CN108118086A - For detecting RPA primers, probe and the method for pork content in meat products - Google Patents
For detecting RPA primers, probe and the method for pork content in meat products Download PDFInfo
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Abstract
The present invention is provided to detect the RPA primer pairs of pork content in meat products and probe, primer pair to be：Sense primer：5 ' TAAATCTYGTGCCAGCCACCGCGGTCATAC 3 ', anti-sense primer：5’‑CATAGTGGGGTATCTAATCCCAGTTTGGGYC‑3’；Wherein, the Y in primer is degeneracy base, and Y is C or T；Probe is：5’‑CTAGTTAAAATAAAATAACCCACGAAAG[dT‑Fam]G[THF]C‑[dT‑BHQ1]CTAATAATCCTGAC[C3 spacer]‑3’.The present invention has the following advantages：The isothermal reaction at 39 40 DEG C is only needed, additional equipment is not required；Reaction time only needs 20min；As a result it is easy to judge；Specific good, high sensitivity.
Description
Technical field
The present invention relates to for detecting the RPA primers of pork content in meat products and method, it is particularly based on meat-processing
Food.This method be particularly suitable for identification halal food certification processed food in the presence or absence of pork (Sus scrofa) into
Point.
Background technology
Food adulteration, especially meat adulteration have become an international question, and China also has very early " hangs sheepshead to sell
The saying of dog meats ".Meat adulteration is not only an economic problems, it is also possible to damage consumer health and even bring a series of religions
Problem.Therefore, the detection technique established rapidly and efficiently is to hit the joint demand of the adulterated crime of meat and market surveillance law enforcement.
Meat product nutritive value is high, is the important component of people's conventional food consumption, due to expensive, becomes
The important object of food adulteration.With cheap meat replace or be mixed into the meat adulteration carried out in the meat of higher price, especially with
The phenomenon that pork replacement beef or mutton, happens occasionally.With the fast development of modern food industry and the variation of food variety, mix
False object also penetrates into the product of multiple species from original cold fresh meat and simple processing meat, especially modern deep processing meat
Product, adulterated means are more hidden, it is difficult to judge its ingredient by the appearance of food.In order to hit these food adulteration crimes, tie up
Protect consumers' rights and interests, supervision department and researcher are also evolving Adulteration identification technology and perfect.The mirror of early stage meat
Sense organ and morphological examination are not depended on, but these methods of inspection are all by instrument, personnel's experience and meat processing procedure etc.
Multifactor influence, and limitation is big, accuracy is low, is difficult to differentiate for the animal derived materials added in deep processing meat.
With the development of molecular biology, quick hair has been obtained in meat and species identification application based on the round pcr of DNA analysis
Exhibition becomes the major technique of Meat ingredients analysis.But PCR reactions have to pass through denaturation, annealing, 3 steps of extension it is multiple
Xun Huan, each step carry out the control gradient of temperature equipment, it is necessary to complicated at different temperature, it is necessary in the experiment of specialty
Room can just be realized, limit its application and popularization to a certain extent.
Recombinase polymeric enzymatic amplification technology (recombinase polymerase amplification, RPA) technology is
A kind of novel in vitro nucleic acid isothermal amplification technology of replicanism exploitation based on T4 phage genomes DNA.Its principle is weight
The compound that group enzyme and the nucleic acid fragment (primer) of appropriate length combine to form can find homologous sequence in template, and be sent out with it
The displacement reaction of raw chain, polymerase is added in system can start DNA synthesis, be can be realized under constant temperature to mesh in template
The exponential amplification of standard film section.Compared with traditional PCR (PCR), RPA technologies can expand under constant temperature
DNA eliminates heating and cooling step repeatedly, has broken away from the dependence to nucleic acid augmentative instrument, nucleic acid amplification is made no longer to be set by instrument
The limitation of standby and place, becomes more convenient.The product of RPA can be directly detected by agarose gel electrophoresis, can also be combined
Fluorescence probe carries out Real_time quantitative detection or is combined with the methods of flow measurement chromatograph test strip, micro-fluidic chip to be detected.Mesh
Before, the detection method established based on RPA technologies is widely used in Pathogen test and medical diagnosis on disease, food safety detection and transgenosis
Make the multiple fields such as analyte detection.
