Primer pair and kit for detecting quantity of lactobacillus brevis thalli in metagen preparation and application of primer pair and kit

Abstract

The invention relates to the technical field of thallus quantity detection, in particular to a primer pair and a kit for detecting the number of lactobacillus brevis thalli in a postbiotic preparation and application of the primer pair and the kit. The primer pair provided by the invention has the advantages of strong specificity and high amplification efficiency, and can be used for carrying out qPCR quantitative detection on the quantity of lactobacillus brevis in the metastatic preparation. As the traditional flow cytometry and the plate cell counting method are not suitable for detecting the number of thalli in the metagen preparation, the qPCR quantitative method provided by the invention can be used for rapidly and accurately carrying out quantitative analysis on the number of lactobacillus brevis thalli in the metagen in a relatively short time, and the result is compared with a plate counting result; compared with the prior art, the qPCR counting method has the advantages that the detection result difference does not exceed one order of magnitude, counting does not distinguish viable bacteria and dead bacteria, and the qPCR counting method has the advantages of high specificity and high speed and can make up the limitation that a plate counting method is suitable for viable bacteria counting, so that the qPCR counting method provided by the invention has more advantages in the application of metagen counting.

Description

Technical Field

[0001] The present invention relates to the technical field of bacterial quantity detection, and in particular to a primer pair, a kit and applications thereof for detecting the bacterial quantity of Lactobacillus brevis in a postbiotic preparation. Background Art

[0002] In 2022, the International Scientific Association of Probiotics and Prebiotics (ISAPP) defined postbiotics as preparations that are beneficial to the host and consist of inanimate microorganisms and their related components. Since postbiotics is a newly proposed concept, although there is sufficient evidence to prove that postbiotics contain substances that are beneficial to health and that some proteins, peptides, cell wall components, etc. are involved in the health effects of postbiotics, whether other components in postbiotics also have health benefits and whether there are interactions between different components in postbiotics in the health effects of postbiotics are still in the exploration stage. Therefore, postbiotic preparations still lack scientific quality control standards, and exploring scientific and fast postbiotic quality control technologies is still a difficult problem facing the current development of postbiotic products.

[0003] Since the material basis for the effect of postbiotics is bacterial components or bacterial metabolites, the number of bacteria in postbiotics must be positively correlated with the content of effective ingredients in the postbiotics. Therefore, counting the number of bacteria in postbiotics can be used as a core indicator to objectively evaluate the quality of postbiotics. Also, since postbiotics are inactivated probiotic preparations, it is impossible to achieve bacterial counting through traditional colony counting techniques, and the plate colony counting technology can only record the number of live bacteria. The number of dead bacteria in the preparation cannot be confirmed by the plate colony counting technology. The detection of probiotics using flow cytometry combined with fluorescent dye technology can count the number of probiotics, and can distinguish between live and dead bacteria through the difference in fluorescent staining. [8] . However, it is difficult to accurately quantify each specific probiotic using current flow cytometry. The prerequisite for using flow cytometry to count bacteria is that the bacterial cells are intact. Some postbiotic preparations undergo sterilization and other processes, and the bacteria may not be able to maintain their integrity. At the same time, the bacteria need to be labeled with specific target molecules with fluorescent substances, which places high demands on instruments and equipment, and the operating procedures are relatively cumbersome. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a technology for detecting the number of bacteria in a postbiotic preparation using qPCR. This technology can be used for the quality control of postbiotic preparations, and can also be used for the production of related kits. The primer pairs provided by the present invention can specifically amplify Lactobacillus brevis (dead and live bacteria) in postbiotic preparations, and have the advantages of strong specificity and high amplification efficiency. The primer pairs can be used for qPCR quantitative detection of the number of Lactobacillus brevis bacteria in postbiotic preparations, and compared with flow cytometry and plate cell counting, it has higher accuracy and sensitivity, and can quickly and accurately quantify Lactobacillus brevis in postbiotics in a shorter time.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The invention provides a primer pair for detecting the number of Lactobacillus brevis bacteria, comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2.

