CN110241056A - Bacillus, method for detecting antibiotic residues using the bacillus and application thereof - Google Patents

Abstract

The invention belongs to the technical field of microbial detection of antibiotics, and in particular relates to a bacillus, a method for detecting antibiotic residues using the bacillus and an application thereof. The results of this study provide a convenient and easy-to-obtain indicator bacteria for the popularization and application of TTC method test. The bacterium is a Gram-positive bacterium, specifically Bacillus licheniformis, which can completely replace Streptococcus thermophilus for TTC test, and obtain the same result as Streptococcus thermophilus. At 46°C, when the concentration of the bacteria used solution is 1.0 mg/mL, culture for 1 hour, add TTC solution and react for 20 minutes, which is convenient for judging the test results.

Description

A kind of bacillus and the method for using the bacillus to detect antibiotic residues and its application

technical field

The invention belongs to the technical field of microbial detection of antibiotics, and in particular relates to a bacillus, a method for detecting antibiotic residues using the bacillus and an application thereof.

Background technique

Antibiotics are a class of metabolites or their derivatives synthesized by microorganisms and other organisms in the course of life activities, which can inhibit or interfere with the life activities of pathogenic bacteria and viruses at very low concentrations, such as penicillin, tetracycline, gold, etc. chloramphenicol, streptomycin, sulfonamides, etc. In medicine, antibiotics are widely used in the prevention and treatment of various microbial infectious diseases and certain cancers. At the source of the dairy industry, antibiotics are used frequently, especially for the treatment of bovine mastitis, and are often used repeatedly in large doses. According to statistics, about ten kinds of antibiotics are applied in one or more combinations in the form of powder, liniment, injection, and milk duct injection, resulting in serious antibiotic residues. In dairy cow feed, there are also antibiotics additive. Many antibiotics such as penicillin, tetracycline, sulfonamides and certain aminoglycoside antibiotics are antigenic and can cause allergic reactions in some susceptible people. Mild cases manifest as urticaria, fever, joint swelling and pain, cellulitis, etc. In severe cases, laryngeal edema, dyspnea, anaphylactic shock, and even life-threatening; The function has a certain damage, and even has carcinogenic, teratogenic, and mutagenic effects; and it can cause the increase of drug-resistant bacteria in the body of long-term intake, and the antibacterial drugs will fail when there is a serious disease that needs to be treated. Antibiotic residues in milk are currently a problem that cannot be ignored in raw milk. To control the occurrence of residues requires not only effective management of milk sources, but also the establishment of advanced antibiotic residue detection methods to strengthen the quality management of milk sources.

There are many methods for detecting antibiotic residues in milk and its products, such as microbiological detection; physical and chemical detection; immune method, etc. The microbiological method is the earliest method used in the detection of antibiotic residues. At present, there are many commercial kits, such as Delvotest, CharmScien, etc., as well as the national standard of thermophilus streptococcus inhibition method (also known as TTC method). However, Streptococcus thermophilus rarely has a single colony, often grows in sheets, is not easy to separate, and it is inconvenient to determine the amount of bacteria in the TTC method by colony counting, which limits the popularization and application of the TTC method test.

Contents of the invention

Aiming at the problems existing in the prior art, the present invention provides a bacillus, a method for detecting antibiotic residues using the bacillus and its application, and the research results provide a convenient and easy-to-obtain indication for the popularization and application of the TTC method test bacteria.

The present invention is achieved in this way, a Bacillus, its 16S rDNA sequence is shown in SEQ ID NO: 3, the preservation number is CGMCD No.18051, and the proposed classification is named Bacillus licheniformis (Bacillus licheniformis), which was published in June 2019 On the 28th, it was preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures.

A kind of method utilizing above-mentioned bacillus to carry out antibiotic residue detection, comprises the following steps:

S1: activating the bacillus strain described in claim 1;

S2: Add the activated bacterial solution to the sample to be tested and incubate at a constant temperature;

S3: Add the TTC indicator to the culture mixture, continue the constant temperature culture, and judge the test result according to the discoloration.

Further, the culture temperature of constant temperature culture in steps S2 and S3 is 37-52°C.

Further, the culture temperature of constant temperature culture in steps S2 and S3 is 46°C.

Further, the constant temperature cultivation time in step S2 is 0.5-2h.

Further, the incubation time at constant temperature in step S2 is 1 h.

Further, in step S3, after adding the TTC indicator, the incubation time at constant temperature is 20 minutes.

Further, the amount of bacillus in the sample to be tested in step S2 is 1.0 mg/mL.

Application of Bacillus in antibiotic detection as mentioned above.

Application of the above-mentioned method for detecting antibiotic residues using Bacillus in antibiotic detection.

