CN108676853A - A kind of real-time fluorescence quantitative PCR kit for mouse detection - Google Patents
A kind of real-time fluorescence quantitative PCR kit for mouse detection Download PDFInfo
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Abstract
The present invention relates to a kind of real-time fluorescence quantitative PCR kits for mouse detection, including PCR reaction solution, enzyme mixation, mouse 12S rRNA genes standard items, negative controls and positive reference substance.The DNA combination real-time fluorescence quantitative PCR detection techniques that the present invention passes through extraction detected sample, it can reach the purpose of mouse content in accurate quantitative analysis detection meat products, have the characteristics that quick, sensitive, specificity is good, to judging that mouse content is of great significance in meat products.
Description
Technical field
The present invention relates to technical field of food detection more particularly to a kind of real time fluorescent quantitatives for mouse detection
PCR kit.
Background technology
In recent years, security issues become increasingly urgent for meat products in food, such as domestic " false ox, false mutton event ", " false duck
Blood event " etc..Wherein in mutton adulterate rat meat become mutton fake a kind of new tool, with the improvement of people ' s living standards and
The enhancing of health perception, people become more concerned with the true or false of food, and animal derived materials differentiate as social concerns in food
One of focus.
The discriminating that past is used for species in meat-based product depends on the protein analyses such as electrophoresis, immunization, ELISA
Method.These technologies can not but analyze processing mixing meat products, and round pcr is short based on its reaction time, has higher
Sensitivity, specificity become the preferential selection for differentiating species in processed food.But traditional round pcr is examined in quantitative and high-volume
Limitation is larger in survey.Therefore, it is eager to find that a kind of new technology can accurately distinguish species and realize quick, large capacity
Real_time quantitative detection.Real-time fluorescence quantitative PCR can detect the spies such as cold cuts and mixing sample by its highly sensitive and specificity
Point has gradually become the mainstream technology that Meat ingredients differentiate.In recent years, existing both at home and abroad to utilize TaqMan probe method to different meat
The report that class kind is differentiated.
The mitochondrial DNA of animal have molecular weight is small, structural conservation, the design feature that thermal stability is good, not degradable and
The genetic characteristics that matrilinear inheritance, copy number are more, evolutionary rate is fast, do not recombinate, provide the sequence analysis of mitochondrial DNA
Information be widely used to research species between and species inside phylogenetic analysis, animal derived materials identification etc. necks
Domain.The size of nearly all domestic animal mtDNA is about between 16.0 ~ 16.5kb.
As the object that phyletic evolution and species specificity are analyzed, studying more mitochondrial DNA (mtDNA) gene has
12SrRNA, 18SrRNA gene, tRNA-Lys genes, ATP enzyme genes, cytochrome b（12S rRNA）Gene etc., D-
Areas loop etc..
Invention content
Technical problem to be solved by the invention is to provide a kind of quick, easy, sensitive, accurate, reliable for mouse source
Property detection real-time fluorescence quantitative PCR kit.
To solve the above problems, a kind of real-time fluorescence quantitative PCR kit for mouse detection of the present invention,
Including
- PCR reaction solution, by 12mM Tris-HCl（pH8.9）, 100mM KCl, 0.12%Triton X-100,0.12% cholic acid
Sodium, 0.6 mg/mL BSA, 1.8mM MgCl2, 50nM dNTP, 500nM sense primers, 500nM downstream primers, 300nM
Taqman probes form；
Wherein upstream primer sequence is 5'- CGGTGTAAAATCCCTTAAAC-3'；Downstream primer sequence is 5'-
ATATAGGCTGAATTAGCAA-3'；Probe sequence is 5 '-ATAAATAAATAAATAGAA-3 '；
- enzyme mixation, by 5U/ μ L Taq archaeal dna polymerases, 50% glycerine, 20mM Tris-HCl (pH8.0,25 DEG C),
100mM KCl, 0.1mM EDTA, 1mM DTT, 0.5% Tween®20 and 0.5% NP-40 composition；
- mouse 12S rRNA gene standard items, by 1.00 × 108copies/ml、1.00×107copies/ml、1.00×
106copies/ml、1.00×105The recombinant plasmid of copies/ml forms；
Wherein 12S rRNA genes standard items sequence is 5 '-CGGCGTAAAACGTGTCAACTATAAATAAATAAATAGAATTAA
AATCCAACTTATATGTGAAAATTCATTGTTAGGACCTAAACTCAATAACGAAAGTAATTCTAGTCATTTATAATACA
CGACAGCTAAGACCCAAACTGGGATTAGATACC-3’；
- negative controls, are made of water；
- positive reference substance, by 1.00 × 107The recombinant plasmid of copies/ml forms.
