Multiple LAMP (loop-mediated isothermal amplification) primer combination for detecting myxobacteria as well as application and kit of multiple LAMP primer combination

Abstract

The invention belongs to a microbiological detection technology, and particularly relates to a multiple LAMP (loop-mediated isothermal amplification) primer combination for detecting myxobacteria as well as application and a kit of the multiple LAMP primer combination. The multiple LAMP primer combination provided by the invention comprises an upstream internal primer FIP, a downstream internal primer BIP, an upstream external primer F3, a downstream external primer B3 and a Loop primer Loop F. The gene has a nucleotide sequence as shown in SEQ ID NO.1-9. According to the multiple LAMP primer combination and the kit prepared from the multiple LAMP primer combination, it is clear that conventionally extracted genome DNA can meet the normal LAMP detection requirement, the detection accuracy is guaranteed, meanwhile, the time for obtaining a result is greatly shortened, the detection efficiency of myliomyxa in the environment is improved, and the detection cost is reduced. The soil health can be efficiently evaluated, scientific basis is provided for optimizing prevention and control means of harmful microorganisms, and the method has important guiding significance and wide application prospects and economic benefits.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, and particularly relates to a multiplex LAMP primer combination for detecting Physarum obscurum, and its application and kit. Background Art

[0002] Myxomycetes belongs to the kingdom Protozoa, has a wide range of ecological adaptability, and most myxomycetes are distributed worldwide. In its vegetative stage, it can phagocytize microorganisms such as bacteria and fungi on the surface of the living matrix, plays a key role in the process of soil material cycling, and also plays an important role in the nutritional cycle of the food chain.

[0003] Myxomycetes will form macroscopic fruiting bodies, and researchers identify its species according to the morphological characteristics of its fruiting bodies. So far, about more than 1,100 species have been described. However, due to the lack of data on the distribution of the vegetative stage of myxomycetes, especially its amoeba stage (swarm cells or myxamoebae), only about 100 species of myxomycetes life histories have been reported so far. It can be seen that the true diversity of myxomycetes in nature is much wider than the results reflected by the appearance of fruiting bodies, and some myxomycetes may even have completely lost the ability to form fruiting bodies.

[0004] The abundance of myxomycetes in soil amoebae exceeds 50%, and it is the most abundant amoeba predator in the soil in some areas. Therefore, the abundance of myxomycetes is an important indicator reflecting the stability of the ecosystem. However, in the investigation of soil microorganisms, myxomycetes have been absent from all environmental surveys due to its large number of Group I introns. Therefore, taking rapid and effective means to detect whether there are myxomycete resources in soil and the environment is of great significance for predicting myxomycete resources, evaluating soil health, and assessing the control conditions of environmental harmful microorganisms.

[0005] Dahl, Fiore-Donno and others [1] designed dozens of primers to amplify the SSU gene of Physarum obscurum, and different primers may match different regions, which makes it difficult to compare and align the amplified sequences for phylogenetic analysis. Wang Wan and others [2] sequenced the genomic DNA of 81 myxomycetes using high-throughput sequencing technology, extracted the reads of the SSU gene, and designed a set of SSU primers after calibration and assembly, avoiding all known Group I intron insertion sites and maintaining high specificity among major myxomycete groups. However, the detection requires the DNA of myxomycetes and can only be carried out after PCR amplification and first-generation sequencing.

[0006] Loop-mediated isothermal amplification (LAMP), as a relatively mature detection technology, has been widely used in the detection of pathogens in humans, animals and plants, playing an important role in the diagnosis of diseases of food crops and cash crops. However, so far, there has been no report on the LAMP detection method for Physarum in the environment.

[0007] [1] Borg Dahl M, Brejnrod AD, Unterseher M, et al. Genetic barcoding of dark-spored myxomycetes (Amoebozoa) - Identification, evaluation and application of a sequence similarity threshold for species differentiation in NGS studies [J]. Mol Ecol Resour. 2018 Mar;18(2):306-318.

