CN106636426A - Glycopeptide antibiotic drug-resistance gene detection kit - Google Patents
Glycopeptide antibiotic drug-resistance gene detection kit Download PDFInfo
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- CN106636426A CN106636426A CN201710024512.5A CN201710024512A CN106636426A CN 106636426 A CN106636426 A CN 106636426A CN 201710024512 A CN201710024512 A CN 201710024512A CN 106636426 A CN106636426 A CN 106636426A
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- 238000001514 detection method Methods 0.000 title claims abstract description 50
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a glycopeptide antibiotic drug-resistance gene detection kit. The glycopeptide antibiotic drug-resistance gene detection kit comprises probes 1 to 23, and corresponding sequences are shown as SEQ ID NO. 1 to SEQ ID NO. 23; the invention further discloses a method for detecting a glycopeptide antibiotic drug-resistance gene by utilizing the kit; when the glycopeptide antibiotic drug-resistance gene detection kit disclosed by the invention is used for detecting, the credibility of an experiment result needs to be evaluated at first; the detection result of the group is credible only when detection results of positive and negative quality control contrasts are positive (+) and negative (-). When the results displayed by the three probes of a detected object are consistent, whether a sample is infected by the glycopeptide antibiotic drug-resistance gene or not can be judged; when the results displayed by the three probes of the detected object are inconsistent, whether the sample to be detected is positive or negative cannot be judged, and the detection kit needs to be used for redetecting or existing other detection methods are used for further identification. The glycopeptide antibiotic drug-resistance gene detection kit is simple to operate, rapid and efficient and high in accuracy and the results are easy to read.
Description
Technical field
The invention belongs to biology field, be related to a kind of parallel probe of glycopeptide antibiotics drug resistant gene detection,
Genetic chip, kit and detection method.
Background technology
In the past few decades, antibiotic is widely used in the treatment of various infectious diseases, but also just because of antibiotic
Widely use, cause new antibiotics resistance form to continuously emerge.Enterococcus faecalis, Much's bacillus, pseudomonas aeruginosa
100 Multiple Classes of Antibiotics to clinically using have drug resistance, become the important bacterium killer for threatening human life at present.Sugar
Peptide antibiotics (G1ycopeptides, Dalbahepides) is D- alanyls-D-alanine associativity and has heptapeptide structure
A class antibiotic, to including the main pathogenic fungi such as coagulase positive or negative Staphylococcus, each group of streptococcus, enterococcus (wrap
Include enterococcus faecalis and VREF), bar bacterium, anaerobic cocci and monocyte Listeria monocytogenes are interior almost all of
Gram-positive bacteria is all active, is clinically usually used in by gram-positive bacteria especially staphylococcus, enterococcus and lung
The treatment of serious infections caused by scorching streptococcus, represents the last line of defense for treating these serious infections.
Since the enterococcus of report glycopeptide class antibiotic resistance in 1988, often go out in hospital in the world
It is existing.Have now found that 7 genotype of Main Drug-Resistant Gene VanA, VanB, VanC, VanH, VanR, VanS and VanX.Glycopeptide class resists
Raw element drug resistant gene can be carried by transposons, plasmid, be transmitted by the conjugation between bacterium, make glycopeptide resistance in enterococcus
Rate is continuously increased, and many vancomycin-resistant enterococcus also to other antibiotic (such as beta-lactam and aminoglycoside
Class antibiotic) resistance, to the clinical treatment of vancomycin-resistant enterococcal infection very big difficulty is caused.On the other hand,
It was found that some methicillin-resistant S Staphylococcus in clinic separation strains subtract to the sensitiveness of vancomycin and teicoplanin
It is weak, transferability vanB resistance determinant is found that in bargen's streptococcus clinical separation strain, it is clinically separated in Bacillus circulans
Find there is vanA gene clusters in strain, illustrate that glycopeptide drug resistant gene is not only propagated between enterococcus, and enterococcus can be transferred to
Other outer bacteriums, to following anti-infective therapy greatly threat is brought, and the glycopeptide to developing and developing efficiently and accurately is resistance to
The detection of medicine gene has great importance in clinical auxiliary treatment.