The content of the invention
The present invention provides accurately, rapidly and sensitively detect the real-time of pork content under constant temperature from meat-based product
Fluorescence RPA detection methods.Reagent including being used for RPA detections, primer and specificity fluorescent label probe.
First purpose of the present invention is to provide the RPA primer pairs for detecting pork content in meat products and probe, institute
Stating primer pair is：
Sense primer：5’-TAAATCTYGTGCCAGCCACCGCGGTCATAC-3’
Anti-sense primer：5’-CATAGTGGGGTATCTAATCCCAGTTTGGGYC-3’；
Wherein, the Y in primer is degeneracy base, and Y is C or T；
The probe is：5’-CTAGTTAAAATAAAATAACCCACGAAAG[dT-Fam]G[THF]C-[dT-BHQ1]
CTAATAATCCTGAC[C3spacer]-3’。
Wherein, dT-Fam represents to carry the thymidylic acid of fluorescein base group, and THF represents tetrahydrofuran connexon,
DT-BHQ1 represents the thymidylic acid with fluorescent quenching group BHQ1, arm between C3spacer expressions.
Second object of the present invention is to provide a kind of RPA reagents for being used to detect pork content in meat products, the RPA
Amplifing reagent includes primer pair described in claim 1 and probe.
Preferably, final concentration of 10 μM in the RPA reagents of the sense primer, anti-sense primer and probe.
Third object of the present invention is to provide the RPA kits for detecting pork content in meat products, the RPA examinations
Agent box includes primer pair described in claim 1 and probe.
Preferably, the RPA kits further include pig standard DNA plasmid, the preparation method of the pig standard DNA plasmid
For：Using standard pig DNA as template, using primer pair described in claim 1 as upstream and downstream primer, PCR amplification is carried out, amplification is produced
Object after purification, connects carrier, is transformed into competent cell culture, extracts plasmid.
Fourth object of the present invention is to provide above-mentioned primer pair and probe, RPA reagents, RPA kits are being appointed as follows
Application in one：
(1) detect or aid in detection pork content；
(2) product of detection or auxiliary detection pork content is prepared；
(3) detect or aid in whether to contain pork content in detection meat products；
(4) prepare detection or auxiliary detection meat products in whether the product containing pork content.
Whether the 5th purpose of the present invention is to provide in a kind of detection or auxiliary detection meat products containing pork content
Method comprises the following steps：The DNA in meat products is extracted as DNA to be checked, using DNA to be detected as template, using claim
Primer pair and probe described in 1 carry out recombinase polymeric enzymatic amplification, read fluorescence values, when fluorescent value is higher than threshold value in reaction tube
When, then judge to contain pork content in meat products.
Preferably, the temperature during recombinase polymeric enzymatic amplification is 39-40 DEG C, constant temperature 20min.
The present invention has the following advantages：
(1) isothermal reaction at 39-40 DEG C is only needed, additional equipment is not required；
(2) reaction time only needs 20min；
(3) result is easy to judge；
(4) specific good, high sensitivity.
Description of the drawings
Attached drawing is used for providing a further understanding of the present invention, and a part for constitution instruction, the reality with the present invention
Example is applied together for explaining the present invention, is not construed as limiting the invention.In the accompanying drawings：
Fig. 1 is respectively using standard pig, ox, sheep, horse, chicken, 1.0 μ L of dog source property DNA and water as template, and it is real-time to carry out constant temperature
Fluorescence reaction.The corresponding sample of S type amplification curves is pig standard DNA.
Fig. 2 is the amplification curve that constant temperature real-time fluorescence reaction is carried out using serial standards as template.
Fig. 3 is using serial standards as template, carries out constant temperature real-time fluorescence reaction, the standard curve drawn according to Cq values.
Specific embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is conventional method unless otherwise specified.In the following embodiments, used main material is detected including RPA exo
Reagent, DNA extraction kit etc. unless otherwise specified, can be bought from company and obtain.
