CN109825606B - Kit for rapidly detecting pig-derived components in food and application thereof - Google Patents
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Abstract
The invention provides a kit for rapidly detecting pig-derived components in food, and relates to the technical field of biological species identification. The invention firstly screens out a universal internal standard gene Actb of pig origin, the nucleotide sequence of which is shown in SEQ ID NO.1, and the Actb is positioned on the 8 th chromosome, has constant copy number in pig species, has no allelic variation, and can be used as a target gene for identifying the pig origin. A PCR amplification primer is designed by taking the gene as a target sequence, and is amplified together with a PCR reaction solution, and a PCR reaction product is used for detecting the platinum-palladium nanoparticle immunochromatographic test strip. The PCR reaction is combined with the immunochromatographic test strip detection based on the platinum-palladium nano particles to form a rapid detection kit, the swine-derived component in food can be rapidly and sensitively detected, and the detection sensitivity can reach 0.8% (w/w). The kit has the advantages of simple use method, low cost, easy observation of reaction results and good specificity, and is very suitable for field real-time detection.
Description
Technical Field
The invention relates to the technical field of biological species identification, in particular to a rapid detection kit for detecting pig-derived components in food by combining Polymerase Chain Reaction (PCR) technology with platinum-palladium nanoparticle immunochromatographic test strip detection.
Background
With the rapid development of economy and the improvement of the living standard of people, the demand of residents in China on meat food is increased year by year. Although many countries have clear regulations that require food labels to truly and clearly identify the type and source of meat, and prohibit adulteration, there are many incidents of meat adulteration in the market, such as the incorporation of pork in donkey meat burns, the incorporation of beef or pork in mutton, the incorporation of other meats in sausages, and so on, for cost reduction. At present, the PCR method is a simple and effective method for detecting food adulteration, and has the advantages of simple operation, short time and high detection accuracy. Therefore, the invention aims to provide a rapid and sensitive detection kit for the pig-derived components in the food.
At present, the internal standard gene is widely used for identifying food adulteration, but how to screen out the proper internal standard gene is very important. At present, meat product adulteration detection technologies at home and abroad are mainly designed aiming at genes on mitochondria to carry out real-time fluorescent quantitative PCR amplification, and because the mitochondria genes are multicopy genes, the detection sensitivity is high, but simultaneously, the detection technology has trouble in distinguishing unconscious cross contamination and conscious illegal addition generated in the processes of sale, transportation and the like. In addition, the concentration of high copy number mitochondrial DNA cannot correspond to the concentration of genomic DNA, so that accurate quantitative analysis of a sample cannot be performed, and qualitative detection by ordinary PCR is difficult because of extremely high homology of mitochondrial genes. Therefore, the method can only realize screening and identification of meat adulteration by means of fluorescent quantitative PCR. If the low-concentration unintentional cross contamination and intentional illegal addition are identified, and the adulteration ratio is determined to realize rapid screening, the low-copy gene on the chromosome is selected as the standard gene in the meat.
The PCR method is a widely applied method at present, and the principle is that a DNA polymerase and a pair of specific primers which are complementarily paired with a template are utilized, and the total amount of the template DNA is continuously increased through a series of steps of denaturation, annealing, extension and the like.
The immunochromatographic test paper is a detection method which is derived for realizing rapid detection, has high precision, low price and easy operation, and generally comprises a sample pad, a combination pad, a detection line, a quality control line, a nitrocellulose membrane, a water absorption pad and a back plate. The detection principle is similar to that of enzyme-linked immunosorbent assay, but the detection process is simpler, more portable and easier to operate. The immunochromatographic test paper has 3 types of sandwich method, competition method and indirect method. The platinum-palladium nanoparticle immunochromatographic test strip belongs to a sandwich method, and is characterized in that a specific antibody of a substance to be detected is crosslinked to nanoparticles and a detection line of a lateral flow chromatography sensor, and after a nanoparticle-antibody compound is combined with an antigen, the compound is combined with the antibody coated on the detection line to form a sandwich structure. Visual qualitative detection is achieved by observing the change in color of the detection lines and control lines.
The platinum-palladium nano particles are formed by introducing a second metal element palladium into a platinum nano catalyst, namely coating a layer of palladium on the nano platinum particles to form platinum with a spherical shell structureA palladium bimetallic particle. The platinum-palladium nanoparticles have excellent horseradish peroxidase-like activity and can be used in H2O2In the presence of the dye, the TMB solution was successfully made to appear visibly blue. Therefore, the platinum-palladium nano particles are applied to the immunochromatographic test paper to play a role in enhancing the detection line, so that the detection sensitivity is greatly improved.
