Rapid detection method and application of tialivin source bacteria

Abstract

The invention relates to a rapid detection method for a Tiivivin source bacterium and application of the rapid detection method for the Tiivivin source bacterium. The detection method comprises the following steps: (1) carrying out enrichment culture on a sample to be detected in enrichment liquid, centrifugally collecting thalli subjected to enrichment culture, and carrying out DNA extraction on the thalli; (2) carrying out PCR (Polymerase Chain Reaction) amplification on the extracted DNA, and determining whether the to-be-detected sample contains the viveromulin source bacteria or not according to an amplification result; the target gene subjected to PCR (Polymerase Chain Reaction) amplification is an A8H26RS01130 gene. According to the detection method disclosed by the invention, the A8H26RS01130 gene is used as a target gene for PCR amplification, so that a small amount of pollution of the viveromulin source bacteria can be accurately identified within 24 hours, and an efficient and reliable quality control scheme can be provided for the cosmetic industry.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a rapid detection method for a bacterium-derived fungus of Rhizoctonia solani and an application thereof. Background Art

[0002] In recent years, the issue of microbial contamination in cosmetics has attracted much attention, among which the contamination risk of Pluralibacter gergoviae has become increasingly prominent. Although this bacterium is not included in the mandatory testing items of the "Technical Specifications for Safety of Cosmetics", as a conditional pathogen, it can cause urinary tract and respiratory tract infections in people with weakened immunity, posing a potential public health threat. Studies have shown that Pluralibacter gergoviae exhibits significant tolerance to traditional preservatives (such as parabens), and is more common in cosmetics such as facial masks and creams, and its harmfulness cannot be ignored.

[0003] At present, the detection of cosmetic microorganisms mainly relies on the traditional plate culture method, which has a long culture cycle and cannot meet the timeliness requirements of production quality control. It also relies on manual interpretation, which is prone to introduce subjective errors. In addition, the current standards do not include specific detection methods for the source bacteria of Japanese bacteria. Although commercially available kits can detect Enterobacteriaceae, their targeted genes are genes that produce carbapenemase, and lack specific detection of the source bacteria of Japanese bacteria. More importantly, compared with other pathogenic strains, the toxicity of the source bacteria of Japanese bacteria is weaker, and companies and regulatory authorities have insufficient prevention and control and attention to it. These problems have led to the inability to timely and accurately detect the source bacteria of Japanese bacteria in cosmetics, which may pose a potential threat to consumers' health.

[0004] The nucleic acid mass spectrometry method disclosed in CN116287342A can simultaneously detect 4 Enterobacteriaceae and 8 carbapenem-resistant genes, but lacks specific identification of the source bacteria of Rhizoctonia solani.

[0005] CN105803064A discloses a detection system based on LAMP technology, which improves the detection efficiency by screening the common gene sequences of pathogenic Enterobacteriaceae, but its target genes are limited to Enterobacteriaceae and still cannot meet the detection needs of Japanese bacterial source bacteria.

[0006] CN116790778A discloses a dual qPCR detection method based on the housekeeping genes (gyrB, infB, ropB and atpD) of the source bacteria of the Rhizoctonia solani. However, multiple amplification may introduce cross-reactions and affect the detection specificity. In addition, the influence of the DNA extraction process on the detection results of the source bacteria of the Rhizoctonia solani is not involved, making it difficult to achieve rapid and high-throughput industrial-grade applications.

[0007] In summary, how to develop a rapid, highly sensitive and specific detection method for detecting the contamination of Japanese gout bacteria in cosmetics has become one of the technical problems that need to be solved urgently. Summary of the invention

[0008] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a rapid detection method for the source bacteria of Japanese gowwedo bacteria and its application. The detection method involved in the present invention uses the A8H26_RS01130 gene as the target gene for PCR amplification, can accurately identify a small amount of contamination of the source bacteria of Japanese gowwedo bacteria within 24 hours, and can provide an efficient and reliable quality control solution for the cosmetics industry.

[0009] In a first aspect, the present invention provides a method for rapid detection of bacterial source bacteria of Japan Gouweiduo, and the detection method comprises the following steps:

[0010] (1) Take the sample to be tested and culture it in the enrichment solution, collect the enriched bacteria by centrifugation, and extract DNA from the bacteria.

