A kit for detecting the transcription level of a gene related to exopolysaccharide synthesis in lactobacillus salivarius

Abstract

The present application relates to the technical field of microorganism detection, and particularly relates to a kit for detecting the transcription level of an extracellular polysaccharide synthesis related gene in lactobacillus salivarius. The kit comprises the following components: RPA amplification reagents, CRISPR-Cas13a detection reagents and reaction buffer; the RPA amplification reagents are RPA amplification reagents containing a target gene 1504 primer pair, a target gene 1506 primer pair, a target gene 1511 primer pair, a target gene 1512 primer pair, a target gene 1516 primer pair and a target gene 1517 primer pair; the CRISPR-Cas13a detection reagents are CRISPR-Cas13a detection reagents containing specific crRNA and single-stranded DNA reporter molecules corresponding to the extracellular polysaccharide synthesis related gene. The present application solves the technical problem that DNA level detection cannot reflect the real expression state of a gene, and provides an effective tool for the activity screening of lactobacillus salivarius with high extracellular polysaccharide yield and the monitoring of a fermentation process.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, and in particular to a kit for detecting the transcriptional level of extracellular polysaccharide synthesis-related genes in Lactobacillus salivarius. Background Technology

[0002] Lactobacillus salivarius is a probiotic widely found in the oral cavity and intestines of humans and animals, possessing various beneficial functions such as immune regulation, pathogen inhibition, and maintenance of intestinal health. Exopolysaccharides (EPS) produced by Lactobacillus salivarius are one of the important active substances responsible for its probiotic functions. Studies have shown that EPS possesses various biological activities, including antioxidant, anti-inflammatory, anti-tumor, and gut microbiota regulation, and has broad application prospects in functional foods and pharmaceuticals.

[0003] The synthesis of extracellular polysaccharides (EPS) in *Lactobacillus salivarius* is encoded by a gene cluster on the chromosome (the *eps* gene cluster), which contains multiple functional genes that jointly regulate the biosynthesis of EPS. The target gene cluster of this invention includes six key genes, whose numbers and functional annotations are as follows: Gene 1504 (flippase): encodes a flippase responsible for transporting intracellularly synthesized EPS precursors to the extracellular space, a crucial step in EPS export. Gene 1506 (glycosyltransferase): encodes a glycosyltransferase responsible for catalyzing the polymerization of glycosyl units to form polysaccharide chains, the core enzyme in EPS synthesis. Gene 1511 (chain-length-determining protein): encodes a chain-length-determining protein, controlling the length and molecular weight distribution of the EPS polysaccharide chain. Gene 1512 (EpsG family protein): encodes an EpsG family protein, involved in the assembly and transport of glycosyl units during EPS synthesis. Gene 1516 (acyltransferase): encodes an acyltransferase responsible for adding acyl modifications to the EPS polysaccharide chain, affecting the physicochemical properties and biological activity of EPS. Gene 1517 (NAD-dependent epimerase / dehydratase family protein): encodes a NAD-dependent epimerase / dehydratase family protein that participates in the isomerization and dehydration reactions of monosaccharide precursors in EPS synthesis, affecting the monosaccharide composition of EPS.

[0004] Unlike genotyping at the DNA level, EPS yield depends not only on the presence of genes but also on their transcriptional expression levels. Therefore, establishing a method to accurately detect the transcriptional level of EPS gene clusters is of great significance for screening high-yielding EPS strains and optimizing fermentation processes.

[0005] Currently, the main method for detecting gene transcription levels is real-time quantitative PCR (RT-qPCR). Although this method can achieve accurate quantification, it still has the following shortcomings: (1) Existing RT-qPCR methods for detecting eps gene transcription levels require thermal cycling instruments and are complex to operate; (2) DNA level detection cannot reflect the true expression status of genes; (3) There is a lack of high-throughput detection methods that can rapidly correlate eps gene cluster transcription levels with EPS yield.

[0006] Therefore, it is crucial to provide a technical solution that can solve the above-mentioned technical problems. Summary of the Invention

[0007] To address the aforementioned problems, the present invention aims to provide a kit for detecting the transcriptional levels of genes related to extracellular polysaccharide synthesis in *Lactobacillus salivarius*. Unlike CRISPR-Cas13a, which detects genomic DNA, this invention employs a CRISPR-Cas13a system targeting RNA, directly detecting the mRNA transcriptional levels of six key genes in the extracellular polysaccharide synthesis gene cluster, thus accurately reflecting the metabolic activity of the EPS synthesis pathway. This invention involves extracting total RNA, reverse transcription to obtain cDNA, and using specific RPA primers with a T7 promoter sequence added to the 5' end of the upstream primer for six-tube parallel amplification. After in vitro transcription by T7 RNA polymerase, the amplified product is specifically recognized and cleaved by the CRISPR-Cas13a system (Cas13a specifically recognizes and binds to single-stranded RNA, and can non-specifically cleave single-stranded RNA after initiation) to release a fluorescent signal. The fluorescence signal intensity is positively correlated with the initial copy number of the target mRNA. This invention solves the technical problem that DNA-level detection cannot reflect the true gene expression state, providing an effective tool for activity screening and fermentation process monitoring of high-yielding extracellular polysaccharide *Lactobacillus salivarius*.