The invention combines a specific PCR technology with platinum-palladium nanoparticle immunochromatographic test strip detection, and provides a rapid and sensitive detection kit for swine-derived components in food. The invention does not need complex instruments, has short time, simple operation and high sensitivity, and can meet the detection requirement only by one common PCR instrument.
Disclosure of Invention
The invention aims to provide a swine-origin universal internal standard gene for detecting swine-origin components in food.
The invention also aims to provide a rapid detection kit for detecting the pig-derived components in the food by combining the specific PCR with the platinum-palladium nanoparticle immunochromatographic test strip, which has high sensitivity, high specificity and simple operation.
A gene for detecting pig-derived components in food is an internal standard gene Actb and has a sequence shown in SEQ ID NO. 1.
The invention provides application of the internal standard gene Actb in detection of swine-derived components.
The invention provides application of the internal standard gene Actb in identification of pig-derived ingredients in food.
The invention provides a specific PCR primer combination for detecting the internal standard gene Actb, which comprises the following 2 primers:
F：5’-GAGATGGCGAAAGGAAACG-3’(SEQ ID NO.2)；
R：5’-CCAAGTGGAACGGGAAGG-3’(SEQ ID NO.2)；
wherein, the 5 'end of the upstream primer F is labeled with Biotin (Biotin), and the 5' end of the downstream primer R is labeled with Fluorescein (FITC).
The invention provides application of the specific PCR primer combination in identifying porcine components.
The invention provides application of the specific PCR primer combination in preparation of a swine-derived component detection kit or a detection reagent.
Furthermore, the invention provides a rapid detection kit for detecting the swine-derived component, which contains the specific PCR primer combination.
The invention provides a method for detecting pig-derived components in food, which comprises the following steps:
(1) extracting DNA from a sample to be detected;
(2) performing PCR detection by using the specific PCR primer combination by using the extracted DNA as a template;
(3) and (5) judging a result: and (4) adopting a platinum-palladium nanoparticle immunochromatographic test strip to judge the result. The detection T line and the quality control C line both have blue bands containing target genes; the quality control C line has a blue band, and the detection T line has no band and does not contain a target gene.
In the above method, the PCR detection in step (2) is specifically configured as follows: 10 × reaction buffer, 0.4mM dNTP, 0.2. mu.M primer F, 0.2. mu.M primer R, 2U Taq PCR polymerase.
In the method, the PCR detection reaction conditions are as follows: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s,53 ℃ for 30s and 72 ℃ for 30 s; 5min at 72 ℃.
In the method, the platinum-palladium nanoparticle immunochromatographic test strip in the step (3) comprises a test line and a quality control line, wherein the test line is marked with an FITC antibody, the quality control line is marked with a biotin secondary antibody, and a platinum-palladium nanoparticle-biotin antibody marker is combined and supported.
The invention screens out the pig source internal standard gene on the chromosome for the first time. The invention uses a plurality of breeds of pigs to verify the internal standard gene, and proves that the internal standard gene is stable and has no allelic variation. The selection of the internal standard gene generally requires low and stable copy number, and the internal standard gene of a general animal is often selected on mitochondria, so that the copy number of the internal standard gene is large and the internal standard gene is not easy to quantify. The invention selects the gene on the 8 th chromosome as the internal standard gene, the copy number is low, the quantification is easy, and the mutation rate is lower compared with the gene on the mitochondria.
FIG. 1 is a schematic view of an immunochromatographic test strip. The invention utilizes PCR reaction to design two primers for carrying out gene amplification aiming at the pig specificity internal standard gene target sequence. And detecting the PCR reaction product by using a platinum-palladium nanoparticle immunochromatographic test strip. A Test Line (TL) and a quality Control Line (CL) of the platinum-palladium nanoparticle immunochromatographic test strip are respectively marked with a FITC antibody and a biotin secondary antibody, and a platinum-palladium nanoparticle-biotin antibody marker is combined and padded.
When the PCR reaction product is dripped into the test strip sample pad, the product sequentially passes through the combination pad, the detection line (TL) and the quality Control Line (CL) due to the capillary action, and when the PCR reaction product is positive due to the specific combination action of the antigen-antibody and the catalytic action of the platinum-palladium nanoparticles, the Test Line (TL) and the quality Control Line (CL) are both provided with blue strips; when the reaction product was negative, only the Control Line (CL) had a blue band.
