A primer set, a kit and a method for detecting adulteration in meat or meat products

Abstract

The present invention relates to the technical field of animal-derived component detection, and specifically to a kit and method for detecting whether duck-derived components and pig-derived components are present in bovine meat and processed meat, and whether duck-derived components and pig-derived components are present in sheep-derived meat and processed meat based on recombinase-mediated isothermal nucleic acid amplification technology (RAA). The present invention designs a specific primer set that can effectively amplify the mitochondrial genome of duck-derived components, the mitochondrial genome of pig-derived components, the mitochondrial genome of bovine-derived components, and the mitochondrial genome of sheep-derived components. The primer set has high specificity and sensitivity and has no cross-reaction with other species. The primer set can be used in combination with a lateral flow test strip to detect duck-derived components and pig-derived components from common adulterated meat sources in meat containing bovine and sheep-derived components and processed meat, and to identify and conduct rapid on-site detection.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety biological detection, and specifically to a primer set, a kit and a method for detecting adulteration in meat or meat products, and in particular to a primer set, a kit and a method for detecting whether duck-derived components or pig-derived components are contained in beef, mutton and processed meat. Background Art

[0002] In recent years, adulteration of beef and mutton has occurred from time to time. In many places, there have been illegal cases of mixing pork, duck, fox meat and other low-value meats into mutton, or directly mixing pork and duck with mutton fat, flavoring, mutton powder, etc. These consecutive exposures of fake mutton production and sales have aroused widespread social concern. There are three main reasons for the adulteration of beef and mutton:

[0003] my country's livestock and poultry meat and meat products consumption market is huge. Since the founding of the People's Republic of China, my country's annual per capita meat consumption has increased by nearly 13 times. The demand for meat consumption has undergone a change from quantity to quality and is currently on a steady upward trend.

[0004] China has a huge livestock and poultry breeding market, which is scattered and difficult for regulatory departments to cover. The supervision is not up to standard. According to relevant statistics, there are more than 3,000 beef cattle trading markets hidden in various small towns and rural streets in my country. Most of these markets are small in scale, with irregular trading methods and inadequate market supervision. There are many loopholes in supervision, which allow unscrupulous vendors to take advantage of them.

[0005] Adulteration of beef and mutton can generate huge profits. Affected by factors such as the increase in feeding costs, the market prices of beef, mutton and other meat products have gradually increased. There are many cases of illegal vendors at home and abroad mixing low-priced meat with high-priced beef and mutton for sale, such as the "horse meat scandal" in the UK and Ireland where horse meat was mixed into beef burgers for sale; in 2013, a case of manufacturing and selling fake mutton was cracked in Wuxi, Jiangsu.

[0006] In view of the frequent incidents of pork and duck meat being mixed with beef and mutton and sold as inferior products, which disrupts the market order, it not only seriously infringes on the rights and interests of consumers, but also threatens the health of consumers. The method of judging the animal source based solely on sensory judgment and morphology (such as color, smell, elasticity, and other senses, as well as muscle texture, etc.) has great limitations and cannot fully meet the current meat product market, especially the rapid detection needs of processed meat products. Therefore, how to use modern molecular biology technology to improve the level of animal source detection and develop a rapid, fast and effective detection system is of great significance to protecting consumer interests and maintaining food safety.

[0007] Currently, the common detection methods for duck-derived components, pig-derived components, bovine-derived components, and sheep-derived components are mainly based on protein chromatography, enzyme-linked immunosorbent assay, and nucleic acid-based molecular biology assays (RT-qPCR, PCR-restriction fragment length polymorphism, and loop-mediated isothermal amplification technology detection, etc.), among which RT-PCR is the most widely used.

[0008] Recombinase-mediated isothermal nucleic acid amplification (RAA) is a nucleic acid amplification technology developed in recent years. It can perform highly specific DNA amplification at a constant low temperature of 39°C and reach a detectable level within minutes. RAA can perform two-step operations of reverse transcription and amplification in the same amplification tube. The product can be detected by agarose gel electrophoresis or visualized by lateral flow strips (LFS). RAA-LFS has the advantages of simple operation, small sample volume, rapid detection, and low cost. It has been widely used in many fields such as medicine, food, and environment. Summary of the invention

[0009] Problem to be solved

[0010] At present, there are many molecular biological detection methods for whether meat and processed meat contain duck-derived ingredients, pig-derived ingredients, bovine-derived ingredients and sheep-derived ingredients, such as RT-PCR and RT-qPCR, which have become conventional means of detection. However, these means and methods are either highly dependent on well-trained laboratory technicians, or require complex and expensive laboratory instruments, or the detection operation process is cumbersome and difficult to be used outside the laboratory environment and under non-laboratory conditions. Duck-derived ingredients or pig-derived ingredients are often added to beef and processed meat for fraud, or duck-derived ingredients or pig-derived ingredients are added to mutton and processed meat. However, due to the limitations of instruments and equipment, the current detection methods are difficult to quickly and accurately identify duck-derived ingredients and bovine-derived ingredients, pig-derived ingredients and bovine-derived ingredients, duck-derived ingredients and sheep-derived ingredients, and pig-derived ingredients and sheep-derived ingredients at the same time under non-laboratory conditions. Therefore, it is urgent to establish a rapid, simple and accurate dual animal-derived component visualization detection method. The RAA dual isothermal nucleic acid amplification system for duck-derived and bovine-derived ingredients, porcine-derived and bovine-derived ingredients, duck-derived and sheep-derived ingredients, and porcine-derived and sheep-derived ingredients has been established and combined with visual LFS detection. This can meet the needs of market supervision departments and local food safety service departments for rapid on-site detection of duck-derived and pig-derived ingredients that are common sources of adulterated meat in beef, mutton and processed meat, and strengthen the supervision of food distribution channels, which is also of great significance to ensuring food safety and consumer rights.

[0011] Technical Solution

[0012] In order to solve the above technical problems, the object of the present invention is to provide a primer set, a kit and a method for detecting duck-derived components, pig-derived components, bovine-derived components and sheep-derived components based on RAA.

[0013] In one aspect of the present invention, the present invention provides a RAA detection primer set for detecting duck-derived components, pig-derived components, bovine-derived components and sheep-derived components, the primer set includes forward detection primers and reverse detection primers for duck-derived components, pig-derived components, bovine-derived components and sheep-derived components, and detection probes for detecting duck-derived components, pig-derived components, bovine-derived components and sheep-derived components; wherein,

[0014] The duck-derived component detection primers include a forward primer Duck Derived-F and a reverse primer Duck Derived-R.

[0015] The sequence of the forward primer Duck Derived-F is 5′-CCACTTCTTAGAGTACTCCCAG-3′

[0016] (SEQ ID NO.3),

[0017] The sequence of the reverse primer Duck Derived-R is 5′-GGGTTGTAACTGTTGATAGTG-3′ (SEQ ID NO. 4);

[0018] The porcine-derived component detection primers include a forward primer Pig Derived-F and a reverse primer Pig Derived-R, wherein the sequence of the forward primer Pig Derived-F is 5′-CCAATAAGCAATGATCAACC-3′ (SEQ ID NO.9),

[0019] The sequence of the reverse primer Pig Derived-R is 5′-CTATGGCTACTGAGATGTATCC-3′ (SEQ ID NO. 10);

[0020] The bovine-derived component detection primers include a forward primer Bovine Derived-F and a reverse primer Bovine Derived-R.

[0021] The sequence of the forward primer Bovine Derived-F is: 5′-CGTATCCCTACCCATCCTTACA-3′ (SEQ ID NO. 21),

[0022] The sequence of the reverse primer Bovine Derived-R is: 5′-ACCCGATTCAGACAAGTAGT-3′ (SEQ ID NO. 22).

[0023] The sheep-derived component detection primers include a forward primer Ovine Derived-F and a reverse primer Bovine Derived-R.

[0024] The sequence of the forward primer Ovine Derived-F is: 5′-CAACACCACACTTCACAGCTTGC-3′ (SEQ ID NO. 25),

[0025] The sequence of the reverse primer Ovine Derived-R is: 5′-GGATCCTGCTAGTGTGTAA-3′ (SEQ ID NO. 26).

