CN107312840A - Duck derived component PCR detections positive criteria molecule, preparation and detection method - Google Patents
Duck derived component PCR detections positive criteria molecule, preparation and detection method Download PDFInfo
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- CN107312840A CN107312840A CN201710538286.2A CN201710538286A CN107312840A CN 107312840 A CN107312840 A CN 107312840A CN 201710538286 A CN201710538286 A CN 201710538286A CN 107312840 A CN107312840 A CN 107312840A
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Abstract
The invention discloses a kind of duck derived component PCR detections positive criteria molecule, preparation and detection method, positive criteria molecule includes duck 18SrRNA gene orders and duck Mitochondrial gene sequence to be capable of in the plasmid molecule of autonomous replication, plasmid molecule.The duck derived component PCR detection positive criteria molecules that the present invention is prepared, with preparation process it is simple, can preserve for a long time, high specificity and the high advantage of sensitivity, the positive criteria molecule needs not rely on the supply of standard duck material, can substitute Duck genome DNA be used for duck composition PCR is qualitative, quantitative measurment and analysis, ensure the reliability of laboratory test results, solve the problem for lacking positive control in foods supervision work.
Description
Technical field
The invention belongs to bioengineering field, more particularly to a kind of duck derived component PCR detections positive criteria molecule, system
Standby and detection method.
Background technology
With the continuous improvement of living standard, people also accordingly improve to the attention degree of Safety of Food Quality.Some are not
Method molecule is seeks exorbitant profit, and the meat of poor quality that not clear animal sources are bought at a low price makes false to make meat products, or using essence etc.
Meat products is emitted, very disruptive meat products market damages consumer's just rights and interests, brings great potential safety hazard.Therefore, meat is strengthened
Product is detected, accurately detects the content of corresponding animal derived components in food, is to prevent and treat the key that meat of poor quality product comes into the market
Measure.
Fluorescent quantitative poly chain reaction (real-time Fluorescent Quantitative Polymerase
Chain Reaction, FQ-PCR) method is PCR (Polymerase Chain Reaction, PCR) skill
One kind of art, is the most frequently used quantitative detecting method of generally acknowledged current detection of nucleic acids.Real-Time Fluorescent Quantitative PCR Technique is anti-in PCR
The fluorophor that energy specific mark PCR primer is added in system is answered, whole PCR processes are monitored in real time using fluorescence signal accumulation,
Quantitative analysis is carried out to sample finally by standard curve, leap of the round pcr by qualitative to quantitative is realized.
Positive reference material is needed when being detected using FQ-PCR technologies to sample as control, to ensure experimental result
Reliability.Therefore, the quality of positive reference material and the accuracy of experimental result are closely related.At this stage, for animal sources
Property composition detection process in used positive reference material be typically Animal genome DNA.Wherein, duck composition detection process
In, Duck genome DNA processes complexity is extracted, is prepared very inconvenient.Because Duck genome DNA stability can not be met for a long time
The need for storage and regular use, therefore, when carrying out duck composition detection every time, it is required for preparing Duck genome DNA works again
For positive reference material, the stability of duck composition measurement is influenceed.
The content of the invention
The invention provides duck derived component PCR detections positive criteria molecule, preparation and detection method, to solve PCR
The positives reference material of detection process prepares the problem of complicated, storage is difficult.
In a first aspect, being independently to answer the invention provides a kind of duck derived component PCR detections positive criteria molecule
Duck 18SrRNA gene orders and duck Mitochondrial gene sequence are included in the plasmid molecule of system, the plasmid molecule.