The immunochromatographic test paper is based on a platinum-palladium nanoparticle-antibody compound and an antigen-antibody recognition system to construct a three-layer sandwich structure. In a standard assay procedure, a sample containing porcine-derived components is mixed with system buffer and applied to a sample pad, and the solution travels up the lateral flow strip and reaches a conjugate pad under capillary force. On the combination pad, the specific PCR product containing pig source and the antibody in the dissolved platinum-palladium nano-particle-antibody complex are subjected to an immune reaction based on the antigen-antibody, and a new complex of the platinum-palladium nano-particle-antibody and the antigen is formed. The complex then moves up the immunochromatographic strip, and when it reaches the detection line, the PCR product and the corresponding antibody on the detection line undergo a second "antigen-antibody" based immune reaction. Thus, the complex containing platinum-palladium nanoparticles is captured and deposited on the detection line, resulting in a characteristic black band. Then, the excessive platinum-palladium nanoparticle-antibody complex will migrate toward the quality control line, and when it reaches the quality control line, the antibody in the platinum-palladium nanoparticle-antibody functional probe will be captured by goat anti-mouse IgG on the quality control line, forming a second characteristic black band. Because the platinum-palladium nano particles also have the activity of horseradish peroxidase, the detection line is used for detecting the activity of the horseradish peroxidaseOr the platinum palladium nanoparticles stacked by the finger control line can be mixed with horseradish peroxidase substrate and H2O2The reaction generates specific blue non-soluble products, thereby improving the signal intensity of the detection line and further improving the detection sensitivity.
The platinum-palladium nanoparticle immunochromatographic test strip depends on the specific binding of an antigen and an antibody, so that the platinum-palladium nanoparticle immunochromatographic test strip has extremely high sensitivity. According to the invention, pork minced meat and non-pork minced meat are mixed in a gradient manner by 5 times of equal mass, and the sensitivity of the detection method is researched by combining PCR reaction with platinum-palladium nanoparticle immunochromatography test strip detection. The results show that the detection limit of the detection kit of the invention is 0.8% (w/w).
Based on the internal standard gene Actb for detecting the pig-derived component in the food, which is determined by the invention, the invention designs the PCR primer combination for detecting the gene, and the PCR reaction is combined with the platinum-palladium nanoparticle immunochromatography test strip to detect whether the pig-derived component exists in the sample to be detected.
Drawings
FIG. 1 is a schematic view of an immunochromatographic test strip;
FIG. 2 shows the specific PCR amplification of the porcine specific internal standard gene in 16 animals, lane 1 shows the porcine PCR product, 1: a pig; 2: cattle; 3: sheep; 4: goat: 5: chicken; 6: deer; 7: duck: 8: goose meat; 9: a horse; 10: donkey; 11: a yak; 12: buffalo; 13: mink; 14: a camel; 15: fish; 16: a rat; 17: negative; m is marker DL 2000;
FIG. 3 is a diagram showing the judgment of positive and negative results in the detection of a swine PCR product platinum palladium nanoparticle immunochromatographic test strip; the Test Line (TL) and the quality Control Line (CL) of the sample 1 both have blue strips, which proves that the sample is positive, and the Test Line (TL) and the quality Control Line (CL) of the sample 2 only have blue strips, which proves that the sample is negative;
FIG. 4 is a test of the detection sensitivity of the PCR-Pt-Pd nanoparticle immunochromatographic test strip; 1: the mixing gradient is 0 time, namely 100 percent of the original mass; 2: the mixing gradient is 5 times, namely 20 percent of the original mass; 3: the mixing gradient is 25 times, namely the original mass is 4%; 4: the mixed gradient is 125 times, namely the original mass is 0.8%; 5: the mixed gradient is 625 times, namely the original mass is 0.16%; 6: and (4) negativity.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Pigs (Sus scrofa), cattle (Bos taurus), sheep (Ovis aries), general chickens (Gallus gallous), pheasants (Phasianus colchicus), turkeys (Meleagris gallopavo), black-boned chickens (Gallus domesticus brisson), ducks (Anas platyrhynchos), geese (Goose calicivirus), dogs (Canis auricularis), rabbits (Oryctolagus cuniculus), yaks (Bos mutus), and yellow croakers (Pseudosciaena polyactis) are purchased from supermarkets. Horse (Equus caballus), donkey (Equus asinus) was purchased from the farmer market in beijing. Mice (Mus musculus) were provided by the food safety and molecular biology laboratory of the university of agriculture, china. Buffalo (Bubalus bubalis), mink (Martes zibellina), camel (Camelus ferus), deer (Cervus) were provided by Dr Limon of the Tianjin Ex-in-Place laboratory.
Example 1 screening of the porcine Universal internal Standard Gene Actb
The target genome was downloaded from NCBI by searching GenBank for genetic information about the pig and saved in ". FASTA" format. The complete genome information of the pig is analyzed, and homology analysis is carried out by utilizing BLAST and DNAMAN version4.0 software, so that an Actb gene is screened, is positioned on the 8 th chromosome and is one of cytoskeleton actin. 20 meats (respectively, chicken of the common family (Gallus gallicus), pheasant (Phasianus colicuus), turkey (Melegagris gallipavo), black-bone chicken (Gallus domesticus brisson), pig (Sus scrofa), cow (Bos taurus), sheep (Ovis aries), duck (Anas platyrhynchos), Goose (Goose calicivirus), dog (Canis lupis familiaris), rabbit (Oryctolagus cunatus), yak (Bos mutus), yellow croaker (Pseudosciaena polyactis), horse (Equus caballus), donkey (Equus asinus), mouse (Mus musculus), buffalo (Bubalus), mink (Martes zibellis), camel (Camellia), and the result of comparison is expressed as "0. tbq", and the sequence is analyzed in a format of 0. v.0.4. tbq. And selecting the fragments with high specificity, and performing BLAST analysis to search sequence homology and specificity in the database. And finally, determining that the final specific target gene beta-actin (Actb) gene can be used as an internal standard gene by integrating the sequence. The nucleotide sequence of the Actb fragment is shown in SEQ ID NO. 1.
Example 2 establishment of PCR detection method for pig-derived ingredients
PCR primers were designed for the Actb gene determined in example 1 using primer premier5.0 software, with Biotin (Biotin) labeled at the 5 'end of the upstream primer F and Fluorescein (FITC) labeled at the 5' end of the downstream primer R.
See table 1.
TABLE 1 PCR primer sequences
The pig sample is rapidly detected by PCR, and the reaction system is 25 mu L and comprises 10x reaction buffer, 0.4mM dNTP, 0.2 mu M primer F, 0.2 mu M primer R and 2U Taq PCR polymerase. The reaction program is 95 ℃ for 5 min; 30 cycles of 95 ℃ for 30s,53 ℃ for 30s and 72 ℃ for 30 s; 5min at 72 ℃. After the amplification is finished, the product is judged by using 2% agarose gel electrophoresis, and a specific band appears to prove that the amplification is successful and contains the target gene. The results are shown in fig. 2, PCR amplification of the pig specific internal standard gene in 16 animals, lane 1 is the pig PCR product, 1: a pig; 2: cattle; 3: sheep; 4: goat: 5: chicken; 6: deer; 7: duck: 8: goose meat; 9: a horse; 10: donkey; 11: a yak; 12: buffalo; 13: mink; 14: a camel; 15: fish; 16: a rat; 17: negative; m is Maker DL 2000. Only the pig sample shows a bright band, which indicates that the Actb gene and the corresponding PCR system can be used for the rapid detection of the pig species.
Example 3 establishment of Immunochromatographic test strip detection method for platinum-palladium nanoparticles as PCR product of swine-derived component
The platinum-palladium nanoparticle labeled antibody is prepared by a sandwich method, and is stored at 4 ℃ for later use after being prepared. FITC antibody was diluted with buffer to optimal concentration, respectively. The distance between the Test Line (TL) and the quality Control Line (CL) was 4.5mm, and the Test Line (TL) and the quality Control Line (CL) were sprayed on the NC film at 1.0. mu.L/cm, respectively. And drying the sprayed NC membrane at 37 ℃ overnight for later use. The test strip was cut to a width of 3.8 mm.
And fully mixing the PCR reaction product with a buffer solution, then dropwise adding the mixture on a sample pad of the platinum-palladium nanoparticle immunochromatographic test strip, allowing the mixed solution to pass through the combination pad and an NC membrane under the power of a capillary tube, and continuously moving towards the water absorption pad, wherein the detection result can be observed after 3 min. As shown in FIG. 3, the Test Line (TL) and the quality Control Line (CL) of sample 1 both have blue bands, which is considered as a positive sample, and the quality Control Line (CL) of sample 2 only has blue bands, which is considered as a negative sample.
