CN104962625A - Diagnostic method for rapidly detecting bacterium metal beta-lactamase drug resistance gene VIM - Google Patents
Diagnostic method for rapidly detecting bacterium metal beta-lactamase drug resistance gene VIM Download PDFInfo
- Publication number
- CN104962625A CN104962625A CN201510353189.7A CN201510353189A CN104962625A CN 104962625 A CN104962625 A CN 104962625A CN 201510353189 A CN201510353189 A CN 201510353189A CN 104962625 A CN104962625 A CN 104962625A
- Authority
- CN
- China
- Prior art keywords
- primer
- vim
- seq
- metallo
- lamp
- 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.)
- Pending
Links
- 241000894006 Bacteria Species 0.000 title claims abstract description 41
- 108090000204 Dipeptidase 1 Proteins 0.000 title abstract description 4
- 102000006635 beta-lactamase Human genes 0.000 title abstract description 4
- 239000002184 metal Substances 0.000 title abstract description 4
- 206010059866 Drug resistance Diseases 0.000 title abstract 2
- 238000002405 diagnostic procedure Methods 0.000 title description 3
- 238000006243 chemical reaction Methods 0.000 claims abstract description 26
- 238000001514 detection method Methods 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 14
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims abstract description 13
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims abstract description 13
- 239000003814 drug Substances 0.000 claims description 40
- 229940079593 drug Drugs 0.000 claims description 40
- 108090000623 proteins and genes Proteins 0.000 claims description 38
- 108060004734 metallo-beta-lactamase Proteins 0.000 claims description 34
- 102000020235 metallo-beta-lactamase Human genes 0.000 claims description 34
- 239000000243 solution Substances 0.000 claims description 10
- 230000004544 DNA amplification Effects 0.000 claims description 8
- 239000013612 plasmid Substances 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 8
- 101150018417 VIM gene Proteins 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 239000012634 fragment Substances 0.000 claims description 6
- 239000013642 negative control Substances 0.000 claims description 6
- 239000013641 positive control Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 238000005498 polishing Methods 0.000 claims description 3
- 239000011535 reaction buffer Substances 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- QCYOIFVBYZNUNW-UHFFFAOYSA-N 2-(dimethylazaniumyl)propanoate Chemical class CN(C)C(C)C(O)=O QCYOIFVBYZNUNW-UHFFFAOYSA-N 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims description 2
- -1 developer Substances 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 239000003795 chemical substances by application Substances 0.000 abstract 1
- 238000006073 displacement reaction Methods 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 15
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000013461 design Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 230000003321 amplification Effects 0.000 description 2
- 150000003952 β-lactams Chemical class 0.000 description 2
- WZPBZJONDBGPKJ-UHFFFAOYSA-N Antibiotic SQ 26917 Natural products O=C1N(S(O)(=O)=O)C(C)C1NC(=O)C(=NOC(C)(C)C(O)=O)C1=CSC(N)=N1 WZPBZJONDBGPKJ-UHFFFAOYSA-N 0.000 description 1
- 108020005210 Integrons Proteins 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- WZPBZJONDBGPKJ-VEHQQRBSSA-N aztreonam Chemical compound O=C1N(S([O-])(=O)=O)[C@@H](C)[C@@H]1NC(=O)C(=N/OC(C)(C)C(O)=O)\C1=CSC([NH3+])=N1 WZPBZJONDBGPKJ-VEHQQRBSSA-N 0.000 description 1
- 229960003644 aztreonam Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003782 beta lactam antibiotic agent Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- YZBQHRLRFGPBSL-RXMQYKEDSA-N carbapenem Chemical compound C1C=CN2C(=O)C[C@H]21 YZBQHRLRFGPBSL-RXMQYKEDSA-N 0.000 description 1
- 229940041011 carbapenems Drugs 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 1
- 230000005571 horizontal transmission Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000002132 β-lactam antibiotic Substances 0.000 description 1
- 229940124586 β-lactam antibiotics Drugs 0.000 description 1
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
- C12Q1/6844—Nucleic acid amplification reactions
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical 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)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an LAMP kit and method used for rapidly detecting bacterium metal beta-lactamase drug resistance gene VIM. The kit comprises a pair of outer primers, a pair of inner primers and a pair of loop primers. The method is implemented as SEQ ID NO.1-6 and includes the steps that DNA of a to-be-detected bacterium genome is extracted, six specific primers and a kind of DNA polymerase with the strand displacement activity are adopted, the DNA sample is amplified at 60 DEG C to 65 DEG C, and a user adds color developing agents to observe changes in the color in a reaction tube to judge whether amplifying is achieved or not. The LAMP kit and method have the advantages of being rapid, efficient, easy and convenient to operate, high in specificity, high in sensitivity, easy and convenient to detect, suitable for site detection and the like, and is suitable for application and popularization.
