CN105400875B - A set of polynucleotides, method and kit for bacterial resistance gene NDM-1 detection - Google Patents
A set of polynucleotides, method and kit for bacterial resistance gene NDM-1 detection Download PDFInfo
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- CN105400875B CN105400875B CN201510875803.6A CN201510875803A CN105400875B CN 105400875 B CN105400875 B CN 105400875B CN 201510875803 A CN201510875803 A CN 201510875803A CN 105400875 B CN105400875 B CN 105400875B
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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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- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
Bacterial resistance gene is used for the invention discloses a kind of --- polynucleotides, method and the kit of -1 gene of New Delhi metallo-β-lactamase (NDM-1) detection.Specifically the polynucleotides and DNA construction pXL08 that can be used as bacterial resistance gene NDM-1 real-time fluorescence PCR detection standard molecule are disclosed in the present invention.Plasmid control molecule of the invention solves the problems, such as bacterial resistance gene NDM-1 real-time fluorescence PCR detection Plays material want, guarantee the comparativity of bacterial resistance gene NDM-1 real time fluorescent PCR method testing result, provides reliable quality control method for the detection of bacterial resistance gene NDM-1 real time fluorescent PCR method.
Description
Technical field
The present invention relates to the plasmid molecules of a set of technical field of bioengineering, and in particular to a set of to be used for bacterial resistance base
Cause --- polynucleotides, method and the kit of -1 gene of New Delhi metallo-β-lactamase (NDM-1) detection.
Background technique
In recent years, with the extensive use of antibiotic, there is different degrees of drug resistance in various bacteria.In September, 2010, print
Degree, Britain, Sweden, Pakistan and Australian scholar, which combine, reports a kind of bacterium that can almost resist all antibiotic,
Referred to as " superbacteria ", the bacterium can generate a kind of new metallo-β-lactamase, and hydrolysing activity is much higher than existing metal
Beta-lactamase.Since first cases of infection are found in India New Delhi, which is referred to as New Delhi Metallo-β-lactamases
Enzyme -1 (New Delhi metallo-blactamase-1, NDM-1).NDM-1 has powerful hydrolysis, can almost resist
All antibiotic used at present, have seriously threatened human health.The mrna length of NDM-1 is 813bp, coding NDM-1 enzyme
Gene is located on the plasmid that 1 length is 140000bp.Plasmid is to be free on except DNA of bacteria genome, is moveable hereditary
Original part can shift between different genera bacterium, so as to cause diffusion of the drug resistance in bacterium.Carry NDM-1 plasmid
Bacterium be mainly the relevant bacterium of nosocomial infection.In addition, NDM-1 infected patient passes through the contact of environmental dissemination, medical staff
The diffusive infection that can cause drug-fast bacteria is propagated, nosocomial infection is made to face unprecedented challenge.
Plasmid DNA standard substance is a kind of recombinant plasmid molecule of specific fragment containing testing goal gene, in PCR
It can be used as positive control in qualitative detection, can be used as the standard items of quantitative analysis in PCR quantitative analysis, building is quantitative to divide
The standard curve of analysis.But due to being limited by DNA quantitative detection magnitude tracing level, the Plasmid DNA standard that can be looked at present
Substance is also fewer, for the Plasmid DNA standard substance or blank of NDM-1 drug resistant gene PCR detection.In biometric science
Field, the research of Plasmid DNA standard substance is in widespread attention, and the international and domestic unit for participating in the area research includes the state in the U.S.
Standard technique research institute of family (NIST), examination criteria group of British government (LGC), Joint Research Centre of EU Committee reference substance
Matter and measuring study institute (IRMM) and Chinese measuring science Institute for Research and Technology etc., have had 5 plasmids to have card reference substance so far
Matter is researched and developed successfully.Development or sky due to the Plasmid DNA standard substance for NDM-1 drug resistant gene PCR related detecting method
It is white, practical NDM-1 drug resistant gene PCR detection when, constituent parts use it is mostly be designed, designed building plasmid DNA molecule make
For standard items, target gene specific fragment therein is different, while lacking unified valued methods, plasmid DNA molecule
Definite value poor accuracy causes the testing result very different between each laboratory, lacks comparativity, validity and reliability.
Summary of the invention
It is suitable for what bacterial resistance gene NDM-1 real-time fluorescence quantitative PCR detected the purpose of the present invention is to provide a set of
Plasmid control molecule and its application.
The first aspect of the present invention, provides a kind of isolated polynucleotides, and the polynucleotides include bacterial resistance base
Because of the sequence of NDM-1.
In another preferred example, the sequence of the bacterial resistance gene NDM-1 is selected from the group:
(a) sequence polynucleotide sequence as shown in SEQ ID NO.:1；
(b) multicore of nucleotide sequence and homology >=95% (preferably >=98%) of sequence shown in SEQ ID NO.:1
Nucleotide sequence；
(c) sequence is with the exact matching of polynucleotides shown in SEQ ID NO.:1 or complete complementary and length is SEQ ID
20%-100% (the preferably 50%-100%, more preferably 80%-100%, most preferably 90%- of sequence length shown in NO.:1
100%, polynucleotide sequence such as 95%)；
(d) polynucleotide sequence complementary with any polynucleotide sequence of (a)-(c).
