CN107012229A - Pig derived component quick determination method and kit in food - Google Patents
Pig derived component quick determination method and kit in food Download PDFInfo
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- CN107012229A CN107012229A CN201710269183.0A CN201710269183A CN107012229A CN 107012229 A CN107012229 A CN 107012229A CN 201710269183 A CN201710269183 A CN 201710269183A CN 107012229 A CN107012229 A CN 107012229A
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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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Abstract
The present invention relates to animal derived food detection field, it is pig derived component quick determination method and kit method in a kind of food, is characterized in, the animal DNA extracts reagent prepared by the present invention extracts measuring samples DNA；According to pig source property mitochondria specific gene (cytb) primers and detection probe in GenBank, enter performing PCR amplification to cytb genes under the PCR amplification conditions of optimization；By the hybridization of amplified production and probe, paper slip is chromatographed by lateral flow and detected.Use quick determination method its detection time only 5 minutes of the present invention, as a result naked eyes interpretation；With existing pig derived component often with species specific PCR detection technique compared with, with easy to operate, quick, equipment and the low advantage of testing cost, the qualitative detection for carrying out pig derived component for developing country, laboratories and market self-checking mechanism provides new approaches.
Description
Technical field
The present invention relates to a boar derived component quick determination method and kit for animal derived food detection field.
Background technology
Meat product is the important component of human diet structure, due to the meat difference in selling prices of in the market separate sources
Larger, to reap staggering profits, the illegal enterprise in part and trade company are produced by adding the modes such as ox, sheep oil in the relatively low pork of price
Various false beef, mutton product.Above-mentioned behavior not only compromises the interests of consumer, very disruptive market order, Er Qieke
Religion and national disputation, influence national unity and harmony can be brought.Therefore, the science of foundation, quickly and accurately detection method are needed badly,
Differentiate the pork content adulterated in beef or mutton product.
Since 1980s, starting to study the Species estimation of meat both at home and abroad mainly has chromatography, electrophoresis,
Enzyme-linked immunosorbent assay etc., due to the complexity and the diversity of processing mode of food ingredients, these traditional methods are to some
The food of deep processing and heat treatment can not be detected accurately.The characteristics of DNA has heat endurance and species specificity, more in recent years
Detection technique based on DNA level, such as DNA hybridization, PCR sequencings, species specific PCR are used for the research of Animal species identification.
Wherein species specific PCR, because detection is quick, with higher specificity and sensitivity, be increasingly becoming in food meat into
Divide the main stream approach differentiated.But round pcr has certain limitation simultaneously, Standard PCR-gel electrophoresis technology in the detection will be through
Multiple steps such as glue, electrophoresis, imaging are crossed, and apply the nucleic acid staining agent such as ethidium bromide (EB) to easily cause the dirt to environment
Dye；Although real-time fluorescence quantitative PCR (qPCR) technology overcomes drawbacks described above, qPCR needs higher technical requirements and costliness
Instrument, still can not extensive use.Therefore, simple, fast and accurately animal derived food detection technique, and extensive use is set up
It is still key subjects of the pendulum in face of researcher in developing country and laboratories.
Colloidal gold immunity chromatography is the product that colloidal gold-labeled method and immunochromatography technique are combined, and general principle is
Diafiltration concentration and capillarity using miillpore filter, make antigen-antibody react, and in detection line position, positive reaction exists
Occur red stripes on lateral flow chromatograph test strip, negative reaction is without band.The present invention establishes pig derived component quick detection
Method and kit.The spy that special primer enters performing PCR amplification, amplified matter and a modification is designed according to porcine mtdna cytb genes
Specific probes hybridize, and are detected finally by lateral flow chromatograph test strip.PCR terminate after hybridization and detection time only need 5 minutes,
And result naked eyes can interpretation.The pig derived component quick determination method and kit that the present invention is set up can for developing country,
The qualitative detection that laboratories and market self-checking mechanism carry out pig derived component provides new approaches.
The content of the invention
Present invention aims to overcome that there is provided a kind of easy to operate, quick, sensitivity is high, special for the deficiencies in the prior art
Property it is strong, effect is good, pig derived component quick determination method in the high food of application value；And its detection kit is provided.
