CN107523620A - PCR detection kit and its application comprising production NDM drug-fast bacterias - Google Patents
PCR detection kit and its application comprising production NDM drug-fast bacterias Download PDFInfo
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- CN107523620A CN107523620A CN201710695728.4A CN201710695728A CN107523620A CN 107523620 A CN107523620 A CN 107523620A CN 201710695728 A CN201710695728 A CN 201710695728A CN 107523620 A CN107523620 A CN 107523620A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
Abstract
The invention discloses include blaNDMThe PCR detection kit of the drug-fast bacteria of gene and its application, belong to technical field of biological.The detection primer of the present invention and the nucleotide sequence such as SEQ ID NO of probe：Shown in 13；PCR detection kit, including fluorescent PCR detection mixed liquor, described detect in mixed liquor contain foregoing primer and probe.Gone out using the kit and primed probe energy quick detection of the present invention including blaNDM1To blaNDM16Totally 16 kinds of blaNDMThe microorganism of high drug resistant gene, has the advantages that easy to operate, high sensitivity, specificity is good, the degree of accuracy is high, can find and make a definite diagnosis in time doubtful case, improve the level monitoring to all kinds of infectious diseases.
Description
Technical field
The present invention relates to include blaNDMThe PCR detection kit of the drug-fast bacteria of gene and its application, and in particular to Yi Zhongneng
Enough known bla all at present of quick detection simultaneouslyNDMGenotype, including blaNDM1To blaNDM16Totally 16 kinds of blaNDMHigh resistance base
PCR primer, probe and the kit of the microorganism of cause, belong to technical field of biological.
Background technology
NDM is New Delhi metallo-β-lactamase (New Delhi metallo- β-lactamase) english abbreviation, by
The famous medical journal of Britain《The Infectious Diseases》Report first, the base of a fruit nurse Wal of card Defo university of Britain
Assorted that the superbacteria is named as into " New Delhi metalloproteinases ", it is a kind of special that the bacterium due to carrying the gene can produce
Beta-lactamase, and active site is that metal ion occurs so gaining the name in India capital New Delhi first again：NDM.Produce NDM
Bacterium typically based on Escherichia coli and Klebsiella pneumoniae, these bacteriums can both cause inside-hospital infection, Ye You communities
Infection, including urinary tract infections, bloodstream infection, pneumonia, catheter-related Infections: Clinical Study, wound infection etc..So far, there is blaNDM1Arrive
blaNDM16The microorganism of totally 16 kinds of high drug resistant genes is found.Nearly 200 kinds of antibiotic of whole world listing are thin to this kind of novel super
Bacterium is almost felt simply helpless.
Up to the present, it is clinical to producing blaNDMThe understanding of drug-fast bacteria to sample still mainly by carrying out selective training
Support after counting clump count, then contrasted by traditional drug sensitive test and PCR amplifications and with standard library.With molecular biology
Development and Protocols in Molecular Biology are in the application of microorganism, particularly Fluorescence PCR assay appearance, because it has time saving province
Power, sensitiveness height, high specificity, it is simple to operate quick the features such as, and the time restriction that is not put to the test, and be widely used in facing
Bed diagnosis, disease research and pathogen detection etc..However, PCR antidiastoles bla is used so farNDMThe method of drug-fast bacteria
Still it is extremely complex, not only take, be laborious, and also the degree of accuracy is unlike traditional drug sensitive test more preferably, especially since
blaNDMDrug resistant gene species is various, has there is 16 kinds of resistance bla so farNDMIt is found, existing blaNDMDrug-fast bacteria detection examination
Agent box is all to be merely able to detect single drug resistant gene, such as is merely able to detect blaNDM1Drug resistant gene, cause definitely to reflect one by one
These other different blaNDMGenotype has the defects of efficiency is low, cost is high.
Therefore, it is extremely urgent to find a kind of method that can effectively differentiate " superbacteria " that produces various NDM.
The content of the invention
Differentiate it is an object of the invention to provide one kind and diagnose various production blaNDMThe fluorescence PCR detection reagent of drug-fast bacteria
Box and preparation method thereof, detection method, bla is produced available for detecting and diagnosingNDMThe infection of drug-fast bacteria, for clinic to suspicious sense
Contaminate patient and carry out aetology antidiastole.PCR primer, probe and the kit of the present invention, which can be detected easily, to be currently known
All 16 kind blaNDMDrug resistant gene, be not only simple, quickly, it is accurate, sensitive, and at present both at home and abroad there is not yet similar
Technology is reported.