[0008] [2] Wang W, Wang W, Wei S, et al. Design of potentially universal SSU primers in myxomycetes using next-generation sequencing [J]. Microbiol Methods. 2021 May;184:106203. Summary of the Invention

[0009] Based on the above deficiencies, the present invention provides a multiplex LAMP primer combination for detecting Physarum, its application and kit. The multiplex LAMP primer combination provided by the present invention includes an upstream inner primer FIP, a downstream inner primer BIP, an upstream outer primer F3, a downstream outer primer B3 and a Loop primer Loop F. It has the nucleotide sequences shown in SEQ ID NO.1-9. The multiplex LAMP primer combination provided by the present invention and the kit prepared therefrom clarify that the conventionally extracted genomic DNA can meet the normal LAMP detection requirements, greatly shorten the time to obtain results while ensuring the detection accuracy, improve the detection efficiency of Physarum in the environment and reduce the detection cost. It can efficiently evaluate soil health, provide a scientific basis for optimizing the control means of harmful microorganisms, and has important guiding significance, broad application prospects and economic benefits.

[0010] The technical solution of the present invention includes:

[0011] In a first aspect, the present invention provides a multiplex LAMP primer combination for detecting Physarum melakomum, and the multiplex LAMP primer combination includes internal primers, external primers, and Loop primers.

[0012] Specifically, the internal primers consist of an upstream internal primer FIP and a downstream internal primer BIP.

[0013] Preferably, the nucleotide sequence of the upstream internal primer FIP is as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0014] Preferably, the nucleotide sequence of the downstream internal primer BIP is as shown in SEQ ID NO.3 or SEQ ID NO.4.

[0015] Specifically, the external primers consist of an upstream external primer F3 and a downstream external primer B3.

[0016] Preferably, the nucleotide sequence of the upstream external primer F3 is as shown in SEQ ID NO.5 or SEQ ID NO.6.

[0017] Preferably, the nucleotide sequence of the downstream external primer B3 is as shown in SEQ ID NO.7 or SEQ ID NO.8.

[0018] Specifically, the Loop primer is Loop F.

[0019] Preferably, the nucleotide sequence of the Loop F is as shown in SEQ ID NO.9.

[0020] In a second aspect, the present invention provides a reagent of the multiplex LAMP primer combination, and the reagent contains the above-mentioned multiplex LAMP primer combination.

[0021] Specifically, the final concentration of the internal primers in the reagent is 3.2 μM; the final concentration of the external primers is 0.8 μM; the final concentration of the Loop primers is 0.8 μM.

[0022] Preferably, the final concentration of the upstream internal primer FIP in the internal primers is 1.6 μM; the final concentration of the downstream internal primer BIP is 1.6 μM.

[0023] Preferably, the final concentration of the upstream external primer F3 in the external primers is 0.4 μM, and the final concentration of the downstream external primer B3 is 0.4 μM.

[0024] In a third aspect, the present invention provides the application of the above-mentioned multiplex LAMP primer combination or reagent in the preparation of a kit for detecting Physarum melakomum.

[0025] Fourth aspect, the present invention provides a kit for detecting Physarum perichaetiale, and the kit includes the above-mentioned multiplex LAMP primer combination or reagent.

[0026] Preferably, the kit further includes one or more of DNA polymerase, reaction buffer, colorimetric agent, and nuclease-free water.

[0027] Fifth aspect, the present invention provides the application of the above-mentioned kit in the visual detection of Physarum perichaetiale.

[0028] Sixth aspect, the present invention provides a method for visual detection of Physarum perichaetiale, and the method includes using the above-mentioned kit.

[0029] Specifically, the method includes the following steps:

[0030] S1. Extract nucleic acid from the sample to be tested using the kit;

[0031] S2. Perform loop-mediated isothermal amplification using the multiplex LAMP primer combination;

[0032] S3. Make a determination according to the color change of the loop-mediated isothermal amplification product.

[0033] Preferably, the sample to be tested in step S1 includes environmental samples.

[0034] Further preferably, the environmental samples include, but are not limited to, one or more of water samples, soil samples, atmospheric samples, and biological samples.

[0035] Preferably, the conditions for the loop-mediated isothermal amplification are: 60 - 65°C, amplification reaction for 30 min; or 60 - 65°C, amplification reaction for 30 min, and amplification reaction at 80°C for 10 min.

[0036] Preferably, the determination according to the color change of the loop-mediated isothermal amplification product in step S3 is: if the loop-mediated isothermal amplification product shows yellow, the sample to be tested is positive; if the loop-mediated isothermal amplification product shows magenta, the sample to be tested is negative.

