A nucleic acid detection primer set and visual detection method for rapid detection of Saccharomyces boulardii based on loop-mediated isothermal amplification technology

Abstract

The present invention belongs to the field of biological detection technology, and discloses a nucleic acid detection primer set and a visual detection method for rapid detection of Saccharomyces boulardii based on loop-mediated isothermal amplification technology, and specifically discloses a reagent for detecting yeast, including LAMP primer set 1 and / or LAMP primer set 2. The reagent for detecting yeast provided by the present invention contains LAMP primer set 1 and / or LAMP primer set 2, wherein LAMP primer set 1 is designed for the ITS sequence of the genome of Saccharomyces boulardii or Saccharomyces cerevisiae, and can be used to detect ITS1-5.8S-ITS2 of yeast and / or Saccharomyces boulardii and other fungi to distinguish yeast from other fungi. LAMP primer set 2 is designed for the conserved sequence HXT9 sequence of Saccharomyces cerevisiae, and can effectively identify and distinguish Saccharomyces boulardii and Saccharomyces cerevisiae.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to a nucleic acid detection primer set and a visual detection method for rapidly detecting Saccharomyces boulardii based on a loop-mediated isothermal amplification technology. Background Art

[0002] Saccharomyces boulardii is a probiotic that can treat diarrhea and prevent antibiotic-related complications. Clinical trials have shown that Saccharomyces boulardii has therapeutic effects on several types of diarrhea and enteritis, especially diarrhea associated with overgrowth of Clostridium difficile. In addition, Saccharomyces boulardii preparations are also used as adjuvant treatments for Vibrio cholerae, rotavirus, traveler's diarrhea, and Helicobacter pylori. Freeze-dried preparations of Saccharomyces boulardii have been widely used.

[0003] The biological characteristics of Saccharomyces boulardii are not exactly the same as those of Saccharomyces cerevisiae. It is a new species of the genus Saccharomyces and is considered to be a subspecies of Saccharomyces cerevisiae. Currently, there are still many difficulties in the identification and typing of Saccharomyces boulardii. The traditional experimental scheme for phenotypic identification takes a long time, and the difficulty of molecular identification is that the genomes of Saccharomyces boulardii and Saccharomyces cerevisiae are similar, which makes it difficult.

[0004] According to the identification and phylogenetic analysis of Saccharomyces cerevisiae and Saccharomyces boulardii, through the analysis of the gene database, the gene region of the intergenic transcribed region (ITS) in 26SrDNA showed high interspecific differences and low intraspecific polymorphism, which can be used to distinguish yeast from other species. In addition, the HXT9 gene is a member of the hexose transporter gene family in the Saccharomyces cerevisiae genome. The HXT9 sequence was determined to be a conserved sequence of Saccharomyces cerevisiae, which can be used to distinguish Saccharomyces boulardii / Saccharomyces cerevisiae. In recent years, the use of multiplex PCR, MLST or gene sequencing methods can achieve the distinction between Saccharomyces boulardii and Saccharomyces cerevisiae, but for the quality inspection of probiotic preparations such as Saccharomyces boulardii powder, the above methods are technically feasible but require a long time and more data analysis, and often cannot meet the needs of timely identification of a large number of finished drugs.

[0005] Loop-mediated isothermal amplification (LAMP) technology is a new isothermal nucleic acid amplification method that mainly uses different specific primers to identify specific regions of the target gene and uses a polymerase with chain displacement activity to rapidly amplify nucleic acids under constant temperature conditions (around 65°C), ensuring high specificity and efficiency of amplification.

[0006] In response to the current quality inspection requirements for Saccharomyces boulardii preparations, the present invention will establish a rapid and accurate identification scheme for Saccharomyces boulardii and / or Saccharomyces cerevisiae based on LAMP technology. Summary of the invention

[0007] The first aspect of the present invention aims to provide a reagent for detecting yeast.

[0008] The purpose of the second aspect of the present invention is to provide use of the reagent of the first aspect of the present invention in preparing a kit.

[0009] The third aspect of the present invention aims to provide a kit for detecting yeast.

[0010] The purpose of the fourth aspect of the present invention is to provide the use of the reagent of the first aspect of the present invention and / or the kit of the third aspect of the present invention.

[0011] The fifth aspect of the present invention aims to provide a LAMP visualization detection method for detecting Saccharomyces boulardii and / or Saccharomyces cerevisiae.