Second aspect, present invention also offers a kind of preparation side to prepare positive criteria molecule described in first aspect
Method, comprises the following steps：
（1）Separately design amplimer 18S-F, 18S-R of duck 18SrRNA gene orders and the amplification of duck Mitochondrial gene sequence
Primer ANAS-F, ANAS-R；
Amplimer 18S-F, 18S-R of duck 18SrRNA gene orders be：
18S-F ：5'-GCGTCGACAGCCTGAGAAACGGCTACC-3',
18S-R ：5'-AACTGCAGTGCTGGCACCAGACTTGC-3'；
Amplimer ANAS-F, ANAS-R of duck Mitochondrial gene sequence be：
ANAS-F：5'-CCATGAAGCCCCATTCTCA-3',
ANAS-R：5'-CCGGTGGCAACAAAGAAAGT-3'；
（2）Using Duck genome as templet gene group, with the amplimer and duck Mitochondrial gene sequence of duck 18SrRNA gene orders
Amplimer, enter performing PCR amplification, obtain duck 18SrRNA gene orders and duck Mitochondrial gene sequence；
（3）Duck 18SrRNA gene orders and duck mtDNA sequence are respectively connecting to pMD18-T carriers, duck 18SrRNA weights are obtained
Group and duck mitochondria recon；
（4）By duck 18SrRNA recons and duck mitochondria recon is by digestion with restriction enzyme and splices, positive mark is obtained
Quasi-molecule.
Alternatively, the restriction enzyme isSal I enzymes andPstI enzymes.
Alternatively, step（2）In, the reaction system of PCR amplifications is the μ L of 1 μ L, 10uM anti-sense primer of 10uM sense primers 1,
The μ L of templet gene group 3, the μ L of Taq enzyme 12.5, plus ddH2The volume of O adjustment reaction systems is 25 μ L；The reaction bar of the PCR amplifications
Part is 94 DEG C of denaturation 5 min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 30s, and 40 circulations are carried out altogether, PCR is obtained
Product；
PCR primer is separated by agarose gel electrophoresis, purpose product glue reclaim kits obtain PCR pure
Change product, as duck 18SrRNA gene orders and duck Mitochondrial gene sequence.
Alternatively, step（3）In, duck 18SrRNA gene orders and duck Mitochondrial gene sequence are carried with pMD18-T respectively
Body is attached, and is connected overnight at 4 DEG C, is obtained duck 18SrRNA recons 18S-pMD18-T and duck mitochondria recon ANAS-
pMD18-T。
Alternatively, step（4）In, duck 18SrRNA recons and duck mitochondria recon are used respectivelySal I enzymes andPstI
Enzyme carries out double digestion, and 37 DEG C of water-baths 2 hours, agarose gel electrophoresis, gel extraction will reclaim fragment connection, and 4 spend night company
Connect, shaking table uniformly mixes 2 hours and carries out plasmid conversion, obtain converting plasmid；Conversion plasmid is subjected to gel electrophoresis separation, reclaimed
Macromolecular plasmid band carries out the screening of recombinant clone plasmid and reclaimed, and obtains recombinant clone；Recombinant clone is tested by Standard PCR
Card,SalI andPstI digestion verification and digestion products sequence verification, obtained plasmid molecule 18S-ANAS-pMD18-T
As positive criteria molecule.
The third aspect, present invention also offers a kind of inspection to detect positive criteria molecular specificity described in first aspect
Survey method, specifically, using specific detection primer and probe, real-time fluorescence PCR is carried out by template of positive criteria molecule anti-
Should, specific detection primer and probe include following two groups：
Specific detection primer 18S-F, 18S-R and probe 18S-P of duck 18SrRNA gene orders：
18S-F：5'-GCGTCGACAGCCTGAGAAACGGCTACC-3',
18S-R：5'-AACTGCAGTGCTGGCACCAGACTTGC-3',
18S-P：5'-TGCGCGCCTGCTGCCTTCCT-3'；
Specific detection primer ANAS-F, ANAS-R and probe ANAS-P of duck Mitochondrial gene sequence：
ANAS-F：5'-CCATGAAGCCCCATTCTCA-3',
ANAS-R：5'-CCGGTGGCAACAAAGAAAGT-3',
ANAS-P：5'-TCGCCGACAGCGTCTACGGCT-3'.
Alternatively, the 5' of probe is terminal modified fluorescent reporter group, and 3' is terminal modified fluorescent quenching group.