In summary, the advantages and positive effects of the present invention are: when the present invention uses lactic acid bacteria medium to isolate Streptococcus thermophilus from yogurt, a Gram-positive large bacillus is obtained instead of Streptococcus; Instead of Streptococcus thermophilus, the TTC method test was completed. The gram-positive large bacillus was isolated, purified, cultured, identified by PCR and biochemically identified. The effects of the two bacteria on the test results of the TTC method were compared, and an indicator bacterium was provided for the application of the TTC method.

Description of drawings

Figure 1 is the colony morphology of Streptococcus thermophilus;

Fig. 2 is the colony morphology of bacillus licheniformis;

Fig. 3 is the microscopic detection result of unknown bacteria;

Fig. 4 is unknown bacteria PCR electrophoresis figure;

Figure 5 is the phylogenetic tree of the 16S rDNA gene sequence of unknown bacteria.

Detailed ways

In order to make the purpose of the present invention, technical solutions and advantages clearer, the present invention will be described in further detail below in conjunction with the examples, and the equipment and reagents used in each embodiment and test example can be obtained from commercial sources unless otherwise specified. The specific embodiments described here are only used to explain the present invention, not to limit the present invention.

The invention discloses a bacillus, a method for detecting antibiotic residues using the bacillus and its application, as shown in the following examples. The used control bacteria Streptococcus thermophilus standard bacterial strain (ACCC 10213) of the present invention is purchased from China Industrial Microorganism Culture Collection Management Center (CICC); TTC indicator 2,3,5-triphenyltetrazolium chloride is purchased from Shanghai Lanji Biology; other biochemical reagents are commercially available.

Isolation screening and biochemical identification of embodiment 1 bacterial strain

According to reports, Streptococcus thermophilus can be isolated from yoghurt using lactic acid bacteria medium. The inventor bought fresh milk in the 31 district of Shihezi City. Streptococcus thermophilus was isolated, but an unknown strain of Bacillus was isolated and identified as follows.

Streptococcus thermophilus rarely has a single colony, which often exists in sheets and is not easy to separate, as shown in Figure 1; while Bacillus licheniformis has many single colonies, which are easy to separate, as shown in Figure 2.

1. Staining and microscopic examination of unknown bacteria

Smear the unknown bacillus, Gram stain, and observe the morphological characteristics of the bacteria under the microscope. The results are shown in Figure 3. The strains were smeared, stained, and microscopically examined, and the results were Gram-positive. Most of the bacteria were rod-shaped and existed in pairs, chains, or individually, and some of them had formed spores.

2. PCR identification of unknown bacteria

Strain culture conditions: inoculate the unknown bacteria into the lactic acid bacteria liquid medium, and culture the bacteria liquid at 46°C for more than 12 hours.

Preparation of the template: Take 1 mL of the above-mentioned cultured bacterial solution and place it in an EP tube, centrifuge at 12,000 r for 10 min, discard the supernatant, then suspend the precipitate with 1 mL of sterile water, and evenly flick the bottom sediment with your fingers. Boil in a boiling water bath for 10 min, centrifuge at 12000 rpm for 10 min, collect the supernatant in a PCR tube as a template, and use 3 μL to 5 μL as a template. Store at -20°C for later use.

Synthesis of 16S rDNA primers: Synthesized by Shanghai Sangon Bioengineering Co., Ltd. Forward primer: 5′-AGAGTTTGATCMTGGCTCAG-3′, reverse primer: 5′-TACGGTTACCTTGTTACGACTT-3′, see SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

PCR reaction system (30 μL): PCR Mix 15 μL, ddH ₂ O 10.2 μL, forward primer 0.9 μL, reverse primer 0.9 μL, template 3 μL. PCR reaction parameters: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 40 s, annealing at 54.5°C for 30 s, extension at 72°C for 2 min, 35 cycles, final extension at 72°C for 10 min, storage at 4°C. PCR products were taken for agarose gel electrophoresis.

Determination and comparison of unknown bacterial sequences: The amplified products were sent to Shanghai Bioengineering Technology Co., Ltd. for purification and sequencing, and the unknown bacterial sequences were submitted to the Genbank database for homology comparison. The sequences were generated using MEGA5.0 software to generate a phylogenetic tree.

The results are shown in Figure 4. The DNA of the unknown bacteria was amplified by PCR to obtain a target fragment with 500 bp. In Figure 4, A is the 2000 super DNA mark, B is the unknown bacteria, C is the lactic acid bacteria culture medium, and D is the negative control. Use the BLAST function in NCBI to compare the 16S rDNA sequences of the above unknown bacteria, see SEQ ID NO: 3, and compare the unknown bacteria with accession numbers MH424451.1, MG753545.1, KJ842640.1, MH373540.1, JX402428.1, EU257697. 1. The homology of Bacillus licheniformis of MH373542.1, KU877642.1, KJ842633.1, EU256501.1, KY401428.1 is 99.50-100%; the phylogenetic tree of 16S rDNA was downloaded from the Nucleoticle database, which is 7 See Figure 5 for the standard strains of Bacillus species. It can be preliminarily determined that the unknown bacteria may be Bacillus licheniformis.