[0011] (2) Perform PCR amplification on the extracted DNA, and determine whether the sample to be tested contains the source bacteria of the Japanese gout bacteria based on the amplification results.

[0012] The target gene for PCR amplification is the A8H26_RS01130 gene (encoding a hypothetical protein, GeneBank accession number: NZ_CP020388).

[0013] The present invention creatively selects the A8H26_RS01130 gene as the target gene for PCR amplification, and is verified by BLAST comparison to be unique to the source bacteria of the Japanese Gouweiduo bacteria. The gene is highly conserved in the source bacteria of the Japanese Gouweiduo bacteria and has no homologous sequence, which avoids cross-reaction and improves the specificity of detection.

[0014] At the same time, the present invention enriches the sample to be tested in the enrichment solution before DNA extraction of the bacteria, which can significantly improve the enrichment efficiency of low-concentration bacteria and reduce the detection limit.

[0015] Preferably, in step (1), the mass volume ratio of the sample to be tested to the enrichment solution is 1:(50-150) g / mL.

[0016] The specific point values ​​between 50-150 can be selected as 50mL, 60mL, 70mL, 80mL, 90mL, 100mL, 110mL, 120mL, 130mL, 140mL, 150mL, etc.

[0017] In the present invention, the mass ratio of the sample to be tested to the enrichment solution is controlled at 1:(50-150), which can be beneficial to the growth of specific Rigouweiduo bacteria source bacteria and increase the bacterial concentration of bacteria in the enrichment solution after enrichment culture.

[0018] Preferably, the enrichment solution in step (1) comprises SCDLP culture medium.

[0019] Preferably, the enrichment solution in step (1) further comprises histidine.

[0020] In the present invention, histidine is added to the enrichment solution, which can reduce the adverse effects of interfering substances such as preservatives in the sample to be tested on the enrichment effect of the Japanese Gouweiduo bacterial source bacteria.

[0021] Preferably, the amount of histidine added to the enrichment solution is 3.0-6.0 g / L.

[0022] Among them, the specific point values ​​in 3.0-6.0g / L can be selected as 3.0g / L, 3.5g / L, 4.0g / L, 4.5g / L, 5.0g / L, 5.5g / L, 6.0g / L, etc.

[0023] Preferably, the DNA extraction in step (1) comprises the following steps:

[0024] (a) The bacterial cells are mixed with a lysis solution and mechanically disrupted to perform a lysis reaction.

[0025] (b) The product of bacterial lysis is adsorbed on magnetic beads, washed, and eluted to obtain DNA.

[0026] In the present invention, during the DNA extraction process, mechanical disruption can facilitate the release of nucleic acids in bacteria.

[0027] Preferably, the temperature of the cleavage reaction in step (a) is 60-70° C. and the time is 15-30 min.

[0028] Among them, the specific point values ​​in 60-70℃ can be selected as 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, 69℃, 70℃, etc., and the specific point values ​​in 15-30min can be selected as 15min, 17min, 19min, 21min, 23min, 25min, 27min, 30min, etc.

[0029] In the present invention, the lysis temperature is controlled at 60-70° C., which can improve the lysis efficiency and increase the concentration and quality of the extracted nucleic acid.

[0030] Preferably, in the process of mixing the bacterial cells with the lysis solution in step (a), the mass ratio of the growth solution after growth culture to the lysis solution is (2-5):1.

[0031] Among them, the specific point values ​​​​in 2-5 can be selected as 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.

[0032] Preferably, the nucleic acid sequence of the forward primer amplified by PCR in step (2) includes the sequence shown in SEQ ID NO: 1, and the specific sequence of SEQ ID NO: 1 is: GCGGCGACAATATAGACCGAC.

[0033] Preferably, the nucleic acid sequence of the reverse primer for PCR amplification includes the sequence shown in SEQ ID NO: 2, and the specific sequence of SEQ ID NO: 2 is: CGAGATTAGCGCTGCTGGAG.

