CN116287290B - Fluorescent quantitative PCR method for identifying tilapia source component in fish meal - Google Patents
Fluorescent quantitative PCR method for identifying tilapia source component in fish meal Download PDFInfo
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Abstract
The invention discloses a fluorescent quantitative PCR method for identifying tilapia source components in fish meal, and belongs to the technical field of feed raw material detection. According to the specific gene KPL4 of tilapia mossambica, a fluorescent quantitative PCR primer is designed, the nucleotide sequence of the primer is shown as SEQ ID NO.1-2, and the specificity of the primer is verified. And (3) carrying out fluorescent quantitative PCR amplification on DNA of the fish meal sample to be detected by using the primer, calculating the DNA content of the tilapia source component in the sample to be detected according to an amplification curve, and judging whether the fish meal contains the tilapia source component and the actual content range thereof. The identification method is simple, convenient and quick, has high sensitivity, can lower the detection limit of the tilapia source component to 0.01 percent, and is a technical index of the detection sensitivity of the tilapia source component in the fish meal which is firstly proposed at home and abroad. The method is also suitable for detecting tilapia-derived components in other animal raw materials.
Description
Technical Field
The invention relates to the technical field of feed raw material detection, in particular to a fluorescent quantitative PCR method for identifying tilapia source components in fish meal.
Background
Fish meal is an indispensable, high quality animal feed material in the feed industry. Because of the high price of fish meal, the market is flooded with adulterated fish meal, including the incorporation of various animal and plant raw materials into the fish meal, thereby reducing the nutritive value of the fish meal and also causing serious trouble for judging the quality of the fish meal. At present, fish meal adulteration identification is still mainly carried out by a microscopic method, namely, a tissue of a suspected adulteration is searched under a microscope, the adulteration is roughly identified according to tissue characteristics, sometimes, the identification result is required to be supplemented and judged by a physical and chemical method, and the problems of high omission factor, low sensitivity, poor repeatability, incapability of quantification, dependence on personal experience and the like exist. In recent years, near infrared spectrum technology has been developed to identify substances that are significantly different from standard substances to identify adulterants, but near infrared calibration models of fish meal and other raw materials have been established. Meanwhile, the recognition result of the near infrared spectrum is easy to be influenced by factors such as the instrument type, the measuring environment, the sample loading mode and the like, the sample state and the like, so that the effect and the applicability of the built model are influenced, and the universal applicability is not realized. Therefore, there is a need for a simple, fast, sensitive and accurate adulteration identification method.
The real-time fluorescence quantitative PCR detection technology is applied, and specific primers which are designed and obtained aiming at mitochondrial genes of different species are used for identifying target species, so that the method is widely applied to the authenticity identification of processed foods and traditional Chinese medicines. The PCR technology is utilized to identify the feed species composition or the raw material adulteration, and related research reports exist, for example, italian researchers use the PCR technology to detect bovine mitochondrial DNA fragments so as to detect bovine-derived components in animal-derived feeds, and the PCR products are sequenced and confirmed, so that the detection sensitivity can reach 0.1125%; the method for rapidly detecting the bovine-derived components in animal-derived feeds by using the PCR technology is also established in the United states, and detection reports on the bovine, sheep, pig, chicken and horse-derived components in feeds are also reported in China. High quality fish meal is typically made from sea fish caught in the sea. Among the adulterants, tilapia powder produced by using tilapia offal as a raw material is a common one, but no report of fluorescent quantitative PCR identification of tilapia-derived components in the tilapia powder is currently seen at home and abroad.
Disclosure of Invention
The invention aims to provide a fluorescence quantitative PCR method for identifying tilapia source components in fish meal, so as to solve the problems in the prior art. The method provided by the invention can rapidly and sensitively detect whether the tilapia source component exists or not and the content range of the tilapia source component in the fish meal to be detected, and the minimum detection limit can reach 0.01%.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a primer combination for quantitatively identifying tilapia-derived components by fluorescence, which comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.1 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 2. The primer has good specificity to tilapia through carrying out fluorescent quantitative PCR amplification on 50 common Perciformes, cyprinus fishes and shrimps and crabs including tilapia.
The primer group is used for carrying out fluorescent quantitative PCR amplification on the DNA of the fish meal to be detected, and the content of tilapia source components in the fish meal to be detected is judged according to a fluorescent quantitative detection curve.
