CN107338316A - Positive criteria molecule, preparation and detection method are used in calf-derived Cyclospora PCR detections - Google Patents
Positive criteria molecule, preparation and detection method are used in calf-derived Cyclospora PCR detections Download PDFInfo
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Abstract
The invention discloses calf-derived Cyclospora PCR detections positive criteria molecule, preparation and detection method, the positive criteria molecule is the plasmid molecule for being capable of autonomous replication, and ox 18SrRNA gene orders and ox mitochondrial COX I gene sequence are included in the plasmid molecule.The calf-derived Cyclospora PCR detection positive criteria molecules that the present invention is prepared; with preparation process it is simple, can preserve for a long time, the advantage of high specificity and high sensitivity; the positive criteria molecule needs not rely on the supply of standard ox material; can substitute cow genome group DNA be used for Niu Chengfen 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 calf-derived Cyclospora PCR detections positive criteria molecule, system
Standby and detection method.
Background technology
The accurate content for detecting corresponding animal derived components in food, it is to prevent and treat the key that meat of poor quality product comes into the market to arrange
Apply.
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, it 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, realizes leap of the round pcr by qualitative to quantitative.
Due to needing positive reference material to ensure reality as control when being detected using FQ-PCR technologies to sample
Test the reliability of result.And at this stage, it is typically for positive reference material used in the detection process of animal derived materials
Animal genome DNA.Wherein, during ox composition detection, extraction cow genome group DNA processes are complicated, prepare very inconvenient.By
It can not meet long-term storage and the regular needs used in cow genome group DNA stability, therefore, carry out ox every time into go-on-go
During survey, it is required for preparing cow genome group DNA again as positive reference material, influences the stability of ox composition measurement.
The content of the invention
The invention provides calf-derived Cyclospora 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, the invention provides a kind of calf-derived Cyclospora PCR detections positive criteria molecule, the positive criteria
Molecule is the plasmid molecule for being capable of autonomous replication, and ox 18SrRNA gene orders and ox mitochondria are included in the plasmid molecule
COXI gene orders.
Second aspect, present invention also offers a kind of to prepare the preparation side of positive criteria molecule described in first aspect
Method, comprise the following steps：
（1）Separately design amplimer 18S-F, 18S-R and ox mitochondrial COX I gene sequence of ox 18SrRNA gene orders
Amplimer BCOXI-F, BCOXI-R；
Amplimer 18S-F, 18S-R of ox 18SrRNA gene orders be：
18S-F ：5'-GCGTCGACAGCCTGAGAAACGGCTACC-3'；
18S-R ：5'-AACTGCAGTGCTGGCACCAGACTTGC-3'；
Amplimer BCOXI-F, BCOXI-R of ox mitochondrial COX I gene sequence be：
BCOXI-F：5'- ACACTTAGCAGGAGTTTCCTC-3'；
BCOXI-R：5'- TGATATAGAATAGGGTCTCCTCC-3'；
（2）Using cow genome group as templet gene group, with the amplimer and ox mitochondrial COX I gene of ox 18SrRNA gene orders
The amplimer of sequence, enter performing PCR amplification, obtain ox 18SrRNA gene orders and ox mitochondrial COX I gene sequence；
（3）The ox 18SrRNA gene orders and ox mitochondrial COX I gene sequence are respectively connecting to pMD18-T carriers, obtained
To ox 18SrRNA recons and ox mitochondrial COX I recons；
（4）The ox 18SrRNA recons and ox mitochondrial COX I recons by digestion with restriction enzyme and are spliced, obtained
To positive criteria molecule.
Alternatively, the restriction enzyme isSal I enzymes andPstI enzymes.