[0026] Preferably, the duck-derived component detection probe Duck Derived-P in the present invention has a sequence of 5′-GCACAAGCTCCAACACAACAAATAAAGTCAHCAAGAGCCCTCAACC-3′ (SEQ ID NO.33); the pig-derived component detection probe PigDerived-P has a sequence of 5′-CGAATCACCC GTATCATAAATTACTCAATCCHCAAGCCCATTAAACTT-3′ (SEQID NO.40); the bovine-derived component detection probe Bovine Derived-P has a sequence of 5′-CTCTTCGTGCTCCCAATTACACCAAATTCHATTAGTAAGGTCAGC-3′ (SEQ ID NO.41); the sheep-derived component detection probe Ovine Derived-P has a sequence of 5′-CCATATGACTTCTACCCCT AATACTCATAHCTAGCCAACATCATC-3′ (SEQ ID NO.45); the reverse primers Duck Derived-P for detecting duck-derived components and pig-derived components are The 5′ ends of Derived-R and Pig Derived-R are modified with FITC, the 5′ ends of the detection probes Duck Derived-P and Pig Derived-P are modified with biotin, the 3′ ends of DuckDerive dP and Pig Derived-P are phosphorylated, and H is tetrahydrofuran; the 5′ ends of the bovine-derived component and sheep-derived component reverse primers Bovine Derived-R and Ovine Derived-R are modified with digoxigenin, the 5′ ends of the probe primers Bovine Derived-P and Ovine Derived-P are modified with rhodamine, the 3′ ends of Bovine Derived-P and OvineDerived-P are phosphorylated, and H is tetrahydrofuran.

[0027] In one aspect of the present invention, the present invention provides a kit for detecting duck-derived components, pig-derived components, bovine-derived components and sheep-derived components, wherein the kit comprises the aforementioned primer set.

[0028] In one aspect of the present invention, a RAA detection primer set for detecting duck-derived components, pig-derived components, bovine-derived components and sheep-derived components provided by the present invention can be used to prepare a kit for determining whether the meat and processed meat samples to be tested contain duck-derived components, pig-derived components, bovine-derived components and sheep-derived components.

[0029] In one aspect of the present invention, the kit of the present invention may also contain other reagents, including DNA extraction reagent, sample lysis solution, buffer, PCR sample amplification reagent, and kit instructions.

[0030] In one aspect of the present invention, a RAA detection primer set for detecting duck-derived components and / or bovine-derived components, pig-derived components and / or bovine-derived components provided by the present invention can be used to determine whether the meat to be tested and processed meat contains duck-derived components, pig-derived components and bovine-derived components.

[0031] In one aspect of the present invention, a RAA detection primer set for detecting duck-derived components and / or sheep-derived components, pig-derived components and / or sheep-derived components provided by the present invention can be used to determine whether the meat to be tested and processed meat contains duck-derived components, pig-derived components and sheep-derived components.

[0032] In one aspect of the present invention, the kit provided by the present invention can be used to detect whether the meat to be tested and processed meat contain duck-derived components and / or bovine-derived components, pig-derived components and / or bovine-derived components, as well as duck-derived components and / or sheep-derived components, pig-derived components and / or sheep-derived components.

[0033] In one aspect of the present invention, the present invention provides a method for determining whether beef or mutton and processed meat contain duck-derived ingredients or pig-derived ingredients, comprising the following steps:

[0034] 1) Extracting the total DNA of the meat and processed meat to be tested, or rapidly lysing the meat and processed meat samples to release nucleic acids;

[0035] 2) using the total DNA extracted in step 1) or the supernatant of the sample lysate as a template, and using the forward primer and the reverse primer in the primer set to perform RAA amplification to obtain an amplified product;

[0036] 3) Detecting the amplification product, and judging whether the test sample contains duck-derived components, pig-derived components, sheep-derived components and cattle-derived components based on the detection result of the amplification product.

[0037] Preferably, in the present invention, if the amplified product contains a 287bp DNA fragment, the sample to be tested contains duck-derived components; if the amplified product does not contain the 287bp DNA fragment, the sample to be tested does not contain duck-derived components; if the amplified product contains a 274bp DNA fragment, the sample to be tested contains pig-derived components; if the amplified product does not contain the 274bp DNA fragment, the sample to be tested does not contain pig-derived components; if the amplified product contains a 351bp DNA fragment, the sample to be tested contains bovine-derived components; if the amplified product does not contain the 351bp DNA fragment, the sample to be tested does not contain bovine-derived components; if the amplified product contains a 397bp DNA fragment, the sample to be tested contains sheep-derived components; if the amplified product does not contain the 397bp DNA fragment, the sample to be tested does not contain sheep-derived components.

[0038] In one aspect of the present invention, the present invention provides a method for detecting whether a sample to be tested contains duck-derived components, pig-derived components, sheep-derived components and cattle-derived components, comprising:

[0039] Detect whether the nucleic acid lysate or total DNA amplification product of the tested meat and processed meat contains specific DNA fragments. If the specific DNA fragments are contained, the tested sample contains duck-derived components, pig-derived components, sheep-derived components and bovine-derived components. If the specific DNA fragments are not contained, the tested sample does not contain duck-derived components, pig-derived components, sheep-derived components and bovine-derived components; wherein the specific DNA fragments are the target sequences of the forward primer and the reverse primer described in the present invention.

[0040] In one aspect of the present invention, the present invention provides a method for detecting duck-derived components and / or bovine-derived components, porcine-derived components and / or bovine-derived components, as well as duck-derived components and / or sheep-derived components, porcine-derived components and / or sheep-derived components based on RAA amplification.

[0041] Preferably, the method comprises the following steps:

[0042] 1) Lyse the meat sample according to the method provided by the nucleic acid release agent; or follow the instructions of the DNA extraction reagent to extract total DNA and dissolve it in TE solution or pure water;

[0043] 2) Using the total DNA or meat and processed meat lysate extracted in step 1) as a template, RAA amplification is performed using the forward primer and reverse primer in the primer set. If the amplified product contains a 287 bp DNA fragment, the sample to be tested contains duck-derived components; if the amplified product does not contain the 287 bp DNA fragment, the sample to be tested does not contain duck-derived components; if the amplified product contains a 274 bp DNA fragment, the sample to be tested contains pig-derived components; if the amplified product does not contain the 274 bp DNA fragment, the sample to be tested does not contain pig-derived components; if the amplified product contains a 351 bp DNA fragment, the sample to be tested contains bovine-derived components; if the amplified product does not contain the 351 bp DNA fragment, the sample to be tested does not contain bovine-derived components; if the amplified product contains a 397 bp DNA fragment, the sample to be tested contains sheep-derived components; if the amplified product does not contain the 397 bp DNA fragment, the sample to be tested does not contain sheep-derived components.

[0044] The sequence of the forward primer Duck Derived--F is 5′-CCACTTCTTAGAGTACTCCCAG-3′ (SEQ ID NO. 3),

[0045] The sequence of the reverse primer Duck Derived--R is 5′-GGGTTGTAACTGTTGATAGTG-3′ (SEQ ID NO. 4);

[0046] The sequence of the forward primer Pig Derived-F is 5′-CCAATAAGCAATGATCAACC-3′ (SEQ ID NO. 9),

[0047] The sequence of the reverse primer Pig Derived-R is 5′-CTATGGCTACTGAGATGTATCC-3′ (SEQ ID NO. 10);

[0048] The sequence of the forward primer Bovine Derived-F is: 5′-CGTATCCCTACCCATCCTTACA-3′ (SEQ ID NO. 21),

[0049] The sequence of the reverse primer Bovine Derived-R is: 5′-ACCCGATTCAGACAAGTAGT-3 (SEQ ID NO. 22)′.

[0050] The sequence of the forward primer Ovine Derived-F is: 5′-CAACACCACACTTCACAGCTTGC-3′ (SEQ ID NO. 25),

[0051] The sequence of the reverse primer Ovine Derived-R is: 5′-GGATCCTGCTAGTGTGTAA-3′ (SEQ ID NO. 26).

