CN105950773A - Primer, probe, method and kit for detecting VRE (Vancomycin-resistant Enterococcus spp.) gene - Google Patents
Primer, probe, method and kit for detecting VRE (Vancomycin-resistant Enterococcus spp.) gene Download PDFInfo
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Abstract
The invention relates to the technical field of molecular biology, and discloses a primer, a probe, a method and a kit for detecting a VRE (Vancomycin-resistant Enterococcus spp.) gene. Primers and fluorescent labeled probes are respectively designed aiming at a nucleic acid conservative region of 16S rDNA (ribosome Deoxyribose Nucleic Acid), vanA and vanB by adopting a Taqman probe real-time fluorescence PCR (Polymerase Chain Reaction) method; the 5' ends of 3 gene probes are all labeled by a fluorescent report group FAM, and 3' ends are all labeled by a fluorescent quenching group TAMRA. After the primer and the probe are prepared into a PCR detection mixed solution, enzyme and sample nucleic acid are added, an FAM channel on a fluorescent PCR instrument is selected for amplification, and detection on a target gene is realized through change of a fluorescent signal. The kit disclosed by the invention has the characteristics of high accuracy, strong specificity ad high sensitivity, and enterococcus and VRE in a urine sample can be rapidly and accurately detected.
Description
Technical field
The present invention relates to technical field of molecular biology, particularly relate to a kind of detection vancomycin-resistant enterococcus base
The primer of cause, probe, method and test kit.
Background technology
Vancomycin-resistant enterococcus (Vancomycin-Resistant Enterococcus spp., VRE) energy
Causing urinary tract, abdominal cavity, pelvic cavity, Postoperative infection and the disease such as endocarditis, meningitis, case fatality rate reaches
To 21.0%～27.5%, it is hospital conditions pathogenic bacterium main in global range.Wherein, to human disease
Predominantly enterococcus faecalis (Enterococcus faecalis) and enterococcus faecalis (Enterococcus
Faecium), modal Resistant genetype is vanA and vanB.Enterococcus is as common nosocomial infection
Multi-drug resistant bacteria, to multiple antibacterials skies such as cephalosporins, part fluoroquinolones, aminoglycosides
So drug resistance.Its drug resistant gene can be transferred to other pathogenic microorganism by vancomycin-resistant enterococcus.Therefore, right
What the mankind constituted a serious threat is not only VRE itself, also includes that the transfer of its drug resistant gene is produced
It is difficult to the new Resistant strain treated.Therefore, strengthen VRE detection and not only contribute to instruct clinical application, and
And beneficially VRE resistance transfer detection and control.
VRE mainly identifies by antibacterial culturing that the method adding drug sensitive test detects the most clinically, this inspection
Survey method not only complex operation, waste time and energy, sensitivity relatively low, poor repeatability, traditional method is difficult to differentiate between
Different genotype, and the Molecular Biology Mechanism that resistance causes cannot be understood.
Summary of the invention
The present invention provides a kind of and detects the primer of vancomycin-resistant enterococcus gene, probe, method and test kit,
Solve the detection not only complex operation of VRE in prior art, waste time and energy, sensitivity relatively low, repeatability
Difference, traditional method is difficult to differentiate between different genotype, and cannot understand the molecular biology machine that resistance causes
The technical problem of system.
It is an object of the invention to be achieved through the following technical solutions:
A kind of primer detecting vancomycin-resistant enterococcus gene and probe, detect enterococcus gene 16S
The primer nucleotide sequences of rDNA, as shown in SEQIDNO:2～3, detects enterococcus gene 16S rDNA
The nucleotide sequence of Taqman probe as shown in SEQIDNO:4；Detection vancomycin-resistant enterococcus is special
The primer nucleotide sequences of specific gene vanA, as shown in SEQIDNO:6～7, detects vancomycin resistance
The nucleotide sequence of the Taqman probe of enterococcus specific gene vanA is as shown in SEQIDNO:8；
The primer nucleotide sequences of detection vancomycin-resistant enterococcus specific gene vanB is such as
Shown in SEQIDNO:10～11, the Taqman of detection vancomycin-resistant enterococcus specific gene vanB
The nucleotide sequence of probe is as shown in SEQIDNO:12.
