CN109136330A - The kit of common livestock meat is quickly detected based on species specificity PCR method - Google Patents
The kit of common livestock meat is quickly detected based on species specificity PCR method Download PDFInfo
- Publication number
- CN109136330A CN109136330A CN201811022275.XA CN201811022275A CN109136330A CN 109136330 A CN109136330 A CN 109136330A CN 201811022275 A CN201811022275 A CN 201811022275A CN 109136330 A CN109136330 A CN 109136330A
- Authority
- CN
- China
- Prior art keywords
- kit
- added
- dna
- adsorption column
- primer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of kits that common livestock meat is quickly detected based on species specificity PCR method, it includes extraction part and the detection part of DNA, includes specific primer, ddH corresponding to the general chemistry reagents such as molecular biology reagents, the lauryl sodium sulfate such as Proteinase K, 2xTaq Plus Master Mix and each poultry kind in reaction system2O etc..The kit for beef, mutton, pork, dog meats, horseflesh and donkey meat identification quick and precisely；In addition to donkey primer, the specificity of other poultry kind primers is relatively strong, cross reaction does not occur with other species；Sensitivity is higher；It can complete to detect in 2.5h, it is quickly time saving and at low cost, multiple samples can be detected simultaneously.Kit of the invention is fully available for the detection of cooked meat product, provides guide for method and technical support for each Food Inspection mechanism, and the detection for raiseeing derived component in food is of great significance.
Description
Technical field:
The present invention relates to a kind of gene testers, in particular to a kind of quickly to be detected often based on species specificity PCR method
See the kit of livestock meat.
Background technique:
With the improvement of people ' s living standards with the transformation of consumption idea, the demand of meat products rises year by year, in interests
Driving under, many retailers mix cheap meat to improve commercial profit in high price meat and meat products, while being added using various
Add agent adjustment product taste and color, to achieve the effect that mix the spurious with the genuine.The especially processing system such as beef or mutton volume and mutton cubes roasted on a skewer
Product, consumer are difficult to screen it from texture and mouthfeel.The appearance of these adulterated meat products, which has not only constituted a serious infringement, to disappear
The person's of expense equity more causes the health of consumer and seriously affects.Therefore, a kind of inspection precisely, quickly, high-throughput is established
Survey method provides strong technical support for animal derived food supervision, is the major issue of current urgent need to resolve.
In daily life, meat by cutting, blend and cut and mix, autoclaving and fire-cure equal deep processings processing, and
After a large amount of condiment and additive are added in gastronomical process, its original morphological feature is lost, which increase provenance identifications
Difficulty, the conventional method identified by sense organ and experience to meat is far from meeting needs.To the meat in food
The technology that ingredient carries out discriminatory analysis can totally be classified as spectral detection, protein detection and detection of nucleic acids three categories.Wherein, spectrum
Detection method is easy to operate and bad to sample nondestructive, but it is quantitative not accurate enough；Though protein assay can be carried out qualitative, quantitative inspection
It surveys, but its product for not being suitable for protein denaturation；Detection method based on nucleic acid is with hereditary information between animal species
Difference as detection target spot, the advantages such as limit with high specificity, not by tissue class, it has also become Meat ingredients identification
Main stream approach.
Summary of the invention:
The purpose of the present invention is to provide a kind of reagents that common livestock meat is quickly detected based on species specificity PCR method
Box, be able to solve the adulterated identification of livestock meat in the prior art it is at high cost, detection time is long, error is big the problems such as.
The present invention is to solve technical solution used by its technical problem:
It include extraction part and the detection of DNA based on the kit that species specificity PCR method quickly detects common livestock meat
Part includes the general chemistry reagents, 2xTaq such as molecular biology reagents, the lauryl sodium sulfate such as Proteinase K in reaction system
Plus Master Mix (Dye Plus) and each poultry plant a corresponding specific primer, ddH2O etc..