The fluorescent reporter group of 5 ' end labels is FAM in the probe, and the fluorescent quenching group of 3 ' end labels is TAMRA.
Compared with the prior art, the present invention has the following advantages：
1, the present invention uses gene clone technology, and conservative fragments in the 12S rRNA gene kinds of mouse DNA are inserted into carrier
In pMD18-T, the recombinant plasmid containing 12S rRNA genetic fragments is obtained, in this, as standard items.According to mouse 12S
The genetic fragment coding gene sequence of rRNA designs and synthesizes a group-specific primers and probe, optimizes PCR reaction conditions, establishes
Using real-time fluorescence quantitative polymerase chain reaction as the detection method of platform, and the method established is assessed.
2, for the present invention by the DNA combination real-time fluorescence quantitative PCR detection techniques of extraction detected sample, it is accurate to can reach
The purpose for quantitatively detecting mouse content in meat products will be to judging that mouse content is of great significance in meat products
Microbiological detection of foods field plays a significant role,
3, the present invention is quick, sensitive, specificity is good.
Description of the drawings
Specific embodiments of the present invention will be described in further detail below in conjunction with the accompanying drawings.
Fig. 1 is primer and probe system optimization experimental result of the present invention.A represents primer as 1.6ul, and probe is 1.6 ul；b
Primer is represented as 1.0ul, probe is 1.6 ul；C represents primer as 1.0ul, and probe is 0.8 ul；D represents primer as 2.0ul,
Probe is 0.8 ul；E represents primer as 1.6ul, and probe is 0.8 ul；F represents primer as 2.0ul, and probe is 1.0 ul；G generations
Table primer is 1.0ul, and probe is 0.8 ul；H represents primer as 1.0ul, and probe is 1.0 ul；I represents primer as 2.0ul, visits
Needle is 1.6 ul.
Fig. 2 is sensitivity experiment result of the present invention.It is respectively 1.00 × 10 that a, which represents plasmid concentration,7Copies/ml, b are represented
Plasmid concentration is 1.00 × 106Copies/ml, c represent plasmid concentration as 1.00 × 105Copies/ml, d represent plasmid concentration as
1.00×104Copies/ml, e represent plasmid concentration as 1.00 × 103Copies/ml, f represent plasmid concentration as 1.00 ×
102Copies/ml and negative control.
Fig. 3 is specificity experiments result of the present invention.It is mouse standard amplification curve wherein occur, does not occur standard amplification song
Line is ox, sheep, pig, fish, chicken, duck, goose, rabbit, donkey, the frog, dog, negative control.
Fig. 4 is mouse standard curve of the present invention.
