Method for rapidly detecting cell adhesiveness of probiotics and application of method

Abstract

The invention relates to a method for rapidly detecting the cell adhesiveness of probiotics and application of the method. The method comprises the following steps: adding the probiotics into adherent culture cells, incubating, discarding liquid, digesting and collecting the cells by pancreatin, extracting total DNA (Deoxyribonucleic Acid), performing qPCR (Quantitative Polymerase Chain Reaction) amplification, and calculating the adhesion quantity of the probiotics on a single cell according to a Ct value. The invention provides a method for rapidly detecting the cell adhesion rate of probiotics by using a qPCR (quantitative polymerase chain reaction) technology, which comprises the following steps of: extracting total genome DNA (deoxyribonucleic acid) after the probiotics are adhered to cells on the basis of the gene expression difference of prokaryotic and eukaryotic cells, and detecting the specific gene expression level of the probiotics and the cells by using the qPCR technology. The cell adhesion ability of the probiotics is evaluated by calculating the ratio of the gene expression quantities of the two, and the method has the characteristics of high efficiency, rapidness, high accuracy and the like.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a method for rapid detection of probiotic cell adhesion and application thereof. Background Art

[0002] Probiotics are a type of active microorganisms that colonize the human intestine. When a certain amount is ingested, they are beneficial to the health of the body. They have the functions of promoting nutrient absorption, regulating intestinal flora, improving the body's immunity, inhibiting intestinal inflammation, and protecting the intestinal mucosa. For this reason, they are widely used in food and medicine. Lactobacillus rhamnosus, as a member of the genus Lactobacillus, is one of the most studied probiotics. Since it was first isolated from a healthy human in 1983, Lactobacillus rhamnosus has been widely used in dietary supplements, food ingredients and medicines. A large number of studies have shown that Lactobacillus rhamnosus has high gastrointestinal tolerance, can effectively inhibit pathogens, enhance the intestinal immune barrier, and prevent and treat diarrhea. In view of its excellent safety and functionality, Lactobacillus rhamnosus is widely used in the adjuvant treatment of diarrhea in children and has been certified for use in infant food. Lactobacillus acidophilus belongs to the genus Lactobacillus and is one of the important probiotics in the intestine, which is closely related to human health. It has the functions of regulating intestinal flora, inhibiting the invasion of pathogens, enhancing the body's immunity, and lowering blood lipids. Therefore, it has been increasingly used in the fields of medicine, food, feed, etc.

[0003] The intestine is the main place for probiotics to proliferate. After entering the digestive tract from the mouth, probiotics adhere to the intestinal mucosa or intestinal epithelial cells, forming a biological barrier on the cell surface, proliferating and secreting a variety of metabolites with immunomodulatory effects, intervening in the intestinal environment. Therefore, evaluating whether probiotics have the ability to adhere to the intestinal mucosa or epithelial cells is also one of the main criteria for screening probiotics. Given the difficulty of examining cell adhesion in vivo, the adhesion evaluation of probiotics is currently often carried out using in vitro cultured intestinal cells. Human colon adenocarcinoma cells Caco-2 and HT29 are widely used for in vitro adhesion evaluation of probiotics because they have the morphological and functional characteristics of mature intestinal epithelial cells.

[0004] Real-time fluorescence quantitative polymerase chain reaction (qPCR) is a method that adds fluorescent groups to DNA or RNA amplification reactions, uses fluorescent signals to monitor the number of cycles during the amplification reaction, and performs quantitative analysis on unknown templates. Compared with traditional PCR technology, it can not only perform quantitative analysis on products, but also has short time consumption, high sensitivity, and strong specificity. Therefore, it is widely used in clinical diagnosis, drug development, and disease research. qPCR detection methods are mainly divided into fluorescent probe method and fluorescent dye method. SYBR Green method is a commonly used fluorescent dye method. SYBR GreenI can bind to the minor groove of double-stranded DNA and then produce fluorescent signals. With the accumulation of amplified products, the fluorescence intensity increases, and the fluorescent signal is proportional to the content of double-stranded DNA. Therefore, the target sequence in the sample to be tested can be quantitatively analyzed by detecting the fluorescence value of the reaction solution.

[0005] At present, the detection technologies used for probiotic cell adhesion mainly include direct detection method and indirect detection method. Direct detection method includes plate colony counting method and direct microscopy method, while indirect detection method includes spectrophotometry, fluorescence labeling method and radioisotope method.

[0006] The plate spreading method is the simplest and most intuitive detection method. The adhered bacteria are spread on a solid culture medium plate, and the cell adhesion is calculated by counting the number of colonies on the plate. The operation is simple. However, the plate colony counting method requires the exploration of the dilution multiple of the mixed solution of cells and bacteria, which is time-consuming and labor-intensive, greatly affected by subjective factors, and has poor repeatability. In addition, for anaerobic microorganisms, the culture conditions must be strictly required, which increases the difficulty of the experiment.

