CN110066882A - Detect the specific primer of five kinds of glycopeptide class Drug-resistant genes and probe combinations and application in Enterococcus - Google Patents
Detect the specific primer of five kinds of glycopeptide class Drug-resistant genes and probe combinations and application in Enterococcus Download PDFInfo
- Publication number
- CN110066882A CN110066882A CN201910346784.6A CN201910346784A CN110066882A CN 110066882 A CN110066882 A CN 110066882A CN 201910346784 A CN201910346784 A CN 201910346784A CN 110066882 A CN110066882 A CN 110066882A
- Authority
- CN
- China
- Prior art keywords
- gene
- probe
- primer
- vang
- kinds
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- 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 the specific primer sets and probe combinations of five kinds of glycopeptide class Drug-resistant genes vanC, vanE, vanG, vanL and vanN in a kind of detection Enterococcus, primer including five kinds of glycopeptide class Drug-resistant genes are carried out with specific amplification combines, and further includes the specific probe for detecting five kinds of glycopeptide class Drug-resistant genes.The present invention further simultaneously discloses the method for carrying out five kinds of glycopeptide class Drug-resistant genes of detection using the specific primer and probe combinations.The present invention has high specific and degree of covering, and cannot be only used for the detection to vanC, vanE, vanG, vanL and vanN drug resistant gene in human infection sample, prompts the drug-resistant intensity to vancomycin etc.；It can also be used in animal and environmental samples the distribution of five kinds of glycopeptide class Drug-resistant genes and the monitoring of transfer case.
Description
Technical field
The present invention relates in field of biotechnology, the spy of five kinds of glycopeptide class Drug-resistant genes in Enterococcus is detected
Specific primer probe combinations and its application.The resistance mechanism for VanC, VanE, VanG, VanL and VanN operon being related to is logical
Cross by peptide glycan precursor C-terminal d-ala residue replace with d-ser make host generate drug resistance, only to vancomycin show in low journey
The drug resistance of degree, and it is invalid to teicoplanin.
Background technique
Glycopeptide antibiotics (Glycopeptides, GAPs) are a kind of antibiotic with heptapeptide structure, by amino acid with
Saccharide residue, chlorine atom, methyl and/or fat chain composition.Important glycopeptide antibiotics for treating human infection include first
For vancomycin (Vancomycin) and teicoplanin (Teicoplanin), although the two is respectively early in 1958 and 1988
It is just approved for clinic, but is still widely used in clinical practice at present.Second generation of glycopeptide antibiotics Telavancin,
Dalbavancin and Oritavancin is approved for clinic respectively at 2013 and 2014.With first generation glycopeptide class antibiosis
Element is compared, and second generation antibiotic shows stronger antibacterial ability and more superior pharmacokinetic properties.
Sugared peptide medicament inhibits peptide glycan by being integrated to the end d-ala-d-ala of bacteria cell wall peptide glycan precursor
(PG) synthesis, prevents bacterium from normally synthesizing cell wall, to reach antibacterial effect.GAPs is treatment by Gram positive
The last resort of severe infections caused by substance (such as staphylococcus aureus, enterococcus spp and clostridium difficile).Europe in 1988
Vancomycin-resistant enterococcus clinical separation strain is reported for the first time.From that time, the enterococcus of vancomycin resistance is with unexpected
Speed is propagated, and is found now in the hospital of most countries.
The study found that bacterium is due to there is a series of manipulation for encoding albumen in bacterium to the resistance of sugared peptide medicament
Son, product are used to synthesize the peptide glycan precursor with sugared peptide medicament low-affinity, including peptide glycan precursor C-terminal d-ala residue
Replaced by d-lac or d-ser, makes its failure by modifying sugared peptide medicament combination target spot；Either due to bacterial identification albumen,
The high-affinity precursor usually generated by host is eliminated, sugared peptide medicament combination target spot is removed, to generate drug resistance.
13 class glycopeptide class drug resistance operons are found in gram-positive bacteria at present, wherein 9 kinds are present in enterococcus,
4 kinds are present in other gram-positive bacterias.Though VanA, VanB, VanD and VanM operon composition found in enterococcus has
Difference, but the resistance mechanism of its core is all to reduce glycopeptide class by the way that peptide glycan precursor C-terminal d-ala residue is replaced with d-lac
The affinity of drug and peptide glycan precursor is eventually exhibited as the height drug resistance to vancomycin, and (VanB is removed to teicoplanin
Height drug resistance outside).And the resistance mechanism of VanC, VanE, VanG, VanL and VanN operon slightly has with above-mentioned several operons
Difference makes host generate drug resistance, but only shows vancomycin by the way that peptide glycan precursor C-terminal d-ala residue is replaced with d-ser
The drug resistance of middle low degree out, and it is invalid to teicoplanin.
There are two classes, i.e. Phenotypic examination and genotype detection to the detection method of glycopeptide class drug resistance at present.Phenotype inspection
It surveys by cultivating pathogen in the presence of sugared peptide medicament, passes through inhibition zone size, or measurement semilethal drug concentration
(MIC50) carry out.Phenotypic examination step is complicated, and time-consuming, and whole process at least needs 48-72 hours, pathogenicbacteria separation incubation
In, due to there is no antibiotic selective pressure, the plasmid etc. for carrying Van operon may be made to lose, lead to subsequent drug
Sensitivity Detection obtains the result of false negative.In view of this, more and more bacterial drug resistance researchers are proposed in the world
Genotype detection method.By the methods of polymerase chain reaction (PCR), sequencing, whether direct detection bacterium carries and glycopeptide
The relevant gene of class drug resistance, its advantage is that can provide the drug resistance information of gene level, it can be 24 hours after sample collection
It inside obtains as a result, technical step is less, expense is relatively inexpensive under normal circumstances.But there is also according only to amplified production piece for PCR method
The disadvantages of uncertainty and the sequencing data analysis of Duan great little judging result are more complex influences testing result reliability and wide
General popularization.
Existing genotype detection method spininess is directed to involved by the present invention VanA, VanB type glycopeptide class drug resistant gene
And the detection method of VanC, VanE, VanG, VanL and VanN type glycopeptide class drug resistant gene seldom report.