A kind of method detecting vancomycin-resistant enterococcus gene, including:
Prepared by sample nucleic acid, nucleic acid-templated to obtain；
Be respectively directed to enterococcus gene 16S rDNA, vancomycin-resistant enterococcus specific gene vanA,
Vancomycin-resistant enterococcus specific gene vanB design specific primer and fluorescence labeling probe, wherein,
The primer nucleotide sequences of detection enterococcus gene 16S rDNA, as shown in SEQIDNO:2～3, detects
The nucleotide sequence of the Taqman probe of enterococcus gene 16S rDNA is as shown in SEQIDNO:4；
Detection vancomycin-resistant enterococcus specific gene vanA primer nucleotide sequences such as SEQIDNO:6～
Shown in 7, the nucleotides sequence of the Taqman probe of detection vancomycin-resistant enterococcus specific gene vanA
Row are as shown in SEQIDNO:8；The primer nucleoside of detection vancomycin-resistant enterococcus specific gene vanB
Acid sequence, as shown in SEQIDNO:10～11, detects vancomycin-resistant enterococcus specific gene vanB
The nucleotide sequence of Taqman probe as shown in SEQIDNO:12；
Take urine specimen, positive reference substance, negative controls are placed in centrifuge tube, are separately added into nucleic acid extraction
Liquid fully mixes, and carries out heating and centrifugal treating, takes supernatant and detects for fluorescent PCR；
Configuration enzyme reagent, wherein, described enzyme is by thermus aquaticus archaeal dna polymerase (Taq enzyme) and uracil-N-
Glycosylase (UNG enzyme) mixing composition；
By 16S rDNA gene PCR detection mixed liquor and the concussion mixing of enzyme reagent, it is centrifuged processing；
By vanA gene PCR detection mixed liquor and the concussion mixing of enzyme reagent, it is centrifuged processing；By vanB
Gene PCR detection mixed liquor and the concussion mixing of enzyme reagent, be centrifuged processing
Take the detection mixed liquor of the PCR after enzyme-added mixing respectively to be placed in fluorescent PCR pipe, take sample nucleic acid
Extracting supernatant, negative controls nucleic acid extraction supernatant, positive reference substance nucleic acid extraction supernatant have added
Having in the fluorescent PCR pipe of PCR detection mixed liquor, carry out fluorescent PCR amplification, reaction condition is:
37 DEG C × 2min, 94 DEG C × 2min, circulate 1 time；93 DEG C × 15s, 60 DEG C × 60s, circulate 40 times；Single
Point fluoroscopic examination is at 60 DEG C, and gathers fluorescence signal at this.
For 16S rDNA gene, vanA, vanB gene, the FAM on fluorescent PCR instrument is used to lead to
Road expands, and is determined the genotype of detection site by data collected by quantitative real time PCR Instrument.
A kind of test kit detecting vancomycin-resistant enterococcus gene, visits including nucleic acid extraction liquid, the first primer
Pin mixed liquor, the second primed probe mixed liquor, three-primer probe mixed liquor, PCR reaction enzymes system, the moon
Property reference substance, positive reference substance and separation these reagent bottle of union intermediate package or the packing box of pipe, wherein, institute
State the first primed probe mixed liquor by dideoxyribonucleotide triphosphate dN (U) TP, 16S rDNA gene
Upstream and downstream primer and a fluorescence labeling probe composition；Described second primed probe mixed liquor is by deoxyribose
The upstream and downstream primer of ribonucleoside triphosphote dN (U) TP, vanA gene and a fluorescence labeling probe composition；
Described three-primer probe mixed liquor by dideoxyribonucleotide triphosphate dN (U) TP, vanB gene upper,
Downstream primer and a fluorescence labeling probe composition；
Wherein, the primer nucleotide sequences such as SEQIDNO:2～3 of enterococcus gene 16S rDNA is detected
Shown in, the nucleotide sequence of the Taqman probe of detection enterococcus gene 16S rDNA is such as
Shown in SEQIDNO:4；The prime nucleotide sequence of detection vancomycin-resistant enterococcus specific gene vanA
Row, as shown in SEQIDNO:6～7, detect vancomycin-resistant enterococcus specific gene vanA's
The nucleotide sequence of Taqman probe is as shown in SEQIDNO:8；Detection vancomycin-resistant enterococcus is special
Property gene vanB primer nucleotide sequences as shown in SEQIDNO:10～11, detect vancomycin resistance
The nucleotide sequence of the Taqman probe of enterococcus specific gene vanB is as shown in SEQIDNO:12.
