CN109825606A - Pig derived components quick detection kit and its application in a kind of food - Google Patents
Pig derived components quick detection kit and its application in a kind of food Download PDFInfo
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Abstract
The present invention provides pig derived components quick detection kit in a kind of food, is related to biological species identification technology field.The present invention filters out the general internal standard Gene A ctb in a pig source first, and nucleotide sequence is located on the 8th chromosome as shown in SEQ ID NO.1, and copy number is constant in pig species, and allelic variation is not present, and can be used as the target gene in identification pig source.PCR amplification primer is devised by target sequence of the gene, is expanded together with PCR reaction solution, PCR reaction product is detected for platinum palladium nano-particles immuno-chromatographic test paper strip.PCR reaction bonded constitutes quick detection kit based on the immuno-chromatographic test paper strip detection of platinum palladium nano-particles, can rapidly and sensitively detect the pig derived components in food, detection sensitivity is up to 0.8% (w/w).Kit application method of the present invention is simple, low in cost, and reaction result is easy to observe, and specificity is good, is highly suitable for live real-time detection.
Description
Technical field
The present invention relates to biological species identification technology field, more particularly in a kind of detection food pig derived components it is poly-
Polymerase chain reaction technology (PCR) combines the quick detection kit of platinum palladium nano-particles immuno-chromatographic test paper strip detection.
Background technique
With rapid development of economy, the raising of living standards of the people, demand of China resident to meat product increases year by year
Add.Although many country's clear stipulaties mark type, the source of meat with requiring food labelling true, unambiguous, forbid adulterated behavior,
But still there is the event of many meat adulterations in the market, such as in order to reduce cost, pork, sheep are mixed in donkey fire
Beef or pork are mixed in meat, adulterate other meats etc. behavior in sausage.Currently, PCR method is that detection food adulteration is simple
Effective method, it is easy to operate, the time is short, detection accuracy is high.Therefore, it is an object of the present invention to provide pig sources in a kind of food
The rapid sensitive detection kit of ingredient.
Currently, internal standard gene is widely used for identifying food adulteration, but how to filter out suitable internal standard base
Because being particularly important.Current meat products detection of adulterations technology both domestic and external is specifically drawn for the gene design on mitochondria
Object carries out real-time fluorescence quantitative PCR amplification, and since chondriogen is multi-copy gene, detection sensitivity is high, but simultaneously
There is puzzlement when distinguishing as unconscious cross contamination caused by processes and the conscious illegal addition such as selling, transporting.
In addition, the concentration of high copy number mitochondrial DNA can not be corresponding with the concentration of genomic DNA, therefore essence can not be carried out to sample
True quantitative analysis, simultaneously because chondriogen homology is high, it is difficult to realize qualitative detection by regular-PCR.Therefore, the party
Method can only realize that the screening of meat adulteration identifies by quantitative fluorescent PCR.To identify the cross contamination unintentionally of low concentration and having
The illegal addition of meaning and clearly adulterated ratio realization rapid screening, then want the low copy gene on selective staining body as meat internal standard
Quasi- gene.
PCR method is current using more extensive method, and principle is to utilize a kind of archaeal dna polymerase and a pair of and template
The specific primer of complementary pairing, by it is a series of denaturation, annealing, extend and etc., keep template DNA total amount ever-increasing
Process.
Immune chromatography test paper is to realize a kind of high-precision, the low price, easy-operating inspection that quickly detect and derive
Survey method is usually made of sample pad, bonding pad, detection line, nature controlling line, nitrocellulose filter, water absorption pad, several parts of backboard.
Testing principle is similar with enzyme linked immunosorbent assay (ELISA), but detection process is more simple, portable, easily operated.Immunity-chromatography test
Paper has sandwich method, competition law and 3 kinds of indirect method.Platinum palladium nano-particles immuno-chromatographic test paper strip belongs to sandwich method, principle be by
The specific antibody of test substance is linked on nano particle and in the detection line of Sidestream chromatography sensor, when nano particle-
After antibody complex and antigen binding, which combines with antibody coated in detection line again, forms sandwich knot
Structure.Visual qualitative detection is realized by observation detection line and the variation for controlling line color.