Summary of the invention
The technical problem to be solved in the present invention is to provide five kinds of glycopeptide class medicines in group-specific detection Enterococcus
The primer combination of probe and its application of object drug resistant gene vanC, vanE, vanG, vanL and vanN.
In order to solve the above technical problems, present invention firstly provides five kinds of glycopeptide class Drug-resistants in detection Enterococcus
The specific primer sets and probe combinations of gene: including to five kinds of glycopeptide class Drug-resistant gene vanC, vanE, vanG,
VanL and vanN carries out the primer combination of specific amplification, and primer is as shown in sequence 1-10；It further include five kinds of glycopeptide class medicines of detection
The specific probe of object drug resistant gene vanC, vanE, vanG, vanL and vanN combine, and probe is as shown in sequence 11-15.
Primer sets 1 include vanC gene-specific primer, specially vanC gene forward primer (sequence 1) and vanC gene
Reverse primer (sequence 2)；Primer sets 2 include vanE gene-specific primer, specially vanE gene forward primer (sequence 3) and
VanE gene reverse primer (sequence 4)；Primer sets 3 include vanG gene-specific primer, specially vanG gene forward primer
(sequence 5) and vanG gene reverse primer (sequence 6)；Primer sets 4 include vanL gene-specific primer, specially vanL gene
Forward primer (sequence 7) and vanL gene reverse primer (sequence 8), primer sets 5 include vanN gene-specific primer, specially
VanN gene forward primer (sequence 9) and vanN gene reverse primer (sequence 10).
The primer that the present invention designs combines parameter area having the same, to guarantee each group primer in identical reaction condition
Under can work normally.Specifically: for primer length range between 15-25 oligonucleotides, optimal is 20 oligonucleotides；Draw
For object Tm value range between 45-65 DEG C, optimal is 56 DEG C；Tm value at most differs 3 DEG C between positive, reverse primer, and optimal is 0
℃；For PCR product length range between 100-1000bp, optimal is 200-400bps.
The probe combinations are made of probe 1-5.Probe 1 includes vanC gene-specific probe (sequence 11)；Probe 2
Including vanE gene-specific probe (sequence 12)；Probe 3 includes vanG gene-specific probe (sequence 13)；Probe 4 includes
VanL gene-specific probe (sequence 14)；Probe 5 includes vanN gene-specific probe (sequence 15).
The probe that the present invention designs parameter area having the same, to guarantee each probe under identical reaction conditions all
It can work normally.Specifically: for probe length range between 15-30 oligonucleotides, optimal is 20 oligonucleotides；Probe
For hybridization temperature range between 48-70 DEG C, optimal is 56 DEG C；The G/C content range of probe is optimal to be between 20%-80%
50%.
Described in table 1 specific as follows:
Table 1
In the primer combination, the molar ratio of the primer sets 1,2,3,4,5 can be 1:1.5:1.5:1:1.
In the probe combinations, the probe 1,2,3,4,5 answers equivalent application in hybridization reaction.
The present invention goes back while providing the examination of five kinds of glycopeptide class Drug-resistant genes in specific detection Enterococcus
Agent box, including primer as described above combination (as shown in sequence 1-10) and specific probe combination (as shown in sequence 11-15).
Improvement as kit of the invention: further including pcr amplification reaction liquid, hybridization reaction solution.
Note: pcr amplification reaction liquid and/or hybridization reaction solution can be pcr amplification reaction liquid and/or the hybridization of commercialization
Liquid is also possible to meet the self-control reaction solution of PCR amplification and hybridization reaction principle.
It is following any purposes as the application of primer combination of probe, kit of the invention:
1), identify at least one of drug resistant gene vanC, vanE, vanG, vanL and vanN (that is, be any one, or
Any 2 kinds, 3 kinds, 4 kinds of combination)；
2) whether bacterium to be measured, is detected containing in glycopeptide class Drug-resistant gene vanC, vanE, vanG, vanL and vanN
It is at least one；
3) it, detects in sample to be tested and whether contains glycopeptide class Drug-resistant gene vanC, vanE, vanG, vanL and vanN
At least one of.
The improvement of application as primer combination of probe, kit of the invention:
1) sample to be tested/bacterium to be measured total DNA, is extracted；
2), the total DNA extracted using step 1) carries out PCR amplification using specific primer sets as template；
3), by step 2) obtain pcr amplification product hybridize respectively with the specific probe, then as follows into
Row judgement:
If the results of hybridization of probe is the positive, drug resistant gene corresponding to the positive control probe is present in this to test sample
In sheet/bacterium to be measured.
That is: if having the results of hybridization of one or more probe for the positive, then the sun in described specific probe combination
Drug resistant gene corresponding to property probe is present in the bacterium (or sample to be tested) to be measured；If what the specific probe combined
Results of hybridization is feminine gender, then five kinds of glycopeptide class Drug-resistant genes are not present in the bacterium (or sample to be tested) to be measured
Any one of vanC, vanE, vanG, vanL and vanN.
The sample to be tested can be containing germy environmental samples (soil, water body etc.), be also possible to from people
Body or the sample (excrement, fester etc.) of animal.
Specific primer sets provided by the invention and probe combinations are for detecting five kinds of glycopeptide class Drug-resistant genes
VanC, vanE, vanG, vanL and vanN have high specific, high sensitivity, quick, synchronization detection and degree of covering, not only may be used
For the detection to five kinds of glycopeptide class Drug-resistant genes in enterococcal infection sample (human infection sample), prompt to mould through the ages
The drug-resistant intensity of element etc. provides reference frame for clinical anti-infective therapy's medication, at the same to the development of glycopeptide class Drug-resistant into
Row monitoring；It can also be used in animal and environmental samples the distribution of five kinds of glycopeptide class Drug-resistant genes and the monitoring of transfer case.
The present invention has great promotional value.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 is the specific probe dot matrix layout viewing of five kinds of glycopeptide class Drug-resistant genes；
Fig. 2 is the specific detection result figure of five kinds of glycopeptide class Drug-resistant genes (after hybridization).
Specific embodiment
The present invention is described further combined with specific embodiments below, and the embodiment provided is only for illustrating this hair
It is bright, rather than limit the present invention.
Test method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples commercially obtains unless otherwise specified.
