CN102321621A - Molecular standard sample for Muscina stabulans and preparation method for molecular standard sample - Google Patents
Molecular standard sample for Muscina stabulans and preparation method for molecular standard sample Download PDFInfo
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- CN102321621A CN102321621A CN201110219706A CN201110219706A CN102321621A CN 102321621 A CN102321621 A CN 102321621A CN 201110219706 A CN201110219706 A CN 201110219706A CN 201110219706 A CN201110219706 A CN 201110219706A CN 102321621 A CN102321621 A CN 102321621A
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Abstract
The invention relates to a molecular standard sample for Muscina stabulans and a preparation method for the molecular standard sample, and belongs to the field of the research on vector organism quarantine technologies. The molecular standard sample for the Muscina stabulans is obtained by the following steps of: raising adults of the Muscina stabulans which are collected in the open under laboratory conditions, extracting high-purity deoxyribonucleic acid (DNA), split-charging, and freeze-drying. The standard sample has the following physical and chemical properties: (1) the standard sample is pure white powder in shape; (2) the content in each tube is 1+/-0.1mu g; (3) the purity of DNA is that OD260/280 is equal to 1.8 to 2.0; and (4) the standard sample is easily soluble in water or a TE (Tris-EDTA) buffer solution. The standard sample can be detected by using a specific primer by a polymerase chain reaction (PCR) method, and the sensitivity is 3 to 10 times. The standard used as a positive reference substance is applicable to entry and exit health quarantine departments and other industries, can be used for detecting and identifying the Muscina stabulans, and improves the accuracy of identification results.
Description
Technical field
The present invention relates to false stable fly molecular criteria sample, belong to health quarantine technical study field.
Background technology
Then still be in the stage of starting both at home and abroad about the research of media molecule DNA standard model, a lot of problems all need to be resolved hurrily.Owing to the reasons such as imperfection of sample in the restriction that receives the biological development stage and the biomophic similarity of sibling species, the real work, the traditional morphological means are carried out species and are identified and run into a lot of practical difficulties.Existing molecular dna technology is proved to be effective biological assay means.At present, China also not through authorized by state, unified, normalized false stable fly molecular dna standard model, does not have such standard model in the world yet.Therefore, the false stable fly standard model of this project development will provide reference and foundation for the work of false stable fly Molecular Identification, for certain pushing effect is played in the research work of China's molecular criteria sample.
Summary of the invention
The object of the invention provides false stable fly molecular criteria sample, and homogeneity has good stability, and the present invention also provides the preparation method of standard model in addition, and technology is simple, is easy to preparation.
False stable fly molecular criteria sample of the present invention; After raising through laboratory condition with field acquisition false stable fly adult, extract high purity DNA, carry out packing, freeze-drying; Obtain the molecular criteria article of false stable fly, it has following physico-chemical property: (1) shape: pure white is Powdered; (2) every pipe content 1 μ g ± 0.1 μ g; (3) DNA purity: OD260/280=1.8～2.0; (4) in soluble in water or TE (Tris-EDTA) damping fluid.
Said DNA purity: OD260/280=1.85.
The preparation method of false stable fly molecular criteria article of the present invention may further comprise the steps: the preparation method of false stable fly molecular criteria sample is characterized in that: comprise the steps:
The first step, false stable fly DNA extraction: field acquisition false stable fly adult, under laboratory condition, raise, extract false stable fly DNA, adopt test kit to extract DNA:
(1) carry out homogenate with grinding rod:
The false stable fly tissue of getting 1 ~ 100mg moves in the centrifuge tube of ice bath precooling, grinds to form pasty state fast with grinding rod;
Grind to form even fast with grinding rod after adding Solution I and the 0.9uLRNase A1 of 350 μ L
Slurry;
The Solution I that adds 350 μ L pours the homogenate on the grinding rod in the centrifuge tube, and after fully vibration mixed, 65 ℃ were incubated 5 minutes;
(2) the Solution II of adding 400 μ L, vibration mixes;
(3) the Solution III of 4 ℃ of precoolings of adding 1mL, behind the abundant mixing, centrifugal 2 minutes of 12,000 rpm;
(4) discard upper organic phase, add the Solution III of 4 ℃ of precoolings of 1mL again, behind the abundant mixing, centrifugal 2 minutes of 12,000 rpm;
(5) discard upper organic phase, then aqueous phase solution (colourless lower floor) is transferred to the filter bowl that places on the centrifuge tube, centrifugal 1 minute of 12,000 rpm; (annotate: organic phase (upper strata) has color, do eliminate, and is attached to DNA and prepares on the film otherwise can hinder DNA, and the deposition of meeting each other need not be considered, when filtering, will be removed.）
(6) abandon filter bowl, in filtrating, add the DB damping fluid of 400 μ L, mix;
(7) column spinner in the test kit is placed on the centrifuge tube.Aforesaid operations 6 mixing solutionss are transferred in the column spinner, and centrifugal 1 minute of 12,000 rpm abandon filtrating;
(8) the Rinse A with 500 μ L is added in the column spinner, 12,000 rpm centrifugal 30 seconds, abandons filtrating;
(9) the Rinse B with 700 μ L is added in the column spinner, 12,000 rpm centrifugal 30 seconds, abandons filtrating; (annotate: 100% ethanol that has added designated volume among
PLSCONFM Rinse B.
