CN107630071A - The detection kit of SHV type extended spectrumβ-lactamase drug resistant genes - Google Patents
The detection kit of SHV type extended spectrumβ-lactamase drug resistant genes Download PDFInfo
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- CN107630071A CN107630071A CN201610565017.0A CN201610565017A CN107630071A CN 107630071 A CN107630071 A CN 107630071A CN 201610565017 A CN201610565017 A CN 201610565017A CN 107630071 A CN107630071 A CN 107630071A
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Abstract
The invention discloses a kind of detection kit of SHV types extended spectrum β lactamases drug resistant gene, include special primer pair and Taqman fluorescence probe, the kit passes through real-time fluorescence quantitative PCR, bacterium SHV type extended spectrum β lactamases drug resistant genes can be detected exactly, instruct clinical application, convenient and swift, high sensitivity is specific good.
Description
Technical field
The present invention relates to the detection of pathogenic microorganism, and in particular to one kind uses real-time fluorescence quantitative PCR amplification technique (FQ-PCR
Technology), kit, primer pair and its application method of quick detection bacterium SHV type extended spectrumβ-lactamase drug resistant genes.
Background technology
Beta-lactam antibiotic (β-lactams) means a major class antibiotic in chemical constitution with beta-lactam nucleus, including clinic
The most frequently used penicillin and cynnematin, and other are non-for the cephamycin-type of new development, thiomycin class, monobactams etc.
Typical beta-lactam class antibiotic.Such antibiotic has that bactericidal activity is strong, toxicity is low, indication is wide and that clinical efficacy is good is excellent
Point.
The generation of beta-lactamase (β-lactamase) is bacterium to the most common mechanism of beta-lactam antibiotic resistance, be by
The enzyme family of a variety of enzyme compositions, can hydrolyze beta-lactam antibiotic, account for 80% in various resistance mechanisms, be broadly directed to
Many Community Acquired Infections and the important pathogen of inside-hospital infection.
Extended spectrumβ-lactamase (ESBL), it is a kind of energy hydrolyzing penicillin enzyme, cephalosporins and monocyclic class antibiotic
Beta-lactamase, the bacterium that can produce ESBL is ESBL (+) bacterium, can to above-mentioned Multiple Classes of Antibiotics produce resistance.According to
ESBLs genetic homologies are encoded, multiple classifications such as TEM types, SHV types, CTX-M types, OXA types can be classified as,
Each classification contains a variety of hypotypes, and every kind of classification and its characteristic of hypotype hydrolysis medicine can difference.
Traditional culture of microorganism and drug sensitive test method can clinically provide preferable value, but because of the behaviour that time-consuming, cumbersome
The reason for making process, needing expensive instrument and equipment etc. different fails clinically large-scale promotion use.Thus research and develop a kind of letter
Just, the detection method of quick bacterium SHV types extended spectrumβ-lactamase drug resistant gene has great application prospect.
Real-Time Fluorescent Quantitative PCR Technique is a kind of nucleic acid Fast Detection Technique quickly grown in recent years, and fluorescence is carried using one kind
The PCR amplification instrument of detection means, fluorescence detection device can send the exciting light of specific wavelength according to certain routines periodically,
Detection fluorescence signal is collected, the dynamic change by detecting fluorescence signal reflects the level of amplification of PCR each circulation in real time, tries
Acquisition amplification curve can be automatically analyzed by software after testing end, according to amplification curve and the intersection point (i.e. Ct values) of fluorescence threshold line
And the shape of amplification curve, it can be determined that yin and yang attribute result, and learn the definite value result of concentration of specimens.Therefore, the technology exists
In the detection and quantitative analysis of nucleic acid samples, gradually substitute traditional PCR method, obtain quite varied application.
The present invention utilizes real-time fluorescence quantitative PCR for the relatively common and important SHV genotype of extended spectrumβ-lactamase
Technology, designs specific primer and probe, the real-time fluorescence that bacterium SHV type extended spectrumβ-lactamase drug resistant genes are made are determined
PCR detection kit is measured, and describes its application method.
The content of the invention
It is an object of the invention to provide a kind of reality for quick detection bacterium SHV type extended spectrumβ-lactamase drug resistant genes
When fluorescent quantificationally PCR detecting kit and its primer pair, it is and a kind of super wide using the kit quick detection bacterium SHV types
The method for composing beta-lactamase drug resistant gene.
