CN115612721A - Digital PCR (polymerase chain reaction) identification method for large yellow croaker, small yellow croaker and products thereof - Google Patents

Digital PCR (polymerase chain reaction) identification method for large yellow croaker, small yellow croaker and products thereof Download PDF

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CN115612721A
CN115612721A CN202211400297.1A CN202211400297A CN115612721A CN 115612721 A CN115612721 A CN 115612721A CN 202211400297 A CN202211400297 A CN 202211400297A CN 115612721 A CN115612721 A CN 115612721A
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yellow croaker
digital pcr
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products
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邵彪
管祁
高利亭
汪少芸
陈刚
蔡茜茜
张霞
王振涛
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NANTONG PRODUCT QUALITY SUPERVISION AND INSPECTION INSTITUTE
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Abstract

The invention discloses a digital PCR identification method for large yellow croaker, small yellow croaker and products thereof, which utilizes a digital PCR technology to detect the components of the large yellow croaker and the small yellow croaker and comprises the following steps: (1) Designing and modifying reference gene primers and fluorescent probes of large yellow croakers, small yellow croakers and fishes; (2) extracting sample DNA; (3) Preparing a digital PCR reaction system, generating microdroplets, setting amplification parameters and reading the microdroplets; and (4) judging the digital PCR result. The invention provides an accurate and reliable analysis method for identifying the authenticity of the fish-derived components of the large yellow croaker, the small yellow croaker and the products thereof, can realize the simultaneous and rapid detection of the two components of the large yellow croaker and the small yellow croaker, has absolute quantitative capability and can directly obtain the copy number of the target gene in the sample without the help of a standard product.

Description

Digital PCR (polymerase chain reaction) identification method for large yellow croaker, small yellow croaker and products thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a digital PCR (polymerase chain reaction) counterfeit identification method for large yellow croakers, small yellow croakers and products thereof.
Background
The large yellow croaker (Larimichthys crocea) and the small yellow croaker (Larimichthys polyactis) are collectively called yellow croakers, also called yellow croakers, are important marine economic fishes in China, are distributed in coastal sea areas, are delicious in taste and rich in nutrition, and are well favored by consumers.
However, the phenomena of false mark, false mark and adulteration in the marine fish market are very common in the global range, and the first place of the adulteration of foods is listed. On the one hand, the reason is that the phenomenon of false labeling and virtual labeling is caused by the fact that fish varieties are various, part of the fish varieties are similar in form and tissue structure and are difficult to distinguish and identify; on the other hand, since the quality of the fishes is different and the price difference is large, illegal vendors intentionally fake the fishes with lower quality and price and adulterate the fishes in a mixed way under the drive of benefits. In recent years, incidents of "stained yellow croakers", fake yellow croakers, have been frequently exposed. In the aspect of selling iced fresh fish, spotted maigre and the like similar to the body forms and appearances of big and small spotted maigre are common substitutes, the phenomena of labeling the spotted maigre, yellow croaker, spotted maigre and the like on the same product of an electronic shopping mall and a network sales platform are frequent, and yellow azo dyes with carcinogenic risks, such as basic yellow and basic orange and the like, are used for dyeing in order to make the color and the appearance more similar. In addition, in the aspect of fish deep-processing products, such as fillets, minced fillet, fish balls, fish sausages, fish steaks, dried fish, shark fins, fish maws, fish cans and the like, the fish deep-processing products are serious disaster areas of counterfeit and inferior quality because the fish deep-processing products do not have the original appearance characteristics and tissue structure forms. Therefore, the phenomenon of adulteration of fishes including big and small yellow croakers not only disturbs market order, influences social image and damages legal rights and interests of consumers, but also has potential food safety hazards, and the market supervision is urgently needed to be implemented to attack illegal behaviors. Therefore, the development of accurate and reliable fish-derived component identification technology is of great significance.
At present, fish product true and false identification methods mainly comprise a morphology observation method, a wave spectrum analysis method, a protein analysis method, a molecular biology method and the like, wherein the molecular biology method taking genetic material DNA as an analysis object is the most ideal scheme and is the most common method for fish true and false identification, and because DNA molecules have high stability and species specificity, the method is less influenced by external factors compared with other methods. Currently, in molecular biology PCR analysis technologies, digital PCR technology is regarded as a new generation PCR technology due to its absolute quantitative performance, and has greater advantages in analysis of food adulteration ratio.
