Primer for detecting lactobacillus acidophilus and application thereof

Abstract

The invention relates to a primer for detecting lactobacillus acidophilus and application thereof, the primer comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer comprises a sequence as shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer comprises a sequence as shown in SEQ ID NO: 2. The primer disclosed by the invention is high in specificity and low in detection limit, and lactobacillus acidophilus in a product can be specifically identified.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and relates to a primer for detecting Lactobacillus acidophilus and application thereof. Background Art

[0002] "Probiotic" sanitary napkins are sanitary napkin products that use a specific production process to introduce inactivated probiotics (postbiotics). "Inactivated probiotics (postbiotics)" usually refers to a probiotic product that has been inactivated. In this product, the probiotics are treated with special methods, such as heat treatment or chemical treatment, to inactivate them, but retain their structure, which can extend the shelf life of the probiotics. Through the inherent characteristics and advantages of postbiotics, the purpose of enhancing the efficacy of sanitary napkins is achieved. This type of product claims to be able to reduce odor, relieve itching, prevent infection, etc., and may be beneficial to the health of women's private parts.

[0003] Due to the low content and inactivity of probiotics in sanitary napkins, it is impossible to use methods for identifying active probiotics, such as sequencing after cultivation, to identify strains. There is currently no method that can effectively identify probiotic strains. The strains currently added to sanitary napkins include Lactobacillus acidophilus. Lactobacillus acidophilus belongs to the genus Lactobacillus, a Gram-positive bacillus, with a rounded end of the rod, mainly present in the small intestine, releasing lactic acid, acetic acid and some antibiotics that work on harmful bacteria, but the antibacterial effect is relatively weak. Lactobacillus acidophilus belongs to beneficial bacteria. As a probiotic, it is an important microorganism for survival and colonization in the intestines of humans and animals. Because the bacteria are non-toxic and harmless, they are often made into bioactive foods and microecological preparations, which can improve the balance of animal intestinal microecology, promote growth and prevent diseases; they can also tolerate acid and bile, eliminate lactose intolerance, lower cholesterol, inhibit and treat diarrhea and enhance body immunity.

[0004] The detection of probiotics is of great significance for the development and production of new foods and daily chemical raw materials, as well as the quality and safety control of subsequent products. Therefore, it is very important to provide a primer and detection method for rapid and specific detection of Lactobacillus acidophilus. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a primer for detecting Lactobacillus acidophilus and application thereof.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a primer for detecting Lactobacillus acidophilus, the primer comprising an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer comprising the sequence shown in SEQ ID NO:1, and the nucleotide sequence of the downstream primer comprising the sequence shown in SEQ ID NO:2.

[0008] SEQ ID NO: 1: CCTACCAAGGTGATGATAGG.

[0009] SEQ ID NO: 2: TAATCCTGTTTGCTACCCAC.

[0010] The primers of the present invention can specifically detect Lactobacillus acidophilus, and can quickly and accurately identify probiotics in products through a PCR method, with strong specificity, low detection limit, simple operation, low requirements on equipment, and can be widely used.

[0011] In a second aspect, the present invention provides a use of the primers for detecting Lactobacillus acidophilus according to the first aspect in preparing a product for detecting Lactobacillus acidophilus.

[0012] In a third aspect, the present invention provides a kit for detecting Lactobacillus acidophilus, wherein the kit comprises the primers for detecting Lactobacillus acidophilus according to the first aspect.

[0013] Preferably, the kit further comprises a positive control, a negative control, PCRmix and ddH2O.

[0014] Preferably, the PCR mix includes DNA polymerase, PCR buffer, Mg 2+ and dNTPs.

[0015] Preferably, the positive control is Lactobacillus acidophilus genomic DNA, and the negative control is ddH2O.

[0016] Preferably, the Lactobacillus acidophilus includes the Lactobacillus acidophilus LA88 strain with a preservation number of CGMCC No.24109, the LA85 strain with a preservation number of CGMCC No.1.12735, the Lactobacillus acidophilus SY23 strain with a preservation number of CGMCC No.30465, the Lactobacillus acidophilus LA05 strain with a preservation number of CGMCC No.23546, the Lactobacillus acidophilus BNCC185344 strain, the Lactobacillus acidophilus BNCC138598 strain, the Lactobacillus acidophilus CICC 20244 strain or the Lactobacillus acidophilus CICC 6075 strain.