[0007] The present invention provides a kit for detecting the number of Lactobacillus brevis cells, comprising the primer pair described in the above technical solution and a reagent used in qPCR.

[0008] Preferably, the reagent is a detection reagent for dye-based quantitative PCR.

[0009] The present invention provides an application of the primer pair described in the above technical solution or the kit described in the above technical solution in detecting the number of Lactobacillus brevis cells, wherein the Lactobacillus brevis cells include live cells and / or dead cells of Lactobacillus brevis.

[0010] Preferably, the sample to be tested includes a postbiotic preparation prepared using Lactobacillus brevis.

[0011] Preferably, the Lactobacillus brevis includes Lactobacillus brevis YM 1301.

[0012] The present invention provides a method for detecting the number of Lactobacillus brevis cells in a postbiotic preparation, comprising the following steps:

[0013] Using the genomic DNA of the sample to be tested as a template, a primer pair is used to perform a qPCR reaction, and the bacterial concentration of Lactobacillus brevis in the sample to be tested is calculated according to formula I; the primer pair is the primer pair described in the above technical solution, or the primer pair in the kit described in the above technical solution;

[0014] y = -2.613x + 46.102 Formula Ⅰ;

[0015] Wherein, y is the Cq value of the qPCR reaction, and x is the logarithmic value of the Lactobacillus brevis cell concentration.

[0016] Preferably, the reaction procedure of the qPCR reaction is: pre-denaturation at 95°C for 3 min, amplification for 40 cycles; the conditions of each cycle are: 95°C, 15 s; 60°C, 30 s, and collection of fluorescence signals.

[0017] Preferably, the reaction system of the qPCR reaction is: 0.5 μL of upstream primer, 0.5 μL of downstream primer, 1 μL of template, 3 μL of double distilled water and 5 μL of FastStart Essential DNA Green Master; the concentrations of the upstream primer and the downstream primer are both 2 μM.

[0018] The present invention provides an application of the primer pair described in the above technical solution or the method described in the above technical solution in the quality control of a postbiotic preparation, wherein the postbiotic preparation includes a postbiotic preparation prepared using Lactobacillus brevis.

[0019] Beneficial effects:

[0020] The present invention provides a primer pair for detecting the number of Lactobacillus brevis bacteria, comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2. The primer pair provided by the present invention has the advantages of strong specificity and high amplification efficiency. The band is the brightest when Lactobacillus brevis DNA is used as a template, and there is no amplified band when amplification is performed with Lactobacillus plantarum DNA as a template. The primer pair can be used for qPCR quantitative detection of the number of Lactobacillus brevis bacteria in a postbiotic preparation, and has higher accuracy and sensitivity (the minimum detection limit is 10 3 CFU / mL), which can quickly and accurately quantify Lactobacillus brevis in postbiotics in a short time.

[0021] Since traditional flow cytometry and plate cell counting methods are not suitable for detecting the number of bacteria in postbiotic preparations, the present invention also provides a qPCR quantitative method, which can quickly and accurately quantify the number of Lactobacillus brevis bacteria in postbiotics in a relatively short time. The detection results of the qPCR quantitative method provided by the present invention are similar to those of the plate counting, and the data are in the same order of magnitude, indicating that the qPCR counting method of the present invention can be used to replace the traditional plate counting method. In addition, because qPCR counting does not distinguish between live bacteria and dead bacteria and has the advantages of high specificity and fast speed of detection, it can make up for the limitations of the plate counting method that is suitable for live bacteria counting. Therefore, the qPCR counting method provided by the present invention is more advantageous in the application of postbiotic counting. There are certain application prospects in the fields of food and medicine, and technical support is provided for the detection of bacterial counts in Lactobacillus brevis postbiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0023] Figure 1 The results of agarose gel electrophoresis of Lactobacillus brevis and Lactobacillus plantarum;

[0024] Figure 2 This is the qPCR standard curve of Lactobacillus brevis. DETAILED DESCRIPTION

[0025] The invention provides a primer pair for detecting the number of Lactobacillus brevis cells, comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2.