The invention have the benefit that have accuracy rate height, clinical test results is total with DNA sequencing result
Coincidence rate more than 99%；Common pathogen escherichia coli, kerekou pneumonia primary in high specificity, with urine
Salmonella, staphylococcus aureus, Pseudomonas aeruginosa, Bacillus proteus, proteus mirabilis, Streptococcus sanguis,
Staphylococcus epidermidis, Candida albicans, Klebsiella oxytoca, Burkholderia cepacia, Bao Man not lever
Bacterium, diphtheria corynebacterium, Salmonella typhimurium, hemophilus influenza, the equal no cross reaction of Neisseria meningitidis；
Highly sensitive, the lowest detection determined according to plasmid copy number is limited to 103Copies/mL, cultivates according to bacterium
The lowest detection that method determines is limited to 103CFU/mL.This product is in external qualitative detection human urine sample
Enterococcus gene 16S rDNA and vancomycin-resistant enterococcus specific gene vanA and vanB, for facing
Bed determines that enterococcus and VRE infect the method that provides assistance in diagnosis, and provides reference for clinician's medication.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to enforcement
In example, the required accompanying drawing used is briefly described, it should be apparent that, the accompanying drawing in describing below is only
Some embodiments of the present invention, for those of ordinary skill in the art, are not paying creative work
On the premise of, other accompanying drawing also can be obtained according to these accompanying drawings.
Fig. 1 is the quantitative fluorescent PCR curve chart of the enterococcal infection sample of the embodiment of the present invention four；
Fig. 2 is the quantitative fluorescent PCR curve chart of the vanA type VRE infection sample of the embodiment of the present invention four；
Fig. 3 is the quantitative fluorescent PCR curve chart of the vanB type VRE infection sample of the embodiment of the present invention four.
Detailed description of the invention
Understandable for enabling the above-mentioned purpose of the present invention, feature and advantage to become apparent from, below in conjunction with the accompanying drawings and
The present invention is further detailed explanation for detailed description of the invention.
Embodiment one
Embodiments provide a kind of primer detecting vancomycin-resistant enterococcus gene and probe, detection
The primer nucleotide sequences of enterococcus gene 16S rDNA, as shown in SEQIDNO:2～3, detects intestinal ball
The nucleotide sequence of the Taqman probe of bacterium gene 16S rDNA is as shown in SEQIDNO:4；Detection
Primer nucleotide sequences such as SEQIDNO:6～7 institute of vancomycin-resistant enterococcus specific gene vanA
Showing, the nucleotide sequence of the Taqman probe of detection vancomycin-resistant enterococcus specific gene vanA is such as
Shown in SEQIDNO:8；The prime nucleotide sequence of detection vancomycin-resistant enterococcus specific gene vanB
Row, as shown in SEQIDNO:10～11, detect vancomycin-resistant enterococcus specific gene vanB's
The nucleotide sequence of Taqman probe is as shown in SEQIDNO:12；
Wherein, the sequence of the specific primer of 16S rDNA gene design is as follows:
Forward primer P-16S rDNA-F:5 '-CGCTAGACCGCGAGGTCAT-3 '
Downstream primer P-16S rDNA-R:5 '-ACTAGCGATTCCGGCTTCAT-3 '
TaqMan fluorescence labeling probe sequence for 16S rDNA gene is as follows:
T-16SrDNA:5 '-FAM-CAAATCTCTTAAAGCTTCTCTCAGTTCG-TAMR
A-3’
Sequence for the specific primer of vanA gene design is as follows:
Forward primer P-vanA-F:5 '-AGTGCCGCGTTAGCTGTTG-3 '
Downstream primer P-vanA-R:5 '-GCGTTTTCAGAGCCTTTTTCC-3 '
TaqMan fluorescence labeling probe sequence for vanA gene is as follows:
T-vanA:5 '-FAM-ATCAGGCTGCAGTACGGAATCTTTCGTA-TAMRA-3 '
The sequence of the specific primer of vanB gene design is as follows:
Forward primer P-vanB-F:5 '-CGTTTAGTTCTTCCGTACT-3 '
Downstream primer P-vanB-R:5 '-GAGGACGCTTACCTACCCT-3 '
TaqMan fluorescence labeling probe sequence for vanB gene is as follows:
T-vanB:5 '-FAM-TTACGCCAAAGGACGAACCTGACCGT-TAMRA-3 '
The nucleotide sequence 5 ' of 16S rDNA, vanA, vanB gene probe holds the fluorescence report of equal labelling
Group is FAM, and 3 ' to hold the quenching group of equal labelling be TAMRA.