Alternatively, step（2）In, the reaction system of PCR amplification is 10 μM of μ L of sense primer 1,10 μM of anti-sense primers 1
μ L, the μ L of templet gene group 3, the μ L of Taq enzyme 12.5, add ddH2The volume of O adjustment reaction systems is 25 μ L；The PCR is expanded anti-
Condition is answered 94 DEG C of 30 s of denaturation, 58 DEG C of 30 s of annealing, 72 DEG C of 30 s of extension, to carry out 40 circulations altogether for 94 DEG C of 5 min of denaturation,
Obtain PCR primer；The PCR primer is separated by agarose gel electrophoresis, purpose product is pure with glue reclaim kit
Change, obtain PCR purified products, as ox 18SrRNA gene orders and ox mitochondrial COX I gene sequence.
Alternatively, step（3）In, by the ox 18SrRNA gene orders and ox mitochondrial COX I gene sequence respectively with
PMD18-T carriers are attached, and are connected overnight at 4 DEG C, obtain ox 18SrRNA recons 18S-pMD18-T and ox mitochondria
COXI recons BCOXI-pMD18-T.
Alternatively, step（4）In, the ox 18SrRNA recons and ox mitochondrial COX I recons are used respectivelySal I
Enzyme andPstI enzymes carry out double digestion, 37 DEG C of water-baths 2 hours, agarose gel electrophoresis, gel extraction, will recovery fragment connection, 4
Night connection is spent, shaking table uniformly mixes 2 hours and carries out plasmid conversion, obtains converting plasmid；The conversion plasmid is subjected to gel
Electrophoretic separation, recovery macromolecular plasmid band carry out the screening recovery of recombinant clone plasmid, obtain recombinant clone；By the restructuring gram
It is grand verified by Standard PCR,SalI andPstI digestion verification and digestion products sequence verification, obtained plasmid molecule
18S-BCOXI-pMD18-T are positive criteria molecule.
The third aspect, present invention also offers a kind of to detect the inspection of positive criteria molecular specificity described in first aspect
Survey method, specifically, using specific detection primer and probe, real-time fluorescence is carried out by template of the positive criteria molecule
PCR reacts, and the specific detection primer and probe include following two groups：
Specific detection primer 18S-F, 18S-R and probe 18S-P of ox 18SrRNA gene orders：
18S-F：5'-GCGTCGACAGCCTGAGAAACGGCTACC-3'；
18S-R：5'-AACTGCAGTGCTGGCACCAGACTTGC-3'；
18S-P：5'-TGCGCGCCTGCTGCCTTCCT-3'
Specific detection primer BCOXI-F, BCOXI-R and probe BCOXI-P of ox mitochondrial COX I gene sequence：
BCOXI-F：5'- ACACTTAGCAGGAGTTTCCTC-3'；
BCOXI-R：5'- TGATATAGAATAGGGTCTCCTCC-3'；
BCOXI-P：5'- ACCAAACCCCTCTGTTCGTATGATCCG-3'.
Alternatively, the 5' of the probe is terminal modified fluorescent reporter group, and 3' is terminal modified fluorescent quenching group.
The calf-derived Cyclospora PCR detection positive criteria molecules that the present invention is prepared, have that preparation process is simple, can grow
The advantage of time preservation, high specificity and high sensitivity, the positive criteria molecule need not rely on the supply of standard ox material,
Can substitute cow genome group DNA be used for Niu Chengfen PCR is qualitative, quantitative measurment and analysis, ensure the reliable of laboratory test results
Property, solve the problem for lacking positive control in foods supervision work.
It should be appreciated that the general description and following detailed description of the above 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.
Fig. 1 is the structural representation of positive criteria molecule prepared by the present invention；
Fig. 2 is ox 18SrRNA gene order real-time fluorescent PCR amplification curve comparison figures；
Fig. 3 is the gene order real-time fluorescent PCR amplification curve comparison figure of ox mitochondrial COX 1；
Fig. 4 is the sensitivity technique result figure of positive criteria molecule prepared by 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 describes, it is clear that described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other under the premise of creative work is not made
Embodiment, belong to the scope of protection of the invention.
Embodiment one
1st, DNA is extracted：Beef genomic DNA is extracted using QIAGEN DNeasy Blood and Tissue Kit, with light splitting
Photometer detects DNA purity and concentration.It is 1.8-1.9 or so to determine OD260/OD280 values, and concentration is said more than 10ng/ μ L
Bright DNA purity is higher, moderate concentration, meets PCR amplifications and requires.
2nd, primer and specific probe design：To searching for ox 18SrRNA gene orders and ox mitochondria in GeneBank
COXI gene orders, according to the ox 18SrRNA gene orders of acquisition and ox mitochondrial COX I gene sequence, utilize Primer 5.0
Two pairs of PCR primers are designed, are respectively used for amplifying ox 18SrRNA gene orders and ox mitochondrial COX I gene sequence, design two kinds
Specific probe, to detect whether the positive criteria molecule finally prepared successfully constructs.The 5' of probe is terminal modified fluorescence report
Group is accused, 3' is terminal modified fluorescent quenching group.Primer and probe is as shown in the table：
Table 1 is used to expand（Specific detection）The primer and spy of ox 18SrRNA gene orders and ox mitochondrial COX I gene sequence
Pin
3rd, the clone of ox 18SrRNA gene orders and ox mitochondrial COX I gene sequence
Using cow genome group as templet gene group, enter performing PCR amplification using the primer pair 18S-R and 18S-P in table 1, obtain ox
18SrRNA gene orders.Using cow genome group as templet gene group, entered using the primer pair BCOXI-F and BCOXI-R in table 1
Performing PCR expands, and obtains ox mitochondrial COX I gene sequence.Specifically, the reaction system of PCR amplifications is 10 μM of μ L of sense primer 1,
10 μM of μ L of anti-sense primer 1, the μ L of templet gene group 3, the μ L of Taq enzyme 12.5, add ddH2The volume of O adjustment reaction systems is 25 μ L；Institute
The reaction condition for stating PCR amplifications is 94 DEG C of 5 min of denaturation, and 94 DEG C are denatured 30 s, 58 DEG C of annealing 30 s, 72 DEG C of 30 s of extension, altogether
40 circulations are carried out, obtain PCR primer；PCR primer is separated by agarose gel electrophoresis, purpose product glue reclaim
Kits, obtain PCR purified products, as ox 18SrRNA gene orders and ox mitochondrial COX I gene sequence.
Ox 18SrRNA gene orders and ox mitochondrial COX I gene sequence are attached with pMD18-T carriers respectively, 4
Connected overnight at DEG C, obtain ox 18SrRNA recons 18S-pMD18-T and ox mitochondrial COX I recons BCOXI-pMD18-T.
4th, the structure of positive criteria molecule
Ox 18SrRNA recons and the recon of ox mitochondrial COX 1 are used respectivelySal I enzymes andPstI enzymes progress double digestion, 37 DEG C
Water-bath 2 hours, agarose gel electrophoresis, gel extraction, night connection is spent into recovery fragment connection, 4, it is small that shaking table uniformly mixes 2
Shi Jinhang plasmids convert, and obtain converting plasmid；Conversion plasmid is subjected to gel electrophoresis separation, recovery macromolecular plasmid band is carried out
The screening recovery of recombinant clone plasmid, obtains recombinant clone；Recombinant clone is verified by Standard PCR,SalI andPstI's
Digestion verification and digestion products sequence verification, obtained plasmid molecule 18S-BCOXI-pMD18-T are positive criteria molecule.
18S-BCOXI-pMD18-T concrete structure is as shown in Figure 1.
