CN102251047A - Technique for detecting medicine resistance genes of various bacteria and detection kit - Google Patents
Technique for detecting medicine resistance genes of various bacteria and detection kit Download PDFInfo
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- CN102251047A CN102251047A CN2011102206344A CN201110220634A CN102251047A CN 102251047 A CN102251047 A CN 102251047A CN 2011102206344 A CN2011102206344 A CN 2011102206344A CN 201110220634 A CN201110220634 A CN 201110220634A CN 102251047 A CN102251047 A CN 102251047A
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 43
- 239000003814 drug Substances 0.000 title claims abstract description 40
- 241000894006 Bacteria Species 0.000 title claims abstract description 37
- 238000001514 detection method Methods 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 10
- 108020004711 Nucleic Acid Probes Proteins 0.000 claims abstract description 13
- 239000002853 nucleic acid probe Substances 0.000 claims abstract description 13
- 230000003321 amplification Effects 0.000 claims abstract description 12
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 12
- 229940079593 drug Drugs 0.000 claims description 32
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 206010034133 Pathogen resistance Diseases 0.000 claims description 5
- 238000009396 hybridization Methods 0.000 claims description 5
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 238000000137 annealing Methods 0.000 claims description 3
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000003752 polymerase chain reaction Methods 0.000 abstract 2
- 230000001580 bacterial effect Effects 0.000 description 6
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 238000009792 diffusion process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 241000191967 Staphylococcus aureus Species 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 206010066901 Treatment failure Diseases 0.000 description 1
- 238000007846 asymmetric PCR Methods 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 101150008979 mecA gene Proteins 0.000 description 1
- 101150114434 vanA gene Proteins 0.000 description 1
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Abstract
The invention discloses a technique for detecting medicine resistance genes of various bacteria and a detection kit, which mainly relate to the field of biotechnology. A detection method adopted by the technique for detecting medicine resistance genes of various bacteria comprises: performing real-time fluorescent polymerase chain reaction (PCR) by using specific primers; amplifying the medicine resistance genes of the bacteria to be detected; crossing the products of the amplification with locked nucleic acid probes; detecting fluorescent signals; and determining the medicine resistance genes of the bacteria to be detected. When the technique and the detection kit are used, the detection is quick and accurate, the sensitivity is high, and the storage period of the kit is long.
Description
Technical field:
The present invention relates to biological technical field, be specifically related to a kind of technology and detection kit of utilizing locked nucleic acid fluorescent PCR method to detect the various bacteria drug resistant gene.
Background technology:
Microbiotic is most widely used antibacterials in the hospital clinical, is controlling, is preventing and treat in the various infectious diseases to play a significant role.Yet along with being extensive use of of antibacterials, pathogenic bacteria constantly increases the resistance of common antibacterials, and the treatment of drug-fast bacteria infection has become a global difficult problem.
The continuous appearance of various resistant organisms can cause serious consequence, causes that operative treatment failure, complication increase, infection and recurrence, hospital stays prolong, the usage quantity increase of expensive microbiotic and other medicine etc.Resistant organism also spreads in the world along with the high speed development of international trade and tourism.Understand appearance reason, the mechanism of drug tolerant bacteria, grasping and using correct detection method is in time to find Resistant strain, effectively prevent and control the key of drug tolerant bacteria diffusion.
Bacterial drug resistance detection at present mainly is by based on the drug susceptibility experiment of cultivating, and comprises disk diffusion method, minimal inhibitory concentration (MIC) method etc.Wherein MIC is owned by France in gold standard, can show the susceptibility of bacterium to medicine, thus guiding clinical treatment.Because the experiment of said medicine susceptibility is based on cultivation, and is therefore consuming time longer, could obtain the result in about 2-3 days, and sensitivity is relatively poor, and therefore, the clinical experimental technique of needing rapid sensitive badly comes the bacterial detection resistance, the guiding clinical treatment scheme, the propagation and the diffusion of control drug-resistant bacteria.
Summary of the invention:
The purpose of this invention is to provide a kind of technology and detection kit that detects the various bacteria drug resistant gene, it has detection quick and precisely, and sensitivity is good, and the long characteristics of the retention period of test kit.
In order to solve the existing problem of background technology, the present invention takes following technical scheme: the technology detecting method of many bacterial resistance genes is: utilize special primer to carrying out real-time fluorescence PCR, amplification tested bacteria drug resistant gene, the product and the locked nucleic acid probe of amplification are hybridized, detect fluorescent signal, determine the tested bacteria drug resistant gene.
Two forward and reverse primer concentrations of described special primer centering are identical.
Concentration and forward and reverse primer concentration described and the locked nucleic acid probe that amplified production is hybridized are 1: 2.
Described pcr amplification temperature cycle comprises 40 temperature cycle, is made up of sex change, annealing and three steps of extension.
Described elongating temperature is 60-80 ℃.
It is right that detection kit of the present invention comprises forward and reverse primer of amplification bacterium specific gene and drug resistant gene, with the amplified production of bacterium specific gene and drug resistant gene locked nucleic acid probe of hybridizing and the reaction solution that carries out PCR reaction and molecular hybridization.
The present invention has following beneficial effect: it has detection quick and precisely, and sensitivity is good, and the long characteristics of the retention period of test kit.
Description of drawings:
The structural representation of the primer that bacterial detection drug resistant gene test kit comprises among Fig. 1 the present invention;
The structural representation of the locked nucleic acid probe that bacterial detection drug resistant gene test kit comprises among Fig. 2 the present invention;
Fig. 3 is the structural representation of multiple asymmetric PCR amplification thermal cycling program among the present invention;
Fig. 4 is streptococcus aureus ATCC 43300 drug resistant gene amplification curve diagrams in the present embodiment.
