CN116445628A - Primer group, kit and method for detecting adulteration in meat or meat products - Google Patents
Primer group, kit and method for detecting adulteration in meat or meat products Download PDFInfo
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- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- 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
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- C12Q1/6844—Nucleic acid amplification reactions
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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Abstract
The invention relates to the technical field of animal-derived component detection, in particular to a kit and a method for detecting bovine-derived meat, whether duck-derived components and pig-derived components exist in processed meat, sheep-derived meat and whether duck-derived components and pig-derived components exist in processed meat based on a recombinase-mediated isothermal nucleic acid amplification (RAA). The invention designs a specific primer group which can effectively amplify the mitochondrial genome of the duck-origin component, the mitochondrial genome of the pig-origin component, the mitochondrial genome of the cow-origin component and the mitochondrial genome of the sheep-origin component, and has higher specificity and sensitivity and no cross reaction with other species. The primer group can be combined with a lateral flow test strip to be used for detecting meat of bovine and sheep-derived components, and identifying and rapidly detecting duck-derived components and pig-derived components which are common in meat processing and are derived from adulterated meat on site.
Description
Technical Field
The invention relates to the technical field of biological detection of food safety, in particular to a primer group, a kit and a method for detecting adulteration in meat or meat products, and particularly relates to a detection primer group, a kit and a method for detecting whether beef or mutton and processed meat contain duck-origin components and pig-origin components.
Background
In recent years, beef and mutton adulteration events have occurred. Other low-value meats such as pork, duck, fox and the like are mixed into mutton, or illegal cases such as mutton oil, essence, mutton powder and the like are mixed into pork and duck directly. These continuously exposed, sales-making aril cases have attracted widespread social attention. The causes of beef and mutton-mixing events are mainly three:
the consumer market of livestock and poultry meat and meat products in China is huge. Since the country is built, the daily meat consumption of China is increased by about 13 times, and the meat consumption demand is changed from the quantity to the quality, so that the meat consumption is in a steadily rising trend.
The Chinese livestock and poultry breeding amount is huge, the market distribution is scattered, the supervision departments are difficult to cover the market distribution completely, and the supervision strength cannot meet the requirements. With respect to data statistics, the existing beef cattle in China are hidden in more than 3000 trading markets at the street sides of various villages and towns, and the trading markets are not only large in scale and small in scale, but also are not standard in trading mode, so that market supervision is not in place, and a plurality of supervision holes exist, and black-heart vendors find a multiplicative machine.
Cattle, mutton and the like can obtain a great deal of profits through adulteration. The market price of beef, mutton and the like is gradually increased under the influence of factors such as the increase of the raising cost. While no illegal vendors are not spent at home and abroad and driven by benefits, the event of selling the low-price meat by mixing the low-price meat into the high-price beef and mutton, such as the explosion of British and Ireland, the "horse meat wind wave" of selling the horse meat by mixing the horse meat into the beef hamburger; in 2013 Jiangsu tin-free forensic market-making fake mutton cases.
In view of the fact that pork and duck are mixed into beef and mutton for sale, the pork and the duck are inflated for a second time, and the event frequency which disturbs the market order is frequent, the rights and interests of consumers are seriously infringed, and even the physical health of the consumers is threatened. The method for judging animal sources by means of sensory judgment, morphology (such as color, smell, elasticity, and other sensory and muscle textures) and the like is limited greatly, and cannot completely meet the current market of meat products, and particularly, the requirement for rapid detection of meat product processed products is met. Therefore, how to utilize modern molecular biology technology to improve animal source detection level, develop a set of rapid, quick and effective detection system, and have important significance for protecting consumer benefits and maintaining food safety.
The detection methods of the duck-origin component, the pig-origin component, the cattle-origin component and the sheep-origin component which are commonly used at present are mainly based on protein chromatography, enzyme-linked immunoassay and nucleic acid-based molecular biological assay (RT-qPCR, PCR-restriction fragment length polymorphism, loop-mediated isothermal amplification technology detection and the like), wherein the RT-PCR method is most widely applied.
Recombinase-mediated isothermal nucleic acid amplification (recombase AidedAmplification, RAA) is a recently developed technique for amplifying DNA with high specificity at low temperatures of 39 ℃ and at detectable levels within minutes. The RAA can simultaneously carry out two-step operation of reverse transcription and amplification in the same amplification tube, the product can be detected by agarose gel electrophoresis, or a lateral flow test strip (Lateral flow strip, LFS) is used for realizing the visualization of the nucleic acid amplification product, and the RAA-LFS has the advantages of simple operation, small sample amount, rapid detection, low cost and the like, and has been widely applied in a plurality of fields such as medicine, food, environment and the like.
Disclosure of Invention
Problems to be solved
Currently, molecular biological detection methods for whether meat and processed meat contains duck-origin components, pig-origin components, cow-origin components and sheep-origin components, such as RT-PCR, RT-qPCR and the like, are also becoming conventional means for detection, but these means and methods are either highly dependent on trained laboratory technicians, require complicated and expensive laboratory instruments, or have complicated detection operation procedures, are difficult to separate from laboratory environments, and are applied under non-laboratory conditions. The duck-origin component or pig-origin component is often added into beef and processed meat to make a false, or the duck-origin component or pig-origin component is added into mutton and processed meat, but the current detection method is difficult to simultaneously and rapidly and accurately identify the duck-origin component and the cattle-origin component, the pig-origin component and the cattle-origin component, the duck-origin component and the sheep-origin component, and the pig-origin component and the sheep-origin component under non-laboratory conditions due to the limitations of instruments and equipment, so that a rapid, simple, convenient and accurate dual animal-origin component visual detection method is needed to be established. The RAA dual isothermal nucleic acid amplification system of the duck-origin component, the cattle-origin component, the pig-origin component and the cattle-origin component, and the duck-origin component, the sheep-origin component, the pig-origin component and the sheep-origin component is established, and is combined with visual LFS detection, so that the requirements of market supervision departments and food safety service departments on site rapid detection of the duck-origin component, the pig-origin component which are common in beef-mutton and processed meat and adulterated with meat can be met, the supervision of food distribution channels is enhanced, and the method has important significance for guaranteeing food safety and consumer rights.
Technical proposal
In order to solve the technical problems, the invention aims to provide a primer group, a kit and a method for detecting duck-origin components, pig-origin components, cattle-origin components and sheep-origin components based on RAA.
In one aspect, the invention provides a RAA detection primer set for detecting duck-derived components, pig-derived components, cattle-derived components and sheep-derived components, wherein the primer set comprises a forward detection primer and a reverse detection primer for the duck-derived components, pig-derived components, cattle-derived components and sheep-derived components, and detection probes for the duck-derived components, pig-derived components, cattle-derived components and sheep-derived components; wherein,,
the Duck-origin component detection primer comprises a forward primer DuckDeved-F and a reverse primer DuckDeved-R, the sequence of the forward primer DuckDeved-F is 5'-CCACTTCTTAGAGTACTCCCAG-3',
the reverse primer DuckDerived-R has the sequence of 5'-GGGTTGTAACTGTTGATAGTG-3';
the Pig-Derived component detection primer comprises a forward primer Pig developed-F and a reverse primer Pig developed-R, the sequence of the forward primer Pig developed-F is 5'-CCAATAAGCAATGATCAACC-3',
the sequence of the reverse primer Pic developed-R is 5'-CTATGGCTACTGAGATGTATCC-3';
The Bovine-Derived component detection primer comprises a forward primer Bovine-improved-F and a reverse primer Bovine-improved-R, wherein the sequence of the forward primer Bovine-improved-F is 5'-CGTATCCCTACCCATCCTTACA-3', and the sequence of the reverse primer Bovine-improved-R is 5'-ACCCGATTCAGACAAGTAGT-3'.
The sheep-Derived component detection primer comprises a forward primer Ovine-improved-F and a reverse primer Bovine-improved-R, wherein the sequence of the forward primer Ovine-improved-F is 5'-CAACACCACACTTCACAGCTTGC-3', and the sequence of the reverse primer Ovine-improved-R is 5'-GGATCCTGCTAGTGTGTAA-3'.
Preferably, the Duck Derived component detection probe Duck Derived-P has a sequence of 5'-GCACAAGCT CCAACACAACAAATAAAGTCAHCAAGAGCCCTCAACC-3'; the sequence of the Pig-derived component detection probe Pig development-P is 5'-CGAATCACCCGTATCATAAATTACTCAATCCHCAAGCCCATTAAACTT-3'; bovine-Derived component detection probe Bovine developed-P with sequence of 5'-CTCTTCGTGCTCCCAATTA CACCAAATTCHATTAGTAAGGTCAGC-3'; sheep-Derived component detection probe Ovine developed-P with sequence of 5'-CCATATGACTTCTACCCCTAATACTCATAHCTAGCCAACATCATC-3'; FITC is modified at the 5' ends of the reverse primers Duck developed-R and Pig developed-R of the Duck-Derived component and Pig-Derived component, biotin is modified at the 5' ends of the detection probes Duck developed-P and Pig developed-P, 3' ends of the Duck developed-P and Pig developed-P are subjected to phosphorylation modification, and H is tetrahydrofuran; digoxin is modified at the 5' ends of reverse primers Bovine-R and Ovine-D-R of Bovine-Derived components and sheep-Derived components, rhodamine is modified at the 5' ends of probe primers Bovine-P and Ovine-D-P, phosphorylation modification is carried out at the 3' ends of Bovine-P and Ovine-D-P, and H is tetrahydrofuran.
In one aspect, the invention provides a kit for detecting duck-derived components, pig-derived components, cattle-derived components and sheep-derived components, comprising the primer set.
In one aspect of the invention, the RAA detection primer set for detecting duck-origin components, pig-origin components, cow-origin components and sheep-origin components provided by the invention can be used for preparing a kit for judging whether meat to be detected and processed meat samples contain duck-origin components, pig-origin components, cow-origin components and sheep-origin components.
In one aspect of the invention, the kits of the invention may further comprise other reagents, including DNA extraction reagents, sample lysates, buffers, PCR sample amplification reagents, and kit instructions.
In one aspect of the invention, the primer set for detecting duck-origin components and/or bovine-origin components, pig-origin components and/or bovine-origin components RAA provided by the invention can be used for judging whether meat to be detected and processed meat contain duck-origin components, pig-origin components and bovine-origin components.
In one aspect of the invention, the primer set for detecting duck-derived components and/or sheep-derived components, pig-derived components and/or sheep-derived components RAA provided by the invention can be used for judging whether meat to be detected and processed meat contain duck-derived components, pig-derived components and sheep-derived components.
In one aspect of the invention, the kit provided by the invention can be used for detecting whether the meat to be detected and processed meat contain duck-derived components and/or bovine-derived components, pig-derived components and/or bovine-derived components, and duck-derived components and/or sheep-derived components, pig-derived components and/or sheep-derived components.
In one aspect, the present invention provides beef or mutton to be tested and a method for processing meat to determine whether the meat contains duck-derived components and pig-derived components, comprising the steps of:
1) Extracting total DNA of meat to be detected and processed meat, or carrying out rapid cracking and nucleic acid releasing treatment on meat and processed meat samples;
2) Taking the total DNA extracted in the step 1) or the supernatant of the sample lysate as a template, and adopting forward primers and reverse primers in the primer group to carry out RAA amplification to obtain an amplification product;
3) Detecting the amplified product, and judging whether the detected sample contains duck-origin components, pig-origin components, sheep-origin components and cow-origin components based on the detection result of the amplified product.
Preferably, in the present invention, if the amplification product contains a 287bp DNA fragment, the sample to be tested contains a duck-origin component, and if the amplification product does not contain a 287bp DNA fragment, the sample to be tested does not contain a duck-origin component; if the amplification product contains 274bp DNA fragments, the sample to be detected contains swine-origin components, and if the amplification product does not contain 274bp DNA fragments, the sample to be detected does not contain swine-origin components; if the amplification product contains 351bp DNA fragments, the sample to be detected contains bovine-derived components, and if the amplification product does not contain 351bp DNA fragments, the sample to be detected does not contain bovine-derived components; if the amplification product contains a 397bp DNA fragment, the sample to be detected contains sheep-derived components, and if the amplification product does not contain a 397bp DNA fragment, the sample to be detected does not contain sheep-derived components.
In one aspect, the invention provides a method for detecting whether a sample to be tested contains duck-derived components, pig-derived components, sheep-derived components and cow-derived components, comprising:
detecting whether the nucleic acid lysate or the total DNA amplification product of the meat to be detected and the processed meat contains specific DNA fragments, if the specific DNA fragments are contained, the sample to be detected contains duck-origin components, pig-origin components, sheep-origin components and cattle-origin components, and if the specific DNA fragments are not contained, the sample to be detected does not contain duck-origin components, pig-origin components, sheep-origin components and cattle-origin components; wherein the specific DNA fragment is the target sequence of the forward primer and the reverse primer.
