CN114686600B - Primer group and method for meat detection based on seven-fold PCR technology - Google Patents

Primer group and method for meat detection based on seven-fold PCR technology Download PDF

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CN114686600B
CN114686600B CN202210176211.5A CN202210176211A CN114686600B CN 114686600 B CN114686600 B CN 114686600B CN 202210176211 A CN202210176211 A CN 202210176211A CN 114686600 B CN114686600 B CN 114686600B
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primer
specific primer
primer pair
plastic
meat
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CN114686600A (en
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蔡振东
蓝航镇
栾卉垚
潘道东
曾小群
吴振
周松
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Ningbo University
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Abstract

The invention discloses a primer group and a method for meat detection based on a seven-fold PCR technology, which are characterized by comprising a camel specific primer pair, a pigeon specific primer pair, a chicken specific primer pair, a duck specific primer pair, a horse specific primer pair, a beef specific primer pair and a pig specific primer pair, wherein the primer group and the method are used for simultaneously identifying 7 meat-source foods and comprise the following steps of 1) sample DNA extraction; 2) Respectively designing species-specific primers according to camel cytochrome c oxidase subunit III, pigeon NADH dehydrogenase 6 subunit, chicken D-loop, duck ATP enzyme 6 subunit, horse NADH dehydrogenase subunit 4, beef cytochrome c oxidase subunit II and pig 16SrRNA gene sequences; 3) Establishing a multiplex PCR reaction; 4) The agarose gel electrophoresis detection is carried out to judge whether the meat is doped or not and the doped meat has the advantages of simple operation, high sensitivity and good specificity.

Description

Primer group and method for meat detection based on seven-fold PCR technology
Technical Field
The invention belongs to the technical field of food quality safety detection, and particularly relates to a primer group and a method for meat detection based on a heptad PCR technology.
Background
Meat adulteration is a food safety problem commonly existing at home and abroad, becomes an important global problem in recent years, and receives extensive attention from the international society. Today, the technological means of adulteration are very different, and the adulterated product can be maximally close to pure meat in aspects of morphology, physical characteristics and the like, and is difficult to distinguish by visual observation and sensory evaluation alone. Frequent meat product adulteration events can jeopardize food safety, violate market rules, even threaten public health, and cause serious food safety accidents. Therefore, development of a practical technology with rapid, sensitive and accurate identification of animal sources is of great importance.
In recent years, with the development of molecular biology, techniques for identifying sources of meat species have been developed. The DNA molecule exists in each cell, has extremely high stability and can resist high temperature, high pressure, chemical method and other treatments. Thus, molecular diagnostics based on DNA level technology in combination with Polymerase Chain Reaction (PCR) are more reliable analytical methods. Currently, multiplex PCR and real-time fluorescent quantitative PCR (RT-PCR) techniques are both considered reliable methods with high sensitivity and specificity. RT-PCR can provide quantitative analysis of the meat content of the adulteration, however, accurate quantification depends on suitable internal parameters, which greatly limits the accuracy of RT-PCR analysis for the adulteration of diverse meat types, and RT-PCR requires specialized instrumentation and specialized skill to operate. In contrast, multiplex PCR can intuitively judge the doping of a large variety of meats by means of a simple agarose gel electrophoresis experiment. Due to the high degree of homology of meat species, the selection of target genes is critical for the construction of multiplex PCR systems. Mitochondrial DNA sequences in animal cells have variable regions of intra-and inter-species polymorphisms, which are well suited for the differentiation of closely related animal species. In addition, mitochondrial DNA sequences have multiple copies within the circular structure, which are more stable during meat processing. Thus, polymorphic sites of mitochondrial sequences can be selected as targets for designing species-specific primers in meat sources. Because multiplex PCR can detect multiple targets simultaneously, species-specific primers need to be analyzed, screened, and optimized to eliminate interactions between animal species, thereby eliminating cross-reactions between meat sources. Therefore, based on mitochondrial DNA sequences, the establishment of a rapid, accurate and sensitive multiplex PCR meat source detection technology is one of the basis and keys for strictly monitoring the safety of meat products in the market.