Embodiment:
With reference to Fig. 1-3, this embodiment is taked following technical scheme: the technology detecting method of many bacterial resistance genes is: utilize special primer to carry out real-time fluorescence PCR, amplification tested bacteria drug resistant gene, the product and the locked nucleic acid probe of amplification are hybridized, detect fluorescent signal, determine the tested bacteria drug resistant gene.
Two forward and reverse primer concentrations of described special primer centering are identical.
Concentration and forward and reverse primer concentration described and the locked nucleic acid probe that amplified production is hybridized are 1: 2.
Described pcr amplification temperature cycle comprises 40 temperature cycle, is made up of sex change, annealing and three steps of extension.
Described elongating temperature is 60-80 ℃.
It is right that detection kit of the present invention comprises forward and reverse primer of amplification bacterium specific gene and drug resistant gene, with the amplified production of bacterium specific gene and drug resistant gene locked nucleic acid probe of hybridizing and the reaction solution that carries out PCR reaction and molecular hybridization.
This embodiment has detection quick and precisely, and sensitivity is good, and the long characteristics of the retention period of test kit.
Embodiment: utilize the bacterial resistance gene detection kit to detect the mecA drug resistant gene, detecting target is shv, ctx-m-1-type, ctx-m-9-type, cmy, dha, kpc, ndm, oxa, vim, imp, mecA and vanA, use streptococcus aureus reference culture ATCC43300 to test, in Bechtop with the bacterium streak inoculation in the LB culture medium flat plate to separate single bacterium colony, flat board is inverted in incubator, hatch 24h for 35 ℃, pick a single bacterium colony from the LB flat board of culturing bacterium, be added among 100 μ l, 1 * TE, then with 95 ℃ of water-bath 5min of centrifuge tube, put-20 ℃ standby, fluorescent PCR reaction system composed as follows: 1 * MasterMix (Roche); Prepare 13 reaction systems respectively with locked nucleic acid probe 27-39 among primer 1-26 and Fig. 2 among Fig. 1, the right concentration of primer is respectively 0.4 μ mol/L, and the concentration of probe is 0.2 μ mol/L; Add 1 μ L bacterium liquid as template.The reaction cumulative volume is 10 μ L.Simultaneously, with carry out the amplified reaction of mecA gene behind 10 times of gradient dilutions of bacterium liquid as template, determine detection sensitivity; PCR carries out on 7900HT (ABI) thermal cycler, adopts the pcr amplification thermal cycling program of Fig. 3.
The result shows, locked nucleic acid fluorescence probe PCR method detects ATCC 43300 bacterial strains and contains the mecA drug resistant gene, the Ct value is 19, other drug resistant genes are amplification not, this result conforms to the information that ATCC provides, and the locked nucleic acid probe that utilizes that this explanation is provided carries out bacterial resistance gene detection kit contained target drug resistant gene in the bacterial detection specifically.(see figure 4).
Claims (6)
1. detect the technology and the detection kit of various bacteria drug resistant gene, the technology detecting method that it is characterized in that many bacterial resistance genes is: utilize special primer to carrying out real-time fluorescence PCR, amplification tested bacteria drug resistant gene, the product and the locked nucleic acid probe of amplification are hybridized, detect fluorescent signal, determine the tested bacteria drug resistant gene.
2. the technology and the detection kit of detection various bacteria drug resistant gene according to claim 1, it is right to it is characterized in that described detection kit comprises forward and reverse primer of amplification bacterium specific gene and drug resistant gene, with the locked nucleic acid probe of the amplified production hybridization of bacterium specific gene and drug resistant gene with carry out the reaction solution of PCR reaction and molecular hybridization.
3. the technology and the detection kit of detection various bacteria drug resistant gene according to claim 1 is characterized in that two forward and reverse primer concentrations of described special primer centering are identical.
4. the technology and the detection kit of detection various bacteria drug resistant gene according to claim 1 is characterized in that concentration and forward and reverse primer concentration of the locked nucleic acid probe of described and amplified production hybridization is 1: 2.
5. the technology and the detection kit of detection various bacteria drug resistant gene according to claim 1 is characterized in that described pcr amplification temperature cycle comprises 40 temperature cycle, are made up of sex change, annealing and three steps of extension.
6. the technology and the detection kit of detection various bacteria drug resistant gene according to claim 5 is characterized in that described elongating temperature is 60-80 ℃.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MD4218C1 (en) * | 2012-06-07 | 2013-11-30 | Национальный Центр Общественного Здоровья Министерства Здравоохранения Республики Молдова | Method for diagnosis of infections caused by enterobacteria producers of beta-lactamases |
| CN109735639A (en) * | 2019-02-28 | 2019-05-10 | 宁波基内生物技术有限公司 | It is a kind of for detecting the primer and probe composition and kit of Klebsiella Pneumoniae and three kinds of main carbapenems |
Citations (1)
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MD4218C1 (en) * | 2012-06-07 | 2013-11-30 | Национальный Центр Общественного Здоровья Министерства Здравоохранения Республики Молдова | Method for diagnosis of infections caused by enterobacteria producers of beta-lactamases |
| CN109735639A (en) * | 2019-02-28 | 2019-05-10 | 宁波基内生物技术有限公司 | It is a kind of for detecting the primer and probe composition and kit of Klebsiella Pneumoniae and three kinds of main carbapenems |
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Application publication date: 20111123 |