[0026] The primer pair provided by the present invention has the advantages of strong specificity and high amplification efficiency when amplifying Lactobacillus brevis (dead bacteria and / or live bacteria). The band is the brightest when Lactobacillus brevis DNA is used as a template, and no amplified band is present when amplifying with Lactobacillus plantarum DNA as a template, indicating that the primers have good amplification specificity, can accurately identify the target strain, will not be affected by other bacteria, and have high accuracy. The primer pair can be used to perform qPCR quantitative detection of the number of Lactobacillus brevis cells in a postbiotic preparation.

[0027] Based on the above advantages, the present invention provides a kit for detecting the number of Lactobacillus brevis cells, comprising the primer pair described in the above technical solution and the reagent used for qPCR.

[0028] As an embodiment, the reagent is a detection reagent for dye-based quantitative PCR.

[0029] Based on the above advantages, the present invention provides the use of the primer pair described in the above technical solution or the kit described in the above technical solution in detecting the number of Lactobacillus brevis cells, wherein the Lactobacillus brevis cells include live and / or dead Lactobacillus brevis cells.

[0030] As an embodiment, the sample to be tested includes a postbiotic preparation prepared using Lactobacillus brevis.

[0031] As an embodiment, the Lactobacillus brevis includes Lactobacillus brevis YM 1301. The Lactobacillus brevis YM1301 of the present invention is stored in the Biochemistry Laboratory of the School of Life Sciences of Shaanxi Normal University and is disclosed in Chinese Patent CN113384600A.

[0032] Based on the above advantages, the present invention provides a method for detecting the number of Lactobacillus brevis cells in a postbiotic preparation, comprising the following steps:

[0033] Using the genomic DNA of the sample to be tested as a template, a primer pair is used to perform a qPCR reaction, and the bacterial concentration of Lactobacillus brevis in the sample to be tested is calculated according to formula I; the primer pair is the primer pair described in the above technical solution, or the primer pair in the kit described in the above technical solution;

[0034] y = -2.613x + 46.102 Formula Ⅰ;

[0035] Wherein, y is the Cq value of the qPCR reaction, and x is the logarithmic value of the Lactobacillus brevis cell concentration.

[0036] As an embodiment, the reaction procedure of the qPCR reaction is: pre-denaturation at 95°C for 3 minutes, amplification for 40 cycles; the conditions of each cycle are: 95°C, 15 seconds; 60°C, 30 seconds, and collection of fluorescence signals.

[0037] As an embodiment, the reaction system of the qPCR reaction is: 0.5 μL of upstream primer, 0.5 μL of downstream primer, 1 μL of template, 3 μL of double distilled water and 5 μL of FastStartEssential DNAGreenMaster; the concentrations of the upstream primer and the downstream primer are both 2 μM. As an embodiment, the FastStart Essential DNAGreenMaster is purchased from Roche, catalog number: 06924204001.

[0038] As an embodiment, the Lactobacillus brevis includes Lactobacillus brevis YM 1301.

[0039] The method provided by the present invention is a method for quantifying the number of Lactobacillus brevis in a postbiotic by using a qPCR counting method. The method utilizes the complementary pairing amplification principle of primers and target genes, measures the number of Lactobacillus brevis in a postbiotic sample according to the collected fluorescent signals, and thereby quantifies the number of Lactobacillus brevis. The qPCR counting does not distinguish between live bacteria and dead bacteria. Simultaneously, due to the specificity of the primers, the qPCR counting can accurately identify the target strain and will not be affected by other bacteria, and has high accuracy. Compared with flow cytometry and plate cell counting, the qPCR counting has higher accuracy and sensitivity, can quickly and accurately quantify the Lactobacillus brevis in a postbiotic in a short time, can be used for quality control of postbiotic preparations, and the number of bacteria in the postbiotic is counted as a core indicator for objectively evaluating the quality of the postbiotic.