Biochip technology is the emerging biometric technology that the nineties grow up.Classify according to detection object,
The big class of genetic chip and protein-chip etc. two can be divided into.Compared with protein-chip, what genetic chip was utilized is nucleotides
Complementation between chain, high specificity, property is good.Glycopeptide antibiotics drug resistant gene chip provided by the present invention with it is traditional
Susceptibility of bacteria detection method is compared, and is directed to pathogen glycopeptide antibiotics drug resistant gene and is detected, its detection flux
The advantage such as greatly, required sample size is few, sensitivity is high, accuracy is high and specificity is good.
The content of the invention
It is an object of the present invention to provide the glycopeptide class that a species specificity is good, result is accurate, false positive rate is low, detection efficiency is high resists
Parallel probe, genetic chip, kit and detection method that raw element drug resistant gene is detected.
The present invention realizes that its purpose technical scheme is:
A kind of glycopeptide antibiotics drug resistant gene detects parallel probe, including probe 1 ~ 23, its corresponding sequence such as SEQ ID
Shown in NO.1 ~ 23.
A kind of glycopeptide antibiotics drug resistant gene detection chip, including solid phase and the probe groups described in claim 1.
Preferably, the solid phase carrier is amido modified glass substrate, nylon membrane or nitrocellulose filter.
A kind of glycopeptide antibiotics drug resistant gene detection kit, including aforesaid genetic chip.
In above-mentioned glycopeptide antibiotics drug resistant gene detection kit, also expand including the resistance to drug target gene of glycopeptide antibiotics
Increase primer pair, primer sequence is as shown in SEQ ID NO.24 ~ 39.
Preferably, CY3 fluorochrome labels are contained at 5 ' ends of the downstream primer of the primer pair.
Preferably, above-mentioned glycopeptide antibiotics drug resistant gene detection kit, also including PCR reaction systems, hybridization
Buffer solution, cleaning solution.
In above-mentioned technical proposal, the PCR reaction systems of 20 μ L include:The μ L of amplification template 2 of 50 ~ 150 ng/ μ L,
The μ L of Premix Taq 10, concentration is 8 ~ 12 μM of each 1 μ L of upstream and downstream primer, the μ L of distilled water 6.
A kind of glycopeptide antibiotics drug resistant gene detection method, is tried using the detection of aforesaid glycopeptide antibiotics drug resistant gene
Agent box detected, may determine that whether sample infects glycopeptide when the testing result that three probes of detection object show is consistent
Class antibiotic resistance genes;When the testing result that three probes of detection object show is inconsistent, it is impossible to judge that this treats test sample
Product are positive or negative, it is proposed that detected again using the detection kit or further tested using existing other detection methods
Card.
Preferably, pcr amplification reaction program is 95 DEG C of 5 min;95℃10 s、55℃ 30 s、72℃30 s、30
Individual circulation;72℃ 10 min.
The invention has the beneficial effects as follows:Each drug resistant gene designs the probe of 3 high specifics, when the detection of 3 probes
The index is just judged when being as a result the positive for positive, the accuracy and accuracy of product can be greatly improved, other are reduced
The false positive issue that method easily occurs;The present invention is carried out by round pcr to pathogen glycopeptide antibiotics drug resistant gene group
Amplification, the specificity of detection is high, and amplified production is hybridized with the oligonucleotide probe on biochip, is marked by CY3
The reverse amplimers of PCR detect results of hybridization.Simple to operate, result is readable, meets the Site Detection demand such as medical treatment, is clinical doctor
That the whether infected glycopeptide antibiotics drug resistant gene of raw quick diagnosis patient is provided is convenient, theoretical foundation fast and accurately,
With wide clinical practice market potential.
Description of the drawings
Fig. 1 detects the genetic chip schematic diagram of glycopeptide antibiotics drug resistant gene.
PCR primer electrophoretic band figure after the amplification of Fig. 2 testing sample glycopeptide antibiotics drug resistant gene;Marker DL2000
(TAKARA).
Specific embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.It is unreceipted concrete in embodiment
The experimental technique of condition, generally according to normal condition, such as Molecular Cloning:A Laboratory guide(The third edition, J. Pehanorm Brookers etc.
Write)Described in condition, or according to the condition proposed by manufacturer.Following examples are used to illustrate the present invention, but are not used in
Limit the scope of the present invention.