Disclosure of Invention
The invention aims to solve the technical problem of providing a primer group and a method for meat detection based on a seven-fold PCR technology, which are simple to operate, high in sensitivity and good in specificity.
The technical scheme adopted for solving the technical problems is as follows:
1. a primer group for meat detection based on a seven-fold PCR technology comprises a camel specific primer pair, a pigeon specific primer pair, a chicken specific primer pair, a duck specific primer pair, a horse specific primer pair, a cow specific primer pair and a pig specific primer pair,
wherein, the specific primer pair sequence of the camel is as follows:
an upstream primer: 5'-GCTCCACTTTCCTAACCGTGT-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-ATAGAGGAACAGCCAGACGACA-3';
the specific primer pair sequences of pigeons are as follows:
an upstream primer: 5'-CACCGCCCGAATCGCACCAC-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-AGGGATGTTTTCTGTCCGGTT-3';
the specific primer pair sequences of the chickens are as follows:
an upstream primer: 5'-CCCTACTTGCCTTCCACCGTA-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-CTTGAATAGCACTCCGCACCC-3';
the specific primer pair sequence of the duck is as follows:
an upstream primer: 5'-TCCCAGCCCTATTGTTCCCAT-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-TGTTAGTAGGGTAGCAAGCCACA-3';
the sequences of the specific primer pairs of the horses are as follows:
an upstream primer: 5'-TAGAAGCCCCAATTGCCGGAT-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-TATTGATGATGTAAGGCCGTGAG-3';
the specific primer pair sequences of the cattle are as follows:
an upstream primer: 5'-GCTGACCCATACAAGCACGA-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-CGTAATATAAGCCTGGACGGGAC-3';
the sequences of the specific primer pairs of the pigs are as follows:
an upstream primer: 5'-GAAGCCTTTCTCCTCGCACAC-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-CCCAACCGAAATTGCTAGTCCA-3'.
2. A meat detection method based on a seven-fold PCR technology comprises the following steps:
step one, sample DNA extraction:
according to the genome DNA kit instruction, separating and extracting meat genome DNA, and measuring the DNA concentration by using a Nanodrop2000 spectrophotometer;
step two, designing a species-specific primer: species-specific primers were designed based on camel cytochrome c oxidase subunit iii sequence (No. mh109991.1), pigeon NADH dehydrogenase 6 subunit (No. kp1688712.1), chicken D-loop (No. mk 163565.1), duck atpase 6 subunit (No. mk 770342.1), horse NADH dehydrogenase subunit 4 (No. mn 187574.1), bovine cytochrome c oxidase subunit ii (No. mn 714191015.1) and pig 16SrRNA gene (No. kj746666.1) gene sequences, respectively, using Oligo7.0 software in combination with bioinformatics alignment analysis:
wherein, the specific primer pair sequence of the pig is as follows:
an upstream primer: 5'-GAAGCCTTTCTCCTCGCACAC-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-CCCAACCGAAATTGCTAGTCCA-3';
the specific primer pair sequences of the cattle are as follows:
an upstream primer: 5'-GCTGACCCATACAAGCACGA-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-CGTAATATAAGCCTGGACGGGAC-3';
the sequences of the specific primer pairs of the horses are as follows:
an upstream primer: 5'-TAGAAGCCCCAATTGCCGGAT-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-TATTGATGATGTAAGGCCGTGAG-3';
the specific primer pair sequence of the duck is as follows:
an upstream primer: 5'-TCCCAGCCCTATTGTTCCCAT-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-TGTTAGTAGGGTAGCAAGCCACA-3';
the specific primer pair sequences of the chickens are as follows:
an upstream primer: 5'-CCCTACTTGCCTTCCACCGTA-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-CTTGAATAGCACTCCGCACCC-3';
the specific primer pair sequences of the pigeons are as follows:
an upstream primer: 5'-CACCGCCCGAATCGCACCAC-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-AGGGATGTTTTCTGTCCGGTT-3';
the sequence of the camel specific primer pair is as follows:
an upstream primer: 5'-GCTCCACTTTCCTAACCGTGT-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-ATAGAGGAACAGCCAGACGACA-3';