First purpose of the present invention is to provide one kind and is directed to various production blaNDMThe detection primer of gene drug-fast bacteria, it is described
Primer includes sequence such as SEQ ID NO：1 and SEQ ID NO：2 two nucleotide sequences.
In one embodiment, it is described for production blaNDMThe composition of the detection primer of gene drug-fast bacteria is as follows：
Sense primer：5’-GACCGCCCAGATCCTCAA-3’(SEQ ID NO：1)
Anti-sense primer：5’-ATTGGCATAAGTCGCAATCC-3’(SEQ ID NO：2).
Second object of the present invention is to provide one kind and includes blaNDMThe detection kit of gene drug-fast bacteria, the detection
Kit includes the detection primer of the present invention.
In one embodiment, in the detection kit, in addition to sequence such as SEQ ID NO：Probe shown in 3.
In one embodiment, one end of the probe is marked with fluorescent reporter group, and the probe other end is marked with glimmering
Optical quenching group.
In one embodiment, 5 ' ends of the probe include fluorescent reporter group, and 3 ' ends include fluorescent quenching group.
In one embodiment, the probe is 5 '-fluorescent reporter group-AGATCAACCTGCCGGTCGCG- fluorescence
(the SEQ ID NO of quenching group -3 '：3).
In one embodiment, the fluorescent reporter group is FAM, VIC, ROX, Cy5, JOE or Quasar705
Any one in fluorophor.
In one embodiment, the fluorescent quenching group is BHQ or ECLIPSE series.
In one embodiment, regular-PCR or the PCR of fluorescent PCR amplification are also included in the detection kit
Buffer and H2O。
In one embodiment, the dication buffer solution containing optimization in the PCR buffer, dNTPS, PCR increase
Strong agent, PCR stabilizers.
In one embodiment, also include in the detection kit in PCR enzymes, negative controls and positive reference substance
At least one.
In one embodiment, the enzyme is archaeal dna polymerase, such as taq archaeal dna polymerases.
In one embodiment, the positive reference substance compares to inactivate or being attenuated the DNA of positive strain or corresponding
Plasmid.
In one embodiment, the negative controls are DEPC-H2O。
In one embodiment, the detection kit is fluorescence detection reagent kit, including the primer of the present invention, spy
Pin, qPCR master mix, taq archaeal dna polymerases.Preferably, in addition to water or TE.
Third object of the present invention is to provide described detection primer and includes bla in preparationNDMThe drug-fast bacteria detection of gene
Application in reagent.
Fourth object of the present invention is to include blaNDMThe detection method of gene drug-fast bacteria, it is to utilize the described of the present invention
Primer or detection kit are detected.
In one embodiment, the drug-fast bacteria for it is following any one or more：Acinetobacter baumannii
Acinetobacterbaumannii, clostridium perfringen Enterobacteraerogenes, enterobacter cloacae
Enterobactercloacae, Escherichia coli Escherichiacoli, klebsiella oxytoca
KlebsiellaOxytoca, abel's bacillus Klebsiellaozaenae, Friedlander's bacillus
Klebsiellapneumoniae, the root that rubs (family name) bacterium Morganellamorganii, proteus mirabilis
Proteusmirabilis, providencia rettgeri Providenciarettgeri, pseudomonas aeruginosa
Pseudomonasaeruginosa, serratia marcescens Serratiamarcescens, candida albicans Candidaspecies.
In one embodiment, the detection method, detected using fluorescent PCR.
In one embodiment, fluorescent PCR amplification system includes (20 μ l systems)：8~12 μ l qPCR master
Mix (2X), certain density primer and probe, 0.1~1 μ l taqDNA polymerases, certain density product to be tested, remaining be
Water.
