CN107400729A - A kind of kit and detection method for detecting many animals derived component - Google Patents
A kind of kit and detection method for detecting many animals derived component Download PDFInfo
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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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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The invention provides a kind of kit for detecting many animals derived components, including membrane DNA chip, the membrane DNA chip is made up of support film and the probe being fixed on support film；The probe is the specific probe of detection pig, sheep, yak, donkey and mouse, and for its sequence as shown in Seq No.1 5,5 ' ends of each probe carry amino labeled；Kit also includes the membrane DNA chip for detecting the internal reference probe of reference gene, positive control probe and negative probes, and for its sequence as shown in Seq No.6 8,5 ' ends of each probe carry amino labeled；And 5 ' end the positive oligonucleotides single stranded DNA with biotin labeling, its sequence is as shown in Seq No.9.The kit of the present invention can be detected disposably to whether containing 5 kinds of animal derived materials such as pig, sheep, donkey, yak, mouse in variety classes meat products in large quantities；The testing result of acquisition has the characteristics that special strong, high sensitivity, false positive rate is low, visualization is high and cost is low.
Description
Technical field
The invention belongs to detect product technical field, more particularly to a kind of kit for detecting many animals derived component and
Detection method.
Background technology
In recent years, China's grocery trade enters the gold period of rapid development, and poultry class meat products and its product enrich
The dining table of broad masses of the people, but there is adulterated doping phenomenon repeatedly in meat products.Fox meat, duck pretend to be mutton, the beef Chinese
Fort meat pie is mixed the events such as horseflesh and emerged in an endless stream, and meat products, which mixes pseudo- fake, also turns into the focus and difficult point in food safety Regulation field.
With modern meat products industrial expansion, got over to weigh factor, the in the markets such as meat products taste, production and processing cost benefit
It is processed into come more meat products by the animal derived meat of multiple types, generally requires to carry out many animals simultaneously with a sample
The detection of derived component.
China's foodstuffs animal derived materials examination criteria, carried out using round pcr, real-time fluorescence PCR technology
The detection of animal derived materials target gene, these technologies are only capable of detecting 1 to 3 kind of animal derived index every time, can not meet 3 kinds
Detected while above animal derived materials.Existing detection method is to operating personnel's technology, Laboratory Instruments equipment configratioin requirement
Higher, testing cost is expensive, it is difficult to meet of large quantities, efficient quick examination requirements, can not meet market surpervision, enterprise's raw material
And the active demand that final product quality controls in real time.
The content of the invention
The problems such as detecting the testing cost costliness of many animals derived components simultaneously at present, the present invention provide a kind of detection
The kit of many animals derived component.
To achieve the above object, the present invention adopts the following technical scheme that.
A kind of kit for detecting many animals derived components, including membrane DNA chip, the membrane DNA chip is by support film and is fixed on
Support the probe composition on film；The probe is the specific probe of detection pig, sheep, yak, donkey and mouse, and internal reference probe is positive
Probe and negative probes, for its sequence as shown in Seq No. 1-8,5 ' ends of each probe carry amino labeled.The pig, sheep, yak
The specific probe of ox, donkey and mouse is combined with the chondriogen of each composition.
Alternatively, support that film is nitrocellulose filter, nylon membrane or polypropylene screen；Most preferably nylon membrane.
As optimization, mentioned reagent box also includes detecting the internal reference probe of reference gene, positive control probe and negative probes
Membrane DNA chip, for its sequence as shown in Seq No. 6-8,5 ' ends of each probe carry amino labeled.Internal reference probe in detecting 18S rRNA
Gene.
More optimally, mentioned reagent box also includes 5 ' positive oligonucleotides single stranded DNAs of the end with biotin labeling, its sequence
Row are as shown in Seq No. 9.Positive oligonucleotides single stranded DNA can be specifically bound with positive control probe, in experimentation
Quality control.
As optimization, mentioned reagent box also includes alkali phosphatase enzyme mark Streptavidin, substrate nitrite ion, deactivated
Liquid, hybridization solution, enzyme Incubating Solution.
The substrate nitrite ion includes the 0.15 chloro- 3- indoylphosphates of the bromo- 4- of mg/mL 5-（BCIP）, 0.30 mg/mL chlorine
It is blue to change nitro tetrazole（NBT）, 5 mmol/L MgCl2With 100 mmol/L Tris-HCl, its pH is 9.5, and wherein substrate is
BCIP and NBT.