[0012] In order to achieve the above object, the technical solution adopted by the present invention is:

[0013] The first aspect of the present invention provides a reagent for detecting yeast, comprising LAMP primer set 1 and / or LAMP primer set 2; the LAMP primer set 1 and LAMP primer set 2 respectively comprise a pair of outer primers, a pair of inner primers and a pair of loop primers; the LAMP primer set 1 comprises at least one of primer sets ITS-1 to ITS-3;

[0014] The sequences of the primer set ITS-1 are as follows:

[0015] External primers: The sequence of ITS-F3-1 is shown in SEQ ID NO: 1, and the sequence of ITS-B3-1 is shown in SEQ ID NO: 2;

[0016] Internal primers: The sequence of ITS-FIP-1 is shown in SEQ ID NO:3, and the sequence of ITS-BIP-1 is shown in SEQ ID NO:4;

[0017] The sequence of loop primer: ITS-LF-1 is shown in SEQ ID NO: 5, and the sequence of loop primer ITS-LB-1 is shown in SEQ ID NO: 6;

[0018] The sequences of the primer set ITS-2 are as follows:

[0019] External primer: The sequence of ITS-F3-2 is shown in SEQ ID NO:7, and the sequence of ITS-B3-2 is shown in SEQ ID NO:8; Internal primer: The sequence of ITS-FIP-2 is shown in SEQ ID NO:9, and the sequence of ITS-BIP-2 is shown in SEQ ID NO:10;

[0020] Loop primer: The sequence of ITS-LF-2 is shown in SEQ ID NO: 11, and the sequence of ITS-LB-2 is shown in SEQ ID NO: 12;

[0021] The sequences of the primer set ITS-3 are as follows:

[0022] External primers: the sequence of ITS-F3-3 is shown in SEQ ID NO: 13, and the sequence of ITS-B3-3 is shown in SEQ ID NO: 14;

[0023] Internal primers: the sequence of ITS-FIP-3 is shown in SEQ ID NO: 15, and the sequence of ITS-BIP-3 is shown in SEQ ID NO: 16;

[0024] Loop primer: The sequence of ITS-LF-3 is shown in SEQ ID NO: 17, and the sequence of ITS-LB-3 is shown in SEQ ID NO: 18;

[0025] The LAMP primer set 2 includes at least one of the primer sets HXT9-1 to HXT9-3:

[0026] The sequences of the primer set HXT9-1 are as follows:

[0027] External primers: The sequence of HXT9-F3-1 is shown in SEQ ID NO: 19, and the sequence of HXT9-B3-1 is shown in SEQ ID NO: 20;

[0028] Internal primers: The sequence of HXT9-FIP-1 is shown in SEQ ID NO: 21, and the sequence of HXT9-BIP-1 is shown in SEQ ID NO: 22;

[0029] Loop primer: The sequence of HXT9-LF-1 is shown in SEQ ID NO: 23, and the sequence of HXT9-LB-1 is shown in SEQ ID NO: 24;

[0030] The sequences of the primer set HXT9-2 are as follows:

[0031] External primers: The sequence of HXT9-F3-2 is shown in SEQ ID NO: 25, and the sequence of HXT9-B3-2 is shown in SEQ ID NO: 26;

[0032] Internal primers: The sequence of HXT9-FIP-2 is shown in SEQ ID NO: 27, and the sequence of HXT9-BIP-2 is shown in SEQ ID NO: 28;

[0033] Loop primer: The sequence of HXT9-LF-2 is shown in SEQ ID NO: 29, and the sequence of HXT9-LB-2 is shown in SEQ ID NO: 30;

[0034] The sequences of the primer set HXT9-3 are as follows:

[0035] External primer: The sequence of HXT9-F3-3 is shown in SEQ ID NO:31, and the sequence of HXT9-B3-3 is shown in SEQ ID NO:32; Internal primer: The sequence of HXT9-FIP-3 is shown in SEQ ID NO:33, and the sequence of HXT9-BIP-3 is shown in SEQ ID NO:34;

[0036] Loop primer: The sequence of HXT9-LF-3 is shown in SEQ ID NO:35, and the sequence of HXT9-LB-3 is shown in SEQ ID NO:36.

[0037] Preferably, the yeast comprises Saccharomyces boulardii and / or Saccharomyces cerevisiae.

[0038] The second aspect of the present invention provides use of the reagent of the first aspect of the present invention in preparing a kit.

[0039] Preferably, the uses of the kit include a1 and / or a2;

[0040] a1 Detecting whether the sample to be tested contains Saccharomyces boulardii and / or Saccharomyces cerevisiae;

[0041] a2 Identification of Saccharomyces boulardii and / or Saccharomyces cerevisiae.

[0042] In a third aspect, the present invention provides a kit for detecting yeast, wherein the kit comprises the reagent according to the first aspect of the present invention.

[0043] Preferably, the kit further comprises at least one of DNA polymerase, amplification reaction solution, betaine, magnesium sulfate, dNTPs, HNB dye, positive quality control substance and blank control substance.