[0040] Based on the above advantages, the present invention provides an application of the primer pair described in the above technical solution, the kit described in the above technical solution, or the method described in the above technical solution in the quality control of a postbiotic preparation, wherein the postbiotic preparation includes a postbiotic preparation prepared using Lactobacillus brevis.

[0041] As an embodiment, the Lactobacillus brevis includes Lactobacillus brevis YM 1301.

[0042] To further illustrate the present invention, the primer pairs, kits and applications thereof for detecting the number of Lactobacillus brevis cells in a postbiotic preparation provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1

[0044] 1. Test strains: Lactobacillus brevis YM 1301 (Lb) and Lactobacillus plantarum CICC24202 (Lp) were stored in the Biochemistry Laboratory of the School of Life Sciences of Shaanxi Normal University and disclosed in Chinese patent CN113384600A.

[0045] 2. Preparation of postbiotic samples

[0046] Lactobacillus brevis YM 1301 was inoculated into MRS liquid culture medium, and was activated by static culture in a 37°C constant temperature incubator for 12 hours. The activation was continued for two generations to obtain a fermentation seed liquid.

[0047] After drying, the smilax glabra is crushed and passed through a 100-mesh sieve for later use. Take 60g of smilax glabra powder and place it in a fermentation bottle. After adding 300mL of distilled water, add α-amylase for enzymatic hydrolysis (enzyme addition: 0.2%, temperature: 60°C, pH: 6.5). After the enzymatic hydrolysis is completed, adjust the pH to neutral, and sterilize with high-pressure steam (121°C, 101KPa, 15min) to obtain the Chinese medicine stock solution. The fermentation seed liquid is inoculated into the Chinese medicine stock solution at an inoculation rate of 8%. After fermentation at 37°C for 48h, the concentration of Lactobacillus brevis YM1301 in the fermentation product is confirmed to be 10 by plate counting. 7 CFU / g, the fermented product was sterilized by high pressure and then dried by microwave vacuum drying equipment to obtain fermented Smilax glabra postbiotics.

[0048] 3. The test strains were streaked on MRS solid medium for activation and cultured at 37°C for 24 h. Single colonies were picked and cultured in MRS liquid medium at 37°C for 14 h. The Lactobacillus brevis culture solution was diluted 10 times and counted. The Lactobacillus brevis count result was 2.06×10 9CFU / mL. At the same time, the 14h cultured Lactobacillus brevis culture was divided into multiple portions, each of which was 1mL, centrifuged at 2000r / min for 5min, the supernatant was discarded, and then the volume was adjusted to 1mL with 70% ethanol. Finally, the treated culture was stored at -20℃. Take 2mL of the plant lactobacillus culture and centrifuge, discard the supernatant, and adjust the volume to 2mL with 70% ethanol and store at -20℃. The collected culture samples were used for subsequent experiments.

[0049] 4. DNA acquisition

[0050] (1) Extraction of genomic DNA from Lactobacillus brevis YM1301 bacterial culture samples

[0051] The prepared Lactobacillus brevis bacterial liquid sample was diluted with physiological saline to a concentration of 10 9 CFU / mL, 10 9 CFU / mL was the starting concentration, and the concentration was diluted 10-fold to 10 5 CFU / mL, a total of 5 gradient dilutions of Lb bacterial solution samples were obtained, and then 1 mL of bacterial solution was drawn from each dilution gradient of bacterial solution sample, and the genomic DNA of each dilution gradient was extracted using a bacterial genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.), and the concentration of the extracted DNA was measured using a micro rapid nucleic acid quantifier. 5 CFU / mL extracted DNA 1μL was diluted 10 times to 10% of the original DNA concentration -3 times, as the concentration is 10 4 , 10 3 , 10 2 The DNA extracted from Lactobacillus brevis at CFU / mL was finally marked and stored at -20℃ for subsequent experiments.

[0052] (2) Extraction of genomic DNA from Lactobacillus plantarum CICC24202

[0053] The Lactobacillus plantarum genomic DNA was extracted from the prepared Lactobacillus plantarum bacterial solution using a bacterial genomic DNA extraction kit, and the DNA concentration was measured using a micro rapid nucleic acid quantification instrument.