[0007] CN106754641A discloses a cell model of Escherichia coli O157:H7 infection of porcine small intestinal epithelium, which is prepared by inoculating porcine small intestinal epithelial cells into a cell culture dish, adding Escherichia coli bacterial solution, incubating and washing to remove non-adherent bacteria, adding Triton X-100 to lyse the cells to release live bacteria adhered to the cell surface, diluting the bacterial solution and coating it on a solid LB plate, and calculating the adhesion rate based on the number of colonies on the plate.

[0008] The direct microscopic method is to stain the bacteria after they adhere, and calculate the cell adhesion rate by counting the number of bacteria adhering to 100 cells under a microscope. However, the selection of the observation field of this method is easily affected by subjective factors, the accuracy is low, and the experimental repeatability is poor.

[0009] The spectrophotometric method uses dyes to stain bacteria, and based on the ultraviolet absorption characteristics of the dyes, the bacterial adhesion rate is obtained by measuring the change in absorbance before and after adhesion. However, this method requires the strain to have strong adhesion and quantity to meet the detection requirements of the spectrophotometer, and is not universally applicable.

[0010] The fluorescent labeling method uses fluorescent dyes to specifically label bacteria. After incubating the substrate with the marker, the cells are fixed and the number of bacteria is detected using a fluorescent microscope or flow cytometer. The fluorescence of fluorescent substances is easily degraded over time, and the markers may change the surface characteristics of bacteria and affect the survival ability of bacteria. Therefore, the fluorescent labeling method is rarely used in actual detection processes.

[0011] Similar to the fluorescent labeling method, the radioisotope method uses a label on bacteria, such as H 3 The thymidine nucleus (TdR) of the labeled bacteria is used to measure the ratio of the radioactivity before and after adhesion to obtain the cell adhesion rate, which has a high detection sensitivity. However, this method has a high experimental cost and a great safety hazard.

[0012] In summary, the traditional direct detection method of probiotic adhesion is time-consuming, labor-intensive, and has poor repeatability. In recent years, many indirect detection technologies have been developed, but these technologies also have problems such as high cost and equipment requirements and great damage to bacteria. Therefore, how to develop a fast, effective, and highly accurate cell adhesion rate detection method has become one of the problems that need to be solved in this field. Summary of the invention

[0013] In order to solve the above technical problems, the present invention provides a method for rapid detection of probiotic cell adhesion and its application. The present invention is based on the gene expression differences between prokaryotic and eukaryotic cells, and uses qPCR technology to quickly detect the adhesion rate of probiotic cells. Compared with traditional methods, it saves manpower and time, and has the characteristics of high efficiency, rapidity and high accuracy.

[0014] To achieve this object, the present invention adopts the following technical solutions:

[0015] In a first aspect, the present invention provides a method for rapid detection of probiotic cell adhesion, the method comprising adding the probiotics to cells cultured on a wall, discarding the liquid after incubation, collecting the cells by trypsin digestion, extracting total DNA, performing qPCR amplification, and calculating the number of probiotics adhered to a single cell based on the Ct value.

[0016] The present invention provides a method for rapidly detecting the cell adhesion rate of probiotics by using qPCR technology. The specific gene expression levels of probiotics and cells are detected by using qPCR technology. In view of the gene expression differences between prokaryotic and eukaryotic cells, the total genomic DNA after the probiotics adhere to the cells is extracted, and the cell adhesion ability of the probiotics is evaluated by calculating the ratio of the gene expression amounts of the two. Compared with previous methods, this method has the characteristics of high efficiency, rapidity, high accuracy, etc.

[0017] The method provided by the present invention can be used to quickly detect the cell adhesion of a certain probiotic, so as to evaluate the intestinal colonization ability of the bacterium; it can also be used to quickly detect the cell adhesion of two or more probiotics, so as to examine the site competition between probiotics and provide guidance for the compatibility of composite probiotics.

[0018] Preferably, the cells comprise human colon adenocarcinoma cells.

[0019] Preferably, the human colon adenocarcinoma cells include Caco-2 and / or HT29.

[0020] Preferably, the ratio of the number of probiotics to cells is (400-600):1.

[0021] The specific point values ​​of the above (400-600) can be selected as 400, 450, 480, 500, 520, 550 or 600, etc.

[0022] Preferably, the incubation time is 1.5 to 2.5 h (for example, it may be 1.5 h, 1.7 h, 1.9 h, 2 h, 2.1 h, 2.3 h or 2.5 h, etc.), and the incubation temperature is 35 to 40 ° C (for example, it may be 35 ° C, 36 ° C, 37 ° C, 38 ° C, 39 ° C or 40 ° C, etc.).

[0023] Preferably, the incubation specifically includes: incubating at 35-40°C (for example, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C, etc.) in an incubator containing 4% to 6% (for example, 4%, 4.5%, 5%, 5.5% or 6%, etc.) CO2 for 1.5 to 2.5h (for example, 1.5h, 1.7h, 1.9h, 2h, 2.1h, 2.3h or 2.5h, etc.).