3. Biochemical identification of unknown bacteria and Streptococcus thermophilus

Refer to the identification table for Bacillus and Streptococcus in the Bergey's Bacterial Identification Manual for the following biochemical identification.

Sugar fermentation test: Add 5% glucose, maltose and mannitol to the sugar fermentation medium, and then distribute them into small tubes with inverted tubes. Autoclave at 121°C for 15 minutes, inoculate S bacteria and Y bacteria into the sugar fermentation medium through aseptic operation. At the same time, do three parallels, and observe the results after 12 hours in the incubator. When the inoculated bacteria decompose the sugar in the medium to produce acid, the indicator in the medium becomes acidic, and the color of the medium turns yellow and positive. If air bubbles appear in the inverted tube in the liquid medium, indicating that the bacteria can produce gas, it is positive; otherwise, it is negative.

Gelatin liquefaction test: The gelatin liquefaction medium was placed in a water bath and dissolved by heating. Calibrate to pH 7.1, filter through gauze, and dispense in test tubes. Do three parallels at the same time, autoclave at 115°C for 15 minutes, and inoculate S bacteria and Y bacteria respectively in gelatin medium at 20°C for 5-7 days after cooling the slope. Observe, if there is no coagulation, it means that the gelatin has been hydrolyzed, and it is positive.

Methyl red (MR) test: Inoculate S bacteria and Y bacteria in glucose-peptone water medium, and do three parallel cultures at 35°C for 2-3 days, add 8-12 drops to each 8mL culture solution 0.04% methyl red alcohol solution, and observe the results. Red is positive, orange is weakly positive, and yellow is negative.

V-P test: Inoculate S bacteria and Y bacteria in glucose-peptone water medium respectively, and do three parallels at the same time. After culturing at 35°C for 48 hours, add 0.6mL of 5% a-naphthol to each 2.5mL of culture solution, and then add 0.2 mLKOH solution, shake well, and observe the result. Red is positive, no red is negative.

The unknown bacteria and purchased standard Streptococcus thermophilus were identified by 6 biochemical cultures, and the results are shown in the table below.

Table 1 Biochemical identification results of unknown bacteria and Streptococcus thermophilus

Note: S bacteria is Streptococcus thermophilus, Y bacteria is Bacillus licheniformis. The same below.

Analyzing the biochemical identification results in Table 1 is consistent with the Bergey's bacterial identification manual, thus confirming that the unknown bacteria is Bacillus licheniformis.

Embodiment 2 Bacillus licheniformis replaces Streptococcus thermophilus and carries out TTC method test

Wash the colonies of Streptococcus thermophilus and Bacillus licheniformis on the lactic acid bacteria solid medium respectively into 1.5mL EP tubes with sterile water, centrifuge at a speed of 5000r/min for 10min, pour off the supernatant, and record the weight as m _1. Use 1mL sterile water to wash the bacteria deposited at the bottom of the tube into a small triangle flask twice in a row, suck up the liquid in the EP tube, then weigh the empty EP tube and record it as m ₂ , m ₁ -m ₂ is is the bacterial weight. Dilute the bacterial solution in the test tube to obtain the bacterial use solution with a concentration of 0.5mg/mL, 1.0mg/mL, 1.50mg/mL, and 2mg/mL, and set aside.

The repurchased raw milk was divided into five triangular flasks, each 120mL, one of which was used as a negative control without adding antibiotics. The other four were added different doses of penicillin potassium 0.004IU/mL, gentamicin 0.4IU/mL, kanamycin 5IU/mL, and streptomycin 0.5IU/mL according to the minimum detection limit as positive controls. Add five portions of milk to test tubes, add 4.5mL to each tube and mark it, put it in a constant temperature water bath at 80°C for 10 minutes, take it out from the water bath, cool it to below 45°C, and set aside.

Add 0.5mL of different concentrations of bacteria solution to 5mL milk sample test tubes, place them in a constant temperature incubator, and culture them at 37°C, 40°C, 43°C, 46°C, 49°C, and 52°C. After culturing for 0.5h, 1h, 1.5h, and 2h respectively, add 0.15mL of 4% TTC indicator, continue culturing, observe the results every 10 minutes, and record them. Simultaneously do parallel experiments.

The results are shown in the table below. When Bacillus licheniformis and Streptococcus thermophilus are tested by TTC method, the same results can be obtained under the same temperature, bacterial quantity and culture time.