Description
Technical field
The present invention relates to the detection of pathogenic micro-organism, be specifically related to one ring mediated isothermal gene amplification (LAMP technology) rapid detection bacterium metallo-β-lactamase drug resistant gene
vIMtest kit and method.
Background technology
In recent years, along with the widespread use clinically of the Carbapenem antibiotics such as imipenum, bacterium creates metal β-lactamase that a class extensively can be hydrolyzed β-lactam antibiotics, the bacterium producing this fermentoid produces resistance to all microbiotic of β-lactams, extreme difficulties is caused to clinical anti-infective therapy, and produce at present the Pseudomonas aeruginosa kind of metallo-β-lactamase and quantity in continuous increase, that has reported mainly contains
iMPwith
vIMtwo types.VIM shaped metal β-lactamase is a kind of β-lactam antibitics that can be hydrolyzed nearly all (except the monocycles such as aztreonam), and along with carbapenems and the antibiotic continuous use of other classes, makes
vIMmultiple hypotype has been there is in the more than ten years, up to now,
vIMfind nearly 30 kinds of hypotypes, removed
vIM-7outside maximum with the difference of other hypotypes, other hypotype amino acid identities are higher.Due to
vIMbetween bacterium not of the same race, carry out horizontal transmission by integron mechanism, bring serious threat to clinical treatment, also do not find clinical effective inhibitor so far.Therefore, find one suitable
vIMthe detection method of gene can provide good basis for clinical treatment.
Traditional traditional culture of microorganism and drug sensitive test method can provide good value clinically, but because of length consuming time, loaded down with trivial details operating process, need the different reasons such as expensive plant and instrument to fail large-scale promotion clinically to use.Thus a kind of easy, bacterium metallo-β-lactamase drug resistant gene fast of research and development
vIMdetection method there is great application prospect.
Summary of the invention
The object of the present invention is to provide a kind of for rapid detection bacterium metallo-β-lactamase drug resistant gene
vIMlAMP kit and a kind of with ring mediated isothermal gene amplification (LAMP technology) rapid detection bacterium metallo-β-lactamase drug resistant gene
vIMmethod.
The technical solution used in the present invention is:
A kind of for rapid detection bacterium metallo-β-lactamase drug resistant gene
vIMlAMP primer group, it comprises a pair inner primer, a pair outer primer and a pair ring primer, and nucleotide sequence is respectively as follows:
Inner primer 1:5 '-GGTAGACCGCGCCATCAACAGATTGCCGATGGTGTT-3 ' (SEQ ID No.1);
Inner primer 2:5 '-TGTCCGTGATGGTGATGAGTTGCAATCTCCGCGAGAAGTG-3 ' (SEQ ID No.2);
Outer primer 1:5 '-GTGAGTATCCGACAGTCAAC-3 ' (SEQ ID No.3);
Outer primer 2:5 '-CGTTACGGGAAGTCCAATT-3 ' (SEQ ID No.4);
Ring primer 1:5 '-ACGACTGCGTTGCGATAT-3 ' (SEQ ID No.5);
Ring primer 2: 5 '-CTTTTGATTGATACAGCGTGGG-3 ' (SEQ ID No.6).
A kind of for rapid detection bacterium metallo-β-lactamase drug resistant gene
vIthe LAMP kit of M, it comprises above-mentioned for rapid detection bacterium metallo-β-lactamase drug resistant gene
vIMlAMP primer group.
As preferably, in described primer sets, the mol ratio of inner primer, outer primer, ring primer is 6-8:1:3-4.
As preferably, also contain in described test kit: archaeal dna polymerase, LAMP reaction solution, developer, positive control and negative control.
