CN104928300B - A kind of polynucleotides, method and kit for the detection of comma bacillus real-time fluorescence quantitative PCR - Google Patents
A kind of polynucleotides, method and kit for the detection of comma bacillus real-time fluorescence quantitative PCR Download PDFInfo
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- CN104928300B CN104928300B CN201410606449.2A CN201410606449A CN104928300B CN 104928300 B CN104928300 B CN 104928300B CN 201410606449 A CN201410606449 A CN 201410606449A CN 104928300 B CN104928300 B CN 104928300B
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Abstract
The invention discloses a kind of polynucleotides, method and kits for the detection of comma bacillus real-time fluorescence quantitative PCR.Specifically, the present invention provides the polynucleotides that can be used in comma bacillus PCR detection and primer pairs with matching, the polynucleotide sequence is prepared as by standard plasmid molecule PLW07 using suitable skeleton plasmid, 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.
Description
Technical field
The present invention relates to a kind of plasmid molecules of technical field of bioengineering, and in particular to it is real that one kind is suitable for comma bacillus
When fluorescence quantitative PCR detection plasmid control molecule and its construction method, quantitative approach and application.
Background technique
Comma bacillus (Vibrio cholerae) is can cause Yi Lei using diarrhea as the deadly infectious disease of cardinal symptom one
Kind pathogenic bacteria, the quick and precisely identification of comma bacillus, to the prevention and treatment important role of the disease.People send out for a long time
Existing, 0l group cholera vibrio can cause the prevalence of cholera.0139 group of cholera is as emerging infectious disease, and 1992 in India and Meng Jia
Draw state that cholera is caused to be very popular.Also occur caused by 0139 group cholera vibrio in various degree since 1993 in many areas in China
Epidemic situation, in recent years, the ratio that 0139 group of cholera is broken out still constantly are rising.O1 groups and O139 group cholera vibrio it is pathogenic
It is adjusted altogether pili (TCP) depending on its bacterial strain coded product relevant to toxin, such as vibrio cholera toxin, toxin expression regulation albumen
(toxR) etc..The detection method of cholera includes biochemical culture, serological test and bacteriophage typing etc., and there is inspections for these methods
Extracting rate is relatively low, and the needs of epidemic situation control are often not achieved in the disadvantage of review time length.Fluorescence quantifying PCR method then can quickly,
Delicately according to the detection situation of the special gene sequence of comma bacillus, the detection of bacterial strain can be completed within a few hours.Plasmid
DNA standard substance is a kind of recombinant plasmid molecule of specific fragment containing testing goal gene, can in PCR qualitative detection
Can be used as the standard items of quantitative analysis in PCR quantitative analysis as positive control, the standard for constructing quantitative analysis is bent
Line.Plasmid DNA molecule has obtained more and more further investigations and application as the standard substance of genetic test at present, but needle
To the Plasmid DNA standard substance or blank of comma bacillus real-time fluorescence PCR detection method.In practical comma bacillus real-time fluorescence
When PCR, for the plasmid DNA molecules for being designed, designed building mostly that constituent parts use as standard items, target gene therein is special
Property segment is different, while lacking unified valued methods, plasmid DNA molecule definite value poor accuracy, cause each laboratory it
Between testing result very different, lack comparativity.
Summary of the invention
The purpose of the present invention is to provide a kind of polynucleotides for the detection of comma bacillus real-time fluorescence quantitative PCR, side
Method and kit.
The first aspect of the present invention, provides a kind of isolated polynucleotides, and the polynucleotides include comma bacillus poison
The gene order of power regulator toxR.
In another preferred example, the gene order of the comma bacillus virulence regulator toxR 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 100bp-
The polynucleotide sequence of 654bp (preferably 150-645bp, more preferably 200-500bp)；
(d) polynucleotide sequence complementary with any polynucleotide sequence of (a)-(c).
In another preferred example, the sequence of the polynucleotides is selected from the group:
(a) sequence polynucleotide sequence as shown in SEQ ID NO.:2 or SEQ ID NO.:1；
(b) nucleotide sequence and homology >=95% of sequence shown in SEQ ID NO.:2 or SEQ ID NO.:1 are (preferable
Ground >=98%) polynucleotide sequence；
(c) polynucleotide sequence complementary with any polynucleotide sequence of (a)-(b).