Further, the method comprises the following steps:
(1) Preparing a fluorescence quantitative standard curve;
(2) Taking fish meal DNA to be detected as a template, and performing fluorescent quantitative PCR amplification;
(3) Qualitatively identifying tilapia source components in the fish meal to be tested according to a fluorescence quantitative PCR amplification curve; if a fluorescence amplification curve appears and the Ct value is less than or equal to 34, judging that the fish meal to be tested contains tilapia source components; if the fluorescence amplification curve does not exist or the Ct value is more than or equal to 34, judging that the fish meal to be tested does not contain tilapia-derived components;
(4) And (3) calculating the DNA content of the tilapia source component in the fish meal to be detected according to the fluorescence quantitative standard curve manufactured in the step (1), and judging the actual content range of the tilapia in the fish meal to be detected according to the DNA content.
Further, the reaction system of the fluorescent quantitative PCR amplification is 2X ChamQ SYBR qPCR Master Mix mu L,10 mu mol/L of upstream primer is 0.4 mu L,10 mu mol/L of downstream primer is 0.4 mu L, 2 mu L of template is 2 mu L, ultra-pure sterile water is 7.2 mu L, and the total reaction volume is 20 mu L; the template is selected fish meal DNA.
Further, the reaction condition of the fluorescent quantitative PCR amplification is that the reaction is pre-denatured for 30s at 95 ℃;95℃for 5s,60℃for 30s,40 cycles.
The invention also provides application of the primer combination in identifying tilapia-derived components in animal feed raw materials.
The invention discloses the following technical effects:
the invention designs the tilapia fluorescent quantitative primer and verifies the specificity of the primer. And then carrying out fluorescent quantitative PCR amplification on the tilapia DNA with different concentration gradients in the sample by using the primer to obtain a standard curve, and establishing a regression equation between the concentration of the tilapia DNA and the Ct value. On the basis, whether the fish meal to be detected contains tilapia source components or not is judged by detecting Ct values of fish meal samples to be detected, the content (mass concentration) range is clear, and the lowest detection limit can reach 0.01%. The identification method is simple, convenient and quick, has high sensitivity and good repeatability, and can realize qualitative and rough quantitative detection of adulterated components. The detection process does not depend on personal experience and is not influenced by factors such as sample form, detection environment and the like. The method is also suitable for quantitative detection of tilapia source components in other animal feed raw materials.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a fluorescent quantitative PCR amplification curve diagram of tilapia primer specificity verification;
FIG. 2 is a fluorescence quantitative PCR amplification curve of different DNA concentration gradients of tilapia;
FIG. 3 is a fluorescence quantitative PCR amplification curve of homemade fish meal with different amounts of tilapia meal;
FIG. 4 is a fluorescent quantitative PCR amplification curve of DNA of seven fish meal samples.
Detailed Description
1. Primer design
The tilapia-derived gene selected by the invention is a tilapia specific gene (KPL 4 gene, geneBank, accession number AH 013711), and a specific amplification primer is designed according to the gene, wherein the specific primer sequence is as follows: upstream primer (SEQ ID NO: 1): 5'-CCCCACTTGAAAGGGACTGA-3', downstream primer (SEQ ID NO: 2): 5'-ATGGCTGAAAGGAGGATTGA-3'. Synthesized by Shanghai Biolimited.
2. Specificity verification of primers
And selecting nearly 50 conventional fishes such as weever, carp and the like, and carrying out specificity verification on the related primers.
DNA sample extraction
The method takes nearly 50 conventional fishes, such as tilapia, other perciformes, cyprinus and the like, which are sold in the market as DNA sample sources, and extracts DNA of a sample to be detected by using an animal tissue DNA extraction kit (purchased from Tiangen biochemical technology (Beijing) and the kit model: DP 304), and comprises the following steps: firstly, crushing a sample, adding 200 mu L of buffer GA and protease K, shaking and mixing uniformly, and completely cracking at 56 ℃; 200. Mu.L of buffer GB is added and the mixture is left at 70 ℃ for 10min; adding 200 mu L of absolute ethyl alcohol, and fully oscillating and uniformly mixing for 15sec; adding the obtained solution into an adsorption column CB3, centrifuging at 12000rpm for 30sec, and discarding the waste liquid; adding 500 μl of buffer GD, centrifuging at 12000rpm for 30sec, discarding the waste liquid, adding 600 μl of rinse PW, centrifuging at 12000rpm for 30sec, pouring the waste liquid, repeating for 2 times, and air drying the residual rinse liquid of the adsorption material; transferring the adsorption column CB3 into a clean centrifuge tube, dripping 100 mu L of sterilized water, standing at room temperature for 2-5min, and centrifuging at 12000rpm for 2min, wherein the solution in the centrifuge tube is the DNA extraction.
