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 PDFInfo
- Publication number
- 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
- Authority
- CN
- China
- Prior art keywords
- turkey
- derived components
- detecting
- seq
- probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 56
- 235000013305 food Nutrition 0.000 title claims abstract description 30
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 20
- 239000000523 sample Substances 0.000 claims abstract description 91
- 230000003321 amplification Effects 0.000 claims abstract description 22
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 22
- 238000012408 PCR amplification Methods 0.000 claims abstract description 10
- 238000006243 chemical reaction Methods 0.000 claims abstract description 10
- 238000003753 real-time PCR Methods 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 18
- 238000012360 testing method Methods 0.000 claims description 6
- 239000012807 PCR reagent Substances 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 15
- 108020004414 DNA Proteins 0.000 description 34
- 241001465754 Metazoa Species 0.000 description 15
- 235000013372 meat Nutrition 0.000 description 14
- 241000894007 species Species 0.000 description 10
- 235000015278 beef Nutrition 0.000 description 8
- 238000013461 design Methods 0.000 description 8
- 241000196324 Embryophyta Species 0.000 description 7
- 108020005196 Mitochondrial DNA Proteins 0.000 description 7
- 238000000605 extraction Methods 0.000 description 6
- 241000282472 Canis lupus familiaris Species 0.000 description 5
- 241000287828 Gallus gallus Species 0.000 description 5
- 241000288147 Meleagris gallopavo Species 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 235000013330 chicken meat Nutrition 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 241000282326 Felis catus Species 0.000 description 4
- 239000000796 flavoring agent Substances 0.000 description 4
- 235000019634 flavors Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000012795 verification Methods 0.000 description 4
- 241000283690 Bos taurus Species 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 241000286209 Phasianidae Species 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000003018 immunoassay Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 235000013622 meat product Nutrition 0.000 description 3
- 235000015277 pork Nutrition 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 241000272517 Anseriformes Species 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 241000283074 Equus asinus Species 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 238000011304 droplet digital PCR Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000001823 molecular biology technique Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 125000006853 reporter group Chemical group 0.000 description 2
- 235000013580 sausages Nutrition 0.000 description 2
- 238000011895 specific detection Methods 0.000 description 2
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 241001519451 Abramis brama Species 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 241000272525 Anas platyrhynchos Species 0.000 description 1
- 241000272808 Anser Species 0.000 description 1
- 240000007087 Apium graveolens Species 0.000 description 1
- 235000015849 Apium graveolens Dulce Group Nutrition 0.000 description 1
- 235000010591 Appio Nutrition 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 241000282832 Camelidae Species 0.000 description 1
- 241000282836 Camelus dromedarius Species 0.000 description 1
- 241000283705 Capra hircus Species 0.000 description 1
- 241000288030 Coturnix coturnix Species 0.000 description 1
- 108010075028 Cytochromes b Proteins 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000283087 Equus Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 241000287826 Gallus Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000017020 Ipomoea batatas Species 0.000 description 1
- 235000002678 Ipomoea batatas Nutrition 0.000 description 1
- 241001596950 Larimichthys crocea Species 0.000 description 1
- 108700036248 MT-RNR1 Proteins 0.000 description 1
- 241000220225 Malus Species 0.000 description 1
- 235000011430 Malus pumila Nutrition 0.000 description 1
- 235000015103 Malus silvestris Nutrition 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 244000062793 Sorghum vulgare Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 241000630524 Taractes rubescens Species 0.000 description 1
- 240000004922 Vigna radiata Species 0.000 description 1
- 235000010721 Vigna radiata var radiata Nutrition 0.000 description 1
- 235000011469 Vigna radiata var sublobata Nutrition 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000012805 animal sample Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 229940036811 bone meal Drugs 0.000 description 1
- 239000002374 bone meal Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 230000037029 cross reaction Effects 0.000 description 1
- 235000021438 curry Nutrition 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 235000019688 fish Nutrition 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 235000015220 hamburgers Nutrition 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000007403 mPCR Methods 0.000 description 1
- 235000019713 millet Nutrition 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 235000014122 turkey meat Nutrition 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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
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.