In one aspect, the invention provides a method for detecting components of duck origin and/or bovine origin, components of pig origin and/or components of bovine origin, and components of duck origin and/or components of sheep origin, components of pig origin and/or components of sheep origin based on RAA amplification.
Preferably, the method comprises the steps of:
1) Lysing the meat sample according to the method provided by the nucleic acid releasing agent; or extracting total DNA according to DNA extraction reagent instruction, and dissolving in TE solution or pure water;
2) Taking the total DNA or meat extracted in the step 1) and processed meat lysate as templates, adopting forward primers and reverse primers in a primer group to carry out RAA amplification, if the amplification product contains 287bp DNA fragments, the sample to be detected contains duck-origin components, and if the amplification product does not contain 287bp DNA fragments, the sample to be detected does not contain duck-origin components; if the amplification product contains 274bp DNA fragments, the sample to be detected contains swine-origin components, and if the amplification product does not contain 274bp DNA fragments, the sample to be detected does not contain swine-origin components; if the amplification product contains 351bp DNA fragments, the sample to be detected contains bovine-derived components, and if the amplification product does not contain 351bp DNA fragments, the sample to be detected does not contain bovine-derived components; if the amplification product contains a 397bp DNA fragment, the sample to be detected contains sheep-derived components, and if the amplification product does not contain a 397bp DNA fragment, the sample to be detected does not contain sheep-derived components.
The forward primer DuckDerived- -F has the sequence of 5'-CCACTTCTTAGAGTACTCCCAG-3',
the reverse primer DuckDerived- -R has the sequence of 5'-GGGTTGTAACTGTTGATAGTG-3';
the forward primer PIg developed-F has a sequence of 5'-CCAATAAGCAATGATCAACC-3',
The sequence of the reverse primer Pic developed-R is 5'-CTATGGCTACTGAGATGTATCC-3';
the forward primer Bovine developed-F has the sequence of 5'-CGTATCCCTACCCATCCTTACA-3',
the reverse primer Bovine developed-R has the sequence of 5'-ACCCGATTCAGACAAGTAGT-3'.
The forward primer Ovine developed-F has the sequence of 5'-CAACACCACACTTCACAGCTTGC-3',
the reverse primer Ovine developed-R has the sequence of 5'-GGATCCTGCTAGTGTGTAA-3'.
Preferably, the reaction system for detecting the amplification of the duck-origin component and/or the bovine-origin component RAA is as follows:
| composition of the components | Volume (mu L) |
| Meat sample lysate or total DNA solution | 2 |
| Base buffer | 29.4 |
| Duck Derived-F(5μM) | 0.88 |
| Duck Derived-R(5μM) | 1.26 |
| Bovine Derived-F(5μM) | 1.17 |
| Bovine Derived-R(5μM) | 1.68 |
| ddH 2 O | To a total system of 47.5 |
Preferably, the reaction system for detecting the amplification of the porcine-derived component and/or bovine-derived component RAA is as follows:
| composition of the components | Volume (mu L) |
| Meat sample lysate or total DNA solution | 2 |
| Base buffer | 29.4 |
| Pig Derived-F(5μM) | 0.73 |
| Pig Derived-R(5μM) | 1.05 |
| Bovine Derived-F(5μM) | 0.44 |
| Bovine Derived-R(5μM) | 0.63 |
| ddH 2 O | To a total system of 47.5 |
Preferably, the reaction system for detecting the amplification of the duck-derived component and/or the sheep-derived component RAA is as follows:
| composition of the components | Volume (mu L) |
| Meat sampleLysis solution or total DNA solution | 2 |
| Base buffer | 29.4 |
| Duck Derived-F(5μM) | 0.58 |
| Duck Derived-R(5μM) | 0.84 |
| Ovine Derived-F(5μM) | 1.03 |
| Ovine Derived-R(5μM) | 1.48 |
| ddH 2 O | To a total system of 47.5 |
Preferably, the reaction system for detecting the amplification of the porcine-derived component and/or the ovine-derived component RAA is as follows:
| Composition of the components | Volume (mu L) |
| Meat sample lysate or total DNA solution | 2 |
| Base buffer | 29.4 |
| Pig Derived-F(5μM) | 0.51 |
| Pig Derived-R(5μM) | 0.73 |
| Ovine Derived-F(5μM) | 0.88 |
| Ovine Derived-R(5μM) | 1.26 |
| ddH 2 O | To a total system of 47.5 |
Preferably, the reaction conditions for the RAA amplification are set as follows: after completion of the preparation of the reaction solution, 2.5. Mu.L of 280mM MgAc was added to each of the 0.2mL eppendorf tubes 2 Fully and uniformly mixing; the eppendorf tube (0.2 mL) was placed on a PCR apparatus with a thermal cover function, and incubated at 39deg.C for 25min.
Preferably, the amplified product can be detected by agarose gel electrophoresis, and the minimum copy number of the initial amplified template for detecting the target sequence by agarose gel electrophoresis of the duck-origin component RAA amplified product is 10 1 Copy number; the minimum copy number of the initial amplification template for detecting the target sequence by agarose gel electrophoresis of the porcine-derived ingredient RAA amplification product is 10 1 Copy number; the lowest copy number of the initial amplification template for detecting the target sequence by agarose gel electrophoresis of the bovine-derived ingredient RAA amplification product is 10 1 And copies.
Wherein, the detection process of the RAA amplification product is as follows: after completion of the RAA reaction, the reaction tube was taken out. Each reaction tube was charged with 100. Mu.L of phenol/chloroform (1:1), thoroughly homogenized by shaking, and centrifuged at 12000rpm for 10min (this step was vigorously mixed by shaking using a vortex shaker). Mixing 10 μL supernatant with 2 μL 6×loading Buffer, loading onto 1.5% agarose gel, and electrophoresis at 200V for 15min; ending electrophoresis when bromophenol blue moves to 2/3 of the position under gel; EB staining for 5min, observing under an ultraviolet lamp and photographing.
In one aspect, the invention provides a method for detecting whether a sample to be tested contains duck-derived components, pig-derived components, cow-derived components and sheep-derived components.
Preferably, the method comprises: detecting whether a specific DNA fragment is contained in a lysate of a sample to be detected or an amplification product of a nucleic acid sample, wherein if the specific DNA fragment is contained, the sample to be detected contains duck-derived components, pig-derived components, cattle-derived components and sheep-derived components, and if the specific DNA fragment is not contained, the sample to be detected does not contain duck-derived components, pig-derived components, cattle-derived components and sheep-derived components; wherein the specific DNA fragment is the target sequence of the primer group in the total DNA of the sample to be detected.
In one aspect, the invention provides a method for detecting duck-derived components, pig-derived components, cow-derived components and sheep-derived components based on RAA-lateral flow chromatography.
Preferably, the method comprises the steps of:
1) Lysing the meat sample according to the method provided by the nucleic acid releasing agent; or extracting total DNA according to DNA extraction reagent instruction, and dissolving in TE solution or pure water;
2) Carrying out RAA amplification by using the lysate obtained in the step 1) or the extracted total DNA or the template and adopting a primer group;
The forward primer Duck developed-F has the sequence of 5'-CCACTTCTTAGAGTACTCCCAG-3',
the sequence of the reverse primer Duck developed-R is 5'-GGGTTGTAACTGTTGATAGTG-3';
the forward primer PIg developed-F has a sequence of 5'-CCAATAAGCAATGATCAACC-3',
the sequence of the reverse primer Pic developed-R is 5'-CTATGGCTACTGAGATGTATCC-3';
the forward primer Bovine developed-F has the sequence of 5'-CGTATCCCTACCCATCCTTACA-3',
the reverse primer Bovine developed-R has the sequence of 5'-ACCCGATTCAGACAAGTAGT-3'.
The forward primer Ovine developed-F has the sequence of 5'-CAACACCACACTTCACAGCTTGC-3',
the reverse primer Ovine developed-R has the sequence of 5'-GGATCCTGCTAGTGTGTAA-3'.
The Duck developed-P of the Duck-origin component detection probe has a sequence of 5'-GCACAAGCTCCAACACAAC AAATAAAGTCAHCAAGAGCCCTCAACC-3'; the Pig-Derived component detection probe Pig developed-P has a sequence of 5'-CGAATCACCCGTATCATAAATTACTCAATCCHCAAGCCCATTAAACTT-3'; the Bovine-Derived component detection probe Bovine developed-P has a sequence of 5'-CTCTTCGTGCTCCCAATTACACCAAA TTCHATTAGTAAGGTCAGC-3'; the sheep-Derived component detection probe Ovine developed-P has a sequence of 5'-CCATATGACTTCTACCCCTAATACTCATAHCTAGCCAACATCATC-3'; FITC is modified at the 5' ends of the reverse primers Duck developed-R and Pig developed-R of the Duck-Derived component and Pig-Derived component, biotin is modified at the 5' ends of the detection probes Duck developed-P and Pig developed-P, and 3' ends of the Duck developed-P and Pig developed-P are subjected to phosphorylation modification, and H is tetrahydrofuran; digoxin is modified at the 5' ends of reverse primers Bovine-R and Ovine-D-R of Bovine-Derived components and sheep-Derived components, rhodamine is modified at the 5' ends of probe primers Bovine-D-P and Ovine-D-P, phosphorylation modification is carried out at the 3' ends of Bovine-D-R and Ovine-D-P, and H is tetrahydrofuran.
Preferably, the reaction system for detecting the amplification of the duck-origin component and/or the bovine-origin component RAA is as follows:
| composition of the components | Volume (mu L) |
| Meat and processed meat sample lysate or DNA solution | 2 |
| Base buffer | 29.4 |
| Duck Derived-F(5μM) | 0.88 |
| Duck Derived-R(5μM) | 1.26 |
| Duck Derived-P(1μM) | 1.26 |
| Bovine Derived-F(5μM) | 1.17 |
| Bovine Derived-R(5μM) | 1.68 |
| Bovine Derived-P(1μM) | 1.68 |
| ddH 2 O | upto47.5 |
Preferably, the reaction system for detecting the amplification of the porcine-derived component and/or bovine-derived component RAA is as follows
| Composition of the components | Volume (mu L) |
| Meat and processed meat sample lysate or DNA solution | 2 |
| Base buffer | 29.4 |
| Pig Derived-F(5μM) | 0.73 |
| Pig Derived-R(5μM) | 1.05 |
| Pig Derived-P(1μM) | 1.05 |
| Bovine Derived-F(5μM) | 0.44 |
| Bovine Derived-R(5μM) | 0.63 |
| Bovine Derived-P(1μM) | 0.63 |
| ddH 2 O | upto47.5 |
Preferably, the reaction system for detecting the amplification of the duck-derived component and/or the sheep-derived component RAA is as follows: :
| composition of the components | Volume (mu L) |
| Meat and processed meat sample lysate or DNA solution | 2 |
| Base buffer | 29.4 |
| Duck Derived-F(5μM) | 0.58 |
| Duck Derived-R(5μM) | 0.84 |
| Duck Derived-P(1μM) | 0.84 |
| Ovine Derived-F(5μM) | 1.03 |
| Ovine Derived-R(5μM) | 1.48 |
| Ovine Derived-P(1μM) | 1.48 |
| ddH 2 O | upto47.5 |
Preferably, the reaction system for detecting the amplification of the porcine-derived component and/or the ovine-derived component RAA is as follows
| Composition of the components | Volume (mu L) |
| Meat and processed meat sample lysate or DNA solution | 2 |
| Base buffer | 29.4 |
| Pig Derived-F(5μM) | 0.51 |
| Pig Derived-R(5μM) | 0.73 |
| Pig Derived-P(1μM) | 0.73 |
| Ovine Derived-F(5μM) | 0.88 |
| Ovine Derived-R(5μM) | 1.26 |
| Ovine Derived-P(1μM) | 1.26 |
| ddH 2 O | upto47.5 |
Preferably, the reaction conditions for the RAA amplification are set as follows: after completion of the preparation of the reaction solution, 2.5. Mu.L of 280mM MgAc was added to each of the 0.2mL eppendorf tubes 2 Fully and uniformly mixing; the eppendorf tube (0.2 mL) was placed on a PCR apparatus with a thermal cover function, and incubated at 39℃for 16min.
3) Detecting duck-origin components and/or bovine-origin components RAA amplification products by using a lateral flow chromatography test strip, and if the test strip only has a color development of a quality control area, the detection area does not have a color development of the strip, and the detection result is negative, so that the sample does not contain the duck-origin components and the bovine-origin components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T1, and the detection result is positive, which indicates that the detection sample contains bovine-derived components but does not contain duck-derived components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T2, and the detection result is positive, which indicates that the detection sample contains duck-origin components but does not contain bovine-origin components; if the test strip has three strip color development and two strip color development, one strip is positioned in the quality control area, one strip is positioned in the detection area T1, and the other strip is positioned in the detection area T2, the detection result is positive, and the sample contains bovine-derived components and duck-derived components at the same time; if the quality control area and the two detection areas of the test strip are not developed, the detection is invalid, and the test strip needs to be replaced for retesting.