The duck derived component PCR detection positive criteria molecules that the present invention is prepared, with preparation process it is simple, can grow
Time preservation, high specificity and the high advantage of sensitivity, the positive criteria molecule need not rely on the supply of standard duck material,
Can substitute Duck genome DNA be used for duck composition PCR is qualitative, quantitative measurment and analysis, it is ensured that laboratory test results it is reliable
Property, solve the problem for lacking positive control in foods supervision work.
It should be appreciated that the general description of the above and detailed description hereinafter are only exemplary and explanatory, not
Can the limitation present invention.
Brief description of the drawings
In order to illustrate more clearly of technical scheme, letter will be made to the required accompanying drawing used in embodiment below
Singly introduce, it should be apparent that, for those of ordinary skills, without having to pay creative labor,
Other accompanying drawings can also be obtained according to these accompanying drawings.
The structural representation for the positive criteria molecule that Fig. 1 is prepared for the present invention；
Fig. 2 is duck 18SrRNA genome sequence real-time fluorescent PCR amplification curve comparison figures；
Fig. 3 is duck mitochondrial genomes sequence real-time fluorescent PCR amplification curve comparison figure；
The sensitivity technique result figure for the positive criteria molecule that Fig. 4 is prepared for the present invention.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation is described, it is clear that described embodiment is only a part of embodiment of the invention, rather than whole embodiments.It is based on
Embodiment in the present invention, it is every other that those of ordinary skill in the art are obtained under the premise of creative work is not made
Embodiment, belongs to the scope of protection of the invention.
Embodiment one
1st, DNA is extracted：Duck genomic DNA is extracted using QIAGEN DNeasy Blood and Tissue Kit, light splitting is used
Photometer detects DNA purity and concentration.Determine OD260/OD280Value is 1.8-1.9 or so, and concentration is in more than 10ng/ μ L, explanation
DNA purity is higher, moderate concentration, meets PCR amplification requirements.
2nd, primer and specific probe design：To searching for duck 18SrRNA gene orders and duck mitochondria base in GeneBank
Because of sequence, according to the duck 18SrRNA gene orders and duck Mitochondrial gene sequence of acquisition, two pairs are designed using Primer 5.0
PCR primer, is respectively used for amplifying duck 18SrRNA gene orders and duck Mitochondrial gene sequence, designs two Species specific probes, uses
To detect whether the positive criteria molecule finally prepared successfully constructs.The 5' of probe is terminal modified fluorescent reporter group, and 3' is repaiied at end
It is decorated with fluorescent quenching group.Primer and probe is as shown in the table：
Table 1 is used to expand（Specific detection）The primer and probe of 18SrRNA gene orders and Mitochondrial gene sequence
3rd, the clone of duck 18SrRNA gene orders and duck Mitochondrial gene sequence
Using Duck genome as templet gene group, enter performing PCR amplification using the primer pair 18S-R and 18S-P in table 1, obtain duck
18SrRNA gene orders.Using Duck genome as templet gene group, carried out using the primer pair ANAS-F and ANAS-R in table 1
PCR is expanded, and obtains duck Mitochondrial gene sequence.Specifically, the reaction system of PCR amplifications is under 10uM sense primers 1 μ L, 10uM
The volume for swimming the μ L of primer 1, the μ L of templet gene group 3, the μ L of Taq enzyme 12.5, plus ddH2O adjustment reaction system is 25 μ L；The PCR
The reaction condition of amplification is 94 DEG C of denaturation 5 min, 94 DEG C of denaturation 30 s, 58 DEG C of annealing 30 s, 72 DEG C of 30 s of extension, and 40 are carried out altogether
Secondary circulation, obtains PCR primer；PCR primer is separated by agarose gel electrophoresis, purpose product glue reclaim kit
Purifying, obtains PCR purified products, as duck 18SrRNA gene orders and duck Mitochondrial gene sequence.