Food quality and safety have been the focus of social attention. At present, no effective and high-reliability method for detecting the authenticity of the components of the big yellow croaker, the small yellow croaker and the products thereof exists at home and abroad for the quality and the safety of the food. In order to solve the phenomena of adulteration and counterfeit and badness of yellow croaker products, maintain the rights of consumers and ensure the food safety, the development of an accurate and reliable method for identifying the authenticity of big and small yellow croakers and products thereof is very important.
Disclosure of Invention
The invention aims to provide a digital PCR (polymerase chain reaction) counterfeit identification method for large yellow croakers, small yellow croakers and products thereof aiming at the defects in the prior art. The digital PCR has absolute quantitative capability, and the copy number of the target gene in the sample can be directly obtained without the help of a standard substance. The invention provides a new method for identifying the authenticity of the big yellow croaker, the small yellow croaker and the products thereof.
In order to realize the aim, the method for identifying the pseudosciaena crocea and the small yellow croaker and the products thereof by digital PCR detects the components of the pseudosciaena crocea and the small yellow croaker by the digital PCR technology, and comprises the following steps:
s1, designing an upstream primer, a downstream primer and a probe respectively aiming at the conserved sequence segments of mitochondrial cytochrome b (Cytb) of large yellow croakers and small yellow croakers;
s2, sample DNA extraction: after selecting a sample, shearing or grinding and uniformly mixing, extracting DNA by adopting a CTAB method or extracting by adopting a kit;
s3, adopting a digital PCR reaction system, wherein the total amount of the reaction system is 20 mu L, generating a micro-droplet by using 70 mu L of micro-droplet generating oil through a micro-droplet generator, transferring the micro-droplet to a 96-well plate, sealing and amplifying, wherein the digital PCR amplification parameters are as follows:
the first step is as follows: denaturation at 95 deg.C for 5min;
the second step is that: denaturation at 94 ℃ for 30s, annealing/extension at 60 ℃ for 60s, and circulation for 40 times;
the third step: denaturation at 98 ℃ for 10min, preservation at 4 ℃, transferring to a microdroplet reader, and selecting a corresponding fluorescence channel for reading;
s4, judging a result: setting a threshold limit value of fluorescence according to an end-point fluorescence value of a negative amplification system in the system; the threshold limit value is required to clearly distinguish the negative microdroplet and the positive microdroplet in the amplification system, and if the fluorescence signal of the sample amplification end point exceeds the threshold limit value, the detection of the target component is indicated; if the fluorescence signal at the end of amplification of the sample is below a threshold limit, it indicates that the target component is not detected.
As a further improvement of the scheme, the sequences of the primers and the probes in the step S1 are shown as follows:
large yellow croaker cytb gene:
an upstream primer: 5 '-GGCCTGAATCGGATAGA-3';
a downstream primer: 5 '-AAGAGAGCTAGGGGTGGTAAG-3';
and (3) probe: 5 '-AGACCTTCTAGGCTTTGCAATCCTCA-3';
the small yellow croaker cytb gene:
an upstream primer: 5 '-TCATCCGTTGCACACATC-3';
a downstream primer: 5 '-TGGAGGTAGGGCAGATAAA-3';
and (3) probe: 5 '-TGACTCATCCGAAAACCTTCATGCCAA-3'.
As a further improvement of the above scheme, the system composition with the total amount of 20 μ L in step S3 comprises: digital PCR reaction solution dd PCR Supermix for Probes (No dUTP) 10. Mu.L, two upstream primers of 20. Mu. Mol large yellow croaker and small yellow croaker mitochondrial cytochrome b (Cytb) 0.9. Mu.L respectively, two downstream primers of 20. Mu. Mol 0.9. Mu.L respectively, two Probes of 10. Mu. Mol 0.9. Mu. L respectively5 μ L, add template DNA1-3 μ L, supplement ddH 2 O to 20. Mu.L.
As a further improvement of the scheme, the 5 'end is labeled by 1 of two groups of FAM and HEX/VIC, and the 3' end is labeled by BHQ1 or BHQ 2.
The invention has the beneficial effects that: the digital PCR identification method for the large yellow croaker, the small yellow croaker and the products thereof adopts a multiple fluorescence quantitative PCR identification method, designs primers and Taqman probes aiming at the conserved DNA sequence segments of the large yellow croaker and the small yellow croaker and general reference genes of fishes, realizes the simultaneous detection of the two components of the large yellow croaker and the small yellow croaker in the same reaction system by using the corresponding primers and the corresponding probes marked with different fluorescence signals, and has the advantage of high flux; the digital PCR has absolute quantitative capability, so that the copy number of the target gene in a sample can be directly obtained without a standard substance.
