Method for rapidly detecting and quantifying bifidobacterium longum CCFM1077 with high sensitivity

Abstract

The invention discloses a method for rapidly detecting and quantifying bifidobacterium longum CCFM1077 with high sensitivity, and belongs to the technical field of microorganisms. The invention provides a specific primer capable of being used for screening and / or identifying the bifidobacterium longum CCFM1077 strain, and a rapid, sensitive and efficient method can be provided for detection and quantification of the bifidobacterium longum CCFM1077 in complex samples such as excrement and yoghurt samples by utilizing a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) technology. A reference basis is provided for detection and quantification of bacteria in the field of oral administration or dairy products by using the bacteria as probiotics.

Description

Technical Field

[0001] The invention relates to a method for rapid and highly sensitive detection and quantification of Bifidobacterium longum CCFM1077, belonging to the technical field of microorganisms. Background Art

[0002] Bifidobacterium longum is a member of the genus Bifidobacterium. It is widely colonized in the human intestine and is recognized as a safe host-beneficial bacterium. It has been widely used as a probiotic in food and medicine. Bifidobacterium longum has evolved multiple mechanisms to resist acid stress in the gastrointestinal environment and has bile salt tolerance. In vivo studies have shown that Bifidobacterium longum can effectively improve gastrointestinal diseases such as irritable bowel syndrome, constipation, and diarrhea. Bifidobacterium longum preparations can safely and effectively treat functional dyspepsia in children, promote the digestion and absorption of nutrients, and effectively inhibit the infection of pathogenic bacteria by producing acetic acid, competing with pathogens for ecological niches, and forming an intestinal chemical protective barrier. In addition, Bifidobacterium longum can relieve constipation by regulating the balance of intestinal flora and promoting intestinal peristalsis, and can effectively reduce the cholesterol content in the blood and relieve inflammation. Studies have confirmed that Bifidobacterium longum CCFM1077 has a good therapeutic effect in regulating blood lipids, improving constipation, and relieving cholestasis.

[0003] The vitality and quantity of probiotics reaching the intestine are one of the factors that affect their efficacy. Existing methods for detecting bacterial counts include: traditional plate colony counting method, turbidimetry, cell weight measurement, flow cytometry, etc. The limitations of these technologies are mainly reflected in: (1) The plate counting method can only measure the total amount of live bacteria in the food system, and cannot distinguish the content of specific strains; (2) The identification is carried out by isolating and culturing pure strains, and the detection cycle is long; if a multi-bacteria system (especially the same strain) is involved, it is difficult to isolate and purify a single strain; (3) Accuracy and reproducibility are easily affected by factors such as temperature, experimental environment and human operation; (4) The use of flow cytometry has the disadvantages of high equipment cost, complex operation, limited sample concentration, and the possibility that fluorescent dyes have non-specific binding and toxic effects on the metabolism of probiotics.

[0004] The prior art has reported methods for designing specific primers and quantitatively detecting specific strains through qPCR technology. Patent CN112063619A designs primers based on 16S rRNA sequences for the detection and quantification of animal Bifidobacterium BB12, but due to the poor strain specificity of 16S rRNA and housekeeping gene sequences, the quantified values ​​are much larger than the actual values, making it difficult to achieve accurate quantification. Patent CN112391484A discloses a method for absolute quantification of Bifidobacterium longum strains in a sample, using a bioinformatics method to obtain a "strain-specific" sequence with a wider confidence interval, and based on this, design specific primers for strain quantification. However, the pan-genome used in the inventive method is based on differential genes found within the species of Bifidobacterium longum. The uniqueness of the genes is limited to the species of Bifidobacterium longum. The accuracy of gene positioning in other species / genera is low, and it is susceptible to background interference from complex flora. The specificity in complex flora environments (such as feces) is difficult to ensure. In addition, when the method is quantitative, the CFU of the strain is obtained by plate colony counting, which is limited to the count of live bacteria of microorganisms that can form colonies. The number of dead bacteria formed due to the long growth time of the strain cannot be counted. The quantitative range is limited to the technical range of live bacteria, and it is impossible to count <1×10 5 CFU or >1×10 7 The CFU range can be accurately quantified.