[0017] In a fourth aspect, the present invention provides a use of the primer for detecting Lactobacillus acidophilus according to the first aspect or the kit according to the third aspect in detecting Lactobacillus acidophilus.

[0018] In a fifth aspect, the present invention provides a method for detecting Lactobacillus acidophilus, the method comprising:

[0019] The DNA of the sample to be tested is extracted, and PCR amplification is performed using the primers described in the first aspect as amplification primers. If a DNA band of 524 bp appears, the sample to be tested contains Lactobacillus acidophilus.

[0020] Preferably, the PCR amplification system comprises: the primers described in the first aspect, PCR mix, DNA template and water.

[0021] DNA template refers to the DNA of the sample to be tested or the positive control.

[0022] Preferably, the PCR amplification procedure includes:

[0023] (1) Preheat at 94-98°C (e.g., 94°C, 95°C, 96°C, 97°C, 98°C, etc.) for 4-6 min (e.g., 4 min, 4.2 min, 4.4 min, 4.6 min, 4.8 min, 5 min, 5.2 min, 5.4 min, 5.6 min, 5.8 min, 6 min, etc.);

[0024] (2) denaturation at 92-95° C. (e.g., 92° C., 93° C., 94° C., 95° C., etc.) for 30-60 s (e.g., 30 s, 32 s, 35 s, 38 s, 40 s, 42 s, 45 s, 48 ​​s, 50 s, 52 s, 55 s, 58 s, 60 s, etc.);

[0025] (3) annealing at 55-60° C. (e.g., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., etc.) for 30-60 seconds (e.g., 30 seconds, 32 seconds, 35 seconds, 38 seconds, 40 seconds, 42 seconds, 45 seconds, 48 ​​seconds, 50 seconds, 52 seconds, 55 seconds, 58 seconds, 60 seconds, etc.);

[0026] (4) 70-75° C. (e.g., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., etc.) for extension for 50-60 s (e.g., 50 s, 51 s, 52 s, 53 s, 54 s, 55 s, 56 s, 57 s, 58 s, 59 s, 60 s, etc.);

[0027] (5) 70-75° C. (e.g., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., etc.) for extension for 4-7 min (e.g., 4 min, 4.2 min, 4.5 min, 4.8 min, 5 min, 5.2 min, 5.5 min, 5.8 min, 6 min, 6.2 min, 6.5 min, 6.8 min, 7 min, etc.);

[0028] Wherein (2) to (4) are repeated 25 to 35 times (e.g., 25 times, 26 times, 27 times, 28 times, 29 times, 30 times, 31 times, 32 times, 33 times, 34 times, 35 times, etc.).

[0029] Other specific point values ​​within the above numerical range can be selected and will not be described in detail here.

[0030] Preferably, the sample to be tested includes a sanitary napkin.

[0031] The method of the present invention fills the gap in the prior art by extracting the DNA of probiotics in sanitary napkins and using specific primers of specific probiotic strains for PCR amplification. The method can be used to quickly and accurately identify probiotics in probiotic sanitary napkins with strong specificity.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] Since what is added to the probiotic sanitary napkin is inactivated bacteria, it is impossible to use the method of identifying active probiotics such as sequencing after cultivation to identify the strains, and the probiotic content added to the sanitary napkin is relatively small (about 0.1%), so there is no detection technology in the field of probiotic sanitary napkins at present. The primers of the present invention can specifically detect Lactobacillus acidophilus, and the probiotics in the product can be quickly and accurately identified by the PCR method, with strong specificity and low detection limit. At the same time, the minimum detection limit of the method in the probiotic sanitary napkin is 0.01%, with high sensitivity and stable method. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the gel electrophoresis result of amplification effect of different primers.

[0035] Figure 2 This is the result of agarose gel electrophoresis for detecting different Lactobacillus acidophilus strains.

[0036] Figure 3 This is the result of agarose gel electrophoresis for specific detection of Lactobacillus acidophilus.