please adds Rinse B around the column spinner tube wall, help complete flushing to be built-up in the salt on the tube wall like this.）
(10) repetitive operation step (9);
(11) column spinner is placed on the centrifuge tube of new 1.5mL, adds sterile purified water or the elution buffer of 50 ~ 200 μ L in the centre of column spinner film, and room temperature left standstill 1 minute.(annotate: be heated to 65 ℃ to sterile purified water or elution buffer and help improving eluting rate when using.）
(12) 12, centrifugal 1 minute eluted dna of 000 rpm;-20 ℃ of preservations are subsequent use;
The preparation of second step, nucleic acid standard model:
DNA after the mensuration concentration is carried out packing by 1 μ g ± 0.1 μ g/pipe, every kind of nucleic acid molecule standard specimen packing 150 pipes, freeze-drying then is the purpose standard substance.
Said the standard model that makes is carried out qualitative evaluation, carry out pcr amplification or qualitative analysis is carried out in order-checking, confirm that prepared nucleic acid is target DNA through Auele Specific Primer; Get the nucleic acid standard model of preparation, add 50 μ LTE, use as template the dissolving back, carries out PCR and order-checking through adopting Auele Specific Primer 28SrRNA-1 and 28SrRNA-2, carries out qualitative analysis, confirms that prepared nucleic acid standard model is the purpose nucleic acid samples;
Upstream primer: 5 '-GACTACCCCCTGAATTTAAGCAT-3 '
Downstream primer: 5 '-GACTCCTTGGTCCGTGTTTCAAG-3 '
Reaction system:
Template 1.0 μ L
Upstream primer 1.0 μ L
Downstream primer 1.0 μ L
Taq enzyme 0.5U
dNTP 2.0?μL
10 * damping fluid, 2.5 μ L
Total 25.0?μL
Reaction conditions:
94℃ 5min
94℃ 30s
50℃ 30s
72 ℃ of 2min circulate 35 times
72℃ 7min。
After false stable fly molecular criteria sample of the present invention is raised through laboratory condition with field acquisition false stable fly adult; Extract high purity DNA, carry out packing, freeze-drying, obtain the molecular criteria article of false stable fly; The good Auele Specific Primer that adopts of its physico-chemical property that has passes through the PCR method detection, and sensitivity reaches 10
-3Doubly.These standard substance are applicable to entry and exit health quarantine department and other industries as positive reference substance, can be used for the detection of false stable fly is identified, improve the accuracy of qualification result.
Four, description of drawings
Fig. 1 is the total DNA electrophorogram of false stable fly.
Fig. 2 is primer amplified electrophoresis result figure, and 1-6 is a target DNA among the figure.
Fig. 3 is sensitivity experiment figure as a result, and by left-to-right, each lattice is a concentration among the figure, is 10 successively
-1, 10
-2, 10
-3, 10
-4, 10
-5
Five, embodiment
Below in conjunction with specific embodiment the present invention is further specified, but the present invention is not limited to specific embodiment.
