CN106191241A - A kind of primer, probe, method and test kit detecting Acinetobacter bauamnnii Carbapenem-resistant class antibiotic resistance gene - Google Patents
A kind of primer, probe, method and test kit detecting Acinetobacter bauamnnii Carbapenem-resistant class antibiotic resistance gene Download PDFInfo
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Abstract
The present invention relates to technical field of molecular biology, disclose a kind of primer, probe, method and test kit detecting Acinetobacter bauamnnii Carbapenem-resistant class antibiotic resistance gene, use Taqman probe for real-time fluorescence PCR method, be respectively directed to OXA51, OXA23 nucleic acid conserved regions design primer and fluorescence labeling probe.After primer, probe are made into PCR detection mixed liquor, add enzyme and sample nucleic acid, select the FAM passage on fluorescent PCR instrument to expand, realized the detection of genes of interest by the change of fluorescence signal.The present invention has accuracy rate height, high specificity, highly sensitive feature, it is possible to the Acinetobacter bauamnnii in clinical sample and CRAB are carried out quick, accurately detect.
Description
Technical field
The present invention relates to technical field of molecular biology, particularly relate to a kind of detection Acinetobacter bauamnnii Carbapenem-resistant class
The primer of antibiotic resistance gene, probe, method and test kit.
Background technology
Acinetobacter bauamnnii is widely distributed in hospital environment, is the important pathogen causing nosocomial infection at present, mainly
Cause respiratory tract infection, it is possible to cause septicemia, urinary system infection, Secondary cases meningitis etc..Feel in Acinetobacter bauamnnii institute
Contaminating modal position is pulmonary, is the important pathogenic bacterium of Nosocomial Pneumonia, especially mechanical ventilationassociatepneumonia pneumonia.Bao Man is not
Lever bacterium has long-term surviving ability in vitro, easily causes clone and sends out.
Acinetobacter bauamnnii infection risk factors includes: is in hospital for a long time, moves in ICU, accepts mechanical ventilation, intrusion
Property operation, antibacterials expose and the Acinetobacter bauamnnii such as serious underlying diseases infects the treatment time that can extend patient.Closely
Due to the increase of multiple antibiotic resistant strain over Nian, Acinetobacter bauamnnii has become the Nosocomial infection being most difficult to treatment and controlling.
Acinetobacter bauamnnii infects and easily occurs at intensive care unit (ICU) and burn ward.And owing to using antibiotic to make Bao Man not
Lever bacterium has drug resistance and the multi-drug resistant of height, detects it and contributes to protecting its harm to patient.
Domestic hospitals mainly uses antibacterial culturing to identify and drug sensitive test method identifies that Acinetobacter bauamnnii and resistance to carbon are blue or green at present
Mould alkenes Acinetobacter bauamnnii CRAB, this detection method not only complex operation, waste time and energy, sensitivity relatively low, poor repeatability,
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 Acinetobacter bauamnnii Carbapenem-resistant class antibiotic resistance gene, probe, method
And test kit, solve prior art to use antibacterial culturing identify and drug sensitive test method identifies Acinetobacter bauamnnii and the penicillium sp of resistance to carbon
Alkenes Acinetobacter bauamnnii CRAB, not only complex operation, waste time and energy, sensitivity relatively low, poor repeatability, it is impossible to understand resistance draw
The technical problem of the Molecular Biology Mechanism risen.
It is an object of the invention to be achieved through the following technical solutions:
A kind of primer detecting Acinetobacter bauamnnii Carbapenem-resistant class antibiotic resistance gene and probe, detect Bao Man not lever
The primer nucleotide sequences of bacterium Oxacillinase OXA51 gene, as shown in SEQIDNO:2～3, detects Acinetobacter bauamnnii benzene azoles
The nucleotide sequence of the Taqman probe of XiLin enzyme OXA51 gene is as shown in SEQIDNO:4；Detection Carbapenem-resistant class antibiotic
The primer nucleotide sequences of Oxacillinase OXA23 gene, as shown in SEQIDNO:6～7, detects Carbapenem-resistant class antibiotic
The nucleotide sequence of the Taqman probe of Oxacillinase OXA23 gene is as shown in SEQIDNO:8.