[0037] Figure 4 This is the result of agarose gel electrophoresis for testing sanitary napkins with different added amounts of Lactobacillus acidophilus. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0039] The Lactobacillus acidophilus LA88 strain involved in the following content is classified and named Lactobacillus acidophilus, the preservation number is CGMCC No.24109, the preservation date is December 15, 2021, the preservation unit is the General Microbiology Center of China Culture Collection Administration, and the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0040] The Lactobacillus acidophilus LA85 strain involved in the following content is classified and named Lactobacillus acidophilus, with a preservation number of CGMCC No.1.12735, a preservation date of July 20, 2020, and a preservation unit of the General Microbiology Center of the China Culture Collection Administration. The preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0041] The Lactobacillus acidophilus SY23 strain involved in the following content is classified and named Lactobacillus acidophilus, with a preservation number of CGMCC No.30465, a preservation date of April 28, 2024, and a preservation unit of the General Microbiology Center of the China Culture Collection Administration. The preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0042] The Lactobacillus acidophilus LA05 strain involved in the following content is classified and named Lactobacillus acidophilus, with a preservation number of CGMCC No.23546, a preservation date of October 9, 2021, and a preservation unit of the General Microbiology Center of the China Culture Collection Administration. The preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0043] The Lacticaseibacillus paracasei FB97 strain involved in the following content is classified and named Lacticaseibacillus paracasei, with a preservation number of CGMCC No. 30464, a preservation date of April 28, 2024, and a preservation unit of the General Microbiology Center of the China Culture Collection Administration. The preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0044] The Lactobacillus salivarius LS97 strain involved in the following content is classified and named Lactobacillus salivarius, with a deposit number of CGMCC No.16922, a deposit date of December 10, 2018, and a deposit unit of the General Microbiology Center of the China Culture Collection Administration. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0045] The components of the MRS culture medium involved in the following content are: yeast powder 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, dipotassium hydrogen phosphate trihydrate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, Tween-80 1 mL / L and water as the balance, pH is about 6.2-6.4, and sterilized at 121°C for 20 min.

[0046] The DNA extraction kit involved in the following content was purchased from Shanghai Mubai Biotechnology, patent number CN112195177B.

[0047] Example 1

[0048] Screening of specific primers for Lactobacillus acidophilus

[0049] Download multiple known Lactobacillus acidophilus tuf gene sequences from the NCBI database, screen out the conservative bases in the target sequence that are specific to non-target strains, and mark them. According to the primer design principles, follow the base matching rules during the 3' end extension process to ensure that the non-target sequence bases do not match and thus terminate the amplification. At the same time, take full account of the influencing factors such as the dimer and hairpin structure of the primers, and use Primer Premier 5.0 for auxiliary design. Finally, amplified fragments of 524bp, 407bp, and 244bp were designed. The designed primers were compared on the NCBI database primer Blast to verify specificity, and the screened primer sequences are as follows:

[0050] Primer pair 1

[0051] SEQ ID NO: 1: CCTACCAAGGTGATGATAGG.

[0052] SEQ ID NO: 2: TAATCCTGTTTGCTACCCAC.

[0053] Primer pair 2

[0054] SEQ ID NO: 3: CGTATCTGACTGCTTAGACG.

[0055] SEQ ID NO: 4: TCGTGAGATGTTGGGTTAAG.

[0056] Primer pair 3

[0057] SEQ ID NO:5: CTTTCAATCCAGCCGGTAACTTG.

[0058] SEQ ID NO: 6: ATGATGCCACACATAGCCTCC.

[0059] Subsequently, three pairs of primers were used to amplify Lactobacillus acidophilus, Lactobacillus paracasei, and Lactobacillus salivarius, and its amplification effect and specificity were preliminarily verified. Following bacterial strains Lactobacillus acidophilus LA88, Lactobacillus paracasei FB97 (CGMCC No.30464), Lactobacillus salivarius LS97 (CGMCC No.16922) were cultivated, and 1mL was taken and was in the logarithmic growth phase bacterial liquid, and was placed in a 1.5mL centrifuge tube. 12000rpm was centrifuged for 1min, and supernatant was abandoned. DNA extraction kit was used to extract (Shanghai Mubai Biology, CN112195177B), and after extraction was completed, -20 ℃ were preserved for standby use.

[0060] The PCR amplification system is shown in Table 1.