[0005] No method or specific primers have been found that can efficiently and accurately detect Bifidobacterium longum CCFM1077 in complex environments such as yogurt and feces. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of existing strain quantification technology in view of the current health effects of Bifidobacterium longum CCFM1077 on the human body as well as the economic benefits and prospects it possesses, and to provide a method for rapid and highly sensitive detection and quantification of Bifidobacterium longum CCFM1077 in feces and yogurt samples and specific primers used in quantitative detection.

[0007] To solve the above problems, the present invention provides a method for obtaining strain-specific genes of Bifidobacterium longum CCFM1077 and using them for identification or absolute quantification. The method comprises the following steps: firstly obtaining genomic nucleic acid information of the strain, obtaining a unique gene sequence of the strain by homologous gene comparison, and specifically verifying the obtained core gene. The successfully verified gene is a specific gene that can be used to screen and / or identify the Bifidobacterium longum strain; then, an overexpression vector of the target gene is constructed to obtain a plasmid of Bifidobacterium longum CCFM1077 and construct a standard curve; and finally, real-time fluorescence quantitative PCR technology is used to achieve rapid and highly sensitive detection and quantification of Bifidobacterium longum CCFM1077 in feces and yogurt samples.

[0008] In one embodiment of the present invention, the method is to first obtain the genomic nucleic acid information of Bifidobacterium longum CCFM1077, then perform homologous gene comparison on the obtained genomic sequence within the Bifidobacterium longum species to obtain the gene0642 of the specific gene sequence of Bifidobacterium longum CCFM1077, and then construct a database with the genome of the obtained Bifidobacterium longum strain, and perform BLAST comparison. The sequence with no homology or low homology is its specific sequence, and then use Primer Premier 6 to design primers for the specific sequence obtained by screening, and upload it to the NCBI database for Primer-BLAST comparison to verify the specificity of the specific sequence of Bifidobacterium longum CCFM1077 strain in the background of other microbial species, and select genes with no homology results as specific genes that can be used to screen and / or identify Bifidobacterium longum CCFM1077.

[0009] In one embodiment of the present invention, the homologous gene alignment and verification are completed using FastANI software, PGAP software, MATTF software, OrthoMCL software, Primer Premier 6 software and NCBI database.

[0010] In one embodiment of the present invention, the unique gene name of Bifidobacterium longum CCFM1077 strain is gene0642 obtained by homologous gene comparison, and the nucleotide sequences of the specific primers of the strain are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0011] In one embodiment of the present invention, the means for verifying the primers include PCR technology and agarose gel electrophoresis technology.

[0012] The present invention provides a specific primer for identifying or quantitatively detecting Bifidobacterium longum CCFM1077. The specific primer consists of nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2.

[0013] The present invention provides a kit for identification, quantitative detection or auxiliary identification / quantitative detection of Bifidobacterium longum CCFM1077, and the kit comprises the specific primers.

[0014] In one embodiment of the present invention, the kit further comprises a PCR reaction buffer.

[0015] The present invention provides a method for identifying Bifidobacterium longum CCFM1077, comprising the following steps:

[0016] (1) Extracting DNA from the sample to be tested;

[0017] (2) using the sample DNA obtained in step (1) as a template and performing PCR amplification using the specific primers described in claim 1;

[0018] If a gene fragment with a nucleotide sequence as shown in SEQ ID NO. 3 can be amplified, the sample to be tested contains Bifidobacterium longum CCFM1077.

[0019] In one embodiment of the present invention, in step (2), the annealing conditions for PCR amplification are annealing at 60-65° C. for 15-30 seconds.