The purpose fragment primer of embodiment 1 is designed with probe
Search gene order VanA, VanB of glycopeptide antibiotics drug resistant gene in ncbi database, VanC, VanH, VanR,
VanS and VanX, using chip probe design software(ArrayDesigner4.2)3 probes are designed for each drug resistant gene,
The probe of another design 16sRNA genes is used as positive quality control probe(PC)With one section of probe of random synthesis as negative Quality Control probe
(NC), correspondence probe sequence is as shown in SEQ ID NO .1 ~ 23.
Using NCBI Photographing On-line softwares(Primer-BLAST)Design VanA, VanB, VanC, VanH, VanR, VanS and
The pcr amplification primer thing of VanX genes, while designing the amplimer of 16sRNA genes.In order that chip results are more directly perceived,
Reverse amplimer(R)5 ' ends are marked with CY3 fluorescent dyes, corresponding primer sequence such as SEQ ID NO .24 ~ 39 institute
Show.
The preparation of the chip of embodiment 2
By the parallel probe in embodiment 1 according to the order point sample of table 1 in amido modified glass substrate, obtain containing probe
Genetic chip(As shown in Figure 1).Concentration and probe concentration is 30 μM, 0.2 μ L of each point, then 80 DEG C of 1.5 h of incubation.
The detection method of embodiment 3
1st, testing sample genome is extracted
Using bacterial genomes DNA extraction kit, according to the genome conduct that testing sample is extracted the step of operational manual
Amplification template.
2nd, PCR amplifications purpose fragment
Grope through many experiments, determine the target gene PCR amplification system and PCR response procedures of Van detections.By each Van base
Because of the primer of VanA, VanB, VanC, VanH, VanR, VanS, VanX and 16SrRNA amplification, PCR amplification buffers are prepared respectively
System, cumulative volume be 20 μ L PCR amplification system in be respectively containing reagent:μ L, F primers of Premix LATaq 10(10μM)
1 μ L, F primers(10 μM)1 μ L, the amplification μ L of template 2, the μ L of distilled water 6.
PCR response procedures are:95℃ 5 min;95 DEG C of 10 s, 55 DEG C of 30 s, 72 DEG C of 30 s, 30 circulations;72℃ 10
min。
3rd, PCR primer and chip hybridization
Operate in accordance with the following steps:
A. on the genetic chip for preparing in example 2, by the VanA of testing sample, VanB, VanC, VanH, VanR,
The pcr amplification product of VanS, VanX and 16SrRNA is mixed and point sample is in the detect tank of chip, the PCR primer point of VanA primers
Other point on VanA-1, VanA-2, VanA-3 probe, divide by the PCR primer of VanB, VanC, VanH, VanR, VanS, VanX primer
To on probe, the PCR primer of 16sRNA primers puts respectively positive quality control probe to other corresponding points(PC)With negative Quality Control probe
(NC).Concretely comprise the following steps:95 DEG C of PCR primer is unwind 5 minutes, is immediately placed on ice, PCR primer and hybridization buffer(50% formyl
Amine, 10 × SSC, 0.2%SDS)Equal-volume mixes, and selects 10 μ L mixed liquors per hole, in wet box, in 60 DEG C of hybridization incubations 2 hours;
B. the genetic chip after incubation is taken out, cleaning solution is used(0.3 × SSC buffer solutions)Cleaning 3 times;
C. chip is put into laser co-focusing detector(GenePix)Chip is scanned at 650 nm;Software analysis fluorescence signal
Intensity level, provides result.
Embodiment 4 detects testing sample
5 parts of the sample of glycopeptide antibiotics drug-resistant bacteria infection is obtained from Third Military Medical University(Effusion of knee joint), sugar-free peptides
Antibiotic resistance genes infect 1 part of sample(Control group).Extract treat test sample respectively according to the primer and method in embodiment 1 and 3
The genome of product, after entering performing PCR amplification, is detected using the genetic chip in embodiment 2.Product electrophoresis after PCR amplifications
Band shows that the amplimer specificity of present invention design is high, can special, delicately amplify target gene(As shown in Figure 2).