A kind of method detecting Acinetobacter bauamnnii Carbapenem-resistant class antibiotic resistance gene, including:
Prepared by sample nucleic acid, nucleic acid-templated to obtain；
It is respectively directed to Acinetobacter bauamnnii Oxacillinase OXA51 gene and Carbapenem-resistant class antibiotic Oxacillinase
OXA23 gene design specific primer and fluorescence labeling probe, wherein, the primer nucleotide sequences of detection Acinetobacter bauamnnii is such as
Shown in SEQIDNO:2～3, the nucleotide sequence of the Taqman probe of detection Acinetobacter bauamnnii is as shown in SEQIDNO:4；Detection
The primer nucleotide sequences of Carbapenem-resistant class antibiotic resistance gene, as shown in SEQIDNO:6～7, detects Carbapenem-resistant class antibiosis
The nucleotide sequence of the Taqman probe of plain gene is as shown in SEQIDNO:8；
Configuration enzyme reagent, wherein, described enzyme is by thermus aquaticus archaeal dna polymerase (Taq enzyme) and uracil-N-glycosylase
(UNG enzyme) mixing composition；
Take positive reference substance, negative controls is placed in centrifuge tube, is separately added into nucleic acid extraction liquid and fully mixes, and carries out
Heating and centrifugal treating, take supernatant and detect for fluorescent PCR；
By OXA51 gene PCR detection mixed liquor and the concussion mixing of enzyme reagent, it is centrifuged processing；By OXA23 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 the DNA profiling after extracting, feminine gender
Reference substance and positive reference substance supernatant add in the fluorescent PCR pipe of existing PCR detection mixed liquor, carry out fluorescent PCR amplification, instead
Answer the condition to be: 37 DEG C 2 minutes, 94 DEG C 2 minutes, circulate 1 time；93 DEG C 15 seconds, 56 DEG C 15 seconds, 60 DEG C 30 seconds, circulate 40 times,
And fluorescence signal is gathered at this；
Use the FAM passage on fluorescent PCR instrument to expand, determine detection by data collected by quantitative real time PCR Instrument
The genotype in site.
A kind of test kit detecting Acinetobacter bauamnnii Carbapenem-resistant class antibiotic resistance gene, including nucleic acid extraction liquid,
One primed probe mixed liquor, the second primed probe mixed liquor, PCR reaction enzymes system, negative controls, positive reference substance and separation are also
Collection these reagent bottle of intermediate package or the packing box of pipe, wherein, the first primed probe mixed liquor is by dideoxyribonucleotide triphosphate dN
(U) TP, the upstream and downstream primer of Acinetobacter bauamnnii Oxacillinase OXA51 gene and a fluorescence labeling probe composition, second
Primed probe mixed liquor is by dideoxyribonucleotide triphosphate dN (U) TP, Carbapenem-resistant class antibiotic Oxacillinase OXA23 base
The upstream and downstream primer of cause and a fluorescence labeling probe composition；
Wherein, the sequence of the upstream and downstream primer of OXA51 gene specific as shown in SEQIDNO:2～3, OXA51 base
The Taqman probe sequence of cause is as shown in SEQIDNO:4, and the sequence of the upstream and downstream primer of OXA51 gene specific is such as
Shown in SEQIDNO:6～7, the Taqman probe sequence of OXA23 gene is as shown in SEQIDNO:8, and 5 ' ends of two probes combine
Fluorescent reporter group FAM, 3 ' end conjunctions is had to have quenching group TAMRA.