[0034] Preferably, the nucleic acid sequence of the Taqman probe amplified by PCR includes the sequence shown in SEQ ID NO: 3, and the specific sequence of SEQ ID NO: 3 is: CAGGATCAGGCCGCCGAAATAGACCA.

[0035] Preferably, the 5' end of the Taqman probe is labeled with a fluorescent group, and the 3' end is labeled with a quencher group.

[0036] Preferably, the fluorescent group includes a FAM fluorescent group, and the quencher group includes a BHQ1 quencher group.

[0037] In the present invention, the primer sequence has good gene specificity to the target gene A8H26_RS01130. The use of the primer sequence for PCR amplification and detection can improve the accuracy of the detection result.

[0038] Preferably, the PCR amplification in step (2) includes any one of qPCR, digital PCR or ddPCR.

[0039] Preferably, in the PCR amplification system of step (2), the final concentrations of the forward primer, reverse primer and Taqman probe are independently 0.1-0.5 μM.

[0040] Among them, the specific point values ​​in 0.1-0.5μM can be selected as 0.1μM, 0.2μM, 0.3μM, 0.4μM, 0.5μM, etc.

[0041] Preferably, the procedure of the PCR reaction in step (2) includes:

[0042] Step 1: 94-96°C, 20-50s; Step 2: 94-96°C, 3-7s; Step 3: 60-62°C, 30-60s; Step 2 and step 3 are repeated for 35-45 cycles.

[0043] Among them, the specific point values ​​in 94-96℃ can be selected from 94℃, 94.5℃, 95℃, 95.5℃, 96℃, etc., the specific point values ​​in 60-62℃ can be selected from 60℃, 60.5℃, 61℃, 61.5℃, 62℃, etc., the specific point values ​​in 20-50s can be selected from 20s, 25s, 30s, 35s, 40s, 45s, 50s, etc., the specific point values ​​in 3-7s can be selected from 3s, 4s, 5s, 6s, 7s, etc., the specific point values ​​in 30-60s can be selected from 30s, 35s, 40s, 45s, 50s, 55s, 60s, etc., and the specific point values ​​in 35-45 can be selected from 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, etc.

[0044] Preferably, the standard for determining whether the sample to be tested contains the source bacteria of the Japanese gout bacteria in step (2) is:

[0045] The DNA purity of the three repeated tests of the sample to be tested is 1.8-2.0, the Ct difference is ≤0.3-0.6, and the Ct value of the qPCR amplification curve is ≤Ct threshold, then it is judged as positive; the Ct value of the qPCR amplification curve is >Ct threshold, then it is judged as positive; the DNA purity is the value of A260 / A280, and the Ct threshold is 32-36.

[0046] Among them, the specific point values ​​in 1.8-2.0 can be selected from 1.8, 1.85, 1.9, 1.95, 2.0, etc., the specific point values ​​in 0.3-0.6 can be selected from 0.3, 0.4, 0.5, 0.6, etc., and the specific point values ​​in 32-36 can be selected from 32, 33, 34, 35, 36, etc.

[0047] In a second aspect, the present invention provides a real-time fluorescence detection kit for detecting bacteria from the Japanese Gouweiduo bacteria, wherein the real-time fluorescence detection kit contains the forward primer, reverse primer and Taqman probe described in the first aspect.

[0048] In a third aspect, the present invention provides an application of the rapid detection method described in the first aspect and / or the real-time fluorescence detection kit described in the second aspect in cosmetics detection.

[0049] Preferably, the cosmetics include any one of toner, lotion, facial mask, cream, shampoo or shower gel.

[0050] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] The present invention provides a rapid detection method for the source bacteria of Rigouweiduo bacteria. The detection method uses the A8H26_RS01130 gene as the target gene for PCR amplification. The gene is highly conserved in the source bacteria of Rigouweiduo bacteria and has no homologous sequence, thus avoiding cross-reaction and improving the specificity of detection. The method can accurately identify a small amount of contamination of the source bacteria of Rigouweiduo bacteria within 24 hours, and the detection sensitivity is 10 2 CFU / mL.