Specific implementation mode
A kind of real-time fluorescence quantitative PCR kit for mouse detection, including
- PCR reaction solution, by 12mM Tris-HCl（pH8.9）, 100mM KCl, 0.12%Triton X-100,0.12% cholic acid
Sodium, 0.6 mg/mL BSA, 1.8mM MgCl2, 50nM dNTP, 500nM sense primers, 500nM downstream primers, 300nM
Taqman probes form；
Wherein upstream primer sequence is 5'- CGGTGTAAAATCCCTTAAAC-3'；Downstream primer sequence is 5'-
ATATAGGCTGAATTAGCAA-3'；Probe sequence is 5 '-ATAAATAAATAAATAGAA-3 '；
- enzyme mixation, by 5U/ μ L Taq archaeal dna polymerases, 50% glycerine, 20mM Tris-HCl (pH8.0,25 DEG C),
100mM KCl, 0.1mM EDTA, 1mM DTT, 0.5% Tween®20 and 0.5% NP-40 composition；
- mouse 12S rRNA gene standard items, by 1.00 × 108copies/ml、1.00×107copies/ml、1.00×
106copies/ml、1.00×105The recombinant plasmid of copies/ml forms；
Wherein 12S rRNA genes standard items sequence is 5 '-CGGCGTAAAACGTGTCAACTATAAATAAATAAATAGAATTA
AAATCCAACTTATATGTGAAAATTCATTGTTAGGACCTAAACTCAATAACGAAAGTAATTCTAGTCATTTATAATAC
ACGACAGCTAAGACCCAAACTGGGATTAGATACC-3’（SEQ ID NO.2）；
- negative controls, are made of water；
- positive reference substance, by 1.00 × 107The recombinant plasmid of copies/ml forms.
Wherein：The fluorescent reporter group of 5 ' end labels is FAM in probe, and the fluorescent quenching group of 3 ' end labels is TAMRA.
Method therefor is conventional method, the primer, probe and sequencing used unless otherwise specified in following experimental examples
Work is by raw work bioengineering（Shanghai）Limited liability company synthesizes and completes.
The preparation of 1 12S rRNA gene standard items of example
Establish real time fluorescence quantifying PCR method it may first have to which the external standards needed for preparation method, standard items should include height
Spend conservative, special sequence, it is ensured that the high specific of reaction.Animal mitochondria DNA has small molecular weight, structural conservation, heat
The heredity that good, the not degradable design feature of stability and matrilinear inheritance, copy number are more, evolutionary rate is fast, do not recombinate is special
Point, Mitochondria 12S rRNA gene orders conservative the most.12S rRNA genetic test mouses, there is higher sensibility
And specificity.This part mainly uses round pcr to expand mouse 12S rRNA genes, is connected using gene recombination technology
Into plasmid vector pMD18-T, construct recombinant plasmid pMD18-T-rat-12S rRNA, and carry out corresponding PCR identification and
Sequencing identification lays the foundation most afterwards through quantitative as the standard items for waiting for method for building up for the method and assessment of next step.
One, the preparation of template DNA
Mouse liver organization genomic DNA is extracted, the template of 12S rRNA gene PCRs amplification is used as.
1. taking the fresh mouse liver organization liquid nitrogen grindings of 1.5g at powder, it is added in 5ml centrifuge tubes, 4ml SE bufferings is added
Liquid, mixing, 4500 r/mim centrifuge 15min.
2. drawing supernatant, 4 °C of 12000 r/min centrifuges 20min, abandons supernatant, collects precipitation.
3. 400 μ l Buffer Digestion are added, mixing is shaken.65 DEG C of water-bath 1-3h are cracked completely to cell（Note
Meaning：1, during water-bath, every 10 minutes reverse mixings are primary, can promote sample dissociation.Mixed liquor becomes clear to have cracked
Entirely.Such as the unchanged clarification of solution, illustrate that cell cracking is not thorough, water bath time should be extended, otherwise will likely reduce the yield of DNA
Or cause the DNA of extraction impure.2, if you need to obtain the DNA of no RNA, the Rnase A of 20 μ l can be added after water-bath, be placed at room temperature for
2-5 minutes）200ul Buffer PA are added, fully reverse mixing is placed in -20 DEG C of refrigerators and places 5min.
4. 4 DEG C of 10000rpm are centrifuged 5 minutes, supernatant is transferred in new pipe.
5. isometric isopropanol is added, overturns 5-8 times and be allowed to mix well -20 DEG C, place 20min.
6. 4 DEG C of 10000rpm are centrifuged 10 minutes, supernatant is abandoned.
7. 75% ethyl alcohol of 1ml precoolings is added, 1-3min is rinsed, 4 DEG C of 10000rpm are centrifuged 2 minutes, abandon supernatant.
8. room temperature of uncapping is inverted 10-20min, until remaining ethyl alcohol volatilizees completely.Obtained DNA 30-50ul pure water
Dissolving.
