CN109517903A - Strand displacement type archaeal dna polymerase induces sex taboo object in pig source in isothermal circulation amplified reaction detection halal food - Google Patents
Strand displacement type archaeal dna polymerase induces sex taboo object in pig source in isothermal circulation amplified reaction detection halal food Download PDFInfo
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Abstract
本发明公开了一种应用链置换型DNA聚合酶诱导等温循环扩增反应检测清真食品中猪源性禁忌物的方法，属于食品安全检测技术领域。本发明通过多重序列对比确定了猪肉的检测靶核酸，设计了特异性的分子信标探针和引物，以肉类基因组DNA为模板，建立实时荧光链置换型DNA聚合酶诱导等温循环扩增反应体系，进行等温核酸扩增，并且验证了方法的特异性、灵敏度和检出限，还通过实际样品的检测确定了本发明建立的方法具有实际可行性。
The invention discloses a method for detecting pig-derived contraindications in halal food by using strand displacement DNA polymerase-induced isothermal cyclic amplification reaction, and belongs to the technical field of food safety detection. The invention determines the detection target nucleic acid of pork through multiple sequence comparison, designs specific molecular beacon probes and primers, and uses the meat genomic DNA as a template to establish a real-time fluorescent strand displacement DNA polymerase-induced isothermal cyclic amplification reaction The system is used for isothermal nucleic acid amplification, and the specificity, sensitivity and detection limit of the method are verified, and the practical feasibility of the method established in the present invention is also confirmed through the detection of actual samples.
Description
Technical field
The present invention relates to pig sources in a kind of strand displacement type archaeal dna polymerase induction isothermal circulation amplified reaction detection halal food
The method of sex taboo object, belongs to technical field of food safety detection.
Background technique
Meat, which identifies to form, at present two predominantly detects system greatly by protein detection and based on detection of nucleic acids.With
Meat essence authentication technique based on protein detection includes immuno analytical method, proteomic techniques.Before different
Under the conditions of mentioning, the advantages of various methods have oneself and limitation, but lack high sensitivity, high specificity, time-consuming short, expense
The method that the features such as low, easy to operate is able to satisfy charge-sheet model requirement again.Meat adulteration based on detection of nucleic acids identifies skill
Art is the molecular biotechnology based on DNA sequence-specific, identifies detection using hereditary information difference between animal species as species
Nowadays the nucleic acid detection method of target is widely used.Compared with protein detection method, the thermal stability and soda acid of DNA is steady
It is qualitative to be superior to protein, therefore have a clear superiority in the identification detection of meat after processing, and the height of nucleic acid detection method
Sensitivity and specificity are also that method of protein detection is incomparable.DNA probe hybridization is earliest for Meat ingredients in food
The nucleic acid detection method of identification.
Real-time fluorescence quantitative PCR is needed through thermal cycling amplification repeatedly, time-consuming more；And ring mediated isothermal amplification method
The primer of (Loop-mediatedisothermalamplification, LAMP) is easy to produce dimer, and complexity is anti-
It answers system to be easy to produce false positive, is restricted in application range.Therefore, at present lack can simultaneously fast quantification
The method for detecting meat adulteration in halal food.
Summary of the invention
To solve the above problems, the present invention provides a kind of methods of pig derived component in quickly detection food.This method
It is, as template, to prepare the induction of strand displacement type archaeal dna polymerase containing the specific molecular beacon complementary with porky genome sequence
Isothermal circulation amplified reaction (Strand-displacement DNA polymerase induces isothermal
Circular amplification reaction, SDPICA) system, it is placed in microplate reader, in specific probe and circulation
Constant-temperature amplification is carried out under the action of primer, is realized to the qualitative of pork and (or) is determined according to amplification curve and fluorescence relative intensity
Amount detection.Specifically, the method for the present invention has determined the detection target nucleic acid sequence for pork by multiple alignment, and
The molecular beacon probe (specific probe) and primer (circulation primer) for devising specificity, using pork DNA as template, are established
Real-time fluorescence SDPICA detection architecture carries out isothermal nucleic acid amplification, according to the fluorescent strength modification SDPICA after amplification
The specificity of method measures sensitivity and the detection limit of real time fluorescent quantitative SDPICA method according to amplification curve, it is determined that this
Inventing established method has practical feasibility.