[0044] Preferably, the yeast comprises Saccharomyces boulardii and / or Saccharomyces cerevisiae.

[0045] Preferably, the positive control comprises Saccharomyces boulardii genomic DNA and / or Saccharomyces cerevisiae genomic DNA.

[0046] The fourth aspect of the present invention provides the use of the reagent of the first aspect of the present invention and / or the kit of the third aspect of the present invention in any one of (1) to (4):

[0047] (1) Identification of Saccharomyces boulardii and / or Saccharomyces cerevisiae;

[0048] (2) preparing products for identifying Saccharomyces boulardii and / or Saccharomyces cerevisiae;

[0049] (3) detecting whether the sample to be tested contains Saccharomyces boulardii and / or Saccharomyces cerevisiae;

[0050] (4) preparing a product for detecting whether a sample to be tested contains Saccharomyces boulardii and / or Saccharomyces cerevisiae;

[0051] The above applications are used for non-disease diagnosis and treatment.

[0052] A fifth aspect of the present invention provides a LAMP visualization detection method for detecting Saccharomyces boulardii and / or Saccharomyces cerevisiae, wherein the detection method is used for: 1) identifying Saccharomyces boulardii and / or Saccharomyces cerevisiae; or 2) detecting whether a sample to be tested contains Saccharomyces boulardii and / or Saccharomyces cerevisiae, and the detection method is used for non-diagnostic purposes.

[0053] Preferably, the detection method comprises the steps of using the reagent of the first aspect of the present invention and / or the kit of the third aspect of the present invention.

[0054] Preferably, the detection method specifically comprises the following steps:

[0055] S1. Extracting nucleic acid from the sample to be tested;

[0056] S2. Using the nucleic acid of step S1 as a template, performing a LAMP amplification reaction using the reagent or kit;

[0057] S3. Determine whether the sample to be tested contains Saccharomyces boulardii and / or Saccharomyces cerevisiae according to the reaction result of step S2.

[0058] Preferably, in step S2, the kit of the third aspect of the present invention is used to perform LAMP amplification reaction.

[0059] Preferably, the final concentration of the DNA polymerase is 0.05-2 U / μL.

[0060] More preferably, the final concentration of the DNA polymerase is 0.0.05 to 1.5 U / μL.

[0061] More preferably, the final concentration of the DNA polymerase is 0.4-1 U / μL.

[0062] Preferably, the final concentration of magnesium sulfate is 5-15 mM.

[0063] More preferably, the final concentration of magnesium sulfate is 6-10 mM.

[0064] More preferably, the final concentration of magnesium sulfate is 8 mM.

[0065] Preferably, the final concentration of betaine is 0.5-1M.

[0066] More preferably, the final concentration of betaine is 0.5-0.8M.

[0067] More preferably, the final concentration of betaine is 0.8M.

[0068] Preferably, the final concentration of the dNTPs is 1-2 mM.

[0069] More preferably, the final concentration of the dNTPs is 1-1.6 mM.

[0070] More preferably, the final concentration of the dNTPs is 1.6 mM.

[0071] Preferably, the LAMP amplification reaction is carried out at 60-70° C. for 30-60 min.

[0072] More preferably, the LAMP amplification reaction is carried out at 65-70° C. for 35-60 min.

[0073] More preferably, the LAMP amplification reaction is carried out at 65-70° C. for 40-60 min.

[0074] Preferably, the molar ratio of the outer primer, inner primer and loop primer in the LAMP amplification reaction system is 1:(2-4):(1-2).

[0075] Further preferably, the molar ratio of the outer primer, inner primer and loop primer in the LAMP amplification reaction system is 1:(3-4):(1-2).

[0076] More preferably, the molar ratio of the outer primer, inner primer and loop primer in the LAMP amplification reaction system is 1:4:2.

[0077] The beneficial effects of the present invention are:

[0078] The reagent for detecting yeast provided by the present invention contains LAMP primer set 1 and / or LAMP primer set 2, wherein LAMP primer set 1 is designed for the ITS sequence of the genome of Saccharomyces boulardii or Saccharomyces cerevisiae, and can be used to detect ITS1-5.8S-ITS2 of yeast and / or Saccharomyces boulardii and other fungi to distinguish yeast from other fungi. LAMP primer set 2 is designed for the conserved sequence HXT9 sequence of Saccharomyces cerevisiae, and can effectively identify and distinguish Saccharomyces boulardii and Saccharomyces cerevisiae.