For the sample of bacterium infection, laser co-focusing detector(GenePix)Chip, probe points are scanned at 650 nm
Fluorescence signal is stronger(More than 20), it is designated as(+), fluorescence signal is weaker(Less than 5), then it is designated as(-), fluorescence signal intensity is difficult to point
Distinguish(5~20)Then it is designated as(*).Positive and negative Quality Control control is designed in detection simultaneously, only when positive and negative Quality Control control
Testing result is divided into not positive simultaneously(+)And feminine gender(-), just think that this group of testing result is credible.
When judging testing result, when 3 probes of VanA, VanB, VanC, VanH, VanR, VanS, VanX gene show
The sample is just judged when showing the positive with the drug resistant gene, if wherein some or two probes show positive, is tackled
The detection sample detects that 3 probe results unanimously just must can accept and believe the testing result again.3 probe in detecting results are inconsistent
When, it is possible to use existing other technologies(Such as elisa methods)Further detection checking.Testing result is as shown in table 2.
The pattern detection result of table 2
As a result it is shown as positive quality control control and is the positive(+), negative Quality Control control is feminine gender(-), illustrate that testing result can
Letter.The infection of glycopeptide antibiotics drug resistant gene is detected in wherein 5 parts samples, sample 1 is by glycopeptide antibiotics drug resistant gene
VanA infects, and sample 2 is infected by glycopeptide antibiotics drug resistant gene VanR, and sample 3 is by glycopeptide antibiotics drug resistant gene VanX
Infection, sample 4 is infected by glycopeptide antibiotics drug resistant gene VanX, and sample 5 is infected by glycopeptide antibiotics drug resistant gene VanR;
Check sample is not detected by glycopeptide antibiotics drug resistant gene, is consistent with actual clinical sample information situation.
Probe disclosed by the invention, genetic chip, kit, specificity is high, result is very accurate, and detection is quick, work
Efficiency high, it is simple to operate, meet the Site Detection demand of hospital etc., providing for the drug-resistant bacteria infection of diagnosis glycopeptide antibiotics can
The experimental basis for leaning on.
SEQUENCE LISTING
<110>Chongqing prestige this rise biological medicine science and technology limited Company
<120>A kind of glycopeptide antibiotics drug resistant gene detection kit
<130> 2017
<160> 39
<170> PatentIn version 3.5
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<213>Artificial sequence
<400> 10
tttttttttt tttttggtgt gaaatatatt tctacccgaa gcatc 45
<210> 11
<211> 45
<212> DNA
<213>Artificial sequence
<400> 11
tttttttttt tttttgtggt gggaacgggc cagataggca aagcg 45
<210> 12
<211> 45
<212> DNA
<213>Artificial sequence
<400> 12
tttttttttt tttttattat cagccacgaa caaatacaga gaatg 45
<210> 13
<211> 45
<212> DNA
<213>Artificial sequence
<400> 13
tttttttttt tttttatatt ggacatcatg cttcccggca caagc 45
<210> 14
<211> 45
<212> DNA
<213>Artificial sequence
<400> 14
tttttttttt tttttgggtt aacaatcggc gcggatgatt atata 45
<210> 15
<211> 45
<212> DNA
<213>Artificial sequence
<400> 15
tttttttttt tttttgaaaa tgttatcgtc cactccggcc ttgtc 45
<210> 16
<211> 45
<212> DNA
<213>Artificial sequence
<400> 16
tttttttttt tttttgatgt acttattcag aacgaagata aacaa 45
<210> 17
<211> 45
<212> DNA
<213>Artificial sequence
<400> 17
tttttttttt tttttggaaa acaggcggtt attcacgccc ccgag 45