Drawings
FIG. 1 is a droplet dispersion diagram of a spotted maigre negative control amplification system in accordance with an embodiment of the present invention;
FIG. 2 is a droplet dispersion diagram of a large and small yellow croaker DNA mixed sample amplification system according to an embodiment of the present invention;
FIG. 3 is a two-dimensional scattergram of DNA mixed sample of big and small yellow croakers according to an embodiment of the present invention;
FIG. 4 is a droplet dispersion diagram of a sample amplification system for large and small minced fillet products of yellow croaker in the example of the present invention;
FIG. 5 is a two-dimensional scatter plot of samples of large and small minced fish products in accordance with an embodiment of the present invention;
wherein: ch1 is large yellow croaker Cytb, and ch2 is small yellow croaker Cytb.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings of the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention, are within the scope of the invention.
Example 1
The digital PCR identification method for large yellow croaker, small yellow croaker and products thereof is used for detecting the components of the large yellow croaker and the small yellow croaker by a digital PCR technology and comprises the following steps:
s1, designing an upstream primer, a downstream primer and a probe aiming at the conserved sequence segments of mitochondrial cytochrome b (Cytb) of large yellow croakers and small yellow croakers respectively, wherein the sequences of the primers and the probe are as follows:
large yellow croaker cytb gene:
an upstream primer: 5 '-GGCCTGAATCCGGATATAGA-3';
a downstream primer: 5 '-AAGAGAGGTAGGTGGTAAG-3';
and (3) probe: 5 '-AGACCTTCTTAGGCTTTGCAATCCTCA-3';
the small yellow croaker cytb gene:
an upstream primer: 5 '-TCATCCGTTGCACACATC-3';
a downstream primer: 5 '-TGGAGGTAGGGCAGATAAA-3';
and (3) probe: 5 '-TGACTCATCCGCGAAAACCTTCATGCCAA-3';
the probes 5 'for mitochondrial cytochrome b (Cytb) of large yellow croaker and small yellow croaker are labeled with FAM and VIC, respectively, and the 3' ends are labeled with BHQ1, respectively.
S2, after selecting a sample, shearing or grinding, and uniformly mixing, extracting DNA by adopting a CTAB method, or extracting by adopting a kit;
s3, the total amount of the digital PCR reaction system is 20 mu L, and the method comprises the following steps: the digital PCR reaction solution dd PCR Supermix for Probes (No dUTP) is 10 mu L, two upstream primers with the concentration of 20 mu mol of large yellow croaker and small yellow croaker mitochondrial cytochrome b (Cyt b) are respectively 0.9 mu L, two downstream primers with the concentration of 20 mu mol are respectively 0.9 mu L, two Probes with the concentration of 10 mu mol are respectively 0.5 mu L, 1-3 mu L of template DNA is added, ddH is supplemented 2 O to 20. Mu.L. Each reaction system was transferred to a 96-well plate and amplified after sealing by generating droplets using a droplet-generating apparatus using 70. Mu.L of droplet-generating oil. Digital PCR amplification parameters: the first step is as follows: denaturation at 95 deg.C for 5min; the second step is that: denaturation at 94 ℃ for 30s, annealing/extension at 60 ℃ for 60s, and circulation for 40 times; the third step: denaturation at 98 deg.C for 10min, and storage at 4 deg.C.Transferring the sample to a droplet reader, and selecting a corresponding fluorescence channel for reading;
s4, judging a result: setting a fluorescence threshold value according to a negative amplification system endpoint fluorescence value in the system; the threshold limit value is required to clearly distinguish the negative microdroplet and the positive microdroplet in the amplification system, and if the fluorescence signal of the sample amplification end point exceeds the threshold limit value, the detection of the target component is indicated; if the fluorescence signal at the end of amplification of the sample is below a threshold limit, it indicates that the target component is not detected.