[0037] The beneficial effects of the present invention are as follows:

[0038] 1. Strong specificity: The present invention designs 4 specific primers targeting the EF1α sequence of Physarum perichaetiale as the target gene. If any region in the 6 regions does not match the primer, the amplification effect cannot be achieved within 50 min.

[0039] 2. High sensitivity: The detection sensitivity of the present invention for Physarum perichaetiale DNA in the environment can reach 10 copies at the DNA level, showing high sensitivity.

[0040] 3. High detection efficiency and speed: By applying the detection method of the present invention, the detection result can be obtained within 40 minutes. In the past, PCR detection required 1.5 - 2 hours for amplification and then agarose gel electrophoresis detection to obtain the detection result. The method of the present invention greatly shortens the operation time, which is convenient and fast.

[0041] 4. Strong applicability and low cost: The LAMP amplification reaction of the present invention is carried out under a constant temperature condition, without the need for an expensive PCR amplifier, and only simple heating equipment such as a water bath and a metal bath is required. Brief Description of the Drawings

[0042] Figure 1 It is the LAMP detection result of the multiplex LAMP primer combination 1; 1 in the figure represents the plasmodium of Fuligogyrosa; 2 represents the spore DNA of Fuligogyrosa; 3 represents the plasmodium DNA of Physarum melleum; 4 represents the spore DNA of Physarum melleum; 5 represents the plasmodium of Didymium nigripes; 6 represents the spore DNA of Didymium nigripse; 7 represents the DNA extracted from a 10g soil sample; 8 represents the DNA extracted from a 1g soil sample; 9 represents the DNA extracted from a 0.5g soil sample; 10 represents the fruiting body DNA of Ganoderma lucidum; 11 represents the DNA of Escherichia coli; 12 represents the blank control with sterile water added.

[0043] Figure 2LAMP detection results for multiplex LAMP primer combination 2; 1 in the figure represents the plasmodium of Fuligogyrosa; 2 represents the spore DNA of Fuligogyrosa; 3 represents the plasmodium DNA of Physarum melleum; 4 represents the spore DNA of Physarum melleum; 5 represents the plasmodium of Didymium nigripes; 6 represents the spore DNA of Didymium nigripse; 7 represents the DNA extracted from a 10 g soil sample; 8 represents the DNA extracted from a 1 g soil sample; 9 represents the DNA extracted from a 0.5 g soil sample; 10 represents the fruiting body DNA of Ganoderma lucidum; 11 represents the DNA of Escherichia coli; 12 represents the blank control with sterile water added.

[0044] Figure 3 Agarose gel electrophoresis detection results for multiplex LAMP primer combination 1; 1 in the figure represents the plasmodium of Fuligogyrosa; 2 represents the spore DNA of Fuligogyrosa; 3 represents the plasmodium DNA of Physarum melleum; 4 represents the spore DNA of Physarum melleum; 5 represents the plasmodium of Didymium nigripes; 6 represents the spore DNA of Didymium nigripse; 7 represents the DNA extracted from a 10 g soil sample; 8 represents the DNA extracted from a 1 g soil sample; 9 represents the DNA extracted from a 0.5 g soil sample; 10 represents the fruiting body DNA of Ganoderma lucidum; 11 represents the DNA of Escherichia coli; 12 represents the blank control with sterile water added.

[0045] Figure 4Agarose gel electrophoresis detection results of multiplex LAMP primer combination 2; 1 in the figure represents the protoplast of Fuligogyrosa; 2 represents the spore DNA of Fuligogyrosa; 3 represents the protoplast DNA of Physarum melleum; 4 represents the spore DNA of Physarum melleum; 5 represents the protoplast of Didymium nigripes; 6 represents the spore DNA of Didymium nigripse; 7 represents the DNA extracted from 10 g of soil sample; 8 represents the DNA extracted from 1 g of soil sample; 9 represents the DNA extracted from 0.5 g of soil sample; 10 represents the fruiting body DNA of Ganoderma lucidum; 11 represents the DNA of Escherichia coli; 12 represents the blank control with sterile water added.