This method can carry out side to target double-stranded DNA in constant temperature using solution strand primer and Bst archaeal dna polymerase and untwist
Amplification edges, release the target single stranded DNA of subsequent reactions, and the privileged site complementary pairing of object chain and molecular beacon probe divides
Sub- beacon probe, which is opened, becomes linear chain structure by cyclic structure, and fluorophor separates immediately with quenching group, generates fluorescence letter
Number.In addition, this method forms double-strand using circulation primer and Klenow enzyme and molecular beacon probe specific part complementary pairing
DNA simultaneously releases target single stranded DNA and enters next round cyclic amplification, to realize highly sensitive detection.
Heretofore described pork refers to the pork that can detect that DNA sequence dna, can be any position with pig
Pork, for example can be fresh meat, pig loin, retreat meat, streaky pork, flank meat etc..
In the above method, the nucleotide sequence (5 ' -3 ') of the molecular beacon probe is by I, II, III three parts successively group
At I and III complementary pairing.I is TCTTGATCTCC, TCTTGCATCAG etc.；III is GGAGATCAAGA, CTGATGCAAGA
Deng；II is the probe ring sequence with pork specific genomic sequences complementary pairing.
The sequence both ends of the molecular beacon probe are modified with fluorophor and quenching group.Fluorophor and quenching group
For group commonly used in the art, for example with the modification of FAM fluorescein in 5 ' ends of sequence, Dabcyl quenching group is modified in sequence
3 ' end.
The DNA containing pork target sequence refers to the nucleotide sequence containing detection target nucleic acid fragment, is pork
Genomic DNA, and it be according to mitochondria Cyt b gene design.
The meat probe ring sequence with target sequence complementary pairing be (5 ' -3 ') CCTCAATGGTATGCCACAAC,
TTACACACATTTGTCGAGACGT etc..
The circulation primer is the circulation primer well known to those skilled in the art that can maintain SDPICA circular response, such as
It can be TCTTGATC, TCTTGCAT etc..
The solution strand primer is the primer well known to those skilled in the art that can be specifically bound with object chain, such as can
To be ATGAAAGAGGCAAATAGATTTTCG, ATTTACGTCTCGACAAATGTGTGTA etc..
Mg in the SDPICA reaction system2+Concentration is 4~10mmol/L, and beet alkali concentration is 0.8~1.5mol/L,
The concentration of dNTPs be 1.2~2mmol/L, unwinding primer concentration be 0.1~0.5 μm of ol/L, Klenow polymerase concentration be 1~
4U, molecular beacon probe concentration are 0.1~0.5 μm of ol/L, and circulation primer concentration is 0.5~1 μm of ol/L, Bst archaeal dna polymerase
For 12~18U.
The microplate reader carries out the endpoint signal detection of real-time fluorescence SDPICA amplification, excitation wavelength 496nm, transmitted wave
A length of 521nm, floor detection, gain 100 postpone 100msec, detect height 7mm.
The proliferation time of the method is 10~30min, and amplification temperature is 37~38 DEG C.In addition, this method further includes pre-
The relationship for first measuring fluorescence signal intensity and reaction time, to establish the recurrence between pork concentration and fluorescence signal intensity
Curved line relation；When detecting sample to be tested, genomic DNA is extracted, carries out SDPICA reaction, fluorescence signal intensity is detected, substitutes into back
Return curve you can learn that in sample to be tested pork concentration.
The extracting method of the genomic DNA is this field general extraction methods, for example can be RNA isolation kit, tradition
DNA extraction process (phenol-chloroform method), strong salty method, CTAB method, SDS method, pyrolysis method；Preferred reagent box method.
Compared with prior art, remarkable advantage is the present invention:
The method of the present invention is reacted based on real-time fluorescence SDPICA, and principle is easily understood, easy to operate and can monitor in real time, right
Reaction condition it is of less demanding.
The selectivity of specific probe and two species-specific primers improves detection method to the special of targeting object
Property recognition capability.
The shortcomings that needing the present invention overcomes normal PCR reaction through thermal cycling amplification repeatedly, avoids and rises repeatedly
The time-consuming process of cooling has easy to operate, quick, high specificity spy, it can be achieved that continuous rapid amplifying under constant temperature
Point.Isothermal circulation amplified reaction is induced to realize nucleic acid amplification by strand displacement type archaeal dna polymerase, targeting object content is lower
When be equally able to achieve detection, improve this method sensitivity, reduce detection limit.
Detailed description of the invention
Fig. 1: strand displacement type archaeal dna polymerase induces isothermal circulation amplified reaction (SDPICA) detection principle diagram.
Fig. 2: the specificity verification figure of pig derived component detection architecture；Wherein (a) is that pig derived component detection architecture is being examined
Fluorescence intensity versus time curve, (b) are pig derived component detection architecture when detecting different meats when surveying different meats
The fluorescence intensity comparison diagram of 20min.