[0061] Table 1

[0062] Additives Addition amount 2 × PhantaMaxMasterMix 12.5μL Upstream primer 1μL Downstream primer 1μL Template DNA 100ng <![CDATA[ddH2O]]> Make up to 25 μL

[0063] PCR amplification procedure:

[0064] (1) Preheat at 95°C for 5 min;

[0065] (2) Denaturation at 94°C for 30 seconds;

[0066] (3) Annealing at 57 °C for 30 s;

[0067] (4) 72°C extension for 50 s;

[0068] (2)-(4) were cycled 30 times;

[0069] (5) Extension at 72°C for 5 min.

[0070] 2% agarose gel electrophoresis, voltage 180V, electrophoresis time 20min. Gel electrophoresis results are as follows Figure 1 As shown, MARKER is 1500bp; Lane 1: negative control (ddH2O), Lane 2: primer pair 1 amplifies LA88, Lane 3: primer pair 1 amplifies FB97, Lane 4: primer pair 1 amplifies LS97, Lane 5: primer pair 2 amplifies LA88, Lane 6: primer pair 2 amplifies FB97, Lane 7: primer pair 2 amplifies LS97, Lane 8: primer pair 3 amplifies LA88, Lane 9: primer pair 3 amplifies FB97, Lane 10: primer pair 3 amplifies FB97. Among the above three pairs of primers, only primer pair 1 has a good amplification effect and strong specificity. Although primer pairs 2 and 3 have good amplification effects, their specificity is not strong. Therefore, primer pair 1 was selected to carry out the following verification experiments.

[0071] Example 2

[0072] Detection of different Lactobacillus acidophilus strains using specific primers

[0073] By following bacterial strains Lactobacillus acidophilus LA88, LA85, LA05, SY23, commercially available strains 1. (Beijing North Na Biological Lactobacillus acidophilus BNCC185344), commercially available strains 2. (Beijing North Na Biological Lactobacillus acidophilus BNCC138598), commercially available strains 3. (Guangdong Province Microbiological Culture Collection Center Lactobacillus acidophilus CICC 20244), commercially available strains 4. (China Industrial Microbiological Culture Collection Management Center Lactobacillus acidophilus CICC 6075) are cultivated, and 1mL is in logarithmic growth phase bacterial liquid, and is placed in 1.5mL centrifuge tube.12000rpm centrifugal 1min, abandons supernatant.Use DNA extraction kit to extract (Shanghai Mubai Biological, CN112195177B), after extraction is complete,-20 ℃ are preserved for standby use.

[0074] The PCR amplification system is shown in Table 2.

[0075] Table 2

[0076]

[0077]

[0078] PCR amplification procedure:

[0079] (1) Preheat at 95°C for 5 min;

[0080] (2) Denaturation at 94°C for 30 seconds;

[0081] (3) Annealing at 57 °C for 30 s;

[0082] (4) 72°C extension for 50 s;

[0083] (2)-(4) were cycled 30 times;

[0084] (5) Extension at 72°C for 5 min.

[0085] 2% agarose gel electrophoresis, voltage 180V, electrophoresis time 20min. Gel electrophoresis results are shown in the attached Figure 2 As shown, MARKER is 1500bp; Lane 1: negative control (ddH2O), Lane 2: LA88, Lane 3: LA85, Lane 4: LA05, Lane 5: SY23, Lane 6: commercial strain ①, Lane 7: commercial strain ②, Lane 8: commercial strain ③, Lane 9: commercial strain ④. The above-mentioned Lactobacillus acidophilus strains were successfully amplified after PCR amplification with primer pair 1 in Example 1, and there were bands of corresponding sizes (524bp) in the gel electrophoresis diagram, indicating that this primer can detect different strains of Lactobacillus acidophilus.

[0086] Example 3

[0087] Detection of primer specificity

[0088] The following strains of Lactobacillus rhamnosus (CICC 22173), Lactobacillus paracasei (CICC 25261), Lactobacillus casei (CICC 6114), Lactobacillus mucosa reuteri (CICC 6119), Lactobacillus salivarius (CICC 23179), Lactobacillus gasseri (BNCC 135322), Lactobacillus crispatus (BNCC 135057), Bifidobacterium longum subsp. longum (BNCC 187962), Escherichia coli (ATCC 8099), Streptococcus mutans (BNCC336931), Staphylococcus epidermidis (BNCC102555), Akkermansia muciniphila (BNCC323275) were cultured, and 1 mL of bacterial solution in the logarithmic growth phase was taken and placed in a 1.5 mL centrifuge tube. The mixture was centrifuged at 12000 rpm for 1 min, and the supernatant was discarded. Use a DNA extraction kit to extract and store at -20℃ for future use.