[0053] Furthermore, the present invention enriches the sample to be tested in the enrichment solution before extracting DNA from the bacteria, which can significantly improve the enrichment efficiency of low-concentration bacteria and reduce the detection limit; and, adding histidine to the enrichment solution can reduce the adverse effects of interfering substances such as preservatives in the sample to be tested on the enrichment effect of the Japanese Gouweiduo bacterial source bacteria.

[0054] Furthermore, in the process of DNA extraction, mechanical disruption can better release nucleic acids in bacteria; in addition, controlling the lysis temperature at 55-65°C can improve the lysis efficiency and improve the concentration and quality of the extracted nucleic acids. Furthermore, by using mechanical disruption and controlling the lysis temperature, combined with magnetic bead adsorption technology, the DNA yield and quality can be significantly improved, and the single batch processing time is ≤1h.

[0055] Furthermore, during the PCR amplification process, the nucleic acid sequence of the forward primer includes the sequence shown in SEQ ID NO:1, the nucleic acid sequence of the reverse primer includes the sequence shown in SEQ ID NO:2, and the nucleic acid sequence of the Taqman probe includes the sequence shown in SEQ ID NO:3. The above primer sequences have good gene specificity to the target gene A8H26_RS01130. Using the above primer sequences for PCR amplification and detection can improve the accuracy of the detection results.

[0056] Furthermore, the detection method involved in the present invention can effectively avoid false positive / false negative results through full-process quality control design, and can process 96 samples in a single batch, providing an efficient and reliable quality control solution for the cosmetics industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 It is the amplification curve of the positive control and negative control samples for target gene detection.

[0058] Figure 2 This is the amplification curve of the target gene detection in shower gel samples contaminated with different concentrations of Rhizoctonia solani bacteria.

[0059] Figure 3 This is the amplification curve of the target gene detection in cream samples contaminated with different concentrations of Rigouweiduo bacteria. DETAILED DESCRIPTION

[0060] 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.

[0061] The Japanese gouweiduo bacteria involved in the following examples are from Shanghai Collection Biotechnology Center, and the strain number is SHBCC D18003.

[0062] Example 1

[0063] Sampling and bacterial enrichment:

[0064] (1) Sampling:

[0065] Weigh 0.10g or 1.0g of sample from the emulsion cosmetics known to be contaminated with the source bacteria of Japanese Gouweiduo bacteria.

[0066] (2) Preparation of enrichment solution:

[0067] (2.1) Preparation of SCDLP enrichment solution containing histidine: 15.0 g casein peptone, 5.0 g soy peptone, 3.0 g sodium chloride, 2.5 g glucose, 3.0 g dipotassium hydrogen phosphate, 4.0 g histidine, 1.5 g lecithin, 8.0 g Tween-80, and distilled water to 1 L. After heating and dissolving the above ingredients, adjust the pH to 7.2, and sterilize at 121°C for 20 min to obtain SCDLP enrichment solution containing histidine.

[0068] (2.2) Preparation of SCDLP enrichment solution without histidine: The only difference between the preparation of step (2.1) and the preparation of step (2.1) is that the preparation of SCDLP enrichment solution without histidine is carried out. The other components are the same as those in step (2.1), and the preparation of SCDLP enrichment solution without histidine is obtained.

[0069] (3) Bacterial culture:

[0070] 0.10 g of sample was mixed with 9.9 mL of sterile SCDLP enrichment solution containing histidine (mass-to-volume ratio of 1:99), 1.00 g of sample was mixed with 9.0 mL of sterile SCDLP enrichment solution containing histidine (mass-to-volume ratio of 1:9), and 0.10 g of sample was mixed with 19.9 mL of sterile SCDLP enrichment solution containing histidine (i.e., mass-to-volume ratio of 1:199), vortexed and cultured at 35 °C and 200 rpm for 20 h.

[0071] Each group tested no less than 3 samples, and the average enrichment concentration of each group was calculated. The enrichment effect of each experimental group is shown in Table 1. It can be seen that after the cosmetics contaminated with the source bacteria of the Japanese Gouweiduo bacteria were enriched in the SCDLP enrichment solution containing histidine, although the initial contamination amount in the enrichment solution was relatively high when the mass volume ratio of the sample to the SCDLP enrichment solution was 1:9, the number of bacteria in the enrichment solution after enrichment was more when the mixed culture ratio was 1:99; however, when the volume of the SCDLP enrichment solution was increased to a mixed culture ratio of 1:199, the enrichment rate would be significantly reduced, the enrichment time would be extended, and the enrichment concentration at 20h was also lower than the enrichment concentration when the mixed culture ratio was 1:99.