The second aspect of the present invention provides a kind of isolated DNA construction, includes the present invention in the DNA construction
First aspect described in polynucleotides and optional sequence label, cleavage sequence, promoter sequence and/or carrier sequence.
It is preferably carried out in mode at of the invention one, the polynucleotides both ends described in first aspect present invention are equipped with linear digestion
Site.
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, standard molecule (the plasmid mark that the plasmid or expression vector are detected as comma bacillus
Quasi-molecule).
In another preferred example, the DNA construction is plasmid or expression vector, and the plasmid or expression vector
Skeleton plasmid is selected from the group: pUC19, pUC18, pUC118, pUC119, pBlueScript II SK and pGEM.
In another preferred example, the sequence of the plasmid is as shown in SEQ ID NO:2.
The third aspect of the present invention provides a kind of kit, includes described in first aspect present invention in the kit
Polynucleotides or second aspect of the present invention described in DNA construction.
In another preferred example, further include primer sequence selected from the group below in the kit:
(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.
It in another preferred example, further include probe sequence shown in SEQ ID NO.:11 in the kit.
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, the detection for comma bacillus.
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 kind of comma bacillus real-time fluorescence quantitative PCR detection method, used mark
Quasi- substance is DNA construction described in polynucleotides described in first aspect present invention or second aspect of the present invention.
In another preferred example, the primer pair used in the method is selected from the group:
(1) primer pair of Sequence composition shown in SEQ ID NO.:3 and SEQ ID NO.:4；
(2) primer pair of Sequence composition shown in SEQ ID NO.:5 and SEQ ID NO.:6；With
(3) primer pair of Sequence composition shown in SEQ ID NO.:7 and SEQ ID NO.:8.
In another preferred example, probe sequence used in the method is as shown in SEQ ID NO.:11.
The sixth aspect of the present invention, provides a kind of polynucleotides product, and the product includes:
(i) comma bacillus standard items, the standard items are selected from: polynucleotides described in first aspect present invention and Ben Fa
DNA construction described in bright second aspect；
(ii) primer pair of specific amplification comma bacillus sequence, the primer pair are selected from the group:
(1) primer pair of Sequence composition shown in SEQ ID NO.:3 and SEQ ID NO.:4；
(2) primer pair of Sequence composition shown in SEQ ID NO.:5 and SEQ ID NO.:6；With
(3) primer pair of Sequence composition shown in SEQ ID NO.:7 and SEQ ID NO.:8.
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.
It in another preferred example, further include probe sequence shown in SEQ ID NO.:11 in the product.
The seventh aspect of the present invention, provides a kind of preparation method of the plasmid control molecule of comma bacillus, and feature exists
In, comprising the following steps:
1. the virulence regulator toxR gene order of artificial synthesized comma bacillus, the virulence regulator toxR expressing gene
Sequence is as shown in SEQ ID NO:1 in sequence table；
2. by step 1. gained comma bacillus virulence regulator toxR expressing gene sequence be cloned on cloning vector, obtain
To the plasmid control molecule of comma bacillus.
In another preferred example, the step 2. described in cloning vector be plasmid pUC19, pUC18, pUC118,
PUC119, pBlueScript II SK or pGEM；Preferably, 2. the cloning vector is pUC19 to 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 PLW07 of the present invention.
Fig. 2 is plasmid control molecule PLW07 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 of the present invention.
Specific embodiment
The present inventor obtains one section of multicore glycosides that can be used in comma bacillus PCR detection by extensive and in-depth research
Acid sequence and primer pair with matching, the experimental results showed that, using suitable skeleton plasmid by the polynucleotides sequence
Column are prepared as standard plasmid molecule, and primer pair of the invention is cooperated to carry out real-time fluorescence PCR detection, have splendid specificity
And sensitivity, and have good stability.
The technical problem to be solved by the present invention is in order to overcome in existing comma bacillus real-time fluorescence PCR detection method
The problem of lacking positive criteria product and positive criteria product configuration, provides a kind of suitable for comma bacillus real-time fluorescence PCR detection
Plasmid control molecule and the plasmid control molecule construction method, quantitative approach and application.
