CN114836523B - Real-time fluorescence PCR detection method for detecting turkey-derived components in foods and feeds by utilizing single copy nuclear genes - Google Patents

Real-time fluorescence PCR detection method for detecting turkey-derived components in foods and feeds by utilizing single copy nuclear genes Download PDF

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CN114836523B
CN114836523B CN202210187422.9A CN202210187422A CN114836523B CN 114836523 B CN114836523 B CN 114836523B CN 202210187422 A CN202210187422 A CN 202210187422A CN 114836523 B CN114836523 B CN 114836523B
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CN114836523A (en
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王强
蔡一村
宁雪
张晨
许镇坚
潘良文
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Technical Center For Animal Plant and Food Inspection and Quarantine of Shanghai Customs
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Abstract

The invention discloses a real-time fluorescence PCR detection method for detecting turkey-derived components in foods and feeds by utilizing single-copy nuclear genes, which comprises the following steps: firstly, taking DNA of a sample to be detected as a template, and carrying out fluorescent quantitative PCR amplification to obtain a PCR amplification product; second, detecting the fluorescent signal of the amplified product; thirdly, judging whether the sample contains turkey-derived components or not according to the Ct value of the detection result; wherein, the reaction system for PCR amplification contains a specific primer pair for amplifying turkey-derived components and a specific probe for amplifying turkey-derived components. The specific primer pair and the probe for the real-time fluorescence PCR amplification of the turkey-derived components have good specificity and high sensitivity, provide a quantitative detection method for rapidly and accurately detecting whether the turkey-derived components are contained in foods and feeds, and have good application prospects.

Description

Real-time fluorescence PCR detection method for detecting turkey-derived components in foods and feeds by utilizing single copy nuclear genes
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to a real-time fluorescence PCR detection method for detecting turkey-derived components in foods and feeds by using single-copy nuclear genes.
Background
The common meat and meat product authenticity identification technology mainly comprises the following steps: (1) Immunoassay techniques based on protein molecular structure, etc.; the immunoassay method based on the protein macromolecular structure has the characteristics of high sensitivity, high flux and simple operation, and can realize rapid detection of a large number of samples. For example, macedo-Silva et al established ELISA methods for identifying inexpensive meat components potentially incorporated in hamburgers by preparing antisera against bovine, chicken, porcine, equine albumin, with a sensitivity of up to 0.6%. However, immunoassay methods based on antigen-antibody specific binding are prone to cross-reactions for closely related species. The fact that the tertiary structure of proteins may be destroyed during food processing to affect antibody recognition is also an important disadvantage of this approach. (2) Nucleic acid-based molecular biology techniques such as PCR, real-time fluorescent PCR, and molecular fingerprinting techniques; molecular biology techniques based on DNA sequence specificity have been widely used with differences in genetic information between animal species as detection targets for species identification. Compared with protein, DNA has high thermal stability, and can extract small fragment DNA for analysis such as PCR although there is a certain degree of degradation in the processing process. Meanwhile, the DNA has the advantages of high specificity, high sensitivity, no restriction by tissue types and the like, and has higher resolution capability for species with relatively close relatives. In recent years, the research on meat and meat product authenticity identification methods based on DNA detection technology is increasing. Currently, PCR-based assays are mainly focused on, including DNA sequencing, multiplex PCR, real-time fluorescent quantitative PCR, and the like. In particular, the real-time fluorescent quantitative PCR technology has become a mainstream detection technology in the field of detection of animal-derived components.
PCR technology synthesizes millions of copies of DNA in vitro by binding a specific oligonucleotide to the target DNA sequence. The amplification of DNA fragments by species-specific primers, separation by agarose electrophoresis and visualization of specific bands are the most basic method for identifying species components by PCR techniques. Mitochondrial DNA has large copy number in cells, high sensitivity, high evolution speed, higher interspecific diversity and lower intraspecies variation, and has been widely used for primer design and amplification of target sequences. Examples of common mitochondrial genes are cytochrome b genes, 12S and 16S ribosomal RNA subunits, and D-loop regions. However, since the copy number of mitochondrial DNA sequences is high and not constant, it can only be applied to qualitative detection of animal-derived components, and it is difficult to apply it to quantitative detection. "Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR) Analytical Methods", C.Floren, I.Wiedemann, B.Brenig, E.Sch utz, J.Beck, food Chemistry 173 (2015) 1054-1058, the conclusion of which suggests that the use of primers and probes of mitochondrial origin at the time of quantification leads to a deviation in the results.