[0008] The objective of this invention can be achieved through the following technical solutions: The first objective of this invention is to provide a kit for detecting the transcriptional level of extracellular polysaccharide synthesis-related genes in *Lactobacillus salivarius*, comprising the following components: RPA amplification reagents, CRISPR-Cas13a detection reagents and reaction buffer; The RPA amplification reagent is an RPA amplification reagent containing primer pairs for target gene 1504, 1506, 1511, 1512, 1516, and 1517. The CRISPR-Cas13a detection reagent is a CRISPR-Cas13a detection reagent containing specific crRNA and single-stranded DNA reporter molecules of the corresponding extracellular polysaccharide synthesis-related genes.

[0009] In one embodiment of the present invention, the primer pair for the target gene 1504 is RPA-F-1504 as shown in SEQ ID NO.2 and RPA-R-1504 as shown in SEQ ID NO.3; The primer pairs for the target gene 1506 are RPA-F-1506 as shown in SEQ ID NO.5 and RPA-R-1506 as shown in SEQ ID NO.6; The primer pairs for the target gene 1511 are RPA-F-1511 as shown in SEQ ID NO.8 and RPA-R-1511 as shown in SEQ ID NO.9; The primer pairs for the target gene 1512 are RPA-F-1512 as shown in SEQ ID NO.11 and RPA-R-1512 as shown in SEQ ID NO.12; The target gene 1516 primer pair is RPA-F-1516 as shown in SEQ ID NO.14 and RPA-R-1516 as shown in SEQ ID NO.15; The primer pairs for the target gene 1517 are RPA-F-1517 as shown in SEQ ID NO.17 and RPA-R-1517 as shown in SEQ ID NO.18.

[0010] In one embodiment of the present invention, the specific crRNA corresponding to the target gene 1504 is crRNA-1504 as shown in SEQ ID NO.1; The specific crRNA corresponding to the target gene 1506 is crRNA-1506 as shown in SEQ ID NO.4; The specific crRNA corresponding to the target gene 1511 is crRNA-1511 as shown in SEQ ID NO.7; The specific crRNA corresponding to the target gene 1512 is crRNA-1512 as shown in SEQ ID NO.10; The specific crRNA corresponding to the target gene 1516 is crRNA-1516 as shown in SEQ ID NO.13; The specific crRNA corresponding to the target gene 1517 is crRNA-1517 as shown in SEQ ID NO.16.

[0011] In one embodiment of the present invention, the single-stranded DNA reporter molecule is an ssDNA reporter molecule as shown in SEQ ID NO.19; Its 5' end has the FAM mark, and its 3' end has the BHQ1 mark.

[0012] In one embodiment of the present invention, the kit includes a positive control and a negative control; The positive control is a mixture containing target sequences of target genes 1504, 1506, 1511, 1512, 1516 and 1517, and a double-stranded DNA promoter probe for verifying Cas13a activity. The negative control is sterile water or PBS buffer that does not contain genes related to extracellular polysaccharide synthesis.

[0013] A second objective of this invention is to provide a method for detecting the transcriptional level of extracellular polysaccharide synthesis-related genes in *Lactobacillus salivarius*. Using the above-mentioned kit, the detection method for any target gene includes the following steps: (S1) RNA standards of target genes with different copy concentrations were reverse transcribed to obtain the corresponding cDNA; The cDNA obtained above was placed in reaction tubes containing RPA reaction systems and amplified at isothermal temperature to obtain corresponding amplification products, which were used as experimental groups with different concentrations. RNase-free water was used as a negative control group; (S2) Mix the amplification products obtained in step (S1) with the corresponding CRISPR-Cas13a detection system, and detect the fluorescence signal value of each group of products after the reaction. A standard curve was plotted using the fluorescence signal values ​​of the experimental group minus those of the negative control group as the fluorescence intensity. (S3) The total RNA extracted from the Lactobacillus salivarius to be tested was reverse transcribed to obtain the cDNA of the Lactobacillus salivarius to be tested; The cDNA of Lactobacillus salivarius to be tested was placed in six independent reaction tubes, each containing an RPA reaction system, and amplified at isothermal temperature to obtain the corresponding amplification products, which were used as the test groups. The amplification product was mixed with the corresponding CRISPR-Cas13a detection system, and the fluorescence signal value of each group of products was detected after the reaction. The fluorescence intensity was calculated by subtracting the fluorescence signal value of the negative control group from the fluorescence signal value of the test group. Substitute the fluorescence signal value into the standard curve obtained in step (S2) to obtain the copy concentration of the target gene in the Lactobacillus salivarius to be tested, and complete the detection of the transcriptional level of extracellular polysaccharide synthesis-related genes in Lactobacillus salivarius.

[0014] In one embodiment of the present invention, during steps (S1) and (S3), the temperature during the isothermal amplification process is 37~39℃ and the time is 20~30min.