The liquid that deactivates contains 100 mmol/L NaOH；Hybridization solution contains 2 × SSPE and 0.1wt% lauryl sodium sulfate
（SDS）；Enzyme Incubating Solution contains 2 × SSPE, 0.5wt% SDS；It is 2 × SSPE that enzyme, which is incubated washing lotion, and 2 × SSPE buffer solutions include
0.3 mol/L NaCl, 20 mmol/L NaH2PO4, 20 mmol/L EDTANa2, its pH is 7.4.
Alternatively, mentioned reagent box also includes amplimer pair and the internal reference amplification of pig, sheep, yak, donkey and mouse composition
Primer pair, its sequence is as shown in Seq No. 10-21；The 5' ends of the amplimer centering reverse primer carry biotin mark
Note.
Alternatively, kit also includes auxiliary material, and the auxiliary material includes deoxyribonucleoside triphosphate（dNTP）, uracil
DNA glycosyl enzymes（UNG enzymes）, archaeal dna polymerase（Taq enzyme）With 10 × PCR buffer solutions.
10 × PCR buffer solutions contain 15 mmol/L MgCl2, 100 mmol/L Tris-HCl, 500 mmol/L
KCl, its pH are 8.0-8.3.
Pig, sheep, donkey, yak, the lowest detection of mouse composition are limited to 0.1wt% in mentioned reagent box detection meat products.
Using the method for mentioned reagent box detection many animals derived components, comprise the following steps：
（1）Extract DNA in sample；
（2）With step（1）Middle DNA is that template carries out multiplex PCR；
（3）By step（2）Middle PCR primer hybridizes and developed the color with the membrane DNA chip in kit；
（4）Result is judged：
a）Blank control：PC develops the color, the detection of remaining target spot no signal；
b）Negative control：PC develops the color, and internal reference has signal detection；
c）Positive control：PC develops the color, and internal reference has signal detection, and target spot has signal detection；
Meet being tested to be effective for three above condition, the animal derived materials according to contained by judging colour developing result simultaneously.
Above-mentioned steps（2）The OD of middle DNA solution260/280It is worth for 1.7-1.9.
Above-mentioned steps（3）Middle hybridization step includes deactivating, and hybridizes, and enzyme is incubated operation.
The present invention has advantages below：
While combination of the animal sources composition detection kit of the present invention by providing a variety of primer pairs carries out multiplexed PCR amplification
Carry out asymmetric PCR technology（Asymmetric PCR）To improve detection sensitivity.Asymmetric PCR technology refers to
The forward primer and reverse primer of inequality are used in PCR amplification procedures（Or the forward primer that amplification extension condition is different
And reverse primer）, PCR amplification after produce substantial amounts of single stranded DNA, the single stranded DNA can effectively and be fixed on support film on
Probe hybridizes, so as to improve detection sensitivity.Animal derived materials detection kit provided by the invention is by 5 ' ends with life
The reverse primer or forward primer of thing element flag sequence, which are attached in template and extend amplification, obtains substantial amounts of 5 ' end with biology
The single stranded DNA of element mark, the single stranded DNA of the 5 ' end with biotin labeling are accordingly miscellaneous with probe by base complementrity principle
Hand over, after color development treatment, show respective color, and then obtain testing result.Positive control probe and negative probes can be used for judging
The reliability for the result that develops the color.Responseless hydroxyl on membrane DNA chip is removed by the effect of alkali before hybridization, reduces background to probe
Absorption, reduce ambient interferences.The testing result that the present invention obtains is with special strong, high sensitivity, false positive rate is low, visualizes journey
The features such as degree height and low cost.
Brief description of the drawings
Fig. 1 is membrane DNA chip probe layout schematic diagram；
Fig. 2 is 5 kinds of pig, sheep, yak, donkey, mouse animal derived materials testing results；
Fig. 3 is 16 kinds of interference animal derived materials testing results；
Fig. 4 is inspection pig, sheep, donkey, yak, mouse composition sensitivity the result；
Fig. 5 is the adulterated sensitivity checking of animal derived materials.
Embodiment
With reference to embodiment and accompanying drawing, the present invention will be further described, but the present invention is not limited by following embodiments
System.
It is prepared by the kit of embodiment 1
It is prepared by 1.1 membrane DNA chips
Pig as shown in Seq No. 1-8, sheep, yak, donkey, five kinds of probes of mouse and positive control probe, negative probes and internal reference are visited
Pin is configured to solution of the concentration as 25 μm of ol/L using ultra-pure water, using micro- quantitative specking formula membrane DNA chip point sample instrument, by each nucleosides
Acid probe is distributed in the specific location area on negatively charged nylon membrane DNA chip, the coated probe layout of chip surface such as Fig. 1 institutes
Show, wherein, PC is positive control probe, and NC is negative probes.