Platinum palladium nano-particles are exactly that the second metallic element palladium is introduced in platinum nano catalyst, i.e., wrap on nano-platinum particle
One layer of palladium is wrapped up in, being formed has spherical shell structure platinum palladium bimetal granule.Platinum palladium nano-particles have superior class horseradish peroxidase
Enzymatic activity, can be in H2O2In the presence of so that TMB solution is shown apparent blue.Therefore, the present invention is by platinum palladium nanometer
Particle is applied on immune chromatography test paper, to play the role of enhancing detection line, greatly improves detection sensitivity.
The present invention combines specific PCR technology with the detection of platinum palladium nano-particles immuno-chromatographic test paper strip, provides one
The rapid sensitive detection kit of pig derived components in kind food.For the present invention without complicated instrument, the time is short, easy to operate, spirit
Sensitivity is high, it is only necessary to which a regular-PCR instrument can meet the needs of detection.
Summary of the invention
The object of the present invention is to provide the general internal standard genes in pig source for detecting pig derived components in food.
Another object of the present invention is to provide a kind of in high sensitivity, high specific, detection food easy to operate
The quick detection kit of the specific PCR combination platinum palladium nano-particles immuno-chromatographic test paper strip detection of pig derived components.
It is a kind of for detecting the gene of pig derived components in food, be internal standard Gene A ctb, there is SEQ ID NO.1 institute
The sequence shown.
The present invention provides application of the above-mentioned internal standard Gene A ctb in detection pig derived components.
The application that the present invention provides above-mentioned internal standard Gene A ctb in food in the identification of pig derived components.
The Specific PCR primers combination that the present invention provides a kind of for detecting above-mentioned internal standard Gene A ctb, including with
Lower 2 primers:
F:5 '-GAGATGGCGAAAGGAAACG-3 ' (SEQ ID NO.2)；
R:5 '-CCAAGTGGAACGGGAAGG-3 ' (SEQ ID NO.2)；
Wherein 5 ' end labels biotin (Biotin) of upstream primer F, 5 ' the end mark fluorescent elements of downstream primer R
(FITC)。
The present invention provides application of the above-mentioned Specific PCR primers combination in the identification of pig derived components.
The present invention provides the combinations of above-mentioned Specific PCR primers in preparation pig derived components detection kit or detection reagent
Application.
Further, the present invention provide it is a kind of containing above-mentioned Specific PCR primers combination detection pig derived components it is quick
Detection kit.
The present invention provides a kind of method for detecting pig derived components in food, comprising the following steps:
(1) sample to be tested extracts DNA；
(2) it to extract DNA as template, is combined using the Specific PCR primers and carries out PCR detection；
(3) result judges: carrying out result judgement using platinum palladium nano-particles immuno-chromatographic test paper strip.Detect T line and Quality Control C
Line has blue bands, contains target gene；Quality Control C line has blue bands, and detects T line without band, does not contain target gene.
In the above method, step (2) the PCR detection, the concrete configuration of 25 μ L PCR detection architectures are as follows: 10x
Reaction buffer, 0.4mM dNTP, 0.2 μM of primers F, 0.2 μM of primer R, 2 U Taq PCR polymerase.
In the above method, PCR detects reaction condition are as follows: 95 DEG C of 5min；95 DEG C of 30s, 53 DEG C of 30s, 72 DEG C of 30s, totally 30
Circulation；72℃5min.
In the above method, platinum palladium nano-particles immuno-chromatographic test paper strip described in step (3) includes p-wire and nature controlling line,
P-wire is marked with FITC antibody, and nature controlling line is marked with biotin secondary antibody, in conjunction with being lined with platinum palladium nano-particles-biotin antibody mark
Remember object.
The present invention filters out pig source internal standard gene on chromosome for the first time.The present invention verifies internal standard with multiple breeding pigs
Gene, it was demonstrated that the internal standard gene is stablized, and allelic variation is not present.The selection of internal standard gene generally requires copy number low
And stablize, general animal internal standard gene often selects on mitochondria, and such reference gene copy number is more, is not easy to quantitative.