Embodiment two
The embodiment of the present invention provides again a kind of method detecting vancomycin-resistant enterococcus gene, including:
Prepared by step 101, sample nucleic acid, nucleic acid-templated to obtain；
Step 102, it is respectively directed to enterococcus gene 16S rDNA, vancomycin-resistant enterococcus specificity base
Because vanA, vancomycin-resistant enterococcus specific gene vanB design specific primer and fluorescent labeling are visited
Pin；
Wherein, the primer nucleotide sequences such as SEQIDNO:2～3 of enterococcus gene 16S rDNA is detected
Shown in, the nucleotide sequence of the Taqman probe of detection enterococcus gene 16S rDNA is such as
Shown in SEQIDNO:4；The prime nucleotide sequence of detection vancomycin-resistant enterococcus specific gene vanA
Row, as shown in SEQIDNO:6～7, detect vancomycin-resistant enterococcus specific gene vanA's
The nucleotide sequence of Taqman probe is as shown in SEQIDNO:8；Detection vancomycin-resistant enterococcus is special
Property gene vanB primer nucleotide sequences as shown in SEQIDNO:10～11, detect vancomycin resistance
The nucleotide sequence of the Taqman probe of enterococcus specific gene vanB is as shown in SEQIDNO:12；
Step 103, take positive reference substance, negative controls is placed in centrifuge tube, is separately added into nucleic acid extraction
Liquid fully mixes, and carries out heating and centrifugal treating, takes supernatant and detects for fluorescent PCR；
Step 104, configuration enzyme reagent；
Wherein, described enzyme is by thermus aquaticus archaeal dna polymerase (Taq enzyme) and uracil-N-glycosylase
(UNG enzyme) mixing composition；
Step 105, by detection mixed liquor and the enzyme reagent concussion mixing of 16S rDNA gene PCR, carry out
Centrifugal treating；By vanA gene PCR detection mixed liquor and the concussion mixing of enzyme reagent, it is centrifuged processing；
By vanB gene PCR detection mixed liquor and the concussion mixing of enzyme reagent, it is centrifuged processing；
Step 106, take enzyme-added mixing respectively after PCR detection mixed liquor be placed in fluorescent PCR pipe,
Sample on this nucleic acid extraction supernatant, negative controls nucleic acid extraction supernatant, positive reference substance nucleic acid extraction
Clear liquid adds in the fluorescent PCR pipe of existing PCR detection mixed liquor, carries out fluorescent PCR amplification；
Wherein, reaction condition is: 37 DEG C × 2min, 94 DEG C × 2min, circulates 1 time；93 DEG C × 15s,
60 DEG C × 60s, circulate 40 times；Single-point fluoroscopic examination is at 60 DEG C, and gathers fluorescence signal at this；
Step 107, for 16S rDNA gene, vanA, vanB gene, use fluorescent PCR instrument
On FAM passage expand, determine detection site by data collected by quantitative real time PCR Instrument
Genotype.
Wherein, testing result is according to CTValue judges, instrument CTHurdle display Undet. (ABI
StepOnePlus) C or is not shownT(Eppendorf Mastercycler ep realplex fluorescence is fixed for value
Amount PCR detection system), represent that detection sample, less than detection limit, is reported as feminine gender；Measuring samples CT≤ 35,
Test results report is positive；Measuring samples CTDisplay, between 35～40, needs replication, CTDisplay
Still between 35～40, and amplification curve is S-type, then be judged as the positive；If amplification is a straight line,
Then it is judged as feminine gender.
The present invention uses Taqman probe for real-time fluorescence PCR method, at sequence analysis Enterococcus 16S
On the basis of rDNA, vancomycin resistance vanA and vanB gene order, choose conservative fragments and separately design
Specific primer and specific fluorescence labeling probe for these 3 genes.Flag F AM held by probe 5 '
Fluorescein is fluorescent reporter group (representing with R), and 3 ' end labelling TAMRA fluoresceins are fluorescent quenching base
Group's (representing with Q), it can absorb the fluorescence signal that 5 ' end fluorescent reporter group send within closely.PCR
When being reacted into annealing stage, the probe first genes of interest in template is combined, primer and genes of interest subsequently
In conjunction with, the fluorescence signal that now on probe, R group sends be can't detect fluorescence by Q group absorptions, instrument
Signal；When reaction proceeds to the extension stage, the exonuclease function of 5 ' → the 3 ' of Taq archaeal dna polymerase
Probe is degraded.So R group on probe is free out, sent fluorescence signal not by Q base
Group absorbs, and detector can detect fluorescence signal.Along with the carrying out of PCR reaction, PCR primer and fluorescence
The growth of signal presents corresponding relation.By fluorescence signal growth curve, it is achieved the detection of genes of interest.