In one embodiment, fluorescent PCR amplification system is：10 μ l qPCR master mix (2X), 1 μ l's draws
Physical prospecting pin mixed liquor, 0.3 μ l taqDNA polymerases, the μ l of DNA profiling 5 to be checked, sterile deionized water is added to supply volume to 20 μ
l。
In one embodiment, fluorescent PCR amplification system preparation method is as follows：Take 10 μ l qPCR master mix
(2X), with 1 μ l NDM primed probes mixed liquor and 3.7 μ l H2O is mixed, and is added 0.3 μ l taqDNA polymerases, is taken 15
The above-mentioned mixed liquors of μ l add 5 μ l DNA samples to be measured, obtain the PCR reaction solutions that reaction total system is 20 μ l.
In one embodiment, the PCR enzymes are by 0.3 μ l Taq enzymes (5U/ μ l), lacking uracil-N- glycosylase systems
Into.
In one embodiment, described NDM primed probes mixed liquor by isometric 10pmol/ μ l NDM upstreams
Primer, 10pmol/ μ l NDM anti-sense primers and 5pmol/ μ l probes composition.
In one embodiment, the product to be tested is DNA profiling solution, positive reference substance or the negative controls of sample
(DEPC-H2O)。
In one embodiment, in the positive reference substance, control plasmid can be packed individually can also be hybrid packed.
The present invention is for differentiating and diagnosing production blaNDMMethod used in the kit of the fluorescent PCR detection of gene drug-fast bacteria,
Using Taqman quantitative fluorescent PCR principles, respectively for blaNDMDrug resistant gene designs specific primer, specific amplification
Nucleotide sequence, while separately design Taqman probes, and mark different fluorescent reporter groups, and positioned at upstream and downstream primer it
Between.Probe 5 ' holds mark fluorescent reporter group, 3 ' end mark non-fluorescence quenching groups.When probe is complete, reporter group
The fluorescent energy launched is quenched group absorptions, and instrument can't detect signal.With PCR progress, Taq enzyme is in chain extension
During run into the probe combined with template, its 5 ' → 3 ' exonuclease activity will cut off probe, and reporter group is remote
Quenching group, its energy can not be absorbed, that is, produce fluorescence signal.Therefore, the fluorescent quantitative PCR technique tool that the present invention uses
There is the features such as detection in real time, quantitative and high flux detect, and easy to operate, high sensitivity, the advantages that specificity is good.
Using the present invention for production blaNDMDuring the fluorescence PCR detection reagent kit detection of gene drug-fast bacteria, its application method is such as
Under：
1) DNA of sample to be tested is extracted, obtains DNA profiling solution；
2) detection mixed solution prepared by the present invention is added, prepares reaction system；
3) PCR is expanded and detected.
The present invention is for differentiating and diagnosing production blaNDMMethod used in the kit of the fluorescent PCR detection of drug-fast bacteria, is used
Be Taqman quantitative fluorescent PCR principles, respectively for blaNDMDrug resistant gene designs specific primer, specific amplification nucleic acid
Sequence, while Taqman probes are separately designed, and different fluorescent reporter groups is marked, and between upstream and downstream primer.Visit
Pin 5 ' holds mark fluorescent reporter group, 3 ' end mark non-fluorescence quenching groups.When probe is complete, reporter group is sent out
The fluorescent energy penetrated is quenched group absorptions, and instrument can't detect signal.With PCR progress, Taq enzyme is in chain extension process
In run into the probe combined with template, its 5 ' → 3 ' exonuclease activity will cut off probe, and reporter group is away from being quenched
Group, its energy can not be absorbed, that is, produce fluorescence signal.Therefore, the fluorescent quantitative PCR technique that the present invention uses has real
When detection, quantitative and high flux the features such as detecting, and easy to operate, high sensitivity, the advantages that specificity is good.
Using the present invention for production blaNDMDuring the fluorescence PCR detection reagent kit detection of drug-fast bacteria, its application method is as follows：
1) DNA of sample to be tested is extracted, obtains DNA profiling solution；
2) detection mixed solution prepared by the present invention is added, prepares reaction system；
3) PCR is expanded and detected.
The sample to be tested of PCR of the present invention detection understands bacterial strain, Disease Control and Prevention Center (CDC), our company's experiment from ATCC is qualitative
Room includes, but are not limited to the bacterial strain of the acquisitions such as excrement, urine, phlegm, blood, tissue fluid, secretion from the patient's sample obtained.
The nucleic acid extraction of sample is existing DNA of bacteria extractive technique, specifically can be different according to sampling mode, by each nucleic acid extraction specification
Operation is carried out.