[0052] Preferably, the reaction system for detecting RAA amplification of duck-derived components and / or bovine-derived components is as follows:

[0053] Element Volume (μL) Meat sample lysate or total DNA solution 2 Basal buffer 29.4 DuckDerived-F (5μM) 0.88 DuckDerived-R(5μM) 1.26 BovineDerived-F(5μM) 1.17 BovineDerived-R(5μM) 1.68 <![CDATA[ddH2O]]> The total system is 47.5

[0054] Preferably, the reaction system for detecting RAA amplification of porcine and / or bovine components is as follows:

[0055] Element Volume (μL) Meat sample lysate or total DNA solution 2 Basal buffer 29.4 PigDerived-F(5μM) 0.73 PigDerived-R(5μM) 1.05 BovineDerived-F(5μM) 0.44 BovineDerived-R(5μM) 0.63 <![CDATA[ddH2O]]> The total system is 47.5

[0056] Preferably, the reaction system for detecting RAA amplification of duck-derived components and / or sheep-derived components is as follows:

[0057] Element Volume (μL) Meat sample lysate or total DNA solution 2 Basal buffer 29.4 DuckDerived-F (5μM) 0.58 DuckDerived-R(5μM) 0.84 OvineDerived-F(5μM) 1.03 OvineDerived-R(5μM) 1.48 <![CDATA[ddH2O]]> The total system is 47.5

[0058] Preferably, the reaction system for detecting RAA amplification of porcine and / or sheep components is as follows:

[0059] Element Volume (μL) Meat sample lysate or total DNA solution 2 Basal buffer 29.4 PigDerived-F(5μM) 0.51 PigDerived-R(5μM) 0.73 OvineDerived-F(5μM) 0.88 OvineDerived-R(5μM) 1.26 <![CDATA[ddH2O]]> The total system is 47.5

[0060] Preferably, the reaction conditions for RAA amplification are set as follows: after the reaction solution is prepared, 2.5 μL of 280 mM MgAc2 is added to each 0.2 mL eppendorf tube and mixed thoroughly; the 0.2 mL eppendorf tube is placed in a PCR instrument with a hot cover function, at 39° C., and kept warm for 25 minutes.

[0061] Preferably, the amplified product can be detected by agarose gel electrophoresis. The minimum copy number of the target sequence of the duck-derived component RAA amplified product detected by agarose gel electrophoresis is 10 1 The minimum number of copies of the initial amplification template for the target sequence detected by agarose gel electrophoresis of the amplified product of porcine RAA was 10 1 The minimum copy number of the target sequence initial amplification template detected by agarose gel electrophoresis of the amplified product of bovine RAA was 10 1 copies.

[0062] The detection process of RAA amplification products is as follows: After the RAA reaction is completed, the reaction tube is taken out. 100 μL of phenol / chloroform (1:1) is added to each reaction tube, fully shaken and evenly mixed, and centrifuged at 12000rpm for 10 minutes (this step can be vigorously shaken and mixed using a vortex shaker). Take 10 μL of supernatant and mix with 2 μL of 6× Loading Buffer, load on 1.5% agarose gel, and electrophoresed at 200V for 15 minutes; end the electrophoresis when bromophenol blue moves to the lower 2 / 3 of the gel; EB stain for 5 minutes, observe and take pictures under ultraviolet light.

[0063] In one aspect of the present invention, the present invention provides a method for detecting whether a sample to be tested contains duck-derived components, pig-derived components, bovine-derived components and sheep-derived components.

[0064] Preferably, the method comprises: detecting whether the lysate of the sample to be tested or the amplified product of the nucleic acid sample contains a specific DNA fragment; if the specific DNA fragment is contained, the sample to be tested contains duck-derived components, pig-derived components, bovine-derived components and sheep-derived components; if the specific DNA fragment is not contained, the sample to be tested does not contain duck-derived components, pig-derived components, bovine-derived components and sheep-derived components; wherein the specific DNA fragment is the target sequence of the primer set described in the present invention in the total DNA of the sample to be tested.

[0065] In one aspect of the present invention, the present invention provides a method for detecting duck-derived components, pig-derived components, bovine-derived components and sheep-derived components based on RAA-lateral flow chromatography technology.

[0066] Preferably, the method comprises the following steps:

[0067] 1) Lyse the meat sample according to the method provided by the nucleic acid release agent; or follow the instructions of the DNA extraction reagent to extract total DNA and dissolve it in TE solution or pure water;

[0068] 2) using the lysate obtained in step 1) or the extracted total DNA or as a template, and performing RAA amplification using a primer set;

[0069] The sequence of the forward primer Duck Derived-F is 5′-CCACTTCTTAGAGTACTCCCAG-3′ (SEQ ID NO. 3),

[0070] The sequence of the reverse primer Duck Derived-R is 5′-GGGTTGTAACTGTTGATAGTG-3′ (SEQ ID NO. 4);

[0071] The sequence of the forward primer Pig Derived-F is 5′-CCAATAAGCAATGATCAACC-3′ (SEQ ID NO. 9),

[0072] The sequence of the reverse primer Pig Derived-R is 5′-CTATGGCTACTGAGATGTATCC-3′ (SEQ ID NO. 10);

[0073] The sequence of the forward primer Bovine Derived-F is: 5′-CGTATCCCTACCCATCCTTACA-3′ (SEQ ID NO. 21),

[0074] The sequence of the reverse primer Bovine Derived-R is: 5′-ACCCGATTCAGACAAGTAGT-3′ (SEQ ID NO. 22).

[0075] The sequence of the forward primer Ovine Derived-F is: 5′-CAACACCACACTTCACAGCTTG C-3′ (SEQ ID NO. 25),

[0076] The sequence of the reverse primer Ovine Derived-R is: 5′-GGATCCTGCTAGTGTGTAA-3′ (SEQ ID NO. 26).

[0077] The duck-derived component detection probe Duck Derived-P has a sequence of 5′-GCACAAGCTCCA ACACAACAAATAAAGTCAHCAAGAGCCCTCAACC-3′ (SEQ ID NO.33); the pig-derived component detection probe PigDerived-P has a sequence of 5′-CGAATCACCCGTATCATA AATTACTCAATCCHCAAGCCCATTAAACTT-3′ (SEQ ID NO.40); the bovine-derived component detection probe Bovine Derived-P has a sequence of 5′-CTCTTCGTGCTCCCAATTAC ACCAAATTCHATTAGTAAGGTCAGC-3′ (SEQ ID NO.41); the sheep-derived component detection probe Ovine Derived-P has a sequence of 5′-CCATATGACTTCTACCCCTAATACTCATAHCTAGCCAACATCATC-3′ (SEQ ID NO.45); the reverse primers DuckDerived-R and Pig Derived-R of the duck-derived component and the pig-derived component are modified with FITC at their 5′ ends, the detection probes Duck Derived-P and PigDerived-P are modified with biotin at their 5′ ends, the 3′ ends of Duck Derived-P and Pig Derived-P are phosphorylated, and H is tetrahydrofuran; the reverse primers Bovine Derived-R and Ovine Derived-R of the bovine-derived component and the sheep-derived component are modified with digoxigenin at their 5′ ends, the probe primers Bovine Derived-P and Ovine Derived-P are modified with rhodamine at their 5′ ends, the 3′ ends of Bovine Derived-R and Ovine Derived-P are phosphorylated, and H is tetrahydrofuran.

[0078] Preferably, the reaction system for detecting RAA amplification of duck-derived components and / or bovine-derived components is as follows:

[0079] Element Volume (μL) Meat and processed meat sample lysate or DNA solution 2 Basal buffer 29.4 DuckDerived-F (5μM) 0.88 DuckDerived-R(5μM) 1.26 DuckDerived-P(1μM) 1.26 BovineDerived-F(5μM) 1.17 BovineDerived-R(5μM) 1.68 BovineDerived-P(1μM) 1.68 <![CDATA[ddH2O]]> upto47.5

[0080] Preferably, the reaction system for detecting RAA amplification of porcine and / or bovine components is as follows:

[0081] Element Volume (μL) Meat and processed meat sample lysate or DNA solution 2 Basal buffer 29.4 PigDerived-F(5μM) 0.73 PigDerived-R(5μM) 1.05 PigDerived-P(1μM) 1.05 BovineDerived-F(5μM) 0.44 BovineDerived-R(5μM) 0.63 BovineDerived-P(1μM) 0.63 <![CDATA[ddH2O]]> upto47.5

[0082] Preferably, the reaction system for detecting RAA amplification of duck-derived components and / or sheep-derived components is as follows:

[0083]

[0084]

[0085] Preferably, the reaction system for detecting RAA amplification of porcine and / or sheep components is as follows:

[0086] Element Volume (μL) Meat and processed meat sample lysate or DNA solution 2 Basal buffer 29.4 PigDerived-F(5μM) 0.51 PigDerived-R(5μM) 0.73 PigDerived-P(1μM) 0.73 OvineDerived-F(5μM) 0.88 OvineDerived-R(5μM) 1.26 OvineDerived-P(1μM) 1.26 <![CDATA[ddH2O]]> upto47.5

[0087] Preferably, the reaction conditions for RAA amplification are set as follows: after the reaction solution is prepared, 2.5 μL of 280 mM MgAc2 is added to each 0.2 mL eppendorf tube and mixed thoroughly; the 0.2 mL eppendorf tube is placed in a PCR instrument with a hot cover function, at 39° C., and kept warm for 16 minutes.