Duck 18SrRNA gene orders and duck Mitochondrial gene sequence are attached with pMD18-T carriers respectively, at 4 DEG C
Connect overnight, obtain duck 18SrRNA recons 18S-pMD18-T and duck mitochondria recon ANAS-pMD18-T.
4th, the structure of positive criteria molecule
Duck 18SrRNA recons and duck mitochondria recon are used respectivelySal I enzymes andPstI enzymes carry out double digestion, 37 DEG C of water-baths
2 hours, agarose gel electrophoresis, gel extraction will reclaim fragment connection, and 4 spend night connection, and shaking table uniformly mixes 2 hours
Row plasmid is converted, and obtains converting plasmid；Conversion plasmid is subjected to gel electrophoresis separation, macromolecular plasmid band is reclaimed and is recombinated
Cloned plasmids screening is reclaimed, and obtains recombinant clone；Recombinant clone is verified by Standard PCR,SalI andPstI digestion
Checking and digestion products sequence verification, obtained plasmid molecule 18S-ANAS-pMD18-T is positive criteria molecule.18S-
ANAS-pMD18-T concrete structure is as shown in Figure 1.
Embodiment two
1st, the structure result of positive criteria molecule
18S-ANAS-pMD18-T using the preparation of embodiment one enters performing PCR with the primer and probe in table 1 to it respectively as template
Amplification, and find that extension increasing sequence is consistent with expected fragment after sequence verification.As a result show that the positive criteria molecule built includes duck
The success of 18SrRNA gene orders and duck Mitochondrial gene sequence, i.e. positive criteria molecule construction.
2nd, the specific detection result of positive criteria molecule
Real-time fluorescent PCR amplification is carried out to Duck genome, negative sample and positive criteria molecule with the primer and probe in table 1,
Amplification curve result as shown in Figures 2 and 3, wherein, Fig. 2 be duck 18SrRNA genome sequence real-time fluorescent PCR amplification curves pair
Than figure, Fig. 3 is duck mitochondrial genomes sequence real-time fluorescent PCR amplification curve comparison figure.Pin is can be seen that with reference to Fig. 2 and Fig. 3
To the primer and probe designed by duck 18SrRNA and duck Mitochondrial gene sequence, no matter to Duck genome or the sun of the present invention
Property standard molecule can detect positive signal.The Ct values of duck 18SrRNA genes detection are 15 circulations of Duck genome, positive mark
11 circulations of quasi-molecule, and negative sample is then without signal；The Ct values of duck chondriogen detection are 15 circulations of Duck genome,
21 circulations of positive criteria molecule, and negative control is then without signal.Illustrate that positive criteria molecule prepared by the present invention can be special
The probe in detecting of the opposite sex is arrived, with good specificity.
3rd, the sensitivity technique result of positive criteria molecule
Ten times of gradient dilutions are carried out to positive criteria molecule（10-1To 10-7）, real-time fluorescence PCR is carried out using the probe in table 1
Detection, reaction system and the detection of reaction condition homospecificity are identical, and each reaction is repeated 6 times.Standard curve is set up, according to detection
The uniformity of amplification efficiency and correlation analysis positive criteria molecule.As a result as shown in figure 4, showing that minimum detected value is to reach
To 10-7Dilution factor, illustrates that positive criteria molecule sensitivity prepared by the present invention is higher.
4th, the Detection of Stability result of positive criteria molecule
General short-term stability Journal of Sex Research 4-8 weeks, long-time stability are carried out 6 months or 1 year.By ISO directive/guides 35, commented on stability
The method of valency is evaluated the long-time stability of standard substance.
The positive criteria molecule of preparation is placed under the conditions of 25 DEG C of room temperature, placed two months, real-time fluorescence PCR inspection is carried out
Survey, the Ct values to acquisition carry out the stability of statistical analysis positive criteria molecule.Each real-time PCR detection is each reacted
The Ct values average value of 6 repetitions carries out relative standard deviation analysis result and is shown in Table 2.As a result positive criteria point under room temperature condition is shown
Son storage just starts significant difference occur after 1 week, and this significant difference is after positive criteria molecular memory exceedes January
Disappear.It is inferred that positive criteria molecule better heat stability prepared by the present invention, not degradable, but as time went on, it is whole
Individual genome also shows unstable, and positive criteria molecule starts to produce significant change after one month.