Detecting the method being loaded is：Each reaction takes 10 μ l qPCR master mix (2X) and 0.3 μ lTaq enzymes (Taq
Enzyme 5U/ μ l), mix, add 1 μ l NDM primed probes mixed liquor and 3.7 μ lddH2O, vibration mix several seconds, 3000rpm
Centrifugation 5 seconds, obtain mixed liquor.The foregoing μ l of mixed liquor 15 are taken to be placed in PCR pipe, it is then that 5 μ l DNA profiling solution, the positive is right
Added according to product or negative controls in PCR reaction tubes, cover PCR reaction lids, carry out pcr amplification reaction immediately.
The pcr amplification reaction condition that the present invention recommends：
ABI7500fastdx instruments：95 DEG C of 2min, (95 DEG C of 15s, 60 DEG C of 30s) 40 circulations
Rich day 9600plus instrument：94 DEG C of 2min, (94 DEG C of 10s, 60 DEG C of 40s) 40 circulations.
The PCR thresholds of reaction are set：Threshold value setting principle is with threshold line just above negative controls detection fluorescence curve
Peak.
Fluorescent PCR quality control of the present invention：Negative controls testing result shows Undetermined on Ct columns
Or N/A (CFX96) or No Ct (SLAN) (ABI7500)；Positive reference substance testing result should be：VIC channel C t values are ≤35
(other same VIC of fluorescence labeling result basis for estimation), otherwise experiment are considered as invalid.Specifically it is shown in Table 1.
The criterion of the result of the test of table 1
If sample Ct values to be checked need replication between 38~40, the Ct values obtained such as replication are still 38~40
Between, then it is judged to be less than test limit, is reported as feminine gender.
The present invention finally discloses foregoing for production blaNDMThe fluorescent PCR detection primer and probe of drug-fast bacteria, foregoing production
Application of the fluorescence PCR detection reagent kit of blaNDM drug-fast bacterias in preparation and/or detection reagent.
Beneficial effects of the present invention：
The kit of the present invention and its application greatly shorten detection time, easy to operate.Specific primer and with probe
Design ensure that the highly conserved and specific of primer and probe, avoid between two pairs of primer and probes without complementary pairing or
Intersect the situation of amplification.
Brief description of the drawings
The amplification curve diagram of Fig. 1 embodiments detection sensitivity of 8 first days；
The amplification curve diagram of 8 second days detection sensitivities of Fig. 2 embodiments；
The amplification curve diagram of the 3rd day detection sensitivity of Fig. 3 embodiments 8；
The amplification curve diagram of batch interior repeatability of Fig. 4 embodiments 10；
Fig. 5 embodiments 10 batch between the repeatability amplification curve diagram of first day；
Fig. 6 embodiments 10 batch between the repeatability amplification curve diagram of second day；
Fig. 7 embodiments 10 batch between the repeatability amplification curve diagram of the 3rd day；
The amplification curve diagram of the linearity of Fig. 8 embodiments 11；
Bla in the detection gram negative strain of Fig. 9 embodiments 11NDMThe standard curve of the linearity of gene；
The specific amplification curve diagram of Figure 10 embodiments 12；
Influence of the tri- kinds of fluorescence labelings of Figure 11 embodiments 13Cy3, JOE, ROX to PCR.
Specific embodiment
The present invention is expanded on further with reference to specific embodiment, it should be appreciated that following examples are merely to illustrate the present invention
Rather than limit the scope of the invention.
Embodiment 1
The design and synthesis 1 of nucleic acid detection kit primed probe
In 5 ' flag F AM fluorophors of probe, 3 ' mark BHQ1 fluorophors of probe.
Synthesis is for producing the primer, probe that the PCR of various NDM drug-fast bacterias is detected：
Sense primer：5’-GACCGCCCAGATCCTCAA-3’(SEQ ID NO：1)
Anti-sense primer：5’-ATTGGCATAAGTCGCAATCC-3’(SEQ ID NO：2)
Probe：5’-FAM-AGATCAACCTGCCGGTCGCG-BHQ1-3’(SEQ ID NO：3)
Embodiment 2
The design and synthesis 2 of nucleic acid detection kit primed probe
VIC fluorophors, 3 ' mark BHQ1 fluorophors of probe are marked the 5 ' of probe.