Embodiment 1, five kind of glycopeptide class Drug-resistant gene primer combination and probe combinations degree of covering and specificity
The present embodiment is as follows to the primer sets of five kinds of glycopeptide class Drug-resistant genes of detection of the present invention
It closes and is illustrated with the degree of covering of probe combinations and specificity.
Step 1: from comprehensive drug resistance database (the The Comprehensive of antibiotics resistance authoritative database-antibiotic
Antibiotic Resistance Database, CARD) downloading five kinds of glycopeptides class Drug-resistant gene of the present invention
The reference sequences of vanC, vanE, vanG, vanL and vanN: AF162694 (vanC), FJ872411 (vanE), DQ212986
(vanG), EU250284 (vanL) and JF802084 (vanN).
Step 2: submitting NCBI to carry out online Blast comparison respectively the reference sequences of above-mentioned five kinds of drug resistant genes, compare
Database is nonredundancy nucleic acid database (20190315 version).Wherein, vanC gene reference sequence AF162694 obtains 35 altogether
Aligned sequences, vanE gene reference sequence FJ872411 obtain 4 aligned sequences, vanG gene reference sequence DQ212986 altogether
23 aligned sequences are obtained altogether, and vanL gene reference sequence EU250284 obtains 3 aligned sequences, vanN gene reference sequence altogether
JF802084 obtains 5 aligned sequences altogether.
Step 3: the aligned sequences that the reference sequences of above-mentioned five kinds of drug resistant genes are retrieved merge, and then carry out core one by one
Real, only retaining Sequence annotation is respectively that the aligned sequences of vanC, vanE, vanG, vanL and vanN and gene annotation are not known
But with aligned sequences of the reference sequences consistency more than 95% (containing), composition data collection A；Remaining aligned sequences group after screening
At data set B.
Before data set A should be considered as cut-off comparison data library version day, all vanC for having found and completing sequencing,
The arrangement set of vanE, vanG, vanL and vanN gene；Before data set B should be considered as cut-off comparison data library version day,
It is all it has been found that and complete sequencing with the highest arrangement set of vanC, vanE, vanG, vanL and vanN gene similarity.
Through screening, data set A includes 35 sequences, comprising: vanC gene aligned sequences 15, vanE gene aligned sequences
2, vanG gene aligned sequences 13, vanL gene aligned sequences 2, vanN gene aligned sequences 3；Data set B includes
35 sequences, comprising: vanC gene aligned sequences 20, vanE gene aligned sequences 2, vanG gene aligned sequences 10,
VanL gene aligned sequences 1, vanN gene aligned sequences 2.
Step 4: by the primer combination and probe combinations of five kinds of glycopeptide class Drug-resistant genes of detection of the present invention
It is compared respectively at above-mentioned data set A and B, as the result is shown:
VanC gene magnification primer contained by primer sets 1 of the present invention and 15 vanC gene ratios in data set A
To sequence exactly match, and with other gene aligned sequences in data set A and all sequences in data set B exist to
Few 3 mispairing, illustrate: (1) vanC gene magnification primer contained by primer sets 1 of the present invention can be covered so far
It was found that all vanC genes and its mutated gene, with height covering property of vanC gene；(2) primer of the present invention
Group 1 contained by vanC gene magnification primer to vanE, vanG, vanL and vanN gene, and it is similar with vanC gene other
Gene can not be expanded effectively, the vanC gene specific with height.
VanC gene-specific probe sequence contained by probe 1 of the present invention and 15 vanC bases in data set A
Because aligned sequences exactly match, and deposited with other gene aligned sequences in data set A and all sequences in data set B
In at least three mispairing, illustrate: (1) vanC gene-specific probe contained by probe 1 of the present invention can be covered is so far
Only it has been found that all vanC genes and its mutated gene, with height covering property of vanC gene；(2) of the present invention
VanC gene-specific probe contained by probe 1 is and similar with vanC gene to vanE, vanG, vanL and vanN gene
Other genes can not effectively hybridize, the vanC gene specific with height.
VanE gene magnification primer contained by primer sets 2 of the present invention is compared with 2 vanE genes in data set A
Sequence exact matching, and have at least 4 with other gene aligned sequences in data set A and all sequences in data set B
A mispairing, illustrates: (1) vanE gene magnification primer contained by primer sets 2 of the present invention can be covered has sent out so far
Existing all vanE genes and its mutated gene, the covering property of vanE gene with height；(2) primer sets of the present invention
VanE gene magnification primer contained by 2 is to vanC, vanG, vanL and vanN gene, and other bases similar with vanE gene
Cause can not be expanded effectively, the vanE gene specific with height.
VanE gene-specific probe sequence contained by probe 2 of the present invention and 2 vanE genes in data set A
Aligned sequences exact matching, and exist with other gene aligned sequences in data set A and all sequences in data set B
At least three mispairing, illustrates: (1) vanE gene-specific probe contained by probe 2 of the present invention can be covered so far
All vanE genes and its mutated gene having found, the covering property of vanE gene with height；(2) spy of the present invention
VanE gene-specific probe contained by needle 2 to vanC, vanG, vanL and vanN gene, and it is similar with vanE gene its
His gene, can not effectively hybridize, the vanE gene specific with height.
VanG gene magnification primer contained by primer sets 3 of the present invention and 13 vanG gene ratios in data set A
To sequence exactly match, and with other gene aligned sequences in data set A and all sequences in data set B exist to
Few 4 mispairing, illustrate: (1) vanG gene magnification primer contained by primer sets 3 of the present invention can be covered so far
It was found that all vanG genes and its mutated gene, with height covering property of vanG gene；(2) primer of the present invention
Group 3 contained by vanG gene magnification primer to vanC, vanE, vanL and vanN gene, and it is similar with vanG gene other
Gene can not be expanded effectively, the vanG gene specific with height.
VanG gene-specific probe sequence contained by probe 3 of the present invention and 13 vanG bases in data set A
Because aligned sequences exactly match, and deposited with other gene aligned sequences in data set A and all sequences in data set B
In at least three mispairing, illustrate: (1) vanG gene-specific probe contained by probe 3 of the present invention can be covered is so far
Only it has been found that all vanG genes and its mutated gene, with height covering property of vanG gene；(2) of the present invention
VanG gene-specific probe contained by probe 3 is and similar with vanG gene to vanC, vanE, vanL and vanN gene
Other genes can not effectively hybridize, the vanG gene specific with height.