9. the DNA extracted can carry out next step experiment or -20 DEG C of preservations.
Two, the PCR amplification of 12S rRNA genetic fragments
1, the design and synthesis of primer
The present invention compares analysis by carrying out bioinformatics to mouse 12S rRNA complete sequences in ncbi database, and selection is suitble to set
The conservative fragments sequence for counting primer and probe is target, using 3 softwares of Primer express, Primer Premier 5
7 software of software and Oligo, devises the periphery of one group of real-time fluorescence quantitative PCR primer and probe and one group of correlated series
Primer.
Selected extension increasing sequence has nucleotide sequence shown in SEQ ID NO.1 the 1st to 549 in sequence table.
SEQ ID NO.1
1 CGGTGTAAAA TCCCTTAAAC ATTTACTTAA AATTTAAGGA GAGGGTATCA AGCACATTAA
61 AATAGCTTAA GACACCTTGC CTAGCCACAC CCCCACGGGA CTCAGCAGTG ATAAATATTA
121 AGCAATAAAC GAAAGTTTGA CTAAGTTATA CCTCTTAGGG TTGGTAAATT TCGTGCCAGC
181 CACCGCGGTC ATACGATTAA CCCAAACTAA TTATCTTCGG CGTAAAACGT GTCAACTATA
241 AATAAATAAA TAGAATTAAA ATCCAACTTA TATGTGAAAA TTCATTGTTA GGACCTAAAC
301 TCAATAACGA AAGTAATTCT AGTCATTTAT AATACACGAC AGCTAAGACC CAAACTGGGA
361 TTAGATACCC CACTATGCTT AGCCATAAAC CTAAATAATT AAATTTAACA AAACTATTTG
421 CCAGAGAACT ACTAGCCATA GCTTAAAACT CAAAGGACTT GGCGGTACTT TATATCCATC
481 TAGAGGAGCC TGTTCTATAA TCGATAAACC CCGCTCTACC TCACCATCTC TTGCTAATTC
541 AGCCTATAT
The periphery primer sequence is as follows：
Sense primer：rat12S rRNA 65F: 5'- CGGTGTAAAATCCCTTAAAC-3'
Downstream primer：rat12S rRNA 595R: 5'-ATATAGGCTGAATTAGCAA-3'
Amplified fragments size is：549bp.
2, PCR reaction systems and reaction condition
Using DNA as template, using above-mentioned periphery primer rat12S rRNA65F/rat12S rRNA595R as amplimer, under
It states system and reaction condition carries out PCR amplification.PCR system such as table 1.
Table 1
Wherein primer uses rat12S rRNA65F/rat12S rRNA595R, Taq enzyme to use hundred Tyke Power Taq of Beijing
Plus DNA Polymerse, PCR amplification instrument are the serial 96 grads PCR instrument of hundred Tyke iCycling of Beijing.
Amplification program/reaction condition：
94 DEG C of pre-degeneration 5min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 35 cycles；72 DEG C of 10min take 5 μ L amplification productions
Object carries out 1% agarose electrophoresis, detects PCR product size, then uses the DNA gel QIAquick Gel Extraction Kit of Axygen companies production
Remaining pcr amplification product is recycled in purifying.
Three, the structure of recombinant plasmid pMD18-T-rat12S rRNA and conversion
1, connection reaction：The pcr amplification product and pMD18-T that above-mentioned purifying is obtained（Dalian treasured biotech firm）It is attached,
Using following linked system（Such as table 2）It is prepared：
Table 2
It prepares to complete to be placed on 16 DEG C and stay overnight connecting and react.
, pMD18-T-rat-12S rRNA plasmids conversion and PCR identification
The DH5 α competent cells frozen are taken out from -70 DEG C of ultra low temperature freezer, being placed on ice chest makes its naturally to thaw.
1. 10 μ L of connection product is taken to be added in the DH5 α competent cells of 50 μ L, postposition ice bath is gently shaken up 30 minutes.
2. heat shock 90 seconds, sets cooled on ice 2min immediately in 42 DEG C of water-baths after heat shock.