Sequence listing
<110> Shanghai customs animals and plants and food inspection and quarantine technology center
<120> real-time fluorescent PCR detection method for detecting turkey-derived components in foods and feeds using single copy nuclear gene
<130> 221010
<141> 2022-02-28
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
tgaacaaatc cacttccctt taacc 25
<210> 2
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
tcatttctgc tgcacaaagt gag 23
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
tgatgacgag ccgcagccac acc 23
<210> 4
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
agaggatagg atggatgtcg atttg 25
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
ccttgctgcg ggaacaaaag 20
<210> 6
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
acatccttcc tccattgcct ccgtctcc 28
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.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210187422.9A CN114836523B (en) | 2022-02-28 | 2022-02-28 | Real-time fluorescence PCR detection method for detecting turkey-derived components in foods and feeds by utilizing single copy nuclear genes |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210187422.9A CN114836523B (en) | 2022-02-28 | 2022-02-28 | Real-time fluorescence PCR detection method for detecting turkey-derived components in foods and feeds by utilizing single copy nuclear genes |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114836523A CN114836523A (en) | 2022-08-02 |
CN114836523B true CN114836523B (en) | 2024-03-26 |
Family
ID=82561889
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210187422.9A Active CN114836523B (en) | 2022-02-28 | 2022-02-28 | Real-time fluorescence PCR detection method for detecting turkey-derived components in foods and feeds by utilizing single copy nuclear genes |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114836523B (en) |
Citations (3)
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 |
-
2022
- 2022-02-28 CN CN202210187422.9A patent/CN114836523B/en active Active
Patent Citations (3)
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)
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 * |
Also Published As
Publication number | Publication date |
---|---|
CN114836523A (en) | 2022-08-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108624659B (en) | Real-time quantitative PCR method for detecting components of meat products | |
CN108060241B (en) | Double-digital PCR method for quantitatively detecting pigeon-derived components | |
CN111334585B (en) | Primer and kit for simultaneously detecting 8 animal components, detection method and application | |
Wu et al. | Species identification of fox-, mink-, dog-, and rabbit-derived ingredients by multiplex PCR and real-time PCR assay | |
CN112921103A (en) | Primer group for quantitatively analyzing bovine-derived components in meat products and application of primer group | |
CN106521016B (en) | The real-time fluorescence PCR detection method of calf-derived Cyclospora in food and feed is detected using single-copy nuclear gene | |
Salamah et al. | The Employment of Real-Time Polymerase Chain Reaction for the Identification of Bovine Gelatin in Gummy Candy. | |
CN107012247B (en) | Real-time fluorescence PCR detection method for detecting goat-derived ingredients in food and feed by using single-copy nuclear gene | |
CN106995852B (en) | Real-time fluorescence PCR detection method for detecting sheep-derived components in food and feed by using single-copy nuclear gene | |
CN109852705B (en) | Method and kit for rapidly detecting horse-derived components in food | |
CN114836523B (en) | Real-time fluorescence PCR detection method for detecting turkey-derived components in foods and feeds by utilizing single copy nuclear genes | |
CN108085374B (en) | Dual digital PCR method for quantitatively detecting turkey-derived components | |
Chen et al. | A novel multiplex xMAP assay for generic detection of avian, fish, and ruminant DNA in feed and feedstuffs | |
CN112501320B (en) | Snake origin component rapid detection kit and application thereof | |
CN114686600A (en) | Primer group and method for meat detection based on seven-fold PCR technology | |
CN113493826A (en) | Animal-derived component detection method and application thereof | |
CN108060242B (en) | Dual digital PCR method for quantitative detection of African ostrich-derived components | |
CN111440881B (en) | Primer group, kit and detection method for detecting pork and application | |
KR101764128B1 (en) | Primer set for determining identity of shrimp material in food, method of determining identity of shrimp material in food using the same and kit comprising the same | |
Porzahmat Shirvan et al. | TaqMan real-time PCR: a reliable method to detect meat species | |
CN116411086B (en) | Murine component RPA nucleic acid isothermal amplification primer probe group, kit, detection method and application thereof | |
CN110452994B (en) | Primer pair, probe and method for synchronously detecting ten animal source components | |
CN116875702B (en) | Primer group for identifying common animal species, kit and application thereof | |
CN108300770B (en) | Dual digital PCR method for quantitative detection of camel-derived components | |
CN114182027B (en) | Fusion curve technology-based animal-derived component universal screening kit and detection method |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |