CN103497964A - Plasmid standard molecular adaptable to sakazakii real-time fluorescent quantitation PCR (polymerase chain reaction) detection - Google Patents

Abstract

The invention discloses a plasmid standard molecular of sakazakii, an establishing method thereof, a quantitation method thereof and application. The plasmid standard molecular comprises an ITS sequence containing the sakazakii , and the ITS sequence is shown as SEQ ID NO: 1 in sequence lists. The plasmid standard molecular is quantitated by the ultraviolet light spectrophotometry method and a high resolution inductively coupled plasma emission mass spectrometry (HR-ICP-MS), and good traceability is guaranteed. By the aid of the plasmid standard molecular, the problem of the lack of standard material during the sakazakii real-time fluorescent quantitation PCR (polymerase chain reaction) detection, comparability of results of the sakazakii real-time fluorescent quantitation PCR detection, and a reliable quality control method is provided for the sakazakii real-time fluorescent quantitation PCR detection.

Description

A kind of plasmid control molecule that is applicable to the detection of Enterobacter sakazakii real-time fluorescence quantitative PCR

Technical field

The present invention relates to a kind of plasmid molecule of technical field of bioengineering, be specifically related to a kind of plasmid control molecule and construction process, quantivative approach and application that the Enterobacter sakazakii real-time fluorescence quantitative PCR detects that be applicable to.

Background technology

Enterobacter sakazakii (Enterobacter sakazakii) is newfound a kind of pathogenic bacterium that cause extensive concern in recent years in milk-product.Since 1961, the case by this microbial neonatal meningitis, microbemia and necrotizing enterocolitis in world wide was in the news in succession, and Enterobacter sakazakii is used as a kind of important foodborne bacterial pathogens and receives publicity.Many parts of research reports have shown that infant formula powder is the main infection channel that current discovery causes baby and premature infant's meningitis, septicemia and necrotizing enterocolitis, and overall mortality rate is up to 80%, therefore caused the attention of the multinational relevant departments in the world.

At present the detection method of Enterobacter sakazakii is divided into two classes, and a class is traditional cultivation and improves one's methods, and depend on biochemistry and Morphological Characteristics, sense cycle is longer, poor operability; One class is polymerase chain reaction (polymerase chain reaction, PCR) methods involving, comprise regular-PCR method and real time fluorescent PCR method, mainly to be different from a plurality of target genes of other bacterium of enterobacteriaceae for Enterobacter sakazakii, select distinguished sequence, set up PCR and real time fluorescent PCR method.Enterobacter sakazakii PCR related detecting method development in recent years is rapid, due to its quick, sensitive, special characteristic, has become the reliable detection method of food borne pathogenic microorganism.

The plasmid DNA reference material is a kind of recombinant plasmid molecule of the specific fragment that contains the testing goal gene, can be used as positive control in the PCR qualitative detection, can be used as the standard substance of quantitative analysis in the PCR quantitative analysis, build the typical curve of quantitative analysis.Plasmid DNA molecule has obtained increasing further investigation and application as the reference material of gene test at present, but still blank for the plasmid DNA reference material of Enterobacter sakazakii real-time fluorescence PCR detection method.When actual Enterobacter sakazakii real-time fluorescence PCR, the plasmid DNA molecule that is mostly the designed, designed structure that constituent parts is used is as standard substance, goal gene specific fragment wherein is different, lack unified valued methods simultaneously, plasmid DNA molecule definite value poor accuracy, cause the detected result difference between each laboratory very big, lack comparability.

Summary of the invention

Technical problem to be solved by this invention is the problem that lacks positive criteria product and the configuration of positive criteria product in order to overcome in existing Enterobacter sakazakii real-time fluorescence PCR detection method, and a kind of the be applicable to plasmid control molecule of Enterobacter sakazakii real-time fluorescence PCR detection and construction process, quantivative approach and the application of this plasmid control molecule are provided.

Real-time fluorescence PCR detects the principle of Enterobacter sakazakii

But district (ITS) sequence between 16S～23S rDNA in employing real-time fluorescence PCR technology specific amplification Enterobacter sakazakii genomic dna.Design is for the primer of above target gene and the probe of two ends mark fluorescent, amplification assay sample DNA.Real-time fluorescence PCR can carry out Real-Time Monitoring PCR product by the increase that detects fluorescent signal.Meanwhile, with the positive criteria material (or positive criteria molecule) of identical primer, probe and condition amplification concentration known.In the regular-PCR method, positive criteria material (or positive criteria molecule) can be used as positive control; In real time fluorescent PCR method, positive criteria material (or positive criteria molecule) can build stable typical curve, can calculate respectively the absolute content (copy number or concentration) of corresponding gene in sample according to typical curve.