The present invention selects the gene on the 8th chromosome as internal standard gene, and copy number is low, is easy to quantitative, compared on mitochondria
Gene, mutation rate is lower.
Fig. 1 is immune chromatography test paper schematic diagram.The present invention is reacted using PCR, designs two primers in pig specificity
Standard gene target sequence carries out gene magnification.Then PCR reaction product is detected with platinum palladium nano-particles immuno-chromatographic test paper strip.Platinum
Palladium nano-particles immuno-chromatographic test paper strip p-wire (TL) and nature controlling line (CL) are marked with FITC antibody and biotin secondary antibody respectively,
In conjunction with being lined with platinum palladium nano-particles-biotin antibody marker.
When PCR reaction product is added drop-wise to test strips sample pad, due to capillarity, product can be successively by combining
Pad, detection line (TL), nature controlling line (CL), due to the specific binding effect of " Ag-Ab " and the catalysis of platinum palladium nano-particles
Effect, when PCR reaction product is positive, p-wire (TL) and nature controlling line (CL) have blue bands；When reaction product is negative,
Then only nature controlling line (CL) has blue bands.
Immune chromatography test paper is basic structure with " platinum palladium nano-particles-antibody " compound and " Ag-Ab " identification system
Three-layer sandwich structure is built.In the detection process of a standard, the sample containing pig derived components and system buffer are mixed
It is added in sample pad, solution can move upward and to bonding pad with Sidestream chromatography test paper under the action of capillary force.It is tying
It closes on pad, the freeze-draw method containing pig source is based on the antibody in " platinum palladium nano-particles-antibody " compound of dissolution
The immune response of " Ag-Ab " forms the new compound of " platinum palladium nano-particles-antibody " and antigen binding.This is compound later
Object can be continued to move up along immune chromatography test paper, the corresponding antibodies when it reaches detection line, in PCR product and detection line
Second of immune response for being based on " Ag-Ab " occurs.Therefore, the compound containing platinum palladium nano-particles will be crawled simultaneously
It is piled up in detection line, generates a characteristic black stripe." platinum palladium nano-particles-antibody " compound meeting excessive later
It is migrated towards nature controlling line, after it reaches nature controlling line, the antibody in " platinum palladium nano-particles-antibody " functional probe can be by nature controlling line
Upper sheep anti-mouse igg capture, forms the characteristic black stripe of Article 2.Because platinum palladium nano-particles also have class horseradish peroxidase
Enzymatic activity in detection line or accuses that the platinum palladium nano-particles of line accumulation can be with horseradish peroxidase substrate and H2O2Reaction produces
The raw blue non-solubility product of specificity, promotes detection line signal strength to play, and then promote detection sensitivity.
Platinum palladium nano-particles immuno-chromatographic test paper strip is the specific binding by antigen and antibody, therefore it is with high
Sensitivity.The present invention by PCR by being reacted the mixing of the quality such as pork minced meat and non-pork minced meat 5 times of gradients of progress
It is detected in conjunction with platinum palladium nano-particles immuno-chromatographic test paper strip to probe into the sensitivity of this detection method.The results show that the present invention examines
The detectable limit of test agent box is 0.8% (w/w).
Based on present invention determine that for detecting the internal standard Gene A ctb of pig derived components in food, the present invention devises use
In the PCR primer combination for detecting the gene, can detect using PCR reaction bonded platinum palladium nano-particles immuno-chromatographic test paper strip to be measured
Whether pig derived components are had in sample, this rapid reaction is time-consuming few, and specificity is good, and high sensitivity is easy to operate, does not need profession
Personnel's operation, is as a result easy to observe, and is very suitable for the use of base's food supervision and inspection.