1.2 preparation of reagents
Prepare 10 × PCR buffer solutions（P1）, containing 15 mmol/L MgCl2, 100 mmol/L Tris-HCl, 500 mmol/L
KCl, its pH are 8.3.
By the pig as shown in Seq No. 10-21, sheep, yak, donkey, the amplimer pair of mouse and internal reference amplimer to
Multiple PCR primer premixed liquid is made（P2）, wherein every primer concentration is 2 μm of ol/L.
Prepare multiplex PCR premixed liquid（P3）：Each 0.4 mmol/L of dATP, dCTP, dGTP, dTTP and dUTP, ura DNA
Glycosyl enzyme（UNG enzymes）0.2 U/ μ L, archaeal dna polymerase（Taq enzyme）0.4 U/μL.
Positive oligonucleotides single stranded DNA is formulated as to 10 μ Lmol/L solution（PC）.
Preparation is deactivated liquid（H1）, containing 100 mmol/L NaOH.
Prepare 2 × SSPE buffer solutions（H4）, containing 0.3 mol/L NaCl, 20 mmol/L NaH2PO4, 20 mmol/L
EDTA·Na2, its pH is 7.4.
Preparing hybrid liquid（H2）, SDS is formulated as 0.1wt% concentration using 2 × SSPE buffer solutions as solvent.
Prepare enzyme Incubating Solution（H3）, SDS is formulated as 0.5wt% concentration using 2 × SSPE buffer solutions as solvent.
Prepare substrate nitrite ion（H5）, containing the 0.15 chloro- 3- indoylphosphates of the bromo- 4- of mg/mL 5-（BCIP）, 0.30 mg/mL
NBT（NBT）, 5 mmol/L MgCl2With 100 mmol/L Tris-HCl, its pH is 9.5.
The kit of embodiment 2 detects the specificity and sensitivity determination of 5 kinds of animal derived components
DNA extraction purification in 2.1 experiments
The sample containing pig, sheep, donkey, yak, mouse composition is subjected to DNA extraction purification after pre-treatment respectively, and utilizes core
Acid albumin analyzer detects its concentration and quality OD260/OD280, when the value is between 1.7-1.9, for multiplexed PCR amplification.With
The DNA of corresponding animal sources species extraction is positive control, using the known species DNA without the animal sources as negative control, to go out
Bacterium water is blank control.
2.2 multiplex PCR
Using 2.1 DNA obtained as template, multiplex PCR is carried out, system is as shown in table 1, and response parameter is as follows, obtains amplified production：
95 DEG C first denaturation 10min, then procedure below circulation 35 times：95 DEG C 30s-55 DEG C 30s-72 DEG C of 30s, last 72
DEG C extension 10min.
The multi-PRC reaction system of table 1
| Reaction solution forms | Detection reaction | Positive quality control | Negative Quality Control | Blank Quality Control |
| P1 | 5µL | 5µL | 5µL | 5µL |
| P2 | 25µL | 25µL | 25µL | 25µL |
| P3 | 5µL | 5µL | 5µL | 5µL |
| DNA profiling | 200ng | / | / | / |
| The known NA of components D containing animal sources | / | 200ng | / | / |
| Without animal sources components D NA | / | / | 200ng | / |
| Nucleic acid extraction blank | / | / | / | 20µL |
| Nuclease free aqua sterilisa | Add to 50 μ L | Add to 50 μ L | Add to 50 μ L | Add to 50 μ L |
2.3 hybridization colour developings
20 μ L pcr amplification products are added into 1 μ L PC liquid and 979 μ L H2 liquid, are formulated as hybridization system liquid.By 0.5 μ L concentration
999.5 μ L H2 liquid are added for 0.1mg/mL alkali phosphatase enzyme mark solution of streptavidin, are configured to enzyme incubation system liquid.
Hybridize, on the automatic hybridizing instrument of cleaning response function with automatic by the flow of table 2, completion is deactivated, hybridized, clearly
Wash, enzyme be incubated, colour developing etc. step, can also manually complete.