The purpose of the present invention is realized by following technical scheme：
1. pig derived component quick determination method in a kind of food, it is characterised in that comprise the following steps：
1) specific primer and probe of pig derived component quick detection are designed and optimize, its sequence is as follows：
Forward primer (PF)：5’-CTATACTACGGATCCTATATATTCCTAG-3’(28bp,Tm:61.7℃)；
Anti-sense primer:5’-Fam-CTACGAGGTCTGTTCCGATATAAGG-3’(25bp,Tm:64.6 DEG C), the 5 ' of primer
End is modified through Fam；
Oligonucleotide probe (P):5’-ATCACAAATCTACTATCA-Biotin-3’(18bp,Tm:48.5 DEG C), probe
3 ' end modified through Biotin；
2) extraction of sample DNA：
By any one in pork sample and detected pork, mutton, beef, chicken, goose, duck, rabbit meat and its
The product of the meat is shredded, and is transferred to after each meat sample 0.1g, liquid nitrogen grinding are accurately weighed respectively in 1.5mL centrifuge tubes；
800 μ L lysates are added, 65 DEG C of incubation 30min, during which vibration is mixed 3-4 times, 12000 × g centrifugations 5min；
Supernatant is moved into a clean centrifuge tube, adds 400 μ L chloroforms：Isoamyl alcohol (24:1) hybrid extraction liquid, shakes
Swing 12000 × g centrifugations 5min after mixing；
Supernatant is taken in another clean centrifuge tube, 320 μ L isopropanols, precipitation at room temperature 30min are added；
12000 × g centrifuges 5min, abandons supernatant, and the rinsing of the ethanol of 500 μ L 70%, 12000 × g centrifugations are added into precipitation
5min；
Supernatant is abandoned, room temperature dries precipitation, with 20 μ L TE buffer solutions, determine -20 DEG C of preservations after OD values；
3) pcr amplification reaction：
PCR reaction systems:Wherein PCR Master Mix 5.2 μ L, each 0.8 μ L of 10 μm of ol/L primers, the μ L of template DNA 1,
ddH2The μ L of O 17.2, the μ L of cumulative volume 25；
PCR reaction conditions are as follows:95 DEG C of pre-degenerations 5min, 95 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C extend 30s, 40
Individual circulation；95 DEG C are denatured 2 minutes, are placed on ice；
4) preparation of lateral flow chromatograph test strip
It is prepared by colloid gold particle-Streptavidin (AuNPs-SA) compound：Adjust collaurum (AuNPs) pH value of solution extremely
7.0,18 μ g streptavidins (SA) are the protein content for stablizing 1mL 30nm collaurums, are under this condition coupled AuNPs and SA,
It is laid in gold standard pad；The anti-Fam antibody of 1.2mg/mL mouse and 1.2mg/mL BSA- biotin conjugates are drawn in nylon membrane table
Face, forms that detection line is T lines and control line is C lines；
The structure composition of lateral flow chromatograph test strip：Sequentially consist of bottom adsorptive pads, sample area, gold standard pad, NC
Film, T lines, C lines and top adsorptive pads；
5) hybridization of PCR primer and probe：
10 μ L are taken through step 3) denaturation PCR primer and 1 μ L oligonucleotide probes 10mmol/L in 50 μ L hybridization buffers
1g/L PEG4000,1.5M MgCl2, in pH7.0, mix；Subsequent 35 DEG C of incubations 1min, carries out the solution hybridization of nucleic acid；
6) the lateral flow chromatograph test strip detection of hybrid product：
10 μ L hybridization mixtures are taken to be splined on the sample pad of lateral flow chromatograph test strip；
Sample pad lower end is immersed in 90 μ L developping solutions, 0.01mol/L PBS, pH 7.4, knot is visually read in 5 minutes
Really.