Detailed description of the invention
Fig. 1 is immune chromatography test paper schematic diagram；
Fig. 2 is specific PCR amplification of the pig specificity internal standard gene in 16 kinds of animals, and swimming lane 1 is that the PCR of pig is produced
Object, 1: pig；2: ox；3: sheep；4: goat: 5: chicken；6: deer；7: duck: 8: goose；9: horse；10: donkey；11: yak；12: buffalo；13:
Ermine；14: camel；15: fish；16: rat；17: negative；M:Maker DL2000；
Fig. 3 is the positive judgement with negative findings in the detection of pig source PCR product platinum palladium nano-particles immuno-chromatographic test paper strip；
The p-wire (TL) and nature controlling line (CL) of sample 1 have blue bands then to prove positive sample, and sample 2 only has nature controlling line (CL)
Having blue bands is then negative sample；
Fig. 4 is that the detection sensitivity of PCR- platinum palladium nano-particles immuno-chromatographic test paper strip is tested；1: 0 times of gradient of mixing, i.e.,
It is the 100% of original quality；2: 5 times of gradient of mixing, as the 20% of original quality；3: 25 times of gradient of mixing, as original matter
Amount 4%；4: 125 times of gradient of mixing, as original quality 0.8%；5: 625 times of gradient of mixing, as original quality 0.16%；6:
It is negative.
Specific embodiment
Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case where spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to
Range.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.
Pig (Sus scrofa), ox (Bos taurus), sheep (Ovis aries), common family chicken (Gallus gallus),
Pheasant (Phasianuscolchicus), turkey (Meleagris gallopavo), Gallus domesticlus brisson (Gallus domesticus
Brisson), duck (Anas platyrhynchos), goose (Goose calicivirus), dog (Canis lupus
Familiaris), rabbit (Oryctolagus cuniculus), yak (Bos mutus), yellow croaker (Pseudosciaena
Polyactis it) is bought for supermarket.Horse (Equus caballus), donkey (Equus asinus) are the purchase of Beijing market of farm produce.Always
Mouse (Mus musculus) is provided by China Agricultural University's food safety and Molecular Biology Lab.Buffalo (Bubalus
Bubalis), ermine (Martes zibellina), camel (Camelus ferus), deer (Cervus) are entered and left the border by Tianjin and are examined
Doctor Li Zongmeng of office provides.
The screening of the general internal standard Gene A ctb in 1 pig source of embodiment
By the gene information in search GenBank about pig, target gene group is downloaded from NCBI, and saves as
" .FASTA " format.Analyzed for the full-length genome information of pig, using BLAST and DNAMAN Version4.0 software into
Row homology analysis filters out Actb gene, and it is one of actin cytoskeleton which, which is located on the 8th chromosome,.By 20
Kind meat (is common family chicken (Gallus gallus), pheasant (Phasianuscolchicus), turkey (Meleagris respectively
Gallopavo), Gallus domesticlus brisson (Gallus domesticus brisson), pig (Sus scrofa), ox (Bos taurus), sheep
(Ovis aries), duck (Anas platyrhynchos), goose (Goose calicivirus), dog (Canis lupus
Familiaris), rabbit (Oryctolagus cuniculus), yak (Bos mutus), yellow croaker (Pseudosciaena
Polyactis), horse (Equus caballus), donkey (Equus asinus), mouse (Mus musculus), buffalo
(Bubalus bubalis), ermine (Martes zibellina), camel (Camelus ferus), deer (Cervus)) Actb
Channel genes DNAMAN Version4.0, carries out the analysis of multisequencing specificity, and sequence alignment result saves as " .seq " format.
The high segment of selection specificity carries out BLAST analysis again, searches sequence homology and specificity in database.Finally by sequence
Column are integrated, and determine that final specific targets gene β-Actin (Actb) gene can be used as internal standard gene.The Actb
The nucleotide sequence of segment is as shown in SEQ ID NO.1.
The foundation of 2 pig derived components PCR detection method of embodiment
PCR primer is designed for the Actb gene that embodiment 1 determines using primer premier5.0 software design, on
5 ' end labels biotin (Biotin) of primers F are swum, 5 ' the end mark fluorescents of downstream primer R are plain (FITC).
It is shown in Table 1.
