CN111961736A - Primer pair for identifying crocodile-derived component in meat product and application thereof - Google Patents
Primer pair for identifying crocodile-derived component in meat product and application thereof Download PDFInfo
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Abstract
The invention discloses a primer pair for identifying alligator-derived components in meat products and application thereof. The primer pair for identifying the alligator-derived components in the meat product is shown as SEQ ID No.1 for the upstream primer and SEQ ID No.2 for the downstream primer. The invention designs specific primers aiming at a gene sequence of crocodile pigment cytob, and establishes a rapid, specific and sensitive method for detecting crocodile-derived components. Common mutton, beef, pork, chicken, duck meat, fish meat and shrimp meat are taken as reference animal species for specific detection; and the feasibility of the test strip is verified through detection limit and sensitivity experiments. The experimental results show that: the method can effectively and rapidly detect crocodile-derived components, has strong specificity, has the detection limit of crocodile component DNA up to 0.001 ng/mu L and the sensitivity up to 0.01 percent, and can be used for detecting the crocodile-derived components in processed meat products.
Description
Technical Field
The invention belongs to the technical field of biological detection, and relates to a primer pair for identifying alligator-derived components in meat products and application thereof.
Background
Crocodile belongs to the phylum chordata of the animal kingdom, the subphylum vertebrates-class of reptilia-order of alligator, and is one of the oldest animals in existence. In recent years, with the strong support of national policies and the rapid development of crocodile artificial breeding technology, crocodile meat gradually changes from a rare food material to a new food material which can be contacted by consumers. The crocodile breeding industry in China is fast in development, the total breeding amount is increased year by year, but the breeding area is mainly concentrated in the south of China at present. The crocodile meat is rich in vitamins and mineral elements, good in taste and excellent in meat food. According to the existing research, the content of crude protein in the crocodile meat is about 18%, the proportion of essential amino acid to total amino acid is higher than 41%, the proportion of total essential amino acid to total non-essential amino acid is close to 73%, and the crocodile meat belongs to the FAO/WHO optimal mode in nutrition. The crocodile meat has multiple health care functions, has the effects of resisting lipid oxidation to a human body after being freeze-dried and ground, and can improve symptoms caused by anoxia, and also has the capacity of scavenging free radicals and resisting aging of the human body. The crocodile meat contains about 30 kinds of fatty acids, wherein the ratio of polyunsaturated fatty acids is about 30%, the crocodile meat is rich in DHA and EPA, the crocodile meat is similar to deep sea fish in terms of fatty acid content and composition, and the crocodile meat has extremely high nutritional and health-care values. However, the crocodile meat serving as emerging high-value meat has a plurality of defects in the aspects of market supervision and the like. In the traditional meat product industry, a plurality of vendors have good time to sell cheap inferior meat added into high-price high-quality meat, and the behavior not only damages the benefit of consumers, but also disturbs the market order. The crocodile meat has a very good market as a high-protein and low-fat good meat quality. With the expansion of the crocodile meat market, it is very important to establish a perfect and efficient food source tracing and anti-counterfeiting mechanism to deal with the problem of the adulteration of the crocodile meat. The real-time fluorescent PCR technology is widely applied to species source identification of meat and processed products thereof due to the advantages of high specificity and high sensitivity. However, few studies are reported to identify crocodile-derived components by real-time fluorescent PCR.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a primer pair for identifying crocodile-derived ingredients in meat products.
Another purpose of the invention is to provide application of the primer pair.
It is yet another object of the present invention to provide a method for identifying alligator-derived components in meat products.
The purpose of the invention can be realized by the following technical scheme:
the primer pair for identifying the alligator-derived components in the meat product is shown as SEQ ID No.1 for the upstream primer and SEQ ID No.2 for the downstream primer.
The primer pair disclosed by the invention is applied to identification of crocodile-derived ingredients in meat products.
The primer pair disclosed by the invention is applied to preparation of a crocodile-derived component identification reagent.
A kit for identifying crocodile-derived ingredients in meat products comprises the primer pair disclosed by the invention.
The kit preferably further comprises other reagents of SYBR Green fluorescent quantitative PCR.