The specific detection process can be as follows: the eppendorf tube lid containing 50. Mu.LRAA amplification product was opened and 2.5. Mu.L of the amplification product was taken into a new 1.5mL eppendorf tube and diluted 20-fold. A new nucleic acid lateral chromatography test strip binding pad end is directly inserted into an eppendorf tube filled with diluted products, and the liquid level does not exceed the immersion upper limit mark of a sample pad. After the read-out area is fully soaked, the test paper is horizontally placed for 1min, the color development result is observed, and the record is carried out within 10 min. At least one quality control line, with or without detection lines, appears for each test sample. If the test strip only has the color development of the strip in the quality control area, the detection area does not have the color development of the strip, and the detection result is negative, which indicates that the sample does not contain duck-origin components and bovine-origin components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T1, and the detection result is positive, which indicates that the detection sample contains bovine-derived components but does not contain duck-derived components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T2, and the detection result is positive, which indicates that the detection sample contains duck-origin components but does not contain bovine-origin components; if the test strip has three strip color development and two strip color development, one strip is positioned in the quality control area, one strip is positioned in the detection area T1, and the other strip is positioned in the detection area T2, the detection result is positive, and the sample contains bovine-derived components and duck-derived components at the same time; if the quality control area and the two detection areas of the test strip are not developed, the detection is invalid, and the test strip needs to be replaced for retesting.
4) Detecting porcine-derived ingredient and/or bovine-derived ingredient RAA amplification products by using a lateral flow chromatography test strip, and if the test strip only has a color development of a quality control area, the detection area does not have a color development of the strip, and the detection result is negative, so that the sample does not contain porcine-derived ingredient and bovine-derived ingredient; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T1, and the detection result is positive, which indicates that the detection sample contains bovine-derived components but does not contain swine-derived components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T2, and the detection result is positive, which indicates that the detection sample contains pig-derived components but does not contain cattle-derived components; if the test strip has three strip color development and two strip color development, one strip is positioned in the quality control area, one strip is positioned in the detection area T1, and the other strip is positioned in the detection area T2, the detection result is positive, and the sample contains bovine-derived components and porcine-derived components at the same time; if the quality control area and the two detection areas of the test strip are not developed, the detection is invalid, and the test strip needs to be replaced for retesting.
The specific detection process can be as follows: the eppendorf tube lid containing 50. Mu.LRAA amplification product was opened and 2.5. Mu.L of the amplification product was taken into a new 1.5mL eppendorf tube and diluted 20-fold. A new nucleic acid lateral chromatography test strip binding pad end is directly inserted into an eppendorf tube filled with diluted products, and the liquid level does not exceed the immersion upper limit mark of a sample pad. After the read-out area is fully soaked, the test paper is horizontally placed for 1min, the color development result is observed, and the record is carried out within 10 min. At least one quality control line, with or without detection lines, appears for each test sample. If the test strip only has the color development of the strip in the quality control area, the detection area does not have the color development of the strip, and the detection result is negative, which indicates that the sample does not contain pig-derived components and cattle-derived components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T1, and the detection result is positive, which indicates that the detection sample contains bovine-derived components but does not contain swine-derived components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T2, and the detection result is positive, which indicates that the detection sample contains pig-derived components but does not contain cattle-derived components; if the test strip has three strip color development and two strip color development, one strip is positioned in the quality control area, one strip is positioned in the detection area T1, and the other strip is positioned in the detection area T2, the detection result is positive, and the sample contains bovine-derived components and porcine-derived components at the same time; if the quality control area and the two detection areas of the test strip are not developed, the detection is invalid, and the test strip needs to be replaced for retesting.
5) Detecting the duck-derived component and/or sheep-derived component RAA amplification products by using a lateral flow chromatography test strip, and if the test strip only has a color development of a quality control area, the detection area does not have a color development of the strip, and the detection result is negative, so that the sample does not contain the duck-derived component and the sheep-derived component; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T1, and the detection result is positive, which indicates that the detection sample contains sheep-derived components but does not contain duck-derived components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T2, and the detection result is positive, which indicates that the detection sample contains duck-origin components but does not contain sheep-origin components; if the test strip has three strip color development and two strip color development, one strip is positioned in the quality control area, one strip is positioned in the detection area T1, and the other strip is positioned in the detection area T2, the detection result is positive, and the sample contains sheep-derived components and duck-derived components at the same time; if the quality control area and the two detection areas of the test strip are not developed, the detection is invalid, and the test strip needs to be replaced for retesting.
The specific detection process can be as follows: the eppendorf tube lid containing 50. Mu.LRAA amplification product was opened and 2.5. Mu.L of the amplification product was taken into a new 1.5mL eppendorf tube and diluted 20-fold. A new nucleic acid lateral chromatography test strip binding pad end is directly inserted into an eppendorf tube filled with diluted products, and the liquid level does not exceed the immersion upper limit mark of a sample pad. After the read-out area is fully soaked, the test paper is horizontally placed for 1min, the color development result is observed, and the record is carried out within 10 min. At least one quality control line, with or without detection lines, appears for each test sample. If the test strip only has the color development of the strip in the quality control area, the detection area does not have the color development of the strip, and the detection result is negative, which indicates that the sample does not contain duck-derived components and sheep-derived components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T1, and the detection result is positive, which indicates that the detection sample contains sheep-derived components but does not contain duck-derived components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T2, and the detection result is positive, which indicates that the detection sample contains duck-origin components but does not contain sheep-origin components; if the test strip has three strip color development and two strip color development, one strip is positioned in the quality control area, one strip is positioned in the detection area T1, and the other strip is positioned in the detection area T2, the detection result is positive, and the sample contains sheep-derived components and duck-derived components at the same time; if the quality control area and the two detection areas of the test strip are not developed, the detection is invalid, and the test strip needs to be replaced for retesting.
6) Detecting the porcine-derived and/or ovine-derived component RAA amplification products by using a lateral flow chromatography test strip, and if the test strip only has a color development of a quality control area, the detection area does not have a color development of the strip, and the detection result is negative, so that the sample does not contain the porcine-derived and ovine-derived components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T1, and the detection result is positive, which indicates that the detection sample contains sheep-derived components but does not contain pig-derived components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T2, and the detection result is positive, which indicates that the detection sample contains pig-derived components but does not contain sheep-derived components; if the test strip has three strip color development and two strip color development, one strip is positioned in the quality control area, one strip is positioned in the detection area T1, and the other strip is positioned in the detection area T2, the detection result is positive, and the sample contains sheep-derived components and pig-derived components at the same time; if the quality control area and the detection area of the test strip are not developed, the detection is invalid, and the test strip needs to be replaced for retesting.
The specific detection process can be as follows: the eppendorf tube lid containing 50. Mu.LRAA amplification product was opened and 2.5. Mu.L of the amplification product was taken into a new 1.5mL eppendorf tube and diluted 20-fold. A new nucleic acid lateral chromatography test strip binding pad end is directly inserted into an eppendorf tube filled with diluted products, and the liquid level does not exceed the immersion upper limit mark of a sample pad. After the read-out area is fully soaked, the test paper is horizontally placed for 1min, the color development result is observed, and the record is carried out within 10 min. At least one quality control line, with or without detection lines, appears for each test sample. If the test strip only has the color development of the strip in the quality control area, the detection area does not have the color development of the strip, and the detection result is negative, which indicates that the sample does not contain pig-derived components and sheep-derived components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T1, and the detection result is positive, which indicates that the detection sample contains sheep-derived components but does not contain pig-derived components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T2, and the detection result is positive, which indicates that the detection sample contains pig-derived components but does not contain sheep-derived components; if the test strip has three strip color development and two strip color development, one strip is positioned in the quality control area, one strip is positioned in the detection area T1, and the other strip is positioned in the detection area T2, the detection result is positive, and the sample contains sheep-derived components and pig-derived components at the same time; if the quality control area and the detection area of the test strip are not developed, the detection is invalid, and the test strip needs to be replaced for retesting.
The lowest copy number of the initial amplification template of the duck-origin component, which can be detected by each reaction of the RAA-lateral flow chromatography technology, is 32 copies, the lowest copy number of the initial amplification template of the pig-origin component is 22 copies, the lowest copy number of the initial amplification template of the bovine-origin component target sequence is 44 copies, and the lowest copy number of the initial amplification template of the sheep-origin component target sequence is 36 copies.
Advantageous effects
The invention provides a specific primer group for detecting duck-derived components, pig-derived components, cattle-derived components and sheep-derived components, and establishes a dual rapid detection method for adulterated duck-derived components or pig-derived components in beef and mutton. The primer designed in the invention can effectively amplify target genes, has higher specificity and sensitivity, has no cross reaction with other species, can be used for on-site rapid detection of duck-origin components, pig-origin components, cattle-origin components and sheep-origin components, and has important significance for food safety supervision.
Drawings
FIG. 1 shows RAA amplification of samples of different species using a duck-derived component amplification primer set 1-4;
FIG. 2 shows RAA amplification of samples of different species using porcine-derived ingredient amplification primer sets 5 to 8;
FIG. 3 shows RAA amplification of samples of different species using bovine-derived component amplification primer sets 9 to 12;
FIG. 4 shows RAA amplification of samples of different species using sheep derived component amplification primer sets 13 to 16;
FIG. 5A shows RAA amplification sensitivity detection of Bovine-Derived component amplification primer sets Bovine-D-F and Bovine-D-R; FIG. 5B shows RAA amplification sensitivity detection of sheep-Derived component amplification primer sets Ovine-F and Ovine-Derived-R; FIG. 5C shows RAA amplification sensitivity detection of Duck developed-F and Duck developed-R of Duck Derived component amplification primer sets; FIG. 5D shows RAA amplification sensitivity detection of Pig-Derived component amplification primer sets Pig developed-F and Pig developed-R; 50 mu L RAA amplification initial template copy number is from 10 6 Copy to 10 0 Copying and gradually decreasing; wherein M isLabeling Plus DNA; "-" is ddH 2 O replaces the same volumeNegative control of plasmid.
FIG. 6 is a schematic diagram of the structure and detection result of a single-target disposable nucleic acid detection test strip (JY 0201);
FIG. 7 shows RAA-LFS detection results of positive plasmids and negative controls using Duck-derived component detection primer set A, primer set B, primer set C, and primer set D;
FIG. 8 shows RAA-LFS detection results of positive plasmids and negative controls using porcine-derived component detection primer set E, primer set F, primer set G, and primer set H;
FIG. 9 shows the results of RAA-LFS detection using bovine-derived component detection primer set I, primer set J, primer set K, and primer set L for positive plasmid and negative control;
FIG. 10 shows RAA-LFS detection results of positive plasmids and negative controls using duck-derived component detection primer set M, primer set N, primer set O, and primer set P;
FIG. 11 detection sensitivity of Duck-derived component detection primer set ARAA-LFS;
FIG. 12 influence of the addition amount of the downstream primer in the duck-origin component detection primer set ARAA amplification system on the detection sensitivity;
FIG. 13 influence of magnesium acetate addition amount in duck-origin component detection primer set ARAA amplification system on detection sensitivity;
FIG. 14 detection sensitivity of porcine-derived ingredient detection primer set H RAA-LFS;
FIG. 15 detection sensitivity of bovine-derived ingredient detection primer set I RAA-LFS;
FIG. 16 detection sensitivity of sheep derived composition detection primer set M RAA-LFS;
FIG. 17 RAA-LFS detection specificity evaluation of Duck-origin component detection primer A, pig-origin component detection primer set H, cattle-origin component detection primer set I and sheep-origin component detection primer set M
FIG. 18 is a diagram showing the structure and detection result of a dual-target disposable nucleic acid detection test strip (JY 0209);
FIG. 19 shows the results of dual RAA-LFS detection of duck-origin and bovine-origin component detection primer sets using plasmids as templates;
FIG. 20 shows the results of dual RAA-LFS detection of porcine-derived and bovine-derived component detection primer sets using plasmids as templates;
FIG. 21 shows the results of dual RAA-LFS detection of duck-derived and sheep-derived component detection primer sets using plasmids as templates;
FIG. 22 shows the results of double RAA-LFS detection of swine-derived and sheep-derived component detection primer sets using plasmids as templates;
FIG. 23 qPCR melting curve analysis of duck-origin component detection primer set, pig-origin component detection primer set, cattle-origin component detection primer set, sheep-origin component detection primer set;
FIG. 24 shows that the qPCR of the duck-origin component detection primer, the pig-origin component detection primer, the cattle-origin component detection primer and the sheep-origin component detection primer corresponds to absolute quantitative amplification curve analysis of the gradient diluted plasmid;
FIG. 25 double RAA-LFS sensitivity detection results of duck-origin component and bovine-origin component detection primer sets;
FIG. 26 results of dual RAA-LFS sensitivity detection of porcine-derived and bovine-derived component detection primer sets;
FIG. 27 shows the detection results of dual RAA-LFS sensitivity of the detection primer sets for duck-derived components and sheep-derived components;
FIG. 28 results of dual RAA-LFS sensitivity detection of porcine-derived and ovine-derived component detection primer sets;
FIG. 29 Duck-derived and bovine-derived component detection primer sets double RAA-LFS reaction primer optimization detection results;
FIG. 30 results of detection of porcine-derived and bovine-derived component detection primer sets with dual RAA-LFS reaction primers;
FIG. 31 shows the optimal detection results of double RAA-LFS reaction primers of the detection primer sets for duck-origin components and sheep-origin components;
FIG. 32 is a diagram of the results of the detection of porcine-derived and ovine-derived component detection primer sets with dual RAA-LFS reaction primers;
FIG. 33 shows the detection of animal-derived components of duck, pork, beef, and mutton after simple lysis, respectively;
FIG. 34 self-made RAA-LFS rapid test results of random sampling of animal-derived adulterated meat at different ratios.