VanL gene magnification primer contained by primer sets 4 of the present invention is compared with 2 vanL genes in data set A
Sequence exact matching, and have at least 4 with other gene aligned sequences in data set A and all sequences in data set B
A mispairing, illustrates: (1) vanL gene magnification primer contained by primer sets 4 of the present invention can be covered has sent out so far
Existing all vanL genes and its mutated gene, the covering property of vanL gene with height；(2) primer sets of the present invention
VanL gene magnification primer contained by 4 is to vanC, vanE, vanG and vanN gene, and other bases similar with vanL gene
Cause can not be expanded effectively, the vanL gene specific with height.
VanL gene-specific probe sequence contained by probe 4 of the present invention and 2 vanL genes in data set A
Aligned sequences exact matching, and exist with other gene aligned sequences in data set A and all sequences in data set B
At least three mispairing, illustrates: (1) vanL gene-specific probe contained by probe 4 of the present invention can be covered so far
All vanL genes and its mutated gene having found, the covering property of vanL gene with height；(2) spy of the present invention
VanL gene-specific probe contained by needle 4 to vanC, vanE, vanG and vanN gene, and it is similar with vanL gene its
His gene, can not effectively hybridize, the vanL gene specific with height.
VanN gene magnification primer contained by primer sets 5 of the present invention is compared with 3 vanN genes in data set A
Sequence exact matching, and have at least 4 with other gene aligned sequences in data set A and all sequences in data set B
A mispairing, illustrates: (1) vanN gene magnification primer contained by primer sets 5 of the present invention can be covered has sent out so far
Existing all vanN genes and its mutated gene, the covering property of vanN gene with height；(2) primer sets of the present invention
VanN gene magnification primer contained by 5 is to vanC, vanE, vanG and vanL gene, and other bases similar with vanN gene
Cause can not be expanded effectively, the vanN gene specific with height.
VanN gene-specific probe sequence contained by probe 5 of the present invention and 3 vanN genes in data set A
Aligned sequences exact matching, and exist with other gene aligned sequences in data set A and all sequences in data set B
At least three mispairing, illustrates: (1) vanN gene-specific probe contained by probe 5 of the present invention can be covered so far
All vanN genes and its mutated gene having found, the covering property of vanN gene with height；(2) spy of the present invention
VanN gene-specific probe contained by needle 5 to vanC, vanE, vanG and vanL gene, and it is similar with vanN gene its
His gene, can not effectively hybridize, the vanN gene specific with height.
Embodiment 2, the detection specificity of specific primer probe of the present invention combination
Primer and probe used in the present embodiment, by Shanghai, Ying Jun Bioisystech Co., Ltd is synthesized.
One, the preparation of sample to be tested
The present embodiment is to separately include the matter of five kinds of glycopeptide class Drug-resistant genes vanC, vanE, vanG, vanL and vanN
Grain DNA is sample to be tested, examines the detection specificity of primer combination of probe of the present invention.
Above-mentioned each plasmid the preparation method is as follows:
Sample to be tested 1 is the plasmid comprising drug resistant gene vanC: will be between the site HindIII and BamHI of pUC18 plasmid
Sequence replace with sequence shown in vanC gene reference sequence AF162694, obtain recombinant plasmid, the plasmid be include resistance to
The plasmid of medicine gene vanC；
Sample to be tested 2 includes the plasmid of drug resistant gene vanE: by the sequence between the site SalI and KpnI of pUC18 plasmid
Sequence shown in vanE gene reference sequence FJ872411 is replaced with, recombinant plasmid is obtained, which includes as drug resistant gene
The plasmid of vanE；
Sample to be tested 3 includes the plasmid of drug resistant gene vanG: will be between the site HindIII and EcoRI of pUC18 plasmid
Sequence replaces with sequence shown in vanG gene reference sequence DQ212986, obtains recombinant plasmid, which includes as drug resistance
The plasmid of gene vanG；
Sample to be tested 4 includes the plasmid of drug resistant gene vanL: will be between the site HindIII and EcoRI of pUC18 plasmid
Sequence replaces with sequence shown in vanL gene reference sequence EU250284, obtains recombinant plasmid, which includes as drug resistance
The plasmid of gene vanL；
Sample to be tested 5 includes the plasmid of drug resistant gene vanN: will be between the site HindIII and EcoRI of pUC18 plasmid
Sequence replaces with sequence shown in vanN gene reference sequence JF802084, obtains recombinant plasmid, which includes as drug resistance
The plasmid of gene vanN；
Sample to be tested 6 is plasmid pUC18, does not include any one of vanC, vanE, vanG, vanL and vanN gene,
For negative control.
Above-mentioned plasmid passes through sequencing, it is determined that sequence is as described above.
Two, the preparation of primer combination
Primer of the present invention combination includes primer sets 1-5, be respectively used to detection vanC, vanE, vanG, vanL and
VanN gene.
Primer used in the present embodiment is reverse primer sequences (the i.e. sequence in specific primer group of the present invention
2,4,6,8,10) 5 '-ends add the unrelated sequences of the preceding paragraph and testing gene absolutely not homology (the present embodiment use
It is derived from the sequence fragment of model plant arabidopsis), and mark fluorescent molecule.So that reverse primer is during PCR amplification
Higher anneal temperature under can still continue to expand, and make amplified production single stranded end carry fluorescent molecule, the single stranded product
After specific probe hybridization, so that specific probe position shows fluorescence.