[0024] Preferably, the primers for qPCR include a first primer set and a second primer set, the first primer set amplifies the specific gene of the cell, and the second primer set amplifies the specific gene of the probiotic bacteria.

[0025] Preferably, the first primer set amplifies the housekeeping gene of the cell, and the second primer set amplifies the 16S rRNA gene of the probiotic bacteria.

[0026] 16S rRNA is a component of the 30S subunit in the prokaryotic ribosome and is present in the chromosome genome of all bacteria. It is a highly conserved gene fragment in bacteria and can be used to identify different genera and species present in microbial communities and evaluate their relative abundance.

[0027] Preferably, the housekeeping genes include genes encoding β-actin, GAPDH, and 18S rRNA.

[0028] The β-actin gene is a housekeeping gene in eukaryotic cells. Its expression level in cells is stable and it is often used as an internal reference in PCR to detect gene expression levels.

[0029] In view of the differences in gene expression between prokaryotic and eukaryotic cells, the present invention extracts the total genomic DNA after probiotics adhere to cells, designs specific primers for probiotic and cell marker genes, uses qPCR technology to detect the specific gene expression levels of the two, and evaluates the cell adhesion ability of probiotics by calculating the ratio of the gene expression amounts of the two.

[0030] Preferably, the nucleic acid sequence of the first primer set includes the sequences shown in SEQ ID NO.1 to SEQ ID NO.2.

[0031] Preferably, the nucleic acid sequence of the second primer set includes the sequence shown in SEQ ID NO.3 to SEQ ID NO.4 or SEQ ID NO.5 to SEQ ID NO.6.

[0032] Preferably, the nucleic acid sequence of the second primer set includes the sequences shown in SEQ ID NO.3 to SEQ ID NO.6.

[0033] SEQ ID NO.1 to SEQ ID NO.2 provided by the present invention are used to amplify the β-actin coding gene of Caco-2 cells and / or HT29 cells, and the sequences are shown below.

[0034] SEQ ID NO. 1: CTACAATGAGCTGCGTGTGGC.

[0035] SEQ ID NO. 2: CAGGTCCAGACGCAGGATGC.

[0036] SEQ ID NO.3 to SEQ ID NO.4 provided by the present invention are used to amplify the 16S rRNA gene of Lactobacillus rhamnosus LRa05 strain, and the sequences are shown below.

[0037] SEQ ID NO. 3: TTGCATCTTGATTTAATTTTG.

[0038] SEQ ID NO. 4: AGTTTCCCAGTTTCCGATGC.

[0039] SEQ ID NO.5 to SEQ ID NO.6 provided by the present invention are used to amplify the 16S rRNA gene of Lactobacillus acidophilus LA85 strain, and the sequences are shown below.

[0040] SEQ ID NO. 5: CATAGCCGAGTTGAGAGATG.

[0041] SEQ ID NO. 6: ATCTAATCCTGTTCGCTACCCA.

[0042] Preferably, the qPCR reaction system includes 1×AceQ Universal SYBR qPCR Master Mix, 0.2-0.3 μM upstream primer, 0.2-0.3 μM downstream primer and 4-6 ng / μL template DNA, and water is added to make up the volume to 20 μL.

[0043] The specific point value of 0.2 to 0.3 μM mentioned above can be selected from 0.2 μM, 0.22 μM, 0.24 μM, 0.25 μM, 0.26 μM, 0.28 μM or 0.3 μM, etc.

[0044] The specific point values ​​of 4 to 6 ng / μL mentioned above can be selected from 4ng / μL, 4.5ng / μL, 4.8ng / μL, 5ng / μL, 5.2ng / μL, 5.5ng / μL or 6ng / μL, etc.

[0045] Preferably, the reaction conditions of the qPCR include 93-97°C for 4-6 min; 93-97°C for 8-12 s, 44-48°C for 18-22 s, 70-74°C for 23-27 s, and 38-42 cycles.

[0046] The specific point values ​​of 93-97°C mentioned above can be selected from 93°C, 94°C, 95°C, 96°C or 97°C, etc.

[0047] The specific point values ​​of 4 to 6 minutes mentioned above can be selected as 4 minutes, 4.5 minutes, 5 minutes, 5.5 minutes or 6 minutes, etc.

[0048] The specific point values ​​of 8 to 12 seconds mentioned above may be 8s, 9s, 10s, 11s or 12s etc.

[0049] The specific point value of 44-48°C can be selected from 44°C, 45°C, 46°C, 47°C or 48°C, etc.

[0050] The specific point values ​​of 18 to 22 seconds mentioned above may be selected as 18s, 19s, 20s, 21s or 22s etc.

[0051] The specific point value of 70-74°C can be selected from 70°C, 71°C, 72°C, 73°C or 74°C, etc.