Synthesis is for producing the primer, probe that the PCR of various NDM drug-fast bacterias is detected：
Sense primer：5’-GACCGCCCAGATCCTCAA-3’(SEQ ID NO：1)
Anti-sense primer：5’-ATTGGCATAAGTCGCAATCC-3’(SEQ ID NO：2)
Probe：5’-VIC-AGATCAACCTGCCGGTCGCG-BHQ1-3’(SEQ ID NO：3)
Embodiment 3
The design and synthesis 3 of nucleic acid detection kit primed probe
ROX fluorophors, 3 ' mark BHQ2 fluorophors of probe are marked the 5 ' of probe.
Synthesis is for producing the primer, probe that the PCR of various NDM drug-fast bacterias is detected：
Sense primer：5’-GACCGCCCAGATCCTCAA-3’(SEQ ID NO：1)
Anti-sense primer：5’-ATTGGCATAAGTCGCAATCC-3’(SEQ ID NO：2)
Probe：5’-ROX-AGATCAACCTGCCGGTCGCG–BHQ2-3’(SEQ ID NO：3)
Embodiment 4
The design and synthesis 4 of nucleic acid detection kit primed probe
Cy5 fluorophors, 3 ' mark BHQ2 fluorophors of probe are marked the 5 ' of probe.
Synthesis is for producing the primer, probe that the PCR of various NDM drug-fast bacterias is detected：
Sense primer：5’-GACCGCCCAGATCCTCAA-3’(SEQ ID NO：1)
Anti-sense primer：5’-ATTGGCATAAGTCGCAATCC-3’(SEQ ID NO：2)
Probe：5’-Cy5-AGATCAACCTGCCGGTCGCG–BHQ2-3’(SEQ ID NO：3)
Embodiment 5
The design and synthesis 5 of nucleic acid detection kit primed probe
Quasar705 fluorophors, 3 ' mark BHQ3 fluorophors of probe are marked the 5 ' of probe.
Synthesis is for producing the primer, probe that the PCR of various NDM drug-fast bacterias is detected：
Sense primer：5’-GACCGCCCAGATCCTCAA-3’(SEQ ID NO：1)
Anti-sense primer：5’-ATTGGCATAAGTCGCAATCC-3’(SEQ ID NO：2)
Probe：5’-Quasar705-AGATCAACCTGCCGGTCGCG–BHQ3-3’(SEQ ID NO：3)
Embodiment 6
The design and synthesis 6 of nucleic acid detection kit primed probe
JOE fluorophors, 3 ' mark DAB fluorophors of probe are marked the 5 ' of probe.
Synthesis is for producing the primer, probe that the PCR of various NDM drug-fast bacterias is detected：
Sense primer：5’-GACCGCCCAGATCCTCAA-3’(SEQ ID NO：1)
Anti-sense primer：5’-ATTGGCATAAGTCGCAATCC-3’(SEQ ID NO：2)
Probe：5’-JOE-AGATCAACCTGCCGGTCGCG–DAB-3’(SEQ ID NO：3)
Embodiment 7
The preparation and use of nucleic acid detection kit
1st, nucleic acid fluorescent PCR detects the preparation of mixed liquor：
Preparation method is as follows：10pmol/ μ l sense primer, anti-sense primer and 5pmol/ μ l probe are prepared, then is taken
Isometric above-mentioned primed probe mixes and a pipe primed probe mixed liquor is made.QPCR master mix (2X) 10 μ l are taken, with 1
μ l primed probe mixed liquor, 0.3 μ l taq archaeal dna polymerases, the μ l of process water 3.7 mixing, obtain the core that volume is 15 μ l
Sour fluorescent PCR detects mixed liquor.Prepare the μ l nucleic acid fluorescents PCR of n × 15 detection mixed liquors (n is reaction tube number).
2nd, it is loaded
The above-mentioned μ l of mixed liquor 15 are taken in every PCR pipe, are then separately added into testing sample DNA systems again into every PCR pipe
The standby μ l of liquid 5, cover PCR pipe lid, carry out pcr amplification reaction immediately.
3rd, PCR is expanded
Reaction tube is placed on quantitative fluorescence PCR instrument, recommends loop parameter to set：
ABI7500fastdx instruments：95 DEG C of 2min, (95 DEG C of 15s, 60 DEG C of 30s) 40 circulations
Rich day 9600plus instrument：94 DEG C of 2min, (94 DEG C of 10s, 60 DEG C of 40s) 40 circulations.