[0046] Figure 5 For the sensitivity detection results of multiplex LAMP primer combination 1 (E11); 1 in the figure represents the sample to be tested at 200 ng / μL; 2 represents the sample to be tested at 100 ng / μL; 3 represents the sample to be tested at 10 ng / μL; 4 represents the sample to be tested at 1 ng / μL; 5 represents the sample to be tested at 100 pg / μL; 6 represents the sample to be tested at 10 pg / μL; 7 represents 1 pg / μL; 8 represents 0.1 pg / μL; 9 represents the blank control with sterile water added.

[0047] Figure 6 For the agarose gel electrophoresis detection results of the sensitivity of multiplex LAMP primer combination 1 (E11); 1 in the figure represents the sample to be tested at 200 ng / μL; 2 represents the sample to be tested at 100 ng / μL; 3 represents the sample to be tested at 10 ng / μL; 4 represents the sample to be tested at 1 ng / μL; 5 represents the sample to be tested at 100 pg / μL; 6 represents the sample to be tested at 10 pg / μL; 7 represents 1 pg / μL; 8 represents 0.1 pg / μL; 9 represents the blank control with sterile water added.

[0048] Figure 7 For the sensitivity detection results of multiplex LAMP primer combination 2 (E12); 1 in the figure represents the sample to be tested at 200 ng / μL; 2 represents the sample to be tested at 100 ng / μL; 3 represents the sample to be tested at 10 ng / μL; 4 represents the sample to be tested at 1 ng / μL; 5 represents the sample to be tested at 100 pg / μL; 6 represents the sample to be tested at 10 pg / μL; 7 represents 1 pg / μL; 8 represents 0.1 pg / μL; 9 represents the blank control with sterile water added.

[0049] Figure 8 The detection results of the sensitivity of multiplex LAMP primer combination 2 (E12) by agarose gel electrophoresis; in the figure, 1 represents the test sample of 200 ng / μL; 2 represents the test sample of 100 ng / μL; 3 represents the test sample of 10 ng / μL; 4 represents the test sample of 1 ng / μL; 5 represents the test sample of 100 pg / μL; 6 represents the test sample of 10 pg / μL; 7 represents 1 pg / μL; 8 represents 0.1 pg / μL; 9 represents the blank control added with sterile water.

[0050] Figure 9 The LAMP detection results of other multiplex LAMP primer combinations; 1 in the figure represents the plasmodium of Fuligogyrosa; 2 represents the spore DNA of Fuligogyrosa; 3 represents the plasmodium DNA of Physarum melleum; 4 represents the spore DNA of Physarum melleum; 5 represents the plasmodium of Didymium nigripes; 6 represents the spore DNA of Didymium nigripse; 7 represents the DNA extracted from 10 g of soil sample; 8 represents the DNA extracted from 1 g of soil sample; 9 represents the DNA extracted from 0.5 g of soil sample; 10 represents the fruiting body DNA of Ganoderma lucidum; 11 represents the DNA of Escherichia coli; 12 represents the blank control added with sterile water.

[0051] Figure 10 The detection results of other multiplex LAMP primer combinations by agarose gel electrophoresis; 1 in the figure represents the plasmodium of Fuligogyrosa; 2 represents the spore DNA of Fuligogyrosa; 3 represents the plasmodium DNA of Physarum melleum; 4 represents the spore DNA of Physarum melleum; 5 represents the plasmodium of Didymium nigripes; 6 represents the spore DNA of Didymium nigripse; 7 represents the DNA extracted from 10 g of soil sample; 8 represents the DNA extracted from 1 g of soil sample; 9 represents the DNA extracted from 0.5 g of soil sample; 10 represents the fruiting body DNA of Ganoderma lucidum; 11 represents the DNA of Escherichia coli; 12 represents the blank control added with sterile water. Detailed implementation mode

[0052] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments; based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0053] The main reagents and instrument information used in the following embodiments are as follows:

[0054] 2.5×pH Sensitive LAMP Reaction Mix, colorimetric agent N-Red Stain, and BstⅡDNA polymerase were all purchased from Beijing TransGen Biotech Co., Ltd., and the PCR instrument was purchased from Thermo Fisher Scientific.