In another preferred example, the polynucleotide sequence is as shown in SEQ ID NO.:1.
The second aspect of the present invention provides a set of isolated DNA construction, includes the present invention in the DNA construction
Polynucleotides described in first aspect and optional sequence label, cleavage sequence, promoter sequence and/or carrier sequence.
It in another preferred example, include the sequence of bacterial resistance gene NDM-1 in the DNA construction.
In another preferred example, the DNA construction is linear DNA construction or cyclic DNA construction.
In another preferred example, the DNA construction is plasmid or expression vector.
In another preferred example, the standard scores that the plasmid or expression vector are detected as bacterial resistance gene NDM-1
Sub (plasmid control molecule).
In another preferred example, the skeleton plasmid of the plasmid or expression vector is selected from the group: pUC19, pUC18,
PUC118, pUC119, pBlueScript II SK and pGEM.
The third aspect of the present invention provides a set of kit, includes described in first aspect present invention in the kit
Polynucleotides or second aspect of the present invention described in DNA construction.
It in another preferred example, further include primer pair in the kit, the primer pair specific amplification bacterial resistance
The gene order of gene NDM-1.
In another preferred example, the primer pair choosing of the gene order of specific amplification bacterial resistance gene NDM-1
From the following group:
Primer pair shown in SEQ ID NO.:3 and SEQ ID NO.:4；
Primer pair shown in SEQ ID NO.:5 and SEQ ID NO.:6；
Primer pair shown in SEQ ID NO.:7 and SEQ ID NO.:8；
Primer pair shown in SEQ ID NO.:9 and SEQ ID NO.:10；
It in another preferred example, further include primer pair in the kit, the primer pair specific amplification bacterial resistance
Gene NDM-1 sequence.
It in another preferred example, further include probe sequence selected from the group below, the probe sequence such as SEQ in the kit
Shown in ID NO.:11.
The fourth aspect of the present invention provides polynucleotides as described in the first aspect of the invention, second aspect of the present invention
The purposes of kit described in the DNA construction or third aspect present invention, which is characterized in that be used for bacterial resistance base
Because of the detection of NDM-1.
In another preferred example, described to be detected as non-diagnostic or therapeutic purposes.
In another preferred example, described to be detected as fluorescence quantitative PCR detection.
The fifth aspect of the present invention provides a set of bacterial resistance gene NDM-1 real-time fluorescence quantitative PCR detection method,
Used standard substance is the building of DNA described in polynucleotides or second aspect of the present invention as described in the first aspect of the invention
Object.
The sixth aspect of the present invention, provides a set of polynucleotides product, and the product includes:
(i) bacterial resistance gene NDM-1 standard items, the standard items are selected from: multicore described in first aspect present invention
DNA construction described in thuja acid or second aspect of the present invention；
(ii) primer pair of specific amplification bacterial resistance gene NDM-1 sequence, the primer pair are as follows:
The primer pair of Sequence composition shown in SEQ ID NO.:3 and SEQ ID NO.:4.
In another preferred example, the product is the combination (combination) of polynucleotides, preferably the component
(i) and (ii) is independent.
In another preferred example, the product is kit form.
The seventh aspect of the present invention provides the preparation method of the plasmid control molecule of a set of bacterial resistance gene NDM-1,
The following steps are included:
1. artificial synthesized bacterial resistance gene NDM-1 sequence, the bacterial resistance gene NDM-1 such as SEQ ID NO.:1 institute
Show；
2. by step, 1. gained gene order is cloned on cloning vector, obtains the matter of bacterial resistance gene NDM-1 detection
Grain standard molecule.
In another preferred example, the step 2. the cloning vector be pUC19, pUC18, pUC118, pUC119,
PBlueScript II SK or pGEM.
In another preferred example, 2. the cloning vector is pUC19 to the step.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 is the map of plasmid control molecule pXL08 of the present invention.
Fig. 2 is plasmid control molecule pXL08 Detection of Stability result figure obtained by the present invention.
Fig. 3 is the real-time fluorescence PCR standard curve established using plasmid control molecule pXL08 of the present invention.
Specific embodiment
For the present inventor by extensive and in-depth research, it is glimmering in real time that one section of acquisition can be used in bacterial resistance gene NDM-1
The polynucleotide sequence of light PCR detection and primer pair with matching, the experimental results showed that, using suitable skeleton plasmid
The polynucleotide sequence is prepared as standard plasmid molecule, and primer pair of the invention is cooperated to carry out real-time fluorescence PCR detection,
With splendid specificity and sensitivity, and have good stability.