[0052] The specific point values ​​of 23 to 27s mentioned above may be selected as 23s, 24s, 25s, 26s or 27s etc.

[0053] The specific point values ​​of 38 to 42 mentioned above can be selected from 38, 39, 40, 41 or 42, etc.

[0054] Preferably, the probiotics are of the same probiotic species.

[0055] Preferably, the probiotics include any one of Lactobacillus rhamnosus, Lactobacillus acidophilus or Lactobacillus paracasei.

[0056] Preferably, the probiotics are at least two probiotic species.

[0057] The present invention can be used for quantitative analysis of multi-bacterial cell adhesion based on the differences in 16S rRNA of different bacteria. By designing specific primers for different bacteria, the cell adhesion abilities of multiple bacteria can be compared, and the site competition between probiotics can be investigated.

[0058] Preferably, the probiotics include a combination of at least two of Lactobacillus rhamnosus, Lactobacillus acidophilus or Lactobacillus paracasei.

[0059] Preferably, the method for rapid detection of probiotic cell adhesion further comprises the step of drawing a standard curve using a standard substance.

[0060] Preferably, the standard curve comprises a probiotic standard curve and a cell standard curve.

[0061] Preferably, the use of a standard to draw a standard curve specifically comprises: performing a 10-fold gradient dilution of the standard plasmid, amplifying by qPCR, and drawing a standard curve with the logarithm of the DNA template copy number as the abscissa and the Ct value as the ordinate.

[0062] Preferably, the method of calculating the number of probiotics attached to a single cell according to the Ct value specifically includes: substituting the Ct value into the standard curve, respectively obtaining the copy numbers of the probiotic marker gene and the cell marker gene, calculating the ratio thereof, and obtaining the number of probiotics attached to a single cell.

[0063] In a second aspect, the present invention provides the use of the method for rapid detection of probiotic cell adhesion as described in the first aspect in evaluating the intestinal colonization ability of probiotics, screening potential probiotics, or providing guidance for probiotic compatibility.

[0064] In a third aspect, the present invention provides a method for evaluating the cell adhesion ability of each bacterial species in a composite probiotic, the method for evaluating the cell adhesion ability of each bacterial species in a composite probiotic comprises: using the method for rapid detection of probiotic cell adhesion described in the first aspect to detect the cell adhesion of each bacterial species in the composite probiotic, and comparing the relative adhesion ability of each bacterial species when they are in a competitive state.

[0065] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0066] Compared with the prior art, the present invention has the following beneficial effects:

[0067] The present invention provides a method for rapid detection of probiotic cell adhesion rate using qPCR technology. Based on the gene expression difference between prokaryotic and eukaryotic cells, the total genomic DNA after probiotics adhere to cells is extracted, and the specific gene expression levels of probiotics and cells are detected using qPCR technology. By calculating the ratio of the gene expression amounts of the two, the cell adhesion ability of probiotics is evaluated. Compared with traditional methods, it saves manpower and time, and has the characteristics of high efficiency, rapidity, and high accuracy. And unlike the current indirect detection technology such as fluorescence detection, it will not affect the activity of probiotics and reduce the accuracy of the test results. And the qPCR experimental operation technology is mature, the equipment requirements are not high, the sensitivity and specificity are higher, and it is a faster detection method.

[0068] In addition, the present invention can be applied to the quantitative analysis of multi-bacterial cell adhesion based on the differences in 16S rRNA of different bacteria. By designing specific primers for different bacteria, the cell adhesion abilities of multiple bacteria can be compared and the site competition between probiotics can be examined. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 This is the standard curve of 16S rRNA of Lactobacillus rhamnosus LRa05.

[0070] Figure 2 This is a standard curve of the β-actin encoding gene in Caco-2 cells and HT29 cells. DETAILED DESCRIPTION

[0071] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0072] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0073] The Lactobacillus rhamnosus used in the following preparation examples, embodiments and test examples is the Lactobacillus rhamnosus (Lactobacillus rhamnosus) LRa05 strain, classified and named Lactobacillus rhamnosus Lactobacillus rhamnosus, and deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 24377, a deposit date of January 24, 2022, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0074] The Lactobacillus acidophilus used in the following preparation examples, embodiments, and test examples is Lactobacillus acidophilus (Lactobacillus acidophilus) LA85 strain, classified and named Lactobacillus acidophilus, deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.1.12735, a deposit date of July 20, 2020, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0075] Preparation Example 1

[0076] This preparation example provides a method for culturing Lactobacillus rhamnosus LRa05, the method comprising the following steps:

[0077] (1) Weigh an appropriate amount of Lactobacillus rhamnosus LRa05 powder in 0.85% saline, and then inoculate it into MRS liquid culture medium at a 10% inoculum and culture for 6 h;

[0078] (2) After the culture is completed, a small amount of bacterial liquid is taken with an inoculating loop and streaked on MRS solid medium. Incubate the medium upside down at 37°C until a single colony grows.