DNA described in mentioned reagent box extract part reaction the following steps are included:
1) 100mg sample is taken, is put into 2mL sterile centrifugation tube, 500 μ LSDS lysates and 20 μ L Proteinase Ks are added, is mixed
Water-bath (raises to solution is transparent and plants difference, the Tissue Lysis time is also different, and raw, cold cuts tissues are split under the conditions of 55 DEG C after even
It is also different to solve the time, the hard meat tissue such as jerky can first rehydration water-bath or water bath time is appropriately extended again, need according to specific
Experimental conditions are adjusted), it is during which mixed by inversion centrifuge tube frequently；
2) isometric Tris saturated phenol is added, is mixed by inversion rear 12000r/min centrifugation 10min, supernatant is transferred to separately
It is primary to repeat this step operation for one centrifuge tube；
3) be then added in centrifuge tube 0.5 times of volume Tris saturated phenol and 0.5 times of volume chloroform/isoamyl alcohol (24:
1), 12000r/min is centrifuged 10min after mixing, draws supernatant to another new centrifuge tube；
4) dehydrated alcohol of the sodium acetate solution of 0.1 times of volume and 4 DEG C of pre-coolings of 2.5 times of volumes is added, after being mixed by inversion
There is white precipitate precipitation, precipitates 2h at -20 DEG C；
5) (adsorption column is put into collecting pipe) is added in adsorption column in solution and flocculent deposit, 12000r/min centrifugation
After 5min, the waste liquid in collecting pipe is outwelled, adsorption column is put into collecting pipe；
6) 70% ethyl alcohol, 500 μ L is added in adsorption column to wash precipitating, 12000r/min is centrifuged 5min and abandons waste liquid, will inhale
Attached column is put into collecting pipe, repeats work of drilling；
7) adsorption column is put into collecting pipe, 12000r/min is centrifuged 2min, abandons waste liquid, adsorption column is put and is dried in the air at room temperature
It is dry, thoroughly remove remaining ethyl alcohol；
8) adsorption column is put into clean centrifuge tube, it is hanging that 100 μ L eluent TE are added dropwise, after being placed at room temperature for 5min
12000r/min is centrifuged 2min, and solution is collected into centrifuge tube；
9) blank control is done with TE, the concentration and purity of extracted DNA is measured with trace dna, determination of protein concentration instrument,
Applied sample amount is 1 μ L.
Mentioned reagent box is used to raise kind the reaction system detected are as follows: 2 × Taq Plus Master Mix, 25 μ L, in system
Primer, upstream and downstream primer (10 μm of olL are added-1) each 2 μ L, 0.5 μ g, dd H of DNA profiling2O supplies system to 50 μ L.
Mentioned reagent box is used to raise kind of a reaction condition for detection are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, * DEG C of annealing
30s, 72 DEG C of extension 60s, totally 35 recycle；72 DEG C of extension 7min.After reaction, take 5 μ L PCR products solidifying through 1% agarose
After gel electrophoresis detects amplified production, result is analyzed on gel imaging system.Annealing temperature " * " in the above reaction system according to
Ox, sheep, pig, dog, horse and donkey sequence be respectively 54 DEG C, 65 DEG C, 62 DEG C, 46 DEG C, 67 DEG C and 66 DEG C.
PCR amplification result is detected with the agarose gel electrophoresis of 1% or (1.5%):
(1) weigh appropriate agarose, be added in the triangular flask for filling 1 × TAE, make its final concentration reach 1% (or
1.5%) it, is completely dissolved with micro-wave oven；
(2) when gel solution is cooled to 50~60 DEG C, EB to final concentration of 0.5 μ g/mL, slow equidirectional rotation is added
Triangular flask is uniformly mixed it, gently pours into the plastic tank for being plugged comb it when non-scald on hand, places at room temperature；
(3) after being gelled admittedly, carefully comb is vertically extracted；
(4) gel made is put into Horizontal electrophoresis tank and (1 × TAE electrophoretic buffer has been added), pay attention to checking point sample
Whether the hole back side is complete, avoids that leakage sample occurs；
(5) PCR product to be detected and loading dye (Loading Buffer) are uniformly mixed point sample with 5:1 ratio,
To detect the integrality of extracted DNA；
(6) when detecting the PCR amplification adaptability of DNA, 10 μ L PCR products are added in each swimming lane, one of swimming
DNA Marker (D2000) is added in road, while doing negative control；
(7) voltage is set as 110V in this experiment, the race glue time is set as 30min (can be appropriately extended according to pillar location
Or shorten)；
(8) Ago-Gel is placed in gel imager after the completion of electrophoresis and carries out imaging analysis.