Absorbance a260 and a280 at 260nm and 280nm were measured using a micro ultraviolet spectrophotometer, and the purity of DNA was determined as a ratio of a260/a280 and the DNA concentration was calculated. DNA with a mass concentration of 100 ng/. Mu.L was selected for subsequent manipulation.
2.2 verification of the specificity of the primers
Except tilapia, the extracted 50 fish DNA are randomly divided into 5 groups, 10 fish in each group are mixed by taking 1 mu L of 10 fish DNA samples in each group to obtain 10 mu L of mixed fish DNA samples, the 5 mixed fish DNA is detected by using KPL4 gene primer sequences for fluorescence quantitative PCR, and the shrimp and crab are detected by using representative samples such as penaeus vannamei, macrobrachium rosenbergii, penaeus monodon, portunid and blue crab.
The sample DNA was used as a template, and real-time fluorescent PCR amplification was performed using the upstream primer (SEQ ID NO: 1) and the downstream primer (SEQ ID NO: 2). The amplification system was ChamQ SYBR qPCR Master Mix (2X) 10. Mu.L, 10. Mu. Mol/L of the upstream primer 0.4. Mu.L, 10. Mu. Mol/L of the downstream primer 0.4. Mu.L, 2. Mu.L of the DNA template, 7.2. Mu.L of ultrapure sterile water and the total reaction volume was 20. Mu.L. The reaction solution of the amplification system was placed on ice. The amplification reaction conditions are 95 ℃ presegeneration for 30s;95℃for 5s,60℃for 30s,40 cycles. The sample was subjected to fluorescent quantitative PCR detection, and the detection results are shown in FIG. 1.
As can be seen from FIG. 1, by using the designed specific primer for tilapia, fluorescent quantitative PCR amplification is carried out on 50 kinds of common Perciformes, cyprinus fishes and shrimps crabs including tilapia, only tilapia (Ct value < 35) can be amplified, but other species (Ct value < 35) cannot be amplified. The designed primer has good tilapia source specificity.
3. Drawing a fluorescent quantitative standard curve of tilapia source DNA
The DNA in the tilapia muscle was extracted and the DNA concentration was calculated according to the method of 2.1 using commercially available tilapia muscle as a source of DNA sample. DNA with a mass concentration of 100 ng/. Mu.L was selected for subsequent manipulation.
Continuously diluting the extracted tilapia DNA (the mass concentration is 100 ng/. Mu.L) by 10 times of sterilized water to ensure that the DNA content respectively reaches 100%, 10%, 1%, 0.1% and 0.01% of the actual sample content, and respectively taking sample DNA with different dilution concentrations as templates and utilizing SEQ ID NO:1-2, and performing real-time fluorescence PCR amplification according to the method of 2.2. The amplification results are shown in FIG. 2.
According to the amplification curve, a regression equation between the DNA concentration and the Ct value is established, the Log DNA concentration is plotted as an abscissa, the Ct value is plotted as an ordinate, an amplification standard curve of the tilapia DNA is drawn, the amplification is repeated for 3 times, and the average Ct value is calculated. The obtained fluorescent quantitative standard curve of the tilapia source component is as follows: y= -2.704x+25.99, where Y is Ct value and X is Log DNA value. Wherein, the Ct value of fluorescence quantitative PCR amplification of different DNA concentration gradients of tilapia is shown in Table 1.