The principle of real-time fluorescence PCR detection comma bacillus
It can virulence regulator toxR gene in specific amplification cholera vibrio gene group DNA using real-time fluorescent PCR technology
Sequence, design is for the primer of the above target gene and the probe of both ends mark fluorescent, amplification assay sample DNA.Real-time fluorescence PCR
It can be by detecting the increase of fluorescence signal come real-time monitoring PCR product.At the same time, with identical primer, probe and condition
Expand the positive criteria substance (or positive criteria molecule) of known concentration.In PCR method, positive criteria substance (or positive criteria
Molecule) it can be used as positive control；In real-time fluorescence PCR, positive criteria substance (or positive criteria molecule) can construct stabilization
Standard curve, the absolute content (copy number or concentration) that gene is corresponded in sample can be calculated separately out according to standard curve.
Standard substance
Standard substance is that there are one or more 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
In the present invention, the specific sequence of the comma bacillus is one of comma bacillus virulence regulator toxR gene
Point, length 885bp.
In order to solve the above technical problems, one of the technical solution that the present invention takes are as follows: a kind of plasmid control of comma bacillus
Molecule, the plasmid control molecule include comma bacillus virulence regulator toxR gene order, the comma bacillus virulence regulator
ToxR gene order is as shown in SEQ ID NO:1 in sequence table:
Plasmid control molecule of the invention, the target gene comma bacillus that preferably PCR containing comma bacillus is detected respectively
Virulence regulator toxR gene order.
Heretofore described comma bacillus virulence regulator toxR gene, rises in the synthesis of DNA, RNA and protein
Effect, it can be used as a kind of target gene of comma bacillus detection, the sequence of the comma bacillus virulence regulator toxR gene compared with
It is good 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
1307 are toxR gene order:
In order to solve the above technical problems, the two of the technical solution that the present invention takes are as follows: a kind of comma bacillus as described above
The quantitative approach of plasmid control molecule 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.
In order to solve the above technical problems, the three of the technical solution that the present invention takes are as follows: a kind of comma bacillus real-time fluorescence is fixed
PCR detection method is measured, used standard substance is plasmid control molecule as described above.
In order to solve the above technical problems, the four of the technical solution that the present invention takes are as follows: a kind of comma bacillus as described above
Plasmid control molecule preparation method, comprising the following steps:
1. the virulence regulator toxR gene order of artificial synthesized comma bacillus, the virulence regulator toxR gene order
As shown in SEQ ID NO:1 in sequence table；
2. by step 1. gained comma bacillus virulence regulator toxR gene order be cloned on cloning vector, obtain suddenly
The plasmid control molecule of random vibrios.
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 the virulence regulator of comma bacillus in the Genbank of NCBI (US National Biotechnology Information center)
ToxR gene；
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 any conventional carrier, it is preferred that cloning vector, it more preferably can be in large intestine
The cloning vector being proliferated in bacillus, 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
Detect the ability of the standard substance of comma bacillus.
The present invention also provides the kit for the above method, contained in the kit polynucleotides of the invention or
Person DNA construction and primer sequence selected from the group below:
(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；
(4) primer sequence shown in SEQ ID NO.:9 and SEQ ID NO.:10；And optionally SEQ ID NO.:11
Shown in probe sequence.
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.
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 comma bacillus real-time fluorescence PCR detection Plays material want, guarantee that comma bacillus is glimmering in real time
The comparativity of light PCR method testing result provides quality control for the detection of comma bacillus real time fluorescent PCR method；
(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 detects comma bacillus, high specificity, high sensitivity, linear stable.
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.
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, the gene virulence regulator toxR gene order of comma bacillus is searched in GenBank,
2, above-mentioned sequence is analyzed, selects suitable sequence and suitable restriction enzyme site, gene virulence regulator
ToxR gene order length is 885bp, 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, gained gene virulence regulator toxR gene
It is (public purchased from TAKARA that sequence is cloned into plasmid vector pUC19 as shown in SEQ ID NO:1 in sequence table, by gained full-length gene
Department) in, building obtains plasmid control molecule PLW07 (the SEQ ID NO:2 in its sequence such as sequence table comprising toxR gene order
It is shown), the plasmid map of building gained plasmid control molecule PLW07 is as shown in Figure 1.