[0079] (3) picking up a single bacterium on the plate and placing it in liquid MRS medium, culturing it at 37° C. to obtain a probiotic seed culture solution;

[0080] (4) The seed culture solution was inoculated into MRS liquid medium containing 8% NaCl at an inoculum size of 3%, and cultured at 37°C for 10 h.

[0081] Preparation Example 2

[0082] This preparation example provides a method for culturing Lactobacillus acidophilus LA85, which is different from Preparation Example 1 only in that Lactobacillus rhamnosus LRa05 is replaced by Lactobacillus acidophilus LA85.

[0083] Preparation Example 3

[0084] This preparation example provides a method for culturing Caco-2 cells, the method comprising the following steps:

[0085] (1) Caco-2 cells stored in liquid nitrogen were taken out, quickly dissolved in a water bath, and then added to DMEM complete medium. DMSO was removed by centrifugation at 1000 rpm for 5 min, and then resuspended in a culture dish with fresh medium and cultured in a 5% CO2 incubator at 37°C;

[0086] (2) After 80% of the cells adhered to the wall, the cells were removed, the old culture medium was discarded, trypsin was added for digestion, the cells were collected by pipetting, and the cells were obtained by centrifugation at 1000 rpm for 5 min;

[0087] (3) Resuspend the cells in a culture dish containing fresh culture medium and subculture in a 5% CO2 incubator until the cells adhere to the wall;

[0088] (4) After the cells adhered, digest and collect the cells in the same manner as in step (2) and adjust the cell concentration to 2×10 5 / mL, and inoculated into 12-well plates for adherent culture.

[0089] Preparation Example 4

[0090] This preparation example provides a method for culturing HT29 cells, which differs from Preparation Example 3 only in that Caco-2 cells are replaced by HT29 cells.

[0091] Example 1

[0092] This embodiment provides a method for probiotics to adhere to cells, comprising the following steps:

[0093] (1) Centrifuging at 5000 rpm for 5 min, collecting the Lactobacillus rhamnosus LRa05 grown in MRS liquid medium containing 8% NaCl using the method provided in Preparation Example 1, and washing the cells three times with 8% NaCl;

[0094] (2) Resuspend the cells in saline and adjust the cell concentration to 1×10 8 CFU / mL;

[0095] (3) adding the bacterial suspension to a 12-well plate containing an adherent monolayer of Caco-2 cells prepared by the method provided in Preparation Example 3, with the ratio of Lactobacillus rhamnosus to Caco-2 cells being 500:1, and incubating in a 5% CO2 incubator at 37°C for 2 h;

[0096] (4) After the incubation, discard the non-adherent bacterial suspension, wash the cells with sterile PBS, and then add trypsin to digest and collect the cells.

[0097] Example 2

[0098] This embodiment provides a method for probiotics to adhere to cells, comprising the following steps:

[0099] (1) Centrifuging at 5000 rpm for 5 min, collecting the Lactobacillus rhamnosus LRa05 grown in MRS liquid medium containing 8% NaCl using the method provided in Preparation Example 1, and washing the cells three times with 8% NaCl;

[0100] (2) Resuspend the cells in saline and adjust the cell concentration to 1×10 8 CFU / mL;

[0101] (3) adding the bacterial suspension to a 12-well plate containing an adherent monolayer of HT29 cells prepared by the method provided in Preparation Example 4, with the ratio of Lactobacillus rhamnosus to HT29 cells being 400:1, and incubating in a 4% CO2 incubator at 35°C for 2.5 h;

[0102] (4) After the incubation, discard the non-adherent bacterial suspension, wash the cells with sterile PBS, and then add trypsin to digest and collect the cells.

[0103] Example 3

[0104] This embodiment provides a method for probiotics to adhere to cells, comprising the following steps:

[0105] (1) Centrifuging at 5000 rpm for 5 min, collecting the Lactobacillus rhamnosus LRa05 grown in MRS liquid medium containing 8% NaCl using the method provided in Preparation Example 1, and washing the cells three times with 8% NaCl;

[0106] (2) Resuspend the cells in saline and adjust the cell concentration to 1×10 8 CFU / mL;

[0107] (3) adding the bacterial suspension to a 12-well plate containing adherent monolayer Caco-2 cells prepared by the method provided in Preparation Example 3, with the ratio of Lactobacillus rhamnosus to Caco-2 cells being 600:1, and incubating in a 6% CO2 incubator at 40°C for 1.5 h;

[0108] (4) After the incubation, discard the non-adherent bacterial suspension, wash the cells with sterile PBS, and then add trypsin to digest and collect the cells.

[0109] Example 4

[0110] This example provides a method for probiotics to adhere to cells, which is different from Example 1 only in that the adherent monolayer Caco-2 cells prepared by the method provided in Preparation Example 3 are replaced by the adherent monolayer HT29 cells prepared by the method provided in Preparation Example 4.