step three, seven PCR reaction system establishment
PCR reaction system: 2.5 mu.L easy Taq (10×), 2. Mu.L dNTPs (2.5 mM), 0.5. Mu.L easy Taq
DNA polymerase (5U. Mu.L) -1 ) Each of the upstream and downstream primers of each mixed species was 0.5. Mu.L (10. Mu.M), 1. Mu.L of the genomic DNA of the species (0.01-10 ng. Mu.L -1 ) And using ddH 2 The system was replenished with O to 25. Mu.L. Conditions for PCR reactions using T100 ™ Thermal Cycler (Bio-Rad, germany): denaturation at 94℃for 5min, then 34 cycles at 94℃for 30s,63℃for 30s, and 72℃for 45s, and finally extension at 72℃for 5min;
step four, agarose gel electrophoresis is carried out to analyze the result
The PCR fragment was amplified, analyzed by agarose Gel at a mass concentration of 4%, and by Gel Doc TM And taking a picture by an XR+System gel imaging System, and judging whether the sample has mixed meat according to the existence of the target strip of the gel pattern. If the meat sample is a pure meat product, i.e. is not doped with other meats, then only one strip matched with the species appears in the gel imaging; if the meat sample is not pure meat, i.e. is doped with other meat species, then the imaging is performed with bands corresponding to the target species, but also bands of other species.
Compared with the prior art, the invention has the advantages that:
1. the invention provides a simple, efficient and high-sensitivity technical method for identifying the meat sources of 7 animal foods at one time.
2. Based on European horse meat wind wave and reported beef doping types, 7 pairs of species-specific primers with different lengths are designed by taking beef doping as a main research object and purposefully utilizing mitochondrial DNA sequences of camel, pigeon, chicken, duck, horse, cow and pig. Single and multiplex PCR assays showed that all primers had specific, sensitive and efficient properties, at least without cross-reactivity with 14 land animals and 3 fish.
3. Compared with the multiple PCR technology at home and abroad, the detection sensitivity is concentrated in the range of 1 pg-0.32 ng; because of technical limitations, most of multiplex PCR detection species are less than 7, the seven-fold PCR method has high detection sensitivity, and the detection limit of DNA of seven meat species is 0.025-0.1 ng.
In summary, the invention provides a reliable multiplex PCR method, a plurality of species-specific candidate primers of target mitochondrial DNA sequences are designed, and finally 7 pairs of species-specific primers of camel (128 bp), pigeon (157 bp), chicken (220 bp), duck (272 bp), horse (314 bp), cow (434 bp) and pig (502 bp) are determined, so that the meat sources of 7 animal foods of camel, pigeon, chicken, duck, horse, beef and pig can be identified simultaneously. The multiplex PCR technology can be widely applied to the identification of the species source of meat in food by analysis of simple agarose gel.
Drawings
FIG. 1 (A) is a gel image of PCR amplified products using species-specific primers of camel (camel), pigeon (pipe), chicken (chicken), duck (duck), horse (horse), bovine (beef) and porcine (gark) origin, respectively, with the corresponding genomic DNA as template; (B) Agarose gel images of PCR products of all meat varieties PCR amplified with pre-mixed universal primers for eukaryotic 12S rRNA,16S rRNA and 18S rRNA genes, respectively; (C) Agarose gel images of products obtained by PCR amplification using individual template DNA of camel, pigeon, chicken, duck, horse, cow and pig; MIX refers to a mixture of 7 pairs of primers including camel, pigeon, chicken, duck, horse, beef and pig; 1-7 are a mixture of 6 pairs of primers of six non-target species; (D) In order to take mixed genome DNA as an amplification template, agarose gel images of products after single PCR amplification are respectively carried out by using specific primer pairs of camel, pigeon, chicken, duck, horse, cow and pig; CM is a mixture of genomic DNA of seven species of camel, pigeon, chicken, duck, horse, cow and pig; 1-7 are DNA mixtures of six meats other than the target species. M is a DNA molecular weight standard Marker (100-1031 bp).