[0079] The LAMP visualization detection method for detecting Saccharomyces boulardii and / or Saccharomyces cerevisiae provided by the present invention distinguishes Saccharomyces boulardii and Saccharomyces cerevisiae through color change comparison, and the detection result is intuitive and easy to judge, and has high sensitivity (the detection limit of Saccharomyces cerevisiae is 10 pg, and the detection limit of Saccharomyces boulardii is 0.01 pg), strong specificity, simple operation, high safety, meets the quality detection requirements of yeast preparations, and has good application advantages in the field of fungal nucleic acid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 This is a graph showing the LAMP detection results of Saccharomyces boulardii and Saccharomyces cerevisiae. In the graph, 1 and 2 represent DNA of Saccharomyces boulardii, 3 and 4 represent DNA of Saccharomyces cerevisiae, and C represents a negative control (DEPC water).

[0081] Figure 2 This is a graph showing the results of amplifying Saccharomyces boulardii DNA or Saccharomyces cerevisiae DNA using an ITS primer set; wherein a represents Saccharomyces boulardii DNA, and b represents Saccharomyces cerevisiae DNA. In the graph, 1 represents a DNA concentration of 100 pg, 2 represents a DNA concentration of 10 pg, 3 represents a DNA concentration of 1 pg, 4 represents a DNA concentration of 0.1 pg, 5 represents a DNA concentration of 0.01 pg, and 6 represents a negative control (DEPC water).

[0082] Figure 3 This is a graph showing the LAMP test results for Saccharomyces boulardii and Saccharomyces cerevisiae. In the graph, 1 and 2 are DNA from Saccharomyces boulardii, 3 and 4 are DNA from Saccharomyces cerevisiae, and C is a negative control (DEPC water).

[0083] Figure 4 This is a graph showing the results of amplifying Saccharomyces boulardii DNA or Saccharomyces cerevisiae DNA using the HXT9 primer set; wherein a is Saccharomyces boulardii DNA, and b is Saccharomyces cerevisiae DNA. In the figure, 1 represents a DNA concentration of 100 pg, 2 represents a DNA concentration of 10 pg, 3 represents a DNA concentration of 1 pg, 4 represents a DNA concentration of 0.1 pg, 5 represents a DNA concentration of 0.01 pg, and 6 represents a negative control (DEPC water).

[0084] Figure 5 This is a result judgment diagram of the LAMP detection method in Example 5.

[0085] Figure 6 This is a diagram showing the optimization results of the amount of DNA polymerase used and the LAMP reaction time in the LAMP detection method of Example 5. In the diagram, + represents a positive control product, and - represents a blank control product.

[0086] Figure 7This is a diagram showing the optimization results of the LAMP reaction time in the LAMP detection method of Example 5, in which + represents a positive quality control and - represents a blank control.

[0087] Figure 8 This is a diagram of the optimization results of the magnesium ion concentration in the LAMP detection method of Example 5, in which + represents the positive quality control and - represents the blank control.

[0088] Fig. 9 Specificity evaluation results of the LAMP detection method of Example 5, wherein 1 to 3 represent three different active granule preparations of Saccharomyces cerevisiae, 4 represents Saccharomyces boulardii capsules, 5 represents a positive reference (Saccharomyces cerevisiae DNA), and 6 represents a blank control (DEPC water).

[0089] Fig.10 Specificity evaluation results of the LAMP detection method of Example 5, where 1 to 4 represent four different probiotic agents, 5 represents Saccharomyces boulardii powder, 6 represents a positive reference (Saccharomyces cerevisiae DNA), and 7 represents a blank control (DEPC water).

[0090] Fig.11 This is a result diagram of the detection of Saccharomyces boulardii by the LAMP detection method of Example 5, in which 1 and 2 represent Saccharomyces boulardii probiotic preparation samples, 3 and 4 represent Saccharomyces cerevisiae DNA, and C represents a blank control (DEPC water). DETAILED DESCRIPTION

[0091] The present invention is further described in detail below through specific examples.

[0092] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0093] Unless otherwise specified, the materials and reagents used in this example were obtained from commercial sources.

[0094] Example 1

[0095] The inventors analyzed the gene database based on Saccharomyces boulardii (GenBank: KP744576.1) and Saccharomyces cerevisiae (GenBank: J01347.1) published in GenBank. The gene region of the intergenic transcribed region (ITS) in 26S rDNA showed high interspecific differences and low intraspecific polymorphism, which can be used to distinguish Saccharomyces boulardii, Saccharomyces cerevisiae and other species. Three sets of specific primers were further designed based on the Saccharomyces cerevisiae ITS sequence (GenBank: MK504363) to distinguish Saccharomyces boulardii from other probiotics. Each set of primers can be divided into three pairs of six, namely outer primers F3 and B3, inner primers FIP and BIP, loop primers LF and LB, and the nucleotide sequences are shown in Table 1.