Beneficial effects of the present invention: it is high that this test kit has accuracy rate, and clinical test results always meets with DNA sequencing result
Rate more than 99%；Pathogenic bacterium diphtheria corynebacterium common in high specificity, with sputum, hemophilus influenza, Bacillus proteus, unusual change
Shape bacillus, enterobacter cloacae, Candida albicans, Neisseria meningitidis, streptococcus pneumoniae, enterococcus faecalis, staphylococcus epidermidis, gold
Staphylococcus aureus, escherichia coli, Klebsiella pneumonia, Klebsiella oxytoca, Burkholderia cepacia, Aerugo vacation list
The equal no cross reaction of born of the same parents bacterium；Highly sensitive, the lowest detection determined according to plasmid copy number is limited to 103Copies/mL, according to bacterium
The lowest detection that culture method determines is limited to 103CFU/mL.The present invention can be used in external qualitative detection people's sputum sample Bao Man not
Lever bacterium Oxacillinase gene OXA51 and Carbapenem-resistant class antibiotic Oxacillinase gene OXA23, determines for clinic
CRAB infects 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 institute in embodiment
The accompanying drawing used is needed to be briefly described, it should be apparent that, the accompanying drawing in describing below is only some enforcements of the present invention
Example, for those of ordinary skill in the art, on the premise of not paying creative work, also can obtain according to these accompanying drawings
Obtain other accompanying drawing.
Fig. 1 is the quantitative fluorescent PCR curve chart of the Acinetobacter bauamnnii infection sample of the embodiment of the present invention four；
Fig. 2 is the fluorescent quantitation of the Carbapenem-resistant class Acinetobacter bauamnnii CRAB infection sample of the embodiment of the present invention four
PCR curve figure.
Detailed description of the invention
Understandable for enabling the above-mentioned purpose of the present invention, feature and advantage to become apparent from, real with concrete below in conjunction with the accompanying drawings
The present invention is further detailed explanation to execute mode.
Embodiment one
Embodiments provide a kind of primer detecting Acinetobacter bauamnnii Carbapenem-resistant class antibiotic resistance gene and
Probe, the primer nucleotide sequences of detection Acinetobacter bauamnnii Oxacillinase OXA51 gene, as shown in SEQIDNO:2～3, is examined
Survey the nucleotide sequence of Taqman probe of Acinetobacter bauamnnii Oxacillinase OXA51 gene as shown in SEQIDNO:4；Detection
The primer nucleotide sequences of Carbapenem-resistant class antibiotic Oxacillinase OXA23 gene, as shown in SEQIDNO:6～7, detects
The nucleotide sequence of the Taqman probe of Carbapenem-resistant class antibiotic Oxacillinase OXA23 gene is as shown in SEQIDNO:8；
Wherein, the sequence for the specific primer of Acinetobacter bauamnnii Oxacillinase OXA51 gene design is as follows:
Forward primer P-OXA51-F:5 '-TTTATCAAGATTTAGCTCGT-3 '
Downstream primer P-OXA51-R:5 '-AATTATCGACTTGGGTACCGAT-3 '
TaqMan fluorescence labeling probe sequence for Acinetobacter bauamnnii Oxacillinase OXA51 gene is as follows:
T-OXA51:5 '-FAM-ACACGCTTCACTTCATTAGACATGAG-TAMRA-3 '
Sequence for the specific primer of Carbapenem-resistant class antibiotic Oxacillinase OXA23 gene design is as follows:
Forward primer P-OXA23-F:5 '-TCTGCAGTCCCAGTCTATCAGG-3 '
Downstream primer P-OXA23-R:5 '-ACCTGCTGTCCAATTTCAGCA-3 '
For Carbapenem-resistant class antibiotic Oxacillinase OXA23 gene TaqMan fluorescence labeling probe sequence such as
Under:
T-OXA23:5 '-FAM-TTGCGCGACGTATCGGTCTTGATCTCAT-TAMRA-3 '
The fluorescent reporter group of nucleotide sequence 5 ' the end labelling of above two Taqman probe is FAM, 3 ' end labellings
Quenching group is TAMRA.
Embodiment two
The embodiment of the present invention provides again a kind of side detecting Acinetobacter bauamnnii Carbapenem-resistant class antibiotic resistance gene
Method, including:
Prepared by step 101, sample nucleic acid, nucleic acid-templated to obtain；
Step 102, it is respectively directed to Acinetobacter bauamnnii Oxacillinase OXA51 gene and Carbapenem-resistant class antibiotic benzene
Azoles XiLin enzyme OXA23 gene design specific primer and fluorescence labeling probe；
Wherein, the primer nucleotide sequences of detection Acinetobacter bauamnnii is as shown in SEQIDNO:2～3, and Bao Man is motionless in detection
The nucleotide sequence of the Taqman probe of bacillus is as shown in SEQIDNO:4；The primer of detection Carbapenem-resistant class antibiotic resistance gene
Nucleotide sequence, as shown in SEQIDNO:6～7, detects the nucleotides sequence of the Taqman probe of Carbapenem-resistant class antibiotic resistance gene
Row are as shown in SEQIDNO:8.