The beneficial effects of the present invention are:
Specific primer used in kit of the present invention can quickly, it is special, delicately detect common poultry beef cattle,
Sheep, pig, dog, horse and donkey derived component, have a characteristic that
(1) high specificity: for beef, mutton, pork, dog meats, horseflesh and donkey meat identification quick and precisely；Except donkey primer
Outside, the specificity of other poultry kind primers is relatively strong, cross reaction does not occur with other species；
(2) high sensitivity: for beef, detection limit reachable 5 × 10 of this method for raw meat-7μ g, and for ripe
The detection of meat is limited to 5 × 10-2μg；For mutton, this method is limited to 5 × 10 for the detection of raw meat-2μ g, and for ripe
The detection of meat is limited to 5 × 10-1μg；For pork and horseflesh, this method is 5 × 10 for the detection limit of raw meat-3μ g,
It and is 5 × 10 for the detection of cold cuts limit-2μg；For dog meats and donkey meat, detection of this method for raw meat and cold cuts
Limit is 5 × 10-1μg；
(3) quickly time saving: it can complete to detect in 2.5h, it is quickly time saving and at low cost；
Meanwhile kit of the invention can simultaneously detect multiple samples, and be fully available for the inspection of cooked meat product
It surveys.
Detailed description of the invention:
Fig. 1 is the ox primer specificity testing result of embodiment 1, and the annealing temperature of reaction is 54 DEG C；
Fig. 2 is the sheep primer specificity testing result of embodiment 1, and the annealing temperature of reaction is 65 DEG C；
Fig. 3 is the pig primer specificity testing result of embodiment 1, and the annealing temperature of reaction is 62 DEG C；
Fig. 4 is the dog primer specificity testing result of embodiment 1, and the annealing temperature of reaction is 46 DEG C；
Fig. 5 is the horse primer specificity testing result of embodiment 1, and the annealing temperature of reaction is 67 DEG C；
Fig. 6 is the donkey primer specificity testing result of embodiment 1, and the annealing temperature of reaction is 66 DEG C；
Fig. 7 is remolding sensitivity when ox DNA (life, cold cuts) is used for PCR in embodiment 1 compared with what 1~No. 8 loading wells represented
Template additive amount is respectively 5 × 10-1、5×10-2、5×10-3、5×10-4、5×10-5、5×10-6、5×10-7、5×10-8μ g,
Left-hand component is from raw meat, right-hand component from cold cuts；
Fig. 8 is remolding sensitivity when sheep DNA (life, cold cuts) is used for PCR in embodiment 1 compared with what 1~No. 8 loading wells represented
Template additive amount is respectively 5 × 10-1、5×10-2、5×10-3、5×10-4、5×10-5、5×10-6、5×10-7、5×10-8μ g,
Left-hand component is from raw meat, right-hand component from cold cuts；
Fig. 9 is remolding sensitivity when pig DNA (life, cold cuts) is used for PCR in embodiment 1 compared with what 1~No. 8 loading wells represented
Template additive amount is respectively 5 × 10-1、5×10-2、5×10-3、5×10-4、5×10-5、5×10-6、5×10-7、5×10-8μ g,
Left-hand component is from raw meat, right-hand component from cold cuts；
Figure 10 is remolding sensitivity when dog dna (life, cold cuts) is used for PCR in embodiment 1 compared with what 1~No. 8 loading wells represented
Template additive amount is respectively 5 × 10-1、5×10-2、5×10-3、5×10-4、5×10-5、5×10-6、5×10-7、5×10-8μ g,
Left-hand component is from raw meat, right-hand component from cold cuts；
Figure 11 is remolding sensitivity when horse dna (life, cold cuts) is used for PCR in embodiment 1 compared with what 1~No. 8 loading wells represented
Template additive amount is respectively 5 × 10-1、5×10-2、5×10-3、5×10-4、5×10-5、5×10-6、5×10-7、5×10-8μ g,
Left-hand component is from raw meat, right-hand component from cold cuts；
Figure 12 is remolding sensitivity when donkey DNA (life, cold cuts) is used for PCR in embodiment 1 compared with what 1~No. 8 loading wells represented
Template additive amount is respectively 5 × 10-1、5×10-2、5×10-3、5×10-4、5×10-5、5×10-6、5×10-7、5×10-8μ g,
Left-hand component is from raw meat, right-hand component from cold cuts.