The preparation method of false stable fly molecular criteria sample, carry out as follows:
The first step, false stable fly DNA extraction: field acquisition false stable fly adult, under laboratory condition, raise, extract false stable fly DNA, adopt test kit to extract DNA:
(1) carry out homogenate with grinding rod:
The false stable fly tissue of getting 1 ~ 100mg moves in the centrifuge tube of ice bath precooling, grinds to form pasty state fast with grinding rod;
Grind to form even fast with grinding rod after adding Solution I (precious biotechnology (Dalian) ltd product) and the 0.9uLRNase A1 of 350 μ L
Slurry;
The Solution I that adds 350 μ L pours the homogenate on the grinding rod in the centrifuge tube, and after fully vibration mixed, 65 ℃ were incubated 5 minutes;
(2) the Solution II (precious biotechnology (Dalian) ltd product) of adding 400 μ L, vibration mixes;
(3) the Solution III of 4 ℃ of precoolings of adding 1mL, behind the abundant mixing, centrifugal 2 minutes of 12,000 rpm;
(4) discard upper organic phase, add the Solution III (precious biotechnology (Dalian) ltd product) of 4 ℃ of precoolings of 1mL again, behind the abundant mixing, centrifugal 2 minutes of 12,000 rpm;
(5) discard upper organic phase, then aqueous phase solution (colourless lower floor) is transferred to the filter bowl that places on the centrifuge tube, centrifugal 1 minute of 12,000 rpm; (annotate: organic phase (upper strata) has color, do eliminate, and is attached to DNA and prepares on the film otherwise can hinder DNA, and the deposition of meeting each other need not be considered, when filtering, will be removed.）
(6) abandon filter bowl, in filtrating, add the DB damping fluid of 400 μ L, mix;
(7) column spinner in the test kit is placed on the centrifuge tube.Aforesaid operations 6 mixing solutionss are transferred in the column spinner, and centrifugal 1 minute of 12,000 rpm abandon filtrating;
(8) the Rinse A with 500 μ L is added in the column spinner, 12,000 rpm centrifugal 30 seconds, abandons filtrating;
(9) the Rinse B with 700 μ L is added in the column spinner, 12,000 rpm centrifugal 30 seconds, abandons filtrating; (annotate: 100% ethanol that has added designated volume among
PLSCONFM Rinse B.
please adds Rinse B around the column spinner tube wall, help complete flushing to be built-up in the salt on the tube wall like this.）
(10) repetitive operation step (9);
(11) column spinner is placed on the centrifuge tube of new 1.5mL, adds sterile purified water or the elution buffer of 50 ~ 200 μ L in the centre of column spinner film, and room temperature left standstill 1 minute.(annotate: be heated to 65 ℃ to sterile purified water or elution buffer and help improving eluting rate when using.）
(12) 12, centrifugal 1 minute eluted dna of 000 rpm;-20 ℃ of preservations are subsequent use;
The preparation of second step, nucleic acid standard model:
DNA after the mensuration concentration is carried out packing by 1 μ g ± 0.1 μ g/pipe, every kind of nucleic acid molecule standard specimen packing 150 pipes, freeze-drying then is the purpose standard substance, and it has following physico-chemical property: (1) shape: pure white is Powdered; (2) every pipe content 1 μ g ± 0.1 μ g; (3) DNA purity: OD260/280=1.8～2.0; (4) in soluble in water or TE (Tris-EDTA) damping fluid;
The 3rd step, get the nucleic acid standard model of preparation, add 50 μ LTE, use as template dissolving back, carries out PCR and order-checking through adopting specificity, carries out qualitative analysis, confirms that prepared nucleic acid standard model is the purpose nucleic acid samples: example:
Upstream primer: 5 '-GACTACCCCCTGAATTTAAGCAT-3 '
Downstream primer: 5 '-GACTCCTTGGTCCGTGTTTCAAG-3 '
Reaction system:
Template 1.0 μ L
Upstream primer 1.0 μ L
Downstream primer 1.0 μ L
Taq enzyme 0.5U
dNTP 2.0?μL
10 * damping fluid, 2.5 μ L
Total 25.0?μL
Reaction conditions:
94℃?5min；?94℃?30s，50℃?30s，72℃?2min，35cycles；72℃?7min。
The above-mentioned standard model that makes is carried out DNA integrity, homogeneity, stability test
(1) DNA quality and integrity detection
Get the DNA of preparation, carry out gel electrophoresis, purity and concentration and detect;
(1) DNA integrity detection: the dna direct electrophoretic examinations that direct method-usefulness is extracted, 120V, 1.5% agarose gel electrophoresis, the integrity of observing band, detected result is seen Fig. 1, and band is complete, and no disperse shape explains that DNA band integrity is better.