Reference material

Reference material is to have one or more enough all even fine characteristic values of having determined, in order to correcting device, estimates measuring method or to material or the material of material assignment.

Plasmid control molecule

Plasmid control molecule is a kind of recombinant plasmid molecule of the specific fragment that contains the testing goal gene, in the PCR qualitative detection, can be used as positive control, can be used as the standard substance of quantitative analysis in the PCR quantitative analysis, builds the typical curve of quantitative analysis.The advantage of plasmid molecule is mainly to cultivate in a large number by microorganism, and DNA is easy to amplification, thus the reference material of unlimited stable quantity can be provided, and purity is higher; And processing ease, stability is high, and same standard molecule can comprise a plurality of external source goal gene simultaneously, and economy is efficient again.

For solving the problems of the technologies described above, one of technical scheme that the present invention takes is: the plasmid control molecule of a kind of Enterobacter sakazakii, and the ITS sequence that this plasmid control molecule comprises Enterobacter sakazakii, described ITS sequence is as shown in SEQ ID NO:1 in sequence table.

Between the 16S described in the present invention～23S rDNA, district (ITS) sequence refers in the rDNA gene, the gene intervening sequence of 16S rDNA and 23S rDNA, its length and sequence variation are larger, its amplified material is carried out to RFLP or sequential analysis, can be used for the evaluation of classifying of different biotypes, bacterial strain, kind, genus to bacterium, even the very near kind for the differentiation relation.It is also target gene commonly used during Enterobacter sakazakii detects, and its sequence is preferably as shown in SEQ ID NO:1.

Plasmid control molecule of the present invention, preferably contain the target gene ITS sequence that the Enterobacter sakazakii fluorescent PCR detects.The sequence of plasmid control molecule of the present invention is preferably as shown in SEQ ID NO:2.

For solving the problems of the technologies described above, two of the technical scheme that the present invention takes is: the quantivative approach of the plasmid control molecule of a kind of Enterobacter sakazakii as mentioned above, and it comprises the following steps:

1. extract plasmid control molecule;

2. according to step 1. based composition and the sequence length of the plasmid control molecule of gained, calculate the content of the phosphoric in plasmid control molecule;

3. prepare the phosphorus standardized solution of gradient concentration, and make the high resolution inductively coupled plasma with this standardized solution and launch mass spectral:mass spectrographic typical curve;

4. high resolution inductively coupled plasma emission mass spectrometric detection plasmid control molecule solution, and according to step 3. the typical curve of gained draw the phosphorus content of plasmid control molecule solution;

5. according to the 4. 2. content of the phosphoric in the plasmid control molecule of gained of the phosphorus content of the plasmid control molecule solution of gained and step of step, calculate the concentration of plasmid control molecule.

Wherein the condition of the described 3. high resolution of step inductively coupled plasma emission mass spectrometric detection is as follows: cooling gas flow is preferably 10L/min～25L/min, be 16.86L/min best, the assisted gas flow is preferably 0.5L/min～3.0L/min, be 0.88L/min best, the atomization gas flow is for being preferably 0.5L/min～1.5L/min, be more preferably 1.123L/min, power is preferably 1200W～1400W, is 1350W best.

The quantivative approach of plasmid control molecule of the present invention preferably comprises ultraviolet spectrophotometry and High resolution-inductive coupled plasma mass spectrometry (HR-ICP-MS).

Wherein ultraviolet spectrophotometry preferably comprises the following steps the plasmid control molecule quantivative approach:

1. extract plasmid control molecule;

2. with the TE damping fluid, ultraviolet spectrophotometer being proofreaied and correct is zero point;

3. get the needs of appropriate DNA(according to instrument) to cuvette (nanodrop directly puts at surveyed area), the recording instrumnet reading;

If 4. can directly read direct record of DNA concentration; If can not, recording the optical density(OD) of sample at 260nm and 280nm, the concentration of DNA sample is OD260 * nucleic acid extension rate * 50, concentration unit is ng/ μ L.

(2) HR-ICP-MS preferably comprises the following steps the plasmid control molecule quantivative approach:

1. extract plasmid control molecule;

2. according to based composition and the sequence length of plasmid control molecule, calculate the content of the phosphoric of plasmid control molecule;

3. prepare the phosphorus standardized solution of gradient concentration, and make the typical curve of HR-ICP-MS with this standardized solution.