Fig. 3: the detection sensitivity of the method for the present invention investigates figure.
Fig. 4: the investigation figure of the detection limit of method.
Specific embodiment
The present invention is described further in conjunction with example:
As shown in Figure 1, expanding schematic illustration for SDPICA.
Resulting genomic DNA is extracted from pork product in the effect of Bst archaeal dna polymerase and specificity solution strand primer
Under, target nucleic acid fragments are released, SDPICA reaction opens molecule with hybridizing for molecular beacon ring sequence by target nucleic acid fragment
The loop-stem structure of beacon probe, and MB-cDNA structure is formed under the action of primer and archaeal dna polymerase, retain point opened
Sub- beacon probe signal, release target nucleic acid fragment triggers next circulation in cDNA forming process, passes through amplification and realizes signal
Amplify step by step, achievees the purpose that Sensitive Detection.
Embodiment 1:
1, pork product extracting genome DNA (improvement sodium chloride method)
0.5g pork fresh meat is weighed, sample is placed on refiner and is mixed, is put in mortar, 1mLTE buffer is added
(10 mM Tris, 1mM EDTA, pH 8.0)；It is spare that homogenate is sub-packed in different 50mL centrifuge tubes；By sample homogenization liquid
It is dissolved in lysis buffer (the 10mM Tris-HCl, pH8.0 of 8mL；2mM EDTA, pH8.0；0.4M NaCl) and 800 μ L
20% SDS is placed on vortex vortex mixer and is uniformly mixed；The Proteinase K of 40 μ L 20mg/mL is added thereto, then sets
1h is incubated in 65 DEG C of water-baths；After incubation, (precipitating proteins, DNA dissolve the NaCl of addition 6mL 5M in this concentration thereto
Degree increases, and protein solubility reduces), it is subsequently placed on vortex vortex mixer and mixes 30s；Above-mentioned mixed liquor is placed in 10,
It is centrifuged 30min on the centrifuge of 000 × g, its supernatant is then transferred to another clean centrifuge tube；It is added in equal volume
Isopropanol (precipitating DNA), mixing of turning upside down is placed on -20 DEG C of refrigerator freezing 20min；It is placed in the centrifuge of 16,000 × g again
Upper centrifugation 20min, supernatant is removed, and adds 1mLTE buffer solution sediment, DNA is transferred to microcentrifugation after dissolution
Pipe, the DNA as extracted in actual sample.
2, the building of strand displacement type archaeal dna polymerase induction isothermal circulation amplified reaction (SDPICA) system
(1) building of reaction system 1
To obtain target pork DNA fragment specific, reaction system 1 is constructed.Its specific building process is as follows: Mg2+It is dense
Degree is 8mmol/L, and beet alkali concentration is 1mol/L, and the concentration of dNTPs is 1.8mmol/L, and unwinding primer concentration is 0.8 μm of ol/
L, Bst archaeal dna polymerase concentration are 16U, and genomic DNA is 5 μ L.
(2) building of reaction system 2
For the Sensitive Detection for realizing pork DNA, reaction system 2 is constructed.Its specific building process is as follows: circulation primer is dense
Degree is 0.5 μm of ol/L, and Klenow polymerase concentration is 0.24U, and the concentration of dNTPs is 1.2mmol/L, and molecular beacon probe is dense
Degree is 0.2 μm of ol/L, and DMSO is that 6 μ L, DNA are 5 μ L.
(3) reaction system 1 and reaction system 2 are coupled
The pork Single-stranded DNA fragments and the privileged site of the molecular beacon probe in reaction system 2 that reaction system 1 obtains are mutual
It recruiting pair, molecular beacon probe, which is opened, becomes linear chain structure by cyclic structure, and fluorophor separates immediately with quenching group,
Generate fluorescence signal.
3, the specific detection of pork DNA
It places reaction liquid into 96 orifice plates, is placed in the endpoint signal detection that microplate reader carries out real-time fluorescence SDPICA amplification,
Excitation wavelength is 480~497nm, launch wavelength is 520~525nm, floor detection, gain 100, delay 100msec, detection
Height 7mm.
Embodiment 2:
1, pork product extracting genome DNA (SDS method)
0.5g pork fresh meat sample to be measured is weighed, tissue block is transferred in centrifuge tube after shredding grinding.Add into sample
Enter 7mL cell pyrolysis liquid, the Proteinase K Solution of 40 μ L is then added, mix, 65 DEG C of digestion 3h-5h are to without obvious tissue block.