Specific embodiment:
To make the objectives, technical solutions, and advantages of the present invention clearer, right below with reference to embodiment and attached drawing
The present invention is described in further details, here, exemplary embodiment and its explanation of the invention is used to explain the present invention, not
As limitation of the invention.
Experimental method used in following examples is unless otherwise specified conventional method；Used material, examination
Agent etc., is commercially available unless otherwise specified.
Embodiment 1
Reaction reagent and consumptive material: liquid nitrogen, Proteinase K, red fluorescence nucleic acid staining liquid, agarose, Tris-Base, nucleic acid
Extracting solution (chloroform/isoamyl alcohol (24:1)), DNA extract phenol reagent (Tris saturated phenol), lauryl sodium sulfate (SDS),
D2000,50 × TAE, HCl, EDTA, NaCl, sodium acetate trihydrate, glacial acetic acid, 95% ethyl alcohol, dehydrated alcohol, (2xTaq Plus
Master Mix (Dye Plus)) (Nanjing promise is only praised), primer, ddH2O, 2mL centrifuge tube, 1.5mL centrifuge tube, adsorption column, receipts
Collector, PCR pipe, 10 μ L pipette tips, 200 μ L pipette tips etc..
Synthesis, pretreatment and the storage requirement of primer: primer is synthesized by the raw work in Shanghai, and newly synthesized primer is solid powder
End, brief centrifugation makes it concentrate on centrifugation bottom of the tube before dissolving, and avoiding uncapping causes its loss to cause damages, and then closes by primer
Illustrate addition EB or TE buffer solution, since multigelation can degrade primer in use, dissolution at report
It is dispensed after the further 10 times of dilutions of primer application same buffer afterwards, mixing saves backup under the conditions of being placed on -20 DEG C.
Its sequence table of the above newly synthesized primer is as follows:
1 primer sequence of table and amplified fragments size
The extraction of 1.1DNA, the measurement of concentration and storage requirement:
(1) clean mortar, pestle and spoon are wrapped with masking foil, dry 2h, taking-up are put to room temperature standby at 250 DEG C
With；
(2) meat piece obtained after multidraw from bulk tissue is cleaned with clear water, removes connective tissue and fat,
It is cut into the fritter of 200mg or so；
(3) Liquid nitrogen precooler mortar, pestle and spoon are first used before grinding, is put into a little meat piece, liquid nitrogen are injected, after organizing adfreezing
It is ground to rapidly powdered, needs to supplement liquid nitrogen frequently during grinding；
(4) digested tankage is put into sample sack after the completion of grinding, 4 DEG C of lower sealings save；
(5) 100mg sample is taken, is put into 2mL sterile centrifugation tube, 500 μ L SDS lysates and 20 μ L Proteinase Ks are added,
Water-bath (raises to solution is transparent and plants difference, the Tissue Lysis time is also different, and raw, cold cuts tissues under the conditions of 55 DEG C after mixing
Pyrolysis time is also different, the hard meat tissue such as jerky can first rehydration water-bath or water bath time is appropriately extended again, need to be according to tool
Body experimental conditions are adjusted), it is during which mixed by inversion centrifuge tube frequently；
(6) isometric Tris saturated phenol then is added, is mixed by inversion rear 12000r/min centrifugation 10min, supernatant is turned
Another centrifuge tube is moved on to, it is primary to repeat this step operation；
(7) the Tris saturated phenol of 0.5 times of volume and chloroform/isoamyl alcohol of 0.5 times of volume are then added in centrifuge tube
(24:1), 12000r/min is centrifuged 10min after mixing, draws supernatant to another new centrifuge tube；