FIG. 2 shows PCR amplified products cloned by using a mixture of seven species genomic DNA of camel, pigeon, chicken, duck, horse, cow and pig as amplification templates and single primer pairs of camel, pigeon, chicken, duck, horse, cow and pig, respectivelypEASY ® -T5 Zero vector, extracting plasmid DNA as template and PCR amplification using vector universal primers M13F and M13R, DNA automatic sequencer sequencing partial results of PCR products;
FIG. 3 (A) is an agarose gel image of amplified PCR fragments by multiplex PCR reactions using a mixture of specific primer pairs of 7 meats in camel, pigeon, chicken, duck, horse, cow and pig, as templates, with DNA mixtures of seven species at concentrations of 20, 10,5,2.5,1,0.5,0.25,0.1,0.05 and 0.025ng, respectively; (B) Using Image Lab from Berle Corp TM Software, drawing gel images corresponding to the electrophoresis patterns; lane1-10 is as abscissa tag (20, 10,5,2.5,1,0.5,0.25,0.1,0.05 and 0.025) in (A); m is a DNA molecular weight standard Marker (100-1031 bp).
FIG. 4 is a gel image of single PCR products of DNA amplification of camel, pigeon, chicken, duck, horse, cow and pig extracted from raw meat, cooked meat and microwave heated meat samples; m is a DNA molecular weight standard Marker (100-1031 bp).
Detailed Description
The present invention will be described in further detail with reference to the following examples, but the present invention is not limited to the following examples.
Detailed description of the preferred embodiments
1. Sample processing and DNA extraction
Fresh camel, pigeon, chicken, duck, horse, cow, pig pure meat is purchased from local retailers and fresh markets, and immediately after purchasing, is transported to a laboratory for processing. All samples were stored at-80℃to inhibit DNA degradation. Genomic DNA from meat samples was isolated using the easy pure genomic DNA kit. And the DNA concentration was measured using a Nanodrop2000 spectrophotometer.
2. Design of species-specific primers
Camel cytochrome c oxidase subunit III sequence (No. MH109991.1), pigeon NADH dehydrogenase 6 subunit (No. KP168712.1), chicken D-loop (No. MK 163565.1), duck ATPase 6 subunit (No. MK 770342.1), horse NADH dehydrogenase subunit 4 (No. MN187574.1), bovine cytochrome c oxidase subunit II (No. MN71419193.1) and pig 16SrRNA gene (No. KJ746666.1) gene sequences were retrieved from the National Center for Biotechnology Information (NCBI) database, respectively, according to the mitochondrial gene accession numbers of the respective species published by GenBank. The conserved regions and variable regions between the analyzed species were aligned using MEGA6 software. By combining oligo7.0 with BLAST programs, species-specific primers are designed based on physical parameters such as melting temperature, cross-reactivity, self-complementarity, and secondary structure. Primers were synthesized by Shanghai Biotechnology Co. To detect if the designed species-specific primers have a mismatch in non-target species, clustalW software was used on 14 land animals: camel, pigeon, chicken, duck, horse, cow, pig, turkey, goose, sheep, rabbit, ostrich, dog, cat, and 3 aquatic fish: and (5) performing sequence screening on the small yellow croaker, the tuna and the snakehead. Using the 17 species as a template, the specificity of the primer pair was further detected by a single PCR method, respectively.