Table 2 hybridizes color operation step
| Program name | Reagent name | Temperature（℃） | Time（min） |
| Deactivate | H1 | 37 | 8 |
| Deactivate cleaning | H2 | 60 | 5 |
| Hybridization | Hybridization system liquid | 42 | 45 |
| Hybridization cleaning 1 | H3 | 52 | 5 |
| Hybridization cleaning 2 | H3 | 52 | 5 |
| Enzyme is incubated | Enzyme incubation system liquid | 42 | 30 |
| It is incubated cleaning 1 | H3 | 42 | 5 |
| It is incubated cleaning 2 | H3 | 42 | 5 |
| It is incubated cleaning 3 | H4 | 37 | 5 |
| It is incubated cleaning 4 | H4 | 37 | 5 |
| Colour developing | H5 | 37 | 15 |
| Colour developing cleaning 1 | Water | 37 | 5 |
| Colour developing cleaning 2 | Water | 37 | 5 |
2.4 results judge
a）Blank control：PC develops the color, the detection of remaining target spot no signal；
b）Negative control：PC develops the color, and internal reference has signal detection；
c）Positive control：PC develops the color, and internal reference has signal detection, and target spot has signal detection；
Meet being tested to be effective for three above condition simultaneously, colour developing result is as shown in Figure 2：Pig, sheep, donkey, yak, mouse five are dynamic greatly
Material resource composition detection, only positive control hybridization signal detects, and negative and blank control does not expand, shows specificity experiments
Testing result is good.Pcr amplification product is subjected to cloning and sequencing respectively, sequencing result obtains DNA sequence dna information and respective five
Kind NCBI sequences are completely the same, it was demonstrated that detection method is solid.
2.5 specificity verification
According to step described in above-mentioned 2.1-2.4, to deer, buffalo, ermine, fox, quail, chicken, goose, racoon dog, horse, duck, rabbit, cavy, bamboo rat,
Cat, rat, ox equal samples carry out DNA extractions, using the DNA of corresponding animal sources species extraction as positive control, are free of with known
The species DNA of the animal sources is negative control, and the specificity of kit is verified using aqua sterilisa as blank control, according to
Step described in 2.1-2.4 detects, as a result as shown in Figure 3：The membrane DNA chip above species and specific color nothing but, it is no intersect it is anti-
Should.
2.6 sensitivity are verified
Extract pig, sheep, donkey, yak, the DNA of mouse, gradient dilution, be separately added into multi-PRC reaction system 10ng, 1ng,
0.1ng, 0.01ng genomic DNA, enter performing PCR amplification and hybridization.Testing result such as Fig. 4：Pig derived component, yak derived component
Detection method sensitivity is up to 0.01ng, and five kinds of animal derived materials detection method sensitivity are up to 0.1ng, therefore, by five
Kind animal derived materials detection method lower sensitivity limit is set to 0.1ng.
2.7 adulterated sensitivity checkings
Adulterated sensitivity checkings are carried out to different five kinds of animal derived components using chicken DNA, prepare respectively five kinds of animals 10%, 1%,
The sample genomic DNA of 0.1% 3 gradient, enter performing PCR amplification and hybridization.Testing result such as Fig. 5：Under five kinds of composition sensitivity
Limit can reach 0.1%.
Composition in the kit of embodiment 3 detection meat products
Collect the market of farm produce, supermarket, 173 parts of market samples in the source such as network, sample type include fresh meat, skewer, burger,
Prefabricated meat, jerky, ham sausage etc., using the method described in 2.1-2.4, the sample gathered is detected, the results detailed in Table
3。
The sample animal derived materials assay of table 3
As a result finding, it is higher that fresh meat sample detection result and its label for labelling meet sex ratio, and about 85%, yak meat, donkey
Fakement phenomena be present in meat, mutton；10 parts of labels only mark the skewer sample of sheep composition, detect sample ingredient, but have 2 parts of detection pigs
Composition, testing result are 80% with its label for labelling accordance；In 17 parts of burger samples, label for labelling has chicken, pig, sheep, duck respectively
Deng animal derived materials, there are 16 parts of detection pig derived components, wherein the product proportion for meeting label for labelling is about 88%, 2 parts are only marked
The burger of sheep composition is noted, sheep derived material is detected, 1 part of detection pig derived component, adulterated situation be present；31 parts of ham sausage samples
In, label for labelling has the animal derived materials such as chicken, pig, sheep, duck respectively, detect pig derived component, wherein 6 parts only mark chicken into
Point sample detect pig source property into adulterated situation being present；In 15 parts of mutton roll samples, sheep derived material is detected, wherein 3 parts
Sample detects pig derived component, adulterated situation be present；In 45 parts of jerky samples, 5 portions of dried porks detect pig derived component, and 100%
It is qualified, 40 parts of dried yak beef samples, only 6 parts detection yak derived components, 2 parts detection pig derived components, 32 parts do not detect pig,
5 kinds of animal derived materials such as sheep, donkey, yak, mouse, adulteration are the most serious.
Whether this kit can be disposably in large quantities to containing pig, sheep, donkey, yak, mouse etc. 5 in variety classes meat products
Kind animal derived materials are detected.