[0015] In one embodiment of the present invention, during steps (S2) and (S3), the temperature is 37~39°C and the time is 30~60 min.

[0016] In step (S2), a standard curve is plotted with the copy concentration of the target gene as the x-axis and the fluorescence intensity as the y-axis.

[0017] A third objective of this invention is to provide a kit for the application of high-extra-polysaccharide-producing Lactobacillus salivarius in activity screening and / or fermentation process monitoring and / or evaluation of Lactobacillus salivarius's extra-polysaccharide production capacity.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) Direct RNA detection: Unlike Cas13a, which detects DNA, Cas13a directly targets RNA, which can reflect the true transcriptional activity of genes and avoid the defect that DNA level detection cannot reflect the expression level.

[0019] (2) High sensitivity: Cas13a has a signal amplification effect, and the detection sensitivity can reach the aM level.

[0020] (3) Isothermal amplification: No thermal cycling instrument is required, making it suitable for rapid on-site detection.

[0021] (4) Quantitative detection: The fluorescence signal intensity is positively correlated with the mRNA copy number, and absolute quantification can be achieved through a standard curve.

[0022] (5) Multiple detection: It can detect the expression levels of six genes simultaneously.

[0023] (6) Time resolution: Samples can be taken at different time points to monitor the expression dynamics of EPS synthesis-related genes during fermentation in real time. Attached Figure Description

[0024] Figure 1 This is a schematic diagram illustrating the technical principle of the present invention. A is a schematic diagram of the total RNA extraction and reverse transcription steps; B is a schematic diagram of the six-tube parallel RPA amplification steps; C is a schematic diagram of the CRISPR-Cas13a detection steps; D is a schematic diagram of fluorescence signal detection and result interpretation.

[0025] Figure 2To validate the feasibility of using the CRISPR-Cas13a detection system to detect six eps genes. In vitro transcribed RNA standards (positive) and RNase-free water (negative) for target genes 1504, 1506, 1511, 1512, 1516, and 1517 were used as samples. Results are expressed as mean ± standard deviation (n=3). Detailed Implementation

[0026] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0027] In this invention, "target gene 1504" is an abbreviation for the gene encoding "flippase"; "target gene 1506" is an abbreviation for the gene encoding "glycosyltransferase"; "target gene 1511" is an abbreviation for the gene encoding "chain-length determining protein"; "target gene 1512" is an abbreviation for the gene encoding "EpsG family protein"; "target gene 1516" is an abbreviation for the gene encoding "acyltransferase"; and "target gene 1517" is an abbreviation for the gene encoding "NAD-dependent epimerase / dehydratase family protein". The sequences involved are shown in Table 1, and are detailed below: Table 1. Summary Table of Sequences The ssDNA reporter molecule has a FAM marker at the 5' end and a BHQ1 marker at the 3' end.

[0028] Lactobacillus salivarius strain (Lactobacillus salivarius AR809, accession number CGMCC 17206) was provided by the Food Microbiology Engineering Technology Research Center of Shanghai University of Science and Technology; the total RNA extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; reverse transcriptase (HiScript III) was purchased from Vazyme; LwCas13a protein was purchased from NEB; RNase inhibitors were purchased from TaKaRa; T7 RNA polymerase and NTPs were purchased from NEB. Unless otherwise specified, all reagents used were commercially available, and the detection methods used were standard in the field. Each experiment was conducted in triplicate.

[0029] Example 1 This embodiment provides the establishment and optimization of the detection system, as detailed below: (1) Total RNA extraction and reverse transcription Lactobacillus salivarius AR809 was inoculated into MRS liquid medium and cultured at 37°C until the logarithmic growth phase (OD200). 600 (The concentration is 0.6~0.8), to obtain bacterial solution; Take 1 mL of bacterial culture, centrifuge at 12000 rpm for 2 min to collect the bacterial cells; extract total RNA according to the instructions of the RNA extraction kit.

[0030] The extracted total RNA was treated with DNase I (37℃ for 30 min) to remove genomic DNA, and then DNase I was inactivated at 85℃ for 5 min. RNA concentration was determined using NanoDrop. 1 μg of total RNA was used for reverse transcription: 1 μL of reverse transcriptase, 1 μL of random primers, 1 μL of dNTPs, and 0.5 μL of RNase inhibitor were added, and RNase-free water was added to a final volume of 20 μL. The reaction was repeated (25℃ for 10 min, 42℃ for 45 min, and 85℃ for 5 min) to obtain the reverse transcription product. The reverse transcription product (cDNA) was stored at -20℃ for later use.

[0031] (2) RPA primer design RPA primer design principles: ① Length 30-35 nt; ② GC content 40-60%; ③ Amplified fragment length 150-250 bp; ④ Avoid primer dimers and hairpin structures. Key design: A T7 promoter sequence is added to the 5' end of each upstream primer pair for subsequent in vitro transcription steps.