[0088] 3) Use lateral flow chromatography test strips to detect the RAA amplification products of duck-derived components and / or bovine-derived components. If only the band in the quality control area of ​​the test strip is colored, and no band in the detection area is colored, the test result is negative, indicating that the sample does not contain duck-derived components and bovine-derived components; if the test strip has two colored bands, one of which is located in the quality control area and the other is located in the detection area T1, the test result is positive, indicating that the test sample contains bovine-derived components but does not contain duck-derived components; if the test strip has two colored bands, its If one of the three strips is located in the quality control area and the other is located in the detection area T2, the test result is positive, indicating that the test sample contains duck-derived components but no bovine-derived components; if the test strip has three colored strips, two of which are colored, one of which is located in the quality control area, one is located in the detection area T1, and one is located in the detection area T2, then the test result is positive, indicating that the sample contains both bovine-derived components and duck-derived components; if the quality control area and the two detection areas of the test strip are not colored, the test is invalid and the test strip needs to be replaced and retested.

[0089] The specific process of the test can be as follows: open the cap of the eppendorf tube containing 50μLRAA amplification product, take 2.5μL of the amplification product into a new 1.5mL eppendorf tube, and dilute it 20 times. Insert the binding pad end of a new nucleic acid lateral chromatography test strip directly into the eppendorf tube containing the diluted product, and the liquid level must not exceed the upper limit mark of the immersion of the sample pad. After the judgment area is completely soaked, lay the test strip flat for 1 minute, observe the color development results, and record them within 10 minutes. At least one quality control line appears for each test sample, with or without a detection line. If only the band in the quality control area of ​​the test strip is colored, and no band in the test area is colored, the test result is negative, indicating that the sample does not contain duck-derived components and bovine-derived components; if the test strip has two bands that are colored, one of which is located in the quality control area and the other is located in the test area T1, the test result is positive, indicating that the test sample contains bovine-derived components but does not contain duck-derived components; if the test strip has two bands that are colored, one of which is located in the quality control area and the other is located in the test area T2, the test result is positive, indicating that the test sample contains duck-derived components but does not contain bovine-derived components; if the test strip has three bands that are colored, two bands are colored, one of which is located in the quality control area, one is located in the test area T1, and one is located in the test area T2, the test result is positive, indicating that the sample contains both bovine-derived components and duck-derived components; if the quality control area and the two test areas of the test strip are not colored, the test is invalid and the test strip needs to be replaced and retested.

[0090] 4) Use lateral flow chromatography test strips to detect RAA amplification products of porcine and / or bovine components. If only the quality control zone band of the test strip is colored and no band is colored in the detection zone, the test result is negative, indicating that the sample does not contain porcine and bovine components; if two bands of the test strip are colored, one of which is located in the quality control zone and the other is located in the detection zone T1, the test result is positive, indicating that the test sample contains bovine components but no porcine components; if two bands of the test strip are colored, the test result is positive. If one of the three strips is located in the quality control area and the other is located in the detection area T2, the test result is positive, indicating that the test sample contains porcine components but no bovine components; if the test strip has three colored strips, two of which are colored, one of which is located in the quality control area, one is located in the detection area T1, and one is located in the detection area T2, then the test result is positive, indicating that the sample contains both bovine and porcine components; if the quality control area and the two detection areas of the test strip are not colored, the test is invalid and the test strip needs to be replaced and retested.

[0091] The specific process of the test can be as follows: open the cap of the eppendorf tube containing 50μLRAA amplification product, take 2.5μL of the amplification product into a new 1.5mL eppendorf tube, and dilute it 20 times. Insert the binding pad end of a new nucleic acid lateral chromatography test strip directly into the eppendorf tube containing the diluted product, and the liquid level must not exceed the upper limit mark of the immersion of the sample pad. After the judgment area is completely soaked, lay the test strip flat for 1 minute, observe the color development results, and record them within 10 minutes. At least one quality control line appears for each test sample, with or without a detection line. If only the band in the quality control area of ​​the test strip is colored, and no band in the test area is colored, the test result is negative, indicating that the sample does not contain porcine and bovine components; if the test strip has two bands that are colored, one of which is located in the quality control area and the other is located in the test area T1, the test result is positive, indicating that the test sample contains bovine components but does not contain porcine components; if the test strip has two bands that are colored, one of which is located in the quality control area and the other is located in the test area T2, the test result is positive, indicating that the test sample contains porcine components but does not contain bovine components; if the test strip has three bands that are colored, two bands are colored, one of which is located in the quality control area, one is located in the test area T1, and one is located in the test area T2, the test result is positive, indicating that the sample contains both bovine and porcine components; if the quality control area and the two test areas of the test strip are not colored, the test is invalid and the test strip needs to be replaced and retested.

[0092] 5) Use lateral flow chromatography test strips to detect the RAA amplification products of duck-derived components and / or sheep-derived components. If only the band in the quality control area of ​​the test strip is colored, and no band in the detection area is colored, the test result is negative, indicating that the sample does not contain duck-derived components and sheep-derived components; if the test strip has two bands that are colored, one of which is located in the quality control area and the other is located in the detection area T1, the test result is positive, indicating that the test sample contains sheep-derived components but does not contain duck-derived components; if the test strip has two bands that are colored, its If one of the three strips is located in the quality control area, and the other is located in the detection area T2, the test result is positive, indicating that the test sample contains duck-derived components but no sheep-derived components; if the test strip has three colored strips, two of which are colored, one of which is located in the quality control area, one is located in the detection area T1, and one is located in the detection area T2, then the test result is positive, indicating that the sample contains both sheep-derived and duck-derived components; if the quality control area and the two detection areas of the test strip are not colored, the test is invalid and the test strip needs to be replaced and retested.

[0093] The specific process of the test can be as follows: open the cap of the eppendorf tube containing 50μL RAA amplification product, take 2.5μL amplification product into a new 1.5mL eppendorf tube, and dilute it 20 times. Insert the binding pad end of a new nucleic acid lateral chromatography test strip directly into the eppendorf tube containing the diluted product, and the liquid level must not exceed the upper limit mark of the immersion of the sample pad. After the interpretation area is completely soaked, lay the test strip flat for 1 minute, observe the color development results, and record them within 10 minutes. At least one quality control line appears for each test sample, with or without a detection line. If only the band in the quality control area of ​​the test strip is colored, and no band in the test area is colored, the test result is negative, indicating that the sample does not contain duck-derived components and sheep-derived components; if the test strip has two bands that are colored, one of which is located in the quality control area and the other is located in the test area T1, the test result is positive, indicating that the test sample contains sheep-derived components but does not contain duck-derived components; if the test strip has two bands that are colored, one of which is located in the quality control area and the other is located in the test area T2, the test result is positive, indicating that the test sample contains duck-derived components but does not contain sheep-derived components; if the test strip has three bands that are colored, two bands are colored, one of which is located in the quality control area, one is located in the test area T1, and one is located in the test area T2, the test result is positive, indicating that the sample contains both sheep-derived components and duck-derived components; if the quality control area and the two test areas of the test strip are not colored, the test is invalid and the test strip needs to be replaced and retested.

[0094] 6) Use lateral flow chromatography test strips to detect RAA amplification products of porcine and / or sheep components. If only the quality control zone band of the test strip is colored and no band is colored in the detection zone, the test result is negative, indicating that the sample does not contain porcine and sheep components; if two bands of the test strip are colored, one of which is located in the quality control zone and the other is located in the detection zone T1, the test result is positive, indicating that the test sample contains sheep components but no porcine components; if two bands of the test strip are colored, If one of the strips is located in the quality control area and the other is located in the detection area T2, the test result is positive, indicating that the test sample contains porcine components but no sheep-derived components. If the test strip has three colored strips and two of them are colored, one of which is located in the quality control area, one is located in the detection area T1, and one is located in the detection area T2, then the test result is positive, indicating that the sample contains both sheep-derived and porcine components. If both the quality control area and the detection area of ​​the test strip are not colored, the test is invalid and the test strip needs to be replaced and retested.