[0072] In addition, adding histidine to the SCDLP enrichment solution can further increase the enrichment concentration.

[0073] This indicates that when the sample to be tested in the present invention is mixed with the enrichment solution containing histidine at a mass volume ratio of 1: (50-150), less sample to be tested can be used for enrichment to obtain a higher concentration of bacterial solution, thereby shortening the detection time while improving the detection ability of the source bacteria of Japanese Gouweiduo bacteria.

[0074] Table 1

[0075]

[0076]

[0077] Example 2

[0078] DNA extraction:

[0079] (1) Sampling: Take 1 mL of the enrichment solution after bacterial culture, centrifuge at 10,000 g for 2 min to collect the bacteria, and wash them three times with sterile PBS.

[0080] (2) DNA extraction: Magnetic bead DNA extraction kit ( Magnetic Bacterial / Fungal DNA Kit) was used to extract bacterial nucleic acid. The specific steps are as follows:

[0081] (2.1) Lysis: Add 300 μL of the lysis solution in the kit to the collected enriched cells, use / do not use mechanical disruption, and shake and lyse in a water bath at 65°C / 55°C / 75°C for 20 min;

[0082] (2.2) DNA extraction: After the lysis is completed, add the magnetic bead suspension according to the instructions of the kit, and vortex at high speed for 10 minutes to allow the magnetic beads to adsorb DNA. Then add the washing solution A in the kit, vortex to break up the magnetic beads, and then transfer to a magnetic stand for magnetic separation until the solution is clear. After aspirating the solution, add washing solutions B and C (the operation is the same as above, vortex to break up the magnetic beads, and then transfer to a magnetic stand for magnetic separation until the solution is clear) to remove impurities. After aspirating the washing solution C, open the lid and dry until the ethanol in the washing solution is completely evaporated. Add 70μL of elution solution, vortex at high speed and warm bath at 60℃ to elute the DNA, transfer to a magnetic stand for magnetic separation until the solution is clear, aspirate the solution and save it to obtain high-purity DNA.

[0083] (2.3) DNA concentration and purity were measured using a Thermo Scientific NanoDrop spectrophotometer.

[0084] Each set of lysis conditions was tested for no less than 3 samples, and the DNA concentration and purity of each set of tests were calculated. The test results are shown in Table 2. It can be seen that mechanical disruption helps to lyse the source bacteria of Rigouweiduo more thoroughly, thereby extracting DNA with higher concentration and better quality. At the same time, controlling the lysis temperature at 60-70°C can further improve the lysis efficiency, increase the nucleic acid concentration and nucleic acid quality, and thus improve the sensitivity of the detection of the source bacteria of Rigouweiduo.

[0085] Table 2

[0086] Experimental conditions Nucleic acid concentration (ng / μL) A260 / 280 Mechanical crushing & 65℃ pyrolysis 216.0±3.4 1.89 No mechanical crushing & 65℃ pyrolysis 90.1±6.8 1.63 Mechanical crushing & 55℃ pyrolysis 113.7±5.6 1.78 Mechanical crushing & 75℃ pyrolysis 207.8±6.2 1.83

[0087] Example 3

[0088] Specific amplification detection:

[0089] (1) Prepare the sample:

[0090] Positive control: Extract the enriched bacterial solution of the Japanese Gouweiduo bacteria source bacteria (bacterial concentration is 2.1×10 9 CFU / mL) of DNA;

[0091] Negative control: DNA of Escherichia coli, Salmonella, Shigella, and Klebsiella pneumoniae were extracted respectively according to the method of Example 1-2, and the four DNAs were mixed at equal nucleic acid concentrations to obtain a negative control DNA mixed solution with a concentration of 228.4 ng / μL.

