Primer group for rapidly detecting amanita ovatus, application of primer group and kit containing primer group

Abstract

The invention relates to a primer group for rapidly detecting amanita ovulata, application of the primer group and a kit containing the primer group, and belongs to the technical field of nucleic acid detection.The nucleotide sequence of the primer group is shown as SEQ ID No 1-5; the invention further discloses application of the primer group, a kit containing the primer group and a method for performing LAMP amplification on the amanita ovatus gene by using the primer group, LAMP amplification is adopted for detecting the amanita ovatus, only 10-20 mg of sample is needed for detection, sample DNA extraction and detection result judgment can be completed within one hour, the detection time is shortened, the detection efficiency is improved, and the detection cost is reduced. The detection sensitivity is high and the result is stable and reliable.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology, specifically, it relates to a primer set for rapid detection of Amanita ovalis, the uses of the primer set, and a kit containing the primer set. Background Technology

[0002] The morphology of *Amanita phalloides* is similar to that of the edible *Amanita muscaria* and *Matsutake*, and they share the same growing season and environment, often growing together and easily contaminated. However, it contains amatoxins, a protein expressed by the ITS gene in the internal transcriptional spacer region, making it highly toxic. It can cause acute renal failure-type poisoning, leading to kidney failure and potentially affecting the liver, resulting in an extremely high mortality rate. The appearance of *Amanita phalloides* is shown in the attached image. Figure 1 .

[0003] In terms of rapid detection methods, molecular biology detection technologies are generally developed based on extracted DNA. The cumbersome nature of nucleic acid extraction methods limits the use of many nucleic acid amplification technologies outside the laboratory environment. The rapid nucleic acid extraction method based on fiber filter paper adsorption effectively avoids the ethanol precipitation, phenol-chloroform extraction, centrifugation, and column chromatography processes in traditional methods, reducing sample loss and contamination and improving sample processing efficiency. Compared with rapid nucleic acid extraction methods based on magnetic materials and silica matrix materials, the fiber filter paper adsorption method can obtain satisfactory DNA within 1 minute, and has the advantages of simple operation, low price, and rapid extraction. Furthermore, the cellulose filter paper carrying nucleic acids does not require elution. A small number of studies have also been conducted on the extraction of genomic DNA from biological samples. Currently, the extraction of genomic DNA from biological samples is highly dependent on the type of sample to be extracted and the application of downstream amplification technologies, requiring the preparation of appropriate lysis and elution buffers. This technology has not been reported in the extraction of genomic DNA from poisonous mushrooms.

[0004] Currently, there are many methods developed for identifying mushrooms based on molecular biology techniques, most of which use PCR amplification. However, these methods have drawbacks such as long detection times and high dependence on detection instruments. Therefore, it is of great significance to provide a method that can quickly identify Amanita ovata. Summary of the Invention

[0005] To address the problems existing in the background art, this invention provides a primer set for rapid detection of *Amanita ovalis*, which can effectively detect *Amanita ovalis*. Using this primer set, the present invention employs loop-mediated isothermal amplification (LAMP) technology for detection, and can identify *Amanita ovalis* samples or edible fungi contaminated with *Amanita ovalis* within 1 hour. The method is simple and rapid.

[0006] Based on this, the first objective of the present invention is to improve the rapid detection of *Amanita ovalis*. The primer set consists of primers A-NEO Primer F3, A-NEO Primer B3, A-NEO Primer FIP, A-NEO Primer BIP, and A-NEO Primer LF. The nucleotide sequence of the primer set is as follows: The nucleotide sequence of A-NEO F3 is shown in SEQ ID No. 1; The nucleotide sequence of A-NEO B3 is shown in SEQ ID No. 2; The nucleotide sequence of A-NEO FIP is shown in SEQ ID No. 3; The nucleotide sequence of A-NEO BIP is shown in SEQ ID No. 4.

[0007] The nucleotide sequence of A-NEO LF is shown in SEQ ID No. 5.

[0008] A second objective of this invention is to provide the application of the above-mentioned primer set in the detection of Amanita ovalis.

[0009] A third objective of this invention is to provide a kit for detecting Amanita ovalis containing the above-described primer set.

[0010] The fourth objective of this invention is to provide a method for rapid detection of Amanita ovalis, wherein the method uses the aforementioned primer set.

[0011] Furthermore, the above primer set was used to amplify Amanita ovalis using the LAMP method, followed by detection.

[0012] Furthermore, the method includes the following steps: (1) Rapid extraction of DNA from Amanita ovalis using the filter paper method; (2) Amplification was performed using sample DNA as a template for LAMP amplification, with the nucleic acid primer set as described in claim 1: (3) Observe the color change of the LAMP amplification product in step (2). After the amplification reaction is completed, the color of the reaction solution changes from violet to sky blue. Then the sample is identified as Amanita ovalis or contains Amanita ovalis.