The technical problem to be solved by the present invention is in order to overcome existing bacterial resistance gene NDM-1 real-time fluorescence PCR
The problem of lacking positive criteria product and positive criteria product configuration in detection method, provides a set of suitable for bacterial resistance gene
It the construction method of the plasmid control molecule of NDM-1 real-time fluorescence PCR detection and the plasmid control molecule, quantitative approach and answers
With.
The principle of real-time fluorescence PCR detection bacterial resistance gene NDM-1
Using real-time fluorescent PCR technology can specific amplification bacterial resistance gene NDM-1 sequence, design be directed to NDM-1 base
The primer of cause and the probe of both ends mark fluorescent, amplification assay sample DNA.Real-time fluorescence PCR can be by detecting fluorescence signal
Increase carry out real-time monitoring PCR product.At the same time, with positive the marking of identical primer, probe and condition amplification known concentration
Quasi- substance (or positive criteria molecule).In PCR method, positive criteria substance (or positive criteria molecule) can be used as positive right
According to；In real-time fluorescence PCR, positive criteria substance (or positive criteria molecule) can construct stable standard curve, according to standard
Curve can calculate separately out the absolute content (copy number or concentration) that gene is corresponded in sample.
Standard substance
Standard substance is that there are a set of or a variety of uniform characteristic values determined very well enough to comment to calibrator (-ter) unit
Valence measurement method or material or substance to material assignment.
Plasmid control molecule
The specific sequence of detection bacterium drug resistant gene NDM-1 involved in the present invention a kind of simultaneously devises one on this basis
Kind plasmid control molecule, i.e. the specific sequence of bacterial resistance gene NDM-1 is NDM-1 full length gene sequence, and length is
813bp。
It is preferably carried out in mode at of the invention one, the present invention provides a set of bacterial resistance gene NDM-1 detections
Plasmid control molecule, the plasmid control molecule include bacterial resistance gene NDM-1 sequence, the bacterial resistance gene NDM-1
Sequence is as shown in SEQ ID NO:1 in sequence table:
Plasmid control molecule of the invention, the full-length gene order preferably containing bacterial resistance gene NDM-1.
Heretofore described bacterial resistance gene NDM-1, can encode a kind of new metallo-β-lactamase, and hydrolysis is lived
Property be much higher than existing metallo-β-lactamase, have powerful hydrolysis, can almost resist all antibiosis used at present
Element.The bacterial resistance gene NDM-1 sequence is preferably as shown in SEQ ID NO:1.
The sequence of plasmid control molecule of the present invention is preferably as shown in SEQ ID NO:2, wherein the 423rd to the
1235 are bacterial resistance gene NDM-1 sequence:
The quantitative approach of the plasmid control molecule of bacterial resistance gene NDM-1 as described above detection in the present invention comprising
Following steps:
1. extracting plasmid control molecule；
2. being calculated in plasmid control molecule according to the step 1. base composition of resulting plasmid control molecule and sequence length
P elements content；
3. preparing the phosphorus standard solution of gradient concentration, and high-resolution inductively coupled plasma body hair is made with this standard solution
Penetrate mass spectrographic standard curve；
4. high-resolution inductively coupled plasma body emit Mass Spectrometer Method plasmid control molecule solution, and according to step 3. gained
Standard curve obtain the phosphorus content of plasmid control molecule solution；
5. according to the step 4. phosphorus content of resulting plasmid control molecule solution and step 2. resulting plasmid control molecule
In P elements content, calculate the concentration of plasmid control molecule.
Wherein 3. the condition of high-resolution inductively coupled plasma body transmitting Mass Spectrometer Method is as follows described in step: cooling gas flow
Preferably 10L/min~25L/min is most preferably 16.86L/min, and secondary air amount is preferably 0.5L/min~3.0L/
Min is most preferably 0.88L/min, and it is more preferably 1.123L/ that atomization gas flow, which is preferably 0.5L/min~2.43L/min,
Min, power are preferably 1200W~1400W, are most preferably 1350W.
The quantitative approach of plasmid control molecule of the present invention is preferably comprised ultraviolet spectrophotometry and high-resolution inductance
Coupled plasma mass spectrometry (HR-ICP-MS).
Specific implementation explanation:
The quantitative approach of plasmid control molecule of the present invention is preferably comprised ultraviolet spectrophotometry and high-resolution inductance
Coupled plasma mass spectrometry (HR-ICP-MS).
Wherein ultraviolet spectrophotometry is preferably comprised following steps to plasmid control molecule quantitative approach:
1. extracting plasmid control molecule；
2. making ultraviolet specrophotometer be corrected to zero point with TE buffer；
3. taking appropriate DNA (according to the needs of instrument) (nanodrop is directly put in detection zone) into cuvette, record
Instrument readings.
4. directly being recorded if it can directly read DNA concentration；If can not, the light for recording sample in 260nm and 280nm is close
Degree, the concentration of DNA sample are OD260 × nucleic acid extension rate × 50, and concentration unit is ng/ μ L.
