CN108148915A - For detecting the primer and probe of mouse meat, kit and method - Google Patents
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- CN108148915A CN108148915A CN201711499702.9A CN201711499702A CN108148915A CN 108148915 A CN108148915 A CN 108148915A CN 201711499702 A CN201711499702 A CN 201711499702A CN 108148915 A CN108148915 A CN 108148915A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses a kind of for detecting the fluorescence PCR primer of mouse meat and probe and kit and method.The present invention is using the fluorescence PCR method for relying on deep-sea fireball bacterium RNase H2 enzymes; non-conventional design is carried out to primer simultaneously to improve the specificity of PCR reactions; so that the fluorescence PCR primer and probe and method using the application can accurately amplify the sequence of MUS (NC 005089.1) gene; so as to accurately judge whether contain mouse meat in sample; and the specificity of fluorescent PCR system detection is significantly improved, provide strong support for monitoring for food hygiene.
Description
Technical field
The present invention relates to technical field of gene detection, in particular for detect the primer and probe of mouse meat, kit and
Method.
Background technology
The division of labor of modern food industry is more and more thinner, and chain is increasingly longer, and adulterated chance becomes more, with non-meat
Pretend meat, potential risk is brought to consumer.Meat adulteration is not conventional detection project, data branch disclosed in shortage yet
It holds, therefore is difficult to judge domestic meat products adulterated situation on earth on the whole at present.
Mitochondria is the main cell device of eucaryote respiration, and mitochondria possesses independent genome, typical dynamic
The mitochondria normal length of object cell encodes 13 protein in 16000 bases or so, 2 rRNAs and 20
tRNA.Several features of chondriogen cause its gene common-use words species identification.First, containing tens to upper in a cell
Thousand mitochondrias, so the gene of mitochondria is easily amplified.Second, mitochondria is matrilinear inheritance, and there are one each genes
Copy, in this way it is easy to determine sequence.Third, chondriogen evolution pressure is small, and variation is fast, is conducive to distinguish empty according to gene
Kind.
The method of identified for genes is mainly polymerase chain reaction method, is commonly called as PCR method.Come by the developing history of PCR
It sees, experienced the PCR stages based on gel electrophoresis, to the stage centered on fluorescent PCR.The method of mainstream at present is
Using conventional fluorescent PCR, effect is preferable.In the long-term practice of prior art detection, find in reagent is applied, fluorescent PCR
The specific deficiency for cooking meat identification is main problem.Even if the probe and primer that devise for certain meat of science, but
It is the cross reaction that other meats are also possible that with low degree, difficulty is brought to detection judgement.Additionally, due to Site Detection
Condition is incomplete, and grass-roots work personnel's lacks experience, to differentiating nonspecific signals into positive signal.
Invention content
Based on this, one kind is provided and can accurately be examined it is an object of the invention to overcome above-mentioned the deficiencies in the prior art part
The method for surveying mouse meat.
To achieve the above object, the technical solution that the present invention takes includes the following aspects：
In one aspect, the present invention provides for detecting the fluorescence PCR primer of mouse meat and probe, the primer and spy
Needle base sequence is as follows：
Mus primers Fs 1：5’-ATTCCGCCCAATCACtCAAAddW-3’(SEQ ID NO.1)；
Mus primers R1：5’-TTGGCCTCCGATTCATGTtAAGAddW-3’(SEQ ID NO.2)；
Mus probes P1：5’-TACTGAATTCTAGTAGCCAAC-3’(SEQ ID NO.3)；
Wherein, t represents thymidine acid, and T represents thymine deoxyribotide, and ddW represents double de-
Oxygen ribonucleotide, the 5 ' ends of probe P1 combine fluorescent reporter group, and the 3 ' ends of probe P1 combine fluorescent quenching group.
It should be noted that the W can be A, T, C or G；Wherein, the bi-deoxyribose nucleotide of 3 ' ends cannot be into
One step is extended by archaeal dna polymerase.Therefore, which can effectively prevent the generation of the non-specific products such as primer dimer, significantly drop
Low nonspecific signals.
The 6th nucleotide at primers F 1 and the 3 ' ends of R1 is ribonucleotide rather than deoxyribonucleotide.It is single
Only primer can't be identified and combined by the RNase H2 of P.abyssi；When primer is combined and just with template in annealing temperature
Really after pairing, 3 ' the 6th, end ribonucleotides in primer-template complex are just identified by P.abyssi RNase H2, are cut
It cuts, forms normal primer-template complex, the extension of PCR can just originate.
The special structure of above-mentioned two effectively reduces non-specific amplification, and the specificity of PCR reactions improves 30~100
Times.