2. pig derived component quick detection kit in a kind of food, it is characterized in that, it includes following system：
1) the pcr amplification reaction system of porcine mtdna cytb genes, specifically includes following components：
(a) pcr template DNA prepares solution
Sample dissociation liquid：1g containing CTAB, EDTA 0.3722g, Tris-HCl 0.7882g, NaCl 4.09g, are adjusted to pH
8.0, autoclave sterilization, 100mL；
Extract：Chloroform is 24 with isoamyl alcohol volume:1 mixed liquor, 60mL；
Isopropanol：50mL；
70% ethanol：200mL；
TE buffer solutions：10mmol/L Tris-HCl, 1mmol/L EDTA (pH8.0), 10mL；
(b) a pair of specific primers of porcine mtdna cytb genes
Optimized design and artificial synthesized primer is as follows：
Forward primer (PF)：5’-CTATACTACGGATCCTATATATTCCTAG-3’(28bp,Tm:61.7℃)；
Anti-sense primer:5’-Fam-CTACGAGGTCTGTTCCGATATAAGG-3’(25bp,Tm:64.6 DEG C), the 5 ' of primer
End is modified through Fam；
Primer and probe is synthesized by Shanghai bioengineering Co., Ltd；
(c) PCR reaction solutions
PCR Master Mix：American AB I companies PCR Master Mix kits, 2mL；ddH2O：5mL；
2) detection architecture of porcine mtdna cytb genes, specifically includes following components：
(a) specific probe of porcine mtdna cytb genetic tests
Oligonucleotide probe (P):5’-ATCACAAATCTACTATCA-Biotin-3’(18bp,Tm:48.5 DEG C), probe
3 ' end modified through Biotin；
(b) hybridization of single-stranded amplicon and probe and detection solution
Hybridization buffer：1g/L PEG4000,1.5M MgCl2, pH7.0,100mL；
Developping solution：0.01mol/L PBS, pH 7.4,200mL.
Pig derived component quick determination method and kit in a kind of food of the present invention, according to porcine mtdna cytb genes
Design a pair of specific primers, by PCR amplification and solution hybridization, as a result by ELISA test strip, can pig derived component it is fast
Speed detection.Compared with the technologies such as existing nucleic acid, the beneficial effects of the present invention are：
1. simple and quick, easy to spread, naked eyes can sentence read result in 5 minutes；
2. accuracy is high, high specificity, it had both avoided the complex detection step of Standard PCR-gel electrophoresis, overcame again
Real-time fluorescence quantitative PCR (qPCR) needs higher technical requirements and the defect of expensive instrument；
3. sensitivity is high, reproducible, effect is good, and application value is high, and primer and probe detects pig derived component after optimization
The more conventional electrophoresis of sensitivity significantly improve and repeatability more preferably, can be widely applied to developing country and laboratories and
Market self-checking mechanism carries out the qualitative detection of pig derived component.
Brief description of the drawings
Fig. 1 experimental principle figures；
Fig. 2 specificity experiments result figures；
Fig. 3 sensitivity experiments result figures.
Embodiment
Below with embodiment, the invention will be further described.
1st, pig derived component quick detection detection method in a kind of food, includes following step：
(1) modification method prepares meat sample product and meat products template DNA
Pork that market is bought, mutton, beef, chicken, goose, duck, rabbit meat are through Morphological Identification；
Pork sample and market are bought into the meat and its meat products is shredded, each meat sample 0.1g, liquid nitrogen are accurately weighed respectively
It is transferred to after grinding in 1.5mL centrifuge tubes, adds 800 μ L lysates, 65 DEG C of incubation 30min, during which vibration is mixed 3-4 times,
12000 × g centrifuges 5min；
Supernatant is moved into a clean centrifuge tube, adds 400 μ L chloroforms：Isoamyl alcohol (24:1) mixed liquor, vibration is mixed
12000 × g centrifugations 5min after even；
Supernatant is taken in another clean centrifuge tube, 320 μ L isopropanols, precipitation at room temperature 30min are added；
12000 × g centrifuges 5min, abandons supernatant, and the rinsing of the ethanol of 500 μ L 70%, 12000 × g centrifugations are added into precipitation
5min；
Abandon after supernatant, precipitation drying at room temperature with 20 μ L TE buffer solutions, determine -20 DEG C of preservations after OD values；