Detailed Description
The following describes the embodiments of the present invention in detail with reference to the drawings.
The experimental methods in the following examples are conventional, unless otherwise specified. The experimental materials described in the examples below, unless otherwise specified, were not purchased from conventional biochemical reagent stores. The quantitative experiments in the following examples were all set up in triplicate and the results averaged.
The meat sample source of the invention:
The meat and processed meat related by the invention are purchased in the universities of Ningbo, and the corresponding total DNA is stored in the national key laboratory of the national defense and control of quality safety hazard factors and risks of agricultural products co-established by the university of Ningbo.
The sample preparation method related by the invention comprises the following steps:
meat and processed meat are used as experimental objects, and for the extraction of the traditional DNA method, a general genomic DNA extraction kit (New scenery, hangzhou) is used, and total DNA is extracted from duck, pork and beef and mutton samples according to the instruction manual operation of the reagent.
To evaluate whether the RAA-LFS system of the rapid-lysis DNA-free extraction method used can be used for rapid detection of duck-derived components, pig-derived components, cattle-derived components, sheep-derived components in meat processedMeat and processed meat samples were placed in 1.5mL eppendorf tubes and 100. Mu.L +.>After the cool flash nucleic acid releasing agent (Tiosbio, beijing Baozhen Yinghui biotechnology Co., ltd.), the sample to be detected is sheared by using an ophthalmic shearing machine, and then the nucleic acid is released and centrifuged to obtain the lysate of the corresponding sample. 1 μl of sample lysate or dilution thereof is used as an amplification template for RAA. Sample crackThe solution can be tested immediately or stored at-70deg.C for more than 3 months.
The nucleic acid lateral chromatography test strip is from Beijing Baozhen Yinghui biotechnology Co., ltd.
Experimental example 1: primer design and screening for detection of duck-origin components, pig-origin components, cattle-origin components and sheep-origin components
The invention uses the genome sequence of duck mitochondria, pig mitochondria, cow mitochondria and sheep mitochondria as target, analyzes the characteristics of target gene sequence, and designs the species-specific amplification Primer by using Primer Premier 5.0 software. Primer design follows the following principle.
(1) The length of the amplified primer is about 20-30 bp, and the annealing temperatures of different primers are as consistent or similar as possible; (2) the length of the amplified product is about 200-500 bp, and the amplified product has species specificity.
The initial identification of primer amplification specificity was accomplished by alignment of primer amplification segment sequences using BLAST function of the National Center for Biotechnology Information (NCBI). The primers identified were synthesized by general biosystems (Anhui) Inc., purity grade HPLC. The specific sequences of the primers are shown in Table 1.
The forward primer Duck developed-F has the sequence of 5'-CCACTTCTTAGAGTACTCCCAG-3',
the sequence of the reverse primer Duck developed-R is 5'-GGGTTGTAACTGTTGATAGTG-3';
The forward primer PIg developed-F has a sequence of 5'-CCAATAAGCAATGATCAACC-3',
the sequence of the reverse primer Pic developed-R is 5'-CTATGGCTACTGAGATGTATCC-3';
the forward primer Bovine developed-F has the sequence of 5'-CGTATCCCTACCCATCCTTACA-3',
the reverse primer Bovine developed-R has the sequence of 5'-ACCCGATTCAGACAAGTAGT-3'.
The forward primer Ovine developed-F has the sequence of 5'-CAACACCACACTTCACAGCTTGC-3',
the reverse primer Ovine developed-R has the sequence of 5'-GGATCCTGCTAGTGTGTAA-3'.
Duck Derived-P as Duck Derived component detection probe with sequence of 5'-GCACAAGCTCCAACACAACAAAT AAAGTCAHCAAGAGCCCTCAACC-3'; the sequence of the Pig developed-P is 5'-CGAATCACCCGTATCA TAAATTACTCAATCCHCAAGCCCATTAAACTT-3', and the sequence of the Pig developed-P is 5'-CTCTTCGTGCTCCCAATTACACCAAATTCHATTAGTAAGGTCAGC-3'; the sheep-Derived component detection probe Ovine developed-P has a sequence of 5'-CCATATGACTTCTACCCCTAA TACTCATAHCTAGCCAACATCATC-3';
TABLE 1 primers for amplification of duck-origin component, pig-origin component, cattle-origin component and sheep-origin component RAA-basis
UsingRAA nucleic acid amplification kit (basic model JY 0203) uses meat and total DNA in processed meat or meat and processed meat sample lysate as templates to respectively carry out RAA amplification of forward primers and reverse primers of groups 1-4, groups 5-8 and groups 9-12, and an amplification system (single sample/reaction) is as follows:
| Composition of the components | Volume (mu L) |
| Meat and processed meat sample lysate or DNA solution | 2 |
| Base buffer | 29.4 |
| Forward primer (10. Mu.M) | 2.1 |
| Reverse primer (10. Mu.M) | 2.1 |
| ddH 2 O | To a total system of 47.5 |
After completion of the preparation of the reaction solution, 2.5. Mu.L of 280mM MgAc was added to each of the 0.2mL eppendorf tubes 2 Fully and uniformly mixing; the eppendorf tube (0.2 mL) was placed on a PCR apparatus with a thermal cover function, and incubated at 39deg.C for 30min.
After completion of the RAA reaction, the reaction tube was taken out. Each reaction tube was charged with 100. Mu.L of phenol/chloroform (1:1), thoroughly homogenized by shaking, and centrifuged at 12000rpm for 10min (this step was vigorously mixed by shaking using a vortex shaker). Mixing 10 μL supernatant with 2 μL 6×loading Buffer, loading onto 1.5% agarose gel, and electrophoresis at 200V for 15min; ending electrophoresis when bromophenol blue moves to 2/3 of the position under gel; EB staining for 5min, observing under an ultraviolet lamp and photographing.
1-4, on the premise of adding 2 mu L of total DNA solution or sample lysate, carrying out RAA amplification in total DNA of 5 species of cattle, sheep, ducks, pigs and chickens by using 1-4 of duck-origin component amplification primer groups, wherein an electrophoresis band of an amplification product of the duck-origin component amplification primer group 2 in total DNA of duck meat is clear and bright, has no impurity band, the size of the amplification band is consistent with that of expected, and no obvious amplification product exists in total DNA of other meat; RAA amplification is carried out in total DNA of different species by using pig-derived component amplification primer groups 5-8 respectively, the electrophoresis strips of amplification products of the pig-derived component amplification primer group 5 in the total DNA of pork are clear and bright, no impurity strips exist, the size of the amplification strips is consistent with that of the expectation, and no obvious amplification products exist in the total DNA of other meats; the primer group 9-12 for amplifying bovine-derived components is used for carrying out RAA amplification in total DNA of different species, the electrophoresis strip of the amplified product of the primer group 11 in the total DNA of beef is clear and bright, no impurity strip exists, the size of the amplified strip is consistent with the expected, and no obvious amplified product exists in the total DNA of other meats, so that the primer group can be used for detecting bovine-derived components. The primer group 13-16 for amplifying the sheep-derived component is used for carrying out RAA amplification in the total DNA of different species, the electrophoresis strip of the amplified product of the primer group 13 in the total DNA of the mutton is clear and bright, no impurity strip exists, the size of the amplified strip is consistent with that of the expected, and no obvious amplified product exists in the total DNA of other meats, so that the primer group can be used for detecting the sheep-derived component.
The amplified products of the optimal primer groups of each primer pair are cloned to a T vector, and the target sequence is determined after sequencing. Determining that the optimal primer combination of each primer pair is respectively a Duck-origin component amplification primer group 2, a pig-origin component amplification primer group 5, a cattle-origin component amplification primer group 11 and a sheep-origin component amplification primer group 13, and renaming the upstream and downstream primers of the Duck-origin component amplification primer group 2 into Duck developed-F and Duck developed-R; the upstream and downstream primers of the Pig-Derived component amplification primer set 5 are renamed as Pig developed-F and Pig developed-R; the upstream and downstream primers of Bovine-Derived component amplification primer set 11 are renamed Bovine-D-F and Bovine-D-R; the upstream and downstream primers of sheep Derived component amplification primer set 13 were renamed Ovine-D-F and Ovine-D-R.
Experimental example 2: preparation and composition of duck-origin component, pig-origin component, bovine-origin component and sheep-origin component test sample comparison tray
Duck-Derived component amplification primer set Duck-D-F and Duck-D-R positive amplification products, pig-Derived component amplification primer set Pig-Derived component amplification products and Pig-Derived component detection primer set Bovine-F and Bovine-D-R positive amplification products, sheep-Derived component detection primer set Ovine-F and Ovine-D-R positive amplification products were cloned and sequenced to correct plasmids, plasmid extraction was performed after shaking bacteria were transformed overnight, recombinant plasmid OD260, OD280 and OD260/OD280 values were measured with an ultraviolet spectrophotometer, and the plasmid DNA concentration and purity were determined 3 times.
The copy number of the plasmid was obtained according to the following formula:
copy number = plasmid concentration x 6.02 x 10 23 /(660X total length of plasmid)
Calculate copy number and dilute to 1×10 8 Copy/. Mu.L, store at-20deg.C for later use. Diluting the recombinant plasmid with a fixed value to 1X 10 6 Copy/. Mu.L, serial 10-fold dilution to obtain 1X 10 0 Copy/. Mu.L, 1X 10 1 Copy/. Mu.L, 1X 10 2 Copy/. Mu.L, 1X 10 3 Copy/. Mu.L, 1X 10 4 Copy/. Mu.L, 1X 10 5 Copy/. Mu.L and 1X 10 6 The copy/. Mu.L plasmid dilution was used as the template for subsequent amplifications.
Experimental example 3: detection of primer sensitivity for Duck-, pig-, cattle-, and sheep-derived components
To further examine the sensitivity of Duck-Derived component amplification primer set Duck-improved-F and Duck-improved-R, pig-Derived component amplification primer set Pic-improved-F and Pic-improved-R, bovine-Derived component detection primer set Bovine-improved-F and Bovine-improved-R and sheep-Derived component detection primer set Ovine-improved-F and Ovine-improved-R, 10-fold serial dilutions (10 per microliter) of the respective amplification product plasmid DNA were prepared 6 、10 5 、10 4 、10 3 、10 2 、10 1 、10 0 Copy) was used as template for RAA using ddH 2 O was used as a negative control template. The same template was compared by 1.5% agarose gel electrophoresis to separately amplify the different primer RAAs.
As can be seen from the experimental results of the amplification sensitivity of the bovine-Derived component amplification primer set (FIG. 5A) and the sheep-Derived component amplification primer set (FIG. 5B), the Duck-Derived component amplification primer set (FIG. 5C), and the pig-Derived component amplification primer set (FIG. 5D) RAA, the Duck-Derived component detection primer set Duck developed-F and Duck developed-R, and the pig-Derived componentThe detection primer group of Pig-Derived-F and Pig-Derived-R, the Bovine-Derived component detection primer group of Bovine-Derived-F and Bovine-Derived-R and the sheep-Derived component detection primer group of Ovine-Derived-F and Ovine-Derived-R RAA can detect as low as 10 1 Duck-derived component plasmids in individual copies.
Experimental example 4: detection of duck-derived component, pig-derived component, cattle-derived component and sheep-derived component RAA-LFS
For the amplification segments of Primer sets 1 to 8 in example 1, a Primer Premier 5.0 software was used to design a species-specific probe sequence located in the middle segment of the amplification Primer, modified with tetrahydrofuran. The primer design follows the principle that: the probe length is about 30-45 bp; (2) the probe sequences are species specific.