4, mass propgation contains the recombination bacillus coli comprising gained plasmid control molecule PLW07, is largely mentioned using plasmid
It takes kit (OMEGA) to extract plasmid, obtains the plasmid control molecule PLW07 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 PLW07
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 423rd to the 1307th be gene virulence regulator toxR gene.
Experimental result are as follows: largely extracted by sequence selection step, the artificial synthesized step of full genome and plasmid and etc.
The plasmid control molecule PLW07 of high-purity is obtained 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 PLW07 of embodiment
Uniformity is to characterize the coherency state of the relevant structure of one or more characteristics or composition in 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 results are shown in Table 1 for plasmid control molecule PLW07 uniformity testing
1 plasmid control molecule pLW07 uniformity testing data of table (unit: ng/ μ L)
| Sample | Repeat 1 | Repeat 2 | Repeat 3 | Average value |
| 1 | 94.9 | 95.4 | 94.4 | 94.90 |
| 2 | 94.9 | 95 | 94.1 | 94.67 |
| 4 | 94.1 | 95.4 | 93.1 | 94.20 |
| 5 | 93.3 | 95.1 | 93 | 93.80 |
| 6 | 93.5 | 93.7 | 93.9 | 93.70 |
| 7 | 93.3 | 94.9 | 93.2 | 93.80 |
| 8 | 93.6 | 92.3 | 95.8 | 93.90 |
| 9 | 92.6 | 94 | 92.9 | 93.17 |
| 10 | 93.8 | 94 | 93.6 | 93.80 |
| 11 | 92.6 | 93 | 92.4 | 92.67 |
| 12 | 92.7 | 95.4 | 92.7 | 93.60 |
| 13 | 93.5 | 94.4 | 92.2 | 93.37 |
| 14 | 93.3 | 94 | 93.8 | 93.70 |
| 15 | 94.1 | 93.7 | 91.8 | 93.20 |
The homogeneity statistical result of 2 plasmid control molecule pLW07 of table
| Plasmid DNA standard substance | Q1 | Q2 | F value | F0.05(14,30) |
| pLW07 | 13.72 | 27.31 | 1.08 | 2.04 |
Homogeneity statistical 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 the plasmid mark
Quasi-molecule PLW07 is uniformly, to meet " JJG1006-94 primary standard substance technical specification " and uniformly examine Eligibility requirements.
The study on the stability of 3 plasmid control molecule PLW07 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 Nanodrops 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, plasmid control molecule prepare after the completion of 0th month, 0.5 month, 1 month, 2 months, 4 months, 6 months it is right
The plasmid control molecule (- 20 DEG C of preservations) of preparation randomly selects 3 bottles, and each sample replication 3 times is averaged, is grown
Phase study on the stability.This research uses ultraviolet spectrophotometry and has carried out tracking investigation to plasmid control molecule.
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 PLW07 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 PLW07 of table
| Time (moon) | Repeat 1 | Repeat 2 | Repeat 3 | Average value | SD |
| 0 | 91.25 | 91.1 | 92.45 | 91.60 | 0.74 |
| 0.5 | 92.3 | 91.2 | 93.3 | 92.27 | 1.05 |
| 1 | 90.9 | 91.2 | 89.7 | 90.6 | 0.80 |
| 2 | 90.7 | 89.1 | 89.5 | 89.77 | 0.83 |
| 4 | 90.5 | 89 | 89.9 | 89.80 | 0.75 |
| 6 | 91.3 | 89.5 | 92.1 | 90.97 | 1.33 |
Statistical result is as shown in table 4.
The stability statistical result of 4 plasmid control molecule PLW07 of table
| Plasmid DNA standard substance | β1 | β0 | s(β1) | t0.95,n-2·s(β1) |
| PLW07 | 0.17 | 91.26 | 0.19 | 0.52 |
By statistical result it is found that plasmid control molecule PLW07 saved under the conditions of -20 DEG C 6 months be stable.
Experimental example 4 quantifies plasmid control molecule PLW07 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 are as follows: pass through ultraviolet spectrophotometry definite value, the concentration of plasmid control molecule PLW07 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 | SD |
| PLW07 | 94.9 | 94.9 | 94.1 | 93.3 | 93.5 | 93.3 | 93.6 | 92.6 | 93.78 | 0.81 |
Embodiment 5 quantifies plasmid control molecule PLW07 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 PLW07 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 PLW07 is diluted 3000 times, the plasmid control molecule after HR-ICP-MS detection dilution, and
The phosphorus content of the plasmid control molecule after dilution is obtained according to standard curve；
5, the concentration of plasmid control molecule PLW07 is calculated according to the phosphorus content and extension rate that measure；
6, each sample test eight times, are averaged.
