liquid chromatography-mass spectrometry (LC-MS). However, these methods have drawbacks, including low specificity, reliance on large instruments, and complex operation. 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 *Amanita fulva*. However, these detection techniques often require laboratories with specialized instruments, reagents, and strict environmental conditions. The instruments and reagents are expensive, the operation is demanding, the process is complex, and the detection time is still relatively long, failing to meet the needs for rapid detection and making them unsuitable for widespread use in grassroots environments.

[0006] Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technique developed in recent years. Due to its advantages such as simplicity, 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 or hydroxynaphthol blue (HNB) to the reaction system allows for visual observation of color changes to determine the amplification process. Because LAMP amplification relies on recognizing six independent regions of the target sequence, the reaction is highly specific. Furthermore, the nucleic acid amplification process is carried out under isothermal conditions; ordinary water baths or equipment with a stable heat source can meet the reaction requirements, significantly reducing detection costs.

[0007] The selection of target genes is one of the important factors in LAMP detection. Currently, the primers designed for LAMP detection are mainly derived from intertranscribed spacers (ITS), but there are not enough sites to distinguish similar species. Therefore, the development of new detection targets has become a hot topic in detection. Summary of the Invention

[0008] Objectives of this invention: The first objective is to provide a set of LAMP primer compositions for the rapid identification of *Amanita lilacinus* and their applications; the second objective is to provide a LAMP method for identifying *Amanita lilacinus*. The third objective is to provide a set of LAMP primer compositions for the rapid identification of *Amanita fulva* and their applications; the third objective is to provide a LAMP method for identifying *Amanita fulva*.

[0009] Technical solution: The LAMP primer composition of the present invention comprises: a forward inner primer FIP as shown in SEQ ID No. 1, a reverse inner primer BIP as shown in SEQ ID No. 2, a forward outer primer F3 as shown in SEQ ID No. 3, a reverse outer primer B3 as shown in SEQ ID No. 4, a forward loop primer LF as shown in SEQ ID No. 5, and a reverse loop primer LB as shown in SEQ ID No. 6.

[0010] Traditional identification methods for *Amanita lilacinus* rely solely on morphological characteristics. However, the growth of *Amanita lilacinus* is affected by temperature, leading to morphological instability, and it is also influenced by closely related species, making accurate identification difficult. This invention addresses this issue by using the genomic sequence of *Amanita lilacinus* and comparing it with the genomic sequences of other fungi using BLAST software. The *Amanita lilacinus* translation elongation factor 1-alpha (tef1) gene sequence was selected, and specific LAMP primers were designed. The LAMP reaction specifically recognizes six independent regions on the target sequence using four primers (FIP, BIP, F3, B3) (Table 1), demonstrating high specificity. 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 identification of *Amanita lilacinus* while ensuring reaction accuracy. The LAMP reaction, with its four primers specifically recognizing six independent regions on the target sequence, exhibits higher specificity and sensitivity compared to the two independent regions recognized by PCR primers.

[0011] The application of the LAMP primer composition described in this invention in the identification of *Agaricus purpureus*, wherein the identification is for non-disease diagnostic purposes.

[0012] The application of the LAMP primer composition described in this invention in the preparation of a kit for identifying *Agaricus purpureus*.

[0013] The present invention discloses a LAMP kit for identifying *Agaricus purpureus*, the kit containing the aforementioned LAMP primer composition.

[0014] Furthermore, the kit also contains 10×ThermoPol Buffer, MgSO4, dNTPs, betaine, BstDNApolymerase, and hydroxynaphthol blue.

[0015] The present invention discloses a LAMP identification method for *Agaricus purpureus*, comprising the following steps: 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 amplification product is sky blue, the identification result is positive, indicating that *Agaricus purpureus* is present in the organism to be identified; if the amplification product is purple, the identification result is negative, indicating that *Agaricus purpureus* is not present in the organism to be identified.

[0016] Furthermore, the LAMP reaction system consisted of: 2.5 μL 10×ThermoPol Buffer, 8 mmol·L⁻¹ - 1 MgSO4, 1.2 mmol·L -11.6 μmol·L⁻¹ of dNTPs, FIP and BIP inner primers -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, 180mmol·L -1 Hydroxynaphthol blue, 2 μL template DNA, total volume of system 26 μL.

[0017] Furthermore, the LAMP reaction procedure is as follows: reaction amplification at 63°C for 60 min.

[0018] The method for identifying *Agaricus purpureus* of this 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 *Hymenopterus lilacinus* 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 *Hymenopterus lilacinus*; purple indicates a negative result, indicating the absence of *Hymenopterus lilacinus*.