The platinum-palladium nanoparticle immunochromatographic test strip depends on the specific binding of an antigen and an antibody, so that the platinum-palladium nanoparticle immunochromatographic test strip has extremely high sensitivity. Pork minced meat and non-pork minced meat (mixed minced meat of cattle, sheep, mice and the like) are mixed in a 5-time gradient equal mass mode, PCR reaction is carried out, and then the PCR reaction is combined with a platinum-palladium nanoparticle immunochromatographic test strip to explore the sensitivity of a platinum-palladium nanoparticle immunochromatographic test strip detection method. The results are shown in FIG. 4, 1: the mixing gradient is 0 time, namely 100 percent of the original mass; 2: the mixing gradient is 5 times, namely 20 percent of the original mass; 3: the mixing gradient is 25 times, namely the original mass is 4%; 4: the mixed gradient is 125 times, namely the original mass is 0.8%; 5: the mixed gradient is 625 times, namely the original mass is 0.16%; 6: and (4) negativity. When the mixing gradient was 125 times (the mass of the mixed minced pork was 125 times that of the minced pork) which was 0.8% of the initial mass, a very faint detection band could still appear. When the mixing gradient is 625 times (the mass of the mixed minced pork is 625 times of that of the minced pork) which is 0.1% of the initial mass, the TL line can be hardly detected, so that the detection limit of the detection kit of the invention is 0.8% (w/w).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university of agriculture in China
<120> quick detection kit for pig-derived ingredients in food and application thereof
<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 (5)
1. A rapid detection method for detecting pig-derived components in food is characterized by comprising the following steps:
(1) extracting DNA from a sample to be detected;
(2) performing PCR detection by using the extracted DNA as a template and adopting a specific PCR primer combination;
(3) and (5) judging a result: adopting a platinum palladium nanoparticle immunochromatographic test strip to judge the result, wherein the detection T line and the quality control C line both have blue strips and contain target genes; the quality control C line has a blue band, and the detection T line has no band and does not contain a target gene;
the gene of the pig-derived component is an internal standard gene and has a sequence shown as SEQ ID NO. 1;
the specific PCR primer combination comprises the following 2 primers:
F：5’-GAGATGGCGAAAGGAAACG-3’；(SEQ ID NO.2)
R：5’-CCAAGTGGAACGGGAAGG-3’；(SEQ ID NO.3)
wherein, the 5 'end of the upstream primer F is labeled with Biotin (Biotin), and the 5' end of the downstream primer R is labeled with Fluorescein (FITC);
the platinum-palladium nanoparticle immunochromatographic test strip comprises a test line and a quality control line, wherein the test line is marked with an FITC antibody, the quality control line is marked with a biotin secondary antibody, and a platinum-palladium nanoparticle-biotin antibody marker is combined and padded.
2. Use of the specific PCR primer combination of claim 1 for identification of porcine-derived components.
3. The use of the specific PCR primer combination of claim 1 in the preparation of a swine-derived component detection kit or detection reagent.
4. The method of claim 1, wherein the PCR assay of step (2) is a 25 μ L PCR assay system specifically configured to: 10 × reaction buffer, 0.4mM dNTP, 0.2. mu.M primer F, 0.2. mu.M primer R, 2U Taq PCR polymerase.
5. The method of claim 1, wherein the PCR detection reaction conditions are: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s,53 ℃ for 30s and 72 ℃ for 30 s; 5min at 72 ℃.
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| CN201910289636.5A CN109825606B (en) | 2019-04-11 | 2019-04-11 | Kit for rapidly detecting pig-derived components in food and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103173558B (en) * | 2013-03-26 | 2015-02-25 | 四川川娇生态猪业股份有限公司 | Identification or prediction method of pork quality |
| CN108220450B (en) * | 2016-12-20 | 2021-08-03 | 河南广电计量检测有限公司 | Identification method and identification kit for animal-derived components 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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Effective date of registration: 20231228 Address after: 462300 intersection of Wenming road and national highway 107, Yicheng District, Luohe City, Henan Province Patentee after: Zhongyuan Food Laboratory Address before: China Agricultural University (East District), 17 Qinghua East Road, Haidian District, Beijing 100083 Patentee before: CHINA AGRICULTURAL University |
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