Embodiment 8
The sensitivity analysis of nucleic acid detection kit
This PCR detection sensitivity experimental programs are determined according to the code of U.S. clinical Laboratory Standard research institute.
(1) pipe 10 is prepared6The bacteria suspension of cfu/ml concentration：blaNDMGene uses bacterial strain CDC88.
(2) above-mentioned bacteria suspension is diluted to the dilution of 8 concentration gradients step by step, each diluted concentration see the table below 2 respectively.Will
The bacterium mixed liquor liquid of diluted concentration about 100CFU/ml concentration carries out count of bacteria according to standard microbiology method.
(3) the bacterium mixed liquor DNA of above-mentioned 8 concentration gradient dilutions is extracted, it is standby.
(4) sensitivity of the real-time PCR detections of real-time PCR analysis sheet.Three repetitions of each dilution factor, are repeated three days, therefore often
9 data are obtained in individual dilution factor one, and using SPSS statistical softwares version 2 2.0 (95% positive rate is horizontal) statistics, this is real-time
PCR detects blaNDMThe sensitivity of gene, 2 are as a result see the table below, the amplification curve of three days detection sensitivities is shown in accompanying drawing 1,2 and 3.It is logical
Cross SPSS software analysis, horizontal upper this real-time fluorescence quantitative PCR detection bla of 95% positive rateNDMThe sensitivity of gene is
829CFU/mL, in fact, this kit is up to 2/3rds to the recall rate of 60cfu/ml extremely low concentration bacteria suspension,
I.e. 67%.
The PCR of table 2 is to blaNDMSensitivity analysis result
Embodiment 9
The Accuracy Analysis of nucleic acid detection kit
This PCR detection sensitivity experimental programs are determined according to the code of U.S. clinical Laboratory Standard research institute.It is logical
Cross and detect following kind of sample, the accuracy of this real-time PCR detection is assessed, the source of bacterial strain：
The clinical samples (n=200) that reference strain is collected from ATCC, CDC (n=88) or this laboratory, and it is single
Or multiple outer addition target genes sample ((n=13), totally 301 plants of samples, including 283 plants of gram negative strains and
5 kinds of candida albicans, it is as follows：
Acinetobacter baumannii Acinetobacterbaumannii (n=17), Freund (family name) citrobacter
Citrobacterfreundii (n=1), clostridium perfringen Enterobacteraerogenes (n=2), enterobacter cloacae
Enterobactercloacae (n=12), Escherichia coli Escherichiacoli (n=165), white (family name) bacillus of Cray of hastening parturition
KlebsiellaOxytoca (n=1), abel's bacillus Klebsiellaozaenae (n=2), e coil k 1 pneumonia
Bacterium Klebsiellapneumoniae (n=55), the root that rubs (family name) bacterium Morganellamorganii (n=1), unusual deformed rod
Bacterium Proteusmirabilis (n=1), providencia rettgeri Providenciarettgeri (n=1), pseudomonas aeruginosa
Pseudomonasaeruginosa (n=21), Bacterium prodigiosum Serratiamarcescens (n=4) and candida albicans
Candidaspecies (n=5).
Whether the bacterial strain obtained from ATCC or CDC contains blaNDMGene, all it is discussed in detail in bacterial strain specification；
And for clinical strains, using Illumina MiSeq or HiSeq to bacterial strain genome sequencing, then in whole genome sequence
On the basis of, using gene, from the beginning package technique determines whether to contain bla in clinical strainsNDMGene, in 301 parts of samples, its
The bla of middle determinationNDMTotally 39 parts of positive sample.
The DNA of this 301 parts of samples is extracted, this 301 parts of sample bla are detected using this real-time fluorescence quantitative PCRNDMGene.
3 are the results are shown in Table, wherein 39 parts of positives, this PCR is to blaNDMThe sensitiveness of gene is 100%, and specificity is 100%, overall essence
Spend for 100%.