Embodiment two
1st, the structure result of positive criteria molecule
18S-the BCOXI-pMD18-T prepared using embodiment one are carried out with the primer and probe in table 1 to it respectively as template
PCR is expanded, and finds that extension increasing sequence is consistent with expected fragment after sequence verification.As a result show that the positive criteria molecule of structure includes
The success of ox 18SrRNA gene orders and ox mitochondrial COX I 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 cow genome group, 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 is ox 18SrRNA gene order real-time fluorescent PCR amplification curve comparisons
Figure, Fig. 3 is ox mitochondrial COX I gene 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 ox 18SrRNA and bovine mitochondrial gene sequence, no matter the sun to cow genome group or the present invention
Property standard molecule can detect positive signal.The Ct values of ox 18SrRNA genes detection are 18 circulations of cow genome group, and the positive is marked
13 circulations of quasi-molecule, and negative sample does not have signal then；The Ct values of ox mitochondrial COX I gene detection are cow genome group 15
Circulation, 8 circulations of positive criteria molecule, and negative control does not have signal then, while base is carried out to 30 common animals and plants species
Because of group extraction and an ox mitochondrial COX I gene specific detection, without signal, illustrate positive criteria molecule prepared by the present invention
It can be arrived by specific probe in detecting, there is specificity well.
3rd, the sensitivity technique result of positive criteria molecule
Ten times of gradient dilutions are carried out to positive criteria molecule（10-1To 10-5）, 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 3 times.Standard curve is established, 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 10-5Dilution factor, illustrate 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.
Under the conditions of the positive criteria molecule of preparation is placed in into 25 DEG C of room temperature, place six months, carry out real-time fluorescence PCR inspection
Survey, the stability of statistical analysis positive criteria molecule is carried out to the Ct values of acquisition.Each real-time PCR detection is each reacted
The Ct values average value of 3 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 one month after just start significant difference occur, and this significant difference in positive criteria molecular memory more than two
Disappeared after month.It is inferred that positive criteria molecule better heat stability prepared by the present invention, not degradable, but prolong over time
Long, whole gene group 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 considering specification and putting into practice the disclosure invented here
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>Positive criteria molecule, preparation and detection method are used in calf-derived Cyclospora PCR detections
<160>6
<210>1
<211>27
<212>DNA
<213>It is artificial synthesized
<220>
<223>
<400>1
GCGTCGACAG CCTGAGAAAC GGCTACC 27
<210>2
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<212>DNA
<213>It is artificial synthesized
<220>
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<400>2
AACTGCAGTG CTGGCACCAG ACTTGC 26
<210>3
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<212>DNA
<213>It is artificial synthesized
<220>
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<400>3
ACACTTAGCA GGAGTTTCCT C 21
<210>4
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<213>It is artificial synthesized
<220>
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<400>4
TGATATAGAA TAGGGTCTCC TCC 23
<210>5
<211>20
<212>DNA
<213>It is artificial synthesized
<220>
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<400>5
TGCGCGCCTG CTGCCTTCCT 20
<210>6
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<212>DNA
<213>It is artificial synthesized
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ACCAAACCCC TCTGTTCGTA TGATCCG 27
Claims (8)
1. positive criteria molecule is used in a kind of calf-derived Cyclospora PCR detections, it is characterised in that the positive criteria molecule is can be certainly
The plasmid molecule of main duplication, ox 18SrRNA gene orders and ox mitochondrial COX I gene sequence are included in 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 and ox mitochondrial COX I gene sequence of ox 18SrRNA gene orders
Amplimer BCOXI-F, BCOXI-R；
Amplimer 18S-F, 18S-R of ox 18SrRNA gene orders be：
18S-F ：5'-GCGTCGACAGCCTGAGAAACGGCTACC-3'；
18S-R ：5'-AACTGCAGTGCTGGCACCAGACTTGC-3'；
Amplimer BCOXI-F, BCOXI-R of ox mitochondrial COX I gene sequence be：
BCOXI-F：5'- ACACTTAGCAGGAGTTTCCTC-3'；
BCOXI-R：5'- TGATATAGAATAGGGTCTCCTCC-3'；
（2）Using cow genome group as templet gene group, with the amplimer and ox mitochondrial COX I gene of ox 18SrRNA gene orders
The amplimer of sequence, enter performing PCR amplification, obtain ox 18SrRNA gene orders and ox mitochondrial COX I gene sequence；
（3）The ox 18SrRNA gene orders and ox mitochondrial COX I gene sequence are respectively connecting to pMD18-T carriers, obtained
To ox 18SrRNA recons and ox mitochondrial COX I recons；
（4）The ox 18SrRNA recons and ox mitochondrial COX I recons by digestion with restriction enzyme and are spliced, obtained
To positive criteria molecule.