Step 103, configuration enzyme reagent, wherein, described enzyme is by thermus aquaticus archaeal dna polymerase (Taq enzyme) and uracil-N-
Glycosylase (UNG enzyme) mixing composition；
Step 104, take positive reference substance, negative controls is placed in centrifuge tube, is separately added into nucleic acid extraction liquid the most mixed
Even, and carry out heating and centrifugal treating, take supernatant and detect for fluorescent PCR；
Step 105, by detection mixed liquor and the enzyme reagent concussion mixing of OXA51 gene PCR, be centrifuged processing；By OXA23
Gene PCR detection mixed liquor and the concussion mixing of enzyme reagent, be centrifuged processing；
Wherein, PCR detection mixed liquor is by 3.2 μ L 2.5mmol/L dN (U) TP, 1.2 μ L 10 μm ol/L primers, 0.4 μ L
10 μm ol/L probes, 4 μ L 10 × PCR Buffer, 4 μ L25mmol/LMgCl2And 20.6 μ L sterilizing purified water composition.
Step 106, take enzyme-added mixing respectively after PCR detection mixed liquor be placed in fluorescent PCR pipe, take the DNA after extracting
Template, negative controls and positive reference substance supernatant add in the fluorescent PCR pipe of existing PCR detection mixed liquor, carry out fluorescence
PCR expand, reaction condition is: 37 DEG C 2 minutes, 94 DEG C 2 minutes, circulate 1 time；93 DEG C 15 seconds, 56 DEG C 15 seconds, 60 DEG C 30 seconds,
Circulate 40 times, and gather fluorescence signal at this；
FAM passage on step 107, employing fluorescent PCR instrument expands, by data collected by quantitative real time PCR Instrument
Determine the genotype of detection site.
Wherein, testing result is according to CTValue judges, instrument CTHurdle display Undet. (ABI StepOnePlus) or not
Display CTValue (Eppendorf Mastercycler ep realplex fluorescence quantitative PCR detection system), represents detection sample
Less than detection limit, it is reported as feminine gender；Measuring samples CT≤ 35, test results report is positive；Measuring samples CTDisplay is 35～40
Between, need replication, CTDisplay is still between 35～40, and amplification curve is S-type, then be judged as the positive；If amplification
It is a straight line, is then judged as feminine gender.
The technical method of the present invention is to use Taqman probe for real-time fluorescence PCR method, in sequence analysis Bao Man not lever
On the basis of bacterium specific gene OXA51, Carbapenem-resistant class antibiotic resistance gene OXA23 sequence, choose conservative fragments and set respectively
Count the specific primer for these 2 genes and specific fluorescence labeling probe.Probe 5 ' end flag F AM fluorescein is fluorescence
Reporter group (represents with R), and 3 ' end labelling TAMRA fluoresceins are fluorescent quenching group (representing with Q), and it can be inhaled within closely
Receive the fluorescence signal that 5 ' end fluorescent reporter group send.When PCR is reacted into annealing stage, probe purpose base first and in template
Because combining, primer is combined with genes of interest subsequently, and the fluorescence signal that now on probe, R group sends is examined by Q group absorptions, instrument
Do not detect fluorescence signal；When reaction proceeds to the extension stage, the exonuclease function of 5 ' → the 3 ' of Taq archaeal dna polymerase will
Probe is degraded.So R group on probe is free out, sent fluorescence signal not by Q group absorptions, detector can be examined
Measure fluorescence signal.Along with the carrying out of PCR reaction, PCR primer presents corresponding relation with the growth of fluorescence signal.Believed by fluorescence
Number growth curve, it is achieved the detection of genes of interest.