At present, primers and probes for detecting animal components at home and abroad are generally designed aiming at mitochondrial genes, and the copy number of the mitochondrial genes in cells is 5000-6000 copies or more. But in actual research work it was found that: when a set of primers and probes are designed by utilizing multiple copies of mitochondrial genes for detection, the Ct value can be as low as about 13, and the Ct value is still about 37 when animal-derived components in a sample are 0.00000001%. At such low detection concentrations of the target analyte, such Ct values occur, making it difficult for the detector to determine whether the actual sample is actually containing the target component or whether the sample is very lightly contaminated. Therefore, the detection is carried out by utilizing the mitochondrial gene design primer and the probe, so that false positive results are easy to appear or the detection result under the condition of low target content is difficult to judge. If a positive detection report is presented based on this low Ct value, a significant trade dispute is elicited.
Therefore, it is necessary to establish a set of real-time fluorescence quantitative PCR detection method for turkey-derived components in foods and feeds, which has good specificity and high sensitivity and can avoid false positive results.
Disclosure of Invention
The invention is found by research: detecting animal-derived components in a certain animal pure meat sample, and detecting by using primers and probes designed by single copy nuclear genes, wherein the Ct value is 22-24; when the animal-derived component in the sample is 0.001%, the Ct value is about 37. Therefore, the invention utilizes the single-copy nuclear gene to carry out species identification detection, and the result finds that the condition that false positive results or difficult judgment results occur can be avoided by utilizing the single-copy nuclear gene detection, and quantitative detection can be carried out by utilizing the single-copy nuclear gene, so that the detection of searching the single-copy nuclear gene and establishing a standard method according to the detection is very necessary.
The primary aim of the invention is to provide a real-time fluorescence PCR detection method for turkey-derived components in foods and feeds, so as to overcome the defects of difficult quantitative detection, easy occurrence of false positive results and the like in the prior art. A second object of the present invention is to provide a detection kit and use of the detection kit. The third object of the invention is to provide a primer pair and a probe for a real-time fluorescence PCR detection method of turkey-derived components in foods and feeds.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
as a first aspect of the present invention, a real-time fluorescent PCR detection method for detecting turkey-derived components in foods and feeds using a single copy of a nuclear gene, the method comprising the steps of:
firstly, taking DNA of a sample to be detected as a template, and carrying out fluorescent quantitative PCR amplification to obtain a PCR amplification product;
second, detecting the fluorescent signal of the amplified product;
thirdly, judging whether the sample contains turkey-derived components or not according to the Ct value of the detection result, and quantitatively detecting the content of the turkey-derived components in the sample;
the reaction system for PCR amplification comprises a specific primer pair for amplifying turkey-derived components and a specific probe for detecting the turkey-derived components, wherein the sequences of the specific primer pair for amplifying the turkey-derived components are shown as SEQ ID NO.1 and SEQ ID NO.2, and the sequences of the specific probe for detecting the turkey-derived components are shown as SEQ ID NO. 3.
According to the invention, the conditions for PCR amplification are as follows:
the reaction system was 25. Mu.L: 12.5. Mu.L of 2 Xreal-time fluorescent PCR reagent, 10. Mu.M each of the primers, 1. Mu.L; 10. Mu.M probe 0.5. Mu.L, 20 ng/. Mu.L DNA template 5. Mu.L;
PCR reaction conditions: 95 ℃ for 10min;95 ℃ for 15s;60 ℃ for 1min; for 45 cycles.
As a second aspect of the present invention, a kit for detecting a turkey-derived component, the kit comprising the following reagents:
(a) A specific primer pair for amplifying turkey-derived components;
(b) A specific probe for detecting a turkey-derived component;
the sequences of the specific primer pairs for amplifying the turkey-derived components are shown in SEQ ID NO.1 and SEQ ID NO.2, and the sequences of the specific probes for detecting the turkey-derived components are shown in SEQ ID NO. 3.
Further, the detection kit further comprises:
(c) And the standard reference substance is used for quantitatively detecting the content of the turkey-derived components in the sample.