[0032] The final RPA primers are as follows: ① Primer pair targeting gene 1504: RPA-F-1504 and RPA-R-1504; ② Primer pairs targeting gene 1506: RPA-F-1506 and RPA-R-1506; ③ Primer pairs targeting gene 1511: RPA-F-1511 and RPA-R-1511; ④ Primer pairs targeting gene 1512: RPA-F-1512 and RPA-R-1512; ⑤ Primer pairs targeting gene 1516: RPA-F-1516 and RPA-R-1516; ⑥ Primer pair targeting gene 1517: RPA-F-1517 and RPA-R-1517.

[0033] (4) Primer specificity verification Using the cDNA obtained by reverse transcription in step (2) as a template, RPA amplification was performed using the six primer pairs obtained in step (3).

[0034] The RPA amplification reaction system (50 μL) formulation is as follows: The RPA enzyme premix used in this embodiment was prepared using lyophilized microspheres. Each microsphere contained recombinant enzyme (final concentration 500 ng / μL), DNA polymerase (final concentration 100 ng / μL), single-stranded DNA binding protein (final concentration 200 ng / μL), accessory protein (final concentration 200 ng / μL), dNTPs (500 μM each), and an ATP regeneration system.

[0035] Target gene 1504: 1 μL of cDNA obtained in step (1) was mixed with 25 μL of RPA enzyme premix (a reagent from the recombinase polymerase amplification kit, purchased from Beijing Saibaisheng Gene Technology Co., Ltd.), 2.5 μL of RPA-F-1504 (10 μM), 2.5 μL of RPA-R-1504 (10 μM), and water was added to a final volume of 47.5 μL. The mixture was then thoroughly mixed to obtain the final solution. Add 2.5 μL of magnesium acetate solution (concentration of 280 mM, the same below) to the mixture to start the reaction (reaction conditions: constant temperature reaction at 39℃ for 25 minutes) to obtain the RPA product of target gene 1504; 5 μL of the RPA product of the target gene 1504 was subjected to 2% agarose gel electrophoresis.

[0036] Target gene 1506: 1 μL of cDNA obtained in step (1) was mixed with 25 μL of RPA enzyme premix, 2.5 μL of RPA-F-1506 (10 μM), 2.5 μL of RPA-R-1506 (10 μM), and water was added to make up to 47.5 μL. The mixture was then mixed to obtain the final solution. Add 2.5 μL of magnesium acetate solution (concentration of 280 mM, the same below) to the mixture to start the reaction (reaction conditions: constant temperature reaction at 39℃ for 25 minutes) to obtain the RPA product of target gene 1506; 5 μL of the RPA product of the target gene 1506 was subjected to 2% agarose gel electrophoresis.

[0037] Target gene 1511: 1 μL of cDNA obtained in step (1) was mixed with 25 μL of RPA enzyme premix, 2.5 μL of RPA-F-1511 (10 μM), 2.5 μL of RPA-R-1511 (10 μM), and water was added to make up to 47.5 μL. The mixture was then mixed to obtain the final solution. Add 2.5 μL of magnesium acetate solution (concentration of 280 mM, the same below) to the mixture to start the reaction (reaction conditions: constant temperature reaction at 39℃ for 25 minutes) to obtain the RPA product of target gene 1511; 5 μL of the RPA product of the target gene 1511 was subjected to 2% agarose gel electrophoresis.

[0038] Target gene 1512: 1 μL of cDNA obtained in step (1) was mixed with 25 μL of RPA enzyme premix, 2.5 μL of RPA-F-1512 (10 μM), 2.5 μL of RPA-R-1512 (10 μM), and water was added to make up to 47.5 μL. The mixture was then mixed to obtain the final solution. Add 2.5 μL of magnesium acetate solution (concentration of 280 mM, the same below) to the mixture to start the reaction (reaction conditions: constant temperature reaction at 39℃ for 25 minutes) to obtain the RPA product of target gene 1512; 5 μL of the RPA product of the target gene 1512 was subjected to 2% agarose gel electrophoresis.

[0039] Target gene 1516: 1 μL of cDNA obtained in step (1) was mixed with 25 μL of RPA enzyme premix, 2.5 μL of RPA-F-1516 (10 μM), 2.5 μL of RPA-R-1516 (10 μM), and water was added to make up to 47.5 μL. The mixture was then mixed to obtain the final solution. Add 2.5 μL of magnesium acetate solution (concentration of 280 mM, the same below) to the mixture to start the reaction (reaction conditions: constant temperature reaction at 39℃ for 25 minutes) to obtain the RPA product of target gene 1516; 5 μL of the RPA product of the target gene 1516 was subjected to 2% agarose gel electrophoresis.

[0040] Target gene 1517: 1 μL of cDNA obtained in step (1) was mixed with 25 μL of RPA enzyme premix, 2.5 μL of RPA-F-1517 (10 μM), 2.5 μL of RPA-R-1517 (10 μM), and water was added to make up to 47.5 μL. The mixture was then mixed to obtain the final solution. Add 2.5 μL of magnesium acetate solution (concentration of 280 mM, the same below) to the mixture to start the reaction (reaction conditions: constant temperature reaction at 39℃ for 25 minutes) to obtain the RPA product of target gene 1517; 5 μL of the RPA product of the target gene 1517 was subjected to 2% agarose gel electrophoresis.