[0013] Furthermore, step (1) specifically includes the following: S1, Select the cap portion of the sample and grind it into powder in liquid nitrogen; S2, take the ground sample into a 1.5 mL centrifuge tube, add the lysis buffer, vortex to mix evenly, and obtain the crude extract; S3, Immerse the nucleic acid binding region of the filter paper strip into the crude extract for 3-5 seconds each time, for a total of 3 times; S4, Immerse the filter paper strip in the eluent to wash away impurities.

[0014] Furthermore, the lysis buffer consists of a mixture of 0.06% SDS, 0.125 mol / L NaCl, 0.01 mol / L Tris-HCl buffer, and 1 mmol / L EDTA, pH=8.0. The elution buffer consists of a mixture of 0.01 mol / L Tris-HCl buffer and 1.5% Tween-20, pH=7.8-8.2. The filter paper strips used have a pore size of 1-3 μm and a specification of (40-50) mm × 2 mm, consisting of a (40-50) mm × 2 mm hydrophobic region formed by immersing one end in melted solid paraffin and a (2-4) mm × 2 mm DNA knot region at the other end.

[0015] Furthermore, the LAMP amplification reaction system in step (2) contains 2 mmol Mg 2+ 1× Isothermal Amplification buffer, MgSO4 4~8 mmol, dNTP mix 1.4 μM, F3 / B3 0.16 μM, FIP / BIP 1.20 μM, LF 0.32 μM, Bst DNA polymerase 320 U / mL, HNB 120 μM, template extracted by DNA filter paper strip method, and ddH2O balance.

[0016] The beneficial effects of this invention: This invention presents a primer set capable of detecting *Amanita ovalis*, which can effectively detect the fungus. Using this primer set with loop-mediated isothermal amplification (LAMP) technology, the detection exhibits excellent specificity and sensitivity, requires no expensive equipment, is easy to operate, has a short detection time, and provides simple and accurate result interpretation, making it suitable for rapid detection at the grassroots level. Only a small sample volume of 10-20 mg is needed, and sample DNA extraction and result interpretation can be completed within 1 hour. Attached Figure Description

[0017] Figure 1 This is an image of the appearance of *Amanita ovata*. Figure 2 This involves sensitivity testing of samples doped with different concentrations of Amanita ovalis. Figure 3 This is a specific result of LAMP amplification of *Amanita ovata*. Figure 4 This is an electrophoresis image showing the LAMP specificity results of *Amanita ovata*. Figure 5 This is a stability test for Amanita ovalis. Detailed Implementation

[0018] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are all within the scope of protection of this invention.

[0019] To illustrate the present invention more clearly, the following embodiments will be described in detail.

[0020] Example 1 This embodiment provides a primer set for detecting Amanita ovalis, and the primer set name and gene sequence are shown in the table below.

[0021] Table 1. Primer names and sequences for detecting Amanita ovalis. Example 2 This embodiment provides a method for detecting Amanita ovalis, which uses the primer set described in Table 1 and employs visual loop-mediated isothermal amplification (LAMP) for detection, as detailed below.

[0022] Reagent and material preparation: The fiber filter paper strips required for rapid DNA extraction using the filter paper method have a pore size of 1-3 μm and a specification of (40-50) mm × 2 mm. They consist of a 40 mm × 2 mm hydrophobic area formed by immersing one end in molten solid paraffin and a (2-4) mm × 2 mm DNA binding area at the other end.

[0023] Lysis buffer: a mixed solution of 0.06% SDS, 0.125 mol / L NaCl, 0.01 mol / L Tris-HCl buffer, and 1 mmol / L EDTA, pH=8.0.

[0024] Elution buffer: a mixture of 0.01 mol / L Tris-HCl buffer and 1.5% Tween-20, pH=7.8-8.2.

[0025] Bst DNA polymerase, MgSO4 10× Isothermal Amplification buffer: NEB Corporation, USA, catalog number M0537S.

[0026] HNB Hydroxynaphthol Blue Indicator: Beijing Solarbio Technology Co., Ltd., Product No. G1218.

[0027] dNTP Solution Mix: NEB product number N0447S (USA).

[0028] The abbreviations of technical terms used in this invention are as follows: LAMP: loop-mediated isothermal amplification; dNTP: deoxynucleoside triphosphate; PAGE: polyacrylamide gel electrophoresis grade.

[0029] Detection methods (1) Sample grinding: Select the cap part of the sample. If there are visible impurities that are not the bacteria themselves, wipe them off with sterile water using a sterile cotton swab. Cut them into the smallest possible pieces with sterile scissors and grind them in liquid nitrogen.