As preferably, described archaeal dna polymerase is
bstarchaeal dna polymerase.
As preferably, described LAMP reaction solution contains: 10 mM dNTP, 10 × ThermoPol reaction buffer, 150 mM MgSO
4the aqueous solution, 5 mM trimethyl-glycines, the volume ratio of four is 6-8:4-5:2-3:8-10.
As preferably, described developer is SYBR GREEN I.
As preferably, described positive control is for containing bacterium metallo-β-lactamase drug resistant gene
vIMthe plasmid of fragment, described bacterium metallo-β-lactamase drug resistant gene
vIMfragment is as shown in SEQ ID NO.7; Described negative control is DEPC water.
A kind of rapid detection bacterium metallo-β-lactamase drug resistant gene
vIMmethod, comprise the steps:
(1) measuring samples DNA is extracted;
(2) utilize above-mentioned for rapid detection bacterium metallo-β-lactamase drug resistant gene
vIMlAMP primer group isothermal duplication is carried out to measuring samples DNA;
(3) result judges: in above-mentioned reaction tubes, add 1-2 μ L developer 10 × SYBR GREEN I, and the color of observing response liquid after mixing, if present green, is
vIMextended spectrumβ-lactamase drug resistant gene, orange is then non-
vIMgene.
As preferably, 25 μ L reaction systems of isothermic gene amplification reaction contain: inner primer each 8pmol/ μ L, each 1 pmol/ μ L of outer primer, ring primer each 4 pmol/ μ L, reaction solution 12.5 μ L, archaeal dna polymerase 8U, DNA 1-100 ng to be checked, with sterilizing deionized water polishing to 25 μ L; The condition of isothermic gene amplification reaction is: 60-65 DEG C of reaction 55-65min.
The invention has the beneficial effects as follows: invention directed toward bacteria metallo-β-lactamase drug resistant gene
vIMspecific designs 6 primers, are combined with 6 regions of goal gene, have higher specificity, can exactly by bacterium metallo-β-lactamase drug resistant gene
vIMwith other non-bacterial metallo-β-lactamase drug resistant genes
vIMmake a distinction.In addition, test kit of the present invention is convenient and swift, can discriminating bacteria metallo-β-lactamase drug resistant gene in the short period of time
vIM, do not need expensive plant and instrument; Highly sensitive; Specificity is good, is applicable to Site Detection.
Accompanying drawing explanation
Fig. 1 is that the present invention is to bacterium extended spectrumβ-lactamase drug resistant gene
vIMand to non-bacterial extended spectrumβ-lactamase drug resistant gene
vIMamplification (-: negative sample ,+: bacterium extended spectrumβ-lactamase drug resistant gene
vIMsample);
Fig. 2 be embodiment 3 specificity verification result (1:DEPC, 2:
cTX-M-1,3:
pME, 4:
sME, 5:
cMY-2, 6:
tEM, 7:
iMP, 8:
vIMand
cMY-2);
Fig. 3 is the LAMP sensitivity experiment result (a:: the electrophoresis result of LAMP amplification of the present invention of embodiment 4; B: the colour developing result of LAMP amplification of the present invention; Wherein, 1 is DEPC, and 2 ~ 10 for containing bacterium extended spectrumβ-lactamase drug resistant gene
vIMpMD19-T plasmid, concentration is followed successively by 1.0 × 10
-1, 1.0 × 10
-0, 1.0 × 10
1, 1.0 × 10
2, 1.0 × 10
3, 1.0 × 10
4, 1.0 × 10
5, 1.0 × 10
6copy/μ L).
Fig. 4 is that (wherein, 1 is DEPC, and 2 ~ 10 for containing bacterium extended spectrumβ-lactamase drug resistant gene for the Standard PCR sensitivity experiment result of embodiment 4
vIMpMD19-T plasmid, concentration is followed successively by 1.0 × 10
-0, 1.0 × 10
1, 1.0 × 10
2, 1.0 × 10
3, 1.0 × 10
4, 1.0 × 10
5, 1.0 × 10
6, 1.0 × 10
7copy/μ L).