Embodiment three
Embodiments provide a kind of test kit detecting vancomycin-resistant enterococcus gene, including nucleic acid
Extract, the first primed probe mixed liquor, the second primed probe mixed liquor, three-primer probe mixed liquor,
PCR reaction enzymes system, negative controls, positive reference substance and separation these reagent bottle of union intermediate package or pipe
Packing box, wherein, described first primed probe mixed liquor by dideoxyribonucleotide triphosphate dN (U) TP,
The upstream and downstream primer of 16S rDNA gene and a fluorescence labeling probe composition；Described second primed probe
Mixed liquor is glimmering by the upstream and downstream primer of dideoxyribonucleotide triphosphate dN (U) TP, vanA gene and one
Signal probe forms；Described three-primer probe mixed liquor by dideoxyribonucleotide triphosphate dN (U) TP,
The upstream and downstream primer of vanB gene and a fluorescence labeling probe composition；
Wherein, the primer nucleotide sequences such as SEQIDNO:2～3 of enterococcus gene 16S rDNA is detected
Shown in, the nucleotide sequence of the Taqman probe of detection enterococcus gene 16S rDNA is such as
Shown in SEQIDNO:4；The prime nucleotide sequence of detection vancomycin-resistant enterococcus specific gene vanA
Row, as shown in SEQIDNO:6～7, detect vancomycin-resistant enterococcus specific gene vanA's
The nucleotide sequence of Taqman probe is as shown in SEQIDNO:8；Detection vancomycin-resistant enterococcus is special
Property gene vanB primer nucleotide sequences as shown in SEQIDNO:10～11, detect vancomycin resistance
The nucleotide sequence of the Taqman probe of enterococcus specific gene vanB is as shown in SEQIDNO:12.
Described nucleic acid extraction liquid is by 3% (M/V) Chelex-100,0.5% (V/V) Tris-HCl, 1M, pH9.0
Form with 0.5% (V/V) TritonX-100.
Described first primed probe mixed liquor is by 4 μ L 10 × PCR Buffer, 4 μ L25mmol/LMgCl2、
3.2 μ L 2.5mmol/L dN (U) TP, 2.4 μ L 10 μm ol/L primers, 0.4 μ L 10 μm ol/L probe and
18.2 μ L sterilizing purified water compositions；Second primed probe mixed liquor by 4 μ L 10 × PCR Buffer,
4μL25mmol/LMgCl2, 3.2 μ L 2.5mmol/L dN (U) TP, 1.2 μ L 10 μm ol/L primers, 0.2 μ L
10 μm ol/L probes and 20.9 μ L sterilizing purified water composition；Three-primer probe mixed liquor is by 4 μ L
10×PCR Buffer、4μL25mmol/LMgCl2、3.2μL 2.5mmol/L dN(U)TP、1.2μL
10 μm ol/L primers, 0.6 μ L 10 μm ol/L probe and 20.4 μ L sterilizing purified water composition.
PCR reaction enzymes system is by 5U/ μ L Taq archaeal dna polymerase and 2U/ μ L UNG enzyme by volume 3: 1
Ratio mixing composition.
The condition of PCR amplification is: 37 DEG C × 2min, 94 DEG C × 2min, circulate 1 time；93 DEG C × 15s,
60 DEG C × 60s, circulate 40 times；Single-point fluoroscopic examination at 60 DEG C, for 16S rDNA gene, vanA,
VanB gene, uses the FAM passage on fluorescent PCR instrument to expand.
Described positive reference substance is E-16S rDNA bacterium, E-vanA bacterium and the E-vanB bacterium containing inactivation
Mixed liquor, bacteria concentration is 106CFU/mL；Negative controls is the escherichia coli solution containing inactivation,
Bacteria concentration is 106CFU/mL, wherein, E-16S rDNA bacterium is containing 16S rDNA genetic fragment
Engineering bacteria, E-vanA bacterium is the engineering bacteria containing vanA genetic fragment, and E-vanB bacterium is containing vanB
The engineering bacteria of genetic fragment.
It is high that this test kit has accuracy rate, clinical test results and the total coincidence rate of DNA sequencing result 99% with
On；Common pathogen escherichia coli, Klebsiella pneumonia, golden yellow Portugal in high specificity, with urine
Grape coccus, Pseudomonas aeruginosa, Bacillus proteus, proteus mirabilis, Streptococcus sanguis, staphylococcus epidermidis,
Candida albicans, Klebsiella oxytoca, Burkholderia cepacia, Acinetobacter bauamnnii, diphtheria corynebacterium,
Salmonella typhimurium, hemophilus influenza, the equal no cross reaction of Neisseria meningitidis；Highly sensitive, press
The lowest detection determined according to plasmid copy number is limited to 103Copies/mL, according to bacterium culture method determine minimum
Detection is limited to 103CFU/mL.This product is enterococcus gene in external qualitative detection human urine sample
16S rDNA and vancomycin-resistant enterococcus specific gene vanA and vanB, determines enterococcus for clinic
Infect, with VRE, the method that provides assistance in diagnosis, provide reference for clinician's medication.