4. HR-ICP-MS detects plasmid control molecule, and draws the phosphorus content of plasmid control molecule according to typical curve.

5. calculate the concentration of plasmid control molecule according to the phosphorus content of plasmid control molecule.

For solving the problems of the technologies described above, three of the technical scheme that the present invention takes is: a kind of Enterobacter sakazakii real-time fluorescence quantitative PCR detection method, its accepted standard material is plasmid control molecule as above.

For solving the problems of the technologies described above, four of the technical scheme that the present invention takes is: the preparation method of the plasmid control molecule of a kind of Enterobacter sakazakii as above comprises the following steps:

1. the ITS sequence of synthetic Enterobacter sakazakii, described ITS sequence is as shown in SEQ ID NO:2 in sequence table;

2. by step 1. the ITS sequence clone of gained Enterobacter sakazakii to cloning vector, obtain the plasmid control molecule of Enterobacter sakazakii.

Wherein step 1. the method for described synthetic preferably comprise synthetic this sequence of full gene or obtain this sequence by the pcr amplification technology.

Plasmid control molecule construction process of the present invention preferably comprises the following steps:

1. in the NCBI(U.S. state-run biotechnology information center) Genbank in the inquiry Enterobacter sakazakii the ITS sequence;

2. above-mentioned sequence is analyzed, selected suitable sequence and suitable restriction enzyme site, and restriction enzyme site is added to 5 ' end and the 3 ' end of selected sequence.

3. the sequence after processing is carried out to the service of full gene synthetic, comprise the work such as synthetic, DNA fragmentation splicing of strand Oligo DNA, and full-length gene is cloned in plasmid vector, obtain plasmid control molecule pXL06.

Described plasmid vector can be any conventional carrier, preferably cloning vector, the cloning vector that more preferably can breed in intestinal bacteria, described cloning vector is more preferably: pUC19, pUC18, pUC118, pUC119, pBlueScript II SK or pGEM serial carrier are pUC19 best.

4. the sequence of sequence verification plasmid control molecule pXL06.

5. the real-time fluorescence PCR detection method of plasmid control molecule pXL06 checking.

Described real-time fluorescence PCR detection method checking, refer to and detect plasmid control molecule pXL06 carrying out the specificity of real-time fluorescence PCR while analyzing and building the characteristic such as typical curve ability, using and identify that this plasmid control molecule detects the ability of the reference material of Enterobacter sakazakii as real time fluorescent PCR method.

On the basis that meets this area general knowledge, above-mentioned each optimum condition, but arbitrary combination obtains the preferred embodiments of the invention.

Agents useful for same of the present invention and raw material be commercially available obtaining all.

Positive progressive effect of the present invention is: the present invention utilizes the method for full gene synthetic to build to contain the Enterobacter sakazakii real-time fluorescence PCR to detect the plasmid control molecule of district (ITS) sequence between target gene 16S～23S rDNA, and utilize ultraviolet spectrophotometry and high resolution inductively coupled plasma emission mass spectrum (HR-ICP-MS) to carry out quantitatively, having guaranteed the traceability that it is good to it.It is strong that plasmid control molecule of the present invention has homogeneity, the advantage that stability is high, the invention solves the difficult problem that the Enterobacter sakazakii real-time fluorescence PCR detects the Plays material want simultaneously, guarantee the comparability of Enterobacter sakazakii real time fluorescent PCR method detected result, for the Enterobacter sakazakii real time fluorescent PCR method detects, provide quality control.

The accompanying drawing explanation

Fig. 1 is the collection of illustrative plates of plasmid control molecule pXL06 of the present invention.

Fig. 2 is gained plasmid control molecule pXL06 Detection of Stability of the present invention figure as a result

Fig. 3 is the real-time fluorescence PCR typical curve that utilizes plasmid control molecule pXL06 of the present invention to set up.

Embodiment

Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, according to ordinary method and condition, or select according to catalogue.

The structure of embodiment 1 plasmid control molecule

Experiment reagent and laboratory apparatus:

Plasmid extracts test kit (OMEGA) in a large number, and other biochemical reagents are import packing or domestic analytical pure biochemical reagents; Laboratory apparatus comprises whizzer, thermostat water bath, constant-temperature shaking incubator, liquid-transfering gun etc.