(2) DNA concentration and purity detecting: ultraviolet spectrophotometer method-absorption 1 μ L DNA is with the absorption value of spectrophotometric determination 260nm and 280nm, then according to OD
260/ OD
280Value is judged DNA purity, and according to OD
260Calculate its concentration.Calculate by following formula:
DNA concentration (ng/ μ L)=OD
260* 50
Work as OD
260/ OD
280＜1.8, the expression protein contnt is higher; OD
260/ OD
280＞2.0, the expression rna content is higher; Work as OD
260/ OD
280=1.8～2.0, DNA is purer in expression.
(2) standard model analysis of Uniformity
For checking the homogeneity of the purpose nucleic acid standard model for preparing; Adopt PicoGreen dna molecular fluorescent quantitation method and ultraviolet spectrophotometry; Get and randomly draw 15 pipe samples; Every pipe sample is divided into 2 one's share of expenses for a joint undertaking appearance to be tested, and the result sees that (table 1) data carry out F check (table 2) confirmatory sample homogeneity.
(3) standard model stability analysis
Originally there is the card standard model to have the stability period limit gauge accepted argument of card standard model bright with reference to external equal band card
(in vacuum, lucifuge ,-20 ℃ of storages down, stable validity period is 2 years) is foundation, its biological nature; Scheduled to last with 2 years; Standard model to preparation is got two duplicate samples stage by stage at every turn, carries out qualitative test according to different preservation conditions, and every duplicate samples is its definite value result to measure 3 sub-values.In whole stability tests, used personnel, instrument, testing method and laboratory are all identical with uniformity test.
According to said determination method calculation result respectively, this false stable fly molecular criteria sample vacuum, lucifuge,
Store down for-20 ℃, stable validity period is 2 years, measures the result and sees table 3.
Slope can be used computes:
In the formula:
Intercept is by computes:
The standard deviation of the point on the straight line can be by computes:
Get its square root s=0.00251%, the uncertainty relevant with slope used computes:
Degree of freedom is that the student of n-2=5 and p=0.95 (95% confidence level) the t-factor that distributes equals 2.57.Because
is so slope is inapparent.Thereby do not observe unstable in the stability experiment.
(4) sensitivity test
The molecular criteria article of this germ are diluted to 10ng/ μ L, as mother liquor, dilute by 10 times of concentration gradients then, four (4) methods are carried out the PCR detection in the by specification.Detected result shows when this diluted sample to 1000 (promptly 10
-3*) doubly, band is invisible, therefore judges that the sensitivity of these standard substance is 10
-3
Table 1 false stable fly molecular criteria article homogeneity detected result
|
| Increment | 1 content (μ g) |
|
| 1 | 1.037 | 1.034 | |
| 2 | 1.039 | 1.03 | |
| 3 | 1.03 | 1.028 | |
| 4 | 1.032 | 1.028 | |
| 5 | 1.038 | 1.027 | |
| 6 | 1.031 | 1.025 | |
| 7 | 1.032 | 1.024 | |
| 8 | 1.037 | 1.029 | |
| 9 | 1.03 | 1.033 | |
| 10 | 1.037 | 1.033 | |
| 11 | 1.029 | 1.026 | |
| 12 | 1.023 | 1.031 | |
| 13 | 1.034 | 1.038 | |
| 14 | 1.031 | 1.037 | |
| 15 | 1.037 | 1.04 | |
| ? | 1.037 | 1.034 |
Table 2 false stable fly molecular criteria article homogeneity F assay
Conclusion: under 95% fiducial probability, with other factors the influence of test result is compared, the ununiformity of sample is acceptable.
Table 3 false stable fly molecular criteria article Detection of Stability result
Claims (4)
1. false stable fly molecular criteria sample; It is characterized in that: after raising through laboratory condition with field acquisition false stable fly adult, extract high purity DNA, carry out packing, freeze-drying; Obtain the molecular criteria article of false stable fly, it has following physico-chemical property: (1) shape: pure white is Powdered; (2) every pipe content 1 μ g ± 0.1 μ g; (3) DNA purity: OD260/280=1.8～2.0; (4) in soluble in water or TE (Tris-EDTA) damping fluid.
2. false stable fly molecular criteria sample according to claim 1 is characterized in that: DNA purity: OD260/280=1.85.
3. the preparation method of false stable fly molecular criteria sample is characterized in that: comprise the steps:
The first step, false stable fly DNA extraction:
Field acquisition false stable fly adult is raised under laboratory condition, extracts false stable fly DNA;
The preparation of second step, nucleic acid standard model:
DNA after the mensuration concentration is carried out packing by 1 μ g ± 0.1 μ g/pipe, every kind of nucleic acid molecule standard specimen packing 150 pipes, freeze-drying then is the purpose standard substance.
4. the preparation method of false stable fly molecular criteria sample according to claim 3 is characterized in that: the first step, extraction false stable fly DNA, and concrete steps are:
(1) carry out homogenate with grinding rod:
The false stable fly tissue of getting 1 ~ 100mg moves in the centrifuge tube of ice bath precooling, grinds to form pasty state fast with grinding rod;
Grind to form homogenate fast with grinding rod after adding Solution I and the 0.9uLRNase A1 of 350 μ L;
The Solution I that adds 350 μ L pours the homogenate on the grinding rod in the centrifuge tube, and after fully vibration mixed, 65 ℃ were incubated 5 minutes;
(2) the Solution II of adding 400 μ L, vibration mixes;
(3) the Solution III of 4 ℃ of precoolings of adding 1mL, behind the abundant mixing, centrifugal 2 minutes of 12,000 rpm;
(4) discard upper organic phase, add the Solution III of 4 ℃ of precoolings of 1mL again, behind the abundant mixing, centrifugal 2 minutes of 12,000 rpm;
(5) discard upper organic phase, then aqueous phase solution is transferred to the filter bowl that places on the centrifuge tube, centrifugal 1 minute of 12,000 rpm;
(6) abandon filter bowl, in filtrating, add the DB damping fluid of 400 μ L, mix;
(7) column spinner in the test kit is placed on the centrifuge tube, and aforesaid operations (6) mixing solutions is transferred in the column spinner, and centrifugal 1 minute of 12,000 rpm abandon filtrating;
(8) the Rinse A with 500 μ L is added in the column spinner, 12,000 rpm centrifugal 30 seconds, abandons filtrating;
(9) the Rinse B with 700 μ L is added in the column spinner, 12,000 rpm centrifugal 30 seconds, abandons filtrating;
(10) repetitive operation step (9);
(11) column spinner is placed on the centrifuge tube of new 1.5mL, adds sterile purified water or the elution buffer of 50 ~ 200 μ L in the centre of column spinner film, and room temperature left standstill 1 minute;
(12) 12, centrifugal 1 minute eluted dna of 000 rpm;-20 ℃ of preservations are subsequent use.
5, according to the preparation method of claim 3 or 4 described false stable fly molecular criteria samples; It is characterized in that: the standard model to making carries out qualitative evaluation; Carry out pcr amplification or qualitative analysis is carried out in order-checking through Auele Specific Primer, confirm that prepared nucleic acid is target DNA; Get the nucleic acid standard model of preparation, add 50 μ LTE, use as template the dissolving back, carries out PCR and order-checking through adopting Auele Specific Primer 28SrRNA-1 and 28SrRNA-2, carries out qualitative analysis, confirms that prepared nucleic acid standard model is the purpose nucleic acid samples;
Upstream primer: 5 '-GACTACCCCCTGAATTTAAGCAT-3 '
Downstream primer: 5 '-GACTCCTTGGTCCGTGTTTCAAG-3 '
Reaction system:
Template 1.0 μ L
Upstream primer 1.0 μ L
Downstream primer 1.0 μ L
Taq enzyme 0.5U
dNTP 2.0?μL
10 * damping fluid, 2.5 μ L
Total 25.0?μL
Reaction conditions:
94℃ 5min
94℃ 30s
50℃ 30s
72 ℃ of 2min circulate 35 times
72℃ 7min。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103013979A (en) * | 2012-11-29 | 2013-04-03 | 王忠举 | Preparation method of aeromonas hydrophila nucleic acid standard substance |
| CN105002166A (en) * | 2015-08-17 | 2015-10-28 | 姜陆 | Molecular standard sample of brown cockroaches and preparation method of molecular standard sample |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103013979A (en) * | 2012-11-29 | 2013-04-03 | 王忠举 | Preparation method of aeromonas hydrophila nucleic acid standard substance |
| CN105002166A (en) * | 2015-08-17 | 2015-10-28 | 姜陆 | Molecular standard sample of brown cockroaches and preparation method of molecular standard sample |
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Application publication date: 20120118 |