3. the LB liquid medium of 400 μ l of precooling is added into 1.5ml EP pipes（Without ampicillin）After mixing,
37 DEG C of 200 turns of jog culture 1h.
4. 100ul is taken to be coated on the LB tablets containing Amp, IPTG, X-gal after above-mentioned culture solution is shaken up, face up
30min is placed, after bacterium solution is cultured base absorption completely, is inverted 37 DEG C of insulating box overnight incubations of culture dish.
5. next day is observed, picking white monoclonal colonies are in 100 μ L LB liquid mediums from tablet（The mould of benzyl containing ammonia
Element）PCR pipe in, 37 °C of shaken cultivations 2-3 hours.It draws 2 μ L and carries out PCR identifications as template, remaining bacterium solution is added to
It carries out expanding in the LB liquid medium of 20ml and shake.
6. expanding above-mentioned dilution bacterium solution with carrier universal primer RV-M/M13-47, PCR product is using 1% Ago-Gel electricity
Swimming identifies positive transformant by detecting PCR product size.
Primer RV-M/M13-47 sequences are as follows：
Primer RV-M: 5’-GAGCGGATAACAATTTCACACAGG-3’
Primer M13-47: 5’-CGCCAGGGTTTTCCCAGTCACGAC-3’
System such as table 3：
Table 3
Amplification program/reaction condition：94 DEG C of pre-degeneration 5min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 35 cycles；72
℃ 10min。
Positive recombinant plasmid, measured concentration and purity are extracted using the Plasmid Preparation kit of Axygen companies production, together
When draw a part of plasmid purification supreme marine growth Engineering Co., Ltd sent to be sequenced, determine the gene order of Insert Fragment with
Aim sequence is consistent.
2 real-time fluorescence quantitative PCR of example detects the preliminary foundation of mouse method
One, the design and synthesis of specific primer and probe
Using the conservative fragments of the 12S rRNA genes of the mouse of above-mentioned selection as target, using 3 softwares of Primer express,
7 software of 5 softwares of Primer Premier and Oligo, devises one group of real-time fluorescence quantitative PCR primer and probe.
As core of the invention, one group of primer and probe nucleotide sequence for being used for mouse real-time fluorescence PCR detection
It is as follows：
Sense primer：rat12S rRNA 282F：5'-CGGCGTAAAACGTGTCAAC-3'
Downstream primer：rat12S rRNA 414R：5'-GGTATCTAATCCCAGTTTTGG-3'
Probe：rat12S rRNA 302P：5'-ATAAATAAATAAATAGAA-3'
The primer and probe expands Target Nucleotide Sequence：
5’-CGGCGTAAAACGTGTCAACTATAAATAAATAAATAGAATTAAAATCCAACTTATATGTGAAAATTCATTG
TTAGGACCTAAACTCAATAACGAAAGTAATTCTAGTCATTTATAATACACGACAGCTAAGACCCAAACTGGGATTAG
ATACC-3’
Two, the acquisition of standard items and quantitative
1, take the 100 μ L transferred speciess of bacillus coli DH 5 alpha containing recombinant plasmid pMD18-T- rat-12S rRNA that will be frozen in 5mL
LB liquid medium, 37 DEG C of 200rpm shake training overnight.
2, take the bacterium solution transferred speciess of 1ml overnight incubations in 30ml LB liquid mediums, 200rpm Zengjing Granules 2-3 hours,
Then the Plasmid Preparation kit of Axygen companies production is used to extract plasmid.
3, hundred Tyke bio tech ltd ultramicron ultraviolet-uisible spectrophotometer of Beijing is utilized（ND5000）To carrying
The plasmid taken is measured, and measures A260, A280, the purity of plasmid is judged according to A260/A280.
4, plasmid pMD18-T- rat-12S rRNA concentration（Copy number）It calculates
（1）Molecular weight=3241bp × 660 of plasmid（The average molecular weight of each pair of base）
（2）It is 131.65ng/ μ l, plasmid purity A260/A280=1.93, A260/A230=1.85 to measure plasmid concentration.Because into
When row real-time fluorescence quantitative PCR, need with " copy number " for unit, it is therefore desirable to by Conversion of measurement unit at copies/ml.