[0089] The PCR amplification system is shown in Table 3.

[0090] Table 3

[0091] Additives Addition amount 2 × PhantaMaxMasterMix 12.5μL Upstream primer 1μL Downstream primer 1μL Template DNA 200ng <![CDATA[ddH2O]]> Make up to 25 μL

[0092] PCR amplification procedure:

[0093] (1) Preheat at 95°C for 5 min;

[0094] (2) Denaturation at 94°C for 30 seconds;

[0095] (3) Annealing at 57°C for 30 seconds;

[0096] (4) 72°C extension for 50 s;

[0097] (2)-(4) were cycled 30 times;

[0098] (5) Extension at 72°C for 5 min.

[0099] 2% agarose gel electrophoresis, voltage 180V, electrophoresis time 20min. Gel electrophoresis results are shown in the attached Figure 3 shown.

[0100] As attached Figure 3 As shown, the above strains had no obvious bands after PCR amplification with primer pair 1 in Example 1, indicating that this primer has strong specificity. Wherein, MARKER is 1500bp; in the figure, lane 1: positive control (Lactobacillus acidophilus LA88), lane 2: negative control (ddH2O); lane 3: Lactobacillus rhamnosus, lane 4: Lactobacillus paracasei, lane 5: Lactobacillus casei, lane 6: Lactobacillus mucosa reuteri, lane 7: Lactobacillus salivarius, lane 8: Lactobacillus gasseri, lane 9: Lactobacillus crispatus, lane 10: Bifidobacterium longum subsp. longum, lane 11: Escherichia coli, lane 12: Streptococcus mutans, lane 13: Staphylococcus epidermidis, lane 14: Akkermansia muciniphila.

[0101] Example 4

[0102] sample:

[0103] (1) Commercially available probiotic sanitary napkins containing Lactobacillus acidophilus LA88 strain.

[0104] (2) Sanitary napkins with different probiotics concentrations are prepared by spraying, specifically including: inoculating the acidophilic LA88 strain into MRS medium at a concentration of 2%, culturing at 37°C for 24 hours to obtain a bacterial solution, adjusting the pH of the bacterial solution to 7.0 using NaOH, centrifuging at 5000rpm for 5 minutes, discarding the supernatant, and collecting the centrifugal precipitate (bacterial mud). Add a heat shock protective agent to the bacterial mud in proportion (bacterial mud: 30% trehalose solution = 1:1, W / W). Preheat the obtained mixture to 80°C, then stir continuously at 80°C and 200rpm for 30 minutes to complete inactivation, and obtain an inactivated emulsion. Spray dry the inactivated emulsion and crush it to obtain an inactivated bacterial powder, which is stored at room temperature and protected from light for later use. Evenly place the spray-dried powder on the inner absorbent layer of the sanitary napkin to obtain a probiotic sanitary napkin containing Lactobacillus acidophilus.

[0105] Detection method:

[0106] (1) The probiotic DNA in the probiotic sanitary napkins was fully released by pretreatment, and the number of sanitary napkins used in each group was the same. Specifically, the tissue was ground by a tissue grinder, and then a DNA lysis solution was added and shaken to dissolve. The raw materials for the preparation of the DNA lysis solution included guanidine isothiocyanate with a molar concentration of 1M, N-lauroyl sarcosine sodium salt with a mass percentage of 0.04%, and a pH buffer solution.

[0107] (2) Use a DNA extraction kit to collect and purify the DNA, use an instrument to test the DNA concentration, and store it at 4°C for later use if it passes the test.

[0108] (3) PCR amplification was performed using the primers described in SEQ ID NO: 1 and SEQ ID NO: 2, and gel electrophoresis was performed to verify the amplification. If a band of corresponding size (524 bp) was observed, the corresponding bacterial species was detected.

[0109] The PCR amplification system is shown in Table 4.