(2) HR-ICP-MS is preferably comprised following steps to plasmid control molecule quantitative approach:
1. extracting plasmid control molecule；
2. calculating separately the P elements of each plasmid control molecule according to the base composition of plasmid control molecule and sequence length
Content；
3. preparing the phosphorus standard solution of gradient concentration, and the standard curve of HR-ICP-MS is made with this standard solution.
4. HR-ICP-MS detects plasmid control molecule, and obtains the phosphorus content of plasmid control molecule according to standard curve.
5. calculating the concentration of plasmid control molecule according to the phosphorus content of plasmid control molecule.
The present invention also provides a set of bacterial resistance gene NDM-1 real-time fluorescence quantitative PCR detection method, used by
Standard substance is plasmid control molecule as described above.
The present invention also provides the preparations of the plasmid control molecule of a set of bacterial resistance gene NDM-1 detection as described above
Method, comprising the following steps:
1. the specific sequence of the artificial synthesized bacterial resistance gene NDM-1, the sequence is as shown in SEQ IDNO:1；
2. the sequence of step 1. gained bacterial resistance gene NDM-1 is cloned on cloning vector, bacterial resistance gene is obtained
The plasmid control molecule of NDM-1 detection.
Wherein 1. the artificial synthesized method is preferably step: the side of full genome synthesis or PCR primer amplification
Method obtains the sequence.
Plasmid control molecule construction method of the present invention is preferably comprised following steps:
1. inquiring bacterial resistance gene NDM-1 in the Genbank of NCBI (US National Biotechnology Information center)；
2. analyzing above-mentioned sequence, suitable sequence and suitable restriction enzyme site are selected, and restriction enzyme site is added to
5 ' the ends and 3 ' ends of selected sequence.
3. by treated, sequence carries out the artificial synthesized service of full genome, synthesis, DNA piece including single-stranded Oligo DNA
The work such as section splicing, and full-length gene is cloned into plasmid vector, obtain plasmid control molecule.
The plasmid vector can be conventional carrier, it is preferred that cloning vector, it more preferably can be in Escherichia coli
The cloning vector of middle proliferation, the cloning vector are preferably: pUC19, pUC18, pUC118, pUC119, pBlueScript
II SK or pGEM serial carrier, it is therefore preferable to pUC19 cloning vector.
4. the sequence of sequence verification plasmid control molecule.
5. the real-time fluorescence PCR detection method of plasmid control molecule is verified.
The real-time fluorescence PCR detection method verifying refers to that detection plasmid control molecule is carrying out real-time fluorescence PCR point
The characteristics such as specificity and building standard curve ability when analysis, to identify the plasmid control molecule as real time fluorescent PCR method
The ability of the standard substance of detection bacterium drug resistant gene NDM-1.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
Kit
The present invention provides the kit of detection bacterium drug resistant gene NDM-1 a kind of, include: in the kit
Standard molecule contains polynucleotides shown in SEQ ID NO.:1 in the standard molecule；
It preferably, further include primer sequence in the kit, the primer sequence is used for specific amplification SEQ ID
Sequence shown in NO.:1, the primer sequence are selected from the group:
(1) primer sequence shown in SEQ ID NO.:3 and SEQ ID NO.:4；
(2) primer sequence shown in SEQ ID NO.:5 and SEQ ID NO.:6；
(3) primer sequence shown in SEQ ID NO.:7 and SEQ ID NO.:8；With
(4) primer sequence shown in SEQ ID NO.:9 and SEQ ID NO.:10.
The plasmid control molecule that detection bacterium drug resistant gene NDM-1 is contained in kit provided by the invention (carries
The pXL08 plasmid control molecule of NDM-1 gene) and the primer pair that is matched with plasmid control molecule of the invention, it can facilitate
Carry out PCR detection, and high specificity, high sensitivity is linear good.
Main advantages of the present invention are:
(1) plasmid control molecule comprising polynucleotide sequence of the present invention has uniformity strong, the high advantage of stability, together
When the present invention solve the problems, such as bacterial resistance gene NDM-1 real-time fluorescence PCR detection Plays material want, guarantee that bacterium is resistance to
The comparativity of medicine gene NDM-1 real time fluorescent PCR method testing result is bacterial resistance gene NDM-1 real time fluorescent PCR method
Detection provides quality control；
(2) product for cooperating primer pair of the invention to prepare using plasmid control molecule of the invention is used for real-time fluorescence
When PCR detection bacterium drug resistant gene NDM-1, high specificity, high sensitivity, linearly well.
(3) plasmid control molecule that detection bacterium drug resistant gene NDM-1 is contained in kit provided by the invention (carries
The pXL08 plasmid control molecule of NDM-1 gene).
Combined with specific embodiments below, further statement is of the invention.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.The experimental method of detailed conditions is not specified in the following example, usually according to conventional strip
Part such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and
Number is calculated by weight.Biomaterial used in the embodiment of the present invention and reagent unless otherwise instructed can be from commercially available channels
It obtains.
The building of 1 plasmid control molecule of embodiment
Experiment reagent and laboratory apparatus:
The a large amount of extracts kits of plasmid (OMEGA), other biochemical reagents are import packing or the pure biochemical examination of domestic analysis
Agent；Laboratory apparatus includes centrifuge, thermostat water bath, constant-temperature shaking incubator, liquid-transfering gun etc..
Experimental method the following steps are included:
1, bacterial resistance gene NDM-1 sequence is searched in GenBank；
2, above-mentioned sequence is analyzed, selects suitable sequence and suitable restriction enzyme site, bacterial resistance gene NDM-1
Sequence length is 813bp, and both ends add BamHI restriction enzyme site；
3, by treated, sequence send most valuable treasure bioengineering (Dalian) Co., Ltd, is responsible for progress full genome by it and manually closes
At service, the work such as synthesis, DNA fragmentation splicing including single-stranded Oligo DNA, the sequence of gained bacterial resistance gene NDM-1
As shown in SEQ ID NO:1 in sequence table, gained full-length gene is cloned into plasmid vector pUC19 (purchased from TAKARA company)
In, building obtains the plasmid control molecule pXL08 (SEQ in its sequence such as sequence table comprising bacterial resistance gene NDM-1 sequence
Shown in ID NO:2), the plasmid map of building gained plasmid control molecule pXL08 is as shown in Figure 1.
4, mass propgation contains the recombination bacillus coli comprising gained plasmid control molecule pXL08, is largely mentioned using plasmid
It takes kit (OMEGA) to extract plasmid, obtains the plasmid control molecule pXL08 of high-purity.By ultraviolet specrophotometer and
Electrophoresis carry out purity analysis, Plasmid DNA standard molecule is placed in -20 DEG C of preservations later, extract the method for plasmid the following steps are included:
A, the bacterium for being incubated overnight 100~200mL is centrifuged 10 minutes in 5000 × g of room temperature, discards supernatant；
B, 12mL Solution I (containing RNase A) is added, oscillation mixes well；
C, 12mL Solution II is added, mild mixing of turning upside down is placed at room temperature for 2 minutes so that thallus sufficiently cracks；
D, 16mL Solution III is added, is sufficiently mixed by inversion up and down immediately for several times, until it is heavy to form uniform white
It forms sediment；
E, >=12000 × g, 4 DEG C are centrifuged 10 minutes；
F, absorption 20mL supernatant, which carefully moves in a clean HiBind Maxi adsorption column, (is placed in 50mL collecting pipe
In), 5000 × g room temperature is centrifuged 5 minutes；
G, the liquid in collecting pipe is discarded, 10mL Buffer HB is added and cleans adsorption column, 5000 × g room temperature is centrifuged 5 points
Clock；
H, the liquid in collecting pipe is discarded, 15mL DNA Wash Buffer (dehydrated alcohol dilution) cleaning absorption is added
Column；
I, above-mentioned cleaning step is repeated；
J, 6000 × g is centrifuged 15 minutes to dry adsorption column；
K, adsorption column is placed in a clean 50mL pipe, 2mL~3mL TE buffer is added, is placed at room temperature for 1~2 point
Clock, 8000 × g are centrifuged 2 minutes with eluted dna, and gained DNA solution is the plasmid control molecule solution extracted, is stored in -20
℃。
5, sequence verification plasmid control molecule pXL08
The plasmid control molecule of extraction is sent to eight sequencing companies and carries out sequence verification, eight sequencing companies are respectively north
Capital six directions Hua Da Gene science limited liability company, Shanghai Bo Shang Bioisystech Co., Ltd, Invitrogen (Shanghai) trade have
Limit company, Shanghai Jie Li Bioisystech Co., Ltd, Shanghai Mei Ji Bioisystech Co., Ltd, the raw work biotechnology in Shanghai are limited
Company, Suzhou Jin Weizhi Biotechnology Co., Ltd, precious biotinylated biomolecule Engineering Co., Ltd.Plasmid DNA standard substance is come
Say sequence length and quantitatively there is a direct relation, 8 different sequencing companies carry out sequencings be in Developments of certified reference samples for
The requirement of definite value.It is detailed in ISO directive/guide 35, the specific requirement of standard substance definite value part.
Sequence investigates formula: sequence accuracy=correct base number/base sum
Sequencing result proves that the sequence accuracy that all units for participating in sequence verification obtain is 100%, the plasmid sequence
In the 417th to the 1241st be bacterial resistance gene NDM-1 sequence.
Experimental result are as follows: largely extracted by sequence selection step, the artificial synthesized step of full genome and plasmid and etc.
Obtain the plasmid control molecule pXL08 of high-purity.Through sequence verification, the plasmid control molecule Insert Fragment sequence and design are complete
It is consistent.