Experimental technique comprises the following steps:

1, the ITS sequence of search Enterobacter sakazakii in GenBank,

2, above-mentioned sequence is analyzed, selected suitable sequence and suitable restriction enzyme site.ITS gene order length is 694bp, and two ends add Hind III restriction enzyme site;

3, the sequence after processing is delivered to precious biotechnology (Dalian) company limited, be responsible for carrying out the service of full gene synthetic by it, comprise the work such as synthetic, DNA fragmentation splicing of strand Oligo DNA, gained ITS sequence is as shown in SEQ ID NO:1 in sequence table.The gained full-length gene is cloned in plasmid vector pUC19, build and obtain the plasmid control molecule that comprises the ITS gene order, by its sequence of gained plasmid control molecule called after pXL06(as shown in SEQ ID NO:2 in sequence table), the plasmid map collection of illustrative plates of this plasmid is as shown in Figure 1.

4, a large amount of cultivation contained the recombination bacillus coli that comprises gained pXL06, utilizes plasmid to extract in a large number test kit (OMEGA) and extracts plasmid, obtains highly purified plasmid control molecule pXL06.Carry out purity check through ultraviolet spectrophotometer and electrophoresis, the plasmid DNA standard molecule is placed in-20 ℃ of preservations afterwards, and the method for extracting plasmid comprises the following steps:

A, by the bacterium of 100～200mL incubated overnight in room temperature 5000 * g centrifugal 10 minutes, supernatant discarded;

B, add 12mL Solution I (containing RNase A), vibration fully mixes;

C, add 12mL Solution II, the gentleness that turns upside down mixes, and room temperature is placed 2 minutes so that the abundant cracking of thalline;

D, add 16mL Solution III, fully put upside down up and down immediately and mix for several times, until form uniform white precipitate;

E, >=12000 * g, 4 ℃ are centrifugal 10 minutes;

F, absorption 20mL supernatant move in a clean HiBind Maxi adsorption column (being placed in the 50mL collection tube) carefully, centrifugal 5 minutes of 5000 * g room temperature;

G, discard the liquid in collection tube, add 10mL Buffer HB to clean adsorption column, centrifugal 5 minutes of 5000 * g room temperature;

H, discard the liquid in collection tube, add the dilution of 15mL DNA Wash Buffer(dehydrated alcohol) clean adsorption column;

I, repeat above-mentioned cleaning step;

Centrifugal 15 minutes of j, 6000 * g are with dry adsorption column;

K, adsorption column is placed in to a clean 50mL pipe, adds 2mL～3mLTE damping fluid, room temperature is placed 1～2 minute, and within centrifugal 2 minutes, with eluted dna, the gained DNA solution is the plasmid control molecule solution of extraction to 8000 * g, is kept at-20 ℃.

5, the sequence verification plasmid control divides pXL06.

The plasmid control molecule of extraction is delivered to eight order-checking companies and carry out the sequence checking, eight order-checking companies are respectively Beijing Liuhe Huada Genomics Technology Co., Ltd, Shanghai Bo Shang Bioisystech Co., Ltd, the English Weihe River prompt base (Shanghai) trade Co., Ltd, Shanghai Jie Li Bioisystech Co., Ltd, Shanghai Mei Ji Bioisystech Co., Ltd, Shanghai Sheng Gong Bioisystech Co., Ltd, Suzhou gold only intelligence bio tech ltd, precious biotinylated biomolecule Engineering Co., Ltd.For the plasmid DNA reference material, sequence length with direct relation is quantitatively arranged, it is for the requirement of definite value in Developments of certified reference samples that 8 different order-checking companies carry out sequencing.Refer to ISO guide rule 35, the specific requirement of reference material definite value part.

Sequence is investigated formula: sequence accuracy=correct base number/base sum

Sequencing result proves, the sequence accuracy that the unit of all participation sequences checkings obtains is 100%.

Experimental result is: through steps such as sequence selection step, full gene synthetic step and a large amount of extractions of plasmid, obtain highly purified plasmid control molecule pXL06 through sequence verification, plasmid control molecule Insert Fragment sequence conforms to fully with design, and in this plasmid sequence, the 450th is the ITS sequence to the 1144th.

The uniformity testing of embodiment 2 plasmid control molecule pXL06

Homogeneity is to characterize structure that in material, one or more characteristics are relevant or the coherency state of composition.Take from Different Package unit (as bottle, bag etc.) or take from the sample of the prescribed level of same packaging unit different positions by measurement, measuring result drops in the regulation range of uncertainty, can think that this reference material is uniform to the characteristic quantity of appointment.Homogeneity is the base attribute of reference material, for the spatial distribution characteristic of description standard substance characteristics.Must carry out the homogeneity assessment in development (production) process of reference material, to prove it, there is good homogeneity.The plasmid control molecule had good uniformity, its value can not be subject to the impact of the factor such as packing, and the value difference between each bottle is little, has therefore guaranteed the reliability of detected result.