TABLE 1 fluorescent quantitative PCR amplified Ct values for different DNA concentration gradients of Tilapia
Example 1 identification of Tilapia Source fractions containing different concentrations in fish meal
The method comprises the steps of preparing a tilapia-free fish meal sample, respectively mixing 10%, 1%, 0.1%, 0.01%, 0.001% and 0.0001% (mass fraction) of tilapia meal into the tilapia-free fish meal sample according to mass percentage, and fully and uniformly mixing to prepare 6 parts of fish meal samples. DNA was extracted from these fish meal samples according to method 2.1 and concentration was measured. The extracted fish meal DNA is used as a template, and the real-time fluorescence PCR amplification is carried out by using the primer shown in SEQ ID NO.1-2, wherein the amplification conditions and the amplification system are the same as 2.2. Three parallel experiments were performed on each sample to obtain a fluorescent quantitative PCR amplification curve (fig. 3) and corresponding Ct values (table 2):
TABLE 2 fluorescence quantitative PCR amplified Ct value of self-made fish meal with different amounts of tilapia meal
As can be seen from fig. 3 and table 2: when 0.01-10% of tilapia powder is mixed in the fish powder, the Ct value range of fluorescence quantitative PCR is 26.81+ -0.14-32.47 + -0.10, which is lower than 34.0, and the tilapia powder mixed with a certain amount can be judged. When 0.001% of tilapia powder is mixed into the fish powder, the Ct value of fluorescence quantitative PCR is (34.04+/-0.28), ct value is more than 34, and the tilapia content is extremely low and can not be detected. Therefore, the lowest detection limit of tilapia is considered to be 0.01%. However, the conventional microscopic examination method in the feed industry can only perform qualitative detection, and once no tilapia characteristic tissue is found in the field of view of a microscope, the tilapia characteristic tissue is judged to be undetected, the probability of missed detection is extremely high, and no detection limit standard exists. Therefore, the minimum detection limit of 0.01% of the invention is a technical index of the detection sensitivity of tilapia source components in the fish meal proposed at home and abroad for the first time, so that quantitative analysis of freshwater fish components in the fish meal becomes possible, and an innovative method is provided for fish meal quality judgment and evaluation in the feed industry.
Example 2 practical application of fluorescent quantitative PCR method for identifying tilapia-derived components in fish meal
The identification of tilapia source components was performed on 7 commercial fish meal. Wherein three fish meal samples are suspected to be doped with tilapia meal (named as doped fish meal 1, 2 and 3) in a certain proportion, and the other four fish meal samples are fish meal samples for preliminary judgment of undoped tilapia, and comprise two domestic fish meal (named as domestic pure fish meal 1 and 2) and two imported fish meal (named as imported pure fish meal 1 and 2). Extracting DNA of 7 fish meal samples according to the method of 2.1, detecting the concentration, and carrying out real-time fluorescence PCR amplification by using the DNA as a template and using the primer of SEQ ID NO.1-2, wherein the amplification conditions and the amplification system are the same as 2.2. Three parallel experiments were performed on each sample to obtain a fluorescent quantitative PCR amplification curve (fig. 4) and corresponding Ct values (table 3):
TABLE 3 Ct values for fluorescent quantitative PCR amplification of seven fish meal samples
As can be seen from FIG. 4 and Table 3, the primer designed by the invention is used for fluorescence quantitative PCR, ct values of domestic pure fish meal 1 and 2 and imported pure fish meal 1 and 2 are above 38 and are greater than a critical value 34, and partial samples even no amplification curve are judged to be undetected tilapia mossambica components, which accords with the initial judgment condition. The Ct values of fluorescence quantitative PCR of the doped fish meal 1, 2 and 3 are 26.22+/-0.50, 29.67+/-0.42 and 31.84+/-2.28 respectively, which are smaller than the critical value 34. According to table 2 in example 3, the doping ratios of tilapia in the doped fish meal 1, 2, 3 were determined as follows: 10%, 1% -10% and 0.01% -0.1%.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (6)
1. A fluorescent quantitative PCR method for identifying tilapia-derived components in fish meal is characterized in that an upstream primer with a nucleotide sequence shown as SEQ ID NO.1 and a downstream primer with a nucleotide sequence shown as SEQ ID NO.2 are used for carrying out fluorescent quantitative PCR amplification on fish meal DNA to be detected, and the content of the tilapia-derived components in the fish meal to be detected is determined according to a fluorescent quantitative detection curve;
the method comprises the following steps:
(1) Preparing a fluorescence quantitative standard curve;
(2) Taking fish meal DNA to be detected as a template, and performing fluorescent quantitative PCR amplification;
(3) Qualitatively identifying tilapia source components in the fish meal to be tested according to a fluorescence quantitative PCR amplification curve; if a fluorescence amplification curve appears and the Ct value is less than or equal to 34, judging that the fish meal to be tested contains tilapia-derived components; if the fluorescence amplification curve does not exist or the Ct value is more than or equal to 34, judging that the fish meal to be tested does not contain tilapia-derived components;
(4) And (3) calculating the DNA content of the tilapia source component in the fish meal to be detected according to the fluorescence quantitative standard curve manufactured in the step (1), and judging the actual content range of the tilapia in the fish meal to be detected according to the DNA content.
