CN110031576A - A kind of method that quick detection produces tetracycline antibiotics degrading enzyme bacterium - Google Patents
A kind of method that quick detection produces tetracycline antibiotics degrading enzyme bacterium Download PDFInfo
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- CN110031576A CN110031576A CN201910291334.1A CN201910291334A CN110031576A CN 110031576 A CN110031576 A CN 110031576A CN 201910291334 A CN201910291334 A CN 201910291334A CN 110031576 A CN110031576 A CN 110031576A
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention belongs to technical field of biological, in particular to a kind of to be able to detect the mass spectrometry method for producing tetracycline antibiotics degrading enzyme bacterium, the following steps are included: S1. bacterium and drug co-culture: tested bacteria is cultivated, obtain culture solution, and tetracycline antibiotics standard solution (final concentration of 140 ~ 160 μ g/mL of tetracycline antibiotics) are added, 6 ~ 10h is incubated for after mixing under the conditions of 30 ~ 40 DEG C；S2. sample pre-treatments: the coculture being incubated for being centrifuged, Aspirate supernatant, carries out a target on target plate, naturally dry, is then covered with alpha-cyano -4- hydroxycinnamic acid and naturally dry again；S3. the survey fixed sum data analysis of sample: the target plate after passing point target in step S2 is detected using mass spectrography, analyzes mass ranges depending on the type of tetracycline antibiotics.This method has the characteristics that high sensitivity, accuracy height and high resolution, suitable for producing the detection of tetracycline antibiotics degrading enzyme bacterium.
Description
Technical field
The invention belongs to technical field of biological, in particular to a kind of quickly detection produces tetracycline antibiotics degrading enzyme
The method of bacterium.
Background technique
Since tetracycline degradation enzyme mechanism in 1984 is reported for the first time, have more than ten tetracycline antibiotics degrading enzyme bases
Cause is detected, including tet (X), tet (34), tet (37) and tet (47)-tet (56) etc..Wherein, tet (X) gene is especially worth
It must pay close attention to, it can pass through hydroxylation the degradation first generation (such as tetracycline, aureomycin and terramycin), the second generation (Ru Duoxi
Ring element and minocycline), the third generation (such as tigecycline) and forth generation (such as Yi Lawa ring is plain) nearly all tetracycline medication
(Jana L.Markley etc., 2018, Frontiers in Microbiology).It is a concern that constantly having in recent years new
Tet (X) hypotype there is (such as tet (X1)-tet (X4)), bacterial drug resistance caused by them even multi-drug resistant is more
Seriously.But still lack effective means for such drug resistant gene and the quick detection for encoding zymoprotein.
Matrix-assisted laser desorption ionization technology (MALDI-TOF-MS) is common in mass spectral analysis
Soft ionization techniques have the characteristics that high sensitivity, accuracy height and high resolution, the survey suitable for mixture and large biological molecule
It is fixed.In recent years, which has been widely used in carbapenem enzyme NDM (Jiajia Yu etc., 2018, Ann Clin
Microbiol Antimicrob) and lipid A modification albumen MCR (Laurent Dortet etc., 2018, J Antimicrob
Chemother quick detection).
Summary of the invention
The present invention provides a kind of detect and produces tetracycline antibiotics degrading enzyme bacterium to overcome the problems of the prior art
Method.
The purpose of the present invention is achieved by the following technical programs:
A method of detection produces tetracycline antibiotics degrading enzyme bacterium, by sample to be tested and tetracycline antibiotics standard items
Coculture as experimental group, contain only the solution of tetracycline antibiotics standard items as positive controls, using matrix
The detection of assisted laser desorption ionisation time-of-flight mass spectrometry (TOFMS)；
If tetracycline antibiotics specific ion peak disappears in experimental group, sample to be tested is determined to produce tetracycline antibiotics
The bacterium of degrading enzyme；
If observing, the specific ion peak corresponding positive control group for representing tetracycline antibiotics obviously weakens, and determines the bacterium
Produce tetracycline antibiotics degrading enzyme；
If observing, the specific ion peak corresponding positive control group for representing tetracycline antibiotics is almost unchanged, determines the bacterium
It will not output tetracycline antibiotics degrading enzyme.
Based on tetracycline degradation enzyme mechanism, MALDI-TOF-MS technology is innovatively applied to Tet by the present invention
(X4) and the quick detection of the tetracycline antibiotics degrading enzyme such as related subtypes.
Preferably, the tetracycline antibiotics are tetracycline, aureomycin, terramycin, Doxycycline, minocycline, replace
Add one of ring element or Yi Lawa ring element or a variety of.
The method that the present invention protects the detection to produce tetracycline antibiotics degrading enzyme bacterium simultaneously, including following step
It is rapid:
S1. bacterium and drug co-culture: tested bacteria being cultivated, culture solution is obtained, tetracycline antibiotics standard is added
Product solution is incubated for 6~10h until the concentration of tetracycline antibiotics is 140~160 μ g/mL under the conditions of 30~40 DEG C after mixing；
S2. sample pre-treatments: the coculture being incubated for is centrifuged, and Aspirate supernatant carries out a target on target plate, dries in the air naturally
It is dry, using alpha-cyano -4- hydroxycinnamic acid as matrix；Matrix and sample form cocrystallization film, matrix absorption after laser irradiation
Energy, subsequent matrix transfer energy to sample, help sample ionization.Suggested use and dosage is the recommended method of instrument manufacturer
And dosage.
S3. the survey fixed sum data analysis of sample: target plate is detected using mass spectrography, by taking tetracycline as an example, analysis
Mass ranges are 300~500m/z.
Preferably, the target plate is with 1% chromatographic grade formic acid, chromatography grade acetone and hplc grade methanol respectively to target plate ultrasound 10
~15min impregnates 15~20min in ultrapure water and is rinsed again with hplc grade methanol, dried spare after ultrasound is complete.
Preferably, the Mass Spectrometry Conditions being arranged in the step S3 are as follows: mass ranges, 1~1000m/z, ion source 20kv, line
Sexual reflex 2.60~2.70kv of device, 5.5~6.5kv of camera lens, laser frequency 50Hz, pulse ion extraction delay 114ns, laser energy
Measure 50~60 linear positive ion modes.
Preferably, each sample chooses 100 test points, and each test point bombards 5 times.
Preferably, the method is to detect the tetracycline antibiotics degrading enzyme of Tet (X4) and related subtypes.
Preferably, in step S1 culture solution OD600It is 2.0, pH=7.0.
Matrix-assisted laser desorption ionization technology detect when, the present invention by taking the detection of tetracycline as an example,
Whether disappear or weaken by analysis tetracycline specific ion peak (444m/z), to determine whether the bacterium generates a kind of Fourth Ring
Plain class Degradation of Antibiotics enzyme.Specific standards are as follows: if observing, the specific ion peak for representing tetracycline weakens or disappears, and determines
The bacterium produces tetracycline antibiotics degrading enzyme；If observing, the specific ion peak for representing tetracycline is almost unchanged, and determining should
Bacterium will not produce tetracycline antibiotics degrading enzyme.
Compared with prior art, the present invention has the following advantages and beneficial effects:
The present invention is by Matrix-assisted laser desorption ionization technology in detection tetracycline antibiotics degrading enzyme
When have the characteristics that high sensitivity, accuracy be high and high resolution；It, can based on tetracycline antibiotics degradation enzyme mechanism
Realize the quick detection of the tetracycline antibiotics degrading enzyme such as Tet (X4) and related subtypes.
Detailed description of the invention
Fig. 1 Tet (X4) encodes three bit space structure chart of albumen；
The amino acid alignment result of Fig. 2 Tet (X4) coding albumen and other homologous proteins；
Fig. 3 present invention detects the result for producing tetracycline antibiotics degrading enzyme bacterium；
Fig. 4 is the result that the detection of comparative example 1 produces tetracycline antibiotics degrading enzyme bacterium.
Specific embodiment
It to make the object, technical solutions and advantages of the present invention clearer, combined with specific embodiments below will be to this hair with attached drawing
Bright technical solution is described in detail.Obviously, described embodiments are only a part of the embodiments of the present invention, rather than
Whole embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not before making creative work
Obtained all other embodiment is put, the range that the present invention is protected is belonged to.
Embodiment 1
1. Escherichia coli LHM10-1 is selected to be detected as experiment with bacterium, Escherichia coli LHM10-1 (experimental group) is isolated from
In July, 2017 Jiangxi Province's Zhangshu City pig farm, tet (X4) genetic test result are positive, and the tetracycline that can significantly degrade, gold are mould
Element, terramycin, Doxycycline, minocycline, tigecycline and Yi Lawa ring element.
Above-mentioned Escherichia coli (Escherichia coli) LHM10-1 bacterial strain is preserved in Guangdong Province on March 28th, 2019
Culture Collection (GDMCC), deposit number are GDMCC No:60622.Preservation address are as follows: Guangzhou, Guangdong is first
5 building, the building of compound the 59th of strong Road 100.
2. genome sequencing and analysis
The above-mentioned purified Escherichia coli LHM10-1 of a ring is scraped, referring to Tiangeng bacterial genomes DNA extraction kit (Tiangeng
Biochemical technology Co., Ltd) using step extract the genome of the bacterial strain.Then, respectively using MiSeq platform (China's silver health)
Genome sequencing is carried out to it with PacBio RSII system (Wuhan future group Biotechnology Co., Ltd).Finally by Μ
Nicycler (version 0.4.1) simultaneously assembles the short long initial sequence information of long and long reading of reading, and passes through RAST
(http://rast.nmpdr.org/) carries out Sequence annotation and analysis.At the same time, according to default parameters by online service
Device Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi? id=index Fourth Ring) is simulated
The three-D space structure of protein coded by plain class Degradation of Antibiotics enzyme gene.
By the further analysis to sequencing data of whole genome, a kind of novel tetracycline antibiotics degrading enzyme is obtained
Gene is named as tet (X4).The full length gene is 1158bp (SEQ ID NO:1), can encode one kind by 385 amino acid
The protein Tet (X4) of composition, the three-D space structure of the albumen are shown in Fig. 1.Amino acid alignment the result shows that (see Fig. 2),
It with report for the first time tetracycline degradation zymoprotein Tet (X) (accession number: M37699) have 94.5% similarity, and and Tet
(X1) (accession number: AJ311171), the phase of Tet (X2) (accession number: AJ311171) and Tet (X3) (accession number: AB097942)
63.9%, 95.1% and 85.7% has also been respectively reached like degree.
3. the foundation of detection method:
S1. the experiment preparation of bacterium
By Escherichia coli LHM10-1 and 25922 streak inoculation of Escherichia coli ATCC in blank LB agar (the triumphant microorganism of Guangdong ring
Science and Technology Ltd.) plate, 37 DEG C of culture 18h.
S2. the co-cultivation of bacterium and drug
Above-mentioned cultured bacterial cultures is scraped respectively, is added to containing 800 μ l ddH2The 1.5mL EP of O is managed, and is vortexed and is mixed
(OD600=2.0；PH=7.0).It is then added appropriate tetracycline standard solution (German Dr.Ehrenstorfer company), protects
Its final concentration of 150 μ g/mL in coculture is demonstrate,proved, is vortexed after mixing and is incubated for 8h jointly under the conditions of 37 DEG C.At the same time, with
The tetracycline aqueous solution (150 μ g/mL) and ddH of same concentrations2O is respectively as positive controls and blank control.It is aforementioned four
Group is respectively arranged 6 in parallel.
S3. the pre-treatment of sample
It is wiped first using surface of the hplc grade methanol to point sample target plate, target plate employed in the present invention can be simultaneously
Detect 384 samples (model 223-25114-05DE1580TA:384PLATE 2.8MM).Then with 1% chromatographic grade formic acid,
Chromatography grade acetone and hplc grade methanol are respectively to target plate ultrasound 15min, finally in ddH220min is impregnated in O and uses chromatography again
Grade methanol rinses.It is spare after natural drying.Above-mentioned chromatographic grade cleaning is purchased from Fisher Chemical company, the U.S. with reagent.
The good coculture of above-mentioned incubation is centrifuged 2min under the conditions of 12000rpm, draws 0.5 μ l supernatant point respectively
Target, naturally dry.Then, alpha-cyano -4- hydroxycinnamic acid (50% second of the final concentration 10mg/mL of 0.5 μ l Fresh is drawn
Nitrile, 2.5% trifluoroacetic acid；Sigma-Aldrich) it is covered on above, machine is gone up after natural drying to it.
S4. the survey fixed sum data analysis of sample
Target plate after above-mentioned point sample is dried is placed in AXIMA Performance mass spectrograph (Japanese Shimadzu Corporation), vacuumizes
And preheat laser light source 5min.It chooses on target plate after tri- alignment target Board positions of A1, A24 and P24, sample to be tested is carried out just
Formula detection.It is as follows that system parameter is set: 1~1000m/z of mass ranges, ion source 20kv, linear reflector 2.62kv, camera lens
6kv, laser frequency 50Hz, pulse ion extraction delay 114ns, laser energy 50~60, each sample choose 100 detections
Point, each test point bombard 5 times, linear positive ion mode.
Whether disappear or weaken by tetracycline specific ion peak (444m/z) in analysis 300-500m/z mass ranges,
To determine whether the experiment generates a kind of tetracycline antibiotics degrading enzyme with bacterium.
Specific standards are as follows: if observing, the specific ion peak for representing tetracycline disappears (other in parallel), determines
The bacterium produces tetracycline antibiotics degrading enzyme；If observing, the specific ion peak corresponding positive control group for representing tetracycline is bright
It is aobvious to weaken (other in parallel), determine that the bacterium produces tetracycline antibiotics degrading enzyme；If observing the spy for representing tetracycline
Counter ions peak corresponding positive control group is almost unchanged (other in parallel), and it is anti-to determine that the bacterium will not produce Tetracyclines
Raw element degrading enzyme.If observe that each parallel result is inconsistent, it is proposed that repetitive operation is verified.
Testing result is and each flat as shown in figure 3, the tetracycline specific ion peak (444m/z) in experimental group almost disappears
Reproducibility is good between row.And positive controls (containing isoconcentration tetracycline standard items) and negative control group (Escherichia coli
ATCC25922 the tetracycline specific ion peak in) is consistent and fairly obvious, blank control group (ddH2O the tetracycline in)
Specific ion peak is not present.Comparing result explanation between experimental group and control group, experimental group bacteria Escherichia coli LHM10-1
Tetracycline antibiotics degrading enzyme can be produced, this matches with the Escherichia coli LHM10-1 feature for carrying tet (X4) gene, yin
Property control group bacteria Escherichia coli ATCC 25922 can not produce tetracycline antibiotics degrading enzyme, and and such undegraded medicine
Object.In addition, 379m/z is matrix peak.
Comparative example 1:
S1. the co-cultivation of bacterium and drug (source of bacterium and culture are with embodiment 1)
Cultured bacterial cultures is scraped respectively, is added to containing 1mL ddH2The 1.5mL EP of O is managed, and is vortexed and is mixed (OD600
=1.0；PH=7.0).Referring to dosage (Sudeshna Ghosh etc., 2015, FEMS Microbiology of report
Ecology), appropriate tetracycline standard solution (German Dr.Ehrenstorfer company) is added, guarantees it in coculture
Final concentration of 30 μ g/mL, be vortexed after mixing and be incubated for 5h jointly under the conditions of 30 DEG C.At the same time, with the tetracycline of same concentrations
Aqueous solution (30 μ g/mL) and ddH2O is respectively as positive controls and blank control.Aforementioned four group is respectively arranged 6 in parallel.
S2. the pre-treatment of sample
The surface of point sample target plate is wiped using hplc grade methanol (Fisher Chemical company, the U.S.) first, this
Target plate employed in invention can detect 384 sample (model 223-25114-05DE1580TA:384PLATE simultaneously
2.8MM).Then in ddH210min is impregnated in O and is rinsed again with hplc grade methanol, it is spare after natural drying.By above-mentioned incubation
Good coculture is centrifuged 3min under the conditions of 8000rpm, draws 0.5 μ l supernatant point target respectively, naturally dry.Then, it inhales
Take sinapic acid (50% acetonitrile, 2.5% trifluoroacetic acid of the final concentration 10mg/mL of 0.5 μ l Fresh；U.S. Sigma-
Aldrich) it is covered on above, machine is gone up after natural drying to it.
S3. the survey fixed sum data analysis of sample
Target plate after above-mentioned point sample is dried is placed in AXIMA Performance mass spectrograph (Japanese Shimadzu Corporation), vacuumizes
And after preheating laser light source 5min, sample to be tested is formally detected.It is as follows that system parameter is set: mass ranges 1~
1000m/z；Ion source 16.8kv；Linear reflector 1.62kv；Camera lens 6kv；Laser frequency 50Hz；Pulse ion extracts 114ns；
Laser energy 80-90；Each sample chooses 100 test points, and each test point bombards 2 times；Linear positive ion mode.
As shown in figure 4, by analysis 1-1000m/z mass ranges in tetracycline specific ion peak (444m/z) it is found that
Although the specific peak of drug is in the presence of detection signal is unobvious, and detection contrast effect is poor in positive controls.Therefore, real
Although testing in group the not specific peak of tetracycline, it still needs to further verify whether degradation, and accuracy reduces, and illustrate appropriately
Testing conditions to testing result comparison have large effect.
Sequence table
<110>Agricultural University Of South China
<120>a kind of method that quickly detection produces tetracycline antibiotics degrading enzyme bacterium
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1158
<212> DNA
<213>Escherichia coli (Escherichia coli)
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atgagcaata aagaaaaaca aatgaattta cttagtgata agaacgttgc aataattggt 60
ggtggacccg ttggactgac tatggcaaaa ttattacagc aaaacggcat agacgtttca 120
gtttacgaaa gagacaacga ccgagaggca agaatttttg gtggaaccct tgacctacac 180
aaaggttcag gtcaggaagc aatgaaaaaa gcgggattgt tacaaactta ttatgactta 240
gccttaccaa tgggtgtaaa tattgctgat gaaaaaggca atattttatc cacaaaaaat 300
gtaaagcccg aaaatcgatt tgacaatcct gaaataaaca gaaatgactt aagggctatc 360
ttgttgaata gtttagaaaa cgacacggtt atttgggata gaaaacttgt tatgcttgaa 420
cctggtaaga agaagtggac actaactttt gagaataaac cgagtgaaac agcagatctg 480
gttattattg ccaatggtgg aatgtctaaa gtaagaaaat ttgttaccga cacggaagtt 540
gaagaaacag gtactttcaa tatacaagcc gatattcatc atccagaggt gaactgtcct 600
ggattttttc agctatgcaa tggaaaccgg ctaatggctg ctcatcaagg taatttatta 660
tttgcgaatc ctaataataa tggtgcattg cattttggaa taagttttaa aacacctgat 720
gaatggaaaa accaaacgca ggtagatttt caaaacagaa atagtgtcgt tgattttctt 780
ctgaaagaat tttccgattg ggacgaacgc tacaaagaac tgattcgtgt gacatcatct 840
tttgtagggt tagcgacacg aatatttccc ttaggtaagt cttggaaaag taagcgtcca 900
ttacccataa cgatgattgg agatgctgct catttgatgc ctccttttgc aggacaaggc 960
gtaaacagcg ggttgatgga tgccttgata ttgtcggata atctgaccaa tgggaaattt 1020
aacagcattg aagaggctat tgaaaattat gaacagcaaa tgtttatcta tggcaaagaa 1080
gcacaagaag aatcaactca aaacgaaatt gaaatgttta aacccgactt tacgtttcag 1140
caattgttaa atgtataa 1158
Claims (9)
1. a kind of method that detection produces tetracycline antibiotics degrading enzyme bacterium, which is characterized in that by sample to be tested and tetracycline
The coculture of class antibiotic standard items contains only the solution of tetracycline antibiotics standard items as positive right as experimental group
According to group, detected using Matrix-assisted laser desorption ionization method；
If tetracycline antibiotics specific ion peak disappears in experimental group, sample to be tested is determined to produce tetracycline antibiotics
The bacterium of degrading enzyme；
If observing, the specific ion peak corresponding positive control group for representing tetracycline antibiotics obviously weakens, and determines the bacterium
Produce tetracycline antibiotics degrading enzyme；
If observing, the specific ion peak corresponding positive control group for representing tetracycline antibiotics is almost unchanged, determines the bacterium
It will not output tetracycline antibiotics degrading enzyme.
2. the method that detection according to claim 1 produces tetracycline antibiotics degrading enzyme bacterium, which is characterized in that described
Tetracycline antibiotics are in tetracycline, aureomycin, terramycin, Doxycycline, minocycline, tigecycline or Yi Lawa ring element
It is one or more.
3. the method that detection according to claim 1 produces tetracycline antibiotics degrading enzyme bacterium, which is characterized in that including
Following steps:
S1. bacterium and drug co-culture: tested bacteria being cultivated, culture solution is obtained, tetracycline antibiotics standard is added
Product solution is incubated for 6 ~ 10h until the concentration of tetracycline antibiotics is 140 ~ 160 μ g/mL under the conditions of 30 ~ 40 DEG C after mixing；
S2. sample pre-treatments: the coculture being incubated for is centrifuged, and Aspirate supernatant carries out a target on target plate, dries in the air naturally
It is dry, then use the covering of alpha-cyano -4- hydroxycinnamic acid and again naturally dry；
S3. the survey fixed sum data analysis of sample: the target plate after passing point target is detected using mass spectrography, analyzes mass spectrum model
It encloses depending on the type of tetracycline antibiotics.
4. the method that detection according to claim 3 produces tetracycline antibiotics degrading enzyme bacterium, which is characterized in that described
Target plate, respectively to target plate 10 ~ 15min of ultrasound, is being surpassed with 1% chromatographic grade formic acid, chromatography grade acetone and hplc grade methanol after ultrasound is complete
15 ~ 20min is impregnated in pure water and is rinsed again with hplc grade methanol, is dried spare.
5. the method that detection according to claim 3 produces tetracycline antibiotics degrading enzyme bacterium, which is characterized in that described
The Mass Spectrometry Conditions being arranged in step S3 are as follows: mass ranges, 1 ~ 1000m/z, ion source 20kv, 2.60 ~ 2.70kv of linear reflector,
5.5 ~ 6.5kv of camera lens, laser frequency 50Hz, pulse ion extraction delay 114ns, 50 ~ 60 linear positive ion mode of laser energy.
6. the method that detection produces tetracycline antibiotics degrading enzyme bacterium described in claim 5, which is characterized in that each sample
100 test points are chosen, each test point bombards 5 times.
7. the method that detection according to claim 1 or 3 produces tetracycline antibiotics degrading enzyme bacterium, which is characterized in that
The method is to detect the tetracycline antibiotics degrading enzyme of Tet (X4) and related subtypes.
8. the method that detection according to claim 3 produces tetracycline antibiotics degrading enzyme bacterium, which is characterized in that step
The OD of culture solution in S1600For 2.0, pH=7.0.
9. the method that detection according to claim 3 produces tetracycline antibiotics degrading enzyme bacterium, which is characterized in that with four
Ring element is that the representative drug of tetracycline antibiotics is detected, and analysis mass ranges are 300 ~ 500m/z.
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| CN113005211A (en) * | 2019-12-20 | 2021-06-22 | 中国农业大学 | LAMP primer and method for detecting tigecycline high-level drug resistance gene tet (X) and variant thereof |
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