The uniformity testing of 2 plasmid control molecule pXL08 of embodiment
Uniformity is the coherency state of the relevant structure of a set of or multifrequency nature or composition in characterization substance.Pass through measurement
It is derived from Different Package unit (such as bottle, packet) or is derived from the sample of the prescribed level of same packaging unit different location, measurement knot
Fruit falls in regulation range of uncertainty, then it is believed that the standard substance is uniform to specified characteristic quantity.Uniformity is mark
The essential attribute of quasi- substance, the spatial distribution characteristic for description standard substance characteristics.In development (production) mistake of standard substance
Uniformity assessment must be carried out in journey, to prove it with good uniformity.The plasmid control molecule having good uniformity, amount
Value not will receive the influence of the factors such as packing, and the magnitude difference between each bottle is little, therefore ensure that the reliability of testing result.
Experiment reagent TE buffer (pH7.5).Laboratory apparatus Nanodrops ND-2000.
Experimental method:
1, sample requirement is extracted according to " JJG 1006-1994 primary standard substance technical specification " uniformity, from each plasmid
15 bottles are randomly selected in DNA standard substance.
2, to taken each sample 3 times, each sampling amount is 1 μ L.Each sampling amount is repeated with uv-spectrophotometric
Measurement 3 times, is averaged.
3, measurement result is counted with F method of inspection, judges uniformity testing result.Method particularly includes: extract m sample
This, measures m group equal precision measurement data under the same conditions, if there was no significant difference for measurement variance, should meet following formula
Statistical requirements.
Wherein sum of squares of deviations calculation formula between group are as follows:
Formula is shown in sum of squares of deviations calculating in group are as follows:
ν1=m-1 (freedom degree between group)
ν2=N-m (the group internal degree of freedom).
Experimental result:
1, the uniformity testing result difference of plasmid control molecule pXL08 is as shown in table 1
1 plasmid control molecule pXL08 uniformity testing data of table (unit: ng/ μ L)
The homogeneity statistical of pXL08 the results are shown in Table 2, and under 95% confidence level, F value is less than F0.05(14,30), it was demonstrated that should
Plasmid control molecule pXL08 is uniformly, to meet " JJG1006-94 primary standard substance technical specification " uniformity testing qualification
It is required that.
The homogeneity statistical result of 2 plasmid control molecule pXL08 of table
The study on the stability of 3 plasmid control molecule pXL08 of embodiment
Stability refers to that under specific time interval and storage requirement, the characteristic value of standard substance is maintained at prescribed limit
Interior ability.Stability is the essential attribute of standard substance, the property that the characteristic for description standard substance changes over time, i.e.,
The Time-distribution of description standard substance characteristics.Stability assessment must be carried out in the development process of standard substance.Stablize
Property assessment can not only assess uncertainty of measurement relevant to stability of material, and suitable storage and transport item can be specified
Part.The plasmid control molecule having good stability, characteristic value will not pushing away with the time under the conditions of suitable storage and transport
Move and change, testing result will not by its it is instable influence, ensure that the reliability of testing result.
Experiment reagent TE buffer (pH7.5).Laboratory apparatus Nanodrop ND-2000.
Experimental method:
It is investigated using stability of the classical stability study to plasmid control molecule, i.e., the sample prepared simultaneously is in phase
It is measured over time under the conditions of.
1,0th month, 0.5 month, 1 month, 2 months, 4 months, 6 months, 9 after the completion of prepared by plasmid control molecule
A month, 12 months plasmid control molecules (- 20 DEG C preservation) to preparation randomly select 3 bottles, each sample replication 3 times takes
Average value carries out long-time stability investigation.This research uses ultraviolet spectrophotometry and is tracked to plasmid control molecule
It investigates.
2, Detection of Stability data are assessed, judges study on the stability result.Specific investigation method is as follows:
Straight slope can be calculated with following formula:
In formula: Xi--- i-th of time point；Yi--- the observation at i-th of time point；--- all time points put down
Mean value；--- the average value of all observations.
Intercept can be calculated by following formula:
Every standard deviation can be calculated with following formula on straight line:
In formula: Xi--- i-th of time point；Yi--- the observation at i-th of time point；β1, β0--- regression coefficient；
N --- measurement coefficient.
β1Standard deviation be given by:
Based on β1Standard deviation, can be detected with t- and carry out following judgement: even | β1| < t0.95,n-2·s(β1), then show
Slope is not significant, and unstability is not observed.
Experimental result:
1, plasmid control molecule pXL08 study on the stability result is as shown in Table 3 and Fig. 2:
The STABILITY MONITORING data (unit: ng/ μ L) of 3 plasmid control molecule pXL08 of table
Statistical result is as shown in table 4.
The stability statistical result of 4 plasmid control molecule pXL08 of table
By statistical result it is found that plasmid control molecule pXL08 saved under the conditions of -20 DEG C 12 months be stable.