[0111] Example 5

[0112] This embodiment provides a method for probiotics to adhere to cells, which is different from Embodiment 1 only in that the ratio of the number of Lactobacillus rhamnosus LRa05 to Caco-2 cells is 350:1.

[0113] Example 6

[0114] This embodiment provides a method for probiotics to adhere to cells, which is different from Embodiment 1 only in that the ratio of the number of Lactobacillus rhamnosus LRa05 to Caco-2 cells is 650:1.

[0115] Example 7

[0116] This embodiment provides a method for probiotics to adhere to cells, which is different from Embodiment 1 only in that the incubation temperature is 33°C.

[0117] Example 8

[0118] This embodiment provides a method for probiotics to adhere to cells, which is different from Embodiment 1 only in that the incubation temperature is 42°C.

[0119] Example 9

[0120] This embodiment provides a method for probiotics to adhere to cells, which is different from Embodiment 1 only in that the incubation time is 1 hour.

[0121] Example 10

[0122] This embodiment provides a method for probiotics to adhere to cells, which is different from Embodiment 1 only in that the incubation time is 3 hours.

[0123] Embodiment 11

[0124] This embodiment provides a method for probiotics to adhere to cells, which is different from Example 1 only in that Lactobacillus rhamnosus LRa05 prepared by the method provided in Preparation Example 1 is replaced by Lactobacillus acidophilus LA85 prepared by the method provided in Preparation Example 2.

[0125] Example 12

[0126] This embodiment provides a method for probiotic adhesion to cells, which is different from Example 1 only in that the Lactobacillus rhamnosus LRa05 prepared by the method provided in Preparation Example 1 is replaced by an equal amount of a combination of Lactobacillus rhamnosus LRa05 prepared by the method provided in Preparation Example 1 and Lactobacillus acidophilus LA85 prepared by the method provided in Preparation Example 2, and the bacterial count ratio of Lactobacillus rhamnosus LRa05 to Lactobacillus acidophilus LA85 is 1:1.

[0127] Comparative Example 1

[0128] The adhesion performance of probiotics was investigated by direct microscopy. Caco-2 cells were cultured and the cells were collected after the cell density reached 80%. The cell concentration was adjusted to 2×10 5 / mL, inoculated into a 12-well plate with a cell slide at the bottom, and cultured at 37°C; after the cells adhered to the wall, Lactobacillus rhamnosus LRa05 was added to the cells for adhesion. After the adhesion was completed, the residual bacterial liquid was washed away with PBS, the cell slide was taken out, naturally dried, and then neutral methanol was added for fixation for 20 minutes, and then Gram staining was performed. The adhesion of the strain to the cells was observed under a microscope, and 10 fields of view were randomly selected under an oil immersion lens, and the bacterial adhesion index was counted and calculated. The adhesion index refers to the average number of bacteria adhered to each Caco-2 cell counted in 10 fields of view under a microscope.

[0129] Test Example 1

[0130] This test example extracts total genomic DNA from the cells obtained in Examples 1-10, and performs qPCR detection to evaluate the cell adhesion ability of Lactobacillus rhamnosus LRa05, including the following steps:

[0131] (1) Total genomic DNA extraction

[0132] a. Collect cells and bacteria in a centrifuge tube, take 2 mL of the suspension, centrifuge at 1000 rpm for 5 min, discard the supernatant, and add 0.5 mL of LTE to resuspend the precipitate;

[0133] b. Add 0.5 mL of lysis buffer (Tris-HCl, pH=8.0, EDTA, NaCl, SDS), 30 μL of 20 mg / mL proteinase K, and 10 μL of 10 mg / mL lysozyme to the centrifuge tube and treat at 37°C for 1 h;

[0134] c. Add phenol:chloroform:isoamyl alcohol in a volume ratio of 25:24:1, mix thoroughly, let stand at -20℃ for 2 minutes, then centrifuge at 4℃ and 12000rpm for 5 minutes, and extract twice;

[0135] d. Transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol, mix thoroughly by inversion, leave at room temperature for 10 minutes to precipitate DNA, and then centrifuge at 12,000 rpm at 4°C for 5 minutes;

[0136] e. Wash the precipitate with 75% ethanol and then centrifuge again at 12000 rpm for 5 min;

[0137] f. Leave the centrifuge tube at room temperature, blow dry the water in the tube, then add 100 μL TE to dissolve the DNA, measure the concentration with a spectrophotometer and set aside.