Experimental result are as follows: be computed, the content of the P elements of plasmid control molecule PLW07 is 10.22%.By HR-
It is as shown in table 6 that ICP-MS definite value obtains plasmid control molecule phosphorus element content data, extension rate 3000, and conversion obtains plasmid
The mass concentration of molecule, as shown in table 7.
The phosphorus element content (unit: μ g/L) of 6 plasmid control molecule PLW07 of table
| Plasmid | Repeat 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | Average value | SD |
| PLW07 | 2.38 | 2.28 | 2.20 | 2.24 | 2.39 | 2.33 | 2.34 | 2.36 | 2.32 | 0.07 |
The mass concentration result (unit: ng/ μ L) of 7 plasmid control molecule PLW07 of table
Application of the plasmid control molecule PLW07 that embodiment 6 is developed in real-time fluorescence PCR detection
Experiment reagent: the present inventor is transferred to the primer and probe of design using tens of pairs of primers of conventional design
It is detected after being synthesized by precious bioengineering (Dalian) Co., Ltd, Master Mix is purchased from Life technology company.It is real
Testing instrument includes: real-time fluorescence quantitative PCR amplification instrument (Life technology) centrifuge, thermostat water bath, incubator, day
Equality.
Application of the plasmid control molecule PLW07 in real-time fluorescence PCR detection:
Primer, probe, PCR detection architecture
PCR is loaded system:
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 PLW07 that resulting plasmid control molecule PLW07 is diluted to various concentration
(2.6×106copies/μL、2.6×105copies/μL、2.6×104copies/μL、2.6×103copies/μL、2.6×
102copies/μL、2.6×101copies/μL、2.6×100Copies/ μ L) by the plasmid control molecule PLW07 of various concentration
Respectively as template, real-time fluorescent PCR amplification is carried out according to the method in document.Each reaction is in triplicate, dense according to difference
The relationship between the Ct value and concentration of template amplification is spent, standard curve is established.
Experimental result
1,4 pairs of primer pairs that can effectively amplify target sequence have been filtered out from tens of pairs of primers, specifically such as 8 institute of table
Show, wherein the expanding effect of primer pair 1 is best,
See Table 8 for details for the primer and probe sequence used in experiment.
The primer and probe sequence used in the experiment of table 8
The detection effect of primer pair 1 is best, high specificity, does not have nonspecific band to occur after amplification, detects sensitive
Spend highest, the target sequence of minimum detectable 2.6copies/ μ L；The sensitivity of primer pair 2 and 3 is lower, is able to detect that 2.6
×102The target sequence of copies/ μ L；Primer pair 4 is able to detect that 2.6 × 101The target sequence of copies/ μ L, but it is special
It is anisotropic poor, there is non-specific band to occur.
2, real-time fluorescence PCR detection is combined using plasmid control molecule PLW07 and primer pair 1
Using above-mentioned PCR detection architecture and reaction condition, respectively with the plasmid control molecule PLW07 of various concentration (such as:
2.6×106copies/μL、2.6×105copies/μL、2.6×104copies/μL、2.6×103copies/μL、2.6×
102copies/μL、2.6×101copies/μL、2.6×100Copies/ μ L) mark of real-time fluorescence PCR is established as standard items
Directrix curve.The standard curve of foundation is shown in that Fig. 3, related coefficient reach 0.9927, and linear good, blank control is minimum without amplified peak
The target sequence of detectable 2.6copies/ μ L, shows that plasmid control molecule PLW07 is suitably applied real-time fluorescence PCR detection,
It can be used as the positive criteria substance of real-time fluorescent PCR amplification comma bacillus.