Experiment reagent TE damping fluid (pH7.5).Laboratory apparatus Nanodrops ND-2000.

Experimental technique:

1, according to " JJG1006-1994 primary standard material technical specifications " homogeneity draw samples requirement, from each plasmid DNA reference material, randomly draw 15 bottles.

2,, to get each sample sampling 3 times, each sampling amount is 1 μ L.Uv-spectrophotometric replication 3 times of each sampling amount, average.

3, measurement result is added up judgement uniformity testing result with the F method of inspection.Concrete grammar is: extract m sample, record under the same conditions m group equally accurate measurement data, if measure the variance there was no significant difference, should meet the statistical requirements of following formula.

$F=\frac{\frac{Q_1}{v_1}}{\frac{Q_2}{{\mathrm{<figure-callout\; id="2"\; label="v"\; filenames="HDA0000396196040000012.png,HDA0000396196040000021.png"\; state="\{\{state\}\}">v</figure-callout>}}_2}}\&le;F_{\mathrm{\&alpha;}}({\mathrm{\&nu;}}_1,{\mathrm{\&nu;}}_2)$

Wherein between group, sum of squares of deviations calculation formula is:

${\mathrm{<figure-callout\; id="1"\; label="Q"\; filenames="HDA0000396196040000011.png,HDA0000396196040000012.png"\; state="\{\{state\}\}">Q</figure-callout>}}_1=\underset{i=1}{\overset{m}{\mathrm{\&Sigma;}}}{n}_i{(\stackrel{\&OverBar;}{x_i}-\stackrel{=}{x})}^2$

In group, the sum of squares of deviations is calculated and is seen that formula is:

${\mathrm{<figure-callout\; id="2"\; label="Q"\; filenames="HDA0000396196040000012.png,HDA0000396196040000021.png"\; state="\{\{state\}\}">Q</figure-callout>}}_2=\underset{i=1}{\overset{m}{\mathrm{\&Sigma;}}}\underset{j=1}{\overset{\mathrm{nj}}{\mathrm{\&Sigma;}}}{(x_{\mathrm{ij}}-\stackrel{\&OverBar;}{x_i})}^2$

ν ₁degree of freedom between=m-1(group)

ν ₂=N-m(organizes the internal degree of freedom)

Experimental result, plasmid control molecule pXL06 uniformity testing result is as shown in table 1:

Table 1 plasmid control molecule pXL06 uniformity testing data (unit: ng/ μ L)

Sample	Repeat	1	Repeat 2	Repeat 3	Mean value
						1	76	76	76.1	76.0
2	76	75.9	75.8	75.9
					3	75.3	76	76.1	75.8
4	75.9	75.7	76.3	76.0
					5	76.2	76.9	76.6	76.6
6	76.2	76.2	76.7	76.4
					7	77.4	76.2	77.2	76.9

8	76	76.6	76.1	76.2
					9	76.5	76.3	76.5	76.4
10	77.1	76.2	76.4	76.6
					11	76.1	76.7	76.5	76.4
12	76.8	76.1	75.8	76.2
					13	76.5	75.5	76.6	76.2
14	76.1	76.4	75.9	76.1
					15	76	76.2	76.1	76.1

The homogeneity statistics of table 2 plasmid control molecule pXL06

The plasmid DNA reference material	Q 1	Q 2	The F value	F 0.05(14，30)
					pXL06	3.78	4.15	1.95	2.04

The homogeneity statistics is in Table 2, and under 95% confidence level, the F value is less than F _0.05(14,30), prove that this plasmid control molecule pXL06 is that requirement evenly is up to the standards to meet " JJG1006-94 primary standard material technical specifications " uniformly.

The study on the stability of embodiment 3 plasmid control molecule pXL06

Stability refers to that the characteristic value of reference material remains on the ability in specialized range under the specific timed interval and storage requirement.Stability is the base attribute of reference material, for the time dependent character of the characteristic of description standard material, the i.e. Time-distribution of description standard substance characteristics.Must carry out stability assessment in the triturating of reference material.Stability assessment not only can be assessed the uncertainty of measurement relevant to stability of material, and storage and transport condition that can be clearly suitable.The plasmid control molecule had good stability, As time goes on its characteristic value can not change under suitable storage and transport condition, and detected result can not be subject to its instable impact, has guaranteed the reliability of detected result.