(2) the porcine mtdna cytb gene orders in GenBank, with DNAStar Software for Design and artificially synthesized pig
The forward primer marked in mitochondria cytb gene orders special primer and oligonucleotide probe, cross experiment and label probe knot
Close, can cause to produce false positive in lateral flow chromatography assay, testing result is impacted, it is therefore necessary to primer and probe sequence
Row are optimized screening, optimized to screen and artificial synthesized primer and probe is as follows：
Forward primer (PF)：5’-CTATACTACGGATCCTATATATTCCTAG-3’(28bp,Tm:61.7℃)；
Anti-sense primer (PR)：5’-Fam-CTACGAGGTCTGTTCCGATATAAGG-3’(25bp,Tm:64.6 DEG C), primer
5 ' end modified through Fam；
Oligonucleotide probe (P):5’-ATCACAAATCTACTATCA-Biotin-3’(18bp,Tm:48.5 DEG C), probe
3 ' end modified through Biotin；
Primer and probe is synthesized by Shanghai bioengineering Co., Ltd；
(3) PCR reaction systems and reaction condition
PCR reaction systems:PCR Master Mix, from the μ L of American AB I companies PCR Master Mix kits 5.2,
Each 0.8 μ L of 10 μm of ol/L primers, template DNA 1 μ L, ddH2The μ L of O 17.2, the μ L of cumulative volume 25；
PCR reaction conditions:95 DEG C of pre-degenerations 5min, 95 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, 40 are followed
Ring；95 DEG C are denatured 2 minutes, are placed on ice；
(4) preparation of lateral flow chromatograph test strip
It is prepared by colloid gold particle-Streptavidin (AuNPs-SA) compound：Adjust collaurum (AuNPs) pH value of solution extremely
7.0,18 μ g streptavidins (SA) are the protein content for stablizing 1mL 30nm collaurums, are under this condition coupled AuNPs and SA,
It is laid in gold standard pad；The anti-Fam antibody of 1.2mg/mL mouse and 1.2mg/mL BSA- biotin conjugates are drawn in nylon membrane table
Face, forms that detection line is T lines and control line is C lines；
The structure composition of lateral flow chromatograph test strip：Sequentially consist of bottom adsorptive pads, sample area, gold standard pad, NC
Film, detection line are that T lines, nature controlling line are C lines and top adsorptive pads；
(5) hybridization and detection of single-stranded amplicon and probe
Referring to Fig. 1, experimental principle：Forward primer PF, 5 ' enter performing PCR through Fam modifications and anti-sense primer PR to target gene
Amplification, after PCR reactions terminate, double stranded amplicon through thermal denaturation into single strand dna, the nucleic acid probe P with mark, 3 ' end warps
Biotin modification hybridization, shape life hybridization complex (Fam and Biotin) mark；Reaction solution containing the compound is splined on laterally
Chromatograph test strip sample pad is flowed, then immerses lateral flow chromatograph test strip bottom adsorptive pads in developping solution, it is miscellaneous in reaction solution
The Biotin sites of compound are handed over to be combined with Streptavidin-nano-Au composite in gold standard pad and with developping solution layer from bottom to top
Analysis, being coated when reaching T lines has the anti-Fam monoclonal antibodies of mouse to be captured, and T lines form meat due to the enrichment of nano-Au composite
Eye red color visible band；Superfluous nanogold-Streptavidin continues to chromatograph the Biotin captures being coated to C lines, is formed red
Vitta band；Testing result is the positive.If only C lines take on a red color, testing result is negative.If T lines and C lines are colourless, test strips are lost
Effect；
Experimentation：The PCR primer and 1 μ L oligonucleotide probes (10mmol/L) of the 10 above-mentioned denaturation of μ L is taken to hybridize in 50 μ L
Buffer solution [(1g/L PEG4000,1.5M MgCl2), pH7.0] middle mixing；35 DEG C of incubations carry out nucleic acid solution after 1 minute mutually miscellaneous
Hand over；
Take 10 μ L hybridization mixtures to be splined on the sample pad of chromatograph test strip, 90 μ L developping solutions are immersed into the lower end of sample pad
In (0.01mol/L PBS, pH 7.4), result is visually read in 5 minutes.