<110>Food and medicine Inspection Research institute of Shandong Province
<120>A kind of kit and detection method for detecting many animals derived component
<130> 20170925
<160> 21
<170> PatentIn version 3.5
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Claims (9)
1. a kind of kit for detecting many animals derived components, including membrane DNA chip, the membrane DNA chip is by support film and is fixed on branch
Hold the probe composition on film；The probe is detection pig, sheep, yak, the specific probe of donkey and mouse, its sequence such as Seq No.
Shown in 1-5,5 ' ends of each probe carry amino labeled.
2. kit according to claim 1, it is characterised in that support film is nitrocellulose filter, nylon membrane or poly- third
Alkene film.
3. kit according to claim 1, it is characterised in that also include internal reference probe, the positive of detection reference gene
The membrane DNA chip of probe and negative probes, for its sequence as shown in Seq No. 6-8,5 ' ends of each probe carry amino labeled.
4. kit according to claim 3, it is characterised in that also including 5 ' positive few nucleosides of the end with biotin labeling
Sour single stranded DNA, its sequence is as shown in Seq No. 9.
5. kit according to claim 1, it is characterised in that also including alkali phosphatase enzyme mark Streptavidin, bottom
Thing nitrite ion, deactivate liquid, hybridization solution, enzyme Incubating Solution；The liquid that deactivates is 100 mmol/L NaOH.
6. kit according to claim 1, it is characterised in that also include the expansion of pig, sheep, yak, donkey and mouse composition
Increase primer pair and internal reference amplimer pair, its sequence is as shown in Seq No. 10-21；The amplimer centering reverse primer
5' ends carry biotin labeling.
7. kit according to claim 6, it is characterised in that also include deoxyribose core including auxiliary material, the auxiliary material
Guanosine triphosphate（dNTP）, ura DNA glycosyl enzyme（UNG enzymes）, archaeal dna polymerase（Taq enzyme）With 10 × PCR buffer solutions.
A kind of 8. method using the kit detection many animals derived components as described in claim 1-7 is any, it is characterised in that
Comprise the following steps：
（1）Extract DNA in sample；
（2）With step（1）Middle DNA is that template carries out multiplex PCR；
（3）By step（2）Middle PCR primer hybridizes and developed the color with the membrane DNA chip in kit；
（4）Result is judged：
a）Blank control：PC develops the color, the detection of remaining target spot no signal；
b）Negative control：PC develops the color, and internal reference has signal detection；
c）Positive control：PC develops the color, and internal reference has signal detection, and target spot has signal detection；
Meet being tested to be effective for three above condition, the animal derived materials according to contained by judging colour developing result simultaneously.
9. according to the method for claim 8, it is characterised in that step（3）Middle hybridization step includes deactivating, and hybridizes, enzyme
It is incubated operation.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107460248A (en) * | 2017-09-27 | 2017-12-12 | 黑龙江珍宝岛药业股份有限公司 | A kind of primer combination for detecting animal derived components and application, detection kit |
| CN107460248B (en) * | 2017-09-27 | 2021-01-12 | 黑龙江珍宝岛药业股份有限公司 | Primer combination for detecting animal-derived components, application and detection kit |
| CN107858443A (en) * | 2017-12-08 | 2018-03-30 | 锡林郭勒职业学院 | Quantitatively detect primer, probe and the kit of sheep, ox and pig source property in meat products |
| CN107858443B (en) * | 2017-12-08 | 2020-08-04 | 锡林郭勒职业学院 | Primer, probe and kit for quantitatively detecting sources of sheep, cattle and pigs in meat products |
| CN108179201A (en) * | 2018-03-22 | 2018-06-19 | 四川华汉三创生物科技有限公司 | A kind of kit for detecting animal derived materials |
| CN108220457A (en) * | 2018-03-22 | 2018-06-29 | 四川华汉三创生物科技有限公司 | A kind of Nucleic acid combinations and its application for animal derived materials detection |
| CN108531611A (en) * | 2018-03-22 | 2018-09-14 | 四川华汉三创生物科技有限公司 | A method of detection animal derived materials |
| CN110106258A (en) * | 2019-05-14 | 2019-08-09 | 西南民族大学 | A kind of yak meat identification kit |
| CN110106258B (en) * | 2019-05-14 | 2022-09-23 | 西南民族大学 | Yak meat identification kit |
| CN110452994A (en) * | 2019-08-26 | 2019-11-15 | 国家卫生健康委科学技术研究所 | Primer pair, probe and method for ten kinds of animal source components of synchronous detection |
| CN110452994B (en) * | 2019-08-26 | 2022-06-07 | 国家卫生健康委科学技术研究所 | Primer pair, probe and method for synchronously detecting ten animal source components |
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