In the detection of practical comma bacillus, using the linear good standard curve, examined by experimental fluorescence PCR method
Measure the copy number of comma bacillus specific gene in sample to be tested, and can copy number according to comma bacillus specific gene and bacterium
Linear relationship between sum converses the specific number of comma bacillus.It is examined due to being directed to comma bacillus real-time fluorescence PCR at present
The research and development of the standard substance of survey method are still blank, in the real-time PCR detection of practical comma bacillus, constituent parts use it is mostly be from
For the plasmid DNA molecule of row design construction as standard items, target gene specific fragment therein is different, while lacking system
One valued methods, plasmid DNA molecule definite value poor accuracy cause the testing result very different between each laboratory, lack
Comparativity, validity and reliability.Therefore, it is lacked when this plasmid control molecule can solve real-time fluorescence PCR detection comma bacillus
The problem of weary standard substance is protected suitable for the real time fluorescent PCR method of a variety of amplification comma bacillus virulence regulator toxR genes
The comparativity of testing result is demonstrate,proved, biometric technology is provided for the detection of comma bacillus real time fluorescent PCR method and 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 (7)
1. a kind of kit, which is characterized in that the kit includes isolated DNA construction, and the DNA construction includes suddenly
The gene order and optional sequence label, cleavage sequence, promoter sequence and/or load of random vibrios virulence regulator toxR
Body sequence；The isolated DNA construction is sequence polynucleotides sequence as shown in SEQ ID NO.:2 or SEQ ID NO.:1
Column；
It and further include the primer pair of specific amplification comma bacillus sequence, the primer pair is SEQ ID NO.:3 and SEQ ID
Primer sequence shown in NO.:4.
2. kit as described in claim 1, which is characterized in that the DNA construction is plasmid or expression vector, and institute
The skeleton plasmid for stating plasmid or expression vector is selected from the group: pUC19, pUC18, pUC118, pUC119, pBlueScript II
SK and pGEM.
3. kit as described in claim 1, which is characterized in that further include shown in SEQ ID NO.:11 in the kit
Probe sequence.
4. the purposes of kit as described in claim 1, which is characterized in that the detection for comma bacillus.
5. a kind of purposes of kit described in claim 1, which is characterized in that it is used to prepare the purposes of detection comma bacillus,
Wherein, the polynucleotides are used as detecting the standard substance of comma bacillus.
6. a kind of polynucleotides product, which is characterized in that the product includes:
(i) comma bacillus standard items, the standard items are a polynucleotides, and the polynucleotides include comma bacillus virulence tune
It controls the gene order of sub- toxR and the polynucleotides is sequence as shown in SEQ ID NO.:2 or SEQ ID NO.:1
Polynucleotide sequence；And
(ii) primer pair of specific amplification comma bacillus sequence, the primer pair are SEQ ID NO.:3 and SEQ ID NO.:4
Shown in Sequence composition primer pair.
7. polynucleotides product as claimed in claim 6, which is characterized in that the polynucleotides are a kind of isolated DNA structure
Object is built, and includes the gene order and optional label sequence of comma bacillus virulence regulator toxR in the DNA construction
Column, cleavage sequence, promoter sequence and/or carrier sequence.
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Citations (2)
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|---|---|---|---|---|
| US20090226895A1 (en) * | 2007-06-15 | 2009-09-10 | The Hong Kong Polytechnic University | Method of detecting vibrio parahaemolyticus via real-time PCR-hybridization |
| CN102719542A (en) * | 2012-06-19 | 2012-10-10 | 上海仁度生物科技有限公司 | Simultaneous amplification and testing reagent kit for VC (vibrio cholerae) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20090226895A1 (en) * | 2007-06-15 | 2009-09-10 | The Hong Kong Polytechnic University | Method of detecting vibrio parahaemolyticus via real-time PCR-hybridization |
| CN102719542A (en) * | 2012-06-19 | 2012-10-10 | 上海仁度生物科技有限公司 | Simultaneous amplification and testing reagent kit for VC (vibrio cholerae) |
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| Cholera Toxin Transcriptional Activator ToxR Is a Transmembrane DNA Binding Protein;Virginia L. Miller et al.;《Cell》;19870130;第48卷;第271-279页 * |
| CP001235 REGION: 1069284..1070168;NCBI;《GenBank》;20140131;第1页 * |
| Detection of Vibrio cholerae by Real-Time Nucleic Acid Sequence-Based Amplification;Else M. Fykse et al.;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;20070331;第73卷(第5期);第1457-1466页 * |
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