2nd, pig derived component quick detection kit in a kind of food of the invention, including following system：
(1) the pcr amplification reaction system of porcine mtdna cytb genes, specifically includes following components：
(a) pcr template DNA prepares solution
Sample dissociation liquid：1g containing CTAB, EDTA 0.3722g, Tris-HCl 0.7882g, NaCl 4.09g, are adjusted to pH
8.0, autoclave sterilization, 100mL；
Extract：Chloroform:Isoamyl alcohol (24:1) mixed liquor, 60mL；
Isopropanol：50mL；
70% ethanol：200mL；
TE buffer solutions：10mmol/L Tris-HCl, 1mmol/L EDTA (pH8.0), 10mL；
(b) a pair of specific primers of porcine mtdna cytb genes
Optimized design and artificial synthesized primer is as follows：
Forward primer：5’-CTATACTACGGATCCTATATATTCCTAG-3’(28bp,Tm:61.7℃)；
Anti-sense primer:5’-Fam-CTACGAGGTCTGTTCCGATATAAGG-3’(25bp,Tm:64.6 DEG C), the 5 ' of primer
End is modified through Fam；
Primer and probe is synthesized by Shanghai bioengineering Co., Ltd；
(c) PCR reaction solutions
PCR Master Mix：American AB I companies PCR Master Mix kits, 2mL；ddH2O：5mL；
(2) detection architecture of porcine mtdna cytb genes, specifically includes following components：
(a) specific probe of porcine mtdna cytb genetic tests
Oligonucleotide probe:5’-ATCACAAATCTACTATCA-Biotin-3’(18bp,Tm:48.5 DEG C), the 3 ' of probe
End is modified through Biotin；
(b) hybridization of single-stranded amplicon and probe and detection solution
Hybridization buffer：1g/L PEG4000,1.5M MgCl2, pH7.0,100mL；
Developping solution：0.01mol/L PBS, pH 7.4,200mL.
Example 1：Pig derived component quick detection kit specific detection in food：
With forward primer PF and anti-sense primer (PR) to pork and other 6 kinds of animal derived materials, mutton, beef, chicken,
Goose, duck, the DNA sample of rabbit meat take product to carry out lateral flow chromatograph test strip detection, only pig after entering performing PCR amplification, hybridization
Meat DNA testing results are positive, and other controls and blank control are feminine gender.
As a result show that this method specificity is good, hybridized using the specific probe and single-stranded PCR amplified matters of a modification,
Hybrid product is detected by lateral flow chromatograph test strip again, the specificity of detection is added.(see Fig. 2)
Example 2：Pig derived component quick detection kit sensitivity is detected in food：
Pork DNA is subjected to 10 times of gradient dilutions, enters performing PCR amplification using the DNA of different dilution factors as template respectively, together
When detected that as a result agarose gel electrophoresis method is in template DNA amount through agarose gel electrophoresis and lateral flow chromatograph test strip
Band is substantially obscured during 100pg, can not correct sentence read result, the detection sensitivity of electrophoresis is 100pg.And ELISA test strip
When, as template DNA amount 100fg, it still can clearly judge positive test symbol (see Fig. 3).
As a result show that the detection sensitivity of kit improves 1000 times compared with PCR- agarose gel electrophoresis.
Example 3：Pig derived component quick detection kit is used for the detection of business sample in food
1) collection of specimens is with preserving：
Large percentage is sold from the local supermarket's purchase in Changchun and indicates 28 portions of kind meat products of main component, the species of sample
Such as table 1, each sample does a Duplicate Samples.
2) modification method prepares sample DNA
(a) meat sample product and meat products business sample are shredded, respectively it is each it is accurate weigh 0.1g, liquid nitrogen grinding after be transferred to
In 1.5mL centrifuge tubes, 800 μ L lysates are added, 65 DEG C of incubation 30min, during which vibration is mixed 3-4 times, 12000 × g centrifugations
5min；
(b) supernatant is moved into a clean centrifuge tube, adds 400 μ L chloroforms：Isoamyl alcohol (24:1) mixed liquor, vibration
12000 × g centrifuges 5min after mixing；
(c) supernatant is taken in another clean centrifuge tube, adds 320 μ L isopropanols, precipitation at room temperature 30min；
(d) 12000 × g centrifuges 5min, abandons supernatant, and the rinsing of the ethanol of 500 μ L 70%, 12000 × g are added into precipitation
Centrifuge 5min；
(e) abandon after supernatant, precipitation drying at room temperature with 20 μ L TE buffer solutions, determine -20 DEG C of preservations after OD values
3) PCR reaction systems and reaction condition
PCR reaction systems:PCR Master Mix (American AB I companies PCR Master Mix kits) 5.2 μ L, 10 μ
Each 0.8 μ L of mol/L primers, template DNA 1 μ L, ddH2The μ L of O 17.2, the μ L of cumulative volume 25；
PCR reaction conditions:95 DEG C of pre-degenerations 5min, 95 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, 40 are followed
Ring；95 DEG C are denatured 2 minutes, are placed on ice；
4) preparation of lateral flow chromatograph test strip
It is prepared by colloid gold particle-Streptavidin (AuNPs-SA) compound：Adjust collaurum (AuNPs) pH value of solution extremely
7.0,18 μ g streptavidins (SA) are the protein content for stablizing 1mL 30nm collaurums, are under this condition coupled AuNPs and SA,
It is laid in gold standard pad；The anti-Fam antibody of 1.2mg/mL mouse and 1.2mg/mL BSA- biotin conjugates are drawn in nylon membrane table
Face, forms that detection line is T lines and control line is C lines.