[0095] The specific process of the test can be as follows: open the cap of the eppendorf tube containing 50μL RAA amplification product, take 2.5μL amplification product into a new 1.5mL eppendorf tube, and dilute it 20 times. Insert the binding pad end of a new nucleic acid lateral chromatography test strip directly into the eppendorf tube containing the diluted product, and the liquid level must not exceed the upper limit mark of the immersion of the sample pad. After the interpretation area is completely soaked, lay the test strip flat for 1 minute, observe the color development results, and record them within 10 minutes. At least one quality control line appears for each test sample, with or without a detection line. If only the band in the quality control area of ​​the test strip is colored, and no band in the test area is colored, the test result is negative, indicating that the sample does not contain porcine and sheep-derived components; if the test strip has two bands that are colored, one of which is located in the quality control area and the other is located in the test area T1, the test result is positive, indicating that the test sample contains sheep-derived components but does not contain porcine components; if the test strip has two bands that are colored, one of which is located in the quality control area and the other is located in the test area T2, the test result is positive, indicating that the test sample contains porcine components but does not contain sheep-derived components; if the test strip has three bands that are colored, two bands are colored, one of which is located in the quality control area, one is located in the test area T1, and one is located in the test area T2, the test result is positive, indicating that the sample contains both sheep-derived and porcine components; if both the quality control area and the test area of ​​the test strip are not colored, the test is invalid and the test strip needs to be replaced and retested.

[0096] Among them, the minimum copy number of the initial amplification template of duck-derived components that can be detected in each reaction of RAA-lateral flow chromatography technology is 32 copies, the minimum copy number of the initial amplification template of pig-derived components is 22 copies, the minimum copy number of the initial amplification template of bovine component target sequence is 44 copies, and the minimum copy number of the initial amplification template of sheep-derived component target sequence is 36 copies.

[0097] Beneficial Effects

[0098] The present invention provides a specific primer set for detecting duck-derived ingredients, pig-derived ingredients, bovine-derived ingredients, and sheep-derived ingredients, and establishes a dual rapid detection method for adulterating duck-derived ingredients or pig-derived ingredients in beef and mutton. The primers designed in the present invention can effectively amplify the target gene, have high specificity and sensitivity, have no cross-reaction with other species, and can be used for on-site rapid detection of duck-derived ingredients, pig-derived ingredients, bovine-derived ingredients, and sheep-derived ingredients, which is of great significance for food safety supervision. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1 This is the case of RAA amplification using duck-derived component amplification primer sets 1 to 4 from different species;

[0100] Figure 2This is the case of RAA amplification using porcine component amplification primer sets 5 to 8 for samples from different species;

[0101] Figure 3 This is the case of RAA amplification using bovine component amplification primer sets 9 to 12 of different species samples;

[0102] Figure 4 This is the case of RAA amplification using sheep-derived component amplification primer set 13 to 16 for samples from different species;

[0103] Figure 5 A in the middle is the RAA amplification sensitivity test of the duck-derived component amplification primer set Duck Derived-F and Duck Derived-R; Figure 5 Middle B is the RAA amplification sensitivity test of the pig-derived component amplification primer set Pig Derived-F and Pig Derived-R; Figure 5 Middle C is the RAA amplification sensitivity test of the bovine-derived component amplification primer set Bovine Derived-F and Bovine Derived-R; Figure 5 D is the sensitivity test of RAA amplification of the sheep-derived component amplification primer set Ovine Derived-F and Ovine Derived-R; the initial template copy number of 50μL RAA amplification increased from 10 6 Copy to 10 0 The copies are in descending order; where M is Plus DNA marker; “-” is a negative control in which ddH2O replaces the same volume of plasmid.

[0104] Figure 6 Schematic diagram of the structure and test results of the single-target disposable nucleic acid test strip (JY0201);

[0105] Figure 7 It is the RAA-LFS test result of positive plasmid and negative control using duck-derived component detection primer set A, primer set B, primer set C, and primer set D;

[0106] Figure 8 It is the RAA-LFS test result of positive plasmid and negative control using primer set E, primer set F, primer set G, and primer set H for detecting porcine-derived components;

[0107] Fig. 9 It is the RAA-LFS test result of positive plasmid and negative control using primer set I, primer set J, primer set K, and primer set L for detecting bovine components;

[0108] Fig.10It is the RAA-LFS test result of positive plasmid and negative control using duck-derived component detection primer set M, primer set N, primer set O, and primer set P;

[0109] Fig.11 The detection sensitivity of the primer set ARAA-LFS for detecting duck-derived components;

[0110] Fig.12 The effect of the amount of downstream primer added in the ARAA amplification system of the duck-derived component detection primer set on the detection sensitivity;

[0111] Fig.13 The effect of the amount of magnesium acetate added in the ARAA amplification system of the duck-derived component detection primer set on the detection sensitivity;

[0112] Fig.14 The detection sensitivity of the primer set H RAA-LFS for detecting porcine-derived components;

[0113] Fig.15 The detection sensitivity of primer set I RAA-LFS for detecting bovine-derived components;

[0114] Fig.16 The detection sensitivity of the primer set M RAA-LFS for sheep-derived components;

[0115] Fig.17 Evaluation of RAA-LFS detection specificity of duck-derived component detection primer set A, pig-derived component detection primer set H, bovine-derived component detection primer set I, and sheep-derived component detection primer set M

[0116] Fig.18 Schematic diagram of the structure and test results of the dual-target disposable nucleic acid test strip (JY0209);

[0117] Fig.19 The results of the dual RAA-LFS test of the primer set for duck-derived components and bovine-derived components using plasmid as template;

[0118] Fig. 20 The results of the dual RAA-LFS test using plasmid as template for the primer set for detecting porcine and bovine components;

[0119] Fig.21 The results of the dual RAA-LFS test of the primer set for duck-derived components and sheep-derived components using plasmid as template;

[0120] Fig. 22 The results of the dual RAA-LFS test using plasmid as template for the primer set for detecting porcine and sheep-derived components;

[0121] Fig.23qPCR melting curve analysis of duck-derived component detection primer set, pig-derived component detection primer set, bovine-derived component detection primer set, and sheep-derived component detection primer set;

[0122] Fig.24 Analysis of absolute quantitative amplification curves of qPCR corresponding to gradient dilution plasmids of duck-derived component detection primers, pig-derived component detection primer sets, bovine-derived component detection primer sets, and sheep-derived component detection primer sets;

[0123] Fig.25 The results of dual RAA-LFS sensitivity test of primer sets for duck-derived and bovine-derived components;

[0124] Fig.26 Results of dual RAA-LFS sensitivity tests for the primer sets for detecting porcine and bovine components;

[0125] Fig. 27 The results of dual RAA-LFS sensitivity test of primer sets for duck-derived and sheep-derived components;

[0126] Fig.28 Results of dual RAA-LFS sensitivity test of primer sets for porcine and sheep-derived components;

[0127] Fig.29 The results of primer optimization for the dual RAA-LFS reaction of the primer set for duck-derived and bovine-derived components;

[0128] Fig.30 Results of primer optimization for dual RAA-LFS reaction of the primer set for detecting porcine and bovine components;

[0129] Fig.31 The results of primer optimization for the double RAA-LFS reaction of the primer set for duck-derived and sheep-derived components;

[0130] Fig.32 The results of the optimization of the primers for the dual RAA-LFS reaction of the primer set for detecting porcine and sheep-derived components;

[0131] Fig.33 The double RAA-LFS detection of animal-derived ingredients in duck, pork, beef, and mutton after simple lysis;

[0132] Fig.34 Homemade RAA-LFS rapid test results of random samples of adulterated meat of animal origin with different proportions. DETAILED DESCRIPTION

[0133] The specific implementation modes of the present invention will be described in detail below in conjunction with the accompanying drawings.

[0134] The experimental methods in the following examples are all conventional unless otherwise specified. The experimental materials described in the following examples are not purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0135] The meat samples involved in the present invention come from:

[0136] The meat and processed meat involved in the present invention were all purchased from major supermarkets in Ningbo, and the corresponding total DNA was stored in the National Key Laboratory of Hazard Factors and Risk Prevention and Control of Agricultural Product Quality and Safety jointly established by the province and the ministry at Ningbo University.