[0019] The LAMP primer composition of the present invention comprises: a forward inner primer FIP as shown in SEQ ID No. 7, a reverse inner primer BIP as shown in SEQ ID No. 8, a forward outer primer F3 as shown in SEQ ID No. 9, a reverse outer primer B3 as shown in SEQ ID No. 10, a forward loop primer LF as shown in SEQ ID No. 11, and a reverse loop primer LB as shown in SEQ ID No. 12.

[0020] The application of the LAMP primer composition described in this invention in the identification of *Amanita fulvidra*, wherein the identification is for non-disease diagnostic purposes.

[0021] The application of the LAMP primer composition described in this invention in the preparation of a kit for identifying *Amanita fulva*.

[0022] The present invention discloses a LAMP kit for identifying *Amanita fulvidraco*, the kit containing the above-described LAMP primer composition.

[0023] Furthermore, the kit also contains 10×ThermoPol Buffer, MgSO4, dNTPs, betaine, BstDNApolymerase, and hydroxynaphthol blue.

[0024] The present invention discloses a LAMP identification method for *Amanita fulvidracoides*, comprising the following steps: 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 amplification product is sky blue, the identification result is positive, indicating that *Amanita fulvidracoides* is present in the organism to be identified; if the amplification product is purple, the identification result is negative, indicating that *Amanita fulvidracoides* is not present in the organism to be identified.

[0025] Furthermore, the LAMP reaction system consisted of: 2.5 μL 10×ThermoPol Buffer, 8 mmol·L⁻¹ - 1 MgSO4, 1.2 mmol·L -1 1.6 μmol·L⁻¹ of dNTPs, FIP and BIP inner primers -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, 180mmol·L -1 Hydroxynaphthol blue, 2 μL template DNA, total volume of system 26 μL.

[0026] Furthermore, the LAMP reaction procedure is as follows: reaction amplification at 63°C for 60 min.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following outstanding advantages: The LAMP primer composition disclosed in this invention can be used for rapid and efficient identification of *Agaricus purpureus*. The identification method has the advantages of high specificity, high accuracy, good sensitivity, simple operation, and visual observation of results. When the DNA reaches 10 pg·μL... -1 It can identify the pale purple ring-stalk mushroom in a short time, making it highly practical.

[0028] The LAMP primer composition disclosed in this invention can be used for rapid and efficient identification of *Amanita fulva*. The identification method has the advantages of high specificity, high accuracy, good sensitivity, simple operation, and visual observation of results. When the DNA concentration reaches 100 pg·μL... -1 It can identify the brown-skinned ring-stalked mushroom immediately, making it highly practical. Attached Figure Description

[0029] Figure 1 The nucleic acid sequence and primer positions of the TEF1 gene from *Agaricus purpureus*.

[0030] Figure 2 The results of LAMP identification of different *Amanita lilacinus* strains and negative controls using the LAMP primer composition in Example 1 are shown.

[0031] Figure 3 The results of LAMP identification of different fungi using the LAMP primer composition in Example 1 are shown.

[0032] Figure 4 This is the sensitivity test result of LAMP in Example 2 for identifying *Agaricus purpureus*.

[0033] Figure 5 The nucleic acid sequence and primer positions of the TEF1 gene from *Agaricus brunneoincarnata*.

[0034] Figure 6 The results of LAMP identification of different *Amanita fulva* strains and negative controls using the LAMP primer composition in Example 3 are shown.

[0035] Figure 7 The results of LAMP identification of different fungi using the LAMP primer composition in Example 3 are shown.

[0036] Figure 8 The results of LAMP sensitivity identification of *Amanita fulva* in Example 4 are shown. Detailed Implementation

[0037] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0038] Example 1: Identification of *Agaricus purpureus* using LAMP

[0039] 1. LAMP primer composition for identifying *Agaricus purpureus*: 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 (Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Detailed information is shown in Table 1, with nucleic acid sequences and primer positions as follows...). Figure 1 (As shown).

[0040] 2. Identification Method: Extract DNA from the sample to be tested. Use the DNA solution as a reaction template and add a reaction solution including the primers mentioned above to perform a LAMP reaction. The LAMP reaction system 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. -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, 180mmol·L -1 Hydroxynaphthol blue, 2 μL template DNA, total system volume 26 μL; LAMP reaction procedure: amplify at 63℃ for 60 min, then observe the color change of the amplification product. If it is sky blue, the identification result is positive and there is *Pleurotus erythropus*. If it is purple, the identification result is negative and there is no *Pleurotus erythropus*.

[0041] 3. To verify the specificity of the LAMP method, three different *Amanita lilacinus* samples and 19 other fungal samples were used as test materials (Table 2). The LAMP identification results showed that only the reaction tube solution using *Amanita lilacinus* as a template exhibited a sky-blue positive reaction. Figure 2 Other tested fungal strains ( Figure 3 ) and negative control (ddH2O) Figure 2 The rightmost one (all of them) showed a purple negative reaction.