<210> 18
<211> 45
<212> DNA
<213>Artificial sequence
<400> 18
tttttttttt tttttgccga acaaagaaaa aatgacgttg ttatg 45
<210> 19
<211> 45
<212> DNA
<213>Artificial sequence
<400> 19
tttttttttt tttttttgaa ggcaaaagaa ctggctgcta cccaa 45
<210> 20
<211> 45
<212> DNA
<213>Artificial sequence
<400> 20
tttttttttt tttttgcttc aaaatcaagc catagccgcg gcagt 45
<210> 21
<211> 45
<212> DNA
<213>Artificial sequence
<400> 21
tttttttttt tttttgcggc aaatggaata tcatgcaatg aagcg 45
<210> 22
<211> 34
<212> DNA
<213>Artificial sequence
<400> 22
tttttttttt tttttccgat agtgaaccag tacc 34
<210> 23
<211> 38
<212> DNA
<213>Artificial sequence
<400> 23
tttttttttt tttttctatc tagctagcta gctagcta 38
<210> 24
<211> 25
<212> DNA
<213>Artificial sequence
<400> 24
cgatcaagcg gtcaatcagt tcggg 25
<210> 25
<211> 24
<212> DNA
<213>Artificial sequence
<400> 25
cctctgagca acccccaaac agta 24
<210> 26
<211> 29
<212> DNA
<213>Artificial sequence
<400> 26
gcggttgctc ggaggaacat gatgtgtcg 29
<210> 27
<211> 24
<212> DNA
<213>Artificial sequence
<400> 27
tatcgccaat gtaatcaggc tgtc 24
<210> 28
<211> 27
<212> DNA
<213>Artificial sequence
<400> 28
attcaccgga atacaccgtt tctttag 27
<210> 29
<211> 26
<212> DNA
<213>Artificial sequence
<400> 29
tcttgatagg ataagccgac cgctgc 26
<210> 30
<211> 25
<212> DNA
<213>Artificial sequence
<400> 30
tcggcattac tgtttatgga tgtga 25
<210> 31
<211> 28
<212> DNA
<213>Artificial sequence
<400> 31
ctcctgtctc ctttcaaaat ccaaacag 28
<210> 32
<211> 26
<212> DNA
<213>Artificial sequence
<400> 32
gtggatgatg aacatgaaat tgccga 26
<210> 33
<211> 25
<212> DNA
<213>Artificial sequence
<400> 33
aaccaagtct ggcatcgctg gaagc 25
<210> 34
<211> 25
<212> DNA
<213>Artificial sequence
<400> 34
attcgtgttg tatattcgtt caatg 25
<210> 35
<211> 25
<212> DNA
<213>Artificial sequence
<400> 35
aaccaagtct ggcatcgctg gaagc 25
<210> 36
<211> 19
<212> DNA
<213>Artificial sequence
<400> 36
gatgaaatag tacacggtg 19
<210> 37
<211> 21
<212> DNA
<213>Artificial sequence
<400> 37
ggggaaatca aaatagctat t 21
<210> 38
<211> 21
<212> DNA
<213>Artificial sequence
<400> 38
gagagtttga tyctggctca g 21
<210> 39
<211> 20
<212> DNA
<213>Artificial sequence
<400> 39
aaggaggtga tccarccgca 20
Claims (10)
1. a kind of glycopeptide antibiotics drug resistant gene detects parallel probe, it is characterised in that:Including probe 1 ~ 23, its corresponding sequence
Row are as shown in SEQ ID NO.1 ~ 23.
2. a kind of glycopeptide antibiotics drug resistant gene detects genetic chip, it is characterised in that:Will including solid phase support and right
Seek the probe groups described in 1.
3. glycopeptide antibiotics drug resistant gene as claimed in claim 2 detects genetic chip, it is characterised in that:The solid phase is carried
Body is amido modified glass substrate.
4. a kind of glycopeptide antibiotics drug resistant gene detection kit, it is characterised in that:Including the base described in Claims 2 or 3
Because of chip.
5. glycopeptide antibiotics drug resistant gene detection kit as claimed in claim 4, it is characterised in that:Also include glycopeptide class
Antibiotics resistance target gene amplimer pair, primer sequence is as shown in SEQ ID NO.24 ~ 39.
6. glycopeptide antibiotics drug resistant gene detection kit as claimed in claim 5, it is characterised in that:The primer pair
Contain CY3 fluorochrome labels in 5 ' ends of downstream primer.
7. the glycopeptide antibiotics drug resistant gene detection kit as described in claim 4-6, it is characterised in that:Also include PCR
Reaction system, hybridization buffer, cleaning solution.