[0138] (2) Preparation of standard samples

[0139] a. Extract genomic DNA from bacteria and cells respectively, amplify the target gene by PCR using specific primers, and purify and recover the target fragment by agarose gel electrophoresis;

[0140] b. Clone the fragment into the pUC57 vector and transform it into E. coli DH5α. Use LB agar plates containing ampicillin resistance to screen positive colonies containing the target gene plasmid. Use a plasmid extraction kit to extract the colony plasmid for verification and sequencing;

[0141] c. Cultivate the strains that have been sequenced successfully, extract the plasmid, determine the concentration and calculate the copy number, and use it as a standard for qPCR. Perform a continuous 10-fold gradient dilution of the standard for qPCR analysis. Draw a standard curve with the logarithm of the DNA template copy number as the horizontal axis and the Ct value as the vertical axis. The standard curve of Lactobacillus rhamnosus LRa05 is as follows: Figure 1 The standard curves of Caco-2 cells and HT29 cells are shown in Figure 2 The target gene fragment PCR program is shown in Table 1, the PCR system is shown in Table 2, and the PCR primers are shown in Table 3.

[0142] Table 1

[0143]

[0144] Table 2

[0145] Component name volume Template DNA 1μL Upstream primer 1μL Downstream primer 1μL PRIMER STAR MIX 10μL <![CDATA[dd H2O]]> 7μL Total volume 20μL

[0146] Table 3

[0147]

[0148]

[0149] (3) qPCR detection

[0150] Real-time fluorescence quantitative PCR was performed using the extracted genomic DNA and the prepared standard as templates. The total reaction system was 20 μL, the primer sequences were shown in Table 3, and the addition amounts of the components of the qPCR reaction system were shown in Table 4. The qPCR reaction conditions were shown in Table 5. After the reaction, a melting curve was drawn from 60°C to 95°C at a rate of 0.5°C / s to detect whether nonspecific amplification products and primer dimers were generated.

[0151] Table 4

[0152] Component name volume concentration 2×AceQ Universal SYBR qPCR Master Mix 10μL 1× Upstream primer 0.5μL 10μM Downstream primer 0.5μL 10μM Template DNA XμL 100ng <![CDATA[dd H2O]]> XμL / Total volume 20μL /

[0153] Table 5

[0154]

[0155]

[0156] (4) Calculation of adhesion rate

[0157] According to the Ct value of each gene in the measured sample, the corresponding standard curve was substituted to obtain the sample DNA copy number, and the number of probiotics adhering to a single cell was calculated by calculating the ratio of the DNA template copy numbers of Lactobacillus rhamnosus and cells.

[0158] (5) Experimental results analysis

[0159] After the reaction, the melting curve of qPCR was confirmed to verify whether nonspecific amplification occurred. According to the amplification curve and the standard curve, the Ct value was analyzed, the concentration and ratio of Lactobacillus rhamnosus LRa05 and the cell marker gene were calculated, and the adhesion ability of Lactobacillus rhamnosus was evaluated by absolute quantitative and relative quantitative analysis. The results are shown in Table 6. At the same time, the adhesion performance of Lactobacillus rhamnosus LRa05 was investigated using the direct microscopic method provided in Comparative Example 1, and the adhesion rate was 12 CFU / cell.

[0160] Table 6

[0161]

[0162]

[0163] It can be seen from Table 6 that:

[0164] (1) By comparing Example 1 with Comparative Example 1, it can be seen that the rapid detection method for probiotic cell adhesion provided by the present invention is consistent with the detection results of the direct microscopy method and has high accuracy.

[0165] (2) Comparing Example 1 with Example 4, it can be seen that the type of cells used does not affect the measurement results of the probiotic cell adhesion rate.

[0166] (3) Comparing Example 1 with Example 5 and Example 6, it can be seen that when the number of probiotics is too small, there are still sites on the cell surface that can adhere to probiotics, and it is impossible to obtain an accurate value of the probiotic cell adhesion rate; when the number of probiotics is too large, the cell surface sites have been occupied by probiotics and tend to be saturated, and the adhesion rate can no longer be increased.

[0167] (4) Comparing Example 1 with Example 7 and Example 8, it can be seen that if the incubation temperature is too low or too high, the cell growth state will be poor, and the probiotics cannot adhere to the cell surface normally, and the measured probiotic cell adhesion rate value is low.

[0168] (5) Comparing Example 1 with Example 9 and Example 10, it can be seen that if the incubation time is too short, the probiotics will not adhere to the cells sufficiently, and the measured probiotic cell adhesion rate will be low; if the incubation time is too long, the cell surface sites are completely occupied by the probiotics, and the adhesion rate will no longer increase.

[0169] Test Example 2

[0170] In this test example, total genomic DNA was extracted from the cells obtained in Examples 1, 11 and 12, and qPCR detection was performed to compare the cell adhesion ability of various bacteria to the cells, referring to Test Example 1, wherein the 16S rRNA gene amplification primers of Lactobacillus acidophilus LA85 are shown in SEQ ID NO.5 to SEQ ID NO.6, and the test results are shown in Table 7.

[0171] SEQ ID NO. 5: CATAGCCGAGTTGAGAGATG.

[0172] SEQ ID NO. 6: ATCTAATCCTGTTCGCTACCCA.