The initial identification of primer amplification specificity was accomplished by alignment of primer amplification segment sequences using BLAST function of the National Center for Biotechnology Information (NCBI). The primers identified were synthesized by general biosystems (Anhui) Inc., purity grade HPLC. The specific primer sequences, probes, and amplified downstream primer labels are shown in Table 2 below.
TABLE 2 primers for amplification of duck-origin component, pig-origin component, cattle-origin component and sheep-origin component RAA test paper clause
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1X 10 amplification products corresponding to the upstream and downstream primers of each primer set 6 Copy/. Mu.L recombinant plasmid solution as template, usingRAA nucleic acid amplification kit (test strip method) (JY 0204) is used for preparing a RAA reaction system (single sample/reaction) of a primer group A to a primer group P, and the system is as follows:
| component (A) | Dosage (mu L) |
| Base buffer | 29.4 |
| Upstream primer (10. Mu.M) | 2.1 |
| Probe primer (10 mu M) | 0.6 |
| Downstream primer (10. Mu.M) | 2.1 |
| 1×10 6 Copy/. Mu.L recombinant plasmid solution | 2 |
| Make up the volume with water | 47.5 |
The sample addition sequence was negative control samples (1X 10) 6 Copy/. Mu.L of recombinant plasmid solution was replaced with the same volume of ultrapure water), 1X 10 6 Copy/μl recombinant plasmid solution, and the tube cap is required to be closed immediately after each sample is added, avoiding aerosol contamination. And uniformly mixing the reaction systems, and adding the mixture into a basic reaction unit. Filling the freeze-dried powderThe components were dissolved and note that this step was not well mixed with vigorous shaking using a vortex shaker. The reaction unit was opened and 2.5. Mu.L of 280mM MgAc was added to each 0.2mL eppendorf tube 2 Thoroughly mixed and collected by centrifugation. Note that this step cannot be mixed by vigorous shaking with a vortex shaker.
The reaction tube was placed at 39℃for 16min. After the RAA reaction is finished, the eppendorf tube is opened, the amplified product is sucked into a new eppendorf tube, marked and diluted by 20-50 times.
A single-target disposable nucleic acid detection test strip (JY 0201) is shown in a structural schematic diagram in FIG. 6, the immersion area end (marked with blue arrow upwards) of the test strip is inserted into an eppendorf tube, the liquid level cannot exceed the MAX indication line of the immersion area, the area to be interpreted is fully immersed (about 30-60 sec is needed), the test strip is flatly placed for 1min, and the appearance of a red strip is waited. And directly reading the detection result according to the color development condition of the test strip. The results were observed within 10min, and the interpretation was not valid after 10 min.
As shown in fig. 7 to 10, a duck-origin component detection primer set B, C, D and a pig-origin component detection primer set E, F, G are used, respectively; the detection primer group J, K, L of bovine-derived component and the detection primer group N, O, P of sheep-derived component are used for carrying out RAA amplification, and after the RAA amplification products of the negative control amplification products of the detection primer group are diluted by 20 times, the detection result of the test strip is positive and can not be used for the sensitivity detection of the subsequent RAA nucleic acid amplification test strip; the detection results of the test strips are negative and 1 multiplied by 10 after the negative control amplification products of the duck-origin component detection primer group A, the pig-origin component detection primer group H, the cattle-origin component detection primer group I and the sheep-origin component detection primer group O are diluted by 20 times 6 The RAA detection results of the copy/mu L recombinant plasmid solution are positive, and the method can be used for the sensitivity detection of the subsequent RAA nucleic acid amplification test strip.
Determining a Duck-origin component detection primer group A, a Pig-origin component detection primer group H, a cattle-origin component detection primer group I and a sheep-origin component detection primer group M as animal-origin detection primer groups, and renaming a probe primer Duck-improved-P1 in the Duck-origin component detection primer group A as Duck-improved-P, a probe primer Pig-origin component detection primer group H as Pig-improved-P, a probe primer Bovine-improved-P1 in the cattle-origin component detection primer group I as Bovine-P and a probe primer Ovine-P1 in the sheep-origin component detection primer group M as Ovine-improved-P.
Experimental example 5: detection sensitivity of duck-origin component detection primer group RAA-LFS
Using the positive plasmid of the duck-origin component detection primer set A for the corresponding amplified fragment as a template, the RAA amplification system of Experimental example 4 was used at 1X 10 6 Copy/. Mu.L, 1X 10 5 Copy/. Mu.L, 1X 10 4 Copy/. Mu.L, 1X 10 3 Copy/. Mu.L, 1X 10 2 Copy/. Mu.L, 1X 10 1 Copy/. Mu.L and 1X 10 0 And (3) taking copy/mu L plasmid diluent as a template, respectively carrying out RAA amplification, diluting an amplified product by 20 times, and then carrying out test strip detection to determine the detection sensitivity of different primer combinations.
The detection result of the sensitivity of the duck-origin component detection primer set ARAA-LFS is shown in figure 11, and the minimum detection threshold of the duck-origin component RAA-LFS is 10 3 And copies.
Example 6: optimized RAALFS detection system using duck-origin component detection primer set A
The probes used in the experiments had the following characteristics: (1) the design direction is forward, i.e. the same direction as the upstream primer; (2) the length is 46-52 nt; (3) the 3' end is modified by phosphorylation to inhibit DNA chain extension; (4) a cleavage site is added in the sequence, and when the probe is combined with the template DNA, exo enzyme is used for cleavage, and extension of the DNA chain is started; (6) the 5' end is modified by biotin, and the double-stranded amplification product of the probe and the downstream (FITC-modified) primer can be detected by a nucleic acid test strip.
The test strip detection product is a double-stranded amplification product of a probe and a downstream primer (modified by FITC) after being cut by exo enzyme, so that theoretically increasing the addition amount of the downstream primer is beneficial to improving the sensitivity of RAA nucleic acid test strip detection. However, when the amount of the downstream primer is excessively added, the probability of formation of a dimer of the downstream primer and the probe primer is also caused, and false positive of the detection result is caused.
UsingThe RAA nucleic acid amplification kit (test strip method) (JY 0204) was used for preparing the RAA reaction system (single sample/reaction), and the amount of the added downstream primer was adjusted as shown in Table 3. Each group of systems is paired with 10 2 Copy/. Mu.L sample plate, 10 1 Copy/. Mu.L sample plate, 10 0 Copy/. Mu.L sample plate, negative quality control samples were tested. The tube cover needs to be buckled immediately after the sample is added, so that aerosol pollution is avoided. And uniformly mixing the reaction systems, and adding the mixture into a basic reaction unit. The lyophilized powder was sufficiently dissolved, and note that this step was not possible to mix by vigorous shaking using a vortex shaker. The reaction unit was opened and 2.5. Mu.L of 280mM MgAc was added to each 0.2mL eppendorf tube 2 Thoroughly mixed and collected by centrifugation. Note that this step cannot be mixed by vigorous shaking with a vortex shaker.
The reaction tube was placed at 39℃for 16min. After the RAA reaction is finished, opening the eppendorf tube, sucking the amplified product into a new eppendorf tube, marking, diluting by 20 times, detecting a test strip, and determining the optimal addition amount.
TABLE 3 Duck-derived component amplification primer set adjustment of the amount of downstream primer addition in ARAA amplification System
The results are shown in FIG. 12, at 10 2 Copy/. Mu.L sample plate, 10 1 Copy/. Mu.L sample plate, 10 0 Copy/mu L sample tray, negative quality control sample as template, adjusting the addition amount of the downstream primer under the condition of the same template addition volume, and when the addition amount of the 50 mu L downstream primer (10 mu M) of the amplification system is adjusted to 3 mu L, the detection sensitivity is optimal, and not only 10 can be detected 1 The copied sample, while the negative quality control sample is negative.
MgAc is added into the amplification system 2 RAA amplification can be started, and MgAc in an amplification system 2 The amount of added(s) directly affects the efficiency of amplification and the yield of product. Theoretically, mgAc is increased 2 The addition amount of (2) is helpful to improve the yield of amplified products and increase the detection sensitivity of the RAA nucleic acid test strip. But MgAc 2 When added in excess, false positives of negative sample detection results are also caused.
UsingRAA nucleic acid amplification kit (test strip method) (JY 0204) for preparing RAA reaction system (single sample/reaction), mgAc 2 The adjustment system of the addition amount is as follows.
TABLE 4 Duck-derived component amplification primer set ARAA amplification System MgAc 2 Adjustment of the amount of addition
Each group of systems is paired with 10 2 Copy/. Mu.L sample plate, 10 1 Copy/. Mu.L sample plate, 10 0 Copy/. Mu.L sample plate, negative quality control samples were tested. The tube cover needs to be buckled immediately after the sample is added, so that aerosol pollution is avoided. And uniformly mixing the reaction systems, and adding the mixture into a basic reaction unit. The lyophilized powder was sufficiently dissolved, and note that this step was not possible to mix by vigorous shaking using a vortex shaker. The reaction unit was opened and different volumes of 280mM MgAc, as indicated in Table 4, were added in each of the 0.2mL eppendorf tubes 2 Thoroughly mixed and collected by centrifugation. Note that this step cannot be mixed by vigorous shaking with a vortex shaker.
The reaction tube was placed at 39℃for 16min. After the RAA reaction is finished, opening the eppendorf tube, sucking the amplified product into a new eppendorf tube, marking, diluting by 20 times, detecting a test strip, and determining the optimal addition amount.
The experimental results are shown in FIG. 13, at 10 2 Copy/. Mu.L sample plate, 10 1 Copy/. Mu.L sample plate, 10 0 Copying/mu L sample disc, taking negative quality control sample as template, and adjusting MgAc under the condition of the same template adding volume 2 When MgAc is added in the amount of 2 Detection sensitivity was adjusted to 2.5. Mu.L and 2.8. Mu.LThe performance is the same, not only 10 can be detected 1 Copy/. Mu.L of sample, while negative control samples were negative. As is clear from the results of the adjustment systems 2 and 3 shown in Table 4, mgAc 2 The addition of excess can result in false positives in negative quality control samples. In view of MgAc per 50. Mu.L of amplification system 2 The addition amount of 2.5 mu L and 2.8 mu L shows the same detection sensitivity, and finally MgAc is determined 2 The optimum addition amount of (2) is 2.5. Mu.L to avoid false positives in the negative samples.
Example 7: pig-derived component, cattle-derived component and sheep-derived component detection primer set RAA-LFS detection sensitivity
The pig-derived component, cattle-derived component and sheep-derived component RAA were amplified as described in example 6RAA nucleic acid amplification kit (test strip method) (JY 0204) amplification system and test strip detection mode to prepare RAA reaction system to complete different primer combinations of 1×10 6 Copy/. Mu.L, 1X 10 5 Copy/. Mu.L, 1X 10 4 Copy/. Mu.L, 1X 10 3 Copy/. Mu.L, 1X 10 2 Copy/. Mu.L, 1X 10 1 Copy/. Mu.L and 1X 10 0 RAA amplification of copy/. Mu.L plasmid dilution, and test strip detection after 20-fold dilution of amplified product. Referring to FIGS. 14 to 16, the pig-origin component detecting primer set H, the cattle-origin component detecting primer set I and the sheep-origin component detecting primer set M can detect as low as 10 after the amplification of RAA 1 Each copy of the target gene.
Example 8: for specificity evaluation of detection primers for duck-origin components, pig-origin components, cattle-origin components and sheep-origin components
In the embodiment, the PCR verification shows that beef, mutton, duck, pork and chicken without exogenous pollution are used as experimental objects, and total DNA is extracted as an amplification template. Using the primers of Duck-origin, pig-origin and cattle-origin components selected in example 4, the primers of example 6 were used The preparation of RAA reaction system is carried out by adopting a RAA nucleic acid amplification kit (test strip method) (JY 0204) amplification system and a test strip detection mode, the RAA amplification is carried out on total DNA respectively, the RAA amplification is completed, the test strip detection is carried out after the amplified product is diluted by 20 times, and the specificity of the detection of different primer combinations is determined.