Embodiment 1 is used to detect RPA primers, probe and the kit of pork content in meat products
First, for detecting the design and synthesis of the RPA primers of pork content in meat products and probe
The mitochondria whole genome sequence of many animals such as pig, ox, sheep, horse, chicken, dog is obtained in GenBank, passes through sequence
Row compare, and according to the requirement to primer in RPA detection methods, (RPA is 30-35nt to primer length requirement, this is easy for causing
Substantial amounts of primer dimer is generated under constant temperature so as to influence experiment effect, RPA experiments need to design from target sequence both ends
Multipair primer optimizes, screening, and the replacement or increase and decrease of Individual base can all have an important influence on experimental result), through comparing
Filter out one section of sequence on 12S rDNA afterwards, design a pair of of versatility primer 12S-F and 12S-R, according to different plant species this
The difference of Duan Xulie screens pig specific probe sequence, probe sequence internal base is carried out necessary fluorophor, mispairing and
3 ' ends terminate modification to be suitble to the testing requirements of RPA exo reagents.Sequence is as follows：
12S-F：5’-TAAATCTYGTGCCAGCCACCGCGGTCATAC-3’
12S-R：5’-CATAGTGGGGTATCTAATCCCAGTTTGGGYC-3’
12S-P：5’-CTAGTTAAAATAAAATAACCCACGAAAG[dT-Fam]G[THF]C-[dT-BHQ1]
CTAATAATCCTGAC[C3spacer]-3’
Wherein, the Y in primer is degeneracy base, and Y is C or T.
DT-Fam represents to carry the thymidylic acid of fluorescein base group, THF expression tetrahydrofuran connexons, dT-
BHQ1 represents the thymidylic acid with fluorescent quenching group BHQ1, and arm between C3spacer expressions is used for and prevents 3 ' ends
Excision enzyme and 3 ' end polymerase effects.
Primer and probe sequence have the synthesis of Shanghai Sheng Gong bioengineering Co., Ltd.
2nd, for detecting the RPA methods of pork content in meat products
1st, for detecting the RPA reagents of pork content in meat products
Include the reaction tube of lyophozyme containing exo, rehydration buffer solution for detecting the RPA reagents of pork content in meat products
(Rehydration Buffer), magnesium acetate solution (280mmol/L), the sense primer 12S-F of the design of above-mentioned steps one, downstream
Primer 12S-R and probe 12S-P, the pig standard DNA plasmid and ddH provided for oneself2O.It is above-mentioned to contain lyophilized enzyme powder reaction tube, rehydration
The RPA detection reagents of buffer solution (Rehydration Buffer) and magnesium acetate solution (280mmol/L) are bought from Britain
TwistDx companies.
Wherein, pig standard DNA Plasmid samples preparation method is：
Using 12S-F and 12S-R as upstream and downstream primer, using standard pig DNA as template, porcine mtdna is expanded with regular-PCR method
The DNA fragmentation of detection zone is included in 12S rDNA.After the PCR product Purification Kit of pcr amplification product commercialization,
The method provided to specifications with pMD18T carriers (TaKaRa) is attached, the competent cell training of conversion bacillus coli DH 5 alpha
It supports, picking monoclonal, shakes bacterium, extract the completely plasmid containing target fragment, which is expanded, sequencing identification is correct
Afterwards, measured concentration calculates the copy number of the DNA fragmentation of detection zone in every microlitre, determines the plasmid of target fragment copy number
For standard items.10 times of doubling dilution is carried out to plasmid standard, prepares serial standards.Specifically, the present invention from 3 ×
107Copies/ μ L carry out 10 times of doubling dilutions, are diluted to 3 × 100copies/μL。
It is above-mentioned using 12S-F and 12S-R as upstream and downstream primer, using standard pig DNA as template, with regular-PCR method expand pig line
The specific method of the DNA fragmentation comprising detection zone is in plastochondria 12S rDNA：
PCR reaction systems are as follows：
PCR reaction conditions are 95 DEG C of pre-degenerations 30s, 94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 30s, if 35
A amplification cycles, 72 DEG C of the reaction was continued 10min, terminate program after amplification cycles.