Embodiment four
The present embodiment combines the concrete application mode of the concrete detection case explanation present invention, first collects clinical sample
Through antibacterial culturing, this, identify that being combined DNA sequencing with drug sensitive test method is defined as enterococcal infection, vanA
Type VRE infects, vanB type VRE infects and each 3 examples of VRE negative sample, uses this test kit to enter
Row detection, statistical result coincidence rate.
1, prepared by sample nucleic acid
Urine: sample this 3mL, 12,000rpm 2min；Abandon supernatant, precipitation is directly added into 50 μ L
Nucleic acid extraction liquid, fully mixes, 98 DEG C of 10min (error is less than 1min).12,000rpm 5min,
Take supernatant 5 μ L and do PCR reaction.
2, reference substance prepares
Take positive reference substance, each 100 μ L of negative controls be respectively placed in 1.5mL (or 0.5mL) from
In heart pipe (after frozen reagent melts, concussion mixes 10s), it is separately added into nucleic acid extraction liquid 50 μ L the most mixed
Even, 98 DEG C of 10min, then 12,000rpm 5min, take supernatant 5 μ L and do PCR reaction.
3, enzyme preparation of reagents
Take n × 35 μ L enterococcus 16S rDNA (16S rDNA) PCR respectively and detect mixed liquor,
Vancomycin-resistant enterococcus A gene (vanA) PCR detects mixed liquor, vancomycin-resistant enterococcus 1 B gene
(vanB) PCR detection mixed liquor mixes with n × 0.4 μ L enzyme (Taq+UNG), the concussion mixing several seconds,
The 3000rpm centrifugal mixing several seconds.
4, sample-adding
Take above-mentioned mixed liquor 35 μ L respectively to be placed in PCR pipe, then by negative controls, sample and sun
The process each 5 μ L of supernatant of property reference substance are separately added in the PCR pipe of each existing mixed liquor, build pipe
Lid, carries out fluorescent PCR amplified reaction immediately.
5, PCR amplification
Reaction tube is placed on fluorescent PCR instrument, it is recommended that loop parameter is arranged:
37 DEG C × 2min, 94 DEG C × 2min, circulate 1 time；93 DEG C × 15sec, 60 DEG C × 60s, circulate 40
Secondary；Single-point fluoroscopic examination is at 60 DEG C, and reaction system is 40 μ L.
Fluorescence channel detection selects: select FAM passage.
6, result judges
After detection terminates, baseline adjustment takes the fluorescence signal of 6-15 circulation, and threshold value setting principle is with threshold value
Line is just above the peak of negative controls detection fluorescence curve.Instrument CTHurdle display Undet. (ABI
StepOnePlus) C or is not shownTValue (Eppendorf Mastercycler ep realplex) fluorescence
Quantitative PCR detection system), represent that detection sample, less than detection limit, is reported as feminine gender；Measuring samples
CT≤ 35, test results report is positive；Measuring samples CTDisplay, between 35～40, needs replication,
CTDisplay is still between 35～40, and amplification curve is S-type, then be judged as the positive；If amplification is
One straight line, then be judged as feminine gender.
7, testing result inspection
The testing result of the present invention is identified consistent with drug sensitive test result with antibacterial culturing, and fluorescent PCR detects
As Figure 1-3, Fig. 1 is the quantitative fluorescent PCR curve chart of enterococcal infection sample to result, it is seen that with
The carrying out of PCR reaction, the PCR primer that 16S rDNA gene pairs is answered presents with the growth of fluorescence signal
Corresponding relation, can determine whether that 16S rDNA gene is the positive, and vanB gene and vanA gene are negative.
Fig. 2 is the quantitative fluorescent PCR curve chart that vanA type VRE infects sample, can determine whether 16S rDNA base
Cause and vanA gene are the positive.Fig. 3 is the quantitative fluorescent PCR song that vanB type VRE infects sample
Line chart, can determine whether that 16S rDNA gene and vanB gene are the positive.
Above the present invention is described in detail, former to the present invention of specific case used herein
Reason and embodiment are set forth, and the explanation of above example is only intended to help to understand the present invention's
Method and core concept thereof；Simultaneously for one of ordinary skill in the art, according to the think of of the present invention
Think, the most all will change, in sum, this specification
Content should not be construed as limitation of the present invention.