The specific evaluation results of the RAA-LFS primers of the duck-origin component detection primer group A, the pig-origin component detection primer group H, the cattle-origin component detection primer group I and the sheep-origin component detection primer group M are shown in FIG. 17, and the LFS detection results of the amplified products of the duck-origin component detection primer group, the pig-origin component detection primer group, the cattle-origin component and the sheep-origin component detection primer group are positive only in the corresponding total DNA, and no cross reaction with other species DNA is found, so that the screened primers have excellent specificity and are suitable for RAA-LFS detection of the duck-origin component, the pig-origin component and the sheep-origin component;
experimental example 9: establishment of double RAA-LFS detection system of different primer sets of duck-origin component, pig-origin component, cattle-origin component and sheep-origin component
The duck-origin component A, the pig-origin component detection primer set H, the bovine-origin component detection primer set I and the sheep-origin component detection primer set M in example 7 are configured as mix, so that the primer addition amount in the RAA double amplification system is optimized, and the primer mix comprises the following components:
Amplification of products with the upper and lower primers of each primer set 1X 10 6 Copy/. Mu.L plasmid dilution was used as templateRAA nucleic acid amplification kit (test strip method) (JY 0)204 Preparation of a dual RAA reaction system (single sample/reaction) of duck-derived and bovine-derived components, the system is as follows: />
Negative control sample (1×10) 6 The copy/. Mu.L recombinant plasmid solution was replaced with the same volume of ultrapure water), and the sample addition sequence was negative quality control samples, 1X 10 6 Copying/mu L of the mixed plasmid solution with the same concentration as the duck-origin component, the bovine-origin component and the like, and immediately fastening a tube cover after each sample is added, so that aerosol pollution is avoided. And uniformly mixing the reaction systems, and adding the mixture into a basic reaction unit. The lyophilized powder was sufficiently dissolved, and note that this step was not possible to mix by vigorous shaking using a vortex shaker. The reaction unit was opened, 2.5. Mu.L of 280mM MgAc2 was added to each of the 0.2mL eppendorf tubes, thoroughly mixed and collected by centrifugation. Note that this step cannot be mixed by vigorous shaking with a vortex shaker.
Amplification of products with the upper and lower primers of each primer set 1X 10 6 Copy/. Mu.L of positive plasmid dilution as template, useThe RAA nucleic acid amplification kit (test strip method) (JY 0204) is used for preparing a pig-derived component and cattle-derived component dual RAA reaction system (single sample/reaction) as follows:
Negative control sample (1×10) 6 The copy/. Mu.L recombinant plasmid solution was replaced with the same volume of ultrapure water), and the sample addition sequence was negative quality control samples, 1X 10 6 The plasmid solution is mixed by copying/mu L pig-derived component and bovine-derived component with the same concentration, and a tube cover is required to be buckled immediately after each sample is added, so that aerosol pollution is avoided. And uniformly mixing the reaction systems, and adding the mixture into a basic reaction unit. Make the freeze-dried powder fully dissolved, pay attention to, theThe step cannot be carried out by using a vortex oscillator to carry out intense vibration and uniform mixing. The reaction unit was opened and 2.5. Mu.L of 280mM MgAc was added to each 0.2mL eppendorf tube 2 Thoroughly mixed and collected by centrifugation. Note that this step cannot be mixed by vigorous shaking with a vortex shaker.
Amplification of products with the upper and lower primers of each primer set 1X 10 6 Copy/. Mu.L plasmid dilution was used as templateThe RAA nucleic acid amplification kit (test strip method) (JY 0204) is used for preparing a dual RAA reaction system (single sample/reaction) of duck-origin components and sheep-origin components, and the system is as follows: />
Negative control sample (1×10) 6 The copy/. Mu.L recombinant plasmid solution was replaced with the same volume of ultrapure water), and the sample addition sequence was negative quality control samples, 1X 10 6 And copying/mu L of the plasmid solution mixed by the duck-derived component and the sheep-derived component with the same concentration, and immediately fastening a tube cover after each sample is added, so that aerosol pollution is avoided. And uniformly mixing the reaction systems, and adding the mixture into a basic reaction unit. The lyophilized powder was sufficiently dissolved, and note that this step was not possible to mix by vigorous shaking using a vortex shaker. The reaction unit was opened, 2.5. Mu.L of 280mM MgAc2 was added to each of the 0.2mL eppendorf tubes, thoroughly mixed and collected by centrifugation. Note that this step cannot be mixed by vigorous shaking with a vortex shaker.
Amplification of products with the upper and lower primers of each primer set 1X 10 6 Copy/. Mu.L plasmid dilution was used as templateThe RAA nucleic acid amplification kit (test strip method) (JY 0204) is used for preparing a pig-derived component and sheep-derived component dual RAA reaction system (single sample/reaction) as follows:
negative control sample (1×10) 6 The copy/. Mu.L recombinant plasmid solution was replaced with the same volume of ultrapure water), and the sample addition sequence was negative quality control samples, 1X 10 6 And copying/mu L of the plasmid solution mixed by pig-derived components and sheep-derived components with the same concentration, and immediately fastening a tube cover after each sample is added, so that aerosol pollution is avoided. And uniformly mixing the reaction systems, and adding the mixture into a basic reaction unit. The lyophilized powder was sufficiently dissolved, and note that this step was not possible to mix by vigorous shaking using a vortex shaker. The reaction unit was opened and 2.5. Mu.L of 280mM MgAc was added to each 0.2mL eppendorf tube 2 Thoroughly mixed and collected by centrifugation. Note that this step cannot be mixed by vigorous shaking with a vortex shaker.
The reaction tube was placed at 39℃for 16min. After the RAA reaction is finished, the eppendorf tube is opened, the amplified product is sucked into a new eppendorf tube, marked and diluted by 20-50 times.
A structural schematic diagram of a double-target disposable nucleic acid detection test strip (JY 0209) is shown in FIG. 18, the end of an immersion area (marked with a blue arrow upwards) of the test strip is inserted into an eppendorf tube, the liquid level cannot exceed the MAX indication line of the immersion area, the area to be interpreted is fully immersed (about 30-60 sec is needed), the test strip is flatly placed for 1min, and a red strip is waited to appear. And directly reading the detection result according to the color development condition of the test strip. The results were observed within 10min, and the interpretation was not valid after 10 min.
From the results of the double RAA-LFS detection of the positive recombinant plasmid and the negative control, the color development results of the duck-origin component detection primer group A and the ox-origin component detection primer group I (figure 19), the pig-origin component detection primer group H and the ox-origin component detection primer group I (figure 20), the duck-origin component detection primer group A and the sheep-origin component detection primer group M (figure 21), and the pig-origin component detection primer group H and the sheep-origin component detection primer group M (figure 22) are consistent with the expected color development results, wherein the test strip detection result after the negative control amplification product is diluted by 20 times is negative, the test strip detection result after the positive plasmid amplification product is diluted by 20 times is positive, and the test strip detection method can be used for the meat detection of the subsequent double RAA-LFS.
Experimental example 10: duck-origin component detection primer set 2, pig-origin component detection primer set 5, cattle-origin component detection primer set 11 and sheep-origin component detection primer set 13 absolute quantitative standard curve drawing
Real-time qPCR was performed using the alignment plate in example 2, and the sensitivity of the primers was verified using the forward primer and the reverse primer in the duck-origin component detection primer set 2, the pig-origin component detection primer set 5, the cow-origin component detection primer set 11, and the sheep-origin component detection primer set 13, and the copy number was absolutely quantified.
As can be seen from fig. 23, qPCR melting curve analysis of the duck-origin component detection primer set 2, the pig-origin component detection primer set 5, the cow-origin component detection primer set 11 and the sheep-origin component detection primer set 13 are all sharp single peaks, which indicates that the duck-origin component detection primer set, the pig-origin component detection primer set, the cow-origin component detection primer set and the sheep-origin component detection primer set screened in this embodiment have high amplification specificity and amplification efficiency.
FIG. 24 shows that the above-mentioned duck-origin component detecting primer set 2, pig-origin component detecting primer set 5, bovine-origin component detecting primer set 11 (10 can be successfully detected) 1 ～10 6 Copy of the initial template) and sheep-derived component detection primer set 13 (which can successfully detect 10) 2 ～10 6 Initial template of copy), spans the dynamic detection range of more than 5 logarithmic values, and the template dilution gradient difference is 10 times and the Cq value difference is similar, thus showing good detection effect.
Duck-derived component detection primer set 2Cq value and logarithmically converted copy number (y= -5.5095x+46.385, R) 2 >0.993 Porcine-derived ingredient detection primer set 5Cq value and log-transformed copy number (y= -5.0431x+37.514, r) 2 >0.9965 Bovine-derived component detection primer set 11Cq value and log-transformed copy number (y= -3.9078x+33.246, r) 2 >0.9974 Sheep-derived component detection primer set 13Cq value and log-transformed copy number (y= -5.47x+51.056, R) 2 >0.9975 With good linear relationship, indicatingThe primer designed by the embodiment has better amplification specificity and sensitivity, and can be used for RT-PCR and RAA analysis of duck-origin components, pig-origin components, cattle-origin components and sheep-origin components.
Experimental example 11 Duplex RAA-LFS sensitivity detection of Duck-derived component, pig-derived component and bovine-derived component and sheep-derived component detection primer set
The plasmids corresponding to the amplified fragments of the respective pairs, which were verified in example 10, were used as templates, and 1X 10 was performed using the RAA amplification system in Experimental example 9, respectively, using the duck-origin component detection primer set A, pig-origin component detection primer set H, bovine-origin component detection primer set I, and sheep-origin component detection primer set M 3 Copy/. Mu.L, 1X 10 2 Copy/. Mu.L, 1X 10 1 Copy/. Mu.L of duck-derived and bovine-derived components at the same concentration, 1×10 3 Copy/. Mu.L, 1X 10 2 Copy/. Mu.L, 1X 10 1 Copy/. Mu.L of porcine-derived and bovine-derived components at the same concentration, 1X 10 3 Copy/. Mu.L, 1X 10 2 Copy/. Mu.L, 1X 10 1 Copy/μl of the same concentration duck-derived component and sheep-derived component, 1×10 3 Copy/. Mu.L, 1X 10 2 Copy/. Mu.L, 1X 10 1 And (3) double RAA amplification of the positive mixed plasmid diluent of the pig-derived component and the sheep-derived component with the same concentration in copy/mu L, and detecting the amplified product by a test strip after 20-time dilution, so as to determine the detection sensitivity of the primer combination.
Referring to FIGS. 25-28, the dual RAA-LFS sensitivity detection results of different primer combinations show that the lowest detection threshold value for simultaneous and accurate detection of the dual RAA-LFS of the duck-origin component detection primer group A and the bovine-origin component detection primer group I is 10 3 Copy number; the minimum detection threshold value for simultaneous and accurate detection of the swine-origin component detection primer group H and the bovine-origin component detection primer group I dual RAA-LFS is 10 4 Copy number; the minimum detection threshold value for simultaneous and accurate detection of the duck-origin component detection primer group A and the sheep-origin component detection primer group M dual RAA-LFS is 10 4 Copy number; the minimum detection threshold value for simultaneous and accurate detection of the swine-origin component detection primer group H and the sheep-origin component detection primer group M is 10 1 And copies.
Experimental example 12 optimization of Duck-derived Components, pig-derived Components and, bovine-derived Components and sheep-derived Components Dual RAA-LFS detection System
UsingThe RAA nucleic acid amplification kit (test strip method) (JY 0204) is used for preparing a dual RAA reaction system (single sample/reaction) of a duck-origin component detection primer group A and a bovine-origin primer group I component, wherein the system is as follows:
setting negative quality control sample (recombinant plasmid solution is replaced by ultrapure water with the same volume), and the sample adding sequence is negative quality control sample, 1×10 1 Copy/. Mu.L, 1X 10 2 Copy/. Mu.L, 1X 10 3 Copy/mu L positive mixed plasmid solution of duck-origin component and bovine-origin component with the same concentration, and immediately buckle a tube cover after each sample is added, so that aerosol pollution is avoided. And uniformly mixing the reaction systems, and adding the mixture into a basic reaction unit. The lyophilized powder was sufficiently dissolved, and note that this step was not possible to mix by vigorous shaking using a vortex shaker. The reaction unit was opened, 2.5. Mu.L of 280mM MgAc2 was added to each of the 0.2mL eppendorf tubes, thoroughly mixed and collected by centrifugation. Note that this step cannot be mixed by vigorous shaking with a vortex shaker.
UsingThe RAA nucleic acid amplification kit (test strip method) (JY 0204) is used for preparing a pig-derived component and cattle-derived component dual RAA reaction system (single sample/reaction) as follows:
Setting negative quality control sample (recombinant plasmid solution is replaced by ultrapure water with the same volume), and the sample adding sequence is negative quality control sample, 1×10 1 Copying/μL、1×10 2 Copy/. Mu.L, 1X 10 3 Copy/mu L of positive mixed plasmid solution of pig-derived component and bovine-derived component with the same concentration, and immediately after each sample is added, a tube cover is buckled, so that aerosol pollution is avoided. And uniformly mixing the reaction systems, and adding the mixture into a basic reaction unit. The lyophilized powder was sufficiently dissolved, and note that this step was not possible to mix by vigorous shaking using a vortex shaker. The reaction unit was opened and 2.5. Mu.L of 280mM MgAc was added to each 0.2mL eppendorf tube 2 Thoroughly mixed and collected by centrifugation. Note that this step cannot be mixed by vigorous shaking with a vortex shaker.