[0030] (2) Sample lysis: Take 50-100mg of fresh sample and 10-20mg of dried sample into a 1.5mL centrifuge tube, add 500μL of lysis buffer, vortex for 30 seconds to mix evenly, and obtain crude extract.

[0031] (3) Sample extraction: Immerse the nucleic acid binding region of the filter paper strip into the crude extract for 3-5 seconds each time, and repeat the immersion 3 times.

[0032] (4) Impurity removal: Quickly immerse the filter paper strip in 1 mL of elution solution and gently wash away the impurities. Repeat the elution 3 times, 1 second each time.

[0033] (5) Preparation of reaction system: 1× Isothermal Amplification buffer (containing 2 mmol Mg) 2+ ), MgSO4 6mmol, dNTP mix 1.4μM, F3 / B3 0.16μM, FIP / BIP 1.20μM, LB 0.32μM, Bst DNA polymerase 320U / mL, HNB 120μM, template extracted by DNA filter paper strip method, ddH2O balance.

[0034] (6) Dispensing: Each tube contains 12.5-25 μL of reaction solution.

[0035] (7) Immersion: Immerse the nucleic acid binding region of the filter paper strip obtained after impurity removal into the reaction solution, repeat 3 times, and stay for 5 seconds each time.

[0036] (8) Blank test: Perform a reagent blank at the same time. Except for not adding the sample, perform the other operation steps as in (2) to (7).

[0037] (9) Add about 20 μL of liquid paraffin to the reaction tube to cover the reaction solution.

[0038] (10) Amplification: LAMP amplification conditions are: 61-65℃ constant temperature water bath for 40-55 min.

[0039] (11) Results observation: Observe and record the color change. The color of the negative amplification result does not change and is violet. The color of the positive amplification result changes from violet to sky blue.

[0040] (12) Enzyme inactivation: The reaction was terminated by bathing in a constant temperature water bath at 85℃ for 5 min.

[0041] Example 3: Detection Sensitivity Test Following the method in Example 2, different concentrations of *Amanita phalloides* were added to *Matsutake*, *Amanita spp.*, *Termitomyces albuminosus*, *Boletus edulis*, and *Russula ovata* lysates (50 μL, 5 μL, 2.5 μL, 0.5 μL, and 0% *Amanita phalloides* lysates were added to the lysates of *Matsutake*, *Amanita spp.*, *Termitomyces albuminosus*, *Boletus edulis*, and *Russula ovata* lysates, respectively). These lysates formed mixed lysates containing 10%, 1%, 0.5%, 0.1%, and 0% *Amanita phalloides*. Sensitivity tests were performed according to steps (3) to (11) of Example 2, and the results are shown in the appendix. Figure 2 Appendix Figure 2 In the table, a to e represent matsutake, Chinese amanita, termite mushroom, boletus, and blue-yellow-red mushroom, respectively. 0 to 6 represent the concentrations of Amanita fulva, reagent blank, 100% (meaning the sample is entirely Amanita fulva), 10%, 1%, 0.5%, 0.1%, and 0%, respectively.

[0042] The results showed that the adulteration of *Amanita phalloides* with a concentration greater than 0.1% in matsutake, *Amanita fulva*, termite mushroom, porcini, and blue-yellow-red mushroom could be detected.

[0043] Example 4 Using the method in Example 2, the following species were tested: Amanita fuliginea, Amanita virgineoides, Amanita subjunquillea, Amanita fritillaria, Amanita citrino annulata, Amanita subparvipantherina, Amanita flavipes, Amanita subglobosa, Amanita neoovoidea, Amanita griseofolia, Amanita parvipantherina, Amanita sinensis, Amanita hemibapha, and Amanita yuaniana. Corresponding negative controls were established for Russula. (cyanoxantha, negative control NTC) was used. After the reaction, the color change of the reaction solution was observed and recorded. To verify the accuracy of the results, agarose gel electrophoresis was performed. The results are attached. Figure 3 and attached Figure 4 As shown.

[0044] Figure 3 and 4 In the primer set, M represents the DL2000 Marker, 1 represents the grey-patterned Amanita, 2 represents the pseudoopithecus aureus, 3 represents the yellow-capped Amanita, 4 represents the checkered Amanita, 5 represents the yellow-ringed Amanita, 6 represents the sub-panther-spotted Amanita, 7 represents the turmeric Amanita, 8 represents the globular Amanita, 9 represents the cone-scaled white Amanita, 10 represents the grey-folded Amanita, 11 represents the sub-panther-spotted Amanita, 12 represents the Chinese Amanita, 13 represents the red-yellow Amanita, 14 represents the Yuan's Amanita, and 15 represents the blue-yellow-red mushroom (NTC). The results showed that only the pseudoopithecus aureus exhibited a sky-blue color, which was clearly distinguishable from the colors of other fungi, indicating that the primer set of this invention had good specificity.

