CN114574400A

CN114574400A - Modified geobacillus stearothermophilus

Info

Publication number: CN114574400A
Application number: CN202210358767.6A
Authority: CN
Inventors: 吕涛; 孙志文; 刘龙飞
Original assignee: Beijing Zhongjian Baotai Biotechnology Co ltd
Current assignee: Beijing Zhongjian Baotai Biotechnology Co ltd
Priority date: 2022-04-07
Filing date: 2022-04-07
Publication date: 2022-06-03
Anticipated expiration: 2042-04-07
Also published as: CN114574400B

Abstract

The invention relates to a modified Geobacillus stearothermophilus, in particular to a Geobacillus stearothermophilus (Geobacillus stearothermophilus) GS102 which is preserved in the China general microbiological culture Collection center at 27 th 1 month in 2022 with the preservation number of CGMCC No. 24380. The strain of the invention has high sensitivity to antibiotics sulfadimidine, enrofloxacin, tilmicosin and thiamphenicol specifically.

Description

Modified geobacillus stearothermophilus

Technical Field

The invention relates to a modified geobacillus stearothermophilus, belonging to the technical field of biology.

Background

Modern breeding industry gradually develops towards large-scale and intensive direction, and antibiotics become a material basis for guaranteeing the healthy growth of livestock and poultry and the normal development of animal husbandry. If the unreasonable use of antibiotics is likely to cause the antibiotic residue in the food, not only is the health of human body damaged, but also the sustainable health development of animal husbandry is influenced. The method is characterized in that animals and animal products are organized to monitor antibiotic residues every year since 1999, and from published spot inspection announcements, although the qualification rate of the products is gradually increased, about 0.5% of samples still have overproof antibiotic residues, wherein the overproof antibiotic residues of sulfadimidine, enrofloxacin, tilmicosin and thiamphenicol are serious. Strengthening antibiotic residue detection is crucial to the healthy development of animal husbandry, and a broad-spectrum detection method capable of simultaneously detecting sulfadimidine, enrofloxacin, tilmicosin and thiamphenicol is urgently needed to be developed.

The existing antibiotic residue detection technology generally comprises an instrument method, an enzyme-linked immunosorbent assay and a colloidal gold method, wherein the instrument method has the advantages of accurate qualitative and accurate quantitative determination, but the instrument has high detection cost, high detection environment requirement and long detection period, and is not suitable for field detection; the enzyme-linked immunosorbent assay has low cost, but has complex operation, large batch difference and low accuracy, can only measure one or a certain type of antibiotics each time, has complex sample treatment and high detection cost, and can not implement field detection; the colloidal gold method has higher cost, can only measure one or one class of antibiotics every time although the operation is simple, and has lower accuracy. In order to reduce the detection cost and have simple operation and more suitability for rapid detection, the microbiological method is the best choice, and mainly utilizes the inhibition effect of the antibiotics on the growth of microorganisms to detect the antibiotics in the sample, the principle is that the growth of the microorganisms can generate metabolites to enable the color indicator in the culture medium to change color, and the antibiotics in the sample can inhibit the growth of the microorganisms and do not generate the metabolites to enable the indicator not to change color, so that whether the sample contains the antibiotics or not can be judged through the change of the color of the culture medium.

The conventional microbiological method is usually low in sensitivity, for example, the conventional detection limit of most antibiotics is 200-.

The inventor fortunately found a strain with broad-spectrum sensitivity to various antibiotics, and the preservation number is CGMCC No.21738(CN 202110243389.2). On the basis, the inventor also carries out a deep digging transformation experiment, designs a plurality of experimental schemes by utilizing a biological mutation technology, and tests on various test materials, so that a strain with high sensitivity which exceeds the original strain CGMCC No.21738 for four antibiotics such as sulfadimidine, enrofloxacin, tilmicosin and thiamphenicol is screened out at present.

Disclosure of Invention

In order to improve the detection sensitivity of four antibiotics, namely sulfadimidine, enrofloxacin, tilmicosin and thiamphenicol, the invention provides a modified geobacillus stearothermophilus which can be used for quickly detecting the antibiotics in samples such as food, tissues, feed, serum, urine and the like.

The invention provides a method for detecting antibiotic residues in a sample, which uses Geobacillus stearothermophilus GS102 as a microorganism sensitive to antibiotics and detects whether the sample contains the antibiotics or not by changing the color of an indicator in a culture medium.

The strain is derived from Geobacillus stearothermophilus (Geobacillus stearothermophilus), has the preservation number of CGMCC No.21738, and is a strain which is very sensitive to various antibiotics generally at the same time.

A plurality of gene mutation, knockout and other modification means are carried out on genes or key regions of Geobacillus stearothermophilus (Geobacillus stearothermophilus) with the preservation number of CGMCC No.21738, dozens of mutants are obtained at present, a modified strain is screened out at first, and the mutation is that the 15 th A base of 16s rDNA of Geobacillus stearothermophilus (Geobacillus stearothermophilus) with the preservation number of CGMCC No.21738 is knocked out through sequencing analysis. The sequence of the knocked-out 16srDNA is shown in SEQ ID NO. 1. The modified strain has higher sensitivity to antibiotics sulfadimidine, enrofloxacin, tilmicosin and thiamphenicol than the existing strain. The inventor has deposited the modified strain in China general microbiological culture Collection center (CGMCC) on 27 days 1 month 2022 with the preservation number of CGMCC No.24380 and named GS 102.

The invention provides Geobacillus stearothermophilus which is GS102, is Geobacillus stearothermophilus and has the registration number of CGMCC No.24380 in the common microorganism center of China Committee for culture Collection of microorganisms.

The invention also provides application of the Geobacillus stearothermophilus GS102 in detection of antibiotic residues in a sample.

The invention also provides a preparation for detecting whether a sample contains antibiotics or not, which comprises the Geobacillus stearothermophilus GS 102.

The invention also provides a method for detecting antibiotic residues in a sample, which is characterized in that Geobacillus stearothermophilus GS102 is used as a microorganism sensitive to antibiotics, and whether the sample contains the antibiotics or not is detected by changing the color of an indicator in a culture medium.

The antibiotic of the invention is sulfadimidine, enrofloxacin, tilmicosin and/or thiamphenicol.

The sample of the invention is for example food, feed, muscle tissue, serum and/or urine.

In certain embodiments, the food product is a meat product or a dairy product.

The method and/or the detection rod disclosed by the invention have high sensitivity to various antibiotics, and have the characteristics of high accuracy, high correctness, good stability, simplicity in operation and rapidness in detection.

Drawings

FIG. 1 is a schematic view of the determination.

Detailed Description

The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. The raw materials and reagents used in the invention are commercially available, and any biological seed material can be provided for scientific research.

Mutations are conventional biotechnology and include the addition, deletion or alteration of bases.

The 16S rRNA gene (16S rDNA) is a DNA sequence on bacteria that encodes a rRNA corresponding to it and is present in the genome of all bacteria.

Example 1

Preparation of Geobacillus stearothermophilus (Geobacillus stearothermophilus) GS 102:

(1) culturing GS102 on a plate at 65 ℃ for 24 hours, inoculating the plate into a test tube filled with a propagation medium, culturing the plate in a shaking way at 65 ℃, performing gradient dilution every 8 hours, counting viable bacteria, observing spore formation, and stopping culturing when 80% of spores are formed.

(2) The culture solution was centrifuged at 5000rpm for 10 minutes, and the supernatant was discarded. The precipitate was suspended in sterile physiological saline, centrifuged with shaking, the supernatant was discarded, and the washing was repeated 3 times.

(3) Suspending the spores in sterile physiological saline to a concentration of about 1X 10⁹cfu/ml。

Example 2

The detection method comprises the following steps:

1) sample preparation (Each sample preparation method is exemplified below)

Kidney and liver sample preparation:

the swab was pulled out of the test stick. The exposed open end of the test stick is inserted into the fresh kidney or liver cortex at a depth of about 1-2cm for circular cutting, and the exudate is taken for testing.

Muscle sample preparation:

the muscle samples were quickly frozen and then thawed and the exudate taken for testing.

Preparing a milk sample:

1mL of fresh milk was removed to an empty centrifuge tube, centrifuged at 1200g for 5 minutes, and the supernatant was removed.

Serum sample preparation:

blood was collected in a 50mL centrifuge tube, allowed to clot for 4 hours, the clot removed from the tube, the remaining liquid centrifuged (1200g, 5 minutes), and the supernatant collected.