UsingThe RAA nucleic acid amplification kit (test strip method) (JY 0204) is used for preparing a dual RAA reaction system (single sample/reaction) of duck-origin components and sheep-origin components, and the system is as follows:
setting negative quality control sample (recombinant plasmid solution is replaced by ultrapure water with the same volume), and the sample adding sequence is negative quality control sample, 1×10 1 Copy/. Mu.L, 1X 10 2 Copy/. Mu.L, 1X 10 3 Copy/mu L positive mixed plasmid solution of duck-origin component and sheep-origin component with the same concentration, and immediately buckle a tube cover after each sample is added, so that aerosol pollution is avoided. And uniformly mixing the reaction systems, and adding the mixture into a basic reaction unit. The lyophilized powder was sufficiently dissolved, and note that this step was not possible to mix by vigorous shaking using a vortex shaker. The reaction unit was opened, 2.5. Mu.L of 280mM MgAc2 was added to each of the 0.2mL eppendorf tubes, thoroughly mixed and collected by centrifugation. Note that this step cannot be mixed by vigorous shaking with a vortex shaker.
UsingDual RAA reaction system for pig-derived component and sheep-derived component by using RAA nucleic acid amplification kit (test strip method) (JY 0204)(single sample/reaction) the system is as follows:
setting negative quality control sample (recombinant plasmid solution is replaced by ultrapure water with the same volume), and the sample adding sequence is negative quality control sample, 1×10 1 Copy/. Mu.L, 1X 10 2 Copy/. Mu.L, 1X 10 3 Copy/mu L positive mixed plasmid solution of pig-derived component and sheep-derived component with the same concentration, and immediately after each sample is added, a tube cover is buckled, so that aerosol pollution is avoided. And uniformly mixing the reaction systems, and adding the mixture into a basic reaction unit. The lyophilized powder was sufficiently dissolved, and note that this step was not possible to mix by vigorous shaking using a vortex shaker. The reaction unit was opened and 2.5. Mu.L of 280mM MgAc was added to each 0.2mL eppendorf tube 2 Thoroughly mixed and collected by centrifugation. Note that this step cannot be mixed by vigorous shaking with a vortex shaker.
The reaction tube was placed at 39℃for 16min. After the RAA reaction is finished, the eppendorf tube is opened, the amplified product is sucked into a new eppendorf tube, marked and diluted by 20-50 times.
The detection result of dual RAA-LFS sensitivity of the detection primer set for the duck-origin component and the cattle-origin component is shown in FIG. 29, the detection result of dual RAA-LFS sensitivity of the detection primer set for the pig-origin component and the cattle-origin component is shown in FIG. 30, the detection result of dual RAA-LFS sensitivity of the detection primer set for the duck-origin component and the sheep-origin component is shown in FIG. 31, and the detection result of dual RAA-LFS sensitivity of the detection primer set for the pig-origin component and the sheep-origin component is shown in FIG. 32. The minimum detection threshold of the dual RAA-LFS of the primer group mix4 for detecting the pig-derived components and the bovine-derived components can reach 10 1 The minimum detection threshold of the dual RAA-LFS of the combined mix2 in the two copies and the rest dual detection systems can reach 10 1 And copies.
Experimental example 13: dual RAA-LFS detection of animal-derived components of simply split meat and meat products
qPCR identification shows that the compositions respectively contain duck-origin components,Meat processed product (halogen-containing processed meat product) of pig-derived component and cattle-derived component and sheep-derived component is prepared by taking about 50mg sample and 100 μlThe cool flash nucleic acid releasing agent (BT 0068) is mixed, and after the sample to be detected is sheared by an ophthalmic scissors, the mixture is centrifuged, and 2 mu L of lysate supernatant is taken for detection.
Amplification by the double RAA amplification System optimized in Experimental example 11, using supernatant of meat and meat processed product lysate as templateThe RAA nucleic acid amplification kit (test strip method) (JY 0204) is used for preparing a dual RAA reaction system (single sample/reaction) of duck-origin components and bovine-origin components, and the system is as follows:
| component (A) | Dosage (mu L) |
| Base buffer | 29.4 |
| Duck Derived-F(5μM) | 0.88 |
| Duck Derived-R(5μM) | 1.26 |
| Duck Derived-P(1μM) | 1.26 |
| Bovine Derived-F(5μM) | 1.17 |
| Bovine Derived-R(5μM) | 1.68 |
| Bovine Derived-P(1μM) | 1.68 |
| Lysate supernatant | 2 |
| Make up the volume with water | 47.5 |
Amplification was performed using the double RAA amplification system optimized in Experimental example 11, using supernatant of meat and meat processed product lysate as template, usingThe RAA nucleic acid amplification kit (test strip method) (JY 0204) is used for preparing a pig-derived component and cattle-derived component dual RAA reaction system (single sample/reaction) as follows:
| Component (A) | Dosage (mu L) |
| Base buffer | 29.4 |
| Pig Derived-F(5μM) | 0.73 |
| Pig Derived-R(5μM) | 1.05 |
| Pig Derived-P(1μM) | 1.05 |
| Bovine Derived-F(5μM) | 0.44 |
| Bovine Derived-R(5μM) | 0.63 |
| Bovine Derived-P(1μM) | 0.63 |
| Lysate supernatant | 2 |
| Make up the volume with water | 47.5 |
Amplification by the double RAA amplification System of Experimental example 11 using supernatant of meat and meat processed product lysate as templateThe RAA nucleic acid amplification kit (test strip method) (JY 0204) is used for preparing a dual RAA reaction system (single sample/reaction) of duck-origin components and sheep-origin components, and the system is as follows:
| component (A) | Dosage (mu L) |
| Base buffer | 29.4 |
| Duck Derived-F(5μM) | 0.58 |
| Duck Derived-R(5μM) | 0.84 |
| Duck Derived-P(1μM) | 0.84 |
| Ovine Derived-F(5μM) | 1.03 |
| Ovine Derived-R(5μM) | 1.48 |
| Ovine Derived-P(1μM) | 1.48 |
| Lysate supernatant | 2 |
| Make up the volume with water | 47.5 |
Amplification by the double RAA amplification System of Experimental example 11 using supernatant of meat and meat processed product lysate as templatePig with RAA nucleic acid amplification kit (test strip method) (JY 0204)Preparation (single sample/reaction) of a dual RAA reaction system of source component and sheep source component, the system is as follows:
the sample adding sequence is negative quality control samples (the supernatant of the lysate is replaced by ultrapure water with the same volume), and the supernatants of meat and meat processed products, and a tube cover is required to be buckled immediately after each sample is added, so that aerosol pollution is avoided. And uniformly mixing the reaction systems, and adding the mixture into a basic reaction unit. The lyophilized powder was sufficiently dissolved, and note that this step was not possible to mix by vigorous shaking using a vortex shaker. The reaction unit was opened and 2.5. Mu.L of 280mM MgAc was added to each 0.2mL eppendorf tube 2 Thoroughly mixed and collected by centrifugation. Note that this step cannot be mixed by vigorous shaking with a vortex shaker.
The reaction tube was placed at 39℃for 16min. After the RAA reaction is finished, the eppendorf tube is opened, the amplified product is sucked into a new eppendorf tube, marked and diluted by 20-50 times.
After simple cracking, the animal-derived component dual RAA-LFS detection results of the meat and meat processed products are shown in figure 33, and duck or beef samples are detected respectively by using a duck-derived component detection primer group and a beef-derived component detection primer group; detecting pork or beef samples respectively by using a pig-derived component and a beef-derived component detection primer set; respectively detecting duck meat or mutton samples by using a duck-derived component and sheep-derived component detection primer group; pork or mutton samples are detected using a pig-derived component and sheep-derived component detection primer set, respectively. After the animal-derived component detection primer groups of each sample are detected by the double RAA-LFS, the color development results of the amplified products are consistent with the qPCR identification results under the condition that negative control shows negative.
Experimental example 14: self-made adulterated beef product containing duck-origin components, adulterated beef product containing pig-origin components, adulterated mutton product containing duck-origin components and dual RAA-LFS rapid detection of adulterated mutton product containing pig-origin components
After the duck and beef samples are quickly frozen by liquid nitrogen, grinding the duck and beef samples into powder by using a mortar respectively, and respectively mixing the duck powder and the beef powder in a frozen state according to a ratio of 1: 1. 1: 9. 1:99 and 1: the 999 ratios were thoroughly mixed. The four proportions of samples were weighed 100mg each and divided into two equal parts, one of which, 50mg, was self-made adulterated meat samples, was used to extract total DNA using a universal genomic DNA extraction kit (new scenery, hangzhou), qPCR analysis was performed, cq values were determined, and sample copy numbers were calculated. Another 50mg sample was prepared according toThe method provided by the cool flash nucleic acid releasing agent is used for completing the self-made rapid splitting of the adulterated meat sample, and the dual RAA amplification system in the experimental example 11 is amplified as follows:
| component (A) | Dosage (mu L) |
| Base buffer | 29.4 |
| Duck Derived-F(5μM) | 0.88 |
| Duck Derived-R(5μM) | 1.26 |
| Duck Derived-P(1μM) | 1.26 |
| Bovine Derived-F(5μM) | 1.17 |
| Bovine Derived-R(5μM) | 1.68 |
| Bovine Derived-P(1μM) | 1.68 |
| Meat and processed meat sample lysate | 2 |
| Make up the volume with water | 47.5 |
After completion of the preparation of the reaction solution, 2.5. Mu.L of 280mM MgAc was added to each of the 0.2mL eppendorf tubes 2 And (5) fully and uniformly mixing.
After pork and beef samples are quickly frozen by liquid nitrogen, grinding the pork and beef samples into powder by using a mortar respectively, and respectively mixing the frozen pork powder and beef powder according to the following weight ratio of 1: 1. 1: 9. 1:99 and 1: the 999 ratios were thoroughly mixed. The four proportions of samples were weighed 100mg each and divided into two equal parts, one 50mg of the home-made adulterated meat samples was used to extract total DNA using a rapid universal genomic DNA extraction kit (new scenery, hangzhou), qPCR analysis was performed, cq values were determined, and sample copy numbers were calculated. Another 50mg sample was prepared according to The method provided by the cool flash nucleic acid releasing agent is used for completing the splitting of self-made adulterated meat samples, and the dual RAA amplification system in the experimental example 11 is amplified as follows:
| component (A) | Dosage (mu L) |
| Base buffer | 29.4 |
| Pig Derived-F(5μM) | 0.73 |
| Pig Derived-R(5μM) | 1.05 |
| Pig Derived-P(1μM) | 1.05 |
| Bovine Derived-F(5μM) | 0.44 |
| Bovine Derived-R(5μM) | 0.63 |
| Bovine Derived-P(1μM) | 0.63 |
| Meat and processed meat sample lysate | 2 |
| Make up the volume with water | 47.5 |
After completion of the preparation of the reaction solution, 2.5. Mu.L of 280mM MgAc was added to each of the 0.2mL eppendorf tubes 2 Is sufficient and fullMixing well.
After the duck and mutton samples are quickly frozen by liquid nitrogen, grinding the duck and mutton samples into powder by using a mortar respectively, and respectively mixing the duck powder and mutton powder in a frozen state according to the following steps of 1: 1. 1: 9. 1:99 and 1: the 999 ratios were thoroughly mixed. The four proportions of samples were weighed 100mg each and divided into two equal parts, one 50mg of the home-made adulterated meat samples was used to extract total DNA using a rapid universal genomic DNA extraction kit (new scenery, hangzhou), qPCR analysis was performed, cq values were determined, and sample copy numbers were calculated. Another 50mg sample was prepared according toThe method provided by the cool flash nucleic acid releasing agent is used for completing the splitting of self-made adulterated meat samples, and the dual RAA amplification system in the experimental example 11 is amplified as follows:
| component (A) | Dosage (mu L) |
| Base buffer | 29.4 |
| Duck Derived-F(5μM) | 0.58 |
| Duck Derived-R(5μM) | 0.84 |
| Duck Derived-P(1μM) | 0.84 |
| Ovine Derived-F(5μM) | 1.03 |
| Ovine Derived-R(5μM) | 1.48 |
| Ovine Derived-P(1μM) | 1.48 |
| Meat and processed meat sample lysate | 2 |
| Make up the volume with water | 47.5 |
After completion of the preparation of the reaction solution, 2.5. Mu.L of 280mM MgAc was added to each of the 0.2mL eppendorf tubes 2 And (5) fully and uniformly mixing.