[0055] Example 1 Multiplex LAMP primer combination for detecting Physarum obscurum

[0056] In the present invention, by determining the EF1α sequence of Physarum obscurum, comparing and analyzing the EF1α sequence, and using the online LAMP primer design software Primersoftware Explorer V5 (https: / / primerexplorer.jp / LAMPv5 / index.html; Eiken Chemical Co., Japan), multiplex LAMP primer combination 1 (hereinafter referred to as E11 for short) and multiplex LAMP primer combination 2 (hereinafter referred to as E12 for short) were designed. Both E11 and E12 consist of upstream internal primer FIP, upstream external primer F3, downstream internal primer BIP, downstream external primer B3, and loop primer Loop F.

[0057] The nucleotide sequences of each primer of E11 are shown in Table 1:

[0058] Table 1 Nucleotide sequences of each primer of E11

[0059]

[0060] The nucleotide sequences of each primer of E12 are shown in Table 2:

[0061] Table 2 Nucleotide sequences of each primer of E12

[0062]

[0063]

[0064] Example 2 Kit for detecting Physarum obscurum and its usage method

[0065] The kit for detecting Physarum peruvianum of the present invention includes: E11 or E12, 2×Lamp Master Mix, DNA Polymerase and ddH2O. The operation method of the specific LAMP detection kit for Physarum peruvianum is as follows:

[0066] Step 1: Extract the genomic DNA of the test substance, where the test substance includes environmental samples such as biofilms, water bodies, and soils.

[0067] Step 2: Using the above genomic as a template, perform loop-mediated isothermal amplification with the specific LAMP detection kit for Physarum peruvianum; the conditions for loop-mediated isothermal amplification are: 60 - 65°C, and the amplification reaction is 30 min. If it is necessary to extend the product storage time, then 60 - 65°C, the amplification reaction is 30 min; 80°C for 10 min. The reaction system of the loop-mediated isothermal amplification is shown in Table 3:

[0068] Table 3 Reaction system of loop-mediated isothermal amplification

[0069]

[0070] The composition of the E11 primer solution is shown in Table 4:

[0071] Table 4 E11 primer solution

[0072]

[0073]

[0074] The composition of the E12 primer solution is shown in Table 5:

[0075] Table 5 E12 primer solution

[0076] Primer Sequence number Final concentration FIP SEQ ID NO.2 1.6 μM BIP SEQ ID NO.4 1.6 μM F3 SEQ ID NO.6 0.4 μM B3 SEQ ID NO.8 0.4 μM LooPF SEQ ID NO.9 0.8 μM

[0077] Step 3: After the loop-mediated isothermal amplification reaction ends, observe the color change.

[0078] Application of the kit for detecting Physarum peruvianum in Example 3

[0079] 1. Source of the test sample:

[0080] Spores of Physarum peruvianum: Fuligogyrosa was collected from Shennongjia, Enshi, Hubei Province; Physarum melleum was collected from Dongshan Park, Tianjia'an District, Huainan City, Anhui Province; Didymium nigripes was collected from the Daba Mountains, Shaanxi Province.

[0081] Plasmodium of Physarum peruvianum: The plasmodium of Physarum peruvianum was obtained by feeding culture of myxomycete spores in the laboratory.

[0082] Soil sample: Collected from Dongshan Park, Tianjia'an District, Huainan City, Anhui Province.

[0083] Macroscopic fungus Ganoderma lucidum: Collected from Dongshan Park, Tianjia'an District, Huainan City, Anhui Province.

[0084] Escherichia coli: ATCC 25922 was purchased from Shanghai Luwei Technology Co., Ltd.

[0085] 2. Treatment of samples to be tested:

[0086] Genomic DNA of Physarum peruvianum spores, Physarum peruvianum plasmodium, and Ganoderma lucidum was extracted using the TiaNgen Plant Genomic DNA Kit. Genomic DNA of the soil sample was extracted using the DNeasy PowerMax Soil Kit. Genomic DNA of Escherichia coli was extracted using the Rapid Bacterial Genomic DNA Isolation Kit. The DNA of Ganoderma lucidum and Escherichia coli was used as a negative control, and sterile water was used as a blank control.

[0087] 3. Respectively use the kit containing E11 or E12, and carry out loop-mediated isothermal amplification according to the method described in Example 2.

[0088] 4. After the loop-mediated isothermal amplification reaction ends, observe the color change.