3 PCR of table are to blaNDMThe testing result of gene accuracy
Embodiment 10
The repeatability analysis of nucleic acid detection kit
1st, repeatability in criticizing
Batch of this PCR experiment is detected using U.S. clinical and the method for qualitative analysis of laboratory standards institute (CLSI) as guide
Interior repeatability.Preparation contains blaNDMMrna concentration is 108Cfu/ml bacteria suspensions, prepare high, normal, basic three dilution factors (dilution factor point
Wei 107、105、103Cfu/ml bacteria suspension extraction DNA) is standby.Batch interior repetition of the real-time PCR detections of real-time PCR analysis sheet
Property, three repetitions every time of each dilution factor, the average value and standard deviation (SD) of three Ct values being calculated the results are shown in Table 4,
Amplification curve is shown in accompanying drawing 4.As a result show, this PCR detections blaNDMRepeatability has reached 100% in gene batch.
4 PCR detections bla of tableNDMRepeated result in gene batch
2nd, it is repeated between criticizing
Batch of this PCR experiment is detected using U.S. clinical and the method for qualitative analysis of laboratory standards institute (CLSI) as guide
Between repeatability.Preparation contains blaNDMThe bacteria suspension of gene, the bacteria suspension extraction DNA for preparing 4 concentration are standby.Each concentration is each
Two repetitions, continuously do three days, then each concentration obtains 6 data.The average value and standard deviation for 6 Ct values being calculated
(SD).5 are the results are shown in Table, amplification curve is shown in accompanying drawing 5,6 and 7.As a result show, this PCR detections blaNDMRepeatability is between gene batch
100% (24/24).
5 PCR detections bla of tableNDMRepeated result between gene batch
Embodiment 11
The linear analysis of nucleic acid detection kit
Preparation contains blaNDMTarget gene 10cfu/ml to 108The dilution of cfu/ml 8 concentration, extract each dilution DNA
It is standby.Detected with this PCR to drug resistant gene blaNDMThe linearity, the amplification curve of the linearity is shown in accompanying drawing 8, the mark of the linearity
Directrix curve is shown in accompanying drawing 9.
As shown in Figure 8,9, this PCR is to blaNDMMore than seven log units of the linearity testing result span of gene.
Embodiment 12
The specificity analysis of this nucleic acid detection kit
In order to study this PCR analysis specificity, this experimental analysis drug resistance of 88 plants of reference strains, which includes
Common multidrug resistant Gram-negative bacteria, such as Acinetobacter baumannii Acinetobacterbaumannii (n=14), aerogenesis
Enterobacteria Enterobacteraerogenes (n=2), enterobacter cloacae Enterobactercloacae (n=9), large intestine bar
Bacterium Escherichiacoli (n=15), klebsiella oxytoca KlebsiellaOxytoca (n=1), ozena Cray
Bai Shi bacillus Klebsiellaozaenae (n=2), Friedlander's bacillus Klebsiellapneumoniae (n=23), rubs
Root (family name) bacterium Morganellamorganii (n=1), proteus mirabilis Proteusmirabilis (n=1), general sieve of Lei Shi
Wei Dengsi bacterium Providenciarettgeri (n=1), pseudomonas aeruginosa Pseudomonasaeruginosa (n=12),
Serratia marcescens Serratiamarcescens (n=2), candida albicans Candidaspecies (n=5).
In this 88 plants of bacterial strains, blaNDMPositive strain is 14 plants, and remaining is drug resistant gene negative strain.
Extract the DNA of this 88 parts of samples, study this real-time PCR to blaNDMSpecificity, as a result amplification curve such as accompanying drawing
Shown in 10, positive findings is 14 plants, and with known consistent, remaining is drug resistant gene negative strain, and correctly detection is all by this PCR
BlaNDMTarget gene, no cross reaction.
For in chaff interference Quality Research, this experiment employs high-caliber human hemoglobin and stool sample is carried out
Analysis, detected without interference with this PCR.
Embodiment 13
Influence of the different fluorescence labelings to quantitative fluorescent PCR
By blaNDMProbe by VIC mark be changed to JOE, Cy3, ROX mark, utilize concentration identical bacteria suspension, extraction
DNA, influence of the three kinds of different fluorescence labelings of the person of comparison to quantitative fluorescent PCR.Amplification curve is as shown in Figure 11.
It can be seen that the Sensitivity and Specificity of the PCR results of three kinds of fluorescence labelings is all fine, Ct values are suitable,
The probe of this kit is adapted to different fluorescence labelings, can obtain preferable result.