The technical solution used in the present invention is：
A kind of kit for quick detection bacterium SHV type extended spectrumβ-lactamase drug resistant genes, including carry out real-time fluorescence
The PCR reaction solutions of quantitative PCR, the PCR reaction solutions contain PCR buffer solutions, four kinds of dNTP, heat-resistant dna polymerization
Enzyme, pair of primers and a Taqman fluorescence probe, the nucleotide sequence of the primer and probe are as follows：
Primer 1：5’-GGGAAACGGAACTGAATGAGGC-3’(SEQ ID No.1)；
Primer 2：5’-TCCACCATCCACTGCAGCAG-3’(SEQ ID No.2)；
Fluorescence probe：5’-FAM-CCACTACCCCGGCCAGCATGGC-BHQ-1-3’(SEQ ID No.3).
The kit that the present invention is used for quick detection bacterium SHV type extended spectrumβ-lactamase drug resistant genes is a kind of real-time fluorescence
Quantitative PCR detection kit, it is preferred that PCR reaction solutions contain PCR buffer solutions, MgCl2、dATP、dUTP、dGTP、
DCTP, hot resistant DNA polymerase, UNG enzymes, primer 1, primer 2 and fluorescence probe.
As preferable, the PCR buffer solutions include Tris-HCl buffer solutions, KCl and PCR reinforcing agents.Reacted in PCR
In liquid, the Tris-HCl buffer solutions preferred concentration is 50~100mM, most preferably 67mM；The KCl preferred concentrations are
10~20mM, most preferably 13mM；The PCR reinforcing agents contain the one or more in following substances：Dimethyl sulfoxide (DMSO)
(DMSO), glycerine, formamide, ammonium sulfate, bovine serum albumin(BSA) (BSA), glycine betaine etc..
As preferable, the MgCl2Concentration in PCR reaction solutions is 2~5mM, more preferably 3.3mM.
As preferable, described dATP, dUTP, dGTP, dCTP concentration are 200~300 μM, more preferably 250 μM.
As preferable, the hot resistant DNA polymerase is Taq archaeal dna polymerases, and concentration is 0.05~0.2U/ μ L, more excellent
Elect 0.1U/ μ L as.
As preferable, the UNG enzyme concentrations are 0.01-0.1U/ μ L, more preferably 0.05U/ μ L.
As preferable, the primer 1, primer 2 concentration are 1~10 μM, more preferably 5 μM.
As preferable, the fluorescence probe concentration is 1~5 μM, more preferably 2 μM.
Further, the kit also includes positive control and/or negative control.
As preferable, the positive control is contains bacterium SHV type extended spectrumβ-lactamase drug resistant gene fragments
PMD-18T plasmids, concentration are preferably 1000 copies/μ L.The bacterium SHV types extended spectrumβ-lactamase drug resistant gene piece
Section is shown in the NCBI sequences that numbering is DQ408259.
As preferable, the negative control is DEPC water.
Kit of the present invention should be stored in -20 DEG C, reduce multigelation as far as possible.
Utilize the method for the kit quick detection bacterium SHV type extended spectrumβ-lactamase drug resistant genes of the present invention, every time detection
Positive control and negative control all should be set up, is comprised the following steps：
(1) measuring samples DNA is extracted；
(2) real-time quantitative fluorescence：Utilize the real-time fluorescence of above-mentioned detection bacterium SHV types extended spectrumβ-lactamase drug resistant gene
Quantitative PCR detection kit enter performing PCR amplification, respectively using measuring samples DNA, positive control and the negative control of extraction as
Template, mixed with the PCR reaction solutions of kit, carry out real-time fluorescence quantitative PCR, detect fluorescence signal；
(3) result judges：1. positive control Ct values (threshold cycle) are between 25~28, negative control Ct >=38,
Subsequently judged, otherwise should carry out trouble-shoots；2. measuring samples DNA amplification curve Ct value≤35 are the positive；3. treat
Amplification curve Ct value >=38 for examining sample DNA are feminine gender；4. the ＜ Ct values ＜ 38 of amplification curve 35 of DNA sample to be checked, is
Gray area, DNA extractions and PCR detections need to be re-started to sample.
In above-mentioned steps (2), it is preferred that the cumulative volume of each PCR reaction systems is 20 μ L, including 15 μ L PCR
Reaction solution and 5 μ L templates；PCR reaction conditions are usually arranged as：50 DEG C are depolluted for 2 minutes, 95 DEG C of 5 minutes pre-degenerations, 95 DEG C 15
Second → 60 DEG C 45 seconds, FAM passages be used for collect detection fluorescence signal.Totally 40 circulations.
The beneficial effects of the invention are as follows：
Invention directed toward bacteria SHV type extended spectrumβ-lactamase drug resistant gene specific designs 2 primers and 1 Taqman
Probe, combined with 3 regions of target gene, there is higher specificity, can exactly by the super wide spectrum β of bacterium SHV types-
Lactamase drug resistant gene detects, and instructs clinical application.In addition, the kit of the present invention is convenient and swift, can be shorter
Discriminating bacteria SHV types extended spectrumβ-lactamase drug resistant gene in time, high sensitivity, specificity is good, is adapted to clinical practice.
Brief description of the drawings
Fig. 1 is the fluoroscopic examination result of the specificity experiments of embodiment 4, and template corresponding to each detection curve is 1:DEPC, 2:TEM,
3:CTX-M-1,3:OXA, 4:NDM, 5:VIM, 6:DHA, 7:IMP, 8:SHV.
Fig. 2 is the fluoroscopic examination result of the sensitivity experiment of embodiment 5, and template corresponding to each detection curve is 1:DEPC；2~8
For the positive plasmid containing bacterium SHV type extended spectrumβ-lactamase drug resistant genes, concentration is followed successively by 1.0 × 103、1.0×104、
1.0×105、1.0×106、1.0×107、1.0×108、1.0×109Copy/μ L；9~11 be containing the super wide spectrum β of bacterium SHV types-
The positive plasmid of lactamase drug resistant gene, concentration are followed successively by 1.0 × 100、1.0×101、1.0×102Copy/μ L.
Fig. 3 is 10 times of gradient dilution canonical plottings of the sensitivity experiment of embodiment 5.
Fig. 4 is the clinical sample fluoroscopic examination result of embodiment 6, and template corresponding to each detection curve is 1:DEPC, 2:Positive control,
3-8:Clinical sample.
Embodiment
In order that those skilled in the art more fully understand the technical scheme in the embodiment of the present invention, and make the embodiment of the present invention
Above-mentioned purpose, feature and advantage can be more obvious understandable, the technical scheme in the embodiment of the present invention is made below in conjunction with the accompanying drawings into
One step is described in detail, but is not limited thereto.
Embodiment 1：Design for the primed probe pair of quick detection bacterium SHV type extended spectrumβ-lactamase drug resistant genes
SHV type extended spectrumβ-lactamase drug resistant gene sequences in NCBI are downloaded, sequence ratio is carried out using Vector NTI softwares
It is right, conservative region is selected, primer pair is designed and probe, sequence is as follows：
Primer 1：5’-GGGAAACGGAACTGAATGAGGC-3’(SEQ ID No.1)；
Primer 2：5’-TCCACCATCCACTGCAGCAG-3’(SEQ ID No.2)；
Fluorescence probe：5’-FAM-CCACTACCCCGGCCAGCATGGC-BHQ-1-3’(SEQ ID No.3)；
Embodiment 2：Real time fluorescent quantitative kit for quick detection bacterium SHV type extended spectrumβ-lactamase drug resistant genes
Foundation
For the real-time fluorescence quantitative PCR kit of quick detection bacterium SHV type extended spectrumβ-lactamase drug resistant genes, bag
Include PCR reaction solutions, positive reference substance, negative controls, specification and box body.
Wherein PCR reaction solutions contain PCR buffer solutions, MgCl2, dATP, dUTP, dGTP, dCTP, heat-resistant dna gather
Synthase, UNG enzymes, primer 1, primer 2 and fluorescence probe.
The PCR buffer solutions include 67mM Tris-HCl buffer solutions, 13mM KCl and PCR reinforcing agents.
The PCR reinforcing agents are 8% glycerine.
MgCl in PCR reaction solutions2Concentration is 3.3mM；DATP, dUTP, dGTP, dCTP concentration are respectively 250 μM.
The hot resistant DNA polymerase is Taq archaeal dna polymerases, and concentration is 0.1U/ μ L；
The UNG enzyme concentrations are 0.05U/ μ L；
Each concentration is 5 μM for the primer 1, primer 2；
The fluorescence probe concentration is 2 μM.
The positive reference substance is the plasmid containing bacterium SHV type extended spectrumβ-lactamase drug resistant gene fragments, the bacterium
SHV type extended spectrumβ-lactamase drug resistant gene fragments are shown in the NCBI sequences that numbering is DQ408259, and concentration is copied for 1000
Shellfish/μ L, corresponding Ct values are 28 or so.
The negative controls are DEPC water.
Embodiment 3：The quick determination method of bacterium SHV type extended spectrumβ-lactamase drug resistant genes
Using the kit quick detection bacterium SHV type extended spectrumβ-lactamase drug resistant genes of embodiment 2, comprise the following steps that：
(1) measuring samples DNA is extracted：Using water-boiling method extraction bacterial genomes DNA；
(2) real-time quantitative fluorescence：Utilize the real-time fluorescence of above-mentioned detection bacterium SHV types extended spectrumβ-lactamase drug resistant gene
Quantification kit enters performing PCR amplification, and each template (measuring samples, positive control and the negative control of extraction) is separately added into
PCR reaction solutions, the cumulative volume of each PCR reaction systems is 20 μ L, including 15 μ L PCR reaction solutions and 5 μ L templates.
PCR reaction conditions：50 DEG C are depolluted for 2 minutes, 95 DEG C of 5 minutes pre-degenerations, 95 DEG C 15 seconds → 60 DEG C 45 seconds, FAM passages
Fluorescence signal is detected for collecting.Totally 40 circulations.After being provided with, file, operation program are preserved.
(3) result judges：1. positive control Ct values (threshold cycle) are between 25-~28, negative control Ct >=38,
Subsequently judged, otherwise should carry out trouble-shoots；2. amplification curve Ct value≤35 of detected sample are the positive；It is 3. to be detected
Amplification curve Ct value >=38 of sample are feminine gender；4. the ＜ Ct values ＜ 38 of amplification curve 35 of detected sample, is gray area,
DNA extractions and PCR detections need to be re-started to sample.
Embodiment 4：Specificity experiments
Contained respectively to identified with facing without bacterium SHV type extended spectrumβ-lactamase drug resistant genes using the method for embodiment 3
Bed separation strains are detected, and DEPC water is negative control.
Testing result is shown in Fig. 1, the results showed that, only SHV types extended spectrumβ-lactamase drug resistant gene pipe is positive, remaining Guan Jun
For feminine gender.As a result show, detection kit of the invention specificity is high, can be resistance to by SHV type extended spectrumβ-lactamases exactly
Medicine gene and other non-SHV types extended spectrumβ-lactamase drug resistant genes make a distinction.
Embodiment 5：Sensitivity experiment
Take the positive reference substance (pMD-18T containing bacterium SHV type extended spectrumβ-lactamase drug resistant gene fragments built
Plasmid), determine its concentration and calculate copy number, diluted according to 10 times of concentration gradients, choose 1.0 × 100~1.0 × 109Copy/μ L
Concentration as sample, with the kit and detection method of the present invention.
Testing result is as shown in Figures 2 and 3, the results showed that, this kit is 1.0 × 10 to the minimum detectability concentration of SHV genes0
Copy/μ L, i.e. minimum detectability are 5 copies；The standard curve of 10 times of gradient dilutions, slope 3.35, coefficient R2For
0.995。
Embodiment 6：The detection of clinical sample
6 clinical samples are taken, positive and negative control, are detected according to kit application method, as a result as shown in figure 4, table
Bright positive and negative control test result is normal, and clinical sample 3,5 is SHV gene masculines, and remaining is feminine gender, with clinical medicine
Quick result of the test is consistent.
Above example shows that kit of the invention has the characteristics of good accuracy, high sensitivity, is adapted to Clinical practice.
Above example is only to introduce the preferred case of the present invention, to those skilled in the art, without departing substantially from essence of the invention
Any obvious changes and improvements carried out in the scope of god, it is regarded as the part of the present invention.
Claims (11)
1. a kind of kit for quick detection bacterium SHV type extended spectrumβ-lactamase drug resistant genes, including carry out real-time fluorescence
The PCR reaction solutions of quantitative PCR, the PCR reaction solutions contain PCR buffer solutions, four kinds of dNTP, heat-resistant dnas gather
Synthase, pair of primers and a Taqman fluorescence probe, the nucleotide sequence of the primer and probe are as follows：
Primer 1：5’-GGGAAACGGAACTGAATGAGGC-3’；
Primer 2：5’-TCCACCATCCACTGCAGCAG-3’；
Fluorescence probe：5’-FAM-CCACTACCCCGGCCAGCATGGC-BHQ-1-3’.
2. kit as claimed in claim 1, it is characterised in that the PCR buffer solutions include Tris-HCl buffer solutions, KCl
With PCR reinforcing agents.
3. kit as claimed in claim 2, it is characterised in that in PCR reaction solutions, the Tris-HCl buffer solutions it is dense
Spend for 50~100mM；The concentration of the KCl is 10~20mM.
4. kit as claimed in claim 2, it is characterised in that the PCR reinforcing agents include one kind or more in following substances
Kind：Dimethyl sulfoxide (DMSO), glycerine, formamide, ammonium sulfate, bovine serum albumin(BSA) and glycine betaine.