[0092] (2) Amplification system:

[0093] The nucleic acid sequence of the forward primer for qPCR amplification is SEQ ID NO: 1, and the specific sequence of SEQ ID NO: 1 is: GCGGCGACAATATAGACCGAC.

[0094] The nucleic acid sequence of the reverse primer for qPCR amplification is SEQ ID NO: 2, and the specific sequence of SEQ ID NO: 2 is: CGAGATTAGCGCTGCTGGAG.

[0095] The nucleic acid sequence of the Taqman probe amplified by qPCR is SEQ ID NO: 3, and the 5' end of the nucleic acid sequence is labeled with a FAM fluorescent group, and the 3' end is labeled with a BHQ1 quenching group. The specific sequence of SEQ ID NO: 3 is: CAGGATCAGGCCGCCGAAATAGACCA.

[0096] use Probe qPCR SuperMix UDG reagent was used to amplify the DNA extracted in step (1). The 20 μL amplification system is shown in Table 3.

[0097] Table 3

[0098] Components volume Final concentration template 1μL / Forward primer (10 μM) 0.4μL 0.2μM Reverse primer (10 μM) 0.4μL 0.2μM Probe (10 μM) 0.4μL 0.2μM qPCR reaction buffer (2×) 10μL 1× Inert reference dye (2×) 0.4μL 1× Nuclease-free water Up to 20 μL - Total volume 20μL -

[0099] (3) Amplification procedure:

[0100] Pre-denaturation: 95°C, 30s;

[0101] Cyclic amplification (40 times): 95°C, 5s (denaturation) → 62°C, 40s (annealing / extension).

[0102] (4) Interpretation of results:

[0103] Result determination: If the sample Ct value is ≤35 and the amplification curve is S-shaped, it is determined to be positive; if the sample Ct value is >35, it is determined to be negative;

[0104] Repeatability verification: The same sample was repeated 3 times, and the difference in Ct value was ≤0.50.

[0105] Test results such as Figure 1 As shown, the positive control Ct value is less than 35, which is a positive result. The negative control Ct value cannot be determined (Ct value>35), which is a negative result. The statistical results of the Ct values ​​of the positive control and the negative control are shown in Table 4. It can be seen that the method of the present invention can well detect the source bacteria of the Japanese gowwedo bacteria, that is, the amplification of the source bacteria of the Japanese gowwedo bacteria is positive, while the DNA amplification of other Enterobacteriaceae strains: Escherichia coli, Salmonella, Shigella and Klebsiella pneumoniae mixed bacteria is negative.

[0106] Table 4

[0107] Sample Type Nucleic acid concentration (ng / μL) A260 / 280 Ct value (mean ± SD) Test results Positive Control 204.5 1.84 15.968±0.23 Positive Negative control 228.4 1.87 Undetermined Negative

[0108] Example 4

[0109] In this embodiment, the Japanese gout bacteria source bacteria in the shower gel are detected.

[0110] (1) Preparation of simulated pollution samples:

[0111] Take 0.09g of uncontaminated shower gel sample and 10μL of gradient diluted enriched bacterial solution (10 2 CFU / mL, 10 3 CFU / mL and 10 4 CFU / mL) mixed (i.e., the contamination level of Japanese gouweiduo bacteria in the shower gel sample was 10 1 CFU / mL, 10 2 CFU / mL and 10 3 CFU / mL), mixed with 9.9 mL of sterilized SCDLP enrichment solution containing histidine, and enriched with bacteria according to the method in Example 1.

[0112] (2) DNA extraction:

[0113] Referring to the method of Example 2, mechanical disruption was used, and the cells were shaken and lysed in a 65°C water bath for 20 minutes to extract the DNA in the enrichment solution.

[0114] (3) Specific amplification detection:

[0115] Referring to the method in Example 3, specific amplification detection was performed.

[0116] The test results show that Figure 2 As shown in Table 5, when the contamination amount of the simulated day-old bacteria source bacteria contaminated shower gel sample was 10 2 CFU / mL and 10 3 When the CFU / mL was 0.04, after bacterial enrichment, DNA extraction and qPCR amplification, the Ct value was <35, which was a positive result. The method of the present invention can effectively detect the source bacteria of Japanese gout bacteria contaminated in shower gel, with a detection sensitivity of 10 2 CFU / mL.