[0041] The results showed that all six primer sets amplified a single bright band (approximately 150–250 bp in size), with no nonspecific amplification, indicating that the six primer sets had good specificity.

[0042] Example 2 This embodiment provides the design of crRNA, as detailed below: (1) crRNA design Cas13a crRNA consists of a conserved backbone sequence at the 5' end and a target recognition sequence at the 3' end. The backbone sequence used in this example is: 5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAA C-3' (approximately 28 nt). The target recognition sequence (approximately 28 nt) is designed based on the specific regions of the six eps genes, following the principles of: (1) targeting specific regions of mRNA; (2) having no homology of more than 15 nt with other eps genes; and (3) having a GC content of 40-60%.

[0043] The final crRNA obtained is as follows: ①crRNA targeting gene 1504: crRNA-1504; ②crRNA targeting gene 1506: crRNA-1506; ③crRNA targeting gene 1511: crRNA-1511; ④crRNA targeting gene 1512: crRNA-1512; ⑤crRNA targeting gene 1516: crRNA-1516; ⑥CrRNA targeting gene 1517: crRNA-1517.

[0044] Example 3 This embodiment provides a kit for detecting the transcriptional level of extracellular polysaccharide synthesis-related genes in Lactobacillus salivarius, as detailed below: ①RNA extraction reagent: The RNA extraction reagent is the reagent included in the total RNA extraction kit; the total RNA extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0045] ② Reverse transcription reagent: The reverse transcription reagent is the reagent included in the reverse transcription kit; the reverse transcription kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0046] ③RPA amplification reagents (six pre-packaged kits): 25 μL RPA enzyme premix, 2.5 μL 10 μM upstream primer, 2.5 μL 10 μM downstream primer, 16.5 μL water; 2.5 μL 280 nM magnesium acetate solution (after mixing the remaining components, add the magnesium acetate solution last to start the reaction). When using, add 1 μL of cDNA template; CRISPR-Cas13a assay kits (six pre-packaged kits) are shown in Table 2: Table 2 Summary of CRISPR-Cas13a detection systems per tube 5× Reaction Buffer: A mixed solution of Tris-HCl (pH 7.9) 200 mM, NaCl 50 mM, MgCl2 30 mM, and DTT 10 mM (solvent is water), diluted 5 times before use; Add 2μL of template when using.

[0047] ④ Positive control: A mixture of in vitro transcribed RNA standards for six target genes (10 7 The copy number of the six target genes was the same (copies / μL); this positive control was then serially diluted 10-fold and used for subsequent experiments (10 copies / μL). 7 ~10 2 (6 concentration gradients in copies / μL) The in vitro transcribed RNA standard was prepared by the following method: DNA fragments containing the target sequences of genes 1504, 1506, 1511, 1512, 1516, and 1517 were synthesized and cloned into the EcoRI and HindIII restriction sites of a vector (pUC57) containing the T7 promoter. After sequencing verification, in vitro transcription was performed using T7 RNA polymerase. The transcription product was digested with DNase I to remove the DNA template, purified, and quantified to obtain the standard.

[0048] ⑤ Negative control: RNase-free water.

[0049] Example 4 This embodiment provides a method for detecting the transcriptional level of extracellular polysaccharide synthesis-related genes in Lactobacillus salivarius (the principle is as follows). Figure 1 (As shown), using the kit described in Example 3, the following steps are included: (S1) 1 μg of solution with a concentration of 10 7 10 6 10 5 10 4 10 3 10 2 In vitro transcribed RNA standards of target gene 1504 (copies / μL) were reverse transcribed using a reverse transcription reagent (25℃ 10 min, 42℃ 45 min, 85℃ 5 min) to obtain cDNA at different concentration gradients targeting gene 1504 (as positive control groups). 1 μL of cDNA at different concentration gradients targeting gene 1504 and 1 μL of LNase-free water (as a negative control) prepared above were respectively mixed with 25 μL of RPA enzyme premix, 2.5 μL of RPA-F-1504, and 2.5 μL of LPA-R-1504, and water was added to a final volume of 47.5 μL to obtain a mixture. 2.5 μL of magnesium acetate solution was added to the mixture to start the reaction (reaction conditions: constant temperature reaction at 39℃ for 25 minutes), resulting in RPA products at different concentration gradients targeting gene 1504 and RPA products of the negative control group. (S2) The RPA products of different concentration gradients targeting gene 1504 obtained in step (S1) and the RPA products of the negative control group (2 μL each) were mixed with 0.32 μL LwCas13a protein, 0.72 μL crRNA-1504, 4 μL T7 RNA polymerase, 0.4 μL NTPs, 0.4 μL ssRNA reporter molecule, 0.25 μL RNase inhibitor, 4 μL 5× reaction buffer, and water (to a final volume of 20 μL) to obtain the reaction solution; the reaction was carried out at 39℃ for 30 min to obtain the reaction product. The fluorescence signal values ​​of each group of reaction products were detected using a fluorescence detector at an excitation wavelength of 485 nm and an emission wavelength of 520 nm.