[0137] The sample preparation method involved in the present invention is:

[0138] Total DNA was extracted from duck, pork, beef and mutton samples using a rapid universal genomic DNA extraction kit (Xinjing Biotechnology, Hangzhou) according to the instructions of the reagent. The nucleic acid lateral flow test strips come from Beijing Baoying Tonghui Biotechnology Co., Ltd.

[0139] In order to evaluate whether the RAA-LFS system used can be used for the rapid detection of duck-derived ingredients, pig-derived ingredients, bovine-derived ingredients, and sheep-derived ingredients in meat and processed meat, meat and processed meat were used as experimental objects, and DNA was extracted as amplification template. 30-100 mg of meat and processed meat samples were placed in a 1.5 mL eppendorf tube, and 100 μL Cool Flash Nucleic Acid Releaser (Tiosbio, Beijing Baoying Tonghui Biotechnology Co., Ltd.) was added, and the cells were lysed with a pipette tip for several times to release nucleic acids and obtain the corresponding sample lysate. 1 μL of sample lysate or its dilution was used as the amplification template of RAA. The sample lysate can be used for experiments immediately or stored at -70°C for more than 3 months.

[0140] Experimental Example 1: Primer Design and Screening for Detection of Duck-derived Components, Pig-derived Components, Bovine-derived Components, and Sheep-derived Components

[0141] After a large number of sequence analyses, the present invention team used duck mitochondrial, pig mitochondrial, cattle mitochondrial and sheep mitochondrial genome sequences as targets, analyzed the characteristics of the target gene sequences, and used Primer Premier 5.0 software to design species-specific amplification primers. The primer design followed the following principles.

[0142] ① The length of the amplification primer is about 20 to 30 bp, and the annealing temperatures of different primers should be as consistent or similar as possible; ②

[0143] The amplified product is approximately 200 to 500 bp in length and is species-specific.

[0144] The primer amplification segment sequences were compared using the BLAST function of the National Center for Biotechnology Information (NCBI) to complete the preliminary identification of primer amplification specificity. The primers that passed the identification were synthesized by General Biosystems (Anhui) Co., Ltd., and the purity level was HPLC. The specific sequences of the primers are shown in Table 1.

[0145] The sequence of the forward primer Duck Derived-F is 5′-CCACTTCTTAGAGTACTCCCAG-3′ (SEQ ID NO. 3),

[0146] The sequence of the reverse primer Duck Derived-R is 5′-GGGTTGTAACTGTTGATAGTG-3′ (SEQ ID NO. 4);

[0147] The sequence of the forward primer Pig Derived-F is 5′-CCAATAAGCAATGATCAACC-3′ (SE Q ID NO.9),

[0148] The sequence of the reverse primer Pig Derived-R is 5′-CTATGGCTACTGAGATGTATCC-3′ (SEQ ID NO. 10);

[0149] The sequence of the forward primer Bovine Derived-F is: 5′-CGTATCCCTACCCATCCTTACA-3′ (SEQ ID NO. 21),

[0150] The sequence of the reverse primer Bovine Derived-R is: 5′-ACCCGATTCAGACAAGTAGT-3′ (SEQ ID NO. 22).

[0151] The sequence of the forward primer Ovine Derived-F is: 5′-CAACACCACACTTCACAGCTTG C-3′ (SEQ ID NO. 25),

[0152] The sequence of the reverse primer Ovine Derived-R is: 5′-GGATCCTGCTAGTGTGTAA-3′ (SEQ ID NO. 26).

[0153] The duck-derived component detection probe Duck Derived-P has a sequence of 5′-GCACAAGCTCCAACACAACAAATAAAGTCAHCAAGAGCCCTCAACC-3′ (SEQ ID NO.33); the pig De rived-P has a sequence of 5′-CGAATCACCCGTATCATAAATTACTCAATCCHCAAGCCC ATTAAACTT-3′ (SEQ ID NO.40); the bovine-derived component detection probe Bovine Deriv ed-P has a sequence of 5′-CTCTTCGTGCTCCCAATTACACCAAATTCHATTAGTAAGG TCAGC-3′ (SEQ ID NO.41); the sheep-derived component detection probe Ovine Derived-P has a sequence of 5′-CCATATGACTTCTACCCCTAATACTCATAHCTAGCCAACATCATC-3′ (SEQ ID NO.45);

[0154] Table 1 Primers for amplification of duck-derived ingredients, pig-derived ingredients, bovine-derived ingredients, and sheep-derived ingredients RAA basic version

[0155]

[0156]

[0157] use RAA Nucleic Acid Amplification Kit (Basic Model JY0203), using total DNA from meat and processed meat or lysate of meat and processed meat samples as template, performs RAA amplification with forward primers and reverse primers of Groups 1-4, 5-8 and 9-12 respectively. The amplification system (single sample / reaction) is as follows:

[0158] Element Volume (μL) Meat and processed meat sample lysate or DNA solution 2 Basal buffer 29.4 Forward primer (10 μM) 2.1 Reverse primer (10 μM) 2.1 <![CDATA[ddH2O]]> The total system is 47.5

[0159] After the reaction solution is prepared, add 2.5 μL of 280 mM MgAc2 to each 0.2 mL eppendorf tube and mix thoroughly; place the 0.2 mL eppendorf tube in a PCR instrument with a hot cover function, and keep warm at 39°C for 30 minutes.

[0160] After the RAA reaction is completed, the reaction tubes are removed. 100 μL of phenol / chloroform (1:1) is added to each reaction tube, fully shaken and centrifuged at 12000 rpm for 10 min (a vortex shaker can be used for vigorous shaking and mixing in this step). 10 μL of supernatant is mixed with 2 μL of 6× Loading Buffer, loaded on 1.5% agarose gel, and electrophoresed at 200V for 15 min; the electrophoresis is terminated when bromophenol blue moves to the lower 2 / 3 of the gel; EB staining is performed for 5 min, and observation and photography are performed under ultraviolet light.

[0161] like Figure 1-4 As shown, under the premise of adding 2 μL of total DNA solution or sample lysate, duck-derived component amplification primer sets 1 to 4 were used to perform RAA amplification in the total DNA of cattle, sheep, duck, pig, and chicken, respectively. The electrophoresis band of the amplification product of duck-derived component amplification primer set 2 in the total DNA of duck meat was clear and bright, without any mixed bands, and the size of the amplification band was consistent with the expectation, while there was no obvious amplification product in the total DNA of other meats; pig-derived component amplification primer sets 5 to 8 were used to perform RAA amplification in the total DNA of different species, respectively, and the electrophoresis band of the amplification product of pig-derived component amplification primer set 5 in the total DNA of pork was clear and bright, without any mixed bands, and the size of the amplification band was consistent with the expectation, while there was no obvious amplification product in the total DNA of other meats; bovine component amplification primer sets 9 to 12 were used to perform RAA amplification in the total DNA of different species, and the electrophoresis band of the amplification product of primer set 11 in the total DNA of beef was clear and bright, without any mixed bands, and the size of the amplification band was consistent with the expectation, while there was no obvious amplification product in the total DNA of other meats, which can be used to detect bovine-derived components. RAA amplification was performed in total DNA of different species using primer sets 13 to 16 for amplifying sheep-derived components. The electrophoresis band of the amplified product of primer set 13 in the total DNA of mutton was clear and bright, without any mixed bands, and the size of the amplified band was consistent with expectations. There was no obvious amplified product in the total DNA of other meats, so it can be used to detect sheep-derived components.

[0162] The amplification products of the best primer set of each primer pair were cloned into T vectors and sequenced to determine the target sequences. The best primer combinations of each primer pair were determined to be duck-derived component amplification primer set 2, pig-derived component amplification primer set 5, bovine-derived component amplification primer set 11, and sheep-derived component amplification primer set 13, and the upstream and downstream primers of duck-derived component amplification primer set 2 were renamed Duck Derived-F and Duck Derived-R; the upstream and downstream primers of pig-derived component amplification primer set 5 were renamed Pig Derived-F and Pig Derived-R; the upstream and downstream primers of bovine-derived component amplification primer set 11 were renamed Bovine Derived-F and Bovine Derived-R; the upstream and downstream primers of sheep-derived component amplification primer set 13 were renamed Ovine Derived-F and Ovine Derived-R.