Preparing a urine sample:

transfer 500 μ L of pooled fresh or thawed urine into an empty centrifuge tube, centrifuge if there are particles in the urine (1200g, 5 min, take supernatant).

Preparing a feed sample:

a sample of 1.0. + -. 0.1g of the representative powder was weighed into a disposable 50ml centrifuge tube. PBS buffer (0.01M pH7.4) was added to the 10ml mark, and the cover was closed and allowed to stand for 5 minutes. Shake vigorously for 1 minute, and precipitate for 1 minute before detection.

2) Sample transfer (Each sample preparation method is exemplified below)

Kidney, liver and muscle samples

The swab shaft was held, the swab head was inserted into the ring incision in the kidney, liver or muscle tissue, moved clockwise while rotating for 30 seconds to fill the swab with juice, and the sample particles on the swab were removed.

Milk, serum, feed and urine samples

Open the centrifuge tube, pull the swab out of the test stick, dip into the sample supernatant, and soak for 10 seconds.

3) Sample detection (for various samples)

The incubator was set to 65 ℃ and detection was performed after the temperature had stabilized.

The swab was reinserted into the test stick by rotating the handle vertically slowly, half-way, allowing the swab to puncture the upper sealing foil and completely submerge into the upper clear liquid reagent, but not the lower sealing foil, and waiting for 2 minutes. The swab was completely unscrewed and was brought directly above the agar at the bottom of the test tube. The bottom of the detection tube is tapped 5 times on the hard surface, so that the liquid completely enters the bottom of the detection tube. The swab was completely unscrewed and the bottom of the tube was tapped again 5 times. Incubate at 65 ℃ for 3 hours. And after the incubation is finished, taking out the detection tube, cooling to room temperature, and then carrying out result analysis and judgment.

4) Interpretation of results

And taking out the detection tube from the incubator. Comparing with the result reading card, the visual judgment schematic diagram is shown in figure 1.

Negative: agar color yellow or yellow/green is a negative result.

And (3) suspicious: the lower half of the agar is yellow or yellowish green, and the upper half is blue/purple or brown as suspicious results.

Positive: the color of the agar is blue/purple, which is a positive result.

When the result of the sample detection is positive or suspicious, the national standard method is adopted for confirmation.

The growth of the strain can produce metabolites, which cause the color indicator in the medium to change color, while the antibiotics in the sample can inhibit the growth of the strain without producing metabolites, so that the indicator does not change color. Therefore, whether the sample contains antibiotics or not can be rapidly detected through the change of the color of the culture medium.

Example 3

This example is to investigate the sensitivity of the strains according to the invention and of Geobacillus stearothermophilus (Geobacillus stearothermophilus) with the accession number CGMCC No.21738 to the antibiotics sulfadimidine, enrofloxacin, tilmicosin and thiamphenicol.

Respectively selecting milk, beef, pig kidney, sheep liver, pig urine and chicken serum with negative veterinary drug residues, labeling by Sulfadimidine (Sulfadimidine), Enrofloxacin (Enrofloxacin), Tilmicosin (Tilmicosin) and Thiamphenicol (Thiamphenicol), dipping a swab in a sample, and detecting by adopting the method disclosed by the invention, wherein the lowest concentration of the detected culture medium without discoloration is the detection limit, and generally, the lower the detection limit, the higher the sensitivity. The types of veterinary drugs and the detection limit which can be detected by the method of the invention are shown in tables 1-4.

TABLE 1 Sulfamethazine detection limits

TABLE 2 detection limits for enrofloxacin

TABLE 3 tilmicosin detection limits

TABLE 4 Thiamphenicol detection limits

As can be seen by those skilled in the art, the detection limit of the strain (CGMCC No.24380) of the invention to sulfadimidine, enrofloxacin, tilmicosin and thiamphenicol in various materials (from food to body fluid and from solid to liquid) is obviously lower than the conventional detection limit and is lower than the detection limit of the strain CGMCC No.21738, namely the CGMCC No.24380 has high sensitivity to the specificity of antibiotics sulfadimidine, enrofloxacin, tilmicosin and thiamphenicol.

Finally, it should be noted that: the above embodiments are only used for illustrating the technical solutions of the present application, and not for limiting the same; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present disclosure, and the present disclosure should be construed as being covered by the claims and the specification. The present application is not intended to be limited to the particular embodiments disclosed herein but is to cover all embodiments that may fall within the scope of the appended claims.

SEQUENCE LISTING

<110> Beijing Zhongbaotai Biotech Co., Ltd

<120> a modified Geobacillus stearothermophilus

<130> 20220406

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1392

<212> DNA

<213> Artificial sequence

<400> 1

ctcccttgcg ggttcctcac cgacttcggg tgttgcaagc tctcgtggtg tgacgggcgg 60

tgtgtacaag gcccgggaac gtattcaccg cggcatgctg atccgcgatt actagcgatt 120

ccggcttatg caggcgagtt gcagcctgca atccgaactg agagcggctt tttgggattc 180

gctccccctc gcgggttcgc agccctttgt accgcccatt gtagcacgtg tgtagcccag 240

gtcataaggg gcatatgatt tgacgtcatc cccaccttcc tccgacttgt cgccggcagt 300

ccctctagag tgcccaaccg aatgctggca actagaggcg agggttgcgc tcgttgcggg 360

acttaaccca acatctcacg aacgagctga cgacaaccat gcaccacctg tcaccctgtc 420

cccccgaagg gggaacgccc aatctcttgg gttgtcaggg gatgtcaaga cctggtaagg 480

ttcttcgcgt tgcttcgaat taaaccactg ctccaccgct tgtgcgggcc cccgtcaatt 540

cctttgagtt tcagccttgc ggccgtactc cccaggcgga gtgcttatcg cgttagctgc 600

agcactaaag ggtgtgaccc ctctaacact tagcatcatc gtttacggcg tggactacca 660

gggtatctaa tcctgtttgc tccccacgct ttcgcgcctc agcgtcaggt gcaggccaga 720

gagccgcctt cgccactggt gttcctccac atctctacgc attcaccgct acacgtggaa 780

ttccgctctc ctctcctgcc ctcaagtccc ccagtttcca atgaccctcc acggttgagc 840

cgtgggcttt cacatcagac ttaagagacc gcctgcgcgc gctttacgcc aataattccg 900

gacaacgctc gccccctacg tattaccgcg gctgctggca cgtagttagc cggggctttc 960

tcgtgaggta ccgtcaccgc gccgccctct tcgaacggcg ctccttcgtc cctcacacag 1020

agctttacga cccgaaggcc ttcttcgctc acgcggcgtc gctccgtcag gctttcgccc 1080

attgcggaag attccctact gctgcctccc gtaggagtct gggccgtgtc tcagtcccag 1140

tgtgccggtc accctctcag gccggctacg catcgtcgcc ttggtgagcc gttacctcac 1200

caactagcta atgcgccgcg ggcccatccg caagtgacag cccaaaggcc gcctttcaac 1260

cgaagaccat cggtcttcgg tgttatccgg tattagctcc ggtttcccgg agttatcccg 1320

gtcttgcggg caggttgccc acgtgttact cacccgtccg ccgctgaccg aatcaaggca 1380

agccccaatc cg 1392

Claims

1. Geobacillus stearothermophilus is characterized in that: the Geobacillus stearothermophilus is GS102, Latin is Geobacillus stearothermophilus, and the registration number of the Geobacillus stearothermophilus in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 24380.

2. Geobacillus stearothermophilus according to claim 1, which is derived from Geobacillus stearothermophilus and has the 16srDNA sequence shown in SEQ ID No. 1.

3. Use of geobacillus stearothermophilus according to claim 1 or 2 for detecting antibiotic residues in a sample.

4. The use according to claim 3, wherein the antibiotic is sulfadimidine, enrofloxacin, tilmicosin and/or thiamphenicol.

5. The use according to claim 3 or 4, wherein the sample is food, feed, muscle tissue, serum and/or urine.

6. Use according to claim 5, wherein the food product is a meat product or a dairy product.

7. A reagent for detecting the presence of an antibiotic in a sample, comprising the Geobacillus stearothermophilus of claim 1.

8. The formulation of claim 7, wherein the antibiotic is sulfadimidine, enrofloxacin, tilmicosin, and/or thiamphenicol.

9. The preparation of claim 7 or 8, wherein the sample is food, feed, muscle tissue, serum and/or urine.

10. A method for detecting antibiotic residues in a sample, characterized in that Geobacillus stearothermophilus according to claim 1 is used as an antibiotic-sensitive microorganism, and whether or not the sample contains an antibiotic is detected by changing the color of an indicator in a medium.