5. kit as claimed in claim 1, it is characterised in that the PCR reaction solutions also contain MgCl2With UNG enzymes.
6. kit as claimed in claim 5, it is characterised in that in PCR reaction solutions, MgCl2Concentration be 2~5mM,
UNG enzyme concentrations are 0.01-0.1U/ μ L.
7. kit as claimed in claim 1, it is characterised in that the hot resistant DNA polymerase is Taq archaeal dna polymerases, dense
Spend for 0.05~0.2U/ μ L；Four kinds of dNTP are dATP, dUTP, dGTP and dCTP, and concentration is respectively 200~300 μM；
The concentration of the primer 1 and primer 2 is respectively 1~10 μM；The concentration of the fluorescence probe is 1~5 μM.
8. kit as claimed in claim 1, it is characterised in that the kit also includes positive control and/or negative control.
9. kit as claimed in claim 1, it is characterised in that the positive control is to contain the super wide spectrum β of bacterium SHV types-interior
The pMD-18T plasmids of amidase drug resistant gene fragment, the negative control are DEPC water.
10. utilize kit quick detection bacterium SHV type extended spectrumβ-lactamase drug resistant genes described in claim 1~9 any one
Method, every time detection set up positive control and negative control, comprise the following steps：
1) measuring samples DNA is extracted；
2) real-time quantitative fluorescence：Utilize the real time fluorescent quantitative of above-mentioned detection bacterium SHV types extended spectrumβ-lactamase drug resistant gene
PCR detection kit enters performing PCR amplification, respectively with measuring samples DNA, positive control and the negative control of extraction
For template, mixed with the PCR reaction solutions of kit, carry out real-time fluorescence quantitative PCR, detect fluorescence signal；
3) result judges：1. positive control Ct values negative control Ct >=38, are subsequently judged between 25~28, otherwise should
Carry out trouble-shoots；2. measuring samples DNA amplification curve Ct value≤35 are the positive；3. measuring samples DNA expansion
Increase curve Ct value >=38 as feminine gender；4. the measuring samples DNA ＜ Ct values ＜ 38 of amplification curve 35, is gray area,
DNA extractions and PCR detections need to be re-started to sample.
11. method as claimed in claim 10, it is characterised in that the cumulative volume of each PCR reaction systems is 20 μ L in step 2),
Including 15 μ L PCR reaction solutions and 5 μ L templates；PCR reaction conditions are arranged to：50 DEG C are depolluted for 2 minutes, 95 DEG C 5
Minute pre-degeneration, 95 DEG C 15 seconds → 60 DEG C 45 seconds, FAM passages are used to collect detection fluorescence signal, totally 40 circulations.
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| CN201610565017.0A CN107630071A (en) | 2016-07-18 | 2016-07-18 | The detection kit of SHV type extended spectrumβ-lactamase drug resistant genes |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110804652A (en) * | 2019-12-03 | 2020-02-18 | 郑州安图生物工程股份有限公司 | Additive, kit and reaction method for rapid detection of real-time quantitative PCR of DNA |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2199144C (en) * | 1994-09-12 | 2010-02-16 | Marc Ouellette | Specific and universal probes and amplification primers to rapidly detect and identify common bacterial pathogens and antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories |
| CN102952875A (en) * | 2011-08-31 | 2013-03-06 | 宁波市第二医院 | Bacterium drug-resistant gene detection method, gene chip and kit |
-
2016
- 2016-07-18 CN CN201610565017.0A patent/CN107630071A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2199144C (en) * | 1994-09-12 | 2010-02-16 | Marc Ouellette | Specific and universal probes and amplification primers to rapidly detect and identify common bacterial pathogens and antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories |
| CN102952875A (en) * | 2011-08-31 | 2013-03-06 | 宁波市第二医院 | Bacterium drug-resistant gene detection method, gene chip and kit |
Non-Patent Citations (1)
| Title |
|---|
| 许静洪等: "《生物化学实验指导》", 31 August 2010 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110804652A (en) * | 2019-12-03 | 2020-02-18 | 郑州安图生物工程股份有限公司 | Additive, kit and reaction method for rapid detection of real-time quantitative PCR of DNA |
| CN110804652B (en) * | 2019-12-03 | 2023-07-25 | 郑州安图生物工程股份有限公司 | Additive, kit and reaction method for real-time quantitative PCR (polymerase chain reaction) for rapidly detecting DNA (deoxyribonucleic acid) |
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