The Detection of Stability result of the positive criteria molecule of table 2
Those skilled in the art will readily occur to other realities of the present invention after the disclosure that specification and practice are invented here is considered
Apply scheme.It is contemplated that cover any modification, purposes or the adaptations of the present invention, these modifications, purposes or suitable
The change of answering property follows the general principle of the present invention and including the undocumented common knowledge or used in the art of the present invention
Use technological means.Description and embodiments are considered only as exemplary, and true scope and spirit of the invention claim is pointed out.
It should be appreciated that the scope of the present invention is only limited by appended claim.
Nucleotides sequence list
<110>Food and medicine Inspection Research institute of Shandong Province
<120>Duck derived component PCR detections positive criteria molecule, preparation and detection method
<160>6
<210>1
<211>27
<212>DNA
<213>It is artificial synthesized
<220>
<223>
<400>1
GCGTCGACAG CCTGAGAAAC GGCTACC 27
<210>2
<211>26
<212>DNA
<213>It is artificial synthesized
<220>
<223>
<400>2
AACTGCAGTG CTGGCACCAG ACTTGC 26
<210>3
<211>19
<212>DNA
<213>It is artificial synthesized
<220>
<223>
<400>3
CCATGAAGCC CCATTCTCA 19
<210>4
<211>20
<212>DNA
<213>It is artificial synthesized
<220>
<223>
<400>4
CCGGTGGCAA CAAAGAAAGT 20
<210>5
<211>20
<212>DNA
<213>It is artificial synthesized
<220>
<223>
<400>5
TGCGCGCCTG CTGCCTTCCT 20
<210>6
<211>21
<212>DNA
<213>It is artificial synthesized
<220>
<223>
<400>6
TCGCCGACAG CGTCTACGGC T 21
Claims (8)
1. positive criteria molecule is used in a kind of duck derived component PCR detections, it is characterised in that the positive criteria molecule is can be certainly
Duck 18SrRNA gene orders and duck Mitochondrial gene sequence are included in the plasmid molecule of main duplication, the plasmid molecule.
2. a kind of preparation method of positive criteria molecule as claimed in claim 1, it is characterised in that the preparation method includes
Following steps：
（1）Separately design amplimer 18S-F, 18S-R of duck 18SrRNA gene orders and the amplification of duck Mitochondrial gene sequence
Primer ANAS-F, ANAS-R；
Amplimer 18S-F, 18S-R of duck 18SrRNA gene orders be：
18S-F ：5'-GCGTCGACAGCCTGAGAAACGGCTACC-3',
18S-R ：5'-AACTGCAGTGCTGGCACCAGACTTGC-3'；
Amplimer ANAS-F, ANAS-R of duck Mitochondrial gene sequence be：
ANAS-F：5'-CCATGAAGCCCCATTCTCA-3',
ANAS-R：5'-CCGGTGGCAACAAAGAAAGT-3' ；
（2）Using Duck genome as templet gene group, with the amplimer and duck chondriogen of the duck 18SrRNA gene orders
The amplimer of sequence, enters performing PCR amplification, obtains duck 18SrRNA gene orders and duck Mitochondrial gene sequence；
（3）The duck 18SrRNA gene orders and duck mtDNA sequence are respectively connecting to pMD18-T carriers, duck is obtained
18SrRNA recons and duck mitochondria recon；
（4）By the duck 18SrRNA recons and duck mitochondria recon is by digestion with restriction enzyme and splices, sun is obtained
Property standard molecule.
3. preparation method according to claim 2, it is characterised in that the restriction enzyme isSal I enzymes andPstI
Enzyme.