[0096] Table 1 Primer sequence information designed for ITS sequence

[0097]

[0098]

[0099] Example 2

[0100] The embodiment of the present invention provides genome analysis of Saccharomyces boulardii and Saccharomyces cerevisiae. The HXT9 sequence is a conserved sequence of Saccharomyces cerevisiae. Three sets of specific primers are designed based on the Saccharomyces cerevisiae HXT9 gene sequence (GenBank: NC_001142) to distinguish Saccharomyces boulardii from Saccharomyces cerevisiae. Each set of primers can be divided into three pairs of six, namely outer primers F3 and B3, inner primers FIP and BIP, loop primers LF and LB, and the specific primer sequences are shown in Table 2.

[0101] Table 2 Primer sequence information designed for HXT9 sequence

[0102]

[0103]

[0104] Example 3 Primer screening

[0105] 1. Primer set for ITS

[0106] Visual detection based on LAMP was performed on total DNA of Saccharomyces boulardii or Saccharomyces cerevisiae. The 25 μL reaction system was as follows: 1 μL total DNA of Saccharomyces boulardii or Saccharomyces cerevisiae (1 ng / μL), 3 μL reaction premix A (containing 8U Bst DNA large fragment polymerase and 1 μL 3mM HNB dye), 21 μL reaction premix B (10× reaction buffer (Isothermal Amplification Buffer, NEB, catalog number B0537S) 2.5 μL, ITS primer set (the molar concentrations of the outer primer, inner primer, and loop primer were 10 μM, 40 μM, and 20 μM, respectively) 2.8 μL, 10 mM dNTP solution 4 μL, 5 M betaine solution 4 μL, 50 mM magnesium sulfate solution 4 μL, and DEPC water was added to 21 μL). The reaction conditions were set to 65°C for 45 min.

[0107] The results are as follows Figure 1 As shown in the figure, the three primer sets designed for the ITS sequence have good amplification results for both Saccharomyces boulardii and Saccharomyces cerevisiae, and the amplification results are in line with expectations. The amplification capabilities of the three primer sets were further tested using 100 pg to 0.01 pg of total DNA of Saccharomyces boulardii or total DNA of Saccharomyces cerevisiae. The results are shown in the figure. Figure 2 As shown, for Saccharomyces boulardii, the amplification sensitivity of the three primer sets is 0.01pg, but the amplification efficiency of the third primer set (ITS-3) is higher (shown as brighter bands); for Saccharomyces cerevisiae, the sensitivity of the first primer set (ITS-1) can reach 1pg, and the sensitivity of the third primer set (ITS-3) is 10pg, which is slightly worse than ITS-1, but the amplification efficiency is higher and it is easy to cause color changes. Therefore, considering comprehensively, the ITS-3 primer set with higher amplification efficiency is preferred.

[0108] 2. Primer set for HXT9

[0109] Visual detection based on LAMP was performed on total DNA of Saccharomyces boulardii or Saccharomyces cerevisiae. The 25 μL reaction system was as follows: 1 μL total DNA of Saccharomyces boulardii or Saccharomyces cerevisiae (1 ng / μL), 3 μL reaction premix A (containing 8U Bst DNA large fragment polymerase and 1 μL 3mM HNB dye), 21 μL reaction premix B (2.5 μL 10× reaction buffer, 2.8 μL HXT9 primer set (the molar concentrations of outer primers, inner primers, and loop primers were 10 μM, 40 μM, and 20 μM, respectively), 4 μL 10mM dNTP solution, 4 μL 5M betaine solution, 4 μL 50mM magnesium sulfate solution, and DEPC water was added to 21 μL). The reaction conditions were set to 65°C for 45 min.

[0110] The results are as follows Figure 3As shown in the figure, the three pairs of primers designed for the HXT9 sequence have good amplification results for both Saccharomyces boulardii and Saccharomyces cerevisiae, and the amplification results are in line with expectations. The amplification capabilities of the three primer sets were further tested using 10 ng to 10 pg of Saccharomyces boulardii DNA or total Saccharomyces cerevisiae DNA. The results are shown in the figure. Figure 4 As shown, for Saccharomyces cerevisiae, the sensitivity of the first primer set (HXT9-1) can reach 100pg, the sensitivity of the second primer set (HXT9-2) is 10pg, and the sensitivity of the third primer set (HXT9-3) is 1ng. Therefore, considering comprehensively, the HXT9-2 primer set with higher sensitivity and amplification efficiency is preferred.