3. preparation method according to claim 2, it is characterised in that the restriction enzyme isSal I enzymes andPst I
Enzyme.
4. preparation method according to claim 2, it is characterised in that step（2）In, the reaction system of the PCR amplifications
For 10 μM of μ L of sense primer 1,10 μM of μ L of anti-sense primer 1, the μ L of templet gene group 3, the μ L of Taq enzyme 12.5, add ddH2O adjusts reactant
The volume of system is 25 μ L；The reaction condition of the PCR amplifications is 94 DEG C of 5 min of denaturation, and 94 DEG C are denatured 30 s, 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, obtained
PCR purified products, as ox 18SrRNA gene orders and ox mitochondrial COX I gene sequence.
5. preparation method according to claim 2, it is characterised in that step（3）In, by the ox 18SrRNA gene sequences
Row and ox mitochondrial COX I gene sequence are attached with pMD18-T carriers respectively, are connected overnight at 4 DEG C, are obtained ox 18SrRNA
Recon 18S-pMD18-T and ox mitochondrial COX I recons BCOXI-pMD18-T.
6. preparation method according to claim 2, it is characterised in that step（4）In, by the ox 18SrRNA recons
Used respectively with ox mitochondrial COX I reconsSalI enzymes andPstI enzymes carry out double digestion, 37 DEG C of water-baths 2 hours, Ago-Gel
Electrophoresis, gel extraction, night connection is spent by recovery fragment connection, 4, shaking table uniformly mixes 2 hours and carries out plasmid conversion, turned
Change plasmid；
The conversion plasmid is subjected to gel electrophoresis separation, recovery macromolecular plasmid band carries out recombinant clone plasmid and screened 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-BCOXI-pMD18-T are 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 ox 18SrRNA gene orders：
18S-F：5'-GCGTCGACAGCCTGAGAAACGGCTACC-3'；
18S-R：5'-AACTGCAGTGCTGGCACCAGACTTGC-3'；
18S-P：5'-TGCGCGCCTGCTGCCTTCCT-3'
Specific detection primer BCOXI-F, BCOXI-R and probe BCOXI-P of ox mitochondrial COX I gene sequence：
BCOXI-F：5'- ACACTTAGCAGGAGTTTCCTC-3'；
BCOXI-R：5'- TGATATAGAATAGGGTCTCCTCC-3'；
BCOXI-P：5'- ACCAAACCCCTCTGTTCGTATGATCCG-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 |
|---|---|---|---|---|
| CN113088531A (en) * | 2021-03-25 | 2021-07-09 | 四川省食品药品检验检测院(四川省药品质量研究所、四川省医疗器械检测中心) | Bovine-derived component quantitative analysis standard plasmid, preparation and detection method and application |
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| CN113088531B (en) * | 2021-03-25 | 2023-10-17 | 四川省药品检验研究院(四川省医疗器械检测中心) | Bovine-derived ingredient quantitative analysis standard plasmid, preparation and detection methods and application |
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Address after: Xinluo Avenue high tech Zone of Ji'nan City, Shandong Province, No. 2749 250101 Applicant after: Food and medicine Inspection Research institute of Shandong Province Address before: Xinluo Avenue high tech Development Zone Ji'nan city Shandong province 250101 No. 2749 drug inspection office building 1-101 Applicant before: Food and medicine Inspection Research institute of Shandong Province |
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Application publication date: 20171110 |