Embodiment three
The embodiment of the present invention additionally provides a kind of reagent detecting Acinetobacter bauamnnii Carbapenem-resistant class antibiotic resistance gene
Box, including nucleic acid extraction liquid, the first primed probe mixed liquor, the second primed probe mixed liquor, PCR reaction enzymes system, negative control
Product, positive reference substance and separation these reagent bottle of union intermediate package or the packing box of pipe, wherein, the first primed probe mixed liquor by
Dideoxyribonucleotide triphosphate dN (U) TP, the upstream and downstream primer of Acinetobacter bauamnnii Oxacillinase OXA51 gene and one
Fluorescence labeling probe forms, and the second primed probe mixed liquor is resisted by dideoxyribonucleotide triphosphate dN (U) TP, Carbapenem-resistant class
The upstream and downstream primer of raw element Oxacillinase OXA23 gene and a fluorescence labeling probe composition；
Wherein, the sequence of the upstream and downstream primer of OXA51 gene specific as shown in SEQIDNO:2～3, OXA51 base
The Taqman probe sequence of cause is as shown in SEQIDNO:4, and the sequence of the upstream and downstream primer of OXA51 gene specific is such as
Shown in SEQIDNO:6～7, the Taqman probe sequence of OXA23 gene is as shown in SEQIDNO:8, and 5 ' ends of two probes combine
Fluorescent reporter group FAM, 3 ' end conjunctions is had to have quenching group TAMRA.
Described nucleic acid extraction liquid is made up of 3% (M/V) Chelex-100,0.5% (V/V) Tris-HCl, 1M, pH9.0.
Described first primed probe mixed liquor and described second primed probe mixed liquor are respectively by 3.2 μ L 2.5mmol/L
DN (U) TP, 1.2 μ L 10 μm ol/L primers and 0.4 μ L 10 μm ol/L probe composition.
Described first primed probe mixed liquor and described second primed probe mixed liquor also include 4 μ L 10 × PCR
Buffer、4μL25mmol/LMgCl2And 20.6 μ L sterilizing purified water.
PCR reaction enzymes system is made up of 5U/ μ L Taq archaeal dna polymerase and 3: the 1 ratios mixing by volume of 2U/ μ L UNG enzyme.
The condition of PCR amplification is: 37 DEG C × 2min, 94 DEG C × 2min, circulate 1 time；93 DEG C × 15s, 56 DEG C × 15s, 60
DEG C × 30s, circulate 40 times；Reaction system is 40 μ L, selects FAM passage to expand.
Described positive reference substance is containing the engineering bacteria E-OXA51 containing OXA51 genetic fragment inactivated, containing OXA23
The bacterium solution of the engineering bacteria E-OXA23 of genetic fragment, bacteria concentration is 106CFU/mL；Negative controls is the large intestine containing inactivation
Bacillus solution, bacteria concentration is 106CFU/mL。
It is high that this test kit has accuracy rate, clinical test results and the total coincidence rate of DNA sequencing result more than 99%；Specificity
By force, common with sputum pathogenic bacterium diphtheria corynebacterium, hemophilus influenza, Bacillus proteus, proteus mirabilis, cloaca intestinal bar
Bacterium, Candida albicans, Neisseria meningitidis, streptococcus pneumoniae, enterococcus faecalis, staphylococcus epidermidis, staphylococcus aureus, big
The uncommon bacterium of intestinal angstrom, Klebsiella pneumonia, Klebsiella oxytoca, Burkholderia cepacia, Pseudomonas aeruginosa are all anti-without intersecting
Should；Highly sensitive, the lowest detection determined according to plasmid copy number is limited to 103Copies/mL, determines according to bacterium culture method
Low detection is limited to 103CFU/mL.The present invention can be used for Acinetobacter bauamnnii oxazacillin in external qualitative detection people's sputum sample
Enzyme gene OXA51 and Carbapenem-resistant class antibiotic Oxacillinase gene OXA23, determining that CRAB infects for clinic provides auxiliary
Diagnostic method, provides reference for clinician's medication.
Embodiment four
In actual application, first collect clinical sample, identify through antibacterial culturing and be defined as Bao Man with drug sensitive test method
Acinetobacter, CRAB infect, each 3 examples of negative sample, use this test kit to detect, statistical result coincidence rate.