Primer sequence used in the present embodiment is as follows:
Primer sets 1 for detecting vanC gene include (5 ' -3 '):
Forward primer (sequence 1):
GTTCGCAATGATACTTGGCT；
Reverse primer (after sequence 2 is modified):
TAMRA-cgcggcgtgctgtggtgtttggtg-CCATACTTCCCATGCAAGAC；
Primer sets 2 for detecting vanE gene include (5 ' -3 '):
Forward primer (sequence 3):
CAGTTGCGATTATCTTTGGC；
Reverse primer (after sequence 4 is modified):
TAMRA-cgcggcgtgctgtggtgtttggtg-TCCGATACCACAACCTACAT；
Primer sets 3 for detecting vanG gene include (5 ' -3 '):
Forward primer (sequence 5):
ATAAACTCGTTAGCCTTGCG；
Reverse primer (after sequence 6 is modified):
TAMRA-cgcggcgtgctgtggtgtttggtg-CCGCTTCTTGTATCCGTTTT；
Primer sets 4 for detecting vanL gene include (5 ' -3 '):
Forward primer (sequence 7):
AGGATGCTTTAACAGAAGCG；
Reverse primer (after sequence 8 is modified):
TAMRA-cgcggcgtgctgtggtgtttggtg-CCTGAACAGCCTAGTAACCT；
Primer sets 5 for detecting vanN gene include (5 ' -3 '):
Forward primer (sequence 9):
GGGTTTTATGACCAAGCAGA；
Reverse primer (after sequence 10 is modified):
TAMRA-cgcggcgtgctgtggtgtttggtg-TGTAGGCGTACTCATCACTC；
The present embodiment carries out above-mentioned modification to specific primer of the present invention, it is therefore an objective to be promoted in pcr amplification product
With the minus strand generation efficiency of specific probe hybridization of the present invention, detection sensitivity is promoted；Mark fluorescent molecule be in order to
Make result visualization, digitization.Above-mentioned modification has no influence to the primer specificity, is this field common technique, and
This is a kind of used by the more than the present embodiment of mode in order to achieve the above objectives.Other do not influence primer specific of the present invention
The improved procedure of property, can be with specific primer connected applications of the present invention.
In primer combination of the present invention, the molar ratio of the primer sets 1,2,3,4,5 is 1:1.5:1.5:1:1.
Above-mentioned primer sets 1,2,3,4,5 can be configured to the mother liquor of higher concentration respectively, individually pack；It can also be according to molar ratio
It is hybridly prepared into the solution of higher concentration.It is mixed when use with pcr amplification reaction liquid, carries out PCR amplification after adding template DNA
Reaction.In the present embodiment, it is configured to the primer mixture solution for standby of 4 times of end reaction concentration.
Three, the preparation of probe
Specific probe used in the present embodiment is at specific probe sequence of the present invention (i.e. sequence 11-15)
5 '-ends connect upper 25 base T, and are chemically modified in end.
Probe sequence used in the present embodiment (5 ' -3 ') is as follows:
For detecting the probe 1 (after sequence 11 is modified) of vanC gene:
NH2-poly(T)25-CACCAGCTGACTTTTTCTAGCCA；
For detecting the probe 2 (after sequence 12 is modified) of vanE gene:
NH2-poly(T)25-TGAGAATGGTGCTATGCAGGGA；
For detecting the probe 3 (after sequence 13 is modified) of vanG gene:
NH2-poly(T)25-TGTTCGTGCAGGCTCTTCCT；
For detecting the probe 3 (after sequence 14 is modified) of vanL gene:
NH2-poly(T)25-TTGGCTGTGCTGTCTTAGGT；
For detecting the probe 4 (after sequence 15 is modified) of vanN gene:
NH2-poly(T)25-ACACGGTGGCACTGGAGAAA。
The present embodiment carries out above-mentioned modification to specific probe of the present invention, it is therefore an objective to lead to specific probe
Cross chemical combination key (NH2) be fixed on the carrier (aldehyde radical substrate) of the present embodiment use；And by increasing probe length
(poly(T)25) spatial dimension that increases hybridization reaction, offset influence of the steric hindrance to hybridization efficiency.Above-mentioned modification is to this hair
The bright probe specificity has no influence, is this field common technique, and this more than reality of mode in order to achieve the above objectives
Apply this one kind used by example.Other do not influence the improved procedure of probe specificity of the present invention, such as: other chemical groups
Modification, other kinds of probe carrier (nitrocellulose filter) etc., can be with probe connected applications of the present invention.
Using GeneMachine point sample instrument, above-mentioned specific probe is dissolved in respectively in 50% (V/V) DMSO solution
(concentration and probe concentration is 10 μM).Probe dot matrix arrangement mode as shown in Figure 1 is by dissolved probe points system in aldehyde group modified base
Piece ( Optical grade aldehyde radical substrate, CapitalBio Corporation, production number: 420022) (every dosage on
About 0.44nl), after drying and fixing, stick 12 sample fence (SmartGridTM, the rich biochip Limited Liability public affairs difficult to understand in Beijing
Department, production number: 430033), being made the chip of five kinds of glycopeptide class Drug-resistant genes of detection, spare.
HybPCP3 shown in Fig. 1 be hybridization positive control probe, using purpose be monitor hybridization reaction it is normal with
It is no.Its sequence is selected from the gene order of the extremely remote plant (arabidopsis) of target species (bacterium) affiliation detected with the present invention
(5 '-gcaaccaccaccggagg) has no influence to probe specificity of the present invention.Other and spy of the present invention
The unrelated sequence of needle specificity can be used.The Normal practice that reference substance is this field is set in hybridization reaction, other can rise
It, can be with probe connected applications of the present invention to the reference substance set-up mode of same purpose.
" 7 " in Fig. 1 are the probes of other drug resistant genes unrelated to the invention.
Four, the detection of sample to be tested
Step 1: PCR amplification being carried out to sample to be tested with specific primer group of the present invention
Respectively using 6 kinds of Plasmid DNA of step 1 preparation as template, the primer for 4 times of end reaction concentration that step 2 is prepared
PCR amplification is carried out after 2 × PCR reaction solution and Plasmid DNA the template mixing of mixture and commercialization.
Single PCR reaction system are as follows: 2 × PCR reaction solution (Tiangeng Products number: KT201) 10 μ l, 4 × primer is mixed
Close 5 μ l of object and the mixing of 1 μ l Plasmid DNA (10-100ng/ μ l), moisturizing to 20 μ l.In reaction system, the end of primer sets 1,4,5 is dense
Degree is 0.1 μM, final concentration of 0.15 μM of primer sets 2,3.6 kinds of plasmids are detected respectively, prepare 6 PCR reaction systems altogether.
Pcr amplification reaction carries out on DNA Engine Tetrad 2 (BIO-RAD) thermal cycler, is followed using following heat
Round trip sequence: 94 DEG C of initial denaturation 3min → 94 DEG C are denaturalized 30sec；48 DEG C of renaturation 30sec；72 DEG C of extension 45sec, circulation 20 times → 95
DEG C denaturation 30sec；58 DEG C of renaturation 30sec；72 DEG C of extension 45sec recycle 20 times → 72 DEG C of extension 7min → and are cooled to 16 DEG C.