[0163] Experimental Example 2: Preparation and composition of the comparison plate for the test samples of duck-derived ingredients, pig-derived ingredients, cattle-derived ingredients and sheep-derived ingredients

[0164] The positive amplification products of duck-derived component amplification primer group DuckDerived-F and Duck Derived-R, the positive amplification products of pig-derived component amplification primer group Pig Derived-F and Pig Derived-R, the positive amplification products of bovine-derived component detection primer group Bovine Derived-F and Bovine Derived-R, and the amplification products of sheep-derived component detection primer group OvineDerived-F and Ovine Derived-R were cloned and sequenced. After transformation and shaking overnight, the plasmids were extracted, and the OD260, OD280 and OD260 / OD280 values ​​of the recombinant plasmids were measured by UV spectrophotometer. The experiment was repeated three times to determine the concentration and purity of the plasmid DNA.

[0165] The copy number of the plasmid was obtained according to the following formula:

[0166] Copy number = plasmid concentration × 6.02 × 10 23 / (660×total length of plasmid)

[0167] Calculate the copy number and dilute to 1 × 10 8 copies / μL, stored at –20℃ for future use. Dilute the recombinant plasmid to 1×10 6 copies / μL, and then serially diluted 10-fold to obtain 1×10 0 copies / μL, 1×10 1 copies / μL, 1×10 2 copies / μL, 1×10 3 copies / μL, 1×104 copies / μL, 1×10 5 copies / μL and 1×10 6 The copies / μL plasmid dilution is used as the template for subsequent amplification.

[0168] Experimental Example 3: Detection of primer sensitivity for duck-derived components, pig-derived components, bovine-derived components, and sheep-derived components

[0169] In order to further test the sensitivity of the primer sets Duck Derived-F and Duck Derived-R for duck-derived components, Pig Derived-F and Pig Derived-R for pig-derived components, Bovine Derived-F and Bovine Derived-R for bovine-derived components, and Ovine Derived-F and Ovine Derived-R for sheep-derived components, 10-fold serial dilutions (10 μl per μl) of the plasmid DNA corresponding to the amplification products were prepared. 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 0 ddH2O was used as a negative control template. The amplification of RAA with different primers on the same template was compared by 1.5% agarose gel electrophoresis.

[0170] Primer set for amplification from duck-derived components ( Figure 5 A), porcine component amplification primer set ( Figure 5 B), bovine component amplification primer set ( Figure 5 C) and the sheep-derived component amplification primer set ( Figure 5 D) The results of RAA amplification sensitivity test showed that the primers for duck-derived component detection, Duck Derived-F and Duck Derived-R, the primers for pig-derived component detection, Pig Derived-F and Pig Derived-R, the primers for bovine-derived component detection, Bovine Derived-F and Bovine Derived-R, and the primers for sheep-derived component detection, Ovine Derived-F and Ovine Derived-R, could detect RAA amplification with a detection rate as low as 10 1 copies of the duck-derived component plasmid.

[0171] Experimental Example 4: RAA-LFS detection of duck-derived ingredients, pig-derived ingredients, cattle-derived ingredients, and sheep-derived ingredients

[0172] For the amplification segments of primer sets 1 to 8 in Example 1, the Primer Premier 5.0 software was used to design species-specific probe sequences, which were located in the middle segment of the amplification primers and modified with tetrahydrofuran. The principles followed in primer design were: ① The probe length was about 30 to 45 bp; ② The probe sequence was species-specific.

[0173] The primer amplification segment sequences were compared using the BLAST function of the National Center for Biotechnology Information (NCBI) to complete the preliminary identification of primer amplification specificity. The primers that passed the identification were synthesized by General Biosystems (Anhui) Co., Ltd., and the purity level was HPLC. The specific sequences of the primers, probes, and downstream primers for amplification are shown in Table 2 below.

[0174] Table 2 Primers for amplification of RAA test strips of duck-derived ingredients, pig-derived ingredients, bovine-derived ingredients and sheep-derived ingredients

[0175]

[0176]

[0177]

[0178] The upstream and downstream primers of each primer set corresponded to the amplification product 1×10 6 Copies / μL recombinant plasmid solution as template, using RAA Nucleic Acid Amplification Kit (Test Strip Method) (JY0204) is used to prepare the RAA reaction system of primer set A to primer set P (single sample / reaction), and the system is as follows:

[0179] Components Dosage (μL) Basal buffer 29.4 Upstream primer (10 μM) 2.1 Probe primer (10 μM) 0.6 Downstream primer (10 μM) 2.1 <![CDATA[1×10 6 Copies / μL recombinant plasmid solution]]> 2 Make up to volume with water 47.5

[0180] The order of adding samples is negative control sample (1×10 6 copies / μL recombinant plasmid solution was replaced with the same volume of ultrapure water), 1×10 6 10000 copies / μL recombinant plasmid solution. After adding each sample, the tube cap must be fastened immediately to avoid aerosol contamination. Mix the above reaction system and add the basic reaction unit. Dissolve the lyophilized powder fully. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator. Open the reaction unit and add 2.5μL 280mM MgAc2 to each 0.2mL eppendorf tube, mix thoroughly and collect by centrifugation. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator.

[0181] The reaction tube was placed at 39°C for 16 minutes. After the RAA reaction was completed, the eppendorf tube was opened, the amplified product was transferred to a new eppendorf tube, marked, and diluted 20 to 50 times.

[0182] The structural diagram of the single-target disposable nucleic acid detection test strip (JY0201) is as follows Figure 6 As shown, insert the immersion area of ​​the test strip (marked with a blue arrow pointing upward) into the eppendorf tube. The liquid level must not exceed the MAX indicator line of the immersion area. Wait until the judgment area is completely soaked (about 30 to 60 seconds), lay the test strip flat for 1 minute, and wait for the red strip to appear. Read the test result directly according to the color development of the test strip. Observe the result within 10 minutes, and the judgment after 10 minutes is invalid.

[0183] like Figure 7-Figure 10 As shown, duck-derived component detection primer groups B, C, and D, pig-derived component detection primer groups E, F, and G; bovine-derived component detection primer groups J, K, and L, and sheep-derived component detection primer groups N, O, and P were used for RAA amplification. After the RAA amplification products of the negative control amplification products of the above detection primer groups were diluted 20 times, the test strip test results were positive and could not be used for subsequent RAA nucleic acid amplification test strip sensitivity detection; while the negative control amplification products of duck-derived component detection primer group A, pig-derived component detection primer group H, bovine-derived component detection primer group I, and sheep-derived component detection primer group O were diluted 20 times, and the test strip test results were all negative, 1×10 6 The RAA test results of the recombinant plasmid solution with 100 copies / μL were all positive and could be used for the subsequent sensitivity test of RAA nucleic acid amplification test strips.

[0184] Duck-derived component detection primer group A, pig-derived component detection primer group H, bovine-derived component detection primer group I and sheep-derived component detection primer group M were determined to be animal-derived detection primer groups, and the probe primer Duck Derived-P1 in duck-derived component detection primer group A was renamed Duck Derived-P, the probe primer Pig Derived-P4 in pig-derived component detection primer group H was renamed Pig Derived-P, the probe primer BovineDerived-P1 in bovine-derived component detection primer group I was renamed Bovine Derived-P, and the probe primer OvineDerived-P3 in sheep-derived component detection primer group M was renamed Ovine Derived-P.

[0185] Experimental Example 5: Detection sensitivity of duck-derived component detection primer set RAA-LFS

[0186] The positive plasmid of the corresponding amplified fragment of duck-derived component detection primer set A was used as a template, and the RAA amplification system in Experimental Example 4 was used at 1×10 6 copies / μL, 1×10 5 copies / μL, 1×10 4copies / μL, 1×10 3 copies / μL, 1×10 2 copies / μL, 1×10 1 copies / μL and 1×10 0 The RAA amplification was performed using 1000 copies / μL plasmid dilution as template, and the amplified products were diluted 20 times before being tested on test strips to determine the detection sensitivity of different primer combinations.

[0187] The sensitivity test results of the duck-derived component detection primer set ARAA-LFS are as follows Fig.11 As shown, the minimum detection threshold of duck-derived component RAA-LFS is 10 3 copies.