The structure composition of lateral flow chromatograph test strip：Sequentially consist of bottom adsorptive pads, sample area, gold standard pad, NC
Film, T lines, C lines and top adsorptive pads；
5) hybridization and detection of single-stranded amplicon and probe
The PCR primer and 1 μ L oligonucleotide probes (10mmol/L) of the 10 above-mentioned denaturation of μ L are taken in 50 μ L hybridization buffers
[(1g/L PEG4000,1.5M MgCl2), pH7.0] middle mixing；35 DEG C of incubations carry out nucleic acid solution and mutually hybridized after 1 minute；
Take 10 μ L hybridization mixtures to be splined on the sample pad of lateral flow chromatograph test strip, 90 μ L are immersed into the lower end of sample pad
In developping solution (0.01mol/L PBS, pH 7.4), in visually reading result in 5 minutes.
As a result：This method can be detected to 19 parts of pork sample standard deviations；In addition, having in the dried products such as 9 parts of pork luncheon meat, beef rolls
5 parts detect pork content；Inconsistent commodity are marked to testing result and commodity its corresponding PCR primer is sequenced,
Sequencing result is consistent with testing result.As a result show, this method is in the detection of business sample and complicated food sample
With higher accuracy；It is not difficult to find out from testing result, the adulterated beef phenomenon of in the market pork compares to be serious, and meat products is mixed
False problem is strictly the common problem of industry.(being shown in Table 1)
The Marketing pork of table 1, beef sample qualification result
Table1 Identification results of commercial pork and beef sample
Conclusion：The present invention optimizes pig source property in primer and probe, the food of exploitation to porcine mtdna cytb genetic tests
Composition quick detection kit has higher sensitivity and specificity, and lateral flow chromatograph test strip quick detection and PCR are expanded
Technological perfectionism is combined, and eliminates the detecting steps such as electrophoresis, the imaging of Standard PCR, remolding sensitivity Standard PCR is high 1000 times, and it is examined
Survey result in 5 minutes can the quick interpretation of naked eyes, it is simple and quick；Simultaneously compared to real-time fluorescence quantitative PCR (qPCR) technology, keep away
The defect compared with high-tech requirement and expensive instrument is exempted to need, it is easy to promote；Simultaneously suitable for developing country, laboratories
And market self-checking mechanism carries out the fast qualitative detection of pig derived component, detection efficiency, reduction testing cost are improved.
Claims (2)
1. pig derived component quick determination method in a kind of food, it is characterised in that comprise the following steps：
1) specific primer and probe of pig derived component PCR quick detections are designed and optimize, its sequence is as follows：
Forward primer (PF)：5’-CTATACTACGGATCCTATATATTCCTAG-3’；
Anti-sense primer (PR):5 '-Fam-CTACGAGGTCTGTTCCGATATAAGG-3 ', 5 ' ends of primer are modified through Fam；
Oligonucleotide probe (P):5’-ATCACAAATCTACTATCA-Biotin-3’(18bp,Tm:48.5 DEG C), the 3 ' of probe
End is modified through Biotin；
2) extraction of sample DNA：
By any one in pork sample and detected pork, mutton, beef, chicken, goose, duck, rabbit meat and its described
The product of meat is shredded, and is transferred to after each meat sample 0.1g, liquid nitrogen grinding are accurately weighed respectively in 1.5mL centrifuge tubes；
800 μ L lysates are added, 65 DEG C of incubation 30min, during which vibration is mixed 3-4 times, 12000 × g centrifugations 5min；
Supernatant is moved into a clean centrifuge tube, adds 400 μ L chloroforms：Isoamyl alcohol (24:1) hybrid extraction liquid, vibration is mixed