[0050] A standard curve was plotted using the fluorescence signal values ​​of different concentration gradients of the target gene 1504 minus the fluorescence signal value of the negative control group as fluorescence intensity (ΔF, as the ordinate) and the logarithm of the copy concentration (C) of the target gene 1504 (lgC) as the abscissa. The standard curve plotting method for other target genes is the same as steps (S1) and (S2) above, except that the RPA primers and crRNA for target gene 1504 are replaced with RPA primers and crRNA for other corresponding target genes.

[0051] Positive control group (10 6 The fluorescence intensity of the negative control group (copies / μL) was compared with that of the negative control group. Figure 2 As shown.

[0052] The standard curves for the six target genes are shown in Table 3: Table 3 Summary of Standard Curves for Each Target Gene The above results show that ΔF and log (RNA copy number) of all six genes have a good linear relationship, indicating that the kit obtained in Example 3 can be used for relative quantitative analysis of eps gene transcription level.

[0053] (S3) Take 1 mL of the bacterial culture of Lactobacillus salivarius AR809 (preservation number CGMCC 17206) to be tested, centrifuge at 12000 rpm for 2 min to collect the bacterial cells; Total RNA from Lactobacillus salivarius strain AR809 was extracted using an RNA extraction reagent. 1 μg of total RNA was reverse transcribed using a reverse transcription reagent (25℃ for 10 min, 42℃ for 45 min, 85℃ for 5 min) to obtain cDNA. ① Mix 1 μL of cDNA with 25 μL of RPA enzyme premix, 2.5 μL of RPA-F-1504, and 2.5 μL of RPA-R-1504, and add water to a final volume of 47.5 μL to obtain a mixture. Add 2.5 μL of magnesium acetate solution to the mixture to start the reaction (reaction conditions: constant temperature reaction at 39℃ for 25 minutes) to obtain RPA products targeting gene 1504. 2 μL of RPA product targeting gene 1504 was mixed with 0.32 μL LwCas13a protein, 0.72 μL crRNA-1504, 4 μL T7 RNA polymerase, 0.4 μL NTPs, 0.4 μL ssRNA reporter molecule, 0.25 μL RNase inhibitor, 4 μL 5× reaction buffer, and water (to a final volume of 20 μL) to obtain the reaction solution. The mixture was incubated at 39°C for 30 min to obtain the reaction product targeting gene 1504. ② Mix 1 μL of cDNA with 25 μL of RPA enzyme premix, 2.5 μL of RPA-F-1506, and 2.5 μL of RPA-R-1506, and add water to a final volume of 47.5 μL to obtain a mixture. Add 2.5 μL of magnesium acetate solution to the mixture to start the reaction (reaction conditions: constant temperature reaction at 39℃ for 25 minutes) to obtain RPA products targeting gene 1506. 2 μL of RPA product targeting gene 1506 was mixed with 0.32 μL LwCas13a protein, 0.72 μL crRNA-1506, 4 μL T7 RNA polymerase, 0.4 μL NTPs, 0.4 μL ssRNA reporter molecule, 0.25 μL RNase inhibitor, 4 μL 5× reaction buffer, and water (to a final volume of 20 μL) to obtain the reaction solution. The mixture was incubated at 39°C for 30 min to obtain the reaction product targeting gene 1506. ③ Mix 1 μL of cDNA with 25 μL of RPA enzyme premix, 2.5 μL of RPA-F-1511, and 2.5 μL of RPA-R-1511, and add water to a final volume of 47.5 μL to obtain a mixture. Add 2.5 μL of magnesium acetate solution to the mixture to start the reaction (reaction conditions: constant temperature reaction at 39℃ for 25 minutes) to obtain RPA products targeting gene 1511. 2 μL of RPA product targeting gene 1511 was mixed with 0.32 μL LwCas13a protein, 0.72 μL crRNA-1511, 4 μL T7 RNA polymerase, 0.4 μL NTPs, 0.4 μL ssRNA reporter molecule, 0.25 μL RNase inhibitor, 4 μL 5× reaction buffer, and water (to a final volume of 20 μL) to obtain the reaction solution. The mixture was incubated at 39°C for 30 min to obtain the reaction product targeting gene 1511. ④ Mix 1 μL of cDNA with 25 μL of RPA enzyme premix, 2.5 μL of RPA-F-1512, and 2.5 μL of RPA-R-1512, and add water to a final volume of 47.5 μL to obtain a mixture. Add 2.5 μL of magnesium acetate solution to the mixture to start the reaction (reaction conditions: constant temperature reaction at 39℃ for 25 minutes) to obtain RPA products targeting gene 1512. 2 μL of RPA product targeting gene 1512 was mixed with 0.32 μL LwCas13a protein, 0.72 μL crRNA-1512, 4 μL T7 RNA polymerase, 0.4 μL NTPs, 0.4 μL ssRNA reporter molecule, 0.25 μL RNase inhibitor, 4 μL 5× reaction buffer, and water (to a final volume of 20 μL) to obtain the reaction solution. The mixture was incubated at 39°C for 30 min to obtain the reaction product targeting gene 1512. ⑤ Mix 1 μL of cDNA with 25 μL of RPA enzyme premix, 2.5 μL of RPA-F-1516, and 2.5 μL of RPA-R-1516, and add water to a final volume of 47.5 μL to obtain a mixture. Add 2.5 μL of magnesium acetate solution to the mixture to start the reaction (reaction conditions: constant temperature reaction at 39℃ for 25 minutes) to obtain RPA products targeting gene 1516. 2 μL of RPA product targeting gene 1516 was mixed with 0.32 μL LwCas13a protein, 0.72 μL crRNA-1516, 4 μL T7 RNA polymerase, 0.4 μL NTPs, 0.4 μL ssRNA reporter molecule, 0.25 μL RNase inhibitor, 4 μL 5× reaction buffer, and water (to a final volume of 20 μL) to obtain the reaction solution. The mixture was incubated at 39°C for 30 min to obtain the reaction product targeting gene 1516. ⑥ Mix 1 μL of cDNA with 25 μL of RPA enzyme premix, 2.5 μL of RPA-F-1517, and 2.5 μL of RPA-R-1517, and add water to a final volume of 47.5 μL to obtain a mixture. Add 2.5 μL of magnesium acetate solution to the mixture to start the reaction (reaction conditions: constant temperature reaction at 39℃ for 25 minutes) to obtain RPA products targeting gene 1517. 2 μL of RPA product targeting gene 1517 was mixed with 0.32 μL LwCas13a protein, 0.72 μL crRNA-1517, 4 μL T7 RNA polymerase, 0.4 μL NTPs, 0.4 μL ssRNA reporter molecule, 0.25 μL RNase inhibitor, 4 μL 5× reaction buffer, and water (to a final volume of 20 μL) to obtain the reaction solution. The mixture was incubated at 39°C for 30 min to obtain the reaction product targeting gene 1517. The fluorescence signal values ​​of each group of reaction products were detected using a fluorescence detector at an excitation wavelength of 485 nm and an emission wavelength of 520 nm.