As a third aspect of the invention, a use of the above-described detection kit for qualitatively or quantitatively detecting a turkey-derived component in a food or feed.
As a fourth aspect of the invention, a specific primer pair and a probe for detecting turkey-derived components are provided, wherein the sequences of the specific primer pair are shown as SEQ ID NO.1 and SEQ ID NO.2, and the sequences of the specific probe are shown as SEQ ID NO. 3.
The beneficial effects of the invention are as follows:
1. the primer pair and the probe which are derived from the nuclear gene and can specifically identify the turkey-derived components are provided, the specificity is good, the specific amplification can be realized for the turkey-derived components, and the specific amplification can not be realized for other components except turkeys; moreover, the primer pair and the probe have good reproducibility and stable and reliable results.
2. The primer pair and the probe can be used for rapidly detecting whether turkey-derived components exist in food or feed in a large scale and quantitatively detecting the content of the turkey-derived components in the food or feed, and the primer pair and the probe have the advantages of small required sample amount, simple operation and high sensitivity.
3. The real-time fluorescence PCR detection method for the turkey-derived components is wide in application range, particularly suitable for detecting the turkey-derived components in various foods or feeds, and therefore can be popularized and applied, provides technical support for guaranteeing the quality of products, protecting the awareness and the option of consumers, maintaining normal economic order and the like, and provides technical support for market supervision departments and inspection and quarantine departments of foods.
Drawings
FIG. 1 is a real-time fluorescence PCR sensitivity test amplification plot for a first set of primer pairs and probes of the examples. Wherein the amplification curves are 40 ng/. Mu.L, 20 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 0.1 ng/. Mu.L, 0.01 ng/. Mu.L turkey DNA samples, respectively, from left to right.
FIG. 2 is a real-time fluorescence PCR sensitivity test amplification plot for the second set of primer pairs and probes of example 1. Wherein the amplification curves are 40 ng/. Mu.L, 20 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 0.1. 0.1 ng/. Mu.L turkey DNA samples, respectively, from left to right.
FIG. 3 is an amplification plot of a real-time fluorescent PCR primer-specific assay for the first set of primer pairs and probes of example 1. Wherein the amplification curve is a turkey standard.
FIG. 4 is a quantitative standard curve of turkey-derived components.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not specify specific conditions in the following examples, is generally followed by conventional conditions, such as "molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or conditions supplied by the manufacturer.
The experimental materials of the invention are as follows:
(1) DNA standards for turkeys (Meleagris gallopavo), pigs (susscufa), cattle (Bos taurus), goats (Capra hircus), sheep (Ovis aries), donkeys (Equus asinus), horses (Equus caballius), chickens (Gallus), ducks (Anas platyrhynchos), geese (Anser), quails (Coturnix coturnix), camels (Camelus dromedarius), domestic cats (Felis catus), and domestic dogs (Canis lupus familiaris) were purchased from american Zyagen Laboratories corporation.
(2) Animal components such as goat meat, beef, bream fish, pomfret fish, yellow croaker, salmon meat and the like are commercially available products.
(3) Rat meat and mouse meat were purchased from Shanghai Sipulel-BiKai laboratory animals Co.
(4) The plant components such as rice, corn, soybean, millet, mung bean, sweet potato, white kidney bean, apple, celery and the like are commercial products.
(5) Turkey, turkey ham, pork luncheon meat, dried pork, pork ham sausage (inlet), mutton flavor dog food, mutton chop, beef flavor cat food, chicken ham sausage (inlet), beef luncheon meat, braised beef can, beef jerky, beef ball, curry beef paste, chicken flavor dog food (inlet), beef flavor dog food (inlet), and swallow fish cat can (inlet) are commercially available products.
(6) Australian pet grade chicken bone meal is provided by Shanghai customs ports.
Example 1 design of primer pairs and probes
(1) Design of first primer pair and probe
Based on the published turkey (Meleagris gallopavo chromosome Z) DNA sequence (accession number: NC_ 015041.2) in NCBI, a suitable sequence fragment was selected for primer probe design.