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated, but be not limited thereto.
embodiment 1 is for rapid detection bacterium metallo-β-lactamase drug resistant gene
vIMthe foundation of LAMP kit
For rapid detection bacterium metallo-β-lactamase drug resistant gene
vIMlAMP kit, comprise following composition: (1) Auele Specific Primer group; (2) archaeal dna polymerase; (3) LAMP reaction solution; (4) developer; (5) positive control and negative control.
(1) design of Auele Specific Primer:
According to bacterium metallo-β-lactamase drug resistant gene
vIMspecific designs 6 Auele Specific Primers of (GenBank accession number is: JX070882), sequence is as follows respectively:
Inner primer 1:5 '-GGTAGACCGCGCCATCAACAGATTGCCGATGGTGTT-3 ' (SEQ ID No.1);
Inner primer 2:5 '-TGTCCGTGATGGTGATGAGTTGCAATCTCCGCGAGAAGTG-3 ' (SEQ ID No.2);
Outer primer 1:5 '-GTGAGTATCCGACAGTCAAC-3 ' (SEQ ID No.3);
Outer primer 2:5 '-CGTTACGGGAAGTCCAATT-3 ' (SEQ ID No.4);
Ring primer 1:5 '-ACGACTGCGTTGCGATAT-3 ' (SEQ ID No.5);
Ring primer 2: 5 '-CTTTTGATTGATACAGCGTGGG-3 ' (SEQ ID No.6).
(2) archaeal dna polymerase is
bstarchaeal dna polymerase;
(3) LAMP reaction solution contains: 10mM dNTP, 10 × ThermoPol reaction buffer, 150mM MgSO
4, 5mM trimethyl-glycine, the volume ratio of four is 8:5:3:10.
(4) developer is SYBR GREEN I.
(5) positive control is for containing bacterium metallo-β-lactamase drug resistant gene
vIMthe pMD19-T plasmid (utilizing conventional plasmid construction method to be inserted in pMD19-T plasmid by SEQ ID NO.7 to obtain) of fragment (SEQ ID NO.7); Negative control is DEPC water.
embodiment 2 bacterium metallo-β-lactamase drug resistant gene
vIMmethod for quick
Utilize the test kit rapid detection bacterium metallo-β-lactamase drug resistant gene of embodiment 1
vIM, concrete steps are as follows:
(1) template DNA is extracted: adopt water-boiling method to extract bacterial genomes DNA;
(2) ring mediated isothermal gene amplification reaction: 25 μ L reaction systems contain: inner primer 1,2 final concentration is respectively 8 pmol/ μ L, outer primer 1,2 final concentration is respectively 1 pmol/ μ L, ring primer 1,2 final concentration is respectively 4 pmol/ μ L, reaction solution 12.5 μ L, archaeal dna polymerase 8U, DNA 50 ng to be checked, with sterilizing deionized water polishing to 25 μ L, then the sealing liquid of 20 μ L is added, by above-mentioned reaction tubes in 63 DEG C of reaction 60min;
(3) result judges: in above-mentioned reaction tubes, add 1 μ L developer 1 × SYBR Green I, and after mixing, the color of observing response liquid, if present green, is bacterium metallo-β-lactamase drug resistant gene
vIM, orange is then non-bacterial metallo-β-lactamase drug resistant gene
vIM (as Fig. 1).
embodiment 3 specificity experiments
The method of embodiment 2 is utilized not contain through qualification respectively
vIMthe clinical separation strain of gene detects, and DEPC water belongs with yin contrasts.
Detected result is shown in
fig. 2.Only bacterium metallo-β-lactamase drug resistant gene
vIMpipe is for green, and all the other pipes are for orange.Result shows, detection kit specificity of the present invention is high, can exactly by
vIMand other are non-
vIMmake a distinction.
embodiment 4 sensitivity experiment
(what namely build contains bacterium metallo-β-lactamase drug resistant gene to get positive reference substance
vIMthe pMD19-T plasmid of fragment (SEQ ID NO.7)), measure its concentration and calculate copy number, according to the concentration gradient dilution of 10 times, choosing 1.0 × 10
-1~ 1.0 × 10
8the concentration of copy/μ L is tested as sample.Detect by detection method of the present invention and conventional PCR method respectively.