Experiment reagent TE damping fluid (pH7.5).Laboratory apparatus Nanodrops ND-2000.

Experimental technique:

Adopt classical stability study to be investigated the stability of plasmid control molecule, As time goes on the sample of preparation is measured under the same conditions simultaneously.

1,0th month after prepared by plasmid control molecule, 0.5 month, 1 month, 2 months, 3 months, 4 months, 6 months are randomly drawed 3 bottles to the plasmid control molecule (20 ℃ of preservations) of preparation, each sample replication 3 times, average, carry out the permanent stability investigation.This research has adopted ultraviolet spectrophotometry to carry out following the tracks of to plasmid control molecule and has investigated.

2, the Detection of Stability data are assessed judgement study on the stability result.Concrete investigation method is as follows:

Straight slope can calculate with following formula:

${\mathrm{\&beta;}}_1=\frac{\underset{i=1}{\overset{n}{\mathrm{\&Sigma;}}}(X_i-\stackrel{\&OverBar;}{X})(Y_i-\stackrel{\&OverBar;}{Y})}{\underset{i=1}{\overset{n}{\mathrm{\&Sigma;}}}{(X_i-\stackrel{\&OverBar;}{X})}^2}$

In formula: X _i---i time point; Y _i---the observed value of i time point;

---the mean value of all time points;

---the mean value of all observed values.

Intercept can be calculated by following formula:

${\mathrm{\&beta;}}_0=\stackrel{\&OverBar;}{Y}-{\mathrm{\&beta;}}_1\stackrel{\&OverBar;}{X}$

On straight line, the standard deviation of every can calculate with following formula:

${\mathrm{<figure-callout\; id="2"\; label="s"\; filenames="HDA0000396196040000012.png,HDA0000396196040000021.png"\; state="\{\{state\}\}">s</figure-callout>}}^2=\frac{\underset{i=1}{\overset{n}{\mathrm{\&Sigma;}}}{(Y_i-{\mathrm{\&beta;}}_0-{\mathrm{\&beta;}}_1{X}_i)}^2}{n-2}$

In formula: X _i---i time point; Y _i---the observed value of i time point; β ₁, β ₀---regression coefficient; N---measurement coefficient.

β ₁standard deviation by following formula, provided:

$s\left({\mathrm{\&beta;}}_1\right)=\frac{s}{\sqrt{\underset{i=1}{\overset{n}{\mathrm{\&Sigma;}}}{(X_i-\stackrel{\&OverBar;}{X})}^2}}$

Based on β ₁standard deviation, available t-detects and carries out judging: even | β ₁|<t _{0.95, n-2}s (β ₁), show that slope is not remarkable, do not observe unstable.

Experimental result: plasmid control molecule pXL06 study on the stability result as shown in Table 3 and Figure 2:

The STABILITY MONITORING data of table 3 plasmid control molecule pXL06 (unit: ng/ μ L)

Time (moon)	Repeat 1	Repeat 2	Repeat 3	Mean value	SD
							0	76.03	75.90	75.80	75.91	0.12
0.5	75.00	74.60	76.30	75.30	0.89
						1	76.00	76.20	75.60	75.93	0.31
2	76.63	75.37	75.03	75.68	0.84
						4	75.20	75.70	74.90	75.27	0.40
6	75.40	74.70	74.70	74.93	0.40

Statistics is as shown in table 4:

The stability statistics of table 4 plasmid control molecule pXL06

The plasmid DNA reference material	β 1	β 0	s(β 1)	t 0.95,n-2·s(β 1)
					pXL06	-0.14	75.77	0.06	0.16

From statistics, it 6 months is stable that plasmid control molecule pXL06 preserves under-20 degree conditions.

Experimental example 4 use ultraviolet spectrophotometries are carried out quantitatively plasmid control molecule pXL06

Experiment reagent TE damping fluid (pH7.5).Laboratory apparatus Nanodrops ND-2000.

Experimental technique comprises the following steps:

1, with the TE damping fluid, ultraviolet spectrophotometer being proofreaied and correct is zero point;

2, get embodiment and prepare gained DNA solution 1 μ L, directly at surveyed area, the recording instrumnet reading;

3, directly read DNA concentration, concentration unit is ng/ μ L.

4, each sample test is eight times, averages.