8. glycopeptide antibiotics drug resistant gene detection kit as claimed in claim 7, it is characterised in that:PCR reaction systems
Including:The μ L of amplification template 2 of 50 ~ 150 ng/ μ L, the μ L of Premix Taq 10, concentration is 10 μM of each 1 μ of upstream and downstream primer
L, the μ L of distilled water 6.
9. a kind of detection method of glycopeptide antibiotics drug resistant gene, it is characterised in that:Usage right requires 4 to 8 any one institutes
The glycopeptide antibiotics drug resistant gene detection kit stated is detected;First to carry out reliable experiment result degree assessment, only when
The testing result of positive and negative Quality Control control is divided into not positive simultaneously(+)And feminine gender(-), just think that this group of testing result can
Letter;May determine that whether sample is felt when the testing result that 3 probes of any one drug resistant gene for detecting sample show is consistent
Dye glycopeptide antibiotics drug resistant gene;When the testing result for detecting that any 3 probes of drug resistant gene of sample show is inconsistent, no
The testing sample can be judged for positive or negative, it is proposed that detect again using the detection kit or using existing other detections
Method is further verified.
10. the detection method of glycopeptide antibiotics drug resistant gene as claimed in claim 9, it is characterised in that:Pcr amplification reaction
Program is 95 DEG C of 5 min;95 DEG C of 10 s, 40 DEG C of 30 s, 72 DEG C of 90 s, 30 circulations;72℃ 10 min.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110055308A (en) * | 2019-04-27 | 2019-07-26 | 浙江大学 | Detect the specific primer of four kinds of glycopeptide class Drug-resistant genes and probe combinations and application in Enterococcus |
CN110066882A (en) * | 2019-04-27 | 2019-07-30 | 浙江大学 | Detect the specific primer of five kinds of glycopeptide class Drug-resistant genes and probe combinations and application in Enterococcus |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001012803A2 (en) * | 1999-08-17 | 2001-02-22 | Beth Israel Deaconess Medical Center, Inc. | Methods and compositions for restoring antibiotic susceptibility in glycopeptide-resistant enterococcus |
CN1246474C (en) * | 2001-07-13 | 2006-03-22 | 刘元 | Detection to drug tolerant gene by gene chip technique |
CN105925700A (en) * | 2016-06-06 | 2016-09-07 | 重庆威斯腾生物医药科技有限责任公司 | Toxoplasma detecting parallel probes, gene chip, kit and detection method |
-
2017
- 2017-01-13 CN CN201710024512.5A patent/CN106636426A/en active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001012803A2 (en) * | 1999-08-17 | 2001-02-22 | Beth Israel Deaconess Medical Center, Inc. | Methods and compositions for restoring antibiotic susceptibility in glycopeptide-resistant enterococcus |
CN1246474C (en) * | 2001-07-13 | 2006-03-22 | 刘元 | Detection to drug tolerant gene by gene chip technique |
CN105925700A (en) * | 2016-06-06 | 2016-09-07 | 重庆威斯腾生物医药科技有限责任公司 | Toxoplasma detecting parallel probes, gene chip, kit and detection method |
Non-Patent Citations (3)
Title |
---|
A MIELE等: "Use of primers selective for vancomycin resistance genes to determine van genotype in enterococci and to study gene organization in VanA isolates", 《ANTIMICROB AGENTS CHEMOTHER》 * |
G KLEIN等: "Exclusion of vanA, vanB and vanC type glycopeptide resistance in strains of Lactobacillus reuteri and Lactobacillus rhamnosus used as probiotics by polymerase chain reaction and hybridization methods", 《J APPL MICROBIOL》 * |
王雪松等: "1株VanA型VRE菌株的鉴定及耐药诱导特性研究", 《军事医学》 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110055308A (en) * | 2019-04-27 | 2019-07-26 | 浙江大学 | Detect the specific primer of four kinds of glycopeptide class Drug-resistant genes and probe combinations and application in Enterococcus |
CN110066882A (en) * | 2019-04-27 | 2019-07-30 | 浙江大学 | Detect the specific primer of five kinds of glycopeptide class Drug-resistant genes and probe combinations and application in Enterococcus |
CN110066882B (en) * | 2019-04-27 | 2020-08-07 | 浙江大学 | Specific primer and probe combination for detecting drug resistance genes of five glycopeptide drugs in enterococcus and application |
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