[0117] Table 5

[0118]

[0119] Example 5

[0120] This embodiment detects the source bacteria of Japanese Gouweiduo bacteria in the cream. The only difference between this embodiment and embodiment 4 is that the shower gel sample in step (1) is replaced with a cream sample of equal mass, and the remaining steps are consistent with embodiment 4.

[0121] The test results show that Figure 3 As shown in Table 6, when the contamination amount of the simulated day-old bacterial source contaminated cream sample is 10 2CFU / mL and 10 3 When the CFU / mL was 0.04, after bacterial enrichment, DNA extraction and qPCR amplification, the Ct value was <35, which was a positive result. The method of the present invention can effectively detect the contaminated Japanese gout bacteria in creams, with a detection sensitivity of 10 2 CFU / mL.

[0122] Table 6

[0123]

[0124] The applicant declares that the present invention illustrates the technical solution of the present invention 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.

[0125] 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.

[0126] 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 rapid detection method for the source bacteria of Japanese gouweiduo bacteria, characterized in that: The detection method comprises the following steps: (1) taking a sample to be tested and culturing it in a bacterial enrichment solution, collecting the bacterial cells obtained by the bacterial enrichment culture by centrifugation, and extracting DNA from the bacterial cells; (2) performing PCR amplification on the extracted DNA, and determining whether the sample to be tested contains the source bacteria of the Japanese gouweiduo bacteria according to the amplification results; The target gene for PCR amplification is the A8H26_RS01130 gene.

2. The rapid detection method according to claim 1, characterized in that: The mass volume ratio of the sample to be tested to the enrichment solution in step (1) is 1:(50-150) g / mL.

3. The rapid detection method according to claim 1 or 2, characterized in that: The enrichment solution in step (1) includes SCDLP culture medium; Preferably, the enrichment solution in step (1) further comprises histidine; Preferably, the amount of histidine added to the enrichment solution is 3.0-6.0 g / L.

4. The rapid detection method according to any one of claims 1 to 3, characterized in that: The DNA extraction in step (1) comprises the following steps: (a) mixing the bacterial cells with a lysis solution, mechanically disrupting them, and performing a lysis reaction; (b) The product of bacterial lysis is adsorbed on magnetic beads, washed, and eluted to obtain DNA.

5. The rapid detection method according to claim 4, characterized in that: The temperature of the cleavage reaction in step (a) is 60-70° C. and the time is 15-30 min.

6. The rapid detection method according to any one of claims 1 to 5, characterized in that: The nucleic acid sequence of the forward primer amplified by PCR in step (2) includes the sequence shown in SEQ ID NO: 1; Preferably, the nucleic acid sequence of the reverse primer for PCR amplification includes the sequence shown in SEQ ID NO: 2; Preferably, the nucleic acid sequence of the Taqman probe amplified by PCR includes the sequence shown in SEQ ID NO: 3; Preferably, the 5' end of the Taqman probe is labeled with a fluorescent group, and the 3' end is labeled with a quencher group; Preferably, the PCR amplification comprises any one of qPCR, digital PCR or ddPCR.

7. The rapid detection method according to any one of claims 1 to 6, characterized in that: In the PCR amplification system of step (2), the final concentrations of the forward primer, reverse primer and Taqman probe are independently 0.1-0.5 μM.

8. The rapid detection method according to any one of claims 1 to 7, characterized in that: The procedure of the PCR reaction in step (2) includes: Step 1: 94-96°C, 20-50s; Step 2: 94-96°C, 3-7s; Step 3: 60-62°C, 30-60s; Step 2 and step 3 are repeated for 35-45 cycles.

9. A real-time fluorescence detection kit for detecting the source bacteria of Japanese gouweiduo bacteria, characterized in that: The real-time fluorescence detection kit contains the forward primer, reverse primer and Taqman probe described in claim 6.

10. Use of the rapid detection method according to any one of claims 1 to 8 and / or the real-time fluorescence detection kit according to claim 9 in cosmetics detection; Preferably, the cosmetics include any one of toner, lotion, facial mask, cream, shampoo or shower gel.

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