Preferably, the fluorescent reporter group is VIC, and the fluorescent quenching group is selected as BHQ or/and MGB.
On the other hand, the present invention provides for detecting the kit of mouse meat, the kit contains above-mentioned
Primer and probe and deep-sea fireball bacterium RNase H2 enzymes.Deep-sea fireball bacterium RNase H2 can be in the double-strand core of pairing as a result,
Acid includes DNA-RNA, RNA-RNA, cuts off RNA molecule；And the thermostabilization of RNase H2 enzymes is high, can be protected in 95 DEG C of high temperature
It is fixed to keep steady, and is suitably applied the fluorescent PCR system of the application；And then in PCR amplification, only when primer correctly combines upper mold
Plate, and after multiple deoxynucleotides by the lower RNA of RNase H2 cuttings and behind, primer could extend, and effectively prevent drawing
The formation of object dimer and the formation of other non-specific amplifications.
Preferably, the kit further includes the primer and probe for internal control, and the primer and probe is for specificity
Eukaryotic 18s rDNA are expanded, the primer and probe sequence is as follows：
18s rDNA primers Fs 2:5’-GGTAGTGACGAAAAATAACAATACgGGACddW-3’ (SEQ ID NO.4)；
18s rDNA primers R2:5’-ATACGCTATTGGAGCTGGAAgTACCddW-3’(SEQ ID NO.5)；
18s rDNA probes P2:5’VIC-AAGTGGACTCATTCCAATTA-BHQ MGB-3’ (SEQ ID NO.6).Its
In, the g of small letter corresponds to ribonucleotide, and the G of capitalization corresponds to deoxyribonucleotide.
Preferably, the RNase H2 enzymes come from deep-sea fireball bacterium.It should be noted that deep-sea fireball bacterium
(Pyrococcus abyssi) is a kind of thermophilic ancient bacterium, is isolated from the submarine volcano spout of northern Fijian 2000 meters of depths.Deep-sea heat
Coccus belongs to fireball Gammaproteobacteria hot-bulb Pseudomonas, is a kind of Gram-negative coccus of anaerobism, and metabolism sulfide is main energy sources.
RNase H2 are a kind of endoribonucleases, if certain positions are RNA in a DNA double chain, RNase H2 can identify this
Structure, and cut from 5 ' ends of RNA molecule, leave DNA molecular and the 5 ' RNA that phosphates is held to dissociate that 3 ' hydroxyls dissociate.
Under the action of archaeal dna polymerase, the DNA that 3 ' hydroxyls dissociate continues to extend, will below the end of ' 5 be free RNA nucleotide fragments take
In generation, forms complete DNA double chain.The RNase H2 of deep-sea fireball bacterium, can at high temperature (96 compared with common RNase H2 enzymes
DEG C) keep activity.RNase H2 properties based on P.abyssi, are applied in PCR system, can improve PCR system
Specificity.
On the other hand, the present invention also provides for detecting the method for mouse meat, include the following steps：
1) DNA is extracted from sample；
2) using the DNA of extraction as template, it is anti-that fluorescent PCR amplification is carried out respectively using the primer and probe of claims 1 or 22
Amplification curve should be obtained；
3) amplification curve is analyzed, determines whether sample contains mouse meat.
Preferably, the fluorescent PCR amplification reaction system in the step 2) is：
Preferably, the fluorescent PCR amplified reaction program in the step 2) is：95 DEG C of pre-degeneration 5min；95 DEG C of denaturation
30s, 60 DEG C of extension 30s；Cycle 50 times.Wherein, p.a. represents deep-sea fireball bacterium (Pyrococcus abyssi).
Preferably, the concrete analysis process of amplification curve is in the step 3)：As F2, R2 as internal control signal and
When the fluorescent PCR amplification curve of P2 forms normal logarithmic amplification " S " type curve,
If 1) sample is in the Fluorescence PCR system using F1, R1 and P1, the fluorescent assay signal shape of acquisition
Into logarithmic amplification " S " type curve, then the sample contains mouse meat；
2) if the fluorescent assay signal obtained does not form logarithmic amplification " S " type curve, which does not contain mouse meat.
In conclusion beneficial effects of the present invention are：
The present invention carries out primer unconventional using the fluorescence PCR method for relying on deep-sea fireball bacterium RNase H2 enzymes
Design is to improve the specificity of PCR reactions, so that the fluorescence PCR primer and probe and method using the application can be accurate
Whether ground amplifies the sequence of MUS (NC-005089.1) gene, so as to accurately judge containing mouse meat in sample, for food
The product monitoring of hygiene provides strong support.
Description of the drawings
Fig. 1 is the amplification of fluorescent PCR system 1, wherein, add showing tremendous enthusiasm coccus RNase H2, primers F 1 and R1 with
Internal control gene all expands success；
Fig. 2 is the amplification of fluorescent PCR system 2, wherein, add showing tremendous enthusiasm coccus RNase H2, primers F 1 and R1 with
Internal control gene all expands success；
Fig. 3 is the amplification curve of mouse meat, wherein, using rat meat DNA as template, primers F 1 and R1 and internal control gene are all
It expands successfully；
Fig. 4 be pork, Carnis Bovis seu Bubali, meat of a sheep amplification, wherein, using pork, Carnis Bovis seu Bubali, meat of a sheep DNA as
Template, primers F 1 and R1 are with internal control gene without the successful signal of amplification.
Specific embodiment
Used term in the present invention unless otherwise indicated, generally there are those of ordinary skill in the art usually to manage
The meaning of solution.The present invention is described in further detail with reference to specific embodiment and with reference to data.It should be understood that these embodiments are only
It is in order to demonstrate the invention rather than limits the scope of the invention in any way.In the examples below, it is not described in detail
Various processes and method be conventional method as known in the art, generally use normal condition such as Sambrook et al., molecule
Clone：Laboratory manual (NewYork：Cold Spring HarborLaboratory Press, 1989) item described in
Part or according to the method proposed by manufacturer.Fluorescence quantitative PCR instrument model ABI 7500 used in the present invention or
BioRad CFX96。
The preparation of 1. primer of embodiment, probe and reference substance
(1) primer and probe designs
According to house mouse disclosed in the GenBank GeneBank of US National Biotechnology Information center NCBI
The reference sequences (NC_005089.1) of chondriogen are detected using 3.0 Software for Design of Primer Express of ABI companies
The primer of muroid specific gene.
It is as follows for the specific primer and probe of mouse meat：
Mus primers Fs 1：5’-ATTCCGCCCAATCACtCAAAddW-3’(SEQ ID NO.1)；
Mus primers R1：5’-TTGGCCTCCGATTCATGTtAAGAddW-3’(SEQ ID NO.2)；
Mus probes P1：5’VIC-TACTGAATTCTAGTAGCCAAC-BHQ MGB 3’(SEQ ID NO.3)；
Wherein, t represents thymidine acid, and T represents thymine deoxyribotide, and ddW represents double de-
Oxygen ribonucleotide, W can be with A, T, C or G.
(2) internal control primer and probe designs
In order to monitor the validity of reaction system, internal control primer and probe are added in detection architecture, the present invention chooses true
Core biology conservative gene 18s rDNA (number by its reference sequences：NR_003278.3), its conserved sequence is analyzed, using ABI public affairs
The 3.0 Software for Design detection primers of PrimerExpress and probe, particular sequence of department are as follows：
Internal control primer pair：
18s rDNA primers Fs 2:5’-GGTAGTGACGAAAAATAACAATACgGGACddW-3’
(SEQ ID NO.4)；
18s rDNA primers R2:5’-ATACGCTATTGGAGCTGGAAgTACCddW-3’(SEQ ID NO.5)；
18s rDNA probes P2:5’VIC-AAGTGGACTCATTCCAATTA-BHQ MGB-3’ (SEQ ID NO.6).
Wherein, the g of small letter corresponds to ribonucleotide, and the G of capitalization corresponds to deoxyribonucleotide.
The preparation of 2 primer and probe of embodiment
By the primer and probe of designed embodiment 1 and gene chemical synthesis company is transferred to synthesize, it is generally automatic using instrument
Chemical synthesis need to provide synthesis qualification test report.
3 rh-PCR of embodiment (PCR that RNase H enzymes rely on) detection kit
A kind of embodiment for being used to detect the kit of mouse meat of the present invention, includes PCR and reacts necessary buffer solution, magnesium
Outside the substances such as ion, dNTPs, also comprising detection primer internal control primer and probe, positive control mouse DNA.Its concrete composition
It is as shown in table 1 below.
The composition of 1 rh-PCR detection kits of table
| Number | Reagent |
| 1 | Tris-HCl (pH=8.4) |
| 2 | KCl |
| 3 | MgCl2 |
| 4 | dNTPs |
| 5 | Triton X100 |
| 6 | SYBR Green I |
| 7 | Taq DNA pol |
| 8 | p.a.RNase H2 |
| 9 | Sense primer |
| 10 | Downstream primer |
| 11 | Fluorescence probe |
| 12 | Mouse DNA |
The primer and the sequence information of probe that the PCR reactions of above-mentioned each specific detection are related to are as follows：mus-F1:5’-
ATTCCGCCCAATCACtCAAAddX-3’(SEQ ID NO.1)； mus-R1:5’-
TTGGCCTCCGATTCATGTtAAGAddW-3’(SEQ ID NO.2)； mus-P1:5’VIC-
TACTGAATTCTAGTAGCCAAC-BHQ MGB 3’(SEQ ID NO.3)；
18s rDNA-F2:5’-GGTAGTGACGAAAAATAACAATACgGGACddW-3’ (SEQ ID NO.4)；
18s rDNA-R2:5’-ATACGCTATTGGAGCTGGAAgTACCddW-3’(SEQ ID NO.5)；
18s rDNA-P2:5’VIC-AAGTGGACTCATTCCAATTA-BHQ MGB-3'(SEQ ID NO.6)。
The Proof-Of Principle experiment of 4 rh-PCR kits of embodiment
The principle of the present invention verification test includes the following steps：
Using the rh-PCR kits in embodiment 3, two sets of fluorescent PCR systems are configured, respectively shown in table 2 and table 3.
2 Fluorescence PCR system 1 of table
| Reagent | Final concentration |
| Tris-HCl (pH=8.4) | 20mM |
| KCl | 50mM |
| MgCl2 | 3.0mM |
| dNTPs | 0.8mM |
| Triton X100 | 0.01% |
| mus-F1 | 200nM |
| mus-R1 | 200nM |
| mus-P1 | 100nM |
| 18s rDNA-F2 | 200nM |
| 18s rDNA-R2 | 200nM |
| 18s rDNA-P2 | 100nM |
| Taq DNA pol | 0.5U |
| p.a.RNase H2 | 0.5mU/μl |
| Mouse DNA | 20ng |
| Total volume | 10μl |
3 Fluorescence PCR system 2 of table
The difference of Fluorescence PCR system 1 and system 2 is that the former uses deep-sea fireball bacterium RNase H2 enzymes
(p.a.RNase H2), the latter do not use the enzyme.
Third walks：Upper machine testing
According to the following table 4 set temperature cycle and signal acquisition program.
4 PCR response procedures of table
4th step：Interpretation of result
1 the result is shown in Figure 1 of fluorescent PCR system, in fluorescent PCR system 1, experimental group and control group have the fluorescent PCR amplification bent
Line forms normal logarithmic amplification " S " type curve；And in fluorescent PCR system 2, equal unstressed configuration PCR amplification curve；Fluorescent PCR system 2
As a result see Fig. 2.
The PCR in the present invention is compared with traditional fluorescent PCR as a result, it is necessary to using deep-sea fireball bacterium RNase H2 enzymes,
After primer is combined with template, the RNA in primer is cut from 5 ' ends from the Template-primer double-strand locally matched；Otherwise
Primer can not extend, and entire PCR reactions cannot carry out.
5 rh-PCR of embodiment detects rat meat
A kind of embodiment for being used to detect the method for mouse meat of the present invention, includes the following steps：
The first step：Prepare DNA
1st, rat meat derives from the Experimental Animal Center of scientific research institution, and pork, Carnis Bovis seu Bubali, meat of a sheep are bought from supermarket.2nd, it takes
Various meat 100-300mg, therefrom obtain DNA, and genomic DNA is extracted using commercialized kit；
3rd, DNA concentration and purity are measured, it is desirable that concentration is adjusted to 20-30ng/ μ l；OD260nm/OD280nm=1.7~
2.0。
Second step：PCR reaction systems are prepared
In the dedicated pipe of quantitative fluorescent PCR PCR reaction systems are prepared according to the following table 5.
5 PCR reaction systems of table form
| Reagent | Final concentration |
| Tris-HCl (pH=8.4) | 20mM |
| KCl | 50mM |
| MgCl2 | 3.0mM |
| dNTPs | 0.8mM |
| Triton X100 | 0.01% |
| mus-F1 | 200nM |
| mus-R1 | 200nM |
| mus-P1 | 100nM |
| 18s rDNA-F2 | 200nM |
| 18s rDNA-R2 | 200nM |
| 18s rDNA-P2 | 100nM |
| Taq DNA pol | 0.5U |
| p.a.RNase H2 | 0.5mU/μl |
| DNA profiling | It adds on demand |
| Total volume | 10μl |
Third walks：Upper machine testing
With embodiment 4.
4th step：Analysis result
The amplification curve of mouse meat is as shown in figure 3, and pork, Carnis Bovis seu Bubali, meat of a sheep are as shown in Figure 4.Specific amplification is bent
Knot fruit analytic process is as follows：
It is glimmering as F2, R2 and P2 as internal control signal under the conditions of above-mentioned PCR reaction systems and temperature cycling program
When light PCR amplification curve forms normal logarithmic amplification " S " type curve,
If 1) sample is in the Fluorescence PCR system using F1, R1 and P1, the fluorescent assay signal shape of acquisition
Into logarithmic amplification " S " type curve, then the sample contains mouse meat；
2) if the fluorescent assay signal obtained does not form logarithmic amplification " S " type curve, which does not contain mouse meat.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of range is protected, although being explained in detail with reference to preferred embodiment to the present invention, those of ordinary skill in the art should
Understand, technical scheme of the present invention can be modified or replaced equivalently, without departing from the essence of technical solution of the present invention
And range.
SEQUENCE LISTING
<110>Star battle array（Guangzhou）Gene Tech. Company Limited
<120>For detecting the primer and probe of mouse meat, kit and method
<130> 2017
<160> 6
<170> PatentIn version 3.5
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ttggcctccg attcatgtta agaw 24
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<400> 3
tactgaatyc tagtagccaa c 21
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ggtagtgacg aaaaataaca atacgggacw 30
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atacgctatt ggagctggaa gtaccw 26
<210> 6
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tactgaatyc tagtagccaa c 21
Claims (8)
1. for detecting the fluorescence PCR primer and probe of mouse meat, which is characterized in that the primer and probe base sequence is such as
Under：
Mus primers Fs 1：5’-ATTCCGCCCAATCACtCAAA ddW-3’(SEQ ID NO.1)；
Mus primers R1：5’-TTGGCCTCCGATTCATGTtAAGA ddW-3’(SEQ ID NO.2)；
Mus probes P1：5’-TACTGAATTCTAGTAGCCAAC-3’(SEQ ID NO.3)；
Wherein, t represents thymidine acid, and T represents thymine deoxyribotide, and ddW represents double deoxidation core
Ribotide, the 5 ' ends of probe P1 combine fluorescent reporter group, and the 3 ' ends of probe P1 combine fluorescent quenching group.
2. primer and probe according to claim 1, which is characterized in that the fluorescent reporter group be VIC, the fluorescence
Quenching group is selected as BHQ or/and MGB.
3. for detecting the kit of mouse meat, which is characterized in that the kit contains the primer described in claims 1 or 2
With probe and deep-sea fireball bacterium RNase H2 enzymes.
4. kit according to claim 3, which is characterized in that the kit further includes the primer and spy for internal control
Needle, the primer and probe are used for the Eukaryotic 18s rDNA of specific amplification, and the primer and probe sequence is as follows：
18s rDNA primers Fs 2:5’-GGTAGTGACGAAAAATAACAATACgGGACddW-3’(SEQ ID NO.4)；
18s rDNA primers R2:5’-ATACGCTATTGGAGCTGGAAgTACCddW-3’(SEQ ID NO.5)；
18s rDNA probes P2:5’VIC-AAGTGGACTCATTCCAATTA-BHQ MGB-3’(SEQ ID NO.6).
5. for the method for detecting mouse meat, which is characterized in that include the following steps：
1) DNA is extracted from sample；
2) using the DNA of extraction as template, fluorescent PCR amplified reaction is carried out respectively using the primer and probe of claims 1 or 22 and is obtained
Obtain amplification curve；
3) amplification curve is analyzed, determines whether sample contains mouse meat.
6. according to the method described in claim 5, it is characterized in that, the fluorescent PCR amplification reaction system in the step 2) is：
7. according to the method described in claim 5, it is characterized in that, the fluorescent PCR amplified reaction program in the step 2) is：
95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 30s, 60 DEG C of extension 30s；Cycle 50 times.
8. according to the method described in claim 5, it is characterized in that, in the step 3) amplification curve concrete analysis process
For：When the fluorescent PCR amplification curve of F2, R2 and P2 as internal control signal form normal logarithmic amplification " S " type curve,
If 1), a sample is in the Fluorescence PCR system using F1, R1 and P1, and the fluorescent assay signal of acquisition is formed pair
Number expands " S " type curve, then the sample contains mouse meat；
2) if the fluorescent assay signal obtained does not form logarithmic amplification " S " type curve, which does not contain mouse meat.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109234368A (en) * | 2018-08-13 | 2019-01-18 | 武汉千麦医学检验所有限公司 | A method of for hepatitis B virus YMDD motif area medicament-resistant mutation |
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