Plasmid copies/ml=Avgadro constant × plasmid molal quantity
Wherein Avgadro constant=6.02 × 1023copies/mol。
Therefore plasmid concentration copies/ml=131.65 × 10 of extraction-9g/ml×6.02×1023copies/mol÷
（3241bp×660g/bp•mol）=3.7×1010copies/m0l
The sterile water of+27 μ l of 10 μ l plasmids just obtains a concentration of 1.00 × 1010The plasmid of copies/ml, then the plasmid is carried out
A series of plasmid of concentration just can be obtained in 10 doubling dilutions, and is saved backup in -20 DEG C.
Three, real-time fluorescence quantitative PCR condition optimizing
With a concentration of 1.00 × 106The positive plasmid of copies/ml is template, the 2 × real- produced using hundred Imtech
time PCR Premixture（probe）Real-time fluorescence quantitative PCR kit is tested, and the real-time fluorescence for testing use is fixed
Measure the ASA-9600 real-time fluorescence quantitative PCR instrument that PCR instrument is Suzhou Bai Yuan gene technology Co., Ltd.Reaction system uses 20ul
System, the wherein amount of primer and probe are reacted using the combination of table 4.
Table 4
Reaction condition：94 DEG C of pre-degenerations 2 minutes, 94 DEG C 15 seconds, 60 DEG C of 40s and collect fluorescence signal, 40 cycles.As a result join
According to Fig. 1, when data show 20ul systems, primer（5uM）And probe（5uM）It is minimum to be separately added into 1.6 μ l, Ct values, fluorescence signal
It is most strong.
【Method is assessed】
1, sensitivity experiment
The above-mentioned plasmid that is prepared is subjected to 10 doubling dilutions and obtains a series of plasmid of concentration, choose a concentration of 1.00 ×
107copies/ml、1.00×106copies/ml、1.00×105copies/ml、1.00×104copies/ml、1.00×
103copies/ml、1.00×102Copies/ml etc. is used as gradient template.Reaction system such as table 5：
Table 5
Reaction condition：94 DEG C of pre-degenerations 2 minutes, 94 DEG C 15 seconds, 60 DEG C of 40s and collect fluorescence signal, 40 cycles.According to instrument
Fluorescence signal detected by device is handled through software obtains fluorescence curve, observes the signal of fluorescence curve, as a result with reference to Fig. 2, number
According to display when plasmid concentration reaches 1.00 × 103Still have fluorescence signal when copies/ml, but plasmid concentration reach 1.00 ×
102Fluorescence signal is not detected when copies/ml, therefore the sensitivity of this method is 1.00 × 103copies/ml。
2, specificity experiments
In order to confirm the specificity of the invention detected to mouse, we have chosen 11 kinds of common animals and do specificity experiments,
The animal of selection includes：Ox, sheep, pig, fish, chicken, duck, goose, rabbit, donkey, the frog, dog.
It is experiment that template carries out that the specific test, which includes with the genomic DNA of above-mentioned sample,.Reaction system such as table 6：
Table 6
Reaction condition：94 DEG C of pre-degenerations 2 minutes, 94 DEG C 15 seconds, 60 DEG C of 40s and collect fluorescence signal, 40 cycles.According to instrument
Fluorescence signal detected by device is handled through software obtains fluorescence curve, observes the signal of fluorescence curve, analysis specificity.As a result
With reference to figure 3, concrete outcome only rat tissue test positive, remaining animal is feminine gender, and it is special well to show that the present invention has
Property.Testing result such as table 7.
Table 7
3, prepared by standard curve
Using the logarithm of the positive template of different copy numbers as abscissa, followed with reaching the initial of fluorescence threshold in PCR reaction process
Number of rings（Ct）The standard curve of mouse is obtained for ordinate, as the reference standard of sample to be tested quantitative determination, is as a result referred to
Fig. 4.Standard curve original equation is y=a+bx, and the equation of this standard curve is y=42.9-3.42x.