After the pork and mutton samples are quickly frozen by liquid nitrogen, grinding the pork and mutton samples into powder by using a mortar respectively, and fully mixing the pork powder and the mutton powder in frozen state according to the proportions of 1:1, 1:9, 1:99 and 1:999 respectively. The four proportions of samples were weighed 100mg each and divided into two equal parts, one 50mg of the home-made adulterated meat samples was used to extract total DNA using a rapid universal genomic DNA extraction kit (new scenery, hangzhou), qPCR analysis was performed, cq values were determined, and sample copy numbers were calculated. Another 50mg sample was prepared according toThe method provided by the cool flash nucleic acid releasing agent is used for completing the splitting of self-made adulterated meat samples, and the dual RAA amplification system in the experimental example 11 is amplified as follows:
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after completion of the preparation of the reaction solution, 2.5. Mu.L of 280mM MgAc was added to each of the 0.2mL eppendorf tubes 2 Fully and uniformly mixing; the eppendorf tube (0.2 mL) was placed on a PCR apparatus with a thermal cover function, and incubated at 39℃for 16min. After the RAA reaction is completed, the eppendorf tube is opened, and the amplified product is sucked into a new eppendorf tube, marked and diluted 20 times.
As can be seen from comparing the RAA-LFS detection results (figure 34) and qPCR detection results of self-made animal-derived adulterated meat samples in different proportions, the RAA-LFS detection system invented by the group of the invention can realize the rapid and accurate detection of duck-derived components and cattle-derived components, pig-derived components and cattle-derived components, duck-derived components and sheep-derived components and pig-derived components and sheep-derived components through rapid splitting of the samples.
TABLE 5 qPCR detection results of random sampling of animal-derived adulterated meat from different proportions
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In summary, the present application, through a series of examples, screened out primer sets capable of detecting both duck-derived and bovine-derived components, swine-derived and bovine-derived components, duck-derived and sheep-derived components, swine-derived and sheep-derived components. The primer group can effectively amplify target genes, has higher specificity and sensitivity, does not have cross reaction with other species, can be used for on-site rapid detection of duck-origin components, pig-origin components, cattle-origin components and sheep-origin components, and has important significance for food safety.
The invention shown and described herein may be practiced without any of the elements, limitations specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the invention. It is therefore to be understood that while the present invention has been specifically disclosed by various embodiments and optional features, modification and variation of the concepts herein described may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
The contents of the articles, patents, patent applications, and all other documents and electronically available information described or documented herein are incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to incorporate any and all materials and information from any such articles, patents, patent applications, or other documents.
Claims (9)
1. The RAA detection primer set for detecting duck-origin components, pig-origin components, cattle-origin components and sheep-origin components is characterized by comprising a duck-origin component detection primer, a pig-origin component detection primer, a cattle-origin component detection primer and a sheep-origin component detection primer, a duck-origin component detection probe, a pig-origin component detection probe, a cattle-origin component detection probe and a sheep-origin component detection probe; wherein,,
the Duck-origin component detection primer comprises a forward primer Duck developed-F and a reverse primer Duck developed-R,
the sequence of the forward primer Duck developed-F is 5 '-CCACTTCTTAGAGTACTCCCAG-3',
the sequence of the reverse primer Duck developed-R is 5 '-GGGTTGTAACTGTTGATAGTG-3';
The Pig-Derived component detection primer comprises a forward primer Pig developed-F and a reverse primer Pig developed-R,
the sequence of the forward primer Pic developed-F is 5 '-CCAATAAGCAATGATCAACC-3',
the sequence of the reverse primer Pic developed-R is 5 '-CTATGGCTACTGAGATGTATCC-3';
the Bovine-Derived component detection primer comprises a forward primer Bovine-improved-F and a reverse primer Bovine-improved-R,
the forward primer Bovine developed-F has the sequence of 5 '-CGTATCCCTACCCATCCTTACA-3',
the sequence of the reverse primer Bovine developed-R is 5 '-ACCCGATTCAGACAAGTAGT-3';
the sheep-Derived component detection primer comprises a forward primer Ovine-improved-F and a reverse primer Bovine-improved-R,
the forward primer Ovine developed-F has the sequence of 5 '-CAACACCACACTTCACAGCTTGC-3',
the sequence of the reverse primer Ovine developed-R is 5 '-GGATCCTGCTAGTGTGTAA-3'.
2. The primer set of claim 1, wherein the Duck-Derived component detection probe Duck developed-P has a sequence of 5 '-GCACAAGCTCCAACACAACAAATAAAGTCAHCAAGAGCCCTCAACC-3';
the Pig-Derived component detection probe Pig-Derived-P has a sequence of 5 '-CGAATCACCCGTATCATAAATTACTCAATCCHCAAGCCCATTAAACTT-3';
The Bovine-Derived component detection probe Bovine developed-P has a sequence of 5 '-CTCTTCGTGCTCCCAATTACACCAAATTCHATTAGTAAGGTCAGC-3';
the sheep-Derived component detection probe Ovine developed-P has a sequence of 5 '-CCATATGACTTCTACCCCTAATACTCATAHCTAGCCAACATCATC-3';
FITC is modified at the 5 ' -end of reverse primers Duck and Pig Derived components required for detection, biotin is modified at the 5 ' -end of detection probes Duck and Pig Derived components, 3 ' -end of Duck and Pig Derived components is phosphorylated, and H is tetrahydrofuran; digoxin is modified at the 5 ' -end of reverse primers Bovine-D-R and Ovine-D-R of Bovine-Derived components and sheep-Derived components, rhodamine is modified at the 5 ' -end of probe primers Bovine-D-P and Ovine-D-P, phosphorylation modification is carried out at the 3 ' -end of Bovine-D-P and Ovine-D-P, and H is tetrahydrofuran.
3. A kit for detecting whether or not beef-derived components, sheep-derived components, duck-derived components and/or pig-derived components are contained in meat and processed meat, comprising the primer set of claim 1 or 2.
4. The use of a primer set according to claim 1 or 2 for the preparation of a reagent or kit for detecting whether or not beef or mutton to be tested and processed meat contains components of duck origin and components of pig origin.
5. Use of a kit according to claim 3 for detecting whether meat and processed meat contains bovine-derived components, sheep-derived components, duck-derived components and/or pig-derived components.
6. A method for detecting whether beef or mutton to be detected and processed meat contain duck-origin components and pig-origin components by using a kit, which is characterized by comprising the following steps:
1) Extracting total DNA of meat to be detected and processed meat, or carrying out rapid cracking and nucleic acid releasing treatment on meat and processed meat samples;
2) Taking the total DNA extracted in the step 1) or supernatant of the sample lysate as a template, and carrying out RAA amplification by adopting the forward primer and the reverse primer in the primer set according to the claim 1 or 2 to obtain an amplification product;
3) Detecting the amplified product, and judging whether the amplified product contains duck-derived components and/or bovine-derived components, pig-derived components and/or bovine-derived components or not based on the detection result of the amplified product, wherein the duck-derived components and/or sheep-derived components, pig-derived components and/or sheep-derived components.
7. The method of claim 6, wherein the sample to be tested contains duck-origin components if the amplified product contains a 287bp DNA fragment, and does not contain duck-origin components if the amplified product does not contain a 287bp DNA fragment; if the amplification product contains 274bp DNA fragments, the sample to be detected contains swine-origin components, and if the amplification product does not contain 274bp DNA fragments, the sample to be detected does not contain swine-origin components; if the amplification product contains 351bp DNA fragments, the sample to be detected contains bovine-derived components, and if the amplification product does not contain 351bp DNA fragments, the sample to be detected does not contain bovine-derived components; if the amplification product contains a 397bp DNA fragment, the sample to be detected contains sheep-derived components, and if the amplification product does not contain a 397bp DNA fragment, the sample to be detected does not contain sheep-derived components.
8. A method for detecting whether beef or mutton to be tested and processed meat contain components of duck origin and components of pig origin, comprising the steps of:
detecting whether specific DNA fragments are contained in the meat to be detected and the lysate or the total DNA amplification product of the processed meat, if the specific DNA fragments are contained, the biological sample to be detected contains duck-origin components, pig-origin components, cattle-origin components or sheep-origin components, and if the specific DNA fragments are not contained, the meat to be detected and the processed meat do not contain duck-origin components, pig-origin components, cattle-origin components or sheep-origin components; wherein the specific DNA fragment is the target sequence of the forward primer and the reverse primer of claim 1 or 2.
9. A method for detecting duck-derived and/or bovine-derived, porcine-derived and/or bovine-derived components, duck-derived and/or sheep-derived components, porcine-derived and/or sheep-derived components in meat and processed meat samples based on RAA-lateral flow chromatography, comprising the steps of:
1) Carrying out rapid cracking and nucleic acid releasing treatment on meat and processed meat samples, or extracting total DNA of biological samples to be detected;
2) Carrying out RAA amplification by using the primer set as claimed in claim 1 or 2 by taking the supernatant of the sample lysate obtained in the step 1) or the extracted total DNA as a template;
3) Detecting duck-origin components and/or bovine-origin components RAA amplification products in meat and processed meat samples by using a lateral flow chromatography test strip, and if the test strip only has a quality control area for color development, the detection area has no color development, and the detection result is negative, indicating that the samples do not contain duck-origin components and bovine-origin components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T1, and the detection result is positive, which indicates that the detection sample contains bovine-derived components but does not contain duck-derived components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T2, and the detection result is positive, which indicates that the detection sample contains duck-origin components but does not contain bovine-origin components; if the test strip has three strip color development and two strip color development, one strip is positioned in the quality control area, one strip is positioned in the detection area T1, and the other strip is positioned in the detection area T2, the detection result is positive, and the sample contains bovine-derived components and duck-derived components at the same time; if the quality control area and the two detection areas of the test strip are not developed, the detection is invalid, and the test strip is required to be replaced for retesting;
4) Detecting pig-derived components and/or bovine-derived components RAA amplification products in meat and processed meat samples by using a lateral flow chromatography test strip, and if the test strip only has a color development of a strip in a quality control area, the detection area does not have the color development of the strip, and the detection result is negative, so that the sample does not contain pig-derived components and bovine-derived components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T1, and the detection result is positive, which indicates that the detection sample contains bovine-derived components but does not contain swine-derived components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T2, and the detection result is positive, which indicates that the detection sample contains pig-derived components but does not contain cattle-derived components; if the test strip has three strip color development and two strip color development, one strip is positioned in the quality control area, one strip is positioned in the detection area T1, and the other strip is positioned in the detection area T2, the detection result is positive, and the sample contains bovine-derived components and porcine-derived components at the same time; if the quality control area and the two detection areas of the test strip are not developed, the detection is invalid, and the test strip is required to be replaced for retesting;
5) Detecting the duck-derived component and/or sheep-derived component RAA amplification products in meat and processed meat samples by using a lateral flow chromatography test strip, and if the test strip only has a color development of a strip in a quality control area, the detection area does not have the color development of the strip, and the detection result is negative, so that the sample does not contain the duck-derived component and the sheep-derived component; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T1, and the detection result is positive, which indicates that the detection sample contains sheep-derived components but does not contain duck-derived components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T2, and the detection result is positive, which indicates that the detection sample contains duck-origin components but does not contain sheep-origin components; if the test strip has three strip color development and two strip color development, one strip is positioned in the quality control area, one strip is positioned in the detection area T1, and the other strip is positioned in the detection area T2, the detection result is positive, and the sample contains sheep-derived components and duck-derived components at the same time; if the quality control area and the two detection areas of the test strip are not developed, the detection is invalid, and the test strip is required to be replaced for retesting;
6) Detecting pig-derived components and/or sheep-derived components RAA amplification products in meat and processed meat samples by using a lateral flow chromatography test strip, and if the test strip only has a color development of a strip in a quality control area, the detection area does not have the color development of the strip, and the detection result is negative, so that the sample does not contain pig-derived components and sheep-derived components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T1, and the detection result is positive, which indicates that the detection sample contains sheep-derived components but does not contain pig-derived components; if the test strip has two strip color development, one strip is positioned in the quality control area, the other strip is positioned in the detection area T2, and the detection result is positive, which indicates that the detection sample contains pig-derived components but does not contain sheep-derived components; if the test strip has three strip color development and two strip color development, one strip is positioned in the quality control area, one strip is positioned in the detection area T1, and the other strip is positioned in the detection area T2, the detection result is positive, and the sample contains sheep-derived components and pig-derived components at the same time; if the quality control area and the detection area of the test strip are not developed, the detection is invalid, and the test strip needs to be replaced for retesting.
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| CN111139302A (en) * | 2020-01-03 | 2020-05-12 | 华中农业大学 | On-site rapid detection method and detection test strip for bovine-derived and duck-derived components |
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| CN112941200A (en) * | 2021-02-08 | 2021-06-11 | 韩山师范学院 | Primer pair and probe for detecting duck-origin components, kit and application thereof |
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| KR20160080858A (en) * | 2014-12-29 | 2016-07-08 | 단국대학교 천안캠퍼스 산학협력단 | Composition for identifying meat |
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