Claims (9)
1. the primer detecting vancomycin-resistant enterococcus gene and probe, it is characterised in that detection intestinal
The primer nucleotide sequences of coccus gene 16S rDNA, as shown in SEQIDNO:2～3, detects enterococcus
The nucleotide sequence of the Taqman probe of gene 16S rDNA is as shown in SEQIDNO:4；Detect resistance to
The primer nucleotide sequences of vancomycin enterococcus specific gene vanA as shown in SEQIDNO:6～7,
The nucleotide sequence of the Taqman probe of detection vancomycin-resistant enterococcus specific gene vanA is such as
Shown in SEQIDNO:8；The prime nucleotide sequence of detection vancomycin-resistant enterococcus specific gene vanB
Row, as shown in SEQIDNO:10～11, detect vancomycin-resistant enterococcus specific gene vanB's
The nucleotide sequence of Taqman probe is as shown in SEQIDNO:12.
The primer of detection vancomycin-resistant enterococcus gene the most according to claim 1 and probe, its
Being characterised by, the nucleotide sequence 5 ' of 16S rDNA, vanA, vanB gene probe holds the glimmering of equal labelling
Light reporter group is FAM, and 3 ' to hold the quenching group of equal labelling be TAMRA.
3. the method detecting vancomycin-resistant enterococcus gene, it is characterised in that including:
Prepared by sample nucleic acid, nucleic acid-templated to obtain；
Be respectively directed to enterococcus gene 16S rDNA, vancomycin-resistant enterococcus specific gene vanA,
Vancomycin-resistant enterococcus specific gene vanB design specific primer and fluorescence labeling probe, wherein,
The primer nucleotide sequences of detection enterococcus gene 16S rDNA, as shown in SEQIDNO:2～3, detects
The nucleotide sequence of the Taqman probe of enterococcus gene 16S rDNA is as shown in SEQIDNO:4；
Detection vancomycin-resistant enterococcus specific gene vanA primer nucleotide sequences such as SEQIDNO:6～
Shown in 7, the nucleotides sequence of the Taqman probe of detection vancomycin-resistant enterococcus specific gene vanA
Row are as shown in SEQIDNO:8；The primer nucleoside of detection vancomycin-resistant enterococcus specific gene vanB
Acid sequence, as shown in SEQIDNO:10～11, detects vancomycin-resistant enterococcus specific gene vanB
The nucleotide sequence of Taqman probe as shown in SEQIDNO:12；
Take positive reference substance, negative controls is placed in centrifuge tube, is separately added into nucleic acid extraction liquid and fully mixes,
And carry out heating and centrifugal treating, take supernatant and detect for fluorescent PCR；
Configuration enzyme reagent, wherein, described enzyme is by thermus aquaticus archaeal dna polymerase (Taq enzyme) and uracil-N-
Glycosylase (UNG enzyme) mixing composition；
By 16S rDNA gene PCR detection mixed liquor and the concussion mixing of enzyme reagent, it is centrifuged processing；
By vanA gene PCR detection mixed liquor and the concussion mixing of enzyme reagent, it is centrifuged processing；By vanB
Gene PCR detection mixed liquor and the concussion mixing of enzyme reagent, be centrifuged processing；
Take the detection mixed liquor of the PCR after enzyme-added mixing respectively to be placed in fluorescent PCR pipe, take sample nucleic acid
Extracting supernatant, negative controls nucleic acid extraction supernatant, positive reference substance nucleic acid extraction supernatant have added
Having in the fluorescent PCR pipe of PCR detection mixed liquor, carry out fluorescent PCR amplification, reaction condition is:
37 DEG C × 2min, 94 DEG C × 2min, circulate 1 time；93 DEG C × 15s, 60 DEG C × 60s, circulate 40 times；Single
Point fluoroscopic examination is at 60 DEG C, and gathers fluorescence signal at this；
For 16S rDNA gene, vanA, vanB gene, the FAM on fluorescent PCR instrument is used to lead to
Road expands, and is determined the genotype of detection site by data collected by quantitative real time PCR Instrument.