[0173] Table 7

[0174]

[0175] It can be seen from Table 7 that when only Lactobacillus rhamnosus LRa05 was present, the adhesion rate was 12 CFU / cell, and when only Lactobacillus acidophilus LA85 was present, the adhesion rate was 9 CFU / cell. When Lactobacillus rhamnosus LRa05 and Lactobacillus acidophilus LA85 existed at the same time, the cell adhesion rates of both bacteria decreased to varying degrees, but in comparison, the cell adhesion ability of Lactobacillus rhamnosus LRa05 was stronger.

[0176] In summary, the present invention provides a method for rapid detection of probiotic cell adhesion rate using qPCR technology. Based on the gene expression difference between prokaryotic and eukaryotic cells, the total genomic DNA after probiotics adhere to cells is extracted, and the specific gene expression levels of probiotics and cells are detected using qPCR technology. By calculating the ratio of the gene expression amounts of the two, the cell adhesion ability of probiotics is evaluated. Compared with traditional methods, it saves manpower and time, and has the characteristics of high efficiency, rapidity, and high accuracy.

[0177] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for rapid detection of probiotic cell adhesion, characterized in that: The method comprises adding the probiotics to cells cultured on a wall, discarding the liquid after incubation, collecting the cells by trypsin digestion, extracting total DNA, performing qPCR amplification, and calculating the number of probiotics adhered to a single cell according to the Ct value.

2. The method for rapid detection of probiotic cell adhesion according to claim 1, characterized in that: The cells include human colon adenocarcinoma cells; Preferably, the human colon adenocarcinoma cells include Caco-2 and / or HT29.

3. The method for rapid detection of probiotic cell adhesion according to claim 1 or 2, characterized in that: The ratio of the number of probiotics to cells is (400-600):1; Preferably, the incubation time is 1.5 to 2.5 hours, and the incubation temperature is 35 to 40°C; Preferably, the incubation specifically comprises: incubating at 35-40° C. for 1.5-2.5 h in an incubator containing 4%-6% CO 2 .

4. The method for rapid detection of probiotic cell adhesion according to any one of claims 1 to 3, characterized in that: The primers for qPCR include a first primer set and a second primer set, wherein the first primer set amplifies the specific gene of the cell, and the second primer set amplifies the specific gene of the probiotic bacteria; Preferably, the nucleic acid sequence of the first primer set includes the sequences shown in SEQ ID NO.1 to SEQ ID NO.2; Preferably, the nucleic acid sequence of the second primer set includes the sequence shown in SEQ ID NO.3 to SEQ ID NO.4 or SEQ ID NO.5 to SEQ ID NO.6; Preferably, the nucleic acid sequence of the second primer set includes the sequences shown in SEQ ID NO.3 to SEQ ID NO.

6.

5. The method for rapid detection of probiotic cell adhesion according to any one of claims 1 to 4, characterized in that: The probiotics are of the same probiotic strain; Preferably, the probiotics include any one of Lactobacillus rhamnosus, Lactobacillus acidophilus or Lactobacillus paracasei.

6. The method for rapid detection of probiotic cell adhesion according to any one of claims 1 to 4, characterized in that: The probiotics are at least two probiotic species; Preferably, the probiotics include a combination of at least two of Lactobacillus rhamnosus, Lactobacillus acidophilus or Lactobacillus paracasei.

7. The method for rapid detection of probiotic cell adhesion according to any one of claims 1 to 6, characterized in that: The method for rapid detection of probiotic cell adhesion also includes the step of using a standard substance to draw a standard curve; Preferably, the standard curve includes a probiotic standard curve and a cell standard curve; Preferably, the use of a standard to draw a standard curve specifically comprises: performing a 10-fold gradient dilution of the standard plasmid, amplifying by qPCR, and drawing a standard curve with the logarithm of the DNA template copy number as the abscissa and the Ct value as the ordinate.

8. The method for rapid detection of probiotic cell adhesion according to any one of claims 1 to 7, characterized in that: The method of calculating the number of probiotics attached to a single cell according to the Ct value specifically includes: substituting the Ct value into the standard curve, respectively obtaining the copy numbers of the probiotic marker gene and the cell marker gene, calculating the ratio thereof, and obtaining the number of probiotics attached to a single cell.

9. Use of the method for rapid detection of probiotic cell adhesion as claimed in any one of claims 1 to 8 in evaluating the intestinal colonization ability of probiotics, screening potential probiotics or providing guidance for probiotic compatibility.

10. A method for evaluating the cell adhesion ability of each species in a composite probiotic, characterized in that: The method for evaluating the cell adhesion ability of each bacterial species in the composite probiotics comprises: using the method for rapid detection of probiotic cell adhesion according to any one of claims 1 to 8 to detect the cell adhesion of each bacterial species in the composite probiotics, and comparing the relative adhesion ability of each bacterial species when they are in a competitive state.

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