Table 2 The main results of the influence of bacterial species, bacterial amount, culture temperature and time on the TTC method

In summary, Bacillus licheniformis can completely replace Streptococcus thermophilus in TTC test, and obtain the same results as Streptococcus thermophilus. The test results show that at 46°C, when the concentration of the bacteria used solution is 1.0 mg/mL, culture for 1 hour, add TTC solution and react for 20 minutes, which is convenient for the judgment of test results. The results of this study provide a convenient and easy-to-obtain indicator bacteria for the popularization and application of TTC method test.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

sequence listing

<110> Shihezi University

<120> A Bacillus, a method for detecting antibiotic residues using the Bacillus and its application

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<170> SIPOSequenceListing 1.0

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<213> Artificial sequence (Forward primer)

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agagtttgat cmtggctcag 20

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<213> Artificial sequence (Reverse primer)

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tacggttacc ttgttacgac tt 22

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<213> 16S rDNA (16S rDNA)

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cggactgagg cgtgcctata ctgcagtcga gcggaccgac gggagcttgc tcccttaggt 60

cagcggcgga cgggtgagta aacacgtgggt aacctgcctg taagactggg ataactccgg 120

gaaaccgggg ctaataccgg atgcttgatt gaaccgcatg gttcaatcat aaaaggtggc 180

ttttagctac cacttacaga tggacccgcg gcgcattagc tagttggtga ggtaacggct 240

caccaaggcg acgatgcgta gccgacctga gagggtgatc ggccaacactg ggactgagac 300

acggcccaga ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg 360

acggagcaac gccgcgtgag tgatgaaggt tttcggatcg taaaactctg ttgttaggga 420

agaacaagta ccgttcgaat agggcggcac cttgacggta cttaaccaga aagccacggc 480

taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gaattattgg 540

gcgtaaagcg cgcgcaggcg gtttcttaag tctgatgtga aagcccccgg ctcaaccggg 600

gagggtcatt ggaaactggg gaacttgagt gcagaagagg agagtggaat tccacgtgta 660

gcggtgaaat gcgtagagat gtggaggaac accagtggcg aaggcgactc tctggtctgt 720

aactgacgct gaggcgcgaa agcgtgggga gcgaacagga ttagataccc tggtagtcca 780

cgccgtaaac gatgagtgct aagtgttaga gggtttccgc cctttagtgc tgcagcaaac 840

gcattaagca ctccgcctgg ggagtacggt cgcaagactg aaactcaaag gaattgacgg 900

gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca 960

ggtcttgaca tcctctgaca accctagaga tagggcttcc ccttcggggg cagagtgaca 1020

ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1080

cgcaaccctt gatcttagtt gccagcattc agttgggcac tctaaggtga ctgccggtga 1140

caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca 1200

cacgtgctac aatgggcaga acaaagggca gcgaagccgc gaggctaagc caatcccaca 1260

aatctgttct cagttcggat cgcagtctgc aactcgactg cgtgaagctg gaatcgctag 1320

taatcgcgga tcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1380

acaccacgag agtttgtaac acccgaagtc ggtgaggtaa cctttggagc cagccgccga 1440

aggtgacaga gattt 1455

Claims (10)

1. a kind of bacillus, 16S rDNA sequence is shown in that SEQ ID NO:3, deposit number are CGMCD No.18051.

2. a kind of method for carrying out antibiotics leftover detection using bacillus described in claim 1, which is characterized in that including Following steps:

S1: Bacillus described in claim 1 is activated；

S2: the bacterium solution after activation is added into test sample, constant temperature incubation；

S3: TTC indicator is added into culture mixed liquor, continues constant temperature incubation, testing result is judged according to discoloration.

3. the method according to claim 2 for carrying out antibiotics leftover detection using bacillus, it is characterised in that: step The cultivation temperature of constant temperature incubation is 37-52 DEG C in S2 and S3.

4. the method according to claim 3 for carrying out antibiotics leftover detection using bacillus, it is characterised in that: step The cultivation temperature of constant temperature incubation is 46 DEG C in S2 and S3.

5. the method according to claim 2 for carrying out antibiotics leftover detection using bacillus, it is characterised in that: step The constant temperature incubation time is 0.5-2h in S2.

6. the method according to claim 5 for carrying out antibiotics leftover detection using bacillus, it is characterised in that: step The constant temperature incubation time is 1h in S2.

7. the method according to claim 2 for carrying out antibiotics leftover detection using bacillus, it is characterised in that: step The constant temperature incubation time is 20min after TTC indicator is added in S3.

8. according to the method according to claim 2 for carrying out antibiotics leftover detection using bacillus, it is characterised in that: The additive amount of bacillus is 1.0mg/mL in sample to be tested in step S2.

9. application of the bacillus as described in claim 1 in antibiotic detection.

10. the method for carrying out antibiotics leftover detection using bacillus as described in claim 2-8 is any is examined in antibiotic Application in survey.