1, prepared by sample nucleic acid
Sputum: take sputum and add the 4%NaOH of 4 times of volumes, shake up ambient temperatare and put more than 30min, to be liquefied completely, take
1.0mL sample, in 1.5mL centrifuge tube, adds 0.5mL 4%NaOH and digests 10min, and 12,000rpm are centrifuged 5min, abandon supernatant；Heavy
Shallow lake adds 1mL physiological saline solution and beats washing, and 12,000rpm are centrifuged 5min, abandon supernatant；With normal saline repeated washing once.
Remove most supernatant, precipitation is directly added into 50 μ L nucleic acid extraction liquid, fully mixes, 98 DEG C of heating 10min (error is less than 1min).
12,000rpm are centrifuged 5min, take supernatant 5 μ L and detect for fluorescent PCR.
2, reference substance prepares
Take positive reference substance, each 100 μ L of negative controls are placed in 1.5mL centrifuge tube that (after frozen reagent melts, concussion is mixed
Even 10s), it is separately added into nucleic acid extraction liquid 50 μ L and fully mixes, 98 DEG C of heating 10min (error is less than 1min).12,000rpm
Centrifugal 5min, takes supernatant 5 μ L and detects for fluorescent PCR.
3, enzyme preparation of reagents
Take n × 35 μ L Acinetobacter bauamnnii Oxacillinase gene 51 (OXA51) PCR detection mixed liquor and n × 0.4 μ L enzyme
(Taq+UNG), concussion mixing, 3000rpm is centrifuged the several seconds.Take n × 35 μ L Acinetobacter bauamnnii Carbapenem-resistant class antibiotic benzene
Azoles XiLin enzyme gene 23 (OXA23) PCR detection mixed liquor and n × 0.4 μ L enzyme (Taq+UNG), concussion mixing, 3000rpm is centrifuged
Several seconds.
4, sample-adding
Take the detection mixed liquor of the PCR after the 35 enzyme-added mixings of μ L respectively to be placed in fluorescent PCR pipe, take the specimen after extracting, the moon
Property reference substance and each 5 μ L of positive reference substance supernatant be separately added into existing PCR detection mixed liquor fluorescent PCR pipe in, build pipe
Lid, carries out fluorescent PCR amplification.
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 × 15s, 60 DEG C × 30s of 56 DEG C × 15s, circulates 40 times；Single-point fluoroscopic examination is at 60 DEG C, and reaction system is 40 μ L, fluorescence
Air conduct measurement 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 just surpasses with threshold line
Cross the peak of negative controls detection fluorescence curve.Instrument CTHurdle shows Undet (ABI StepOnePlus) or does not show CT
Value (Eppendorf Mastercycler ep realplex) fluorescence quantitative PCR detection system, represents that detection sample is less than inspection
Survey limit, be reported as feminine gender；Measuring samples CT≤ 35, test results report is positive；Measuring samples CTShow between 35～40,
Need replication, CTDisplay is still between 35～40, and amplification curve is S-type, then be judged as the positive；If amplification is always
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, fluorescent PCR testing result such as Fig. 1
Shown in Fig. 2, Fig. 1 is the quantitative fluorescent PCR curve chart that Acinetobacter bauamnnii infects sample, it is seen that along with the carrying out of PCR reaction,
PCR primer corresponding for OXA51 presents corresponding relation with the growth of fluorescence signal, can determine whether that OXA51 is positive, and OXA23 is negative.
By fluorescence signal growth curve；Fig. 2 is the quantitative fluorescent PCR song that Carbapenem-resistant class Acinetobacter bauamnnii CRAB infects sample
Line chart, can determine whether that OXA51 and OXA23 is the positive.
Being described in detail the present invention above, specific case used herein is to the principle of the present invention and embodiment party
Formula is set forth, and the explanation of above example is only intended to help to understand method and the core concept thereof of the present invention；Meanwhile, right
In one of ordinary skill in the art, according to the thought of the present invention, the most all can change
Part, in sum, this specification content should not be construed as limitation of the present invention.