Experimental result is: through the ultraviolet spectrophotometry definite value, the concentration of plasmid control molecule pXL06 is as shown in table 5:

Table 5 ultraviolet spectrophotometry definite value result (unit: ng/ μ L)

Plasmid	Repeat	1	2	3	4	5	6	7	8	Mean value	SD
												pXL06	76.0	75.9	75.8	74.6	75.1	75.5	75.7	77.0	75.7	0.70

Embodiment 5 use HR-ICP-MS carry out quantitatively plasmid control molecule pXL06

Experiment reagent is P standardized solution (NIST, SRM3139a).Laboratory apparatus is: high resolution inductively coupled plasma emission mass spectrograph (Thermofisher element2).

Experimental technique comprises the following steps:

1,, according to based composition and the sequence length of plasmid control molecule pXL06, calculate respectively the wherein content of phosphoric;

2, the HR-ICP-MS experiment parameter is: cooling gas flow 16.86L/min, and the atomization gas flow is 1.123L/min, the assisted gas flow is 0.99L/min, power 1350W.

3, prepare the phosphorus standardized solution (0,1,2,3,4,5 μ g/L) of gradient concentration, and make the typical curve of HR-ICP-MS with this standardized solution;

4, by 2000 times of plasmid control molecule pXL06 dilutions, HR-ICP-MS detects the plasmid control molecule after dilution, and draws the phosphorus content of the plasmid control molecule after dilution according to typical curve;

5, the concentration that the phosphorus content that basis records and extension rate calculate plasmid control molecule pXL06;

6, each sample test is eight times, averages.

Experimental result is: obtain the plasmid control molecule phosphorus element content through the HR-ICP-MS definite value, as calculated, the content of the phosphoric of plasmid control molecule pXL06 is 9.6%.Obtain plasmid control molecule phosphorus element content data through the HR-ICP-MS definite value as shown in table 6, extension rate is 2000, converts and obtains the mass concentration of plasmid molecule pXL06, as shown in table 7.

The phosphorus element content of table 6 plasmid control molecule pXL06 (unit: μ g/L)

The mass concentration result of table 7 plasmid control molecule pXL06 (unit: ng/ μ L)

The application of plasmid control molecule pXL06 in real-time fluorescence PCR detects of embodiment 6 developments

Experiment reagent: the primer and probe are synthetic by precious biotechnology (Dalian) company limited, and Master Mix is purchased from Life technology company.Laboratory apparatus comprises: real-time fluorescence quantitative PCR amplification instrument (Life technology) whizzer, thermostat water bath, incubator, day equality.

The primer adopted in experiment and probe sequence, as shown in SEQ ID NO:1-SEQ ID NO:5 in sequence table, specifically refer to table 8 content:

The primer and the probe sequence that in table 8 experiment, adopt

The application of plasmid control molecule pXL06 in real-time fluorescence PCR detects

Adopt document " Realtime PCR using TaqMan and SYBR Green for detection of Enterobacter sakazakii in infant formula " (LIU Yin, CAI Xiaoning, ZHANG Xia, et al.Journal of Microbiological Methods, 2006,65 (1): the primer 21-31), probe, PCR detection system and reaction conditions, respectively with the plasmid control molecule pXL06(of different concns as 2.19 * 10 ⁶copies/ μ L, 2.19 * 10 ⁵copies/ μ L, 2.19 * 10 ⁴copies/ μ L, 2.19 * 10 ³copies/ μ L, 2.19 * 10 ²copies/ μ L, 2.19 * 10 ¹copies/ μ L, 2.19 * 10 ⁰copies/ μ L) carry out the real-time fluorescence PCR amplification as template.Each reacts triplicate, according to the Ct value of different concns template amplification and the relation between concentration, Criterion curve.

The application of plasmid control molecule pXL06 in real-time fluorescence PCR detects

Adopt described PCR detection system and reaction conditions, respectively with the plasmid control molecule pXL06(of different concns as 2.19 * 10 ⁶copies/ μ L, 2.19 * 10 ⁵copies/ μ L, 2.19 * 10 ⁴copies/ μ L, 2.19 * 10 ³copies/ μ L, 2.19 * 10 ²copies/ μ L, 2.19 * 10 ¹copies/ μ L, 2.19 * 10 ⁰copies/ μ L) set up the typical curve of real-time fluorescence PCR as standard substance.The typical curve of setting up is shown in Fig. 3, and relation conefficient reaches 0.999, linear good, shows that plasmid control molecule pXL06 is applicable to being applied to real-time fluorescence PCR and detects, and can be used as the positive criteria material of real-time fluorescence PCR amplification Enterobacter sakazakii ITS gene order.When actual Enterobacter sakazakii detects, can utilize the good typical curve of this linearity, fluorescence PCR method detects the copy number of Enterobacter sakazakii specific gene in testing sample by experiment, and can, according to the copy number of Enterobacter sakazakii specific gene and the linear relationship between total plate count, converse the concrete number of Enterobacter sakazakii.