Wherein, the specific primer pair sequence of the camel is as follows:
an upstream primer: 5'-GCTCCACTTTCCTAACCGTGT-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-ATAGAGGAACAGCCAGACGACA-3';
the specific primer pair sequences of pigeons are as follows:
an upstream primer: 5'-CACCGCCCGAATCGCACCAC-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-AGGGATGTTTTCTGTCCGGTT-3';
the specific primer pair sequences of the chickens are as follows:
an upstream primer: 5'-CCCTACTTGCCTTCCACCGTA-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-CTTGAATAGCACTCCGCACCC-3';
the specific primer pair sequence of the duck is as follows:
an upstream primer: 5'-TCCCAGCCCTATTGTTCCCAT-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-TGTTAGTAGGGTAGCAAGCCACA-3';
the sequences of the specific primer pairs of the horses are as follows:
an upstream primer: 5'-TAGAAGCCCCAATTGCCGGAT-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-TATTGATGATGTAAGGCCGTGAG-3';
the specific primer pair sequences of the cattle are as follows:
an upstream primer: 5'-GCTGACCCATACAAGCACGA-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-CGTAATATAAGCCTGGACGGGAC-3';
the sequences of the specific primer pairs of the pigs are as follows:
an upstream primer: 5'-GAAGCCTTTCTCCTCGCACAC-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-CCCAACCGAAATTGCTAGTCCA-3'.
3. Agarose gel electrophoresis detection of PCR products
For a single PCR reaction system, the EasyTaq DNA polymerase kit is used for PCR amplification. The PCR reaction system consists of 2.5 mu L of 10 XEasyTaq cube buffer solution, 2 mu L of 2.5mM dNTPs and EasyTaq DNA polymerase 0.5. Mu.L, 10mM upstream and downstream primers 0.5. Mu.L each, genomic DNA from each species 0.01-10 ng, and ddH 2 The system was replenished with O to 25. Mu.L. The PCR reaction conditions were: denaturation at 94℃for 5min, followed by 34 cycles at 94℃30s,63℃30s,72℃45s and finally extension at 72℃for 5min.
For multiplex PCR assays, after a single PCR assay, a seven-fold PCR reaction system was established using 7 specific primers and corresponding genomic DNA as templates. All PCR fragments were amplified using a T100 ™ thermal cycler, analyzed on a 4wt% agarose Gel, and using Gel Doc TM Xr+system gel imaging System photographs.
4. Sequencing of PCR products
Taking a mixture of seven species genome DNA of camel, pigeon, chicken, duck, horse, cow and pig as an amplification template,
the single primer pair of camel, pigeon, chicken, duck, horse, cow and pig is used for PCR amplification, and the amplification product of the PCR is Diaspin
The DNA gel purification kit is used for carrying out PCR fragment gel recovery and connectionpEASY ® -T5 Zero vector. Plasmid DNA extraction was performed using the sandrep column plasmid DNA miniprep kit and single PCR amplification was performed using vector universal primers M13F and M13R, and finally PCR product sequencing was performed using a DNA automatic sequencer.
5. Analysis of results
Judging whether to dope or not and the type of the doped meat source according to the electrophoresis strip displayed by imaging in the step 4. The products were in the size of camel (128 bp), pigeon (157 bp), chicken (220 bp), duck (272 bp), horse (314 bp), cow (434 bp) and pig (502 bp), respectively. When other strips which do not belong to the meat product should have pure meat strips appear in the image, the product is indicated to be doped with meat; otherwise, the meat product is pure meat product.