Claims (10)
1. the primer detecting Acinetobacter bauamnnii Carbapenem-resistant class antibiotic resistance gene and probe, it is characterised in that detection
The primer nucleotide sequences of Acinetobacter bauamnnii Oxacillinase OXA51 gene is as shown in SEQIDNO:2～3, and detection Bao Man is not
The nucleotide sequence of the Taqman probe of lever bacterium Oxacillinase OXA51 gene is as shown in SEQIDNO:4；Detect the penicillium sp of resistance to carbon
The primer nucleotide sequences of carbapenem antibiotic Oxacillinase OXA23 gene, as shown in SEQIDNO:6～7, detects the penicillium sp of resistance to carbon
The nucleotide sequence of the Taqman probe of carbapenem antibiotic Oxacillinase OXA23 gene is as shown in SEQIDNO:8.
The primer of detection Acinetobacter bauamnnii Carbapenem-resistant class antibiotic resistance gene the most according to claim 1 and probe,
It is characterized in that, the fluorescent reporter group of nucleotide sequence 5 ' the end labelling of two kinds of Taqman probes is FAM, quenching of 3 ' end labellings
The group that goes out is TAMRA.
3. the method detecting Acinetobacter bauamnnii Carbapenem-resistant class antibiotic resistance gene, it is characterised in that including:
Prepared by sample nucleic acid, nucleic acid-templated to obtain；
It is respectively directed to Acinetobacter bauamnnii Oxacillinase OXA51 gene and Carbapenem-resistant class antibiotic Oxacillinase
OXA23 gene design specific primer and fluorescence labeling probe, wherein, the primer nucleotide sequences of detection Acinetobacter bauamnnii is such as
Shown in SEQIDNO:2～3, the nucleotide sequence of the Taqman probe of detection Acinetobacter bauamnnii is as shown in SEQIDNO:4；Detection
The primer nucleotide sequences of Carbapenem-resistant class antibiotic resistance gene, as shown in SEQIDNO:6～7, detects Carbapenem-resistant class antibiosis
The nucleotide sequence of the Taqman probe of plain gene is as shown in SEQIDNO:8；
Configuration enzyme reagent, wherein, described enzyme is by thermus aquaticus archaeal dna polymerase (Taq enzyme) and uracil-N-glycosylase (UNG
Enzyme) mixing composition；
Take positive reference substance, negative controls is placed in centrifuge tube, is separately added into nucleic acid extraction liquid and fully mixes, and heats
And centrifugal treating, take supernatant and detect for fluorescent PCR；
By OXA51 gene PCR detection mixed liquor and the concussion mixing of enzyme reagent, it is centrifuged processing；OXA23 gene PCR is detected
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 the DNA profiling after extracting, negative control
Product and positive reference substance supernatant add in the fluorescent PCR pipe of existing PCR detection mixed liquor, carry out fluorescent PCR amplification, react bar
Part is: 37 DEG C 2 minutes, 94 DEG C 2 minutes, circulate 1 time；93 DEG C 15 seconds, 56 DEG C 15 seconds, 60 DEG C 30 seconds, circulate 40 times, and adopt at this
Collection fluorescence signal；
Use the FAM passage on fluorescent PCR instrument to expand, determine detection site by data collected by quantitative real time PCR Instrument
Genotype.
4. the test kit detecting Acinetobacter bauamnnii Carbapenem-resistant class antibiotic resistance gene, it is characterised in that include nucleic acid
Extract, the first primed probe mixed liquor, the second primed probe mixed liquor, PCR reaction enzymes system, negative controls, positive control
Product and separation these reagent bottle of union intermediate package or the packing box of pipe, wherein, the first primed probe mixed liquor is by deoxyribose core
Guanosine triphosphate dN (U) TP, the upstream and downstream primer of Acinetobacter bauamnnii Oxacillinase OXA51 gene and a fluorescent labeling are visited
Pin forms, and the second primed probe mixed liquor is by dideoxyribonucleotide triphosphate dN (U) TP, Carbapenem-resistant class antibiotic benzene azoles west
The upstream and downstream primer of woods enzyme OXA23 gene and a fluorescence labeling probe composition；
Wherein, the sequence of the upstream and downstream primer of OXA51 gene specific as shown in SEQIDNO:2～3, OXA51 gene
Taqman probe sequence as shown in SEQIDNO:4, the sequence such as SEQIDNO of the upstream and downstream primer of OXA51 gene specific:
Shown in 6～7, the Taqman probe sequence of OXA23 gene is as shown in SEQIDNO:8, and 5 ' ends of two probes are combined with fluorescence report
Accusing group FAM, 3 ' end conjunctions have quenching group TAMRA.