<110>Suzhou Bai Yuan gene technology Co., Ltd
<120>A kind of real-time fluorescence quantitative PCR kit for mouse detection
<160>2
<210>1
<211>549
<212>DNA
1 CGGTGTAAAA TCCCTTAAAC ATTTACTTAA AATTTAAGGA GAGGGTATCA AGCACATTAA
61 AATAGCTTAA GACACCTTGC CTAGCCACAC CCCCACGGGA CTCAGCAGTG ATAAATATTA
121 AGCAATAAAC GAAAGTTTGA CTAAGTTATA CCTCTTAGGG TTGGTAAATT TCGTGCCAGC
181 CACCGCGGTC ATACGATTAA CCCAAACTAA TTATCTTCGG CGTAAAACGT GTCAACTATA
241 AATAAATAAA TAGAATTAAA ATCCAACTTA TATGTGAAAA TTCATTGTTA GGACCTAAAC
301 TCAATAACGA AAGTAATTCT AGTCATTTAT AATACACGAC AGCTAAGACC CAAACTGGGA
361 TTAGATACCC CACTATGCTT AGCCATAAAC CTAAATAATT AAATTTAACA AAACTATTTG
421 CCAGAGAACT ACTAGCCATA GCTTAAAACT CAAAGGACTT GGCGGTACTT TATATCCATC
481 TAGAGGAGCC TGTTCTATAA TCGATAAACC CCGCTCTACC TCACCATCTC TTGCTAATTC
541 AGCCTATAT
<210>2
<211>152
<212>DNA
1 CGGCGTAAAA CGTGTCAACT ATAAATAAAT AAATAGAATT AAAATCCAAC TTATATGTGA
61 AAATTCATTG TTAGGACCTA AACTCAATAA CGAAAGTAAT TCTAGTCATT TATAATACAC
121 GACAGCTAAG ACCCAAACTG GGATTAGATA CC
Claims (2)
1. a kind of real-time fluorescence quantitative PCR kit for mouse detection, including
- PCR reaction solution, by 12mM Tris-HCl, 100mM KCl, 0.12%Triton X-100,0.12% sodium taurocholate, 0.6
mg/mL BSA、1.8mM MgCl2, 50nM dNTP, 500nM sense primers, 500nM downstream primers, 300nM Taqman visit
Needle forms；
Wherein upstream primer sequence is 5'- CGGTGTAAAATCCCTTAAAC-3'；Downstream primer sequence is 5'-
ATATAGGCTGAATTAGCAA-3'；Probe sequence is 5 '-ATAAATAAATAAATAGAA-3 '；
- enzyme mixation, by 5U/ μ L Taq archaeal dna polymerases, 50% glycerine, 20mM Tris-HCl, 100mM KCl, 0.1mM
EDTA, 1mM DTT, 0.5% Tween®20 and 0.5% NP-40 composition；
- mouse 12S rRNA gene standard items, by 1.00 × 108copies/ml、1.00×107copies/ml、1.00×
106copies/ml、1.00×105The recombinant plasmid of copies/ml forms；
Wherein 12S rRNA genes standard items sequence is 5 '-CGGCGTAAAACGTGTCAACTATAAATAAATAAATAGAATTAA
AATCCAACTTATATGTGAAAATTCATTGTTAGGACCTAAACTCAATAACGAAAGTAATTCTAGTCATTTATAATACA
CGACAGCTAAGACCCAAACTGGGATTAGATACC-3’；
- negative controls, are made of water；
- positive reference substance, by 1.00 × 107The recombinant plasmid of copies/ml forms.
2. a kind of real-time fluorescence quantitative PCR kit for mouse detection as described in claim 1, it is characterised in that：
The fluorescent reporter group of 5 ' end labels is FAM in the probe, and the fluorescent quenching group of 3 ' end labels is TAMRA.
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| CN109536587A (en) * | 2018-12-31 | 2019-03-29 | 吴江近岸蛋白质科技有限公司 | A kind of efficient multiple fluorescence quantitative PCR kit and preparation method thereof |
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