[0054] (3) Extraction of Lactobacillus brevis genomic DNA from postbiotic samples

[0055] Weigh 100 mg and 10 mg of the postbiotic sample respectively and dissolve them in 10 mL of distilled water to obtain postbiotic samples with concentrations of 10 mg / mL and 1 mg / mL. Take five portions of each dilution of the postbiotic sample, each with 1 mL, and extract genomic DNA using a bacterial genomic DNA extraction kit. The extracted DNA samples are used in subsequent qPCR experiments.

[0056] 5. Primer design

[0057] The heat shock protein gene (HSP60) sequence and lactate dehydrogenase gene (LDH) of Lactobacillus brevis were retrieved from the NCBI database (http: / / www.ncbinlm.nih.gov / ), and then five specific primers of Lactobacillus brevis were designed, namely HSP60-1, HSP60-2, HSP60-3, LDH-1, and LDH-2. The 16s rRNA primers of Lactobacillus brevis were obtained from existing literature (MATSUDA K, TSUJI H, ASAHARAT, et al. Establishment of an Analytical System for the Human Fecal Microbiota, Based on Reverse Transcription-Quantitative PCR Targeting of Multicopy rRNA Molecules [J]. Applied and Environmental Microbiology, 2009, 75 (7): 1961-9.), and the primers were synthesized by Beijing Qingke Biotechnology Co., Ltd., and the primer information is shown in Table 1.

[0058] Table 1 Information of different primers

[0059]

[0060] 6. Establishment of qPCR method

[0061] The primers in Table 1 were used to perform qPCR reactions with the extracted DNA of Lb and Lp as templates. The qPCR reaction system was: 0.5 μL (2 μM) of upstream primers and downstream primers, 1 μL of template DNA, 3 μL of double distilled water, and 5 μL of FastStart Essential DNA Green Master (Roche, catalog number: 06924204001); the qPCR reaction program was: pre-denaturation at 95°C for 3 min, amplification for 40 cycles, and the conditions for each cycle were: 95°C, 15 s; 60°C, 30 s (collecting fluorescence signals); after the amplification, the obtained amplified samples were subjected to DNA gel electrophoresis at 120 V, and then the target bands were analyzed using a gel imaging system.

[0062] 7. Establishment of qPCR standard curve

[0063] Take 1mL of gradient concentration of 10 9 -102 qPCR reaction was performed with 1 μL of DNA extracted from CFU / mL Lactobacillus brevis bacterial solution, and the Cq value was recorded. A standard curve between the Cq value and the logarithm of the Lactobacillus brevis concentration was established to determine the detection limit of this method.

[0064] 8. Repeatability of qPCR and comparison with plate count method for enumeration of Lactobacillus brevis in postbiotics

[0065] 1 μL of the extracted DNA of different concentrations of postbiotic samples was used for qPCR reaction, and the Cq value was recorded. The theoretical concentration of Lactobacillus brevis was calculated by the Cq value using the constructed qPCR standard curve. The calculated theoretical concentration was compared with the results of plate count to verify the feasibility of the experiment.

[0066] All experiments were repeated three times, and the data and graphs were processed by GraphPadPrism software.

[0067] 9. Results and Analysis

[0068] 1) Primer specificity test results

[0069] Six pairs of primers were used to perform qPCR amplification using Lactobacillus brevis and Lactobacillus plantarum DNA as templates. Figure 1 As shown, 1 to 6 on the left are the results of amplification using primers HSP60-1, HSP60-2, HSP60-3, 16srRNA, LDH-1, and LDH-2 with Lactobacillus brevis DNA as template, and 1 to 6 on the right are the results of amplification using primers HSP60-1, HSP60-2, HSP60-3, 16srRNA, LDH-1, and LDH-2 with Lactobacillus plantarum DNA as template.