Standard PCR detection method primer sequence used is as follows:
VIM-F：ATGTTCAAACTTTTGAGTAAG （SEQ ID NO.8）
VIM-R：CTACTCAACGACTGAGCG （SEQ ID NO.9）
The detected result of test kit of the present invention as
fig. 3shown in, result shows, this test kit pair
vIMthe minimum detectability of gene is: 1.0 × 10
4copy/μ L.
The detected result of conventional PCR method as
fig. 4shown in.Its minimum detectability is: 1.0 × 10
6copy/μ L.
Above embodiment shows, test kit of the present invention has good, the highly sensitive feature of accuracy, and does not need expensive plant and instrument, is applicable to Site Detection.
Above embodiment is only introduces preferred case of the present invention, to those skilled in the art, not deviating from any apparent changes and improvements of carrying out in the scope of spirit of the present invention, all should be regarded as a part of the present invention.
<110> Zhongshan University
The diagnostic method of a <120> rapid detection bacterium metallo-β-lactamase drug resistant gene VIM
<130>
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 36
<212> DNA
<213> artificial sequence
<400> 1
ggtagaccgc gccatcaaca gattgccgat ggtgtt 36
<210> 2
<211> 40
<212> DNA
<213> artificial sequence
<400> 2
tgtccgtgat ggtgatgagt tgcaatctcc gcgagaagtg 40
<210> 3
<211> 20
<212> DNA
<213> artificial sequence
<400> 3
gtgagtatcc gacagtcaac 20
<210> 4
<211> 19
<212> DNA
<213> artificial sequence
<400> 4
cgttacggga agtccaatt 19
<210> 5
<211> 18
<212> DNA
<213> artificial sequence
<400> 5
acgactgcgt tgcgatat 18
<210> 6
<211> 22
<212> DNA
<213> artificial sequence
<400> 6
cttttgattg atacagcgtg gg 22
<210> 7
<211> 810
<212> DNA
<213> artificial sequence
<400> 7
agtcttgatg ttaaaagtta ttagtagttt attggtctac atgaccgcgt ctgtcatggc 60
tgtcgcaagt ccgttagccc attccgggga gccgagtggt gagtatccga cagtcaacga 120
aattccggtc ggagaggtcc gactttacca gattgccgat ggtgtttggt cgcatatcgc 180
aacgcagtcg tttgatggcg cggtctaccc gtccaatggt ctcattgtcc gtgatggtga 240
tgagttgctt ttgattgata cagcgtgggg tgcgaaaaac acagcggcac ttctcgcgga 300
gattgaaaag caaattggac ttcccgtaac gcgtgcagtc tccacgcact ttcatgacga 360
ccgcgtcggc ggcgttgatg tccttcgggc ggctggggtg gcaacgtacg catcaccgtc 420
gacacgccgg ctagccgagg cagaggggaa cgagattccc acgcattctc tagaaggact 480
ctcatcgagc ggggacgcag tgcgcttcgg tccagtagag ctcttctatc ctggtgctgc 540
gcattcgacc gacaatctgg ttgtatacgt cccgtcagcg aacgtgctat acggtggttg 600
tgccgttcat gagttgtcaa gcacgtctgc ggggaacgtg gccgatgccg atctggctga 660
atggcccacc tccgttgagc ggattcaaaa acactacccg gaagcagagg tcgtcattcc 720
cgggcacggt ctaccgggcg gtctagactt gctccagcac acagcgaacg ttgtcaaagc 780
acacaaaaat cgctcagtcg ccgagtagca 810
<210> 8
<211> 21
<212> DNA
<213> artificial sequence
<400> 8
atgttcaaac ttttgagtaa g 21
<210> 9
<211> 18
<212> DNA
<213> artificial sequence
<400> 9
ctactcaacg actgagcg 18
Claims (10)
1. one kind for rapid detection bacterium metallo-β-lactamase drug resistant gene
vIMlAMP primer group, it comprises a pair inner primer, a pair outer primer and a pair ring primer, and nucleotide sequence is respectively as follows:
Inner primer 1:5 '-GGTAGACCGCGCCATCAACAGATTGCCGATGGTGTT-3 ' (SEQ ID No.1);
Inner primer 2:5 '-TGTCCGTGATGGTGATGAGTTGCAATCTCCGCGAGAAGTG-3 ' (SEQ ID No.2);
Outer primer 1:5 '-GTGAGTATCCGACAGTCAAC-3 ' (SEQ ID No.3);
Outer primer 2:5 '-CGTTACGGGAAGTCCAATT-3 ' (SEQ ID No.4);
Ring primer 1:5 '-ACGACTGCGTTGCGATAT-3 ' (SEQ ID No.5);
Ring primer 2: 5 '-CTTTTGATTGATACAGCGTGGG-3 ' (SEQ ID No.6).