1 PCR primer sequence of table
Pig sample, 25 μ L of reaction system, including 10x reaction buffer, 0.4mM are quickly detected using PCR
DNTP, 0.2 μM of primers F, 0.2 μM of primer R, 2 U Taq PCR polymerase.Response procedures are 95 DEG C of 5min；95℃30s,
53 DEG C of 30s, 72 DEG C of 30s, totally 30 recycle；72℃5min.After amplification, product is carried out using 2% agarose gel electrophoresis
Determine specific band proof occur and expand successfully, contain target gene.As a result as shown in Fig. 2, pig specificity internal standard gene
PCR amplification in 16 kinds of animals, swimming lane 1 be pig PCR product, 1: pig；2: ox；3: sheep；4: goat: 5: chicken；6: deer；7:
Duck: 8: goose；9: horse；10: donkey；11: yak；12: buffalo；13: ermine；14: camel；15: fish；16: rat；17: negative；M:
Maker DL2000.Only there is bright band in pig sample, shows that Actb gene and corresponding PCR system can be used for porcine specy
Quick detection.
The foundation of 3 pig derived components PCR product platinum palladium nano-particles immuno-chromatographic test paper strip detection method of embodiment
Platinum palladium nano-particles labelled antibody is prepared using sandwich method, is stored for future use at 4 DEG C after preparing.Respectively by FITC
Antibody is diluted to optium concentration with buffer.P-wire (TL) between nature controlling line (CL) at a distance from be 4.5mm, by 1.0 μ L/cm divide
It is not sprayed on NC film.It will be spare after 37 DEG C of NC film sprayed drying overnight.Test strips are cut into 3.8mm wide.
The sample in platinum palladium nano-particles immuno-chromatographic test paper strip is added dropwise after PCR reaction product and buffer are sufficiently mixed
On pad, mixed liquor passes through bonding pad and NC film under capillary power at this time, and continues to move to water absorption pad direction, after 3min i.e.
Observable testing result.As shown in figure 3, the p-wire (TL) of sample 1 and nature controlling line (CL) have blue bands then to prove sun
Property sample, sample 2 only has nature controlling line (CL), and to have blue bands be then negative sample.
Platinum palladium nano-particles immuno-chromatographic test paper strip is the specific binding by antigen and antibody, therefore it is with high
Sensitivity.By the way that pork minced meat is carried out the quality such as 5 times of gradients with non-pork minced meat (ox, sheep, mouse etc. mix minced meat)
Mixing, carry out PCR reaction, then combine with platinum palladium nano-particles immuno-chromatographic test paper strip immune to probe into platinum palladium nano-particles
The sensitivity of chromatograph test strip detection method.As a result as shown in figure 4,1: 0 times of gradient of mixing, as the 100% of original quality；2:
5 times of gradient of mixing, as the 20% of original quality；3: 25 times of gradient of mixing, as original quality 4%；4: mixing gradient 125
Times, as original quality 0.8%；5: 625 times of gradient of mixing, as original quality 0.16%；6: negative.When mixing gradient is
125 times (mixing minced meat quality is 125 times of pork minced meat quality) when being the 0.8% of initial mass, still can occur very
Light test strip.It is initial mass when mixing gradient is 625 times (mixing 625 times that minced meat quality is pork minced meat quality)
0.1% when, be nearly no detectable TL line, therefore the detection of detection kit of the present invention is limited to 0.8% (w/w).
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, several improvements and modifications can also be made, these improvements and modifications
Also it should be regarded as protection scope of the present invention.