[0089] The measurement results of the multiplex LAMP primer combination 1 (E11) are as Figure 1 shown, and the measurement results of the multiplex LAMP primer combination 2 (E12) are as Figure 2 shown. The results show that the reaction tubes of the test substances containing the DNA fragment of Physarum peruvianum are yellow, and the reaction tubes of the test substances containing the negative control and the blank control are magenta.

[0090] 5. The amplification products were detected by agarose gel electrophoresis

[0091] Take 2 μL of the product from the loop-mediated isothermal amplification reaction for 1% agarose gel electrophoresis. Check whether there are obvious amplification bands and whether the size of the bands meets the expectations.

[0092] The agarose gel electrophoresis detection results of the multiplex LAMP primer combination 1 (E11) are as Figure 3 shown, and the agarose gel electrophoresis detection results of the multiplex LAMP primer combination 2 (E12) are as Figure 4As shown, the amplified fragments visible in the test substance containing the DNA fragment of Physarum obscurum presented bands, while the reaction tubes of the test substances containing negative controls and the blank control showed no reaction bands.

[0093] All of the above results indicate that the test results of samples with Physarum obscurum are all positive, and the results of samples of other fungi are all negative. The multiplex LAMP primer combination provided by the present invention and the kit prepared therefrom can clearly detect the Physarum obscurum resources in environmental samples, have good specificity, have the advantage of not producing false positive results, and have no cross-amplification reaction with bacteria, fungi, etc. in the environment.

[0094] Example 4 Sensitivity of the Kit for Detecting Physarum obscurum

[0095] 1. Treatment of Test Samples

[0096] The protoplasts of Physarum fuligogyrosa were used to extract genomic DNA with the TiaNgen Plant Genomic DNA Kit (product number DP305). The original DNA concentration was about 200 ng / μL, and the DNA was gradient-diluted into test samples with concentrations of 100 ng / μL, 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 0.1 pg / μL, respectively.

[0097] 2. Respectively use the kits containing E11 or E12, and perform loop-mediated isothermal amplification according to the method described in Example 2. Respectively detect 8 types of gradient-diluted test samples according to the method described in Example 2.

[0098] 3. After the loop-mediated isothermal amplification reaction is completed, observe the color change.

[0099] The minimum detection limits of the multiplex LAMP primer combination 1 (E11) and the multiplex LAMP primer combination 2 (E12) are shown in Table 6 below.

[0100] Table 6 Results of Detection Limit Determination

[0101]

[0102]

[0103] In the table, "+" represents a positive reaction result, and "-" represents a negative reaction result.

[0104] The measurement results showed that when the DNA concentrations of Physarum perichaetiale were 100 ng / μL, 10 ng / μL, 1 ng / μL, 100 pg / μL, and 10 pg / μL respectively, the reaction results of the multiplex LAMP primer combination 1 (E11) were all positive, and the reaction tubes of the test substances containing Physarum perichaetiale DNA fragments showed a gradient change from yellow to orange ( Figure 5 - Figure 6 ). The multiplex LAMP primer combination 2 (E12) could still show orange when the DNA concentration was 1 pg / μL ( Figure 7 - Figure 8 ). It can be seen from this that the multiplex LAMP primer combination provided by the present invention and the kit prepared therefrom can clearly detect Physarum perichaetiale DNA with high sensitivity. The lowest detection limit of the multiplex LAMP primer combination 1 (E11) for Physarum perichaetiale DNA is 10 pg / μL, and the lowest detection limit of the multiplex LAMP primer combination 2 (E12) for Physarum perichaetiale DNA is 1 pg / μL.