[0054] The fluorescence intensity was calculated by subtracting the fluorescence signal value of the negative control group from the fluorescence signal value of the Lactobacillus salivarius strain AR809 in each tube. The fluorescence intensity obtained from each group of reaction products was substituted into the corresponding standard curve obtained in step (S2) to obtain the transcription level (copies / μL RNA) of extracellular polysaccharide synthesis-related genes in Lactobacillus salivarius strain AR809. The results are shown in Table 4.

[0055] Table 4. Results of eps gene transcription level detection in Lactobacillus salivarius isolates Example 5 This embodiment provides the specificity verification and sensitivity of the kit described in Example 3, using the method described in Example 4.

[0056] The results showed that each crRNA produced a significant fluorescent signal only for its corresponding target gene, and the fluorescent signals for the other five non-target genes were not significantly different from those of the negative control, indicating that the six crRNAs have extremely high specificity.

[0057] In summary, the CRISPR-Cas13a detection method and kit provided by this invention have the advantages of simple operation, rapid and efficient operation, high sensitivity, and strong specificity, making them suitable for high-throughput detection of the transcriptional level of the *Lactobacillus salivarius* eps gene cluster. Unlike CRISPR-Cas13a, which targets DNA, this invention directly targets RNA, reflecting the true expression activity of the gene and solving the false positive problem of gene presence but non-expression. This invention establishes for the first time a quantitative correlation model between eps gene transcriptional level and EPS yield, which can be used to rapidly assess the EPS production potential of *Lactobacillus salivarius*, providing an effective tool for high-throughput screening of high-EPS-producing strains and optimization of fermentation processes. This invention has broad application prospects in the fields of functional foods and feed additives.

[0058] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the interpretation of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A kit for detecting the transcriptional level of extracellular polysaccharide synthesis-related genes in *Lactobacillus salivarius*, characterized in that, Includes the following components: RPA amplification reagents, CRISPR-Cas13a detection reagents and reaction buffer; The RPA amplification reagent is an RPA amplification reagent containing primer pairs for target gene 1504, 1506, 1511, 1512, 1516, and 1517. The CRISPR-Cas13a detection reagent is a CRISPR-Cas13a detection reagent containing specific crRNA and single-stranded DNA reporter molecules of the corresponding extracellular polysaccharide synthesis-related genes.