[0188] Example 6: Optimization of RAALFS detection system using duck-derived component detection primer set A

[0189] The probes used in the experiment have the following characteristics: ① The design direction is forward, that is, the same direction as the upstream primer; ② The length is 46-52nt; ③ The 3′ end is phosphorylated to inhibit DNA chain extension; ④ A cleavage site is added inside the sequence, which is cut by the exo enzyme when the probe binds to the template DNA and initiates the extension of the DNA chain; ⑥ The 5′ end is modified with biotin, and the double-stranded amplification product of the probe and the downstream (FITC-modified) primer can be detected by the nucleic acid test strip.

[0190] The test strip test product is a double-stranded amplification product of the probe and downstream primer (FITC modified) after exo enzyme cleavage, so theoretically increasing the amount of downstream primer added will help improve the sensitivity of RAA nucleic acid test strip detection. However, when the downstream primer is added in excess, it will also lead to the formation of dimers between the downstream primer and the probe primer, and cause a false positive test result.

[0191] use The RAA reaction system was prepared using the Nucleic Acid Amplification Kit (Test Strip Method) (JY0204) (single sample / reaction), and the amount of downstream primers added was adjusted as shown in Table 3. 2 Copies / μL sample plate, 10 1 Copies / μL sample plate, 10 0 Copy / μL sample plate, negative quality control samples for testing. After adding the sample, the tube cap must be fastened immediately to avoid aerosol contamination. Mix the above reaction system and add the basic reaction unit. Dissolve the lyophilized powder fully. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator. Open the reaction unit, add 2.5μL 280mM MgAc2 to each 0.2mL eppendorf tube, mix thoroughly and collect by centrifugation. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator.

[0192] The reaction tube was placed at 39°C for 16 minutes. After the RAA reaction was completed, the eppendorf tube was opened, the amplified product was transferred to a new eppendorf tube, marked, and diluted 20 times, and the test strip was tested to determine the optimal addition amount.

[0193] Table 3 Adjustment of the amount of downstream primers added in the ARAA amplification system of duck-derived component amplification primer set

[0194]

[0195] The results are as follows Fig.12 Display, with 10 2 Copies / μL sample plate, 10 1 Copies / μL sample plate, 10 0 The negative control sample was used as the template. Under the same template addition volume, the amount of downstream primers was adjusted. When the amount of downstream primers (10 μM) in the 50 μL amplification system was adjusted to 3 μL, the detection sensitivity was the best. Not only could 10 1 The sample was copied and the negative control sample was negative.

[0196] Adding MgAc2 to the amplification system can initiate RAA amplification, and the amount of MgAc2 added to the amplification system directly affects the efficiency of amplification and the yield of the product. In theory, increasing the amount of MgAc2 added can help improve the yield of the amplification product and increase the sensitivity of the RAA nucleic acid test strip detection. However, when MgAc2 is added in excess, it can also lead to false positive results for negative sample tests.

[0197] use The RAA reaction system was prepared using the Nucleic Acid Amplification Kit (Test Strip Method) (JY0204) (single sample / reaction), and the system for adjusting the amount of MgAc2 added was as follows.

[0198] Table 4 Adjustment of the amount of MgAc2 added in the ARAA amplification system of duck-derived component amplification primer set

[0199]

[0200] Each system is 10 2 Copies / μL sample plate, 10 1 Copies / μL sample plate, 10 0Copy / μL sample plate, negative quality control samples for testing. After adding the sample, the tube cap must be fastened immediately to avoid aerosol contamination. Mix the above reaction system and add the basic reaction unit. Dissolve the lyophilized powder fully. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator. Open the reaction unit and add different volumes of 280mM MgAc2 in Table 4 to each 0.2mL eppendorf tube, mix thoroughly and collect by centrifugation. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator.

[0201] The reaction tube was placed at 39°C for 16 minutes. After the RAA reaction was completed, the eppendorf tube was opened, the amplified product was transferred to a new eppendorf tube, marked, and diluted 20 times, and the test strip was tested to determine the optimal addition amount.

[0202] The experimental results are as follows Fig.13 As shown, 10 2 Copies / μL sample plate, 10 1 Copies / μL sample plate, 10 0 The negative control sample was used as the template. Under the same template addition volume, the amount of MgAc2 was adjusted. When MgAc2 was adjusted to 2.5μL and 2.8μL, the detection sensitivity was the same, and not only 10 1 The sample has a sample size of 100 copies / μL, and the negative control sample is negative. According to the results of adjustment system 2 and adjustment system 3 in Table 4, excessive addition of MgAc2 will lead to false positives in negative control samples. Given that the detection sensitivity of 2.5μL and 2.8μL MgAc2 added per 50μL amplification system is the same, the optimal addition amount of MgAc2 is finally determined to be 2.5μL to avoid false positives in negative samples.

[0203] Example 7: RAA-LFS Detection Sensitivity of the Primer Set for Detecting Porcine, Bovine and Sheep Components

[0204] RAA amplification of porcine, bovine, and sheep components was performed using the method described in Example 6. Nucleic Acid Amplification Kit (Test Strip Method) (JY0204) amplification system and test strip detection method were used to prepare the RAA reaction system and complete different primer combinations 1×10 6 copies / μL, 1×10 5 copies / μL, 1×10 4 copies / μL, 1×10 3 copies / μL, 1×10 2 copies / μL, 1×10 1 copies / μL and 1×10 0Copies / μL plasmid dilution RAA amplification, amplification products after 20 times dilution for test strip detection. Figure 14-16 After RAA amplification, the porcine component detection primer group H, the bovine component detection primer group I and the sheep component detection primer group M can all detect as low as 10 1 copies of the target gene.

[0205] Example 8: Specificity evaluation of primers for detecting duck-derived components, pig-derived components, bovine-derived components, and sheep-derived components

[0206] In this example, beef, mutton, duck, pork and chicken meat that were verified by PCR and free of exogenous contamination were used as experimental subjects, and total DNA was extracted as amplification template. The primers for duck-derived components, pig-derived components detection primer set and cattle-derived components detection primer set selected in Example 4 were used, and the primers for cattle-derived components detection primer set described in Example 6 were used. The RAA reaction system was prepared by using the amplification system of the Nucleic Acid Amplification Kit (Test Strip Method) (JY0204) and the test strip detection method. RAA amplification was performed on the total DNA to complete the RAA amplification. The amplification product was diluted 20 times and then tested on the test strip to determine the specificity of detection with different primer combinations.

[0207] The RAA-LFS primer specificity evaluation results of duck-derived component detection primer group A, pig-derived component detection primer group H, bovine-derived component detection primer group I and sheep-derived component detection primer group M are as follows: Fig.17 As shown, it can be seen that the LFS detection results of the amplified products of the duck-derived component detection primer set, the pig-derived component detection primer set, the bovine-derived component and the sheep-derived component detection primer set all showed positive only in the corresponding total DNA, and no cross reaction with the DNA of other species was found, indicating that the selected primer pairs have excellent specificity and are suitable for RAA-LFS detection of duck-derived components, pig-derived components, bovine-derived components and sheep-derived components;

[0208] Experimental Example 9: Establishment of a dual RAA-LFS detection system with different primer sets for duck-derived components, pig-derived components, bovine-derived components, and sheep-derived components

[0209] The duck-derived component A, the porcine-derived component detection primer set H, and the bovine-derived component detection primer set I in Example 7 were configured as a mix to facilitate the optimization of the primer addition amount in the RAA dual amplification system. The components of the primer mix are as follows:

[0210]

[0211] 1×10 of the product amplified by the upstream and downstream primers of each primer set 6 Copies / μL of plasmid dilution were used as templates. The nucleic acid amplification kit (strip method) (JY0204) is used to prepare a dual RAA reaction system of duck-derived components and bovine-derived components (single sample / reaction), and the system is as follows:

[0212]

[0213]

[0214] Set up negative control samples (1×10 6 The recombinant plasmid solution was replaced with the same volume of ultrapure water (1×10 copies / μL), and the order of sample addition was negative control sample, 1×10 6 Copies / μL of the same concentration of duck-derived components and bovine-derived components were mixed with plasmid solutions. After each sample was added, the tube cap must be fastened immediately to avoid aerosol contamination. Mix the above reaction system and add the basic reaction unit. Dissolve the lyophilized powder fully. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator. Open the reaction unit and add 2.5μL of 280mM MgAc2 to each 0.2mL eppendorf tube, mix thoroughