Method for rapidly and quantitatively detecting viable bacteria of lactobacillus jensenii in composite viable bacteria medicinal preparation

Abstract

The invention belongs to the technical field of biology, discloses a method for rapidly and quantitatively detecting viable bacteria of lactobacillus jensenii in a composite viable bacteria medicinal preparation, and particularly discloses a reagent which comprises a primer pair for detecting the lactobacillus jensenii, and the primer pair comprises an upstream primer L.jens-F1 and a downstream primer L.jens-R1. The reagent provided by the invention has good discrimination degree on common strains such as lactobacillus crispatus, lactobacillus reuteri, lactobacillus gasseri, lactobacillus plantarum, lactobacillus fermentum, lactobacillus johnsonii, lactobacillus rhamnosus and lactobacillus delbrueckii in the same genus, and can detect lactobacillus jensenii with high specificity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for rapid quantitative detection of live Lactobacillus jensenii in a composite live bacteria pharmaceutical preparation. Background Art

[0002] Live biological drugs, also known as live bacterial drugs, are called microecological live bacterial products in my country. They are made from normal bacterial members in the human body or harmless foreign bacteria that promote the growth and activity of normal bacterial flora. After being cultured, collected, dried into bacterial powder, and mixed with appropriate excipients, they are used to prevent and treat related symptoms and diseases caused by dysbiosis. In recent years, with the continuous deepening of microbiome research, the field of live biological drugs has received more and more attention, and there is also a trend of developing composite live bacterial preparations from single bacteria to multiple bacteria.

[0003] For live bacterial drugs, the number of live bacteria is an important indicator of their quality and safety. It can intuitively demonstrate the quality of microecological preparations. A higher number of live bacteria can reflect the effective activity and storage stability of microecological preparations to a certain extent. The traditional counting method, that is, the plate colony culture method, is easily affected by the characteristics of the strains, culture conditions, etc., especially for composite live bacterial preparations composed of very similar species characteristics. It is difficult to find a suitable culture medium to count each single bacterium in the composite preparation. Real-time fluorescence quantitative PCR (Quantitative Real-time PCR, qPCR), with the advantages of strong specificity, good repeatability, accuracy, and rapidity, has become an important method for quantitative detection in the field of molecular biology and microbiology. Propidium monoazide (PMA) is a photosensitive material that can covalently cross-link with DNA and can selectively penetrate the cell membrane of dead bacteria (not permeable to living cells). Illumination can convert the photosensitive group of PMA into nitroxide free radicals, which covalently cross-link with the DNA of dead bacteria, thereby blocking the PCR amplification of the DNA molecules of dead bacteria, and does not affect the amplification of the DNA of live bacteria. Therefore, PMA-qPCR can be used as an effective method for rapid quantitative detection of live bacteria. However, there is currently no rapid quantitative detection method for live Lactobacillus jensenii in compound live bacterial pharmaceutical preparations. Summary of the invention

[0004] The invention aims at Lactobacillus jensenii contained in the composite live bacteria preparation, designs species-specific primers according to the 16S rRNA gene sequence of Lactobacillus jensenii, uses the primers to perform PMA-qPCR quantitative detection on the Lactobacillus jensenii in the composite live bacteria preparation, and establishes a simple, rapid and accurate quantitative determination method of Lactobacillus jensenii in the composite live bacteria preparation by comparison in combination with the colony plate counting result.

[0005] The first aspect of the present invention aims to provide a reagent.

[0006] The second aspect of the present invention aims to provide a detection kit.

[0007] The purpose of the third aspect of the present invention is to provide the use of the reagent of the first aspect of the present invention or the detection kit of the second aspect of the present invention.

[0008] The fourth aspect of the present invention aims to provide a method for quantitatively detecting the number of live Lactobacillus jensenii in a microecological live bacteria product.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] The first aspect of the present invention provides a reagent comprising a primer pair for detecting Lactobacillus jensii, wherein the primer pair comprises an upstream primer L.jens-F1 and a downstream primer L.jens-R1, wherein:

[0011] The nucleotide sequence of the upstream primer L.jens-F1 is: 5'-AAGTCGAGCGAGCTTGCCTATAGA-3',

[0012] The nucleotide sequence of the downstream primer L.jens-R1 is: 5'-CTTCTTTCATGCGAAAGTAGC-3'.

[0013] The present invention uses the NCBI database to retrieve the 16S rRNA gene sequence of Lactobacillus jensenii, and designs a primer pair that can be used to detect Lactobacillus jensenii. The primer pair has good discrimination for common strains of the same genus, such as Lactobacillus crispatus, Lactobacillus reuteri, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus rhamnosus, and Lactobacillus delbrueckii, and can detect Lactobacillus jensenii with high specificity.

[0014] The second aspect of the present invention provides a detection kit, which includes the reagent of the first aspect of the present invention.

[0015] In some embodiments of the present invention, the kit further comprises propidium bromide, dNTP, Mg 2+ , enzyme, and SYBRGreen I.

[0016] In some embodiments of the present invention, the enzyme includes Taq enzyme and DNA polymerase.

[0017] In some embodiments of the present invention, the kit further comprises a positive sample.

[0018] The third aspect of the present invention provides the use of the reagent of the first aspect of the present invention or the detection kit of the second aspect of the present invention in any one of (1) to (4):

[0019] (1) Detection of Lactobacillus jensenii;

[0020] (2) preparing a product for detecting Lactobacillus jensenii;

[0021] (3) quantitatively detecting the number of viable Lactobacillus jensenii in the sample to be tested;

[0022] (4) preparing a product for quantitatively detecting the number of viable Lactobacillus jensenii in a sample to be tested;

[0023] The above applications are for non-disease diagnosis and treatment.

[0024] In some embodiments of the present invention, the sample to be tested in (3) to (4) includes a microecological live bacteria product.

[0025] A fourth aspect of the present invention provides a method for quantitatively detecting the number of live Lactobacillus jensenii in a microecological live bacteria product, comprising the step of using the detection kit of the second aspect of the present invention to detect the microecological live bacteria product to be tested.

[0026] In some embodiments of the present invention, the quantitative detection method specifically comprises the following steps:

[0027] 1) Sample pretreatment: dissolving the microecological live bacteria product to be tested to obtain a microecological live bacteria product solution to be tested;

[0028] 2) PMA treatment: the solution of the microecological live bacteria product to be tested in step 1) is mixed with PMA, reacted in the dark, and photolyzed under LED light;

[0029] 3) Genomic DNA extraction: DNA is extracted from the microecological live bacteria product to be tested after PMA treatment in step 2);

[0030] 4) qPCR detection: using the genomic DNA from step 3) as a template, and performing qPCR detection using the primer pair described in claim 1;

[0031] 5) Substituting the CT value detected by qPCR in step (4) into the standard curve regression equation to obtain the number of viable Lactobacillus jensenii in the microecological live bacteria product to be tested.

[0032] In some embodiments of the present invention, the final concentration of PMA in step 2) is 15-50 μg / mL.

[0033] In some embodiments of the present invention, the final concentration of PMA in step 2) is 35-50 μg / mL.

[0034] In some embodiments of the present invention, the final concentration of PMA in step 2) is 45-48 μg / mL.

[0035] In some embodiments of the present invention, the light-protecting reaction time in step 2) is 15 to 30 minutes.

[0036] In some embodiments of the present invention, the light-protecting reaction time in step 2) is 20 to 30 minutes.

[0037] In some embodiments of the present invention, the LED light irradiation photolysis time is 30 to 50 minutes.

[0038] In some embodiments of the present invention, the LED light irradiation photolysis time is 40 to 50 minutes.

[0039] In some embodiments of the present invention, the LED lamp has a power of 500-700W.

[0040] In some embodiments of the present invention, when the LED light is used for photolysis, in order to prevent the strong light irradiation from causing the temperature of the sample to rise and affect the experimental results, the sample is placed on ice during the photolysis process.

[0041] In some embodiments of the present invention, the method for constructing the standard curve includes: gradiently diluting the DNA solution of the Lactobacillus jensenii standard strain to obtain a standard solution, performing qPCR detection respectively, and drawing a standard curve with the logarithm of the number of viable cells of the Lactobacillus jensenii standard strain in the standard solution as the horizontal axis and the CT value as the vertical axis.

[0042] In some embodiments of the present invention, the regression equation of the CT value standard curve is Y=-3.35X+34.14, r 2 =0.999, where X is the logarithmic value of the number of viable bacteria, Y is the number of amplification cycles, and the detection range is 1×10 3 ~1×10 8 CFU.

[0043] In some embodiments of the present invention, the microecological live bacteria product includes bacterial powder and capsules.

[0044] In some embodiments of the present invention, the reaction procedure of the qPCR detection in 4) is: 94-96°C, 2-5 min; 94-96°C, 25-35 s, 56-58°C 25-35 s, 35-40 cycles.

[0045] The beneficial effects of the present invention are:

[0046] The present invention designs primers for the 16S rRNA gene sequence of Lactobacillus jensenii, and explores PMA treatment and qPCR detection conditions, which is different from the prior art:

[0047] 1. The primers provided by the present invention have a wider coverage, especially better discrimination between multiple vaginal lactobacilli. The primers designed previously can only distinguish 2-3 types of lactobacilli and other types of pathogens other than several types of lactobacilli, while the primers of the present application are aimed at distinguishing more than 10 types of lactobacilli with higher similarity, basically covering the common lactobacilli in the vagina.

[0048] 2. Quantitative methods are more accurate. Existing methods have Ct values ​​or copy numbers, which cannot correspond to the results of plate counts. The method prescribed by the general pharmacopoeia for calculating the number of viable bacteria is plate count. The results of the detection method of the present invention are equivalent to the plate count method and have higher accuracy.

[0049] 3. The existing method cannot distinguish between dead bacteria and live bacteria, and the counting is inaccurate. The detection method of the present invention can specifically detect the number of live bacteria of Lactobacillus jensenii in microecological live bacteria products, and can exclude the influence of other similar species in many live bacteria preparations, and quickly perform quantitative counting detection of Lactobacillus jensenii, which is suitable for quantitative detection of the number of live bacteria of Lactobacillus jensenii in all live bacteria preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a graph showing the PCR specificity of the Lactobacillus jensii primer L. jens-F1 / R1 in Example 1 for 10 different lactobacilli.

[0051] Figure 2 This is a graph showing the PCR specificity of the Lactobacillus jensii primer L. jens-F2 / R2 in Example 1 for 10 different lactobacilli.

[0052] Figure 3 This is a graph showing the PCR specificity of the Lactobacillus jensii primer L. jens-F3 / R3 in Example 1 for 10 different lactobacilli.

[0053] Figure 4 This is a graph showing the PCR specificity of the Lactobacillus jensii primers L. jens-F1 / R1 in Example 1 of the present invention for eight species of Lactobacillus jensii.

[0054] Figure 5 This is a qPCR amplification curve of Lactobacillus jensii primers L. jens-F1 / R1 on Lactobacillus jensii in Example 1 of the present invention.

[0055] Figure 6 This is a qPCR melting curve diagram of Lactobacillus jensii primers L. jens-F1 / R1 for Lactobacillus jensii in Example 1 of the present invention.

[0056] Figure 7 This is an amplification curve diagram of the standard curve of live Lactobacillus jensenii in Example 3 of the present invention.

[0057] Figure 8 It is a standard curve diagram of the viable count of Lactobacillus jensenii in Example 3 of the present invention.

[0058] Fig. 9 This is a diagram showing the inhibitory effects of different concentrations of PMA on dead / live bacteria in Example 3 of the present invention.

[0059] Fig.10 This is an amplification curve diagram of Lactobacillus jensenii in the Lactobacillus jensenii powder in Example 4 of the present invention.

[0060] Fig.11 This is an amplification curve of Lactobacillus jensenii in the quadruple live bacteria preparation in Example 5 of the present invention. DETAILED DESCRIPTION

[0061] The present invention is further described in detail below through specific examples.

[0062] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0063] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0064] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0065] Example 1 Primer design and specificity verification of Lactobacillus jensenii

[0066] 1. Primer Design

[0067] The 16S rRNA gene sequence of Lactobacillus jensenii was retrieved using the NCBI database, and primers were designed online using Primer Blast to obtain three pairs of primer sequences. The specific sequences are shown in Table 1.

[0068] Table 1 Primer sequences

[0069]

[0070]

[0071] 2. Primer specificity PCR verification

[0072] ① Specific PCR for different types of Lactobacillus:

[0073] With 3 pairs of Lactobacillus jensenii primers in Table 1 as amplification primers, DNA of 10 kinds of lactobacilli (Lactobacillus jensenii, Lactobacillus crispatus, Lactobacillus reuteri, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus delbrueckii, strain information is shown in Table 2) was subjected to PCR amplification in sequence to investigate the specificity of each primer pair. Specific as follows:

[0074] Table 2 Information of different lactobacilli

[0075]

[0076] (1) Strain activation: MRS broth medium was used to culture at 37° C. in a carbon dioxide incubator for 8 to 12 h to activate 10 strains of Lactobacillus, including Lactobacillus jensenii, Lactobacillus crispatus, Lactobacillus reuteri, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus rhamnosus, Lactobacillus paracasei, and Lactobacillus delbrueckii.

[0077] (2) Genomic DNA extraction: 1 mL of bacterial solution of each lactobacillus was placed in a 1.5 mL centrifuge tube, and the genomic DNA of the above lactobacillus was extracted using the Bacterial Genomic DNA Rapid Extraction Kit of Sangon Biotech (Shanghai) Co., Ltd., and the DNA solution was eluted in a volume of 100 μL.

[0078] (3) PCR amplification: The genomic DNA solutions of the 10 lactobacilli in Table 2, the forward primer L. jens-F1 of Lactobacillus jensii, the reverse primer L. jens-R1 of Lactobacillus jensii, the forward primer L. jens-F2 of Lactobacillus jensii, the reverse primer L. jens-R2 of Lactobacillus jensii, the forward primer L. jens-F3 of Lactobacillus jensii, the reverse primer L. jens-R3 of Lactobacillus jensii, and Taq PCR Mix premix (Shanghai Sangon Biotech Co., Ltd., Catalog No.: B639295) were prepared on ice. The genomic DNA of Lactobacillus jensii and the DNA of the other 9 lactobacilli strains were amplified using the three pairs of primers. A total of 11 tubes of PCR reaction systems corresponding to each pair of primers were prepared, and one tube was added with nuclease-free water (NF water for short) as a template for amplification as a negative control. The single PCR reaction system is shown in Table 3 below, and the PCR program is shown in Table 4.

[0079] Test results: The primers L. jens-F1 / R1 specifically amplified the genomic DNA of Lactobacillus jens, with a product size of 162 bp. There was no amplification of the genomic DNA of 9 strains of bacteria, including Lactobacillus crispatus, Lactobacillus reuteri, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus jens, Lactobacillus rhamnosus, and Lactobacillus delbrueckii. There was no amplification in the negative control. The primers only showed good specificity for Lactobacillus jens. Figure 1The primers for Lactobacillus jensii, L. jens-F2 / R2, can amplify all eight lactobacilli (Lactobacillus paracasei and Lactobacillus delbrueckii cannot be amplified). The bands are bright and clear, but Lactobacillus jensii cannot be accurately distinguished from other lactobacilli ( Figure 2 ), indicating that the specificity of the Lactobacillus jensii primer is poor. The Lactobacillus jensii primer L.jens-F3 / R3 only amplifies Lactobacillus jensii and Lactobacillus johnsonii, but not the other lactobacilli ( Figure 3 ), the specificity of primer L.jens-F3 / R3 was better than that of primer L.jens-F2 / R2, but it was still unable to accurately distinguish Lactobacillus jensii from more than 10 similar lactobacilli.

[0080] Table 3 PCR reaction system

[0081]

[0082] Table 4 PCR reaction program

[0083]

[0084] ② Specific PCR for various Lactobacillus jensenii:

[0085] Using the Lactobacillus jensii primer L.jens-F1 / R1 in Table 1 as the amplification primer, PCR amplification was performed on DNA of 8 different sources of Lactobacillus jensii (strain information is shown in Table 5) in turn to investigate the specificity of the Lactobacillus jensii primer L.jens-F1 / R1. The details are as follows:

[0086] Table 5 Information of Lactobacillus jensenii from different sources

[0087]

[0088] The strain activation and genomic DNA extraction were the same as ①.

[0089] Prepare on ice the genomic DNA solutions of the eight Lactobacillus jensii strains in Table 5, the forward primer L. jens-F1 of Lactobacillus jensii, the reverse primer L. jens-R1 of Lactobacillus jensii, and the Taq PCR Mix premix, and use the primers to amplify the DNA of the eight Lactobacillus jensii strains respectively; prepare a total of 9 tubes of PCR reaction system, 1 tube is added with NF water as a template for amplification as a negative control, the single PCR reaction system is shown in Table 3 above, and the PCR program is shown in Table 4 above.

[0090] Test results: The primers L. jens-F1 / R1 specifically amplified the genomic DNA of L. jens from 8 different sources, with a product size of 162 bp. The negative control showed no amplification, and the primers had good specificity ( Figure 4 ).

[0091] 3. Primer melting curve analysis

[0092] Prepare on ice the genomic DNA solutions of the 10 lactobacilli in Table 2, the forward primer L. jens-F1 of Lactobacillus jensii, the reverse primer L. jens-R1 of Lactobacillus jensii, and ChamQ SYBR qPCR Master Mix (WithoutROX), and use the primers to amplify the genomic DNA of Lactobacillus jensii and the DNA of the remaining 9 lactobacilli strains, respectively; a total of 11 tubes of qPCR reaction system were prepared, 1 tube was added with NF water as a template for amplification as a negative control, a single qPCR reaction system is shown in Table 6 below, and the qPCR program is shown in Table 7.

[0093] Table 6 qPCR reaction system

[0094]

[0095]

[0096] Table 7 qPCR reaction procedure

[0097]

[0098] Test results: Figure 5-6 As shown, the primers L. jens-F1 / R1 of Lactobacillus jensii specifically amplified the genomic DNA of Lactobacillus jensii, with a good amplification curve and a single peak in the melting curve; there was no amplification for 9 strains of Lactobacillus, including Lactobacillus crispatus, Lactobacillus reuteri, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus rhamnosus, Lactobacillus paracasei, and Lactobacillus delbrueckii, and there was no amplification in the negative control.

[0099] In summary, the Lactobacillus jensii primers L. jens-F1 / R1 provided by the present invention have good amplification specificity, have no amplification for a variety of other lactobacilli, and have good qualitative effects.

[0100] Example 2 Rapid quantitative detection method for live Lactobacillus jensenii in microecological live bacteria products

[0101] A method for rapid quantitative detection of live Lactobacillus jensenii in a microecological live bacteria product (composite live bacteria pharmaceutical preparation) (PMA-qPCR method), comprising the following steps:

[0102] (1) Sample pretreatment: Dissolve 0.1 g of the composite live bacterial drug preparation to be tested in 1 mL of PBS to obtain a sample solution to be tested;

[0103] (2) PMA treatment: add 50 μL of 1 mg / mL PMA solution to the sample solution in step (1), mix well, incubate at room temperature in the dark for 20 min, then place the sample solution on ice and photolyze under a 500 W LED lamp for 40 min;

[0104] (3) Genomic DNA extraction: Use the bacterial genomic DNA rapid extraction kit of Sangon Biofactory (Shanghai) Co., Ltd. to extract the genomic DNA of the sample to be tested after treatment in step (2);

[0105] (4) qPCR detection of viable Lactobacillus jensii: The qPCR detection reaction system was prepared as shown in Table 6, the primers used were L. jens-F1 / R1, and the amplification reaction procedure was shown in Table 7;

[0106] (5) Substitute the CT value obtained by qPCR into the standard curve regression equation to determine the number of viable bacteria. The construction of the standard curve is the same as the construction of the viable bacteria standard curve in Example 3 (1).

[0107] Example 3 Effect of PMA concentration on live bacterial detection of Lactobacillus jensenii in PMA-qPCR method

[0108] This example optimizes the PMA concentration in the PMA-qPCR method in Example 2, as follows:

[0109] (1) Construction of live bacteria standard curve: The standard curve of Lactobacillus jensenii was prepared according to the following steps.

[0110] ① Cultivation of Lactobacillus jensenii in logarithmic phase: Use MRS medium to activate Lactobacillus jensenii culture for 8 to 12 hours until the logarithmic growth phase (the bacterial solution at this time should theoretically contain all live bacteria).

[0111] ② Spreading count: Take 100 μL of the cultured Lactobacillus jensenii, dilute it in a 10-fold gradient, and spread 100 μL of the dilution on MRS solid medium. Incubate it in a carbon dioxide incubator at 37°C for 12 h, and calculate the number of viable bacteria based on the number of colonies. It should be noted that in the embodiment of the present invention, the viable count result of 1 mL of logarithmic phase Lactobacillus jensenii is 1x10 8 CFU / mL.

[0112] ③ Genomic DNA extraction: Take 1 mL of Lactobacillus jensenii culture solution in the logarithmic growth period, use the bacterial genomic DNA rapid extraction kit of Sangon Biofactory (Shanghai) Co., Ltd. to extract the genomic DNA, and elute the dissolved DNA solution in a volume of 100 μL.

[0113] ④ DNA gradient dilution: Take 10 μL DNA solution and add it to 90 μL NF water, and then dilute it five times to obtain 1×10 3 ~1×10 8 CFU corresponding to the DNA solution.

[0114] ⑤ Preparation of standard curve: The diluted DNA solution was subjected to qPCR, and the system used was shown in Table 6.

[0115] It should be noted that, in this embodiment, the live bacteria of Lactobacillus jensenii are Y=-3.35X+34.14, r 2 =0.999 (X is the logarithmic value of the number of viable bacteria, Y is the number of amplification cycles, and the detection concentration range is 1x10 3 ~1x10 8 ), the amplification curve is shown in the attached Figure 7 The standard curve and equation are shown in the attached Figure 8 shown.

[0116] (2) Preparation of dead bacteria: Take 1 mL of Lactobacillus jensenii culture solution cultured to the logarithmic phase, place it in a boiling water bath for 5 min, let it stand on ice for 2 min, take 100 μL of the culture solution and spread it on the solid culture medium. If no colonies grow, completely dead bacteria are obtained.

[0117] (3) Add PMA to treat dead bacteria: add 20 μL, 30 μL, 40 μL, and 50 μL of 1 mg / mL PMA solution to 1 mL of dead bacteria solution, incubate in the dark for 20 min, and photolyze under a 500 W LED light for 40 min. The photolysis process should be carried out on ice.

[0118] (4) DNA extraction of dead bacteria after adding PMA: Genomic DNA was extracted using the Bacterial Genomic DNA Rapid Extraction Kit of Sangon Biotech (Shanghai) Co., Ltd., and the dissolved DNA solution was eluted in a volume of 100 μL.

[0119] (5) Fluorescence quantitative PCR detection: The DNA solution of the bacterial solution after PMA treatment was subjected to qPCR detection (the reaction system and reaction procedure are shown in Tables 6 to 7).

[0120] The results are shown in Table 8 and Fig. 9 As shown in the figure, compared with the samples without PMA treatment, the amplification Ct value of qPCR increased by 8 to 10 cycles after PMA treatment, and the amplification inhibition rate of PMA on dead bacteria could reach 99%. It is worth noting that the inhibition effect on dead bacteria after treatment with 50 μL 1 mg / mL PMA solution was the best, and PMA treatment at this concentration did not affect the amplification of live bacteria.

[0121] Table 8 Changes in Ct values ​​of Lactobacillus jensenii after adding different concentrations of PMA

[0122]

[0123] Example 4: Live bacteria count in Lactobacillus jensenii powder samples

[0124] The live bacteria of Lactobacillus jensenii in the Lactobacillus jensenii powder sample were determined using the detection method of Example 2, as follows:

[0125] (1) Weighing and dissolving bacterial powder: A freeze-dried bacterial powder sample containing Lactobacillus jensenii was selected for measurement. 0.1 g of bacterial powder was taken and dissolved in 1 mL of PBS to obtain a bacterial powder solution.

[0126] (2) PMA treatment of bacterial powder: Add 50 μL of 1 mg / mL PMA solution to the bacterial powder solution in step (1), mix well, and incubate at room temperature in the dark for 20 min (to allow PMA to fully penetrate into the dead bacterial cells). Then, place the bacterial powder solution under a 500 W LED lamp for photolysis for 40 min to complete the covalent cross-linking process between the PMA solution and the dead bacterial DNA. Strong light irradiation may cause the temperature of the sample to rise and affect the experimental results. Therefore, the sample was placed on ice to complete the photolysis reaction process.

[0127] (3) Genomic DNA extraction: Use the Bacterial Genomic DNA Rapid Extraction Kit of Sangon Biotech (Shanghai) Co., Ltd. to extract the genomic DNA from the bacterial solution in step (2), and elute the dissolved DNA solution in a volume of 100 μL.

[0128] (5) Detection of the number of live bacteria in the Lactobacillus jensenii powder sample: Since the DNA concentration of 0.1 g powder is relatively high, qPCR detection was performed after dilution 100 times, as follows: 1 μL of the DNA solution in step (4) was taken, diluted 100 times with PBS, and the diluted DNA solution was subjected to qPCR detection (the reaction system is shown in Table 6, and the reaction procedure is shown in Table 7).

[0129] At the same time, the bacterial powder was diluted and coated on the plate to calculate the number of viable bacteria to verify the accuracy of the test results. The specific process was as follows: 1g of bacterial powder was weighed and dissolved in PBS, 100 μL was taken from it for dilution and then coated on MRS culture medium, and cultured in a carbon dioxide incubator at 37°C for 12 hours, and the number of viable bacteria was calculated based on the number of colonies. In this embodiment, the number of viable bacteria of Lactobacillus jensenii powder was calculated by coating, and the number of viable bacteria was 9.12×10 10 CFU / g.

[0130] The test results showed that the Ct value of 0.1g bacterial powder sample was 8.73 ( Fig.10 ), substitute into the standard equation Y = -3.35X + 34.14, r 2=0.999 (Example 3), the number of viable bacteria in the bacterial powder was calculated to be 7.83×10 10 CFU / g, which is basically consistent with the results of the coating plate counting.

[0131] Example 5 Quantitative Detection of Lactobacillus jensenii in Composite Live Bacteria Preparation

[0132] The live bacteria of Lactobacillus jensenii in the composite live bacteria preparation (quadruple lactobacillus preparation) were determined using the detection method of Example 2, as follows:

[0133] (1) Composite live bacteria preparation sample: 1 g of a four-component lactobacillus preparation (including Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus johnsonii and Lactobacillus reuteri) was selected for use in this example.

[0134] (2) Sample weighing and dissolution: Take 0.1 g of the powder of the quadruple live bacteria preparation and dissolve it in 0.9 mL of PBS to obtain a powder solution;

[0135] (3) PMA treatment: add 50 μL of 1 mg / mL PMA solution to the bacterial powder solution in step (2), mix well, incubate at room temperature in the dark for 20 min, then place the sample solution on ice and photolyze under a 500 W LED lamp for 40 min;

[0136] (4) Genomic DNA extraction: Use the bacterial genomic DNA rapid extraction kit of Sangon Biofactory (Shanghai) Co., Ltd. to extract the genomic DNA from the bacterial powder solution treated in step (3);

[0137] (5) qPCR detection of the number of live Lactobacillus jensenii: Since the DNA concentration of 0.1 g bacterial powder is relatively high, qPCR detection is performed after dilution 100 times, as follows: 1 μL of the DNA solution in step (4) is taken, and 100 times dilution is performed using PBS, and the diluted DNA solution is subjected to qPCR detection (the reaction system is shown in Table 6, and the reaction procedure is shown in Table 7);

[0138] (6) Substitute the CT value obtained by qPCR detection into the standard curve (Example 3) to determine the number of viable bacteria.

[0139] At the same time, the viable count of Lactobacillus jensenii in the quadruple antibacterial preparation was calculated by diluting the bacterial powder on the plate to verify the accuracy of the test results. The specific process is as follows: 1g of the bacterial powder of the quadruple live bacterial preparation was weighed and dissolved and mixed with PBS, 100 μL was taken from it for gradient dilution and then applied to the Lactobacillus jensenii selective culture medium, and cultured for 48 hours in a carbon dioxide incubator (oxygen concentration ≤ 1%) at 37°C, and the viable count was calculated based on the colony count. In this embodiment, the viable count of the Lactobacillus jensenii powder is 1.54×10 11 CFU / g.

[0140] qPCR test results Fig.11 As shown, the Ct value of 0.1g bacterial powder sample is 7.27. Substituting into the standard equation Y = -3.35X + 34.14, r 2 =0.999, the number of viable bacteria in the powder is calculated to be 1.05×10 11 CFU / g, close to the result of plate counting.

[0141] In summary, the PMA-qPCR method provided in Example 2 of the present invention has a good live bacterial count result for Lactobacillus jensenii in bacterial powder, and can be applied to the live bacterial count of Lactobacillus jensenii in composite live bacterial pharmaceutical preparations.

[0142] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A reagent comprising a primer pair for detecting Lactobacillus jensii, wherein the primer pair comprises an upstream primer L.jens-F1 and a downstream primer L.jens-R1, wherein: The nucleotide sequence of the upstream primer L.jens-F1 is: 5'-AAGTCGAGCGAGCTTGCCTATAGA-3', and the nucleotide sequence of the downstream primer L.jens-R1 is: 5'-CTTCTTTCATGCGAAAGTAGC-3'.

2. A detection kit comprising the reagent according to claim 1.

3. The detection kit according to claim 2, characterized in that The kit also includes propidium bromide, dNTP, Mg 2+ , enzyme, SYBR Green I.

4. Use of the reagent according to claim 1 or the detection kit according to claim 2 or 3 in any one of (1) to (4): (1) Detection of Lactobacillus jensenii; (2) preparing a product for detecting Lactobacillus jensenii; (3) quantitatively detecting the number of viable Lactobacillus jensenii in the sample to be tested; (4) preparing a product for quantitatively detecting the number of viable Lactobacillus jensenii in a sample to be tested.

5. The use according to claim 4, characterized in that: The samples to be tested described in (3) to (4) include microecological live bacteria products.

6. A method for quantitatively detecting the number of live Lactobacillus jensenii in a microecological live bacteria product, comprising the step of using the detection kit described in claim 3 to detect the microecological live bacteria product to be tested.

7. The quantitative detection method according to claim 6, characterized in that: The quantitative detection method specifically comprises the following steps: 1) Sample pretreatment: dissolving the microecological live bacteria product to be tested to obtain a solution of the microecological live bacteria product to be tested; 2) PMA treatment: the solution of the microecological live bacteria product to be tested in step 1) is mixed with PMA, reacted in the dark, and photolyzed under LED light; 3) Genomic DNA extraction: DNA is extracted from the microecological live bacteria product to be tested after PMA treatment in step 2); 4) qPCR detection: using the genomic DNA from step 3) as a template, and performing qPCR detection using the primer pair described in claim 1; 5) Substituting the CT value detected by qPCR in step 4) into the standard curve regression equation to obtain the number of live Lactobacillus jensenii in the microecological live bacteria product to be tested.

8. The quantitative detection method according to claim 7, characterized in that: The final concentration of PMA in step 2) is 15-50 μg / mL.

9. The quantitative detection method according to claim 7, characterized in that: The light-protection reaction time in step 2) is 15 to 30 minutes; and / or the LED light irradiation photolysis time is 30 to 50 minutes.

10. The quantitative detection method according to any one of claims 7 to 9, characterized in that: The method for constructing the standard curve comprises: gradiently diluting a DNA solution of a standard strain of Lactobacillus jensenii to obtain a standard solution, performing qPCR detection using the primers described in claim 1, and drawing a standard curve with the logarithmic value of the number of viable bacteria of the standard strain of Lactobacillus jensenii in the standard solution as the abscissa and the CT value as the ordinate.

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