[0111] Example 4 A kit for LAMP detection of Saccharomyces boulardii by visually amplifying genomic DNA samples

[0112] A kit for LAMP detection for identifying Saccharomyces boulardii by visually amplifying genomic DNA samples, comprising a reaction premix A (containing 8U Bst DNA large fragment polymerase and 1μL of 3mM HNB dye), a reaction premix B (2.5μL of 10× reaction buffer, 2.8μL of ITS-3 or HXT9-2 (the molar concentrations of outer primers, inner primers, and loop primers are 10μM, 40μM, and 20μM, respectively), 4μL of 10mM dNTP solution, 4μL of 5M betaine solution, 4μL of 50mM magnesium sulfate solution, and DEPC water supplemented to 21μL), two positive quality control products (Saccharomyces boulardii DNA and Saccharomyces cerevisiae DNA), and a blank control product (DEPC water).

[0113] Example 5

[0114] The present invention provides a LAMP detection method for identifying Saccharomyces boulardii by visually amplifying a genomic DNA sample, comprising the following steps:

[0115] (1) obtaining genomic DNA of the sample to be tested;

[0116] (2) LAMP amplification

[0117] The LAMP amplification reaction system is as follows: 25 μL reaction system is as follows: 1 μL of sample DNA to be tested, 3 μL reaction premix A (containing 8U Bst DNA large fragment polymerase and 1 μL 3mM HNB dye), 21 μL reaction premix B (2.5 μL 10× reaction buffer, 2.8 μL ITS-3 or HXT9-2 (the molar concentrations of the outer primer, inner primer, and loop primer are 10 μM, 40 μM, and 20 μM, respectively), 4 μL 10mM dNTP solution, 4 μL 5M betaine solution, 4 μL 50mM magnesium sulfate solution, and DEPC water is added to 21 μL).

[0118] Amplification reaction conditions: 65°C for 40 min.

[0119] (3) Test result judgment

[0120] After LAMP amplification, the color change of the system determines the test result: if the ITS sequence is amplified (blue), it means that the test sample contains the genome of Saccharomyces boulardii or Saccharomyces cerevisiae. On this basis, if the HXT9 sequence is blue (amplified), it means that Saccharomyces cerevisiae is contained. If the HXT9 sequence is not amplified and the color is purple, the test result is determined to be negative, that is, the test sample is Saccharomyces boulardii ( Figure 5 ).

[0121] Example 6

[0122] This example is used to optimize the reaction conditions such as the amount of Bst DNA large fragment polymerase, reaction time, temperature, and magnesium ion concentration in the LAMP detection method of Example 5.

[0123] 1.Bst DNA Large Fragment Polymerase Dosage and Reaction Time

[0124] The effects of different enzyme dosages and reaction times on the LAMP detection method of Example 5 were investigated using a positive quality control (total DNA of Saccharomyces cerevisiae) and a blank control (DEPC water). Specifically, the dosage of Bst DNA large fragment polymerase in the reaction system of the LAMP detection method of Example 5 was set to 2U, 4U and 8U, respectively, and the reaction time was set to 30 minutes, 35 minutes, 40 minutes, 50 minutes and 60 minutes, respectively. The results are shown in Tables 3 and 4. Figure 6 As shown, for the positive quality control product, when the dosage of Bst DNA large fragment polymerase is 2U, among the five reaction times, amplification effect occurs only at 60 minutes, when the dosage of Bst DNA large fragment polymerase is 4U, amplification effect occurs in all five reaction times, and when the dosage of Bst DNA large fragment polymerase is 8U, amplification effect occurs in all five reaction times; all blank controls have no amplification effect. In summary, the dosage of Bst DNA large fragment polymerase is selected to be 8U, and the reaction time is set to 40 minutes.

[0125] Table 3 Test results of positive quality control products with different reaction times and enzyme dosages

[0126]

[0127] 2. Temperature

[0128] The effects of different reaction temperatures on the LAMP detection method of Example 5 were investigated using a positive reference (Saccharomyces cerevisiae genomic DNA) and a blank control (DEPC water). Specifically, the reaction temperatures in the reaction system of the LAMP detection method of Example 5 were set to 60°C, 65°C, and 70°C, respectively. The results are shown in Table 1. Figure 7 As shown in the figure, the positive reference product did not amplify at 60°C, but amplified at 65°C and 70°C, so 65°C was finally selected as the reaction temperature.

[0129] 3. Magnesium ion concentration

[0130] The 10× reaction buffer used in the reaction system of Example 5 contains a certain amount of magnesium ions, and the magnesium ion concentration in the entire 25 μL reaction system is 2 mM. 4 The addition amount was such that the magnesium ion concentration in the 25 μL reaction system was 6 mM, 10 mM, and 14 mM, respectively. The effect of magnesium ion concentration on the LAMP detection method of Example 5 was investigated by experiments. The experimental results are shown in FIG. Figure 8 As shown in the figure, with total DNA of Saccharomyces cerevisiae as the positive quality control and DEPC water as the blank control, when the magnesium ion concentration is 6mM, both the positive reaction and the negative reaction are blue, that is, the positive quality control and the blank control cannot be distinguished; when the magnesium ion concentration is 14mM, both the positive reaction and the negative reaction are purple, that is, the positive quality control and the blank control cannot be distinguished; when the magnesium ion concentration is 10mM, the negative reaction tube is purple and the positive reaction tube is blue, that is, the positive and negative reactions can be clearly distinguished. Therefore, the magnesium ion concentration of 10mM was finally selected, that is, 4μL of 50mM magnesium sulfate solution was added under the premise of 10× reaction buffer.