2. The kit for detecting the transcriptional level of extracellular polysaccharide synthesis-related genes in *Lactobacillus salivarius* according to claim 1, characterized in that, The primer pairs for the target gene 1504 are RPA-F-1504 as shown in SEQ ID NO.2 and RPA-R-1504 as shown in SEQ ID NO.3; The primer pairs for the target gene 1506 are RPA-F-1506 as shown in SEQ ID NO.5 and RPA-R-1506 as shown in SEQ ID NO.6; The primer pairs for the target gene 1511 are RPA-F-1511 as shown in SEQ ID NO.8 and RPA-R-1511 as shown in SEQ ID NO.9; The primer pairs for the target gene 1512 are RPA-F-1512 as shown in SEQ ID NO.11 and RPA-R-1512 as shown in SEQ ID NO.12; The target gene 1516 primer pair is RPA-F-1516 as shown in SEQ ID NO.14 and RPA-R-1516 as shown in SEQ ID NO.15; The primer pairs for the target gene 1517 are RPA-F-1517 as shown in SEQ ID NO.17 and RPA-R-1517 as shown in SEQ ID NO.

18.

3. The kit for detecting the transcriptional level of extracellular polysaccharide synthesis-related genes in *Lactobacillus salivarius* according to claim 1, characterized in that, The specific crRNA corresponding to the target gene 1504 is crRNA-1504 as shown in SEQ ID NO.1; The specific crRNA corresponding to the target gene 1506 is crRNA-1506 as shown in SEQ ID NO.4; The specific crRNA corresponding to the target gene 1511 is crRNA-1511 as shown in SEQ ID NO.7; The specific crRNA corresponding to the target gene 1512 is crRNA-1512 as shown in SEQ ID NO.10; The specific crRNA corresponding to the target gene 1516 is crRNA-1516 as shown in SEQ ID NO.13; The specific crRNA corresponding to the target gene 1517 is crRNA-1517 as shown in SEQ ID NO.

16.

4. The kit for detecting the transcriptional level of extracellular polysaccharide synthesis-related genes in *Lactobacillus salivarius* according to claim 1, characterized in that, The single-stranded DNA reporter molecule is the ssDNA reporter molecule shown in SEQ ID NO.19; Its 5' end has the FAM mark, and its 3' end has the BHQ1 mark.

5. A kit for detecting the transcriptional level of extracellular polysaccharide synthesis-related genes in *Lactobacillus salivarius* according to claim 1, characterized in that, The kit includes a positive control and a negative control; The positive control is a mixture containing target sequences of target genes 1504, 1506, 1511, 1512, 1516 and 1517, and a double-stranded DNA promoter probe for verifying Cas13a activity. The negative control is sterile water or PBS buffer that does not contain genes related to extracellular polysaccharide synthesis.

6. A method for detecting the transcriptional level of extracellular polysaccharide synthesis-related genes in *Lactobacillus salivarius*, characterized in that, Using the kit described in any one of claims 1 to 5, the method for detecting any target gene includes the following steps: (S1) RNA standards of the target gene with different copy concentrations were reverse transcribed to obtain the corresponding cDNA; The cDNA obtained above was placed in reaction tubes containing RPA reaction systems and amplified at isothermal temperature to obtain corresponding amplification products, which were used as experimental groups with different concentrations. RNase-free water was used as a negative control group; (S2) Mix the amplification products obtained in step (S1) with the corresponding CRISPR-Cas13a detection system, and detect the fluorescence signal value of each group of products after the reaction. A standard curve was plotted using the fluorescence signal values ​​of the experimental group minus those of the negative control group as the fluorescence intensity. (S3) The total RNA extracted from the Lactobacillus salivarius to be tested was reverse transcribed to obtain the cDNA of the Lactobacillus salivarius to be tested; The cDNA of Lactobacillus salivarius to be tested was placed in six independent reaction tubes, each containing an RPA reaction system, and amplified at isothermal temperature to obtain the corresponding amplification products, which were used as the test groups. The amplification product was mixed with the corresponding CRISPR-Cas13a detection system, and the fluorescence signal value of each group of products was detected after the reaction. The fluorescence intensity was calculated by subtracting the fluorescence signal value of the negative control group from the fluorescence signal value of the test group. Substitute the fluorescence signal value into the standard curve obtained in step (S2) to obtain the copy concentration of the target gene in the Lactobacillus salivarius to be tested, and complete the detection of the transcriptional level of extracellular polysaccharide synthesis-related genes in Lactobacillus salivarius.

7. The method for detecting the transcriptional level of extracellular polysaccharide synthesis-related genes in *Lactobacillus salivarius* according to claim 6, characterized in that, In steps (S1) and (S3), the temperature during isothermal amplification is 37~39℃ and the time is 20~30min.

8. The method for detecting the transcriptional level of extracellular polysaccharide synthesis-related genes in *Lactobacillus salivarius* according to claim 6, characterized in that, In steps (S2) and (S3), the temperature is 37~39℃ and the time is 30~60min during the reaction.

9. The method for detecting the transcriptional level of extracellular polysaccharide synthesis-related genes in *Lactobacillus salivarius* according to claim 6, characterized in that, In step (S2), a standard curve is plotted with the copy concentration of the target gene as the x-axis and the fluorescence intensity as the y-axis.

10. The application of a kit as described in any one of claims 1 to 5 in the activity screening and / or fermentation process monitoring of high-yielding extracellular polysaccharide-producing Lactobacillus salivarius.

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