The nucleotides sequence of amplified production is classified as：
TAAATCTCGTGCCAGCCACCGCGGTCATACGATTAACCCAAATTAATAGATCCACGGCGTAAAGAGTGTTTAAGAAA
AAAAAACCACAATAGAGTTAAATTATAACTAAGCTGTAAAAAGCCCTAGTTAAAATAAAATAACCCACGAAAGTGAC
TCTAATAATCCTGACACACGATAGCTAGGACCCAAACTGGGATTAGATACCCCACTATG。
2nd, for detecting the RPA methods of pork content in meat products
(1) RPA is expanded
The standard plasmid of the rDNA segments of 12S containing pig prepared using DNA to be detected or step 1 is expanded as template, instead
It answers system and sequentially adds following reagent to freeze enzyme powder reaction tube to RPA exo：
Specifically, first by Mg2+Other compositions in addition are pre-mixed by volume, are added in RPA exo and are freezed enzyme powder reaction tube
In, Mg is added afterwards2+.Once Mg2+It adds in, DNA amplification reaction can start.Reaction tube is immediately placed in quantitative fluorescent PCR
In instrument, run by the program of setting.The response procedures that fluorescence quantitative PCR instrument is set are read as 39-40 DEG C of constant temperature 20min every 30s
Take fluorescent value 1 time.
Wherein, the extraction of DNA to be detected can be extracted with the kit of commercialization, and the present invention is led to TaKaRa companies
With illustrating exemplified by DNA extraction kit (TaKaRaMiniBEST Universal Genomic DNA Extraction Kit)：
To source clearly, the meat products such as the pig of standard, ox, sheep, horse, chicken, dog, take 100mg, add 180 μ L Buffer
GL adds 10 μ L Proteinase K and 10 μ L RNaseA solution, heating concussion mixing, until sample cracks completely.Mixing
It is to crack completely that liquid, which becomes clear,.200 μ L Buffer BD are added in, fully reverse mixing.200 μ L absolute ethyl alcohols are added in,
Fully reverse mixing.Adsorption column is put back in collecting pipe, is all added in solution and translucent fibre shape suspended matter with pipettor
In adsorption column, 2min, then 12000r/min room temperatures centrifugation 1min are stood, outwells the waste liquid in collecting pipe.Adsorption column is put back into receipts
Collector adds in 500 μ L Buffer WA, 12000r/min centrifugation 1min, outwells filtrate.Adsorption column is put back into collecting pipe, is added in
700 μ L Buffer WB, 12000r/min centrifugation 1min, outwell filtrate.Repeat this step 1 time.Adsorption column is put back into collecting pipe,
2min is centrifuged in 12000r/min room temperatures, leave away remaining Buffer WB.Adsorption column is taken out, is put into a new 1.5mL centrifugation
Guan Zhong, 50 μ L Elution Buffer of addition, standing 3min, 12000r/min room temperatures centrifugation 2min, collection DNA solution, -20
It DEG C saves backup.
(2) reading of result
During the reaction, fluorescence values can be read in real time from fluorescence quantitative PCR instrument or fluorescence detector, work as reaction tube
When middle fluorescent value is higher than threshold value, you can judge the positive, i.e., contain pork content in sample；Threshold value is 40.
The specificity experiments of the RPA primer and probes of 2 present invention of embodiment
Using kit and its application method in the embodiment of the present invention 1, respectively with standard pig, ox, sheep, horse, chicken, dog source property
1.0 μ L of DNA are amplification template, and set the negative control using water as template, are reacted under the conditions of 39 DEG C, coreaction 20min, real
When read result.The results show that in this reaction process, only fluorescent value is detected in the reaction tube of source containing pig property sample,
In typical S types amplification curve, using ox, sheep, horse, chicken, dog source property DNA and water as the reaction tube of template in fluorescence is not detected
Value variation, concrete outcome are shown in Fig. 1.Illustrate that the specificity of the RPA primer and probes of the present invention is high.
The sensitivity experiments of the RPA primer and probes of 3 present invention of embodiment
Prepare the standard items of series concentration：3×107copies/μL、3×106copies/μL、…、3×100copies/μ
L, respectively using the standard items of the series concentration of 1.0 μ L as template, using kit and application method in the embodiment of the present invention 1,
It is reacted under the conditions of 39 DEG C, coreaction 20min reads result on fluorescence quantitative PCR instrument.The results show that each serial dilution
Standard items can detect fluorescence signal, include the standard sample (3 × 10 of concentration minimum0Copies/ μ L), in reactant
The detection segments of 3 copies are contained only in system, see Fig. 2,3, this shows that the method sensitivity of the present invention is high.