2. one kind for rapid detection bacterium metallo-β-lactamase drug resistant gene
vIMlAMP kit, it comprises the primer sets shown in claim 1.
3. LAMP kit according to claim 2, is characterized in that, in described primer sets, the mol ratio of inner primer, outer primer, ring primer is 6-8:1:3-4.
4. according to the LAMP kit shown in claim 2, it is characterized in that, also contain in described test kit: archaeal dna polymerase, LAMP reaction solution, developer, positive control and negative control.
5. LAMP kit according to claim 2, is characterized in that, described archaeal dna polymerase is
bstarchaeal dna polymerase.
6. LAMP kit according to claim 2, is characterized in that, described LAMP reaction solution contains: 10 mM dNTP, 10 × ThermoPol reaction buffer, 150 mM MgSO
4the aqueous solution, 5 mM trimethyl-glycines, the volume ratio of four is 6-8:4-5:2-3:8-10.
7. LAMP kit according to claim 2, is characterized in that, described developer is SYBR GREEN I.
8. LAMP kit according to claim 2, is characterized in that, described positive control is for containing bacterium metallo-β-lactamase drug resistant gene
vIMthe plasmid of fragment, described bacterium metallo-β-lactamase drug resistant gene
vIMfragment is as shown in SEQ ID NO.7; Described negative control is DEPC water.
9. a rapid detection bacterium metallo-β-lactamase drug resistant gene
vIMmethod, comprise the steps:
(1) measuring samples DNA is extracted;
(2) primer sets of claim 1 is utilized to carry out isothermic gene amplification reaction to measuring samples DNA;
(3) result judges: in above-mentioned reaction tubes, add 1-2 μ L developer 10 × SYBR GREEN I, and after mixing, the color of observing response liquid, if present green, is bacterium metallo-β-lactamase drug resistant gene
vIM, orange is then non-
vIMgene.
10. method according to claim 9, it is characterized in that, 25 μ L reaction systems of isothermic gene amplification reaction contain: inner primer each 8pmol/ μ L, the each 1 pmol/ μ L of outer primer, ring primer each 4 pmol/ μ L, reaction solution 12.5 μ L, archaeal dna polymerase 8U, DNA 1 ~ 100 ng to be checked, with sterilizing deionized water polishing to 25 μ L;
the condition of isothermic gene amplification reaction is: 60-65 DEG C of reaction 55-65min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510353189.7A CN104962625A (en) | 2015-06-23 | 2015-06-23 | Diagnostic method for rapidly detecting bacterium metal beta-lactamase drug resistance gene VIM |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510353189.7A CN104962625A (en) | 2015-06-23 | 2015-06-23 | Diagnostic method for rapidly detecting bacterium metal beta-lactamase drug resistance gene VIM |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104962625A true CN104962625A (en) | 2015-10-07 |
Family
ID=54216710
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510353189.7A Pending CN104962625A (en) | 2015-06-23 | 2015-06-23 | Diagnostic method for rapidly detecting bacterium metal beta-lactamase drug resistance gene VIM |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104962625A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108559785A (en) * | 2018-07-19 | 2018-09-21 | 中国疾病预防控制中心传染病预防控制所 | Primer combination, probe, kit and Carbapenems Drug-resistant genes blaVIM detection method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101532050A (en) * | 2008-08-08 | 2009-09-16 | 四川大学 | Triple PCR detection technology for animal source bacteria to Beta-lactam drug resistance gene |
| CN103614465A (en) * | 2013-11-06 | 2014-03-05 | 南方医科大学 | Loop-mediated isothermal amplification (LAMP) primers, kit and detection method for detecting common carbapenemase genes of gram negative bacilli |
-
2015
- 2015-06-23 CN CN201510353189.7A patent/CN104962625A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101532050A (en) * | 2008-08-08 | 2009-09-16 | 四川大学 | Triple PCR detection technology for animal source bacteria to Beta-lactam drug resistance gene |