Step 2: being hybridized with specific probe of the present invention with the PCR product that step 1 obtains
PCR product after taking amplification, carries out hybridization reaction after mixing with hybridization reaction solution.
Single part of hybridization reaction system is formulated as follows and (carries out system amplification according to sample size): the PCR for taking step 1 to obtain
11.5 μ l of amplified production, with 20 × SSPE (Sheng Gong company, production number B548111-0200) 3.75 μ l, 4% (W/V, i.e. 4g/
100ml) SDS (Sheng Gong company, production number B548118-0100) 1.25 μ l, 50 × Denhardt's (Sheng Gong company, production number
B548209-0050) 2.5 μ l, 100% (V/V) deionized formamide (Sheng Gong company, production number A600211-0100), 5 μ l and miscellaneous
Positive control (1nM) 1 μ l mixing is handed over, 6 kinds of pcr amplification products are detected respectively, prepare 6 hybridization reaction systems altogether.
The present embodiment hybridizes positive reference substance used in the above-mentioned hybridization reaction solution, is and friendship positive control above-mentioned is visited
Needle HybPCP3 complementation, the oligonucleotides with fluorescent molecule label, sequence are as follows: TAMRA-cctccggtggtggttgc.Its
The use of purpose hybridized with HybPCP3 probe, with monitor hybridization reaction it is normal whether.As previously mentioned, its use is to the present invention
The probe specificity has no influence.Other hybridization positive reference substances unrelated with probe specificity of the present invention
Using.The Normal practice that reference substance is this field is set in hybridization reaction, other can play the reference substance setting of same purpose
Mode, can be with probe connected applications of the present invention.
By prepare five kinds of glycopeptide class Drug-resistant gene detecting chips and cover plate (SmartCoverTM, Beijing Bo Aosheng
Object chip Co., Ltd, production number: 430044) is placed in hybridizing box in.For every a sample to be tested, 20 μ l is taken to match
The hybridization reaction system made is loaded the good hybridizing box of rear enclosed, is placed in 32 DEG C and hybridizes 2 hours.After hybridization, chip is taken out,
It is placed in washing lotion (2 × SSC and 0.2%SDS), room temperature, which is shaken, washes 5 minutes；Later by chip with deionized water rinse after five minutes, from
Heart drying.
Step 3: testing result acquisition and analysis
Fluorescence signal on chip uses GenePix 4200AL laser confocal scanner and its software kit GenePix
Pro.6.0 (Axon Inc.) scanning collection.Sweep parameter is as follows: excitation wavelength: 532nm；Laser Power:33%；
PMT:400.
The hybridization signal that each probe signals point is extracted with 6.0 software of GenePix Pro, calculates each probe signals
The signal-to-noise ratio (Signal Noise Ratio, SNR=(signal value-background value)/background value) of point, so that it is determined that glycopeptide in sample
Class Drug-resistant gene type prompts drug resistance.When signal-to-noise ratio is more than or equal to 3, genetic test corresponding to the probe is determined
It as a result is the positive, such as all specific probe signal-to-noise ratio are respectively less than 3, then determine not including five kinds of sugar to be measured in the sample to be tested
Peptide medicament drug resistant gene.
In the present embodiment, the testing result of 6 kinds of samples to be tested is as shown in Figure 2.
It the results show that being directed to sample to be tested 1, that is, include the plasmid of drug resistant gene vanC, only specific probe 1 (sequence 11)
Results of hybridization is the positive, shows to include vanC drug resistant gene in sample；For sample to be tested 2, i.e., comprising drug resistant gene vanE
Plasmid, only (sequence 12) results of hybridization of specific probe 2 is the positive, shows to include vanE drug resistant gene in sample；For to be measured
Sample 3 includes the plasmid of drug resistant gene vanG, only (sequence 13) results of hybridization of specific probe 3 is the positive, is shown in sample
Include vanG drug resistant gene；It for sample to be tested 4, that is, include the plasmid of drug resistant gene vanL, only specific probe 4 (sequence 14)
Results of hybridization is the positive, shows to include vanL drug resistant gene in sample；For sample to be tested 5, i.e., comprising drug resistant gene vanN
Plasmid, only (sequence 15) results of hybridization of specific probe 5 is the positive, shows to include vanN drug resistant gene in sample；For to be measured
Sample 6, i.e. negative control plasmids pUC18, all specific probe hybridization results are feminine gender.Show specificity of the present invention
Primer and probe combination has extremely strong detection specificity to vanC, vanE, vanG, vanL and vanN gene.
Embodiment 3, with specific primer probe combine detection Enterococcus strain of the present invention
The present embodiment is to the glycopeptide class Drug-resistant bacterial strain collected, the specially enterococcus faecalis of drug resistance of vancomycin
(Enterococcus faecalis), enterococcus faecium (Enterococcus faecium) carried out vanC, vanE, vanG,
The detection of vanL and vanN drug resistant gene, to illustrate that specific primer probe combination provided by the invention can be used for detecting glycopeptide class
The detection of above-mentioned five kinds of drug resistant genes in Drug-resistant bacterial strain.
It is specific as follows:
One, sample to be tested
Since five kinds of glycopeptides class Drug-resistant gene vanC, vanE, vanG, vanL and vanN according to the present invention are in intestines
Distribution in coccus, more rare for vanA, vanB drug resistant gene, wherein vanE, vanL and vanN Gene Isolation
Rate is lower.The bacterial strain comprising above-mentioned five kinds of glycopeptides class Drug-resistant gene is collected by way of isolated strains, needs to expend big
The time and materials cost of amount, and it is possible to be to may come by something with luck, but not by searching for it.And by having reported that associated sugars peptide medicament is resistance to
Bacterial strain or the clone of non-commercial use are asked in the laboratory of medicine gene, it is also possible to which the diffusion problem for bringing drug resistant gene causes
The diffusion of associated medication therapies invalid situation, it is also difficult to success.In view of this, the present embodiment is drawn with specificity of the present invention
Object probe combinations, respectively to the part drug resistance of vancomycin enterococcus faecalis and manure enterococcin strain and vancomycin collected
Sensitive strain is detected, and illustrates specific primer probe combination of the present invention, can be used for related resistance in sample to be tested
The detection of medicine gene.