[0045] Comparison of nucleic acid extraction yield: The table below compares commercial nucleic acid extraction kits with the filter paper method, demonstrating that the filter paper method yields higher quality nucleic acid extraction.

[0046] Commercially available reagent kit: Biospin Fungal Genomic DNA Extraction Kit (manufactured by Hangzhou Biospin Technology Co., Ltd.)

[0047] Table 2. Nucleic acid extraction at 260nm / 280nm using the commercial kit (control group) and the method of the present invention (experimental group). The results showed that the DNA values ​​at 260nm / 280nm extracted from wild fungi samples in the experimental group were all between 1.7 and 2.1, which was better than the 1.3-2.3 values ​​at 260nm / 280nm extracted from wild fungi samples in the control group, and was more conducive to downstream LAMP amplification.

[0048] Example 5: Stability Verification Different strains of *Amanita ovalis* were randomly selected, and their caps were tested using the method described in Example 2. The test results are shown in the appendix. Figure 5 Appendix Figure 5 In the figures, 1 and 2 represent the blank and NTC test, respectively, both showing a violet color; 3-9 represent the seven parallel tests of *Amanita ovalis*, all showing a sky-blue color. This indicates that the method of the present invention has high stability.

[0049] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A primer set for rapid detection of Amanita ovalis, characterized in that... The primer set consists of primers A-NEO PrimerF3, A-NEO Primer B3, A-NEO Primer FIP, A-NEO Primer BIP, and A-NEO Primer LF, and the nucleotide sequence of the primer set is as follows; The nucleotide sequence of A-NEO F3 is shown in SEQ ID No. 1; The nucleotide sequence of A-NEO B3 is shown in SEQ ID No. 2; The nucleotide sequence of A-NEO FIP is shown in SEQ ID No. 3; The nucleotide sequence of NEO BIP is shown in SEQ ID No. 4; The nucleotide sequence of A-NEO LF is shown in SEQ ID No.

5.

2. The application of the primer set as described in claim 1 in the detection of Amanita fulva.

3. A kit for detecting Amanita ovalis, characterized in that... This includes the primers as described in claim 1.

4. A method for rapid detection of Amanita ovalis, characterized in that... The primer set as described in claim 1 is used.

5. The method according to claim 4, characterized in that... The primer set as described in claim 1 was used to amplify Amanita ovalis using the LAMP method, followed by detection.

6. The method according to claim 4, characterized in that... This includes the following steps: (1) Rapid extraction of DNA from Amanita ovalis using the filter paper method; (2) Amplification was performed using sample DNA as a template for LAMP amplification, with the nucleic acid primer set as described in claim 1: (3) Observe the color change of the LAMP amplification product in step (2). After the amplification reaction is completed, the color of the reaction solution changes from violet to sky blue. Then the sample is identified as Amanita ovalis or contains Amanita ovalis.

7. The method according to claim 6, characterized in that... Step (1) specifically includes the following: S1, Select the cap portion of the sample and grind it into powder in liquid nitrogen; S2, take the ground sample into a 1.5 mL centrifuge tube, add the lysis buffer, vortex to mix evenly, and obtain the crude extract; S3, Immerse the nucleic acid binding region of the filter paper strip into the crude extract for 3-5 seconds each time, for a total of 3 times; S4, Immerse the filter paper strip in the eluent to wash away impurities.

8. The method according to claim 7, characterized in that... , The lysis buffer consisted of a mixture of 0.06% SDS, 0.125 mol / L NaCl, 0.01 mol / L Tris-HCl buffer, and 1 mmol / L EDTA, with a pH of 8.

0. The eluent consists of a mixture of 0.01 mol / L Tris-HCl buffer and 1.5% Tween-20, pH=7.8-8.2; The filter paper strips used have a pore size of 1-3 μm and a specification of (40-50) mm × 2 mm. They consist of a hydrophobic region of (40-50) mm × 2 mm formed by immersing one end in molten solid paraffin and a DNA knot region of (2-4) mm × 2 mm at the other end.

9. The method according to claim 6, characterized in that... The LAMP amplification reaction system in step (2) is as follows: Contains 2 mmol Mg 2+ 1× Isothermal Amplification buffer, MgSO4 4~8 mmol, dNTP mix 1.4 μM, F3 / B3 0.16 μM, FIP / BIP 1.20 μM, LF 0.32 μM, Bst DNA polymerase 320 U / mL, HNB 120 μM, template extracted by DNA filter paper strip method, and ddH2O balance.

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