Example 2
Respectively extracting large yellow croaker DNA, small yellow croaker DNA and spotted maigre DNA by using a marine animal tissue extraction kit, diluting and mixing the large yellow croaker DNA and the small yellow croaker DNA in proportion, and amplifying by using a digital PCR method provided by the invention, wherein the reaction system comprises the following steps: the digital PCR reaction solution dd PCR Supermix for Probes (No dUTP) 10. Mu.L, two upstream primers of 20. Mu. Mol of large yellow croaker and small yellow croaker mitochondrial cytochrome b (Cytb) 0.9. Mu.L respectively, two downstream primers of 20. Mu. Mol 0.9. Mu.L respectively, two Probes of 10. Mu. Mol 0.5. Mu.L respectively, 2. Mu.L of template DNA was added to supplement ddH 2 O to 20. Mu.L. Meanwhile, the spotted maigre DNA is used for setting a negative control. Each reaction system was subjected to amplification after transferring to a 96-well plate and sealing, using 70. Mu.L of a droplet-forming oil to form droplets by means of a droplet-forming apparatus. Digital PCR amplification parameters: the first step is as follows: denaturation at 95 deg.C for 5min; the second step: denaturation at 94 ℃ for 30s, annealing/extension at 60 ℃ for 60s, and circulation for 40 times; the third step: denaturation at 98 ℃ for 10min, and storage at 4 ℃. Transferring to a droplet reader, and selecting a corresponding fluorescence channel for reading. Wherein, FIG. 1 is a microdroplet dispersion diagram of a spotted maigre negative control amplification system, FIG. 2 is a microdroplet dispersion diagram of a large and small yellow croaker DNA mixed sample amplification system, and FIG. 3 is a two-dimensional scatter diagram of a large and small yellow croaker DNA mixed sample.
FIG. 1 shows that the fluorescence value of the two fluorescence channels of the negative amplification system has only one zone and the signal is low; in the sample amplification system of fig. 2, two zones are respectively arranged in two fluorescence channels, wherein a zone with low fluorescence signals is a negative droplet, and a zone with low fluorescence signals is a positive droplet, which indicates that both channel results are detected, i.e. large yellow croaker and little yellow croaker components are detected, and according to the analysis of the number of sample droplets, the number of large yellow croaker droplets is 7128, and the number of small yellow croaker droplets is 3816 in 10255 droplets; in the two-dimensional scattergram of FIG. 3, it is further shown that the distribution of the sample droplets, i.e., the number of droplets containing both signals in the droplet is 2687, the number of droplets containing only the pseudosciaena crocea fluorescent signal is 4441, the number of droplets containing only the pseudosciaena crocea fluorescent signal is 1129, and the number of droplets containing no fluorescent signal is 1998.
Example 3
Taking large and small yellow croaker surimi products which are randomly mixed, extracting DNA according to a CTAB method, diluting by 100 times, and amplifying according to a digital PCR method provided by the invention, wherein the reaction system comprises: 10 mu L of dd PCR Supermix for Probes (No dUTP), 0.9 mu L of two upstream primers of 20 mu mol of large yellow croaker and small yellow croaker mitochondrial cytochrome b (Cytb), 0.9 mu L of two downstream primers of 20 mu mol and 0.5 mu L of two Probes of 10 mu mol are added with 2 mu L of template DNA to supplement ddH 2 O to 20. Mu.L. Each reaction system was subjected to amplification after transferring to a 96-well plate and sealing, using 70. Mu.L of a droplet-forming oil to form droplets by means of a droplet-forming apparatus. Digital PCR amplification parameters: the first step is as follows: denaturation at 95 deg.C for 5min; the second step is that: denaturation at 94 ℃ for 30s, annealing/extension at 60 ℃ for 60s, and circulation for 40 times; the third step: denaturation at 98 deg.C for 10min, and storage at 4 deg.C. Transferred to a droplet reader and the corresponding fluorescent channel is selected for reading. Wherein, fig. 4 is a droplet dispersion diagram of a large and small yellow croaker surimi product sample amplification system, and fig. 5 is a two-dimensional dispersion diagram of a large and small yellow croaker surimi product sample.
FIG. 4 shows that two fluorescence channels in the amplification system for minced fish products from large yellow croaker and small yellow croaker respectively have two zones, wherein the zone with low fluorescence signal is a negative droplet, and the zone with low fluorescence signal is a positive droplet, which indicates that the results of the two channels are detected, i.e., the components of large yellow croaker and small yellow croaker are detected, and according to the analysis of the number of droplets in the sample, the number of droplets in large yellow croaker is 1683, and the number of droplets in small yellow croaker is 1445 among 14693 droplets; fig. 5 is a two-dimensional scattergram of samples of large and small yellow croaker surimi products, which further shows that the distribution of sample droplets, i.e., the number of droplets containing two signals in the droplets is 246, the number of droplets containing only yellow croaker fluorescent signals is 1437, the number of droplets containing only yellow croaker fluorescent signals is 1199, and the number of droplets not containing fluorescent signals is 11811.
The above-described embodiments are not intended to limit the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (4)

1. The digital PCR identification method for large yellow croaker, small yellow croaker and products thereof is characterized in that: the method for detecting the components of the large yellow croaker and the small yellow croaker by the digital PCR technology comprises the following steps:
s1, designing an upstream primer, a downstream primer and a probe respectively aiming at the large yellow croaker and small yellow croaker mitochondrion cytochrome b (Cytb) conserved sequence fragments;
s2, sample DNA extraction: after selecting a sample, shearing or grinding and uniformly mixing, extracting DNA by adopting a CTAB method or extracting by adopting a kit;
s3, adopting a digital PCR reaction system, wherein the total amount of the reaction system is 20 mu L, generating microdroplets by using 70 mu L of microdroplet generating oil in the reaction system through a microdroplet generating instrument, transferring the microdroplets to a 96-well plate, sealing and amplifying, wherein the digital PCR amplification parameters are as follows:
the first step is as follows: denaturation at 95 deg.C for 5min;
the second step is that: denaturation at 94 ℃ for 30s, annealing/extension at 60 ℃ for 60s, and circulation for 40 times;
the third step: denaturation at 98 ℃ for 10min, preservation at 4 ℃, transferring to a microdroplet reader, and selecting a corresponding fluorescence channel for reading;
s4, judging a result: setting a threshold limit value of fluorescence according to an end-point fluorescence value of a negative amplification system in the system; a threshold value is required to definitely distinguish a negative microdroplet and a positive microdroplet in an amplification system, and if a fluorescence signal at a sample amplification end point exceeds the threshold value, the detection of a target component is indicated; if the fluorescence signal at the end of amplification of the sample is below a threshold limit, it indicates that the target component is not detected.
2. The digital PCR identification method for large yellow croaker, small yellow croaker and products thereof according to claim 1, wherein: the sequences of the primers and the probes in the step S1 are shown as follows:
large yellow croaker cytb gene:
an upstream primer: 5 '-GGCCTGAATCGGATAGA-3';
a downstream primer: 5 '-AAGAGAGCTAGGGGTGGTAAG-3';
and (3) probe: 5 '-AGACCTTCTAGGCTTTGCAATCCTCA-3';
the small yellow croaker cytb gene:
an upstream primer: 5 '-TCATCCGTTGCACACATC-3';
a downstream primer: 5 '-TGGAGGTAGGGCAGATAAA-3';
and (3) probe: 5 '-TGACTCATCCGAAAACCTTCATGCCAA-3'.
3. The digital PCR identification method for large yellow croaker, small yellow croaker and products thereof according to claim 1, wherein: the system with the total amount of 20 mu L in the step S3 comprises the following components: the digital PCR reaction solution dd PCR Supermix for Probes (No dUTP) 10. Mu.L, two upstream primers of 20. Mu. Mol of large yellow croaker and small yellow croaker mitochondrial cytochrome b (Cytb) 0.9. Mu.L respectively, two downstream primers of 20. Mu. Mol 0.9. Mu.L respectively, two Probes of 10. Mu. Mol 0.5. Mu.L respectively, template DNA 1-3. Mu.L is added to supplement ddH 2 O to 20. Mu.L.
4. The digital PCR identification method for large yellow croaker, small yellow croaker and products thereof according to claim 2, wherein: the 5 'end is marked by 1 group of FAM and HEX/VIC, and the 3' end is marked by BHQ1 or BHQ 2.
CN202211400297.1A 2022-11-09 2022-11-09 Digital PCR (polymerase chain reaction) identification method for large yellow croaker, small yellow croaker and products thereof Pending CN115612721A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116769883A (en) * 2023-07-13 2023-09-19 湖南师范大学 Method for identifying ploidy of hybrid fish based on DNA sample

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116769883A (en) * 2023-07-13 2023-09-19 湖南师范大学 Method for identifying ploidy of hybrid fish based on DNA sample
CN116769883B (en) * 2023-07-13 2024-03-22 湖南师范大学 Method for identifying ploidy of hybrid fish based on DNA sample

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