The method for identifying the crocodile-derived ingredients in the meat product comprises the step of carrying out real-time fluorescence PCR detection on sample DNA by using the primer pair disclosed by the invention.
The method of the present invention preferably comprises the following steps:
(1) extracting sample DNA;
(2) using the extracted sample DNA as a template, performing real-time fluorescent PCR amplification by using the primer pair of claim 1, and using QuantStudioTMAnd (3) analyzing the amplification curve and/or Ct value of the result by using Real-Time PCR Software.
As a further preferred aspect of the present invention, the reaction system for real-time fluorescence PCR is 20. mu.L, and comprises 10. mu.L of TBGreen Taq II, 2. mu.L of template DNA, 0.8. mu.L of each of the upstream and downstream primers, 6. mu.L of sterilized ultrapure water, and 0.4. mu.L of ROX Reference Dye.
As a further preferred aspect of the present invention, the reaction conditions are: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing and extension at 64 ℃ for 30s, and 40 cycles.
Has the advantages that:
the crocodile original component is identified by SYBR Green fluorescent quantitative PCR, a specific primer is designed through crocodile mitochondrial pigment cell b gene, and the reliability of the SYBR Green fluorescent quantitative PCR in detecting the crocodile original component in food is verified through a specific test, a detection limit test, sensitivity detection and crocodile original detection in a processed product. The experimental results show that: the method can effectively and rapidly detect crocodile-derived components, has strong specificity, and has the detection limit of crocodile component DNA up to 0.001 ng/muL and the sensitivity up to 0.01 percent. Through detection and verification of the processed meat product, the method has the characteristics of strong specificity and high sensitivity, and can be used for detecting crocodile-derived ingredients in the processed meat product.
Drawings
FIG. 1 crocodile primer specificity real-time fluorescence PCR detection result
FIG. 2 crocodile primer detection limit real-time fluorescence PCR detection result
The amplification results of the concentrations of the crocodile DNA template are 100, 10, 1, 0.1, 0.01, 0.001, 0.0001 and 0 ng/. mu.L from left to right 1-8 in sequence.
FIG. 3 crocodile-derived component quantitative determination standard curve
FIG. 4 crocodile primer sensitivity real-time fluorescence PCR detection results
1-7 crocodile component contents of 100%, 10%, 1%, 0.1%, 0.01%, 0.001%, 0
FIG. 5 crocodile-derived real-time fluorescence PCR detection result of processed meat product
1 crocodile sausage, 2 crocodile dried meat slices, 3-7 bacon, dried pork slices, chicken sausage, beef stick and blank control
Detailed Description
The materials, reagents and equipment used in the following examples are as follows:
materials and reagents
Beef, mutton, pork, chicken, duck meat, fish, shrimp meat, beef sticks, dried pork slices, chicken sausages and bacon are purchased from supermarkets, the crocodile meat is purchased from Taixing division of Oceanin crocodile development Limited company in Suzhou, Jiangsu, and the dried crocodile meat and the crocodile sausage are prepared in a processing laboratory and are stored in a refrigerator at the temperature of-20 ℃ before use.
EDTA、Tris-HCl、Na2EDTA and proteinase k (20mg/mL) were purchased from Shanghai Biotech; CTAB is purchased from Aladdin corporation; chloroform, isopropanol and ethanol are all purchased from Shanghai test of Chinese medicine; real-time fluorescent PCR Mixed solution (TB)
Premix Ex TaqTMII) Dalibao bioengineering, Inc.
Apparatus and device
Table top high speed refrigerated centrifuge, Eppendorf corporation, germany; QuantStaudioTM6Flex real-time fluorescent quantitative PCR instrument ABI company; nnanodrop-2000 micro nucleic acid protein Analyzer, USA thermoelectric Corp; constant temperature water bath Nanjing Kol instruments and equipments Ltd.
Example 1
1 design and Synthesis of primers
Specific primers were designed based on the genomic sequence of crocodile (NC008143.1) published in GenBank and using Primer5.0 software, and the Primer sequences were compared and evaluated in the database of National Center for Biotechnology Information (NCBI) by BLAST analysis to ensure the specificity of crocodile primers. The primers used for amplification were synthesized by Shanghai Biotech, and the sequences of the primers are shown in Table 1.
TABLE 1 Alligator primer sequences
2 extraction of DNA
100mg of sample was weighed and 10. mu.L of proteinase K (20mg/mL) and 1.5mL of TAB extraction buffer (20g/L CTAB,1.4mol/L NaCl,0.1mol/L Tris-HCl,0.02mol/L Na2EDTA, pH 8.0), the samples were incubated in a water bath at 60 ℃ for 4 h. The lysate was centrifuged through 13000g for 10min and the supernatant was transferred to a new centrifuge tube. After adding 750. mu.L of chloroform, the mixture was shaken and centrifuged at 13000g for 5 min. After the supernatant was collected, 2 volumes of CTAB precipitation solution (5g/L CTAB,40mmol/L NaCl) was added thereto, and 13000g was centrifuged for 15 min. The supernatant was discarded, 350. mu.L of NaCl solution (1.2mol/L NaCl) and 350. mu.L of chloroform were added thereto, and the mixture was gently shaken and centrifuged at 13000g for 10 min. Collecting supernatant, adding 0.6 volume of isopropanol, and centrifuging at 13000g for 10 min. The supernatant was discarded and 500. mu.L of 70% ethanol solution was added to wash the precipitate, and 13000g was centrifuged for 5 min. Discarding the supernatant, drying, dissolving in 100 mu L of sterilized ultrapure water, and determining the Optical Density (OD) values of the samples at 260nm and 280nm by a trace nucleic acid protein analyzer, wherein the DNA 260/280 values of all the samples are between 1.8 and 2.2. The results show that all the DNA extracted from the samples are suitable for PCR detection.
3 real-time fluorescent PCR
The DNA concentration was adjusted to 20 ng/. mu.LDNA solution with sterilized ultrapure water, and real-time fluorescence PCR amplification was carried out using the above designed primers. The fluorescent PCR reaction system is 20 μ L, and comprises 10 μ L of TBGreen Taq II, 2 μ L of template DNA, 0.8 μ L of each of the upstream and downstream primers, 6 μ L of sterilized ultrapure water, and 0.4 μ L of ROX Reference Dye. The reaction conditions were adjusted according to the kit as follows: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing and extension at 64 ℃ for 30s, and 40 cycles.
4 data processing
Utilizing QuantStaudioTMAnd analyzing the amplification curve and Ct value of the result by using Real-Time PCR Software. Data are presented as mean ± standard deviation.
Example 2
1 specificity test
According to the method in the embodiment 1, the PCR experiment is carried out by taking crocodile, sheep, cattle, pig, chicken, duck, fish and shrimp sample DNA and taking 20 ng/mu LDNA concentration as a template, only the crocodile sample is obviously amplified, the Ct value is 20.92 +/-0.05, and the Ct values of chicken, sheep, cattle, pig, duck, fish and shrimp samples are all larger than 35, so that the primer designed by the method for crocodile is considered to have good specificity (see figure 1).
TABLE 2 Ct values of PCR amplification of DNA templates from different meats
2 detection limit test
Real-time fluorescent quantitative PCR amplification experiments were performed on 7 different value concentration gradients of sample DNA by alligator primer pairs 100, 10, 1, 0.1, 0.01, 0.001, 0.0001 ng/. mu.L according to the method of example 1. The results are shown in FIG. 2, and specific numerical values are shown in Table 3. As can be seen from FIG. 2 and Table 3, the samples with Ct values of less than 35 were amplified at template concentrations of 100, 10, 1, 0.1, 0.01, and 0.001 ng/. mu.L, while the Ct values were 35.85. + -. 1.25 at a template concentration of 0.0001 ng/. mu.L. Experiments show that the detection limit of the method is 0.001 ng/. mu.L crocodile DNA concentration. A series of gradient dilutions (100, 10, 1, 0.1, 0.01, 0.001, 0.0001 ng/mu L) are carried out on crocodile meat sample DNA, the logarithm value of the concentration of the crocodile meat DNA is in a linear relation with the corresponding Ct value, and a linear regression equation is obtained by taking the logarithm value of the template concentration as an X axis and the Ct value as a Y axis and is shown in figure 3. The standard curve equation for this test method is therefore: -2.783x +24.618, R20.9862, the concentration of crocodile DNA contained in the sample can be obtained by the formula of the Ct value of crocodile source in the meat product, and R is20.9862 shows that the crocodile primer designed by the experiment has good detection capability.
TABLE 3 Ct values for PCR amplification of different concentrations of crocodile DNA template
3 sensitivity test
The results of the quantitative fluorescence PCR amplification experiments using the crocodile primer on the crocodile component DNA and chicken component DNA mixed samples (crocodile component contents of 100%, 10%, 1%, 0.1%, 0.01%, 0.001%, 0%) according to the method of example 1 were shown in FIG. 3, and the specific values are shown in Table 4, by diluting the crocodile DNA solution at 20 ng/. mu.L, and finally diluting the solution to samples with crocodile DNA contents of 100%, 10%, 1%, 0.1%, 0.01%, 0.001%, 0%, respectively. According to the analysis of the results in FIG. 4 and Table 4, when the percentage of crocodile component DNA is 0.001% of the mixed DNA, the Ct value is 35.15 + -0.13, and the amplified Ct value is less than 35 when the percentage of crocodile component DNA is 100%, 10%, 1%, 0.1% and 0.01%. The result shows that the sensitivity of the detection method in the mixed DNA sample can reach 0.01%.
TABLE 4 Ct values of PCR amplification of DNA template contents of different crocodile components
4 meat processing product sample identification
DNAs of bacon, dried pork slice, chicken sausage and beef stick purchased from supermarket and laboratory-made dried crocodile meat slice and crocodile meat sausage were extracted, and fluorescence quantitative PCR amplification test was performed according to the method of example 1. The detection result is shown in fig. 5, and it can be seen that only 2 crocodile meat products have specific amplification curves, Ct values are 21.28 ± 0.20 and 20.58 ± 0.07 respectively, and the sources of the rest crocodile-free products are not detected, which indicates that the primer has high applicability and specificity and can be used for detecting crocodile sources in commercial processed products.
TABLE 5 Ct values of PCR amplification of DNA templates from different processed meat products
Sequence listing
<110> Nanjing university of agriculture
<120> primer pair for identifying crocodile-derived component in meat product and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Claims (9)
1. The primer pair for identifying the alligator-derived components in the meat products is characterized in that an upstream primer is shown as SEQ ID NO.1, and a downstream primer is shown as SEQ ID NO. 2.
2. Use of the primer pair of claim 1 for identifying alligator-derived components in meat products.
3. Use of the primer pair of claim 1 in the preparation of an alligator-derived component identification reagent.
4. A kit for identifying alligator-derived ingredients in meat products, characterized by comprising the primer pair of claim 1.
5. The kit according to claim 4, characterized in that it further comprises other reagents of SYBR Green fluorescent quantitative PCR.
6. A method for identifying alligator-derived components in meat products, characterized by comprising the step of performing real-time fluorescence PCR detection on sample DNA with the primer pair of claim 1.
7. The method according to claim 6, characterized by the steps of:
(1) extracting sample DNA;
(2) using the extracted sample DNA as a template, performing real-time fluorescent PCR amplification by using the primer pair of claim 1, and using QuantStudioTMAnd (3) analyzing the amplification curve and/or Ct value of the result by using Real-Time PCR Software.
8. The method of claim 6 or 7, wherein the reaction system of real-time fluorescence PCR is 20 μ L, comprising 10 μ L of TBGreen Taq II, 2 μ L of template DNA, 0.8 μ L of each of the upstream and downstream primers, 6 μ L of sterilized ultrapure water, and 0.4 μ L of ROX Reference Dye.
9. The process according to claim 6 or 7, characterized in that the reaction conditions are: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing and extension at 64 ℃ for 30s, and 40 cycles.
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Application publication date: 20201120 |