700 μ L potassium acetate solutions are added into digestion lysate, mix ice bath 15min, 4 DEG C, 12,000 × g is centrifuged 10min, takes
Clearly to new 50mL centrifuge tube.Isometric Tris- saturated phenol is added, is slowly mixed by inversion 10min, 4 DEG C, 1,200 × g is centrifuged
10min.It takes supernatant to be transferred to new centrifuge tube, is added 0.5 volume of chloroform-isoamyl alcohol (24:1), is slowly mixed by inversion 10min,
4 DEG C, 1,200 × g is centrifuged 10min.Above steps may be repeated multiple times to two-phase it cannot be seen that until denatured protein, takes supernatant
Liquid is into new centrifuge tube.The dehydrated alcohol of diploid -20 DEG C of pre-coolings of product is added, jiggles to flocculent deposit and is precipitated, 1,200
× g is centrifuged 10min, outwells supernatant.6000 μ L70% ethanol washings precipitating is added, 1,200 × g is centrifuged 3min, draws supernatant
Liquid.It repeats previous step 1~2 time.Placing at room temperature makes residual ethanol volatilize completely, and the dissolution of 500 μ LTE buffer solutions is added
DNA precipitating.- 20 DEG C of preservation DNA solutions.
2, the building of strand displacement type archaeal dna polymerase induction isothermal circulation amplified reaction (SDPICA) system
(1) building of reaction system 1
To obtain target pork DNA fragment specific, reaction system 1 is constructed.Its specific building process is as follows: Mg2+It is dense
Degree is 10mmol/L, and beet alkali concentration is 1mol/L, and the concentration of dNTPs is 2mmol/L, and unwinding primer concentration is 0.8 μm of ol/
L, Bst archaeal dna polymerase concentration are 18U, and genomic DNA is 5 μ L.
(2) building of reaction system 2
For the Sensitive Detection for realizing pork DNA, reaction system 2 is constructed.Its specific building process is as follows: circulation primer is dense
Degree is 0.3 μm of ol/L, and Klenow polymerase concentration is 0.3U, and the concentration of dNTPs is 1.5mmol/L, molecular beacon probe concentration
For 0.3 μm of ol/L, DMSO is 5 μ L for 6 μ L, DNA.
(3) reaction system 1 and reaction system 2 are coupled
The pork Single-stranded DNA fragments and the privileged site of the molecular beacon probe in reaction system 2 that reaction system 1 obtains are mutual
It recruiting pair, molecular beacon probe, which is opened, becomes linear chain structure by cyclic structure, and fluorophor separates immediately with quenching group,
Generate fluorescence signal.
3, the specific detection of pork DNA
It places reaction liquid into 96 orifice plates, is placed in the endpoint signal detection that microplate reader carries out real-time fluorescence SDPICA amplification,
Excitation wavelength is 480~497nm, launch wavelength is 520~525nm, floor detection, gain 100, delay 100msec, detection
Height 7mm.
Embodiment 3:
1, pork product extracting genome DNA (RNA isolation kit)
Certain pork fresh meat to be measured is weighed, illustrates to be operated according to kit.
2, the building of strand displacement type archaeal dna polymerase induction isothermal circulation amplified reaction (SDPICA) system
(1) building of reaction system 1
To obtain target pork DNA fragment specific, reaction system 1 is constructed.Its specific building process is as follows: Mg2+It is dense
4~10mmol/L is spent, beet alkali concentration is 1.5mol/L, and the concentration of dNTPs is 1.8mmol/L, and unwinding primer concentration is 0.8
μm ol/L, Bst archaeal dna polymerase concentration are 18U, and genomic DNA is 6 μ L.
(2) building of reaction system 2
For the Sensitive Detection for realizing pork DNA, reaction system 2 is constructed.Its specific building process is as follows: circulation primer is dense
Degree is 0.5 μm of ol/L, and Klenow polymerase concentration is 0.4U, and the concentration of dNTPs is 2.2mmol/L, molecular beacon probe concentration
For 0.2 μm of ol/L, DMSO is 6 μ L for 6 μ L, DNA.
(3) reaction system 1 and reaction system 2 are coupled
The pork Single-stranded DNA fragments and the privileged site of the molecular beacon probe in reaction system 2 that reaction system 1 obtains are mutual
It recruiting pair, molecular beacon probe, which is opened, becomes linear chain structure by cyclic structure, and fluorophor separates immediately with quenching group,
Generate fluorescence signal.
3, the specific detection of pork DNA
It places reaction liquid into 96 orifice plates, is placed in the endpoint signal detection that microplate reader carries out real-time fluorescence SDPICA amplification,
Excitation wavelength is 480~497nm, launch wavelength is 520~525nm, floor detection, gain 100, delay 100msec, detection
Height 7mm.