(8) dehydrated alcohol of the sodium acetate solution of 0.1 times of volume and 4 DEG C of pre-coolings of 2.5 times of volumes is added, after being mixed by inversion
There is white precipitate precipitation, precipitates 2h at -20 DEG C；
(9) (adsorption column is put into collecting pipe) is added in adsorption column in solution and flocculent deposit, 12000r/min centrifugation
After 5min, the waste liquid in collecting pipe is outwelled, adsorption column is put into collecting pipe；
(10) 70% ethyl alcohol, 500 μ L is added in adsorption column to wash precipitating, 12000r/min is centrifuged 5min and abandons waste liquid, will
Adsorption column is put into collecting pipe, repeats work of drilling；
(11) adsorption column is put into collecting pipe, 12000r/min is centrifuged 2min, abandons waste liquid, adsorption column is put at room temperature
It dries, thoroughly removes remaining ethyl alcohol；
(12) adsorption column is put into clean centrifuge tube, it is hanging that 100 μ L eluent TE are added dropwise, after being placed at room temperature for 5min
12000r/min is centrifuged 2min, and solution is collected into centrifuge tube, is stored under conditions of -20 DEG C.
(13) blank control is done with TE, measures the concentration of extracted DNA and pure with trace dna, determination of protein concentration instrument
Degree, applied sample amount is 1 μ L.
1.2 species specificity PCR methods detect the specificity of six kinds of poultry meats such as beef or mutton:
PCR reaction system are as follows: each poultry kind pair synthesized is added in system by 2 × Taq Plus Master Mix, 25 μ L
Primer, upstream and downstream primer (10 μm of olL-1) each 2 μ L are answered, 0.5 μ g, dd H2O of DNA profiling supplies system to 50 μ L.
Following proportional arrangement mixed liquor is pressed in PCR pipe:
PCR reaction condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, each primer annealing temperature is different, according to ox,
Sheep, pig, dog, horse and donkey sequence be respectively 54 DEG C, 65 DEG C, 62 DEG C, 46 DEG C, 67 DEG C and 66 DEG C, annealing time is 30s, 72
DEG C extend 60s, recurring number be 35；72 DEG C of extension 7min.After reaction, take 5 μ L PCR products through 1% Ago-Gel electricity
After swimming, result is analyzed on gel imaging system.
1.3 agarose gel electrophoresis detecting step are as follows:
(1) weigh appropriate agarose, be added in the triangular flask for filling 1 × TAE, make its final concentration reach 1% (or
1.5%) it, is completely dissolved with micro-wave oven；
(2) when gel solution is cooled to 50~60 DEG C, EB to final concentration of 0.5 μ g/mL, slow equidirectional rotation is added
Triangular flask is uniformly mixed it, gently pours into the plastic tank for being plugged comb it when non-scald on hand, places at room temperature；
(3) after being gelled admittedly, carefully comb is vertically extracted；
(4) gel made is put into Horizontal electrophoresis tank and (1 × TAE electrophoretic buffer has been added), pay attention to checking point sample
Whether the hole back side is complete, avoids that leakage sample occurs；
(5) PCR product to be detected and loading dye (Loading Buffer) are uniformly mixed point sample with 5:1 ratio,
To detect the integrality of extracted DNA；
(6) when detecting the PCR amplification adaptability of DNA, 10 μ L PCR products are added in each swimming lane, one of swimming
DNA Marker (D2000) is added in road, while doing negative control；
(7) voltage is set as 110V in this experiment, the race glue time is set as 30min (can be appropriately extended according to pillar location
Or shorten)；
(8) Ago-Gel is placed in gel imager after the completion of electrophoresis and carries out imaging analysis.