2. The method according to claim 1, wherein the reaction system for fluorescent quantitative PCR amplification is 2X SYBR qPCR Master Mix. Mu.L, 10. Mu. Mol/L of upstream primer 0.4. Mu.L, 10. Mu. Mol/L of downstream primer 0.4. Mu.L, 2. Mu.L of template, 7.2. Mu.L of ultrapure sterile water, and the total reaction volume is 20. Mu.L; the template is fish meal DNA to be detected.
3. The method according to claim 1, wherein the reaction conditions for the fluorescent quantitative PCR amplification are 95 ℃ pre-denatured for 30s;95℃for 5s,60℃for 30s,40 cycles.
4. The application of the fluorescent quantitative PCR detection primer combination in identifying tilapia-derived components in animal feed raw materials is characterized in that the fluorescent quantitative PCR detection primer combination is an upstream primer of a nucleotide sequence shown as SEQ ID NO.1 and a downstream primer of a nucleotide sequence shown as SEQ ID NO. 2.
5. The use according to claim 4, wherein the animal feed material comprises fish meal.
6. The use according to claim 4, wherein said identification comprises qualitative and quantitative identification.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667449A (en) * | 2013-11-07 | 2014-03-26 | 中国水产科学研究院淡水渔业研究中心 | Real-time fluorescence PCR (polymerase chain reaction) method and primer pair for identifying gender of oreochromis niloticus |
| CN105368948A (en) * | 2015-11-30 | 2016-03-02 | 中国水产科学研究院淡水渔业研究中心 | Primer for sex identification of Nile tilapia and PCR (polymerase chain reaction) identification method |
| KR20170000866A (en) * | 2015-06-24 | 2017-01-04 | 배재대학교 산학협력단 | Probe for identifying sea bream species and tilapia species and identifying method using the same |
| CN108841944A (en) * | 2018-07-31 | 2018-11-20 | 仲恺农业工程学院 | The method for identifying molecules that raw material fish forms in fish meal |
| CN111485028A (en) * | 2019-01-29 | 2020-08-04 | 广东美格基因科技有限公司 | Fluorescent quantitative PCR method for detecting tilapia streptococcus agalactiae and corresponding kit |
| WO2022180399A1 (en) * | 2021-02-25 | 2022-09-01 | The University Court Of The University Of Edinburgh | Predicting resistance to tilapia lake virus |
-
2023
- 2023-01-18 CN CN202310057100.7A patent/CN116287290B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667449A (en) * | 2013-11-07 | 2014-03-26 | 中国水产科学研究院淡水渔业研究中心 | Real-time fluorescence PCR (polymerase chain reaction) method and primer pair for identifying gender of oreochromis niloticus |
| KR20170000866A (en) * | 2015-06-24 | 2017-01-04 | 배재대학교 산학협력단 | Probe for identifying sea bream species and tilapia species and identifying method using the same |
| CN105368948A (en) * | 2015-11-30 | 2016-03-02 | 中国水产科学研究院淡水渔业研究中心 | Primer for sex identification of Nile tilapia and PCR (polymerase chain reaction) identification method |
| CN108841944A (en) * | 2018-07-31 | 2018-11-20 | 仲恺农业工程学院 | The method for identifying molecules that raw material fish forms in fish meal |
| CN111485028A (en) * | 2019-01-29 | 2020-08-04 | 广东美格基因科技有限公司 | Fluorescent quantitative PCR method for detecting tilapia streptococcus agalactiae and corresponding kit |
| WO2022180399A1 (en) * | 2021-02-25 | 2022-09-01 | The University Court Of The University Of Edinburgh | Predicting resistance to tilapia lake virus |
Non-Patent Citations (1)
| Title |
|---|
| Oreochromis mossambicus (tilapia) corticotropin-releasing hormone: cDNA sequence and bioactivity;F H van Enckevort et al.;J Neuroendocrinol;第12卷(第2期);第177-186页 * |
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