Second embodiment
Primer-specific analysis
To verify the specificity of the primers, a single PCR amplification was performed using a single species of DNA and its primer pair, and shown by gel electrophoresis analysis. Camel, pigeon, chicken, duck, horse,The amplification bands of the cattle and the pig are 128bp, 157bp, 220bp, 272bp, 314bp, 434bp and 502bp respectively. To ensure the quality of genomic DNA of seven species, three pairs of universal eukaryotic primers derived from the 18SrRNA,16SrRNA, 12SrRNA target genes were simultaneously selected as positive controls, respectively (fig. 1A). In the invention, all meat samples generate target PCR fragments (figure 1B) with similar brightness and weakness intensity of bands and 99bp,240bp and 456bp which are consistent with expected sizes, which shows that the quality of extracted genomic DNA of meat resources is equivalent, and the amplification efficiency of PCR is ensured. Using genomic DNA from single species meat as template, single PCR products could be obtained using a primer mix of 7 species, whereas no PCR bands were generated using a 6-pair non-target primer mix that did not include the target counterpart (FIG. 1C), indicating that only the target primer pair was able to amplify target species DNA efficiently. Furthermore, using a mixture of 7 species of DNA as templates, all single primer pairs can amplify PCR bands effectively; however, using a6 non-target species DNA mixture as template, the target primer pair was not able to amplify the PCR band efficiently (FIG. 1D), further indicating that the target species could be specifically amplified by the target primer pair. At the same time, the amplicons of 128-bp, 157-bp, 220-bp, 272-bp, 314-bp, 434-bp and 502-bp in FIG. 1D were purified using a gel recovery kit and cloned into commercial vectorspEASY ® T5 Zero, plasmid DNA extraction using SanPrep column plasmid DNA miniextraction kit and single PCR amplification using vector universal primers M13F and M13R, finally using DNA automatic sequencer for PCR product sequencing. Comparing the sequencing results to the NCBI nucleotide database for analysis, it was further determined that the target primer pair was capable of specifically amplifying the target species from the seven species DNA mixture. FIG. 2 shows the results of partial sequencing of the species camel, pigeon, chicken, duck, horse, cow, pig, as described above. Furthermore, by single PCR amplification experiments with each target primer pair, it was confirmed that the target primers were not able to amplify PCR bands effectively in addition to binding to target species, binding to non-target species in 14 land animals (camel, pigeon, chicken, duck, horse, cow, pig, turkey, goose, sheep, rabbit, ostrich, dog, cat) and 3 aquatic fish (yellow croaker, tuna and snakehead) (too much data, not shown), indicating a new design of specific primersThe object pair has no cross reaction with 16 non-target species, so that the possibility of misjudgment is greatly reduced. Based on the experimental results, the novel primer has high specificity and is suitable for identifying meat species in real foods.
Detailed description of the preferred embodiments
Primer sensitivity analysis
After each set of primers was specifically analyzed, a seven-fold PCR system was finally established using 7 pairs of species-specific primers by optimizing the reaction system and procedure. To analyze the dynamic range and limit of detection (LOD) of multiplex PCR reactions, template DNA from all species was serially diluted from 10ng to 0.01ng (10,5,2.5,1,0.5,0.25,0.1,0.05,0.025,0.01) and the threshold value for detection by the seven-fold PCR technique was assessed. As shown in FIG. 3A, 5 clear bands at the top end can be observed under the condition that the template DNA content is 10 ng-0.01 ng, and pigs, cows, horses, ducks and chickens are arranged in sequence from top to bottom. In contrast, 0.025 and 0.01ng template DNA content produced weak electrophoretic bands. Image Lab TM The software draws an electrophoresis pattern according to the electrophoresis band. The clear band is matched with the complete peak type, and the weak band is corresponding to the peak type of the defect. As shown in FIG. 3B, lanes 1-10 show a gradual decrease in fluorescence intensity with decreasing template DNA content, reflecting a decrease in PCR product. Consistent with the result of the electrophoresis gel pattern, the electrophoresis patterns of pigs, cattle, horses, ducks and chickens have complete peaks in lanes 1-10; pigeon and camel patterns exhibited intact patterns in lanes 1-9 and a defective pattern in lane 10. The limit of detection of genomic DNA from chickens, ducks, horses, cattle and pigs, combined with fig. 3A and 3B, was 0.01 ng; whereas the limit of detection of camel and pigeon genomic DNA is about 0.01-0.025 ng.
Detailed description of the preferred embodiments
Primer reproducibility verification
In order to examine the efficiency of each set of primers in detecting the thermally processed meat, raw meat samples were treated with steam boiling (97-99 ℃ C., 30 min) and microwave heating (750W, 10 min), genomic DNA was extracted as a template, and seven-fold PCR amplification experiments were performed using camel, pigeon, chicken, duck, horse, cow, and pig specific primers. As shown in fig. 4A and 4B, the digested sample DNA was subjected to a seven-fold PCR amplification reaction to produce seven target bands, while the detection limit for detecting seven species was about 0.01 ng; the microwave sample DNA was amplified by seven PCR reactions to produce seven target bands, with a seven species threshold of about 0.05 ng detected. The results show that the novel primers are suitable for realizing the identification of the real-world meat product species.