Because the at present research and development for the reference material of Enterobacter sakazakii real-time fluorescence PCR detection method are still blank, when actual Enterobacter sakazakii PCR in real time detects, the plasmid DNA molecule that is mostly the designed, designed structure that constituent parts is used is as standard substance, goal gene specific fragment wherein is different, lack unified valued methods simultaneously, plasmid DNA molecule definite value poor accuracy, cause the detected result difference between each laboratory very big, lacks comparability, validity and reliability.Therefore, this plasmid standard molecule can solve a difficult problem that lacks reference material when real-time fluorescence PCR detects Enterobacter sakazakii, be applicable to the real time fluorescent PCR method of multiple amplification Enterobacter sakazakii ITS gene, guaranteed the comparability of detected result, for new biometric technology support means and the method that provide is provided the Enterobacter sakazakii real time fluorescent PCR method.

Should be understood that, after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims (10)

1. the plasmid control molecule of an Enterobacter sakazakii, is characterized in that, the ITS sequence that this plasmid control molecule comprises Enterobacter sakazakii, and described ITS sequence is as shown in SEQ ID NO:1 in sequence table.

2. the plasmid control molecule of an Enterobacter sakazakii, is characterized in that, the sequence of this plasmid control molecule is as shown in SEQ ID NO:2 in sequence table.

3. the quantivative approach of the plasmid control molecule of Enterobacter sakazakii as claimed in claim 1 or 2, is characterized in that, it comprises the following steps:

1. extract plasmid control molecule;

2. according to step 1. based composition and the sequence length of the plasmid control molecule of gained, calculate the content of the phosphoric in plasmid control molecule;

3. prepare the phosphorus standardized solution of gradient concentration, and make the high resolution inductively coupled plasma with this standardized solution and launch mass spectral:mass spectrographic typical curve;

4. high resolution inductively coupled plasma emission mass spectrometric detection plasmid control molecule solution, and according to step 3. the typical curve of gained draw the phosphorus content of plasmid control molecule solution;

5. according to the 4. 2. content of the phosphoric in the plasmid control molecule of gained of the phosphorus content of the plasmid control molecule solution of gained and step of step, calculate the concentration of plasmid control molecule.

4. the quantivative approach of the plasmid control molecule of Enterobacter sakazakii as claimed in claim 3, it is characterized in that, the step 3. condition of described high resolution inductively coupled plasma emission mass spectrometric detection is: cooling gas flow is 10L/min～25L/min, the assisted gas flow is 0.5L/min～3.0L/min, the atomization gas flow is 0.5L/min～1.5L/min, and power is 1200W～1400W.

5. the quantivative approach of the plasmid control molecule of Enterobacter sakazakii as claimed in claim 3, it is characterized in that, the step 3. condition of described high resolution inductively coupled plasma emission mass spectrometric detection is: cooling gas flow is 16.86L/min, the assisted gas flow is 0.88L/min, the atomization gas flow is 1.123L/min, and power is 1350W.

6. an Enterobacter sakazakii real-time fluorescence quantitative PCR detection method, is characterized in that, institute's accepted standard material is plasmid control molecule as claimed in claim 1 or 2.

7. the application of plasmid control molecule as claimed in claim 1 or 2 in the Enterobacter sakazakii real-time fluorescence quantitative PCR detects.

8. the preparation method of the plasmid control molecule of an Enterobacter sakazakii as claimed in claim 1 or 2, is characterized in that, comprises the following steps:

1. the ITS sequence of synthetic Enterobacter sakazakii, described ITS sequence is as shown in SEQ ID NO:2 in sequence table;

2. by step 1. the ITS sequence clone of gained Enterobacter sakazakii to cloning vector, obtain the plasmid control molecule of Enterobacter sakazakii.

9. the preparation method of the plasmid control molecule of Enterobacter sakazakii as claimed in claim 8, is characterized in that, step 2. described cloning vector is pUC19, pUC18, pUC118, pUC119, pBlueScript II SK or pGEM.

10. the preparation method of the plasmid control molecule of Enterobacter sakazakii as claimed in claim 8, is characterized in that, step 2. described cloning vector is pUC19.

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