| CN103614465A (en) * | 2013-11-06 | 2014-03-05 | 南方医科大学 | Loop-mediated isothermal amplification (LAMP) primers, kit and detection method for detecting common carbapenemase genes of gram negative bacilli |
Non-Patent Citations (1)
| Title |
|---|
| 谢佳等: "临床常见革兰阴性菌对碳青霉烯类药物耐药性检测", 《热带医学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108559785A (en) * | 2018-07-19 | 2018-09-21 | 中国疾病预防控制中心传染病预防控制所 | Primer combination, probe, kit and Carbapenems Drug-resistant genes blaVIM detection method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Naas et al. | Real-time PCR for detection of NDM-1 carbapenemase genes from spiked stool samples | |
| CN103614465B (en) | For detecting the LAMP primer of the common carbapenem of gram negative bacilli, test kit and detection method thereof | |
| CN105112519A (en) | CRISPR-based Escherichia coli O157:H7 strain detection reagent box and detection method | |
| CN107460242B (en) | Detection kit capable of simultaneously detecting multiple high-drug-resistance genes and application thereof | |
| CN106834506A (en) | A kind of LAMP primer group of quick detection bacterium polymyxins drug resistant gene mcr 1, kit and detection method | |
| CN102230019B (en) | Primer of loop-mediated isothermal amplification (LAMP) for detecting multidrug-resistant cfr gene as well as kit and method for detecting multidrug-resistant cfr gene | |
| CN106399542B (en) | A kind of quick detection primer of rolling ring constant-temperature amplification and kit of mycoplasma pneumoniae | |
| CN102899414A (en) | Superbacterium gene NDM and KPC duplex fluorescence quantitative polymerase chain reaction (PCR) detection method and kit | |
| CN109880896A (en) | A kind of multiple LAMP kit and detection method for the specific parting of quick discriminating bacteria polymyxins drug resistant gene mcr | |
| CN104726581A (en) | Vibrio parahaemolyticus constant-temperature detection primer set, detection kit and detection method capable of avoiding false negative detection result | |
| CN102586438A (en) | LAMP-based (loop-mediated isothermal amplification-based) visual fluorogenic and chromogenic genetic testing method for microorganisms | |
| CN101429539B (en) | Reagent kit for real time fluorescence quantitative PCR detection of bacillus pyocyaneus | |
| Zhang et al. | Direct detection of Staphylococcus aureus in positive blood cultures through molecular beacon-based fluorescence in situ hybridization | |
| CN104846078A (en) | Campylobacter jejuni constant-temperature fluorescence detection primer group, kit, and detection method which are capable of avoiding false negative | |
| CN110734988B (en) | Constant-temperature amplification method for methicillin-resistant staphylococcus aureus (MRSA) nucleic acid | |
| CN104946764A (en) | Diagnostic method for quickly detecting drug resistance gene CMY-2 of germ AmpC enzyme | |
| CN101665827A (en) | Mycoplasma pneumoniae rapid detection kit and use method thereof | |
| CN113416797A (en) | Fluorescent quantitative PCR detection kit for simultaneously detecting 7 types of adenoviruses | |
| CN104962625A (en) | Diagnostic method for rapidly detecting bacterium metal beta-lactamase drug resistance gene VIM | |
| CN102242210B (en) | Primer for LAMP (Loop-mediated Isothermal Amplification) superbacteria NDM-1 (New Delhi Metallo-beta-lactamase-1) gene as well as kit and method for detecting superbacteria NDM-1 gene | |
| CN104962626A (en) | Diagnostic method for rapidly detecting bacterium metal beta-lactamase drug resistance gene IMP | |
| CN104293925B (en) | For the multiple PCR primer group of rapid detection β-lactamase drug resistant gene | |
| CN105219847A (en) | A kind of legionella rapid detection and parting kit and detection method thereof | |
| CN102146453B (en) | Fluorescence quantitative polymerase chain reaction (PCR) detection kit for gene of super-bacteria NDM-1 | |
| CN102417931B (en) | Polymerase chain reaction (PCR) fluorescence detection kit and detection method for candida albicans |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20151007 |