[0131] Example 7 Specificity Assessment

[0132] This example is used to evaluate the specificity of the LAMP detection method of Example 5.

[0133] Three active granule preparations of Saccharomyces cerevisiae and one Saccharomyces boulardii capsule were purchased from the market (for specific information, see Table 3). Saccharomyces boulardii powder and four common probiotic preparations on the market were also purchased (for specific information, see Table 4). The LAMP detection method of Example 5 was used for rapid identification.

[0134] The results are shown in Tables 4, 5 and Fig. 9 , Fig.10 As shown, the LAMP detection method of Example 5 has a good color development effect on Saccharomyces cerevisiae and Saccharomyces boulardii, and has no color development effect on other probiotics, indicating that the LAMP detection method of Example 5 has strong specificity.

[0135] Table 4 Relevant information and test results of active granules of Saccharomyces cerevisiae and Saccharomyces boulardii capsules

[0136]

[0137] Table 5 Detailed information and test results of Saccharomyces boulardii powder and four probiotic preparations

[0138]

[0139]

[0140] Example 7 Detection of actual samples

[0141] A batch of Saccharomyces boulardii powder (BIOCODEX, France, batch number 6386, expiration date 202405) and Saccharomyces cerevisiae were purchased from the market as samples to be tested, and the LAMP detection method of Example 5 was used to detect them. The specific process is as follows:

[0142] (1) DNA extraction of samples to be tested: Under sterile operation, 10 mg of Saccharomyces boulardii was weighed into a 1.5 mL centrifuge tube, resuspended with 100 μL of sterile water, and boiled in a boiling water bath for 10 minutes. Centrifuged at 12,000 rpm for 2 minutes, and the supernatant was collected. In addition, the genomic DNA of Saccharomyces cerevisiae was extracted using a yeast genome extraction kit (Omega Bio-Tek, catalog number D3370) according to its instructions.

[0143] (2) LAMP amplification

[0144] The LAMP amplification reaction system and reaction conditions were the same as those in Example 5.

[0145] (3) Test result judgment

[0146] After the reaction is completed, observe the color change in the reaction system, such as Fig.11 As shown, the ITS sequence in the Saccharomyces boulardii powder was amplified, and the HXT9 sequence appeared blue, that is, the identification result was Saccharomyces boulardii, which was the same as the result of the actual sample annotation; the ITS sequence and HXT9 sequence in the Saccharomyces cerevisiae DNA were both amplified, indicating that the identification result was Saccharomyces cerevisiae, which was the same as the result of the actual sample, indicating that the LAMP detection method provided in Example 5 can effectively identify Saccharomyces boulardii and Saccharomyces cerevisiae.