Experimental example 4 quantifies plasmid control molecule pXL08 with ultraviolet spectrophotometry
Experiment reagent TE buffer (pH7.5).Laboratory apparatus Nanodrops ND-2000.
Experimental method the following steps are included:
1, ultraviolet specrophotometer is made to be corrected to zero point with TE buffer；
2, Example preparation gained 1 μ L of DNA solution, directly point are in detection zone, register instrument reading；
3, DNA concentration is directly read, concentration unit is ng/ μ L.
4, each sample test eight times, are averaged.
Experimental result:
1, by ultraviolet spectrophotometry definite value, the concentration of plasmid control molecule pXL08 is as shown in table 5:
5 ultraviolet spectrophotometry definite value result of table (unit: ng/ μ L)
| Plasmid | Repeat 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | Average value |
| pXL08 | 56.8 | 57.4 | 57.0 | 57.9 | 57.4 | 57.7 | 57.8 | 57.1 | 57.4 |
Embodiment 5 quantifies plasmid control molecule pXL08 with HR-ICP-MS
Experiment reagent is P standard solution (NIST, SRM3139a).Laboratory apparatus are as follows: inductively coupled plasma body emits matter
Spectrometer (Thermofi sher element2).
Experimental method the following steps are included:
1, according to the base composition of plasmid control molecule and sequence length, the content of wherein P elements is calculated separately；
2, HR-ICP-MS experiment parameter are as follows: cooling gas flow 16.86L/min, atomization gas flow is 1.123L/min, auxiliary
Helping throughput is 0.99L/min, power 1350W.
3, the phosphorus standard solution (0,1,2,3,4,5g/L) of gradient concentration is prepared, and makes HR-ICP- with this standard solution
The standard curve of MS；
4, plasmid control molecule is diluted 2000 times, the plasmid control molecule after HR-ICP-MS detection dilution, and according to mark
Directrix curve obtains the phosphorus content of the plasmid control molecule after dilution；
5, the concentration of plasmid control molecule is calculated according to the phosphorus content and extension rate that measure；
6, each sample test eight times, are averaged.
Experimental result:
It detects to obtain plasmid control molecule pXL08 phosphorus element content by HR-ICP-MS, then obtains plasmid control through conversion
The mass concentration of molecule, as shown in table 6.
The mass concentration result (unit: ng/ μ L) of 6 plasmid control molecule pXL08 of table
Application of the plasmid control molecule pXL08 that embodiment 6 is developed in real-time fluorescence PCR detection
Experiment reagent: being directed to plasmid control molecule pXL08, devise tens of pairs of primers, expands in target sequence respectively
The primer and probe of design is transferred to precious bioengineering (Dalian) Co., Ltd to synthesize by genetic fragment, and Master Mix is purchased from
Life technology company.Laboratory apparatus includes: real-time fluorescence quantitative PCR amplification instrument (Life technology) centrifugation
Machine, thermostat water bath, incubator, day equality.
Application of the plasmid control molecule pXL08 in real-time fluorescence PCR detection:
Primer (respectively using primer shown in table 7), probe, PCR detection architecture
Reaction condition PCR program:
95 DEG C 10 minutes；
95 DEG C 15 seconds, 60 DEG C 1 minute, 40 circulation.
Embodiment 1 is prepared into the plasmid control molecule that resulting plasmid control molecule pXL08 is diluted to various concentration
(1.68×106copies/μL、1.68×105copies/μL、1.68×104copies/μL、1.68×103copies/μL、
1.68×102copies/μL、1.68×101copies/μL、1.68×100Copies/ μ L), by the plasmid control of various concentration
Molecule carries out real-time fluorescent PCR amplification respectively as template, according to the method in document.It is each to react in triplicate, according to not
With the relationship between the Ct value and concentration of concentration template amplification, standard curve is established.
Experimental result:
1, tens of pairs of primers are tested in this example, the experimental results showed that have 4 pairs of primers can directed toward bacteria it is resistance to
The sequence of medicine gene NDM-1 is effectively expanded.The primer and probe sequence for capableing of Successful amplification target sequence finally obtained is detailed
It is shown in Table 7.
The primer and probe sequence used in the experiment of table 7
Directed toward bacteria drug resistant gene NDM-1 sequence, the detection effect of primer pair 1 is best, and high specificity is not non-after amplification
The band of specificity occurs, detection sensitivity highest, the target sequence of minimum detectable 1.68copies/ μ L；Primer pair 2 and 3
Sensitivity it is lower, be able to detect that 1.68 × 102The target sequence of copies/ μ L；Primer pair 4 is able to detect that 1.68 ×
101The target sequence of copies/ μ L, but specificity is poor, has non-specific band to occur.