Referring to Fig. 1~6, it is as follows to analyze result:
As shown in Figure 1, ox primer is through detecting: the annealing temperature of reaction is 54 DEG C, determines that it is special under 54 DEG C of annealing temperatures
It is anisotropic good；
As shown in Figure 2, sheep primer is through detecting: the annealing temperature of reaction is 65 DEG C, it was demonstrated that its specificity at 65 DEG C is good；
From the figure 3, it may be seen that pig primer annealing temperature be 62 DEG C when, with good and stable specificity；
As shown in Figure 4, dog primer is tested repeatedly, finally determines that its specificity at 46 DEG C is good, and annealing temperature mentions
The primer is unstable when height is to 47 DEG C；
As shown in Figure 5, horse primer specificity under 67 DEG C of annealing temperature is good；
It will be appreciated from fig. 6 that donkey primer has cross reaction with sheep, dog and horse under 66 DEG C of annealing temperatures, but work as annealing temperature
Occur when being increased to 67 DEG C without any band, due to time relationship, specific donkey primer will be screened no longer, select the primer, only
It is that with sheep primer, dog primer and horse primer while check experiment need to be done can just obtain final conclusion in final detection, subsequent
Research in will further screen specific donkey primer, so as to shorten detection time, simplify detecting step.
1.4 species specificity PCR methods detect six kinds of poultry meat lifes, the sensitivity of cold cuts such as beef or mutton:
The life extracted, cold cuts sample DNA concentration are measured, it is anti-to carry out PCR with the condition groped for 10 times of dilutions of series
Answer, at the same using aseptic double-distilled water be template as negative control, the minimum DNA concentration that method for building up can detect to detect,
Analyze the sensitivity of this method.
By Fig. 7~Figure 12 it is found that for beef, this method limits up to 5 × 10~7 μ g the detection of raw meat, and
5 × 10 are limited to for the detection of cold cuts-2μg；For mutton, this method is limited to 5 × 10 for the detection of raw meat-2μ g, and
5 × 10 are limited to for the detection of cold cuts-1μg；For pork and horseflesh, this method for the detection limit of raw meat be 5 ×
10-3μ g, and be 5 × 10 for the detection of cold cuts limit-2μg；For dog meats and donkey meat, this method is for raw meat and cold cuts
Detection limit be 5 × 10-1Without significant difference between the raw meat of μ g, dog and donkey and the DNA cloning result same concentrations in cold cuts source；
Ox, sheep, pig and horse source property be raw, difference is significant between cold cuts DNA testing result same concentrations, illustrates the PCR of this research foundation
Detection method is applicable not only to raw meat sample detection, while applying also for the detection of cold cuts sample.
Above-described specific embodiment has carried out further the purpose of the present invention, technical scheme and beneficial effects
It is described in detail, it should be understood that being not intended to limit the present invention the foregoing is merely a specific embodiment of the invention
Protection scope, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should all include
Within protection scope of the present invention.
In the present invention, unless stated otherwise, all operations are all carried out by the recommendation step of manufacturer.
Unless otherwise instructed, all technical terms in file all have logical with one skilled in the art of the present invention
Understand identical meaning.
Claims (4)
1. a kind of kit for quickly detecting common livestock meat based on species specificity PCR method, it is characterised in that: the kit
Extract part and detection part including DNA, the DNA extract the reaction of part the following steps are included:
1) 100mg sample is taken, is put into 2mL sterile centrifugation tube, 500 μ L lauryl sodium sulfate lysates and 20 μ L albumen are added
Enzyme K, water-bath is transparent to solution under the conditions of 55 DEG C after mixing, is during which mixed by inversion centrifuge tube frequently；
2) isometric Tris saturated phenol is added, is mixed by inversion rear 12000r/min centrifugation 10min, by supernatant be transferred to it is another from
It is primary to repeat this step operation for heart pipe；
3) chloroform/isoamyl alcohol of the Tris saturated phenol of 0.5 times of volume and the 24:1 of 0.5 times of volume is then added in centrifuge tube,
12000r/min is centrifuged 10min after mixing, draws supernatant to another new centrifuge tube；
4) dehydrated alcohol of the sodium acetate solution of 0.1 times of volume and 4 DEG C of pre-coolings of 2.5 times of volumes is added, has after being mixed by inversion white
Color Precipitation precipitates 2h at -20 DEG C；
5) solution and flocculent deposit are added in the adsorption column being put into collecting pipe, after 12000r/min is centrifuged 5min, are outwelled
Waste liquid in collecting pipe, adsorption column is put into collecting pipe；
6) 70% ethyl alcohol, 500 μ L is added in adsorption column to wash precipitating, 12000r/min is centrifuged 5min and abandons waste liquid, by adsorption column
It is put into collecting pipe, repeats work of drilling；
7) adsorption column being put into collecting pipe, 12000r/min is centrifuged 2min, and waste liquid is abandoned, adsorption column is put and is dried at room temperature,
Thoroughly remove remaining ethyl alcohol；
8) adsorption column is put into clean centrifuge tube, it is hanging that 100 μ L eluent TE are added dropwise, it is placed at room temperature for 12000r/ after 5min
Min is centrifuged 2min, and solution is collected into centrifuge tube；
9) blank control is done with TE, the concentration and purity of extracted DNA, loading is measured with trace dna, determination of protein concentration instrument
Amount is 1 μ L.
2. the kit according to claim 1 for quickly detecting common livestock meat based on species specificity PCR method, feature
It is: the reaction system of the kit are as follows: primer, 10 μ are added in system in 2 × Taq Plus Master Mix, 25 μ L
mol·L-1Each 2 μ L of upstream and downstream primer, 0.5 μ g, dd H of DNA profiling2O supplies system to 50 μ L.
3. the kit according to claim 1 for quickly detecting common livestock meat based on species specificity PCR method, feature
It is: the reaction condition of the kit are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, * DEG C of annealing 30s, 72 DEG C extend
60s, totally 35 recycle；72 DEG C of extension 7min；Wherein, the annealing temperature " * " in the reaction system according to ox, sheep, pig, dog,
The sequence of horse and donkey is respectively 54 DEG C, 65 DEG C, 62 DEG C, 46 DEG C, 67 DEG C and 66 DEG C.
4. the kit according to claim 1 for quickly detecting common livestock meat based on species specificity PCR method, feature
Be: the kit after reaction final amplification using 1% or 1.5% agarose gel electrophoresis and gel at
As instrument is detected.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811022275.XA CN109136330A (en) | 2018-09-04 | 2018-09-04 | The kit of common livestock meat is quickly detected based on species specificity PCR method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811022275.XA CN109136330A (en) | 2018-09-04 | 2018-09-04 | The kit of common livestock meat is quickly detected based on species specificity PCR method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109136330A true CN109136330A (en) | 2019-01-04 |
Family
ID=64826397
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811022275.XA Pending CN109136330A (en) | 2018-09-04 | 2018-09-04 | The kit of common livestock meat is quickly detected based on species specificity PCR method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109136330A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651400A (en) * | 2015-02-13 | 2015-05-27 | 山东大学 | Method for conditional knockout of Lox13 gene of mouse and application |
| CN105985948A (en) * | 2015-02-28 | 2016-10-05 | 天津市农业质量标准与检测技术研究所 | Method for extracting animal muscular tissue DNA efficiently and safely |
| CN107012229A (en) * | 2017-04-24 | 2017-08-04 | 吉林农业科技学院 | Pig derived component quick determination method and kit in food |
-
2018
- 2018-09-04 CN CN201811022275.XA patent/CN109136330A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651400A (en) * | 2015-02-13 | 2015-05-27 | 山东大学 | Method for conditional knockout of Lox13 gene of mouse and application |
| CN105985948A (en) * | 2015-02-28 | 2016-10-05 | 天津市农业质量标准与检测技术研究所 | Method for extracting animal muscular tissue DNA efficiently and safely |
| CN107012229A (en) * | 2017-04-24 | 2017-08-04 | 吉林农业科技学院 | Pig derived component quick determination method and kit in food |
Non-Patent Citations (6)
| Title |
|---|
| 中华人民共和国国家质量监督检验检疫总局 中国国家标准化管理委员会: ""动物源性饲料中狗源性成分定性检测方法 PCR方法"", 《中华人民共和国国家标准 GB/T 21105-2007》 * |
| 刘娜等: ""动物肌肉组织DNA的提取方法及实时荧光定量PCR检测"", 《食品工业科技》 * |
| 徐伟丽等: ""动物肌肉组织基因组DNA两种提取方法比较"", 《食品工业科技》 * |
| 徐瑗聪等: ""猪肉、牛肉和绵羊肉掺伪PCR的检测技术"", 《农业生物技术学报》 * |
| 朱业培等: ""基于基因芯片技术检测 6 种动物源性成分"", 《南京农业大学学报》 * |
| 李通: ""基于分子生物学的肉类鉴定方法研究"", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhao et al. | Alkaline lysis-recombinase polymerase amplification combined with CRISPR/Cas12a assay for the ultrafast visual identification of pork in meat products | |
| CN104774958B (en) | Differentiate the animal derived primer combination of probe thing of donkey, horse, fox, kit and multiple real time fluorescence quantifying PCR detection method | |
| CN104946788A (en) | PCR primer and kit for identifying eight kinds of animal-derived components | |
| Zhao et al. | Direct lysis-multiplex polymerase chain reaction assay for beef fraud substitution with chicken, pork and duck | |
| CN105525022A (en) | Method for identifying squids or highly processed product varieties thereof by virtue of fluorescent quantitative PCR | |
| CN104946790A (en) | PCR method for tracking source identification of origin components of eight kinds of animals | |
| CN105039329A (en) | Dual fluorescent quantitative PCR method for species identification of salmons and highly processed products of salmons | |
| CN105063229B (en) | For detecting quantitative fluorescent PCR specific primer, probe and its kit of sheep derived material in meat products | |
| CN105063028A (en) | SSR (simple sequence repeat) primer group and method for constructing malt fingerprint maps by aid of primer group | |
| CN105671157A (en) | Kit for synchronous detection of animal origin ingredients in meat and meat product and application of kit | |
| CN101962675B (en) | Primer group and kit for detecting origin of meat in food with multiple PCR (Polymerase Chain Reaction) | |
| CN103525908A (en) | Method for rapidly detecting chicken, duck and pig blood components in blood jelly | |
| CN106521016A (en) | Real-time fluorescence PCR detection method for detecting bovine-derived materials in food and feed by use of single-copy nuclear gene | |
| CN103451304B (en) | The primed probe of real-time PCR detection recoon dog derived component and method | |
| CN103114142B (en) | Real-time polymerase chain reaction (PCR) detection primer, kit and detection method for detecting puffer fish ingredients | |
| CN113293216A (en) | Identification method for animal-derived components in salmonidae products based on high-throughput sequencing | |
| CN109136330A (en) | The kit of common livestock meat is quickly detected based on species specificity PCR method | |
| CN105132542B (en) | Method, the primer and probe of broad-leaved yellow wingceltis in redwood are identified using round pcr | |
| CN102559919B (en) | Real-time PCR (Polymerase Chain Reaction) detection method of buffalo components in food and feed | |
| Hu et al. | A simple and rapid DNA extraction method from meat by microneedle patch for detection of adulterated components | |
| CN103397100B (en) | Establishment method of Pinctada martensii MSAP (methylation sensitive amplification polymorphism) technical system | |
| CN102433382B (en) | Real-time fluorescent polymerase chain reaction (PCR) detection method for turkey ingredient in foods and feeds | |
| CN105755116B (en) | Primer and its application with microsatellite marker mutually chain whether Eriocheir sinensis sex premature | |
| CN110343766A (en) | A method of based on calf-derived Cyclospora in QPCR quantitative detection livestock meat | |
| CN102382897B (en) | Real-time fluorescent PCR (Polymerase Chain Reaction) detection method and kit for goose components in food and feedstuff |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190104 |