Confirm through sequencing, sample ECC013 and ECC117 include vanC gene, and sample ECC187 includes vanG gene, sample
ECC042 and ECC085 is glycopeptide class medicaments insensitive bacterial strain, does not include any five kinds of glycopeptides class Drug-resistant gene to be detected.
To above-mentioned enterococcus faecalis and manure enterococcin strain, using Qiagene company DNeasy Blood&Tissue Kit
(production number: 69506) extract bacterium full-length genome nucleic acid.
Two, the preparation of primer combination
The primer combination preparation method and step that the present embodiment uses are the same as embodiment 2.
Three, the preparation of probe
The probe combinations preparation method and step that the present embodiment uses are the same as embodiment 2.
Four, the detection of sample to be tested
Step 1: PCR amplification being carried out to sample to be tested with specific primer group of the present invention
Method and step are the same as embodiment 2.Only the template in PCR amplification system is replaced with by Plasmid DNA from above-mentioned to be measured
The bacterium full-length genome nucleic acid extracted in bacterial strain (concentration is 200ng/ μ l or so).
Step 2: being hybridized with specific probe of the present invention with the PCR product that step 1 obtains.
Method and step are the same as embodiment 2.
Step 3: testing result acquisition and analysis
Method and step are the same as embodiment 2.
Measuring samples are expanded through specific primer sets of the present invention, and miscellaneous with specific probe of the present invention
After friendship, by signal extraction, the signal-to-noise ratio computation result of above-mentioned measuring samples is as shown in table 2 below.
Table 2
For sample ECC013 and ECC117, only vanC specific probe (sequence 11) results of hybridization is the positive, shows sample
It include vanC drug resistant gene in product ECC013 and ECC117；For sample ECC187, only vanG specific probe (sequence 13) is miscellaneous
Knot fruit is the positive, shows to include vanG drug resistant gene in sample ECC187；For sample ECC042 and ECC085, no specificity
Probe results of hybridization is the positive, does not include any five kinds of glycopeptides class Drug-resistant to be measured in display sample ECC042 and ECC085
Gene.
The testing result of all samples to be tested and sequencing result are completely the same, and specific probe with and only with include probe
The sample of corresponding drug resistant gene generates positive signal, and without non-specific miscellaneous between other gene PCR amplified productions
It hands over, shows high probe specificity, illustrate specific primer probe combination provided by the invention suitable for isolated strains
The detection of five kinds of glycopeptide class Drug-resistant genes.
The above list is only a few specific embodiments of the present invention for finally, it should also be noted that.Obviously, this hair
Bright to be not limited to above embodiments, acceptable there are many deformations.Those skilled in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Sequence table
<110>Zhejiang University
<120>specific primer of five kinds of glycopeptide class Drug-resistant genes and probe combinations in Enterococcus are detected and are answered
With
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gttcgcaatg atacttggct 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ccatacttcc catgcaagac 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
cagttgcgat tatctttggc 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tccgatacca caacctacat 20
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
ataaactcgt tagccttgcg 20
<210> 6
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
ccgcttcttg tatccgtttt 20
<210> 7
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
aggatgcttt aacagaagcg 20
<210> 8
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
cctgaacagc ctagtaacct 20
<210> 9
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
gggttttatg accaagcaga 20
<210> 10
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
tgtaggcgta ctcatcactc 20
<210> 11
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
caccagctga ctttttctag cca 23
<210> 12
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
tgagaatggt gctatgcagg ga 22
<210> 13
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
tgttcgtgca ggctcttcct 20
<210> 14
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
ttggctgtgc tgtcttaggt 20
<210> 15
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
acacggtggc actggagaaa 20
Claims (6)
1. five kinds of glycopeptide class Drug-resistant genes in specific detection Enterococcus (vanC, vanE, vanG, vanL and
VanN primer combination of probe), it is characterized in that:
Primer including five kinds of glycopeptide classes Drug-resistant gene vanC, vanE, vanG, vanL and vanN are carried out with specific amplification
Combination, the primer is as shown in sequence 1-10；
It further include the specific probe group for detecting five kinds of glycopeptide class Drug-resistant genes vanC, vanE, vanG, vanL and vanN
It closes, the probe is as shown in sequence 11-15.
2. the kit of five kinds of glycopeptide class Drug-resistant genes in specific detection Enterococcus, it is characterized in that: including such as
Primer combination and specific probe combination described in claim 1.
3. the reagent of five kinds of glycopeptide class Drug-resistant genes in specific detection Enterococcus according to claim 2
Box, it is characterized in that: further including pcr amplification reaction liquid, hybridization reaction solution.
4. the application of primer combination of probe, kit, it is characterized in that being following any purposes:
1) at least one of drug resistant gene vanC, vanE, vanG, vanL and vanN, are identified；
2), detect bacterium to be measured whether containing in glycopeptide class Drug-resistant gene vanC, vanE, vanG, vanL and vanN at least
It is a kind of；
3) it, whether detects in sample to be tested containing in glycopeptide class Drug-resistant gene vanC, vanE, vanG, vanL and vanN
It is at least one.
5. application according to claim 4, it is characterized in that:
1) sample to be tested/bacterium to be measured total DNA, is extracted；
2), the total DNA extracted using step 1) carries out PCR amplification using specific primer sets as template；
3), the pcr amplification product that step 2) obtains is hybridized respectively with the specific probe, is then sentenced as follows
It is disconnected:
If the results of hybridization of probe is the positive, drug resistant gene corresponding to the positive control probe be present in the sample to be tested/to
It surveys in bacterium.
6. application according to claim 5, it is characterized in that: the molar ratio of primer sets 1,2,3,4,5 can be in primer combination
1:1.5:1.5:1:1。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910346784.6A CN110066882B (en) | 2019-04-27 | 2019-04-27 | Specific primer and probe combination for detecting drug resistance genes of five glycopeptide drugs in enterococcus and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910346784.6A CN110066882B (en) | 2019-04-27 | 2019-04-27 | Specific primer and probe combination for detecting drug resistance genes of five glycopeptide drugs in enterococcus and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110066882A true CN110066882A (en) | 2019-07-30 |
| CN110066882B CN110066882B (en) | 2020-08-07 |
Family
ID=67369242
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910346784.6A Expired - Fee Related CN110066882B (en) | 2019-04-27 | 2019-04-27 | Specific primer and probe combination for detecting drug resistance genes of five glycopeptide drugs in enterococcus and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110066882B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110923298A (en) * | 2019-12-27 | 2020-03-27 | 领航基因科技（杭州）有限公司 | Primer probe system, kit and method for simultaneously detecting multiple drug-resistant genes |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1687459A (en) * | 2005-04-15 | 2005-10-26 | 北京博奥生物芯片有限责任公司 | Authenticating gram positive bacteria species and method for testing drug resistant gene and dedicating kit |
| US20150204883A1 (en) * | 2012-08-01 | 2015-07-23 | bioMérieux | Method for detecting at least one mechanism of resistance to glycopeptides by mass spectrometry |
| CN106520921A (en) * | 2016-10-08 | 2017-03-22 | 陕西脉元生物科技有限公司 | Primer pair of vancomycin-resistant enterococcus, detection kit and detection method |
| CN106636426A (en) * | 2017-01-13 | 2017-05-10 | 重庆威斯腾生物医药科技有限责任公司 | Glycopeptide antibiotic drug-resistance gene detection kit |
-
2019
- 2019-04-27 CN CN201910346784.6A patent/CN110066882B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1687459A (en) * | 2005-04-15 | 2005-10-26 | 北京博奥生物芯片有限责任公司 | Authenticating gram positive bacteria species and method for testing drug resistant gene and dedicating kit |
| US20150204883A1 (en) * | 2012-08-01 | 2015-07-23 | bioMérieux | Method for detecting at least one mechanism of resistance to glycopeptides by mass spectrometry |
| CN106520921A (en) * | 2016-10-08 | 2017-03-22 | 陕西脉元生物科技有限公司 | Primer pair of vancomycin-resistant enterococcus, detection kit and detection method |
| CN106636426A (en) * | 2017-01-13 | 2017-05-10 | 重庆威斯腾生物医药科技有限责任公司 | Glycopeptide antibiotic drug-resistance gene detection kit |
Non-Patent Citations (4)
| Title |
|---|
| MATTHEW L. FARON等: "Resistance Mechanisms, Epidemiology, and Approaches to Screening for Vancomycin-Resistant Enterococcus in the Health Care Setting", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
| TAKAHIRO NOMURA等: "Identification of VanN-Type Vancomycin Resistance in an Enterococcus faecium Isolate from Chicken Meat in Japan", 《ANTIMICROBIAL AGENTS AND CHEMOTHERAPY》 * |
| 林东昉等: "肠球菌万古霉素高水平耐药基因vanA、vanB、vanD和vanM快速分型检测", 《中华传染病杂志》 * |
| 陈春辉等: "肠球菌万古霉素耐药基因簇遗传特性", 《遗传》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110923298A (en) * | 2019-12-27 | 2020-03-27 | 领航基因科技（杭州）有限公司 | Primer probe system, kit and method for simultaneously detecting multiple drug-resistant genes |
| CN110923298B (en) * | 2019-12-27 | 2023-09-22 | 领航基因科技（杭州）有限公司 | Primer probe system, kit and method for simultaneously detecting multiple drug-resistant genes |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110066882B (en) | 2020-08-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Geiss et al. | Large-scale monitoring of host cell gene expression during HIV-1 infection using cDNA microarrays | |
| US7906286B2 (en) | Probe set, probe carrier, and method for determining and identifying fungus | |
| CN100427610C (en) | Authenticating gram positive bacteria species and method for testing drug resistant gene and dedicating kit | |
| CN101665824B (en) | Biochip for detecting drug resistant genes of helicobacter pylori clarithromycin and preparation method and application thereof | |
| JPH09508800A (en) | Method for identifying differentially expressed genes | |
| CN106755513B (en) | A kind of lncRNA markers of gastric cancer | |
| CN106701900B (en) | Long-chain non-coding RNA HERC2P3 gene and application thereof in gastric cancer | |
| CN109295221B (en) | Application of circular RNA as colorectal cancer molecular marker | |
| CN110055308A (en) | Detect the specific primer of four kinds of glycopeptide class Drug-resistant genes and probe combinations and application in Enterococcus | |
| CN110066882A (en) | Detect the specific primer of five kinds of glycopeptide class Drug-resistant genes and probe combinations and application in Enterococcus | |
| CN107190052A (en) | The purposes of LOC101928926 genes | |
| CN107190005B (en) | Applications of the lncRNA as biomarker in adenocarcinoma of lung diagnosis and treatment | |
| CN101392285A (en) | Method for detecting multiple myeloma and method for inhibiting the same | |
| CN110079619A (en) | Detect the specific primer of four kinds of glycopeptide class Drug-resistant genes and probe combinations and application in gram-positive bacterium | |
| CN111961763A (en) | Novel gene chip for detecting coronavirus | |
| CN1721554B (en) | Method of testing anticancer agent-sensitivity of tumor cells | |
| KR100926255B1 (en) | Diagnostic methods for male infertility and diagnostic kits for male infertility for detecting Y chromosomal microdeletion | |
| CN108642169A (en) | Detect ten kinds of trimethoprim class Drug-resistant genetic methods and used kit | |
| KR100908691B1 (en) | 남성 Male infertility diagnostic kit for detecting chromosomal microdeletion | |
| CN109929939B (en) | Application of lncRNA11496 in diagnosis and treatment of toxoplasmosis | |
| CN106916887A (en) | Application of the ERH genes in carcinoma of urinary bladder diagnosis and treatment product is prepared | |
| KR101850065B1 (en) | Information providing method for drug treatment decisions of hypothyroidism patients by TSHR mutations and DIO2 T92A single nucleotide polymorphism detection | |
| CN108624696A (en) | Application of the molecular labeling in preparing Intravenous leiomyomatosis diagnosis detection product | |
| CN102575285B (en) | Breast cancer susceptibility gene GT198 and uses thereof | |
| CN111378744B (en) | Application of miR-148a-3p as diagnosis and treatment marker for febrile convulsion |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200807 Termination date: 20210427 |