Finally it should be noted that：The foregoing is only a preferred embodiment of the present invention, is not intended to limit the invention,
Although the present invention is described in detail with reference to the foregoing embodiments, for those skilled in the art, still may be used
To modify to the technical solution recorded in foregoing embodiments or carry out equivalent substitution to which part technical characteristic.
Within the spirit and principles of the invention, any modifications, equivalent replacements and improvements are made should be included in the present invention's
Within protection domain.
Sequence table
<110>Northwest University for nationalities
<120>For detecting RPA primers, probe and the method for pork content in meat products
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 213
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
taaatctcgt gccagccacc gcggtcatac gattaaccca aattaataga tccacggcgt 60
aaagagtgtt taagaaaaaa aaaccacaat agagttaaat tataactaag ctgtaaaaag 120
ccctagttaa aataaaataa cccacgaaag tgactctaat aatcctgaca cacgatagct 180
aggacccaaa ctgggattag ataccccact atg 213
Claims (8)
1. for detecting the RPA primer pairs and probe of pork content in meat products, it is characterised in that：The primer pair is：
Sense primer：5’-TAAATCTYGTGCCAGCCACCGCGGTCATAC-3’
Anti-sense primer：5’-CATAGTGGGGTATCTAATCCCAGTTTGGGYC-3’；
Wherein, the Y in primer is degeneracy base, and Y is C or T；
The probe is：5’-CTAGTTAAAATAAAATAACCCACGAAAG[dT-Fam]G[THF]C-[dT-BHQ1]
CTAATAATCCTGAC[C3spacer]-3’；
Wherein, dT-Fam represents to carry the thymidylic acid of fluorescein base group, and THF represents tetrahydrofuran connexon, dT-
BHQ1 represents the thymidylic acid with fluorescent quenching group BHQ1, arm between C3spacer expressions.
2. a kind of RPA reagents for being used to detect pork content in meat products, it is characterised in that：The RPA amplifing reagents include power
Profit requires the primer pair and probe described in 1.
3. RPA reagents according to claim 2, it is characterised in that：The sense primer, anti-sense primer and probe are described
Final concentration of 10 μM in RPA reagents.
4. for detecting the RPA kits of pork content in meat products, it is characterised in that：The RPA kits will including right
Seek the primer pair and probe described in 1.
5. RPA kits according to claim 4, it is characterised in that：The RPA kits further include pig standard DNA matter
Grain, the preparation method of the pig standard DNA plasmid are：Using standard pig DNA as template, using primer pair described in claim 1 as
Upstream and downstream primer carries out PCR amplification, connects carrier by amplified production after purification, is transformed into competent cell culture, extracts matter
Grain.
6. described in the RPA reagents, claim 4 or 5 described in primer pair described in claim 1 and probe, Claims 2 or 3
RPA kits it is following it is any in application：
(1) detect or aid in detection pork content；
(2) product of detection or auxiliary detection pork content is prepared；
(3) detect or aid in whether to contain pork content in detection meat products；
(4) prepare detection or auxiliary detection meat products in whether the product containing pork content.
7. it is a kind of detection or auxiliary detection meat products in whether the method containing pork content, it is characterised in that：Extract meat products
In DNA as DNA to be checked, using DNA to be detected as template, recombinated using primer pair described in claim 1 and probe
Enzymatic polymerization enzymatic amplification, read fluorescence values, when in reaction tube fluorescent value be higher than threshold value when, then judge in meat products containing pork into
Point.
8. according to the method described in claim 7, it is characterized in that：The temperature during recombinase polymeric enzymatic amplification is 39-40
DEG C, constant temperature 20min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112708681A (en) * | 2021-02-08 | 2021-04-27 | 韩山师范学院 | Primer pair and probe for detecting chicken-derived components, kit and application thereof |
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| CN112708681A (en) * | 2021-02-08 | 2021-04-27 | 韩山师范学院 | Primer pair and probe for detecting chicken-derived components, kit and application thereof |
| CN112708681B (en) * | 2021-02-08 | 2024-01-30 | 韩山师范学院 | Primer pair and probe for detecting chicken-derived components, and kit and application thereof |
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