4. preparation method according to claim 2, it is characterised in that step（2）In, the reaction system of the PCR amplifications
For 1 μ L, 10uM anti-sense primer of 10uM sense primers 1 μ L, the μ L of templet gene group 3, the μ L of Taq enzyme 12.5, plus ddH2O adjusts reactant
The volume of system is 25 μ L；The reaction condition of the PCR amplifications is 94 DEG C of denaturation 5 min, 94 DEG C of 30 s of denaturation, 58 DEG C of annealing 30
S, 72 DEG C of 30 s of extension, carries out 40 circulations, obtains PCR primer altogether；
The PCR primer is separated by agarose gel electrophoresis, purpose product glue reclaim kits are obtained
PCR purified products, as duck 18SrRNA gene orders and duck Mitochondrial gene sequence.
5. preparation method according to claim 2, it is characterised in that step（3）In, by the duck 18SrRNA gene sequences
Row and duck Mitochondrial gene sequence are attached with pMD18-T carriers respectively, are connected overnight at 4 DEG C, obtain duck 18SrRNA restructuring
Sub- 18S-pMD18-T and duck mitochondria recon ANAS-pMD18-T.
6. preparation method according to claim 2, it is characterised in that step（4）In, by the duck 18SrRNA recons
Used respectively with duck mitochondria reconSal I enzymes andPstI enzymes carry out double digestion, 37 DEG C of water-baths 2 hours, agarose gel electrophoresis,
Gel extraction, will reclaim fragment connection, and 4 spend night connection, and shaking table uniformly mixes 2 hours and carries out plasmid conversion, obtains converting matter
Grain；
The conversion plasmid is subjected to gel electrophoresis separation, macromolecular plasmid band progress recombinant clone plasmid is reclaimed and screens back
Receive, obtain recombinant clone；
The recombinant clone is verified by Standard PCR,SalI andPstI digestion verification and digestion products sequencing is tested
Card, obtained plasmid molecule 18S-ANAS-pMD18-T is positive criteria molecule.
7. a kind of detection method of positive criteria molecule as claimed in claim 1, it is characterised in that drawn using specific detection
Thing and probe, real-time fluorescence PCR reaction, the specific detection primer and probe are carried out by template of the positive criteria molecule
Including following two groups：
Specific detection primer 18S-F, 18S-R and probe 18S-P of duck 18SrRNA gene orders：
18S-F：5'-GCGTCGACAGCCTGAGAAACGGCTACC-3',
18S-R：5'-AACTGCAGTGCTGGCACCAGACTTGC-3',
18S-P：5'-TGCGCGCCTGCTGCCTTCCT-3'；
Specific detection primer ANAS-F, ANAS-R and probe ANAS-P of duck Mitochondrial gene sequence：
ANAS-F：5'-CCATGAAGCCCCATTCTCA-3',
ANAS-R：5'-CCGGTGGCAACAAAGAAAGT-3',
ANAS-P：5'-TCGCCGACAGCGTCTACGGCT-3'.
8. detection method according to claim 7, it is characterised in that the 5' of the probe is terminal modified fluorescence report base
Group, 3' is terminal modified fluorescent quenching group.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114369611A (en) * | 2021-12-31 | 2022-04-19 | 暨南大学 | Standard plasmid molecule for identifying species of pomfret and application thereof |
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| CN106244698A (en) * | 2016-08-22 | 2016-12-21 | 四川华汉三创生物科技有限公司 | A kind of animal derived materials detection kit |
| CN106755382A (en) * | 2016-12-13 | 2017-05-31 | 苏州百源基因技术有限公司 | One group of specific primer and probe of the real-time PCR detection for being used for duck source property |
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| VAN TUINEN,M.等: "Anas platyrhynchos 18S ribosomal RNA gene, complete sequence", 《GENBANK DATABASE》 * |
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| CN114369611A (en) * | 2021-12-31 | 2022-04-19 | 暨南大学 | Standard plasmid molecule for identifying species of pomfret and application thereof |
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