[0070] The electrophoresis results show that no DNA bands appeared in the qPCR amplification with Lactobacillus plantarum DNA as template. In the results of amplification with Lactobacillus brevis DNA as template, the HSP60-1 primer product band was the brightest, indicating that the primer specificity was strong and the primer amplification efficiency was very high; the HSP60-2 and HSP60-3 primer amplification bands were darker, indicating that the primer specificity was relatively good, but the amplification efficiency was very low; the LDH-1 primer product band was brighter, but the primer specificity and product amplification efficiency were not as good as the HSP60-1 primer; 16srRNA and LDH-2 primers had no amplification products, indicating that the specificity of the two primers was very poor and could not be amplified with Lactobacillus brevis DNA as template.

[0071] Therefore, in this experiment, HSP60-1 primers were selected as primers for subsequent qPCR experiments with Lactobacillus brevis.

[0072] 2) qPCR standard curve and detection limit

[0073] HSP60-1 primers were 10 2 -10 9 The relationship between the Cq value after qPCR amplification and the logarithm of the Lactobacillus brevis concentration is shown in the figure. Figure 2 As shown, the standard curve equation is y = -2.613x + 46.102, R 2 The detection limit of the qPCR standard curve is 10 3 -10 9 CFU / mL.

[0074] 3) Repeatability of qPCR and comparison with plate count in postbiotics of Lactobacillus brevis

[0075] The comparison of the results of qPCR counting of Lactobacillus brevis YM1301 and plate counting is shown in Table 2. The results showed that the difference between the qPCR counting and the plate counting results was very small, and the difference did not exceed one order of magnitude, indicating that it is feasible to replace the traditional plate counting with qPCR counting.

[0076] Table 2 Comparison of qPCR counts and plate count results in postbiotic samples

[0077]

[0078] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A primer pair for detecting the number of Lactobacillus brevis bacteria, characterized in that: It includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.

2.

2. A kit for detecting the number of Lactobacillus brevis cells, characterized in that: The method comprises the primer pair described in claim 1 and reagents used for qPCR.

3. The kit according to claim 2, characterized in that The reagent is a detection reagent for dye-based quantitative PCR.

4. The application of the primer pair described in claim 1 or the test kit described in claim 2 or 3 in detecting the number of Lactobacillus brevis cells, wherein the Lactobacillus brevis cells include live bacteria cells and / or dead bacteria cells of Lactobacillus brevis.

5. The use according to claim 4, characterized in that: The sample to be tested includes a postbiotic preparation prepared using Lactobacillus brevis.

6. The use according to claim 4 or 5, characterized in that: The Lactobacillus brevis includes Lactobacillus brevis YM 1301.

7. A method for detecting the number of Lactobacillus brevis cells in a postbiotic preparation, characterized in that: The following steps are involved: Using the genomic DNA of the sample to be tested as a template, a primer pair is used to perform a qPCR reaction, and the bacterial concentration of Lactobacillus brevis in the sample to be tested is calculated according to formula I; the primer pair is the primer pair described in claim 1, or the primer pair in the kit described in claim 2 or 3; y = -2.613x + 46.102 Formula Ⅰ; Wherein, y is the Cq value of the qPCR reaction, and x is the logarithmic value of the Lactobacillus brevis cell concentration.

8. The method according to claim 7, characterized in that The reaction procedure of the qPCR reaction is: pre-denaturation at 95°C for 3 minutes, amplification for 40 cycles; the conditions of each cycle are: 95°C, 15 seconds; 60°C, 30 seconds, and collection of fluorescence signals.

9. The method according to claim 7 or 8, characterized in that: The reaction system of the qPCR reaction is: 0.5 μL of upstream primer, 0.5 μL of downstream primer, 1 μL of template, 3 μL of double distilled water and 5 μL of FastStart EssentialDNAGreenMaster; the concentrations of the upstream primer and the downstream primer are both 2 μM.

10. Use of the primer pair according to claim 1, the kit according to claim 2 or 3, or the method according to any one of claims 7 to 9 in the quality control of a postbiotic preparation, wherein the postbiotic preparation includes a postbiotic preparation prepared using Lactobacillus brevis.

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