Detailed description of the preferred embodiments
Commercial meat testing to verify the effectiveness of seven-fold PCR for commercial meat product identification, 67 commercial meat products of swine, cattle, horses, pigeons, and camels were purchased on the market. Genomic DNA was extracted for seven-fold PCR amplification and analyzed for meat species by agarose gel electrophoresis. As shown in Table 1 below, most meat samples contained the same labeling elements without any contamination. However, some samples of what is known as 100% pure meat were found to be doped. 5 parts (doped) of 15 parts pork samples (33.3%), 6 parts (40.0%) of 15 parts beef samples, 3 parts (25%) of 12 parts horse meat samples, 1 part (10%) of 10 parts pigeon meat samples, and 3 parts (20%) of 15 parts camel meat samples were doped with some unnoticed meat components. According to this investigation, inexpensive chicken, duck and pork are still often used as an alternative component to red meat. Most importantly, this investigation further verifies the effectiveness of the seven-PCR method of the present invention in identifying common meat varieties.
。
The above description is not intended to limit the invention, nor is the invention limited to the examples described above. Variations, modifications, additions, or substitutions will occur to those skilled in the art and are therefore within the spirit and scope of the invention.
Sequence list
<110> university of Ningbo
<120> primer set for meat detection based on seven-fold PCR technique and method
<160> 14
<170> PatentIn version 3.3
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<213> camel downstream primer (5'-ATAGAGGAACAGCCAGACGACA-3')
<400> 2
<210> 3
<211> 20
<212> DNA
<213> pigeon upstream primer (5'-CACCGCCCGAATCGCACCAC-3')
<400> 3
<210> 4
<211> 21
<212> DNA
<213> pigeon downstream primer (5'-AGGGATGTTTTCTGTCCGGTT-3')
<400> 4
<210> 5
<211> 21
<212> DNA
<213> Chicken upstream primer (5'-CCCTACTTGCCTTCCACCGTA-3')
<400> 5
<210> 6
<211> 21
<212> DNA
<213> Chicken downstream primer (5'-CTTGAATAGCACTCCGCACCC-3')
<400> 6
<210> 7
<211> 21
<212> DNA
<213> Duck upstream primer (5'-TCCCAGCCCTATTGTTCCCAT-3')
<400> 7
<210> 8
<211> 23
<212> DNA
<213> Duck downstream primer (5'-TGTTAGTAGGGTAGCAAGCCACA-3')
<400> 8
<210> 9
<211> 21
<212> DNA
<213> immediate run primer (5'-TAGAAGCCCCAATTGCCGGAT-3')
<400> 9
<210> 10
<211> 23
<212> DNA
<213> horse downstream primer (5'-TATTGATGATGTAAGGCCGTGAG-3')
<400> 10
<210> 11
<211> 20
<212> DNA
<213> bovine upstream primer (5'-GCTGACCCATACAAGCACGA-3')
<400> 11
<210> 12
<211> 23
<212> DNA
<213> bovine downstream primer (5'-CGTAATATAAGCCTGGACGGGAC-3')
<400> 12
<210> 13
<211> 21
<212> DNA
<213> pig upstream primer (5'-GAAGCCTTTCTCCTCGCACAC-3')
<400> 13
<210> 14
<211> 22
<212> DNA
<213> pig downstream primer (5'-CCCAACCGAAATTGCTAGTCCA-3')
<400> 14

Claims (2)

1. A primer group for meat detection based on a seven-fold PCR technology is characterized by comprising a camel specific primer pair, a pigeon specific primer pair, a chicken specific primer pair, a duck specific primer pair, a horse specific primer pair, a cow specific primer pair and a pig specific primer pair,
wherein, the specific primer pair sequence of the camel is as follows:
an upstream primer: 5'-GCTCCACTTTCCTAACCGTGT-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-ATAGAGGAACAGCCAGACGACA-3';
the specific primer pair sequences of pigeons are as follows:
an upstream primer: 5'-CACCGCCCGAATCGCACCAC-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-AGGGATGTTTTCTGTCCGGTT-3';
the specific primer pair sequences of the chickens are as follows:
an upstream primer: 5'-CCCTACTTGCCTTCCACCGTA-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-CTTGAATAGCACTCCGCACCC-3';
the specific primer pair sequence of the duck is as follows:
an upstream primer: 5'-TCCCAGCCCTATTGTTCCCAT-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-TGTTAGTAGGGTAGCAAGCCACA-3';
the sequences of the specific primer pairs of the horses are as follows:
an upstream primer: 5'-TAGAAGCCCCAATTGCCGGAT-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-TATTGATGATGTAAGGCCGTGAG-3';
the specific primer pair sequences of the cattle are as follows:
an upstream primer: 5'-GCTGACCCATACAAGCACGA-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-CGTAATATAAGCCTGGACGGGAC-3';
the sequences of the specific primer pairs of the pigs are as follows:
an upstream primer: 5'-GAAGCCTTTCTCCTCGCACAC-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-CCCAACCGAAATTGCTAGTCCA-3'.
2. A multiplex PCR assay for simultaneous identification of 7 meat-based foods comprising the steps of:
step one, sample DNA extraction:
isolating genomic DNA from the meat sample according to genomic DNA kit instructions and measuring DNA concentration;
step two, designing a species-specific primer: species-specific primers were designed based on camel cytochrome c oxidase subunit iii sequence, pigeon NADH dehydrogenase 6 subunit, chicken D-loop, duck atpase 6 subunit, horse NADH dehydrogenase subunit 4, bovine cytochrome c oxidase subunit ii and pig 16SrRNA gene sequences, respectively:
wherein, the specific primer pair sequence of the pig is as follows:
an upstream primer: 5'-GAAGCCTTTCTCCTCGCACAC-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-CCCAACCGAAATTGCTAGTCCA-3';
the specific primer pair sequences of the cattle are as follows:
an upstream primer: 5'-GCTGACCCATACAAGCACGA-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-CGTAATATAAGCCTGGACGGGAC-3';
the sequences of the specific primer pairs of the horses are as follows:
an upstream primer: 5'-TAGAAGCCCCAATTGCCGGAT-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-TATTGATGATGTAAGGCCGTGAG-3';
the specific primer pair sequence of the duck is as follows:
an upstream primer: 5'-TCCCAGCCCTATTGTTCCCAT-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-TGTTAGTAGGGTAGCAAGCCACA-3';
the specific primer pair sequences of the chickens are as follows:
an upstream primer: 5'-CCCTACTTGCCTTCCACCGTA-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-CTTGAATAGCACTCCGCACCC-3';
the specific primer pair sequences of the pigeons are as follows:
an upstream primer: 5'-CACCGCCCGAATCGCACCAC-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-AGGGATGTTTTCTGTCCGGTT-3';
the sequence of the camel specific primer pair is as follows:
an upstream primer: 5'-GCTCCACTTTCCTAACCGTGT-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-ATAGAGGAACAGCCAGACGACA-3';
step three, seven PCR reaction conditions are established
The PCR reaction system consists of 2.5 mu L of 10 XEasyTaq cube buffer solution, 2.5mM dNTPs 2 mu L, 0.5 mu L of EasyTaq DNA polymerase, 0.5 mu L of each of the upstream and downstream primers of the mixture species mM, 0.01-10 ng of the genome DNA of each species, and a ddH2O supplementing system to 25 mu L; denaturation at 94℃for 5min, then 34 cycles at 94℃for 30s,63℃for 30s, and 72℃for 45s, and finally extension at 72℃for 5min;
step four, agarose gel electrophoresis is carried out to analyze the result
And after the PCR fragment is amplified, analyzing by using agarose gel with the mass concentration of 4%, photographing in a gel imaging system, and judging whether the sample has mixed meat according to a gel pattern strip.
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