Sequence table
<110>China Agricultural University
<120>pig derived components quick detection kit and its application in a kind of food
<130> MP1907452Z
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 561
<212> DNA
<213>pig (Sus scrofa)
<400> 1
gcacttttgg gtcagagatg gcgaaaggaa acgccgtgca aagcagaaaa cgccagcaac 60
tccccggcta ttttccagcc ctgcaacggg ctgtgggcgc cgagaggctc agggcatctc 120
tgccaggtgc cccccaactg tgcggaggag cagcgctctg ttcgcgggca ccctcggtac 180
caccgccagc ccttctagcg agtggcgcac ggaatcgcaa gcccagacac cctactcggt 240
gctgccccac gggctctcag ctgactcgga acagtagatg ctttgttcca gacaatcccg 300
gaggcagctc ggagcgcagc gcccagctgt gcccagctgt gcccagccgt gcgccataag 360
attcgcgccc agggcccagc ggtgcgccgc cgacgccaac tcaccccagc tccggttgca 420
ccacccgaat gtaagtgtaa catcgcctca ggatgatttc ctgaaaatcg acagtgctgc 480
ccttcccgtt ccacttgggc gaccggcgcc ccatcaggat tccgaccacc accgccacgg 540
tcaccagcac caggagaccc a 561
<210> 2
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gagatggcga aaggaaacg 19
<210> 3
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ccaagtggaa cgggaagg 18
Claims (10)
1. it is a kind of for detecting the gene of pig derived components in food, it is internal standard gene, there is sequence shown in SEQ ID NO.1
Column.
2. application of the gene described in claim 1 in detection pig derived components.
3. the Specific PCR primers combination for detecting gene described in claim 1, including following 2 primers:
F:5 '-GAGATGGCGAAAGGAAACG-3 ' (SEQ ID NO.2)；
R:5 '-CCAAGTGGAACGGGAAGG-3 ' (SEQ ID NO.3)；
5 ' end mark fluorescents of wherein 5 ' end labels biotin (Biotin) of upstream primer F, downstream primer R are plain (FITC).
4. application of the Specific PCR primers combination as claimed in claim 3 in the identification of pig derived components.
5. Specific PCR primers combination as claimed in claim 3 is in preparation pig derived components detection kit or detection reagent
Using.
6. a kind of quick detection kit of the detection pig derived components containing the combination of Specific PCR primers described in claim 3.
7. the rapid detection method of pig derived components in a kind of detection food, which comprises the following steps:
(1) sample to be tested extracts DNA；
(2) it to extract DNA as template, is combined using Specific PCR primers described in claim 3 and carries out PCR detection；
(3) result judges: carrying out result judgement using platinum palladium nano-particles immuno-chromatographic test paper strip, detects T line and Quality Control C line is equal
There are blue bands, contains target gene；Quality Control C line has blue bands, and detects T line without band, does not contain target gene.
8. the method for claim 7, which is characterized in that step (2) the PCR detection, 25 μ L PCR detection architectures
Concrete configuration are as follows: 10 × reaction buffer, 0.4mM dNTP, 0.2 μM of primers F, 0.2 μM of primer R, 2U Taq PCR
polymerase。
9. method as claimed in claim 7 or 8, which is characterized in that PCR detects reaction condition are as follows: 95 DEG C of 5min；95℃30s,
53 DEG C of 30s, 72 DEG C of 30s, totally 30 recycle；72℃5min.
10. method as claimed in claim 7 or 8, which is characterized in that platinum palladium nano-particles immunochromatography described in step (3)
Test strips include p-wire and nature controlling line, and p-wire is marked with FITC antibody, and nature controlling line is marked with biotin secondary antibody, in conjunction with being lined with
Platinum palladium nano-particles-biotin antibody marker.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173558A (en) * | 2013-03-26 | 2013-06-26 | 四川川娇生态猪业股份有限公司 | Identification or prediction method of pork quality |
| 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 |
| CN108220450A (en) * | 2016-12-20 | 2018-06-29 | 河南广电计量检测有限公司 | The identification method and identification kit of animal derived materials in meat products |
| CN108531547A (en) * | 2018-03-29 | 2018-09-14 | 杭州泰熙生物技术有限公司 | A kind of detection method for false meat monitoring |
-
2019
- 2019-04-11 CN CN201910289636.5A patent/CN109825606B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173558A (en) * | 2013-03-26 | 2013-06-26 | 四川川娇生态猪业股份有限公司 | Identification or prediction method of pork quality |
| CN108220450A (en) * | 2016-12-20 | 2018-06-29 | 河南广电计量检测有限公司 | The identification method and identification kit of animal derived materials in meat products |
| 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 |
| CN108531547A (en) * | 2018-03-29 | 2018-09-14 | 杭州泰熙生物技术有限公司 | A kind of detection method for false meat monitoring |
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