[0020] The present invention provides a method for quantitatively detecting Bifidobacterium longum CCFM1077, comprising the following steps:

[0021] (1) Extracting DNA from the sample to be tested;

[0022] (2) using the sample DNA obtained in step (1) as a template and performing PCR amplification using the specific primers described in claim 1;

[0023] (3) Determine the content of Bifidobacterium longum CCFM1077 in the sample to be tested based on the Ct value of the amplification curve.

[0024] In one embodiment of the present invention, in step (3), the DNA shown in SEQ ID NO.3 is diluted 10 times in a gradient manner, and 10 -1 ~10 -7 The gradient standard is subjected to fluorescent PCR amplification, a standard curve is drawn, and the Ct value of the amplification curve is brought into the standard curve to obtain the content of Bifidobacterium longum CCFM1077 in the sample to be tested.

[0025] In one embodiment of the present invention, in step (2), the annealing conditions for PCR amplification are annealing at 60-65° C. for 15-30 seconds.

[0026] In one embodiment of the present invention, in step (2), the PCR amplification conditions are: PCR amplification conditions are: 95°C preheating for 5 to 10 min; the cycle program is 95°C denaturation for 30 s, 60 to 65°C annealing for 15 to 30 s, 72°C extension for 15 to 30 s, a total of 34 cycles; 72°C extension for 10 min; 12°C insulation for 2 min.

[0027] The present invention provides application of the specific primer or the kit in screening or detecting Bifidobacterium longum CCFM1077.

[0028] The present invention provides application of the specific primer or the kit in preparing a product for screening or detecting Bifidobacterium longum CCFM1077.

[0029] Beneficial effects:

[0030] 1. Based on whole genome sequencing and homologous gene comparison, the present invention obtains a specific gene (gene name gene 0642) that can be used to screen and / or identify the Bifidobacterium longum CCFM1077 strain, and provides specific primers that can be used to screen and / or identify the Bifidobacterium longum CCFM1077 strain. By using real-time fluorescence quantitative PCR technology, a rapid, sensitive and efficient method can be provided for the detection and quantification of Bifidobacterium longum CCFM1077 in complex samples such as feces and yogurt samples, and a reference basis can be provided for the detection and quantification of the bacteria as a probiotic for oral administration or in the field of dairy products.

[0031] 2. The present invention provides a method for screening and / or identifying specific genes of Bifidobacterium longum CCFM1077 strain, which is simple to operate and can effectively overcome the difficulty of obtaining large-scale pure bacterial cultures. Primers are designed for strain-specific sequences using the method of the present invention, and the primers have high specificity. By optimizing PCR conditions such as annealing temperature, non-target strains and complex fecal samples that do not contain target strains can be amplified without amplification, and strain-specific colonization amounts in complex bacterial flora backgrounds and strain-specific addition amounts in yogurt samples can be detected.

[0032] 3. The specific primers designed by the present invention based on the whole genome sequence of Bifidobacterium longum CCFM1077 can accurately calculate the content of Bifidobacterium longum CCFM1077 in the sample by constructing a plasmid containing the primer fragment, establishing a standard curve for quantification, and utilizing real-time fluorescence quantitative PCR technology, which provides inspiration for exploring the detection and quantification of bacteria in probiotic products.