[0042] Table 1

[0043]

[0044] Table 2

[0045]

[0046]

[0047] Example 2: Sensitivity of LAMP identification of *Pleurotus eryngii*

[0048] To determine the sensitivity of the LAMP identification method, the concentration of extracted DNA from *Agaricus purpureus* was measured using a spectrophotometer and diluted 10-fold to achieve a final 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 *Pleurotus erythrorhizon* reached 10 pg·μL... -1 The solution in the reaction tube will turn sky blue. Figure 4 ).

[0049] Example 3: Identification of *Agaricus brunneoincarnata* using LAMP

[0050] 1. LAMP primer composition for identifying *Agaricus brunneoincarnata*: forward inner primer FIP as shown in SEQ ID No. 7, reverse inner primer BIP as shown in SEQ ID No. 8, forward outer primer F3 as shown in SEQ ID No. 9, reverse outer primer B3 as shown in SEQ ID No. 10, forward loop primer LF as shown in SEQ ID No. 11, and reverse loop primer LB as shown in SEQ ID No. 12 (details are shown in Table 3, nucleic acid sequences and primer positions are as follows). Figure 5 (As shown).

[0051] 2. Identification Method: Extract DNA from the sample to be tested. Use the DNA solution as a reaction template and add a reaction solution including the primers mentioned above to perform a LAMP reaction. The LAMP reaction system 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. -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, 180mmol·L -1 Hydroxynaphthol blue, 2 μL template DNA, total system volume 26 μL; LAMP reaction procedure: amplify at 63℃ for 60 min, then observe the color change of the amplification product. If it is sky blue, the identification result is positive and there is *Pleurotus erythropterus*. If it is purple, the identification result is negative and there is no *Pleurotus erythropterus*.

[0052] 3. To verify the specificity of the LAMP method, five different *Amanita fulva* samples and 23 other fungal samples were used as test materials (Table 4). The LAMP identification results showed that only the reaction tube solution using *Amanita fulva* as a template exhibited a sky-blue positive reaction. Figure 6 Other tested fungal strains and negative control (ddH2O) Figure 6 (Rightmost) all showed a purple negative reaction. Figure 7 ).

[0053] Table 3

[0054]

[0055] Table 4

[0056]

[0057]

[0058] Example 4: Sensitivity of LAMP identification of *Agaricus brunneoincarnata*

[0059] To determine the sensitivity of the LAMP identification method, the concentration of extracted DNA from *Amanita fulva* was measured using a spectrophotometer and diluted 10-fold to achieve a final 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 a template for LAMP reaction. The reaction program was 63℃ for 60 min. HNB colorimetric results indicated that when the DNA concentration of *Amanita fulva* reached 100 pg·μL... -1 The solution in the reaction tube will turn sky blue. Figure 8 ).

[0060] 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 identifying *Amanita fulva*, characterized in that, The LAMP primer composition includes the forward inner primer FIP as shown in SEQ ID No. 7, the reverse inner primer BIP as shown in SEQ ID No. 8, the forward outer primer F3 as shown in SEQ ID No. 9, the reverse outer primer B3 as shown in SEQ ID No. 10, the forward loop primer LF as shown in SEQ ID No. 11, and the reverse loop primer LB as shown in SEQ ID No.

12.

2. The use of the LAMP primer composition of claim 1 in the identification of *Amanita fulvidra*.

3. The use of the LAMP primer composition of claim 1 in the preparation of a kit for identifying *Amanita fulvidra*.

4. A LAMP kit for identifying *Amanita fulva*, characterized in that, The kit contains the LAMP primer composition of claim 1.

5. The LAMP kit for identifying *Amanita fulva* according to claim 4, characterized in that, The kit also contains 10×ThermoPol Buffer, MgSO4, dNTPs, betaine, Bst DNA polymerase, hydroxynaphthol blue.

6. A LAMP method for identifying *Amanita fulva*, characterized in that, Includes the following steps: DNA was extracted from the microorganism to be tested. The DNA solution was used as a reaction template, and LAMP reaction was performed using the primer composition described in claim 1. The color change of the amplification product was then observed. If the amplification product was sky blue, the identification result was positive, indicating that the microorganism to be tested contained *Amanita fulvidra*. If the amplification product was purple, the identification result was negative, indicating that the microorganism to be tested did not contain *Amanita fulvidra*. The LAMP reaction system consisted of: 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 DNA polymerase, 180 mmol·L -1 Hydroxynaphthol blue, 2 μL template DNA, total volume of the system is 26 μL.

7. The LAMP method for identifying *Amanita fulva* according to claim 6, characterized in that, The LAMP reaction procedure is as follows: reaction amplification at 63℃ for 60 min.

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