2, application of the plasmid control molecule pXL08 in real-time fluorescence PCR detection
Using primer pair 1, respectively (such as: 1.68 × 10 with the plasmid control molecule pXL08 of various concentration6copies/μL、
1.68×105copies/μL、1.68×104copies/μL、1.68×103copies/μL、1.68×102copies/μL、
1.68×101copies/μL、1.68×100Copies/ μ L) standard curve of real-time fluorescence PCR is established as standard items.It establishes
Standard curve see that Fig. 3, related coefficient reach 0.999, it is linear good, show that plasmid control molecule pXL08 is suitably applied reality
When fluorescent PCR detect, can be used as the positive criteria substance of real-time fluorescent PCR amplification bacterial resistance gene NDM-1.
Since the research and development of the standard substance of current directed toward bacteria drug resistant gene NDM-1 real-time fluorescence PCR detection method are still
Blank, in practical bacterial resistance gene NDM-1 real-time PCR detection, constituent parts use it is mostly be designed, designed building plasmid
For DNA molecular as standard items, target gene specific fragment therein is different, while lacking unified valued methods, matter
Grain DNA molecular definite value poor accuracy, causes the testing result very different between each laboratory, lack comparativity, validity and
Reliability.Therefore, lack standard when this plasmid control molecule can solve real-time fluorescence PCR detection bacterial resistance gene NDM-1
The problem of substance guarantees the comparable of testing result suitable for a variety of real time fluorescent PCR methods for expanding bacterial resistance gene NDM-1
Property, biometric technology, which is provided, for the detection of bacterial resistance gene NDM-1 real time fluorescent PCR method supports.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (8)
1. a kind of kit of detection bacterium drug resistant gene NDM-1, which is characterized in that include: in the kit
Standard molecule contains polynucleotides shown in SEQ ID NO.:1 in the standard molecule；
It and further include primer sequence in the kit, the primer sequence is for shown in specific amplification SEQ ID NO.:1
Sequence, the primer sequence be SEQ ID NO.:3 and SEQ ID NO.:4 shown in primer sequence.
2. a kind of kit, which is characterized in that the kit includes isolated DNA construction, includes in the DNA construction
Polynucleotides shown in SEQ ID NO.:1 and optional sequence label, cleavage sequence, promoter sequence and/or carrier sequence
Column, and further include primer sequence in the kit, the primer sequence is for shown in specific amplification SEQ ID NO.:1
Sequence, the primer sequence be SEQ ID NO.:3 and SEQ ID NO.:4 shown in primer sequence.
3. kit as claimed in claim 2, which is characterized in that the DNA construction is plasmid or expression vector, the matter
The sequence of grain or expression vector is as shown in SEQ ID NO:2.
4. kit as claimed in claim 1 or 2, which is characterized in that it further include probe in the kit, the probe sequence
Column are as shown in SEQ ID NO.:11.
5. the purposes of kit as claimed in claim 1 or 2, which is characterized in that the inspection for bacterial resistance gene NDM-1
It surveys, it is described to be detected as non-diagnostic purpose.
6. a kind of bacterial resistance gene NDM-1 real-time fluorescence quantitative PCR detection method, which is characterized in that used kit
As described in any one of claim 1-4, and the detection is non-diagnostic purpose and non-treatment purpose.
7. a kind of polynucleotides product, which is characterized in that the product includes:
(i) bacterial resistance gene NDM-1 standard items, the standard items are DNA construction, include SEQ in the DNA construction
Polynucleotides shown in ID NO.:1；
(ii) primer pair of specific amplification bacterial resistance gene NDM-1 sequence, the primer pair are as follows:
The primer pair of Sequence composition shown in SEQ ID NO.:3 and SEQ ID NO.:4.
8. polynucleotides product as claimed in claim 7, which is characterized in that the product further includes (iii) probe, described
Probe sequence is as shown in SEQ ID NO.:11.
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| CN102534014A (en) * | 2012-01-18 | 2012-07-04 | 泰普生物科学（中国）有限公司 | NDM-1 pan-drug resistant gene polymerase chain reaction (PCR) assay kit |
| CN103540660A (en) * | 2013-10-10 | 2014-01-29 | 中国人民解放军疾病预防控制所 | Loop-mediated isothermal amplification (LAMP) method based on TaqMan probe, and LAMP primer and kit special for same |
| CN104232622A (en) * | 2014-09-24 | 2014-12-24 | 中国人民解放军疾病预防控制所 | Nucleic acid isothermal amplification method and application thereof by polymerase spiral reaction |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102534014A (en) * | 2012-01-18 | 2012-07-04 | 泰普生物科学（中国）有限公司 | NDM-1 pan-drug resistant gene polymerase chain reaction (PCR) assay kit |
| CN103540660A (en) * | 2013-10-10 | 2014-01-29 | 中国人民解放军疾病预防控制所 | Loop-mediated isothermal amplification (LAMP) method based on TaqMan probe, and LAMP primer and kit special for same |
| CN104232622A (en) * | 2014-09-24 | 2014-12-24 | 中国人民解放军疾病预防控制所 | Nucleic acid isothermal amplification method and application thereof by polymerase spiral reaction |
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