Embodiment 4:
Isothermal circulation amplified reaction (SDPICA) system is induced according to the strand displacement type archaeal dna polymerase of above-mentioned foundation, with pig
Template after the random sequence genomic DNA of meat is extracted according to the DNA extraction method of above-described embodiment, as reaction
The specificity of DNA, detection primer and probe.Experimental result is illustrated in fig. 2 shown below, the results show that the genomic DNA in pork source
Amplification is shown as positive, other randomness sequences are without expanding.As it can be seen that being directed to the primer and probe of target species
It is specific good.
Embodiment 5:
Isothermal circulation amplified reaction (SDPICA) system is induced according to the strand displacement type archaeal dna polymerase of above-mentioned foundation, will be mentioned
The porky genome DNA obtained carries out 10 times and is serially diluted, to investigate the sensitivity of this method.The following Fig. 3 institute of experimental result
Show, the results show that existing when template quantity is 1pg in strand displacement type archaeal dna polymerase induction isothermal circulation amplification reaction system
The increase of fluorescence signal, then the Monitoring lower-cut of the detection architecture is 1pg.
Embodiment 6:
Isothermal circulation amplified reaction (SDPICA) system is induced according to the strand displacement type archaeal dna polymerase of above-mentioned foundation, respectively
The pork system (0.01%, 0.1%, 1%, 10%, 100%) and blank system of different quality percentage are detected, it should with measurement
The detection limit of method.Experimental result is illustrated in fig. 4 shown below, the results show that when the mass percent of the object in system is
When 0.1%, there are apparent amplification curves, then the Monitoring lower-cut of the detection architecture is 0.1%.
Claims (9)
1. a kind of method of strand displacement type archaeal dna polymerase induction isothermal circulation amplified reaction detection pork content, it is characterised in that:
This method is, as template, it is poly- to prepare strand displacement type DNA containing the specific molecular beacon complementary with the DNA of pork target sequence
Synthase induce isothermal circulation amplification reaction system, be placed in microplate reader, molecular beacon probe and circulation primer under the action of into
Row constant-temperature amplification realizes the qualitative and/or quantitative detection to pork according to amplification curve and fluorescence relative intensity.
2. according to the method described in claim 1, it is characterized by: the nucleotide sequence (5 ' -3 ') of molecular beacon probe by I,
II, III three parts successively form, I and III complementary pairing, and II is the probe ring with meat specific genomic sequences complementary pairing
Sequence.
3. according to the method described in claim 2, it is characterized by: I and III complementary pairing, I be TCTTGATCTCC,
TCTTGCATCAG；III is GGAGATCAAGA, CTGATGCAAGA.
4. according to the method described in claim 1, it is characterized by: the sequence both ends of the molecular beacon probe are modified with fluorescence
Group and quenching group.
5. according to the method described in claim 1, it is characterized by: this method can using solution strand primer and Bst archaeal dna polymerase
Carry out side to target double-stranded DNA in constant temperature to untwist amplification edges, release the target single stranded DNA of subsequent reactions, object chain with
The privileged site complementary pairing of molecular beacon probe, molecular beacon probe, which is opened, becomes linear chain structure, fluorescence by cyclic structure
Group separates immediately with quenching group, generates fluorescence signal.
6. according to the method described in claim 1, it is characterized by: this method is believed using circulation primer and Klenow enzyme and molecule
Mark probe specific part complementary pairing, which forms double-stranded DNA and releases target single stranded DNA, enters next round cyclic amplification, thus real
Existing highly sensitive detection.
7. according to the method described in claim 1, it is characterized by: the DNA containing pork target sequence refers to containing inspection
Survey the nucleotide sequence of target nucleic acid fragment.
8. according to the method described in claim 1, it is characterized by: described containing pork target sequence is mitochondrial cytochrome b gene
Design.
9. according to the method described in claim 1, it is characterized by: described and target sequence complementary pairing pork probe ring sequence
Be classified as it is below any one: CCTCAATGGTATGCCACAAC, TTACACACATTTGTCGAGACGT.
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| PL444578A1 (en) * | 2023-04-25 | 2024-10-28 | Uniwersytet Kazimierza Wielkiego | Method of detecting pork components in a food product |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113462786A (en) * | 2021-06-22 | 2021-10-01 | 江苏省菩德食品安全检测技术有限公司 | Method for identifying meat adulteration component based on DNA targeting gene fragment |
| PL444578A1 (en) * | 2023-04-25 | 2024-10-28 | Uniwersytet Kazimierza Wielkiego | Method of detecting pork components in a food product |
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