The test kit of detection Acinetobacter bauamnnii Carbapenem-resistant class antibiotic resistance gene the most according to claim 4, it is special
Levying and be, described nucleic acid extraction liquid is made up of 3% (M/V) Chelex-100,0.5% (V/V) Tris-HCl, 1M, pH9.0.
The test kit of detection Acinetobacter bauamnnii Carbapenem-resistant class antibiotic resistance gene the most according to claim 4, it is special
Levying and be, described first primed probe mixed liquor and described second primed probe mixed liquor are respectively by 3.2 μ L 2.5mmol/L dN
(U) TP, 1.2 μ L 10 μm ol/L primers and 0.4 μ L 10 μm ol/L probe composition.
The test kit of detection Acinetobacter bauamnnii Carbapenem-resistant class antibiotic resistance gene the most according to claim 4, it is special
Levy and be, described first primed probe mixed liquor and described second primed probe mixed liquor also include 4 μ L 10 × PCR
Buffer、4μL25mmol/LMgCl2And 20.6 μ L sterilizing purified water.
The test kit of detection Acinetobacter bauamnnii Carbapenem-resistant class antibiotic resistance gene the most according to claim 4, it is special
Levying and be, PCR reaction enzymes system is made up of 5U/ μ L Taq archaeal dna polymerase and 3: the 1 ratios mixing by volume of 2U/ μ L UNG enzyme.
The test kit of detection Acinetobacter bauamnnii Carbapenem-resistant class antibiotic resistance gene the most according to claim 4, it is special
Levying and be, the condition of PCR amplification is: 37 DEG C × 2min, 94 DEG C × 2min, circulate 1 time；93 DEG C × 15s, 56 DEG C × 15s, 60 DEG C
× 30s, circulates 40 times；Reaction system is 40 μ L, selects FAM passage to expand.
The test kit of detection Acinetobacter bauamnnii Carbapenem-resistant class antibiotic resistance gene the most according to claim 4, it is special
Levying and be, described positive reference substance is containing the engineering bacteria E-OXA51 containing OXA51 genetic fragment inactivated, containing OXA23 base
Because of the bacterium solution of the engineering bacteria E-OXA23 of fragment, bacteria concentration is 106CFU/mL；Negative controls is the large intestine bar containing inactivation
Bacterium solution, bacteria concentration is 106CFU/mL。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109266658A (en) * | 2018-10-17 | 2019-01-25 | 昆明理工大学 | The specific gene and its primer of a kind of Acinetobacter bauamnnii and application |
| CN109628620A (en) * | 2019-01-22 | 2019-04-16 | 南方医科大学南方医院 | Primer, method and the kit of the detection of complete sequence fluorescent PCR OXA-23 family and OXA-51 family gene type |
| CN114703257A (en) * | 2022-03-14 | 2022-07-05 | 连云港市第二人民医院(连云港市临床肿瘤研究所) | Primer-probe combination for carbapenem-resistant acinetobacter baumannii RPA-LFS detection method and application thereof |
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| CN105624292A (en) * | 2016-01-22 | 2016-06-01 | 江苏大学 | Baumanii detection kit and detection method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105624292A (en) * | 2016-01-22 | 2016-06-01 | 江苏大学 | Baumanii detection kit and detection method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109266658A (en) * | 2018-10-17 | 2019-01-25 | 昆明理工大学 | The specific gene and its primer of a kind of Acinetobacter bauamnnii and application |
| CN109628620A (en) * | 2019-01-22 | 2019-04-16 | 南方医科大学南方医院 | Primer, method and the kit of the detection of complete sequence fluorescent PCR OXA-23 family and OXA-51 family gene type |
| CN114703257A (en) * | 2022-03-14 | 2022-07-05 | 连云港市第二人民医院(连云港市临床肿瘤研究所) | Primer-probe combination for carbapenem-resistant acinetobacter baumannii RPA-LFS detection method and application thereof |
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