[0110] Table 4

[0111] Additives Addition amount 2 × PhantaMaxMasterMix 12.5μL Upstream primer 1μL Downstream primer 1μL Template DNA 50~400ng <![CDATA[ddH2O]]> Make up to 25 μL

[0112] PCR amplification procedure:

[0113] (1) Preheat at 95°C for 5 min;

[0114] (2) Denaturation at 94°C for 30 seconds;

[0115] (3) Annealing at 57 °C for 30 s;

[0116] (4) 72°C extension for 50 s;

[0117] (2)-(4) were cycled 30 times;

[0118] (5) Extension at 72°C for 5 min.

[0119] 2% agarose gel electrophoresis, voltage 180V, electrophoresis time 20min. Gel electrophoresis results are shown in the attached Figure 4 shown.

[0120] The results are attached Figure 4As shown, the swimming lane on the left side of swimming lane 1 is MARKER, swimming lane 1 is a negative control, swimming lane 2 is a commercially available probiotic sanitary towel, swimming lane 3 is a sanitary towel for adding 1% lactobacillus acidophilus, swimming lane 4 is a sanitary towel for adding 0.1% lactobacillus acidophilus, swimming lane 5 is a sanitary towel for adding 0.01% lactobacillus acidophilus, swimming lane 6 is a sanitary towel for adding 0.001% lactobacillus acidophilus, and swimming lane 7 is a sanitary towel for adding 0.0001% lactobacillus acidophilus. As can be seen from the figure, when adding a bacterial strain with a concentration of 0.01% in a sanitary towel, it can still be detected by the method. Primer and method of the present invention are highly sensitive.

[0121] The applicant declares that the present invention illustrates a primer for detecting Lactobacillus acidophilus and its application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0122] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0123] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A primer for detecting Lactobacillus acidophilus, characterized in that: The primers include an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer includes the sequence shown in SEQ ID NO:1, and the nucleotide sequence of the downstream primer includes the sequence shown in SEQ ID NO:

2.

2. Use of the primer for detecting Lactobacillus acidophilus according to claim 1 in preparing a product for detecting Lactobacillus acidophilus.

3. A kit for detecting Lactobacillus acidophilus, characterized in that: The kit comprises the primers for detecting Lactobacillus acidophilus according to claim 1.

4. The kit for detecting Lactobacillus acidophilus according to claim 3, characterized in that: The kit also includes a positive control, a negative control, a PCR mix and ddH2O.

5. The kit for detecting Lactobacillus acidophilus according to claim 4, characterized in that: The positive control is Lactobacillus acidophilus genomic DNA, and the negative control is ddH2O; Preferably, the Lactobacillus acidophilus includes the Lactobacillus acidophilus LA88 strain with a preservation number of CGMCC No.24109, the LA85 strain with a preservation number of CGMCC No.1.12735, the Lactobacillus acidophilus SY23 strain with a preservation number of CGMCC No.30465, the Lactobacillus acidophilus LA05 strain with a preservation number of CGMCC No.23546, the Lactobacillus acidophilus BNCC185344 strain, the Lactobacillus acidophilus BNCC138598 strain, the Lactobacillus acidophilus CICC 20244 strain or the Lactobacillus acidophilus CICC 6075 strain.

6. Use of the primer for detecting Lactobacillus acidophilus according to claim 1 or the kit according to any one of claims 3 to 5 in detecting Lactobacillus acidophilus.

7. A method for detecting Lactobacillus acidophilus, characterized in that: The method comprises: Extract DNA from the sample to be tested, and perform PCR amplification using the primers described in claim 1 as amplification primers. If a 524 bp DNA band appears, the sample to be tested contains Lactobacillus acidophilus.

8. The method according to claim 7, characterized in that The PCR amplification system comprises: the primers according to claim 1, PCR mix, a DNA template and water.

9. The method according to claim 7 or 8, characterized in that: The procedure of the PCR amplification includes: (1) Preheat at 94-98℃ for 4-6 minutes; (2) Denaturation at 92-95°C for 30-60 seconds; (3) Annealing at 55-60°C for 30-60 seconds; (4) 70-75°C for 50-60 seconds; (5) 70-75°C for 4-7 min; (2)-(4) are repeated 25-35 times.

10. The method according to any one of claims 7 to 9, characterized in that: The sample to be tested includes a sanitary napkin.

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