Conclusion
PCR in this research is detected simultaneously includes blaNDM1To blaNDM16Totally 16 kinds of blaNDMHigh drug resistant gene, have good
Sensitivity, accuracy and repeatability, this is that current other similar PCR kits can not accomplish.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, it can all do various change and modification, therefore the protection of the present invention
What scope should be defined by claims is defined.
SEQUENCE LISTING
<110>Beijing Zi Meng Pharmaceutical Technology Co., Ltd
<120>The PCR detection kit of drug-fast bacteria comprising blaNDM genes and its application
<130> 3
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
attggcataa gtcgcaatcc 20
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence
<400> 2
tggagcatgt ggtttaattc ga 22
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
agatcaacct gccggtcgcg 20
Claims (10)
1. a kind of detection primer, it is characterised in that the primer includes sequence such as SEQ ID NO：1 and SEQ ID NO：The two of 2
Bar nucleotide sequence.
2. one kind includes blaNDMThe detection kit of gene drug-fast bacteria, it is characterised in that the detection kit includes right
It is required that the detection primer described in 1.
3. detection kit according to claim 2, it is characterised in that also include sequence such as in the detection kit
SEQ ID NO：Probe shown in 3.
4. detection kit according to claim 3, it is characterised in that one end of the probe is marked with fluorescence report base
Group, the probe other end are marked with fluorescent quenching group.
5. detection kit according to claim 4, it is characterised in that the fluorescent reporter group be FAM, VIC, ROX,
Any one in Cy5, JOE or Quasar705 fluorophor；The fluorescent quenching group is BHQ or ECLIPSE systems
Row.
6. detection kit according to claim 2, it is characterised in that also contain PCR MIX in the detection kit
And H2O。
7. detection kit according to claim 2, it is characterised in that also include PCR enzymes, the moon in the detection kit
Property at least one of reference substance and positive reference substance.
8. the detection primer described in claim 1 includes bla in preparationNDMApplication in the drug-fast bacteria detection reagent of gene.
9. one kind includes blaNDMThe detection method of gene drug-fast bacteria, it is characterised in that be using the primer described in claim 1,
Detection reagent described in claim 2-7 any described detection kit or claim 8 is detected.
10. detection method according to claim 9, it is characterised in that the drug-fast bacteria for it is following any one or more：
Acinetobacter baumannii Acinetobacterbaumannii, clostridium perfringen Enterobacteraerogenes, enterobacter cloacae
Enterobactercloacae, Escherichia coli Escherichiacoli, klebsiella oxytoca
KlebsiellaOxytoca, abel's bacillus Klebsiellaozaenae, Friedlander's bacillus
Klebsiellapneumoniae, the root that rubs (family name) bacterium Morganellamorganii, proteus mirabilis
Proteusmirabilis, providencia rettgeri Providenciarettgeri, pseudomonas aeruginosa
Pseudomonasaeruginosa, serratia marcescens Serratiamarcescens, candida albicans Candidaspecies.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109628616A (en) * | 2018-12-17 | 2019-04-16 | 博奥生物集团有限公司 | For detecting the LAMP primer composition and its application of Carbapenem-resistant gene NDM1-24 hypotype |
| CN117417989A (en) * | 2023-11-17 | 2024-01-19 | 河北省畜牧兽医研究所 | Multiplex fluorescence quantitative PCR kit and method for detecting drug-resistant genes |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105121656A (en) * | 2012-11-15 | 2015-12-02 | 以色列分子检测有限公司 | PCR reaction mixtures and methods of using same |
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2017
- 2017-08-15 CN CN201710695728.4A patent/CN107523620A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105121656A (en) * | 2012-11-15 | 2015-12-02 | 以色列分子检测有限公司 | PCR reaction mixtures and methods of using same |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109628616A (en) * | 2018-12-17 | 2019-04-16 | 博奥生物集团有限公司 | For detecting the LAMP primer composition and its application of Carbapenem-resistant gene NDM1-24 hypotype |
| CN117417989A (en) * | 2023-11-17 | 2024-01-19 | 河北省畜牧兽医研究所 | Multiplex fluorescence quantitative PCR kit and method for detecting drug-resistant genes |
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Application publication date: 20171229 |