4. the test kit detecting vancomycin-resistant enterococcus gene, it is characterised in that include that nucleic acid is taken out
Extract, the first primed probe mixed liquor, the second primed probe mixed liquor, three-primer probe mixed liquor,
PCR reaction enzymes system, negative controls, positive reference substance and separation these reagent bottle of union intermediate package or pipe
Packing box, wherein, described first primed probe mixed liquor by dideoxyribonucleotide triphosphate dN (U) TP,
The upstream and downstream primer of 16S rDNA gene and a fluorescence labeling probe composition；Described second primed probe
Mixed liquor is glimmering by the upstream and downstream primer of dideoxyribonucleotide triphosphate dN (U) TP, vanA gene and one
Signal probe forms；Described three-primer probe mixed liquor by dideoxyribonucleotide triphosphate dN (U) TP,
The upstream and downstream primer of vanB gene and a fluorescence labeling probe composition；
Wherein, the primer nucleotide sequences such as SEQIDNO:2～3 of enterococcus gene 16S rDNA is detected
Shown in, the nucleotide sequence of the Taqman probe of detection enterococcus gene 16S rDNA is such as
Shown in SEQIDNO:4；The prime nucleotide sequence of detection vancomycin-resistant enterococcus specific gene vanA
Row, as shown in SEQIDNO:6～7, detect vancomycin-resistant enterococcus specific gene vanA's
The nucleotide sequence of Taqman probe is as shown in SEQIDNO:8；Detection vancomycin-resistant enterococcus is special
Property gene vanB primer nucleotide sequences as shown in SEQIDNO:10～11, detect vancomycin resistance
The nucleotide sequence of the Taqman probe of enterococcus specific gene vanB is as shown in SEQIDNO:12.
The test kit of detection vancomycin-resistant enterococcus gene the most according to claim 4, its feature
Being, described nucleic acid extraction liquid is by 3% (M/V) Chelex-100,0.5% (V/V) Tris-HCl, 1M, pH9.0
Form with 0.5% (V/V) TritonX-100.
The test kit of detection vancomycin-resistant enterococcus gene the most according to claim 4, its feature
Being, described first primed probe mixed liquor is by 4 μ L 10 × PCR Buffer, 4 μ L25mmol/LMgCl2、
3.2 μ L 2.5mmol/L dN (U) TP, 2.4 μ L 10 μm ol/L primers, 0.4 μ L 10 μm ol/L probe and
18.2 μ L sterilizing purified water compositions；Second primed probe mixed liquor by 4 μ L 10 × PCR Buffer,
4μL25mmol/LMgCl2, 3.2 μ L 2.5mmol/L dN (U) TP, 1.2 μ L 10 μm ol/L primers, 0.2 μ L
10 μm ol/L probes and 20.9 μ L sterilizing purified water composition；Three-primer probe mixed liquor is by 4 μ L
10×PCR Buffer、4μL25mmol/LMgCl2、3.2μL 2.5mmol/L dN(U)TP、1.2μL
10 μm ol/L primers, 0.6 μ L 10 μm ol/L probe and 20.4 μ L sterilizing purified water composition.
The test kit of detection vancomycin-resistant enterococcus gene the most according to claim 4, its feature
Being, PCR reaction enzymes system is urinated phonetic by 5U/ μ L thermus aquaticus archaeal dna polymerase (Taq enzyme) and 2U/ μ L
Pyridine-N-glycosylase (UNG enzyme) 3: 1 ratio mixing composition by volume.
The test kit of detection vancomycin-resistant enterococcus gene the most according to claim 4, its feature
Being, the condition of PCR amplification is: 37 DEG C × 2min, 94 DEG C × 2min, circulate 1 time；93 DEG C × 15s,
60 DEG C × 60s, circulate 40 times；Single-point fluoroscopic examination at 60 DEG C, for 16S rDNA gene, vanA,
VanB gene, uses the FAM passage on fluorescent PCR instrument to expand.
The test kit of detection vancomycin-resistant enterococcus gene the most according to claim 4, its feature
Being, described positive reference substance is E-16S rDNA bacterium, E-vanA bacterium and the E-vanB containing inactivation
The mixed liquor of bacterium, bacteria concentration is 106CFU/mL；Negative controls is the escherichia coli solution containing inactivation,
Bacteria concentration is 106CFU/mL, wherein, E-16S rDNA bacterium is containing 16S rDNA genetic fragment
Engineering bacteria, E-vanA bacterium is the engineering bacteria containing vanA genetic fragment, and E-vanB bacterium is containing vanB
The engineering bacteria of genetic fragment.
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| 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 |
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| CN111057774A (en) * | 2019-10-08 | 2020-04-24 | 上海市动物疫病预防控制中心(上海市兽药饲料检测所、上海市畜牧技术推广中心) | Fluorescent quantitative PCR kit for detecting vancomycin-resistant vanA gene of enterotoxin |
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