[0147] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A reagent for detecting yeast, comprising LAMP primer set 1 and LAMP primer set 2; The LAMP primer set 1 and the LAMP primer set 2 respectively include a pair of outer primers, a pair of inner primers and a pair of loop primers; The LAMP primer set 1 includes at least one of primer set ITS-1, primer set ITS-2, and primer set ITS-3; The sequences of the primer set ITS-1 are as follows: External primers: The sequence of ITS-F3-1 is shown in SEQ ID NO: 1, and the sequence of ITS-B3-1 is shown in SEQ ID NO: 2; Internal primers: The sequence of ITS-FIP-1 is shown in SEQ ID NO:3, and the sequence of ITS-BIP-1 is shown in SEQ ID NO:4; The sequence of loop primer: ITS-LF-1 is shown in SEQ ID NO:5, and the sequence of loop primer ITS-LB-1 is shown in SEQ ID NO:6; The sequences of the primer set ITS-2 are as follows: External primer: The sequence of ITS-F3-2 is shown in SEQ ID NO:7, and the sequence of ITS-B3-2 is shown in SEQ ID NO:8; Internal primer: The sequence of ITS-FIP-2 is shown in SEQ ID NO:9, and the sequence of ITS-BIP-2 is shown in SEQ ID NO:10; Loop primer: The sequence of ITS-LF-2 is shown in SEQ ID NO: 11, and the sequence of ITS-LB-2 is shown in SEQ ID NO: 12; The sequences of the primer set ITS-3 are as follows: External primers: the sequence of ITS-F3-3 is shown in SEQ ID NO: 13, and the sequence of ITS-B3-3 is shown in SEQ ID NO: 14; Internal primers: The sequence of ITS-FIP-3 is shown in SEQ ID NO: 15, and the sequence of ITS-BIP-3 is shown in SEQ ID NO: 16; Loop primer: The sequence of ITS-LF-3 is shown in SEQ ID NO: 17, and the sequence of ITS-LB-3 is shown in SEQ ID NO: 18; The LAMP primer set 2 includes at least one of primer set HXT9-1, primer set HXT9-2, and primer set HXT9-3: The sequences of the primer set HXT9-1 are as follows: External primers: The sequence of HXT9-F3-1 is shown in SEQ ID NO: 19, and the sequence of HXT9-B3-1 is shown in SEQ ID NO: 20; Internal primers: The sequence of HXT9-FIP-1 is shown in SEQ ID NO: 21, and the sequence of HXT9-BIP-1 is shown in SEQ ID NO: 22; Loop primer: The sequence of HXT9-LF-1 is shown in SEQ ID NO: 23, and the sequence of HXT9-LB-1 is shown in SEQ ID NO: 24; The sequences of the primer set HXT9-2 are as follows: External primers: The sequence of HXT9-F3-2 is shown in SEQ ID NO: 25, and the sequence of HXT9-B3-2 is shown in SEQ ID NO: 26; Internal primers: The sequence of HXT9-FIP-2 is shown in SEQ ID NO: 27, and the sequence of HXT9-BIP-2 is shown in SEQ ID NO: 28; Loop primer: The sequence of HXT9-LF-2 is shown in SEQ ID NO: 29, and the sequence of HXT9-LB-2 is shown in SEQ ID NO: 30; The sequences of the primer set HXT9-3 are as follows: External primer: The sequence of HXT9-F3-3 is shown in SEQ ID NO:31, and the sequence of HXT9-B3-3 is shown in SEQ ID NO:32; Internal primer: The sequence of HXT9-FIP-3 is shown in SEQ ID NO:33, and the sequence of HXT9-BIP-3 is shown in SEQ ID NO:34; Loop primer: The sequence of HXT9-LF-3 is shown in SEQ ID NO:35, and the sequence of HXT9-LB-3 is shown in SEQ ID NO:

36.

2. The reagent according to claim 1, characterized in that The yeast includes Saccharomyces boulardii and / or Saccharomyces cerevisiae.

3. Use of the reagent according to claim 1 or 2 in preparing a kit.

4. A kit for detecting yeast, comprising the reagent according to claim 1 or 2.

5. The kit according to claim 4, characterized in that The kit also includes at least one of DNA polymerase, amplification reaction solution, betaine, magnesium sulfate, dNTPs, HNB dye, positive quality control product and blank control product.

6. Use of the reagent according to claim 1 or 2 and / or the kit according to claim 4 or 5 in any one of (1) to (4): (1) Identification of Saccharomyces boulardii and / or Saccharomyces cerevisiae; (2) preparing products for identifying Saccharomyces boulardii and / or Saccharomyces cerevisiae; (3) Detecting whether the sample contains Saccharomyces boulardii and / or Saccharomyces cerevisiae; (4) preparing a product for detecting whether a sample to be tested contains Saccharomyces boulardii and / or Saccharomyces cerevisiae; The above applications are used for non-disease diagnosis and treatment.

7. A detection method for: 1) identifying Saccharomyces boulardii and / or Saccharomyces cerevisiae; or 2) Detecting whether a sample to be tested contains Saccharomyces boulardii and / or Saccharomyces cerevisiae, wherein the detection method is used for non-diagnostic purposes; the detection method comprises the steps of using the reagent according to claim 1 or 2 and / or the kit according to claim 4 or 5.

8. The detection method according to claim 7, characterized in that: The detection method specifically comprises the following steps: S1. Extracting nucleic acid from the sample to be tested; S2. Using the nucleic acid of step S1 as a template, performing a LAMP amplification reaction using the reagent or kit; S3. Determine whether the sample to be tested contains Saccharomyces boulardii and / or Saccharomyces cerevisiae according to the reaction result of step S2.

9. The detection method according to claim 8, characterized in that: In step S2, the kit described in claim 5 is used to perform LAMP amplification reaction; the final concentration of the DNA polymerase is 0.05-2U / μL; the final concentration of the magnesium sulfate is 5-15mM; and the conditions of the LAMP amplification reaction are 60-70°C for 30-60min.

10. The detection method according to claim 9, characterized in that: The molar ratio of the outer primer, the inner primer and the loop primer in the LAMP amplification reaction system is 1:(2-4):(1-2).

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