[0033] 4. The present invention can accurately quantify bacteria in complex samples such as yogurt and feces. The experimental method has the advantages of good repeatability, simple operation, short time consumption, accurate quantification, high degree of automation and low contamination of samples. In addition, the present invention can complete the detection and quantification of probiotics in a relatively short time. Compared with traditional detection methods, there is no need to perform complex steps such as strain separation, cultivation and plate colony counting, which greatly shortens the detection cycle and makes the detection process more efficient and quick. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1: Agarose gel electrophoresis diagram (from the right, except marker, lane 1 is the negative control, and lane 2 is the specific band of Bifidobacterium longum CCFM1077 strain. Lanes 3-25 are other bacterial controls, from right to left respectively: Bifidobacterium longum subsp. longum 1, Bifidobacterium longum subsp. longum 2, Bifidobacterium longum subsp. infantis 1, Bifidobacterium longum subsp. infantis 2, Bifidobacterium breve, Bifidobacterium bifidum 1, Bifidobacterium bifidum 2, Bifidobacterium adolescentis 1, Bifidobacterium adolescentis 2, Bifidobacterium animalis 1, Bifidobacterium animalis 2, Bifidobacterium pseudocatenulatum 1, Bifidobacterium pseudocatenulatum 2, Lactobacillus paracasei 1, Lactobacillus paracasei 2, Lactobacillus plantarum 1, Lactobacillus plantarum 2, Lactobacillus rhamnosus 1, Lactobacillus rhamnosus 2, Pediococcus acidilactici, Escherichia coli), marked in red is 500bp.

[0035] Figure 2 :Amplification curves and melting curves of plasmid standards with different concentration gradients (10 -1 -10 -7 Amplification curve of plasmid standard with concentration gradient (left), and melting curve (right).

[0036] Figure 3 : Plasmid standard curve for quantification (CCFM1077 plasmid standard curve with Ct value as the ordinate and Lg copy number as the abscissa).

[0037] Figure 4 : Fluorescence quantitative PCR amplification curves of DNA of the test samples (the left picture shows the amplification curves of fecal samples with / without the addition of CCFM1077, and the right picture shows the amplification curves of different yogurt samples with / without the addition of CCFM1077, among which there is basically no RFU change in the samples without the addition of CCFM1077).

[0038] Figure 5 : Quantitative accuracy analysis of specific primers for Bifidobacterium longum CCFM1077 strain in fecal system.

[0039] Figure 6 : Quantitative accuracy analysis of specific primers for Bifidobacterium longum CCFM1077 strain in yogurt system.

[0040] Figure 7 : Electrophoresis of different primer sequences of Bifidobacterium longum CCFM1077. DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0042] The raw materials used in the present invention, unless otherwise specified, are all conventional commercially available products. The methods used in the present invention, unless otherwise specified, are all conventional methods in the art. The quality of each substance used in the present invention is the quality of conventional use.

[0043] The tryptone, yeast powder and other chemicals involved in the present invention were purchased from Sinopharm Group; the agarose (product number: CAS#[9012-36-6]) involved was purchased from Sangon Biotech (Shanghai) Co., Ltd.; the 100bp gene ladder (product number: CW0636S), nucleic acid dye (product number: CW2635S), 2×Tag Plus Master Mix (Dye) (Product No.: CW2849L) was purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd.; the bacterial genome extraction kit (Product No.: DP302) and plasmid extraction kit (Product No.: DP107-02) involved were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; the gel recovery kit (Product No. BWDC3511) involved was purchased from Hangzhou Beiwo Medical Technology Co., Ltd.; the lysozyme involved (Product No.: CAS#[12650-88-3]) was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; the fecal genome extraction kit (Product No.: 116570200) and bacterial genome extraction kit (Product No.: DP302) involved were purchased from Tiangen Biochemical Technology Co., Ltd.; the Taq Pro Universal SYBR qPCR Master Mix involved was purchased from Nanjing Novozyme Biotechnology Co., Ltd., and quantitative detection was performed using a BIO-RADCFX96 GPCR instrument; the BHI culture medium (Product No.: HB8478) involved was purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0044] The Bifidobacterium longum subsp. longum CCFM1077 involved in the present invention has been disclosed in patent CN114561325A, and its deposit number is GDMCC No: 60769.

[0045] The culture medium and formula involved are as follows:

[0046] LB solid medium (g / L): tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 20 g / L.

[0047] LB liquid medium (g / L): tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0048] MRS solid medium (g / L): peptone 10g / L, beef extract 10g / L, glucose 20g / L, sodium acetate 2g / L, yeast powder 5g / L, diammonium hydrogen citrate 2g / L, K 2 PO 4 ·3H 2 O 2.6g / L, MgSO 4 7H 2 O 0.1g / L, MnSO 4 0.05 g / L, Tween 80 1mL / L, agar 20g / L.

[0049] MRS liquid medium (g / L): peptone 10g / L, beef extract 10g / L, glucose 20g / L, sodium acetate 2g / L, yeast powder 5g / L, diammonium hydrogen citrate 2g / L, K 2 PO 4 ·3H 2 O 2.6g / L, MgSO 4 7H 2 O 0.1g / L, MnSO 4 0.05 g / L, Tween 80 1mL / L.

[0050] Example 1: Acquisition of strain-specific genes of Bifidobacterium longum CCFM1077 and primer design

[0051] The method comprises the following steps: firstly obtaining the genomic nucleic acid information of Bifidobacterium longum CCFM1077, then performing homologous gene comparison on the obtained genomic sequence within the Bifidobacterium longum species to obtain a specific gene sequence of Bifidobacterium longum CCFM1077, then constructing a database with the obtained genome of the Bifidobacterium longum strain, performing BLAST comparison, and the sequence with no homology or low homology is the specific sequence thereof, then using Primer Premier 6 to design primers for the specific sequence obtained by screening, and uploading the primers to the NCBI database for Primer-BLAST comparison, verifying the specificity of the specific sequence of the Bifidobacterium longum CCFM1077 strain in the background of other microbial species, and selecting the gene with no homology result as the specific gene that can be used for screening and / or identifying Bifidobacterium longum CCFM1077.

[0052] The homologous gene alignment and verification were completed using FastANI software, PGAP software, MATTF software, OrthoMCL software, Primer Premier 6 software and NCBI database.

[0053] The unique gene of Bifidobacterium longum CCFM1077 strain was obtained by homologous gene comparison as gene 0642 (SEQ ID NO.3), and the nucleotide sequences of its specific primers are shown in SEQ ID NO.1 and SEQ ID NO.2. The sequence is as follows:

[0054] F: 5'-AGCAATTGGAGCATGAGC-3' (SEQ ID NO. 1);

[0055] R: 5'-TGATAGACGTGCTGAGATTC-3' (SEQ ID NO. 2).

[0056] Example 2: Verification of Bifidobacterium longum CCFM1077 strain-specific primers

[0057] (1) Activation, culture and DNA extraction of Bifidobacterium longum CCFM1077

[0058] Take out the long bifidobacterium CCFM1077 tube frozen at -80℃, use an inoculation loop to dip a small amount of bacterial solution and streak on MRS solid medium, place it in an anaerobic incubator at 37℃ and invert for 48h. After the colony grows, pick a single colony and inoculate it in a 5mL MRS liquid tube, culture it anaerobically for 18-24h, repeat 2-3 times, take 1mL of bacterial solution, centrifuge it at 8000rpm for 5min and collect the bacteria. The DNA of the long bifidobacterium CCFM1077 strain was extracted according to the instructions of the bacterial genomic DNA extraction kit.

[0059] (2) Electrophoresis verification of Bifidobacterium longum CCFM1077 strain-specific primers

[0060] First, the strain was amplified by PCR using specific primers. Primers were synthesized according to the sequences shown in SEQ ID NO.1 and SEQ ID NO.2. The bacterial genome extracted in step (1) was used as a template for conventional PCR amplification. The PCR was performed using a 25 μL amplification system, which included 12.5 μL of 2×Taq master mix, 1 μL of upstream and downstream primers (10 μmol / L), 1 μL of template DNA, and ddH 2 O 9.5 μL. PCR conditions were as follows: pre-denaturation: 95°C preheating for 10 min; cycle program: 95°C denaturation for 30 s, 60°C annealing for 15 s, 72°C extension for 30 s, 34 cycles in total; 72°C extension for 10 min; 12°C insulation for 2 min.

[0061] The PCR product was then verified by agarose gel electrophoresis. Prepare agarose gel with a (m / v) concentration of 1.5%. Weigh 0.3 g of agarose powder, measure 20 mL of 1×TAE solution, mix and heat until completely melted, clear and without turbidity; after cooling slightly, use a pipette to absorb 2 μL of nucleic acid dye, shake well, and pour it into the electrophoresis plate tank with a comb inserted along the wall of the plate. After 30 minutes, wait for the agarose gel to solidify, carefully pull out the comb and take out the agarose gel and put it into the electrophoresis tank containing 1×TAE electrophoresis solution. Use a pipette to add 10 μL of sample to the gel well, adjust the voltage to 120 V and the current to 200 mA for electrophoresis experiment; the electrophoresis time is 20 to 30 minutes, place the agarose gel in a gel imager for imaging, observe and record the results. At the same time, other strains within the long Bifidobacterium species, other strains within the Bifidobacterium genus, and intestinal strains of other genera were selected for primer specificity verification. The verification results are as follows Figure 1 As shown in the figure, the strain-specific primers of Bifidobacterium longum CCFM1077 only produce specific amplification for this strain, and the product band length is 89 bp. Therefore, the obtained specific primers of Bifidobacterium longum CCFM1077 strain only produce amplification for this target strain, and have no amplification for other strains of the same species, genus and other genera, and have good strain specificity.

[0062] Example 3: Construction of a standard curve for the standard substance vector of Bifidobacterium longum CCFM1077 strain

[0063] (1) Construction of plasmid containing target gene fragment

[0064] The gel electrophoresis band of Bifidobacterium longum CCFM1077 containing the target gene fragment was recovered, and the DNA was purified using a gel recovery kit to obtain purified DNA. The specific steps for constructing the plasmid containing the target gene are as follows:

[0065] Connect the purified PCR product to the T vector (EcoRV recognition site): prepare a reaction solution in a microcentrifuge tube, including 1μL of T-Vector pMD19 (Simple), 1-3μL (0.1-0.3pmoL) of PCR product, 5μL of DNA Ligation Kit, and add sterile water to 10μL system, and mix thoroughly. React at 16℃ for 30min. After the connection reaction is completed, transfer it all to 100μL E.coli competent cells, mix gently, and react on ice for 30min. Then transfer it to a 42℃ water bath for 45s, and immediately transfer it to ice for 1min.

[0066] The E. coli competent cell solution obtained in the previous step was added to 890 μL of liquid LB medium and cultured in a shaking incubator at 37°C for 60 min.

[0067] Then, 100 μL of the transformation solution was spread on a solid LB medium containing sodium ampicillin and cultured overnight at 37° C. Finally, the white colonies on the culture dish were selected and inoculated into a liquid LB medium containing sodium ampicillin and cultured in a shaking incubator at 37° C. for 12 h.

[0068] (2) Extraction of plasmid DNA

[0069] Collect the cultured bacteria from the previous step and use a kit to extract DNA. Use the PCR method to identify the length of the inserted fragment and detect whether the target gene is fully constructed. Sequencing analysis of DNA further verifies whether the plasmid is successfully constructed.

[0070] (3) Establishment of plasmid calibration curve

[0071] The extracted plasmid DNA was diluted 10-fold in a gradient manner and 10 -1 ~10 -7 The gradient standard samples were used for fluorescence PCR amplification and a standard curve was drawn. Figure 2-3 The linear equation of the constructed standard curve is: y = -3.0967x + 37.087. Figure 3 This indicates that the standard curve was successfully constructed and the primers can make the Ct value and bacterial concentration within 10 4 -10 10 The number of bacteria / mL range showed a good linear relationship, R 2 >0.99 has high credibility. And the amplification melting curve of qPCR product ( Figure 2 ) reached its highest peak at 87℃, and the peak was single without interference from other bands, which proved that the primers had the potential to quantify Bifidobacterium longum CCFM1077.

[0072] Example 4: Absolute quantification of strains in a complex (fecal) microbial system

[0073] In order to further verify the potential of the primers for the identification and quantification of Bifidobacterium longum CCFM1077, the bacterial suspension of Bifidobacterium longum CCFM1077 in the exponential phase was collected, a portion of which was washed twice with saline and then spread on MRS agar medium according to gradient dilution for colony counting. The other portion was added to a healthy human fecal sample (bacterial suspension) without Bifidobacterium longum CCFM1077 according to a concentration gradient to construct a complex system, the samples were collected by centrifugation to collect the precipitate, and the genome was extracted using a fecal DNA extraction kit. The extracted fecal bacterial genome samples were detected based on the qPCR reaction system and procedure using specific primers. The qPCR reaction system was 20μL, including 10μL of 2×ChamQ SYBR qPCRMaster Mix, 0.6μL of upstream and downstream primers, 2μL of template DNA, and ddH 2O 6.8 μL. The qPCR reaction program was: denaturation at 95°C for 10 min; the cycle program was denaturation at 95°C for 10 s, annealing at 62°C at the primer-specific temperature for 15 s, extension at 72°C for 15 s, for a total of 40 cycles; melting curve analysis was performed between 65°C and 95°C.

[0074] Figure 4 It shows that in the fecal system without Bifidobacterium longum CCFM1077, the qPCR amplification results did not show a change in the Ct value, while in the fecal system containing Bifidobacterium longum CCFM1077, the Ct value increased, indicating that the primer sequences shown in SEQ ID NO.1 and SEQ ID NO.2 can specifically detect the presence of Bifidobacterium longum CCFM1077 in fecal samples.

[0075] According to the qPCR results, a linear graph was drawn between the Ct value of the strain to be tested and its concentration ( Figure 3 ), and calculate the absolute quantification (detection limit) of specific primers based on qPCR in fecal samples. Table 1 shows that the standard curve for absolute quantification of Bifidobacterium longum CCFM1077 can be used within 10 4 The sensitivity of the primers was about 10 copies / μL DNA solution, and the detection limit of the primers for CCFM1077 in complex microbial systems was 10 4 The absolute quantitative results were compared with the plate count results to obtain the quantitative accuracy of the method. Figure 5 As shown, the logarithmic value of the bacterial number obtained after plate counting was 8.493, the logarithmic value of the bacterial number quantified by specific primer qPCR was 8.584, and the result of the significant difference analysis was p>0.05, which did not show significant difference, proving that the primers have the potential to quantify Bifidobacterium longum CCFM1077 from a complex fecal system.

[0076] Table 1 Absolute quantification (detection limit) of Bifidobacterium longum CCFM1077 added to fecal samples at different dilution gradients

[0077]

[0078]

[0079] Example 5: Extraction and absolute quantification of DNA in yogurt samples

[0080] The specific steps for DNA extraction and absolute quantification in yogurt samples are as follows:

[0081] Three yogurts sold on the market were collected, and 10 mL of each sample was taken into a sterile centrifuge tube. 100 μL of the bacterial solution of Bifidobacterium longum CCFM1077, which was cultured to the logarithmic phase and diluted 100 times, was added. The mixture was centrifuged at 12,000 rpm / min for 15 minutes, and the supernatant was discarded. Then, the DNA in the yogurt samples was extracted using a bacterial DNA extraction kit. After that, the real-time fluorescence quantitative PCR technology was used to detect Bifidobacterium longum CCFM1077, and its detection limit was determined based on the linearity of the results.

[0082] Figure 4 It showed that in yogurt without Bifidobacterium longum CCFM1077, the qPCR amplification results did not show a significant change in the Ct value, while in yogurt containing Bifidobacterium longum CCFM1077, the Ct value increased, indicating that the primer sequences shown in SEQ ID NO.1 and SEQ ID NO.2 can specifically detect the presence of Bifidobacterium longum CCFM1077 in yogurt samples.

[0083] like Figure 6 As shown, the logarithmic value of the number of bacteria obtained after plate counting was 6.220, and the logarithmic values ​​of the number of bacteria quantified by specific primers qPCR for the three yogurt samples were 6.182, 6.230, and 6.102, respectively. The p values ​​of the results of the significant difference analysis were all greater than 0.05, and no significant differences were shown, which proved that the primers have the potential to quantify Bifidobacterium longum CCFM1077 from yogurt samples.

[0084] Comparative Example 1:

[0085] Specific implementation method As in Example 1, the difference is that the primer sequences shown in Table 2 are used for specific detection of Bifidobacterium longum CCFM1077. The results show that in the electrophoresis diagram of all 5 pairs of primers, considering the bands of the negative control and Bifidobacterium longum CCFM1077, only primer pair 1 (primer sequences shown in SEQ ID NO.1 and SEQ ID NO.2) shows no band in the negative control, and shows obvious amplification for Bifidobacterium longum CCFM1077. Therefore, using the primer sequences shown in SEQ ID NO.1 and SEQ ID NO.2 for the absolute quantification of Bifidobacterium longum CCFM1077 has a better effect.

[0086] Table 2 Screening of specific primer sequences for Bifidobacterium longum CCFM1077

[0087]

[0088]

[0089] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A specific primer for identifying or quantitatively detecting Bifidobacterium longum CCFM1077, characterized in that: The specific primers consist of the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. A kit for identification, quantitative detection or auxiliary identification / quantitative detection of Bifidobacterium longum CCFM1077, characterized in that: The kit comprises the specific primer according to claim 1.

3. The kit according to claim 2, characterized in that The kit also contains a PCR reaction buffer.

4. A method for identifying Bifidobacterium longum CCFM1077, characterized in that: The following steps are involved: (1) Extracting DNA from the sample to be tested; (2) using the sample DNA obtained in step (1) as a template and performing PCR amplification using the specific primers described in claim 1; If a gene fragment with a nucleotide sequence as shown in SEQ ID NO. 3 can be amplified, the sample to be tested contains Bifidobacterium longum CCFM1077.

5. A method for quantitatively detecting Bifidobacterium longum CCFM1077, characterized in that: The following steps are involved: (1) Extracting DNA from the sample to be tested; (2) using the sample DNA obtained in step (1) as a template and performing PCR amplification using the specific primers described in claim 1; (3) Determine the content of Bifidobacterium longum CCFM1077 in the sample to be tested based on the Ct value of the amplification curve.

6. The method according to claim 5, characterized in that In step (3), the plasmid DNA shown in SEQ ID NO.3 was diluted 10 times in a gradient manner, and 10 -1 ~10 -7 The gradient standard is subjected to fluorescent PCR amplification, a standard curve is drawn, and the Ct value of the amplification curve is brought into the standard curve to obtain the content of Bifidobacterium longum CCFM1077 in the sample to be tested.

7. The method according to claim 4 or 5, characterized in that: In step (2), the annealing conditions for PCR amplification are annealing at 60-65° C. for 15-30 seconds.

8. The method according to claim 7, characterized in that In step (2), the PCR amplification conditions are preheating at 95°C for 5 to 10 minutes; the cycle program is denaturation at 95°C for 30 seconds, annealing at 60 to 65°C for 15 to 30 seconds, and extension at 72°C for 15 to 30 seconds, for a total of 34 cycles; extension at 72°C for 10 minutes; and insulation at 12°C for 2 minutes.

9. Use of the specific primers according to claim 1 or the kit according to claim 2 or 3 in screening or detecting Bifidobacterium longum CCFM1077.

10. Use of the specific primers according to claim 1 or the kit according to claim 2 or 3 in preparing a product for screening or detecting Bifidobacterium longum CCFM1077.

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