12000 × g centrifugations 5min after even；
Supernatant is taken in another clean centrifuge tube, 320 μ L isopropanols, precipitation at room temperature 30min are added；
12000 × g centrifuges 5min, abandons supernatant, and the rinsing of the ethanol of 500 μ L 70%, 12000 × g centrifugations are added into precipitation
5min；
Supernatant is abandoned, room temperature dries precipitation, with 20 μ L TE buffer solutions, determine -20 DEG C of preservations after OD values；
3) pcr amplification reaction：
PCR reaction systems:Wherein the μ L of PCR Master Mix 5.2, each 0.8 μ L of 10 μm of ol/L primers, template DNA 1 μ L, ddH2O
17.2 μ L, the μ L of cumulative volume 25；
PCR reaction conditions are as follows:95 DEG C of pre-degenerations 5min, 95 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, 40 are followed
Ring；95 DEG C are denatured 2 minutes, are placed on ice；
4) preparation of lateral flow chromatograph test strip
It is prepared by colloid gold particle-Streptavidin (AuNPs-SA) compound：Collaurum (AuNPs) pH value of solution is adjusted to 7.0,18
μ g streptavidins (SA) are the protein content for stablizing 1mL 30nm collaurums, are under this condition coupled AuNPs and SA, are laid on gold
On mark pad；The BSA- biotin conjugates of the anti-Fam antibody of 1.2mg/mL mouse and 1.2mg/mL are drawn in nylon membrane, formed
Detection line is T lines and control line is C lines；
The structure composition of lateral flow chromatograph test strip：Sequentially consist of bottom adsorptive pads, sample area, gold standard pad, NC films, T
Line, C lines and top adsorptive pads；
5) hybridization of PCR primer and probe：
10 μ L are taken through step 3) denaturation PCR primer and 1 μ L oligonucleotide probes 10mmol/L in 50 μ L hybridization buffers (1g/L
PEG4000,1.5M MgCl2), in pH7.0, mix；Subsequent 35 DEG C of incubations 1min, carries out the solution hybridization of nucleic acid；
6) the lateral flow chromatograph test strip detection of hybrid product：
10 μ L hybridization mixtures are taken to be splined on the sample pad of lateral flow chromatograph test strip；
Described sample pad lower end is immersed in 90 μ L developping solutions, 0.01mol/L PBS, pH 7.4, knot is visually read in 5 minutes
Really.
2. pig derived component quick detection kit in a kind of food, it is characterized in that, it includes following system：
1) the pcr amplification reaction system of porcine mtdna cytb genes, specifically includes following components：
(a) pcr template DNA prepares solution
Sample dissociation liquid：1g containing CTAB, EDTA 0.3722g, Tris-HCl 0.7882g, NaCl 4.09g, are adjusted to pH 8.0,
Autoclave sterilization, 100mL；
Extract：Chloroform is 24 with isoamyl alcohol volume:1 mixed liquor, 60mL；
Isopropanol：50mL；
70% ethanol：200mL；
TE buffer solutions：10mmol/L Tris-HCl, 1mmol/L EDTA (pH8.0), 10mL；
(b) a pair of specific primers of porcine mtdna cytb genes
Optimized design and artificial synthesized primer is as follows：
Forward primer (PF)：5’-CTATACTACGGATCCTATATATTCCTAG-3’(28bp,Tm:61.7℃)；
Anti-sense primer (PR)：5’-Fam-CTACGAGGTCTGTTCCGATATAAGG-3’(25bp,Tm:64.6 DEG C), the 5 ' of primer
End is modified through Fam；
Primer and probe is synthesized by Shanghai bioengineering Co., Ltd；
(c) PCR reaction solutions
PCR Master Mix：American AB I companies PCR Master Mix kits, 2mL；ddH2O：5mL；
2) detection architecture of porcine mtdna cytb genes, specifically includes following components：
(a) specific probe of porcine mtdna cytb genetic tests
Oligonucleotide probe (P)：5’-ATCACAAATCTACTATCA-Biotin-3’(18bp,Tm:48.5 DEG C), the 3 ' of probe
End is modified through Biotin；
(b) hybridization of single-stranded amplicon and probe and detection solution
Hybridization buffer：(1g/L PEG4000,1.5M MgCl2), pH7.0,100mL；
Developping solution：0.01mol/L PBS, pH 7.4,200mL.
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