The length of the target fragment for the turkey-derived component is 118bp. The sequences of the fluorescent PCR primer pairs and probes are as follows:
Turkey-118bp-F:5’-TGAACAAATCCACTTCCCTTTAACC-3’ (SEQ ID NO:1)
Turkey-118bp-R:5’-TCATTTCTGCTGCACAAAGTGAG-3’ (SEQ ID NO:2)
Turkey-118bp-P:FAM-TGATGACGAGCCGCAGCCACACC-TAMRA (SEQ ID NO:3)
wherein FAM represents a fluorescent reporter group and TAMRA represents a quencher group. The invention adopts a fluorescent probe method, and the detection principle is that a fluorescent labeled specific probe is utilized to identify a template. Compared with the SYBR dye method in the prior art, the fluorescent marker specific probe has stronger specificity and lower background interference.
(2) Design of second primer pair and probe
Based on published turkey (Meleagris gallopavo chromosome) gene sequences (accession number: NC_ 015037.2) in NCBI, appropriate fragments were selected to design primer pairs and probes as follows:
Turkey-130bp-F:5’-AGAGGATAGGATGGATGTCGATTTG-3’ (SEQ ID NO:4)
Turkey-130bp-R:5’-CCTTGCTGCGGGAACAAAAG-3’ (SEQ ID NO:5)
Turkey-130bp-P:FAM-ACATCCTTCCTCCATTGCCTCCGTCTCC-TAMRA (SEQ ID NO:6)
wherein FAM represents a fluorescent reporter group and TAMRA represents a quencher group. The invention adopts a fluorescent probe method, and the detection principle is that a fluorescent labeled specific probe is utilized to identify a template. Compared with the SYBR dye method in the prior art, the fluorescent marker specific probe has stronger specificity and lower background interference.
(3) Reaction system and reaction program for real-time fluorescent PCR of first and second primer pairs and probes
The real-time fluorescent PCR reaction system is shown in Table 1:
wherein, the real-time fluorescence PCR reaction program is as follows: 95 ℃ for 10min; 15s at 95℃and 1min at 60 ℃; for 45 cycles.
TABLE 1 real-time fluorescent PCR reaction System for turkey Source components
Example 2 preparation of test samples
The animal meat sample was minced and dried and ground to a powder using a freeze grinder (SPEX 6850). Plant samples and samples for practical verification were ground directly into powder using a freeze mill (SPEX 6850).
EXAMPLE 3 extraction of DNA
The DNA of the animal sample is extracted by using an animal genome extraction kit (Tian Gen Biotechnology Co., ltd.; catalog number: DP 323), the DNA of the plant sample is extracted by using a plant genome extraction kit (Tian Gen Biotechnology Co., ltd.; catalog number: DP 305), the DNA of the mixed sample and the actual verification sample is extracted by using an animal-derived plant feed genome extraction kit (Tian Gen Biotechnology Co., ltd.; catalog number: DP 323), and the extraction method is described in the operation specification of the kit. The DNA solution was kept at-20℃until use.
Example 4 sensitivity detection of primer pairs and probes for first and second sets of turkey-derived Components
Real-time fluorescence PCR was performed with turkey DNA content of 40 ng/. Mu.L, 20 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 0.1 ng/. Mu.L, 0.01 ng/. Mu.L, 0.001 ng/. Mu.L and 0.0001 ng/. Mu.L as templates, and the experiment was repeated 6 times.
Results: (1) a first set of primer pairs and probes: in 6 assays, amplification curves were shown in turkey sample DNA at 40 ng/. Mu.L, 20 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 0.1 ng/. Mu.L, 0.01 ng/. Mu.L, and no amplification curves were shown in turkey sample DNA at 0.001 ng/. Mu.L and 0.0001 ng/. Mu.L, see FIG. 1.
(2) Second primer pair and probe: in 6 assays, amplification curves were shown in turkey sample DNA at 40 ng/. Mu.L, 20 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 0.1 ng/. Mu.L, but the amplification curves were not proportional to DNA concentration, whereas no amplification curves were shown in turkey sample DNA at 0.01 ng/. Mu.L, 0.001 ng/. Mu.L, and 0.0001 ng/. Mu.L, see FIG. 2.
Conclusion: 1. when the template usage amount of the first primer pair and the probe is 0.01 ng/. Mu.L, the first primer pair and the probe have obvious S-shaped amplification curves, and the detection sensitivity reaches 0.01 ng/. Mu.L; the primer pair and the probe of the first turkey source component have better accuracy; 2. the second primer pair and probe are significantly different from the first primer pair and probe. The effect of the second primer pair and probe in the sensitivity detection is not obvious.
Since the primer pairs and probes of the second set of turkey-derived components are not significantly effective in the sensitivity detection, the subsequent experiments were directed to only the primer pairs and probes of the first set of turkey-derived components.
Example 5 specific detection of primer pairs and probes for turkey-derived Components
In this example, the 32 animal and plant material DNA samples tested in example 3 were tested using the primer set and probe set for the turkey-derived component of example 1 to detect the fluorescent signal of the amplified product. The results of the measurements are summarized in Table 2 and FIG. 3.
TABLE 2 results of specific detection of turkey-derived Components
As can be seen from FIG. 3 and Table 2, the primer pairs and probes were positive only for turkey DNA meat samples, had a significant S-type amplification curve, and were negative for DNA samples of a plurality of other species, without a significant S-type amplification curve.
Conclusion: the detection method has good species specificity.
Example 6 practical verification of primer pair and Probe of turkey-derived Components
The detection method of example 4 was used to detect 19 samples of meat-based food, animal feed, etc. collected in the market and in the import and export trade. The results are shown in Table 3.
TABLE 3 detection results of turkey-derived Components of 19 samples to be tested
Conclusion: as can be seen from table 3, turkey-derived components were detected in turkey meat and turkey ham; no turkey-derived components were detected in the remaining food and feed. It is also demonstrated that the detection method of example 5 can be used to detect whether turkey-derived components are present in food or feed in large quantities.
EXAMPLE 7 establishment of quantitative Standard Curve
(1) Quantitative detection of turkey-derived component primer pair and probe
Turkey DNA obtained by the extraction of example 3 was diluted to 100 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 0.1 ng/. Mu.L, 0.01 ng/. Mu.L, and five concentration gradients were added, and the first set of primer pairs and probes of example 1 were used for detection, with 6 replicates for each concentration, to obtain Ct values. The negative control was water. As shown in table 4, wherein Ct value is the average of 6 replicates.
TABLE 4 Ct value for real-time fluorescent PCR quantitative detection assay
Conclusion: as can be seen, the amplified Ct values increase in a gradient with decreasing concentration of turkey-derived components, and have a linear relationship (R 2 =0.998)。
(2) Data processing and computation
As a result, as shown in FIG. 4, the quantitative standard straight line is obtained by curve fitting the points with the logarithmic value of the DNA concentration as the abscissa and the average of Ct values of 6 repeated tests of the sample as the ordinate, and the linear equation is y=3.779x+19.309, and R 2 =0.998. For the sample to be quantified, each Ct value of the sample to be measured is measured each time, and the corresponding x value is obtained by using a standard curve formula. The content of turkey source components of the sample to be detected can be obtained.
(3) Verification of quantitative Standard Curve
DNA was extracted as in example 3, diluted and quantified in 6 independent samples at concentrations of 50 ng/. Mu.L and 0.5 ng/. Mu.L, respectively, and Ct values were measured and the content of turkey-derived components therein was calculated by a quantitative standard curve to verify the reproducibility of the method.
The results showed that the average value of 50ng of the sample measurement values was 49.71ng, and the average value of 0.5ng of the sample measurement values was 0.48ng.
Conclusion: the method of this example is described as having the ability to accurately quantify over a range of dynamic conditions. Therefore, the method proves that the actual measured value accords with the theoretical value and has good repeatability.
The invention establishes a real-time fluorescence PCR method for detecting turkey-derived components in foods and feeds by utilizing single-copy nuclear genes, and designs primer pairs and probes aiming at the nuclear genes of turkey species, and the detection of whether the turkey-derived components exist in a sample can be accurately carried out only by combining a real-time fluorescence amplification curve graph for no more than two hours, wherein the sensitivity is 0.01 ng/mu L.
In conclusion, the specific primer pair and the probe for the real-time fluorescence PCR amplification of the turkey-derived components designed by the invention have good specificity and high sensitivity, provide a good detection method for rapidly and accurately distinguishing whether the turkey-derived components are contained in food or feed, and have good application prospects in the detection aspect of the food safety field. Furthermore, the specific primer pairs and probes may be further formulated into a detection kit using methods known in the art, which may include standard references in addition to the specific primer pairs and probes, as will be apparent to those skilled in the art.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
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Claims (6)

1. A real-time fluorescent PCR detection method for detecting turkey-derived components in foods and feeds using a single copy of a nuclear gene, comprising the steps of:
firstly, taking DNA of a sample to be detected as a template, and carrying out fluorescent quantitative PCR amplification to obtain a PCR amplification product;
second, detecting the fluorescent signal of the amplified product;
thirdly, judging whether the sample contains turkey-derived components or not according to the Ct value of the detection result, and quantitatively detecting the content of the turkey-derived components in the sample;
the reaction system for PCR amplification comprises a specific primer pair for amplifying turkey-derived components and a specific probe for detecting the turkey-derived components, wherein the sequences of the specific primer pair for amplifying the turkey-derived components are shown as SEQ ID NO.1 and SEQ ID NO.2, and the sequences of the specific probe for detecting the turkey-derived components are shown as SEQ ID NO. 3.
2. The method of claim 1, wherein the PCR amplification conditions are as follows:
the reaction system was 25. Mu.L: 12.5. Mu.L of 2 Xreal-time fluorescent PCR reagent, 10. Mu.M each of the primers, 1. Mu.L; 10. Mu.M probe 0.5. Mu.L, 20 ng/. Mu.L DNA template 5. Mu.L;
PCR reaction conditions: 95 ℃ for 10min; 15s at 95℃and 1min at 60 ℃; for 45 cycles.
3. A kit for detecting a turkey-derived component, comprising the following reagents:
(a) A specific primer pair for amplifying turkey-derived components;
(b) A specific probe for detecting a turkey-derived component;
the sequences of the specific primer pairs for amplifying the turkey-derived components are shown in SEQ ID NO.1 and SEQ ID NO.2, and the sequences of the specific probes for detecting the turkey-derived components are shown in SEQ ID NO. 3.
4. The test kit of claim 3, wherein the test kit further comprises:
(c) Standard reference.
5. Use of the detection kit according to claim 3 or 4 for qualitative or quantitative detection of turkey-derived components in food or feed.
6. A specific primer pair and a probe for detecting turkey-derived components are characterized in that the sequences of the specific primer pair are shown in SEQ ID NO.1 and SEQ ID NO.2, and the sequence of the specific probe is shown in SEQ ID NO. 3.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102433382A (en) * 2011-12-07 2012-05-02 上海出入境检验检疫局动植物与食品检验检疫技术中心 Real-time fluorescent polymerase chain reaction (PCR) detection method for turkey ingredient in foods and feeds
CN106521016A (en) * 2016-12-30 2017-03-22 上海出入境检验检疫局动植物与食品检验检疫技术中心 Real-time fluorescence PCR detection method for detecting bovine-derived materials in food and feed by use of single-copy nuclear gene
CN108085374A (en) * 2018-02-13 2018-05-29 广东出入境检验检疫局检验检疫技术中心 A kind of dual digital pcr method that turkey derived component quantitatively detects

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102433382A (en) * 2011-12-07 2012-05-02 上海出入境检验检疫局动植物与食品检验检疫技术中心 Real-time fluorescent polymerase chain reaction (PCR) detection method for turkey ingredient in foods and feeds
CN106521016A (en) * 2016-12-30 2017-03-22 上海出入境检验检疫局动植物与食品检验检疫技术中心 Real-time fluorescence PCR detection method for detecting bovine-derived materials in food and feed by use of single-copy nuclear gene
CN108085374A (en) * 2018-02-13 2018-05-29 广东出入境检验检疫局检验检疫技术中心 A kind of dual digital pcr method that turkey derived component quantitatively detects

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ISO/PRF TS 20224-8:2022;ISO Copyright Office;ISO/PRF TS 20224-8:2022;20220621;第1-11页 *
火鸡源性成分实时荧光 PCR 检测方法 ISO 标准研制;潘良文等;食品科学;20230625;第1-14页 *
食品和饲料中火鸡源性成分的实时荧光PCR检测方法;张舒亚等;食品与生物技术学报;20130215;32(02);第101-105页 *

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