[0105] Measurement results of the kit prepared from other multiplex LAMP primer combinations in Comparative Example 1

[0106] Refer to Example 2 to prepare a kit for detecting Physarum perichaetiale. The difference between Comparative Example 1 and Example 2 is only that: the reaction system of loop-mediated isothermal amplification is different, and the specific composition is shown in Table 7:

[0107] Table 7 Reaction system of loop-mediated isothermal amplification in Comparative Example 1

[0108] Component Volume Template DNA 1 μL Other multiplex LAMP primer combination solution 4 μL 2.5×pH Sensitive LAMP Reaction Mix 10 μL Colorimetric agent N-Red Stain 1.3 μL BstⅡ DNA polymerase 1 μL <![CDATA[ddH2O]]> Make up to 25 μL

[0109] The nucleotide sequences and compositions of the primers of the other multiplex LAMP primer combinations are shown in Table 8:

[0110] Table 8 Sequences and compositions of other multiplex LAMP primer combinations

[0111]

[0112] Refer to the method described in Example 3 to process the test samples and perform loop-mediated isothermal amplification. After the loop-mediated isothermal amplification reaction is completed, observe the color change. The measurement results are as Figure 9 shown. The results show that the reaction tubes of the test substances containing Physarum perichaetiale DNA fragments, the reaction tubes of the test substances containing negative controls, and the blank controls are all magenta. There are no reaction bands in the gel diagrams of the reaction tubes of the test substances and the blank controls ( Figure 10 ).

[0113] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or changes made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. A multiplex LAMP primer combination for detecting Physarum melakomum, characterized in that the multiplex LAMP primer combination includes internal primers, external primers and Loop primers; The internal primers consist of an upstream internal primer FIP and a downstream internal primer BIP; the nucleotide sequence of the upstream internal primer FIP is shown in SEQ ID NO.1 or SEQ ID NO.2; the nucleotide sequence of the downstream internal primer BIP is shown in SEQ ID NO.3 or SEQ ID NO.4; The external primers consist of an upstream external primer F3 and a downstream external primer B3; the nucleotide sequence of the upstream external primer F3 is shown in SEQ ID NO.5 or SEQ ID NO.6; the nucleotide sequence of the downstream external primer B3 is shown in SEQ ID NO.7 or SEQ ID NO.8; The Loop primer is Loop F; the nucleotide sequence of Loop F is shown in SEQ ID NO.

9.

2. A reagent of a multiplex LAMP primer combination, characterized in that, The reagent contains the multiplex LAMP primer combination according to claim 1.

3. The reagent according to claim 1, wherein In the reagent, the final concentration of the internal primers is 3.2 μM; the final concentration of the external primers is 0.8 μM; the final concentration of the Loop primer is 0.8 μM.

4. The reagent according to claim 3, wherein In the internal primers, the final concentration of the upstream internal primer FIP is 1.6 μM; the final concentration of the downstream internal primer BIP is 1.6 μM.

5. The reagent according to claim 3, characterized in that, In the external primers, the final concentration of the upstream external primer F3 is 0.4 μM, and the final concentration of the downstream external primer B3 is 0.4 μM.

6. Use of the multiplex LAMP primer combination according to claim 1 or the reagent according to any one of claims 2-5 in the preparation of a kit for detecting Physarum melakomum.

7. A kit for detecting Physarum nigrum, characterized in that, The kit includes the multiplex LAMP primer combination according to claim 1 or the reagent according to any one of claims 2-5.

8. The kit according to claim 7, characterized in that, The kit further includes one or more of DNA polymerase, reaction buffer, colorimetric agent, nuclease-free water.

9. Use of the kit according to any one of claims 7-8 in the visual detection of Physarum melakomum.

10. A method for visual detection of Physarum obscurum, characterized in that, The method includes using the kit according to any one of claims 7-8.

11. The method according to claim 10, characterized in that, The method includes the following steps: S1. Extract nucleic acid from the sample to be tested using the kit; S2. Perform loop-mediated isothermal amplification using the multiplex LAMP primer combination; S3. Make a determination based on the color change of the loop-mediated isothermal amplification product.

12. The method according to claim 11, wherein In step S1, the sample to be tested includes environmental samples.

13. The method according to claim 12, wherein The environmental samples include one or more of water samples, soil samples, atmospheric samples, biological samples.

14. The method according to claim 11, wherein The conditions for the loop-mediated isothermal amplification in step S2 are: 60-65 °C, amplification reaction for 30 min; or 60-65 °C, amplification reaction for 30 min, 80 °C amplification reaction for 10 min.

15. The method according to claim 11, wherein The determination based on the color change of the loop-mediated isothermal amplification product in step S3 is: if the loop-mediated isothermal amplification product shows yellow, the sample to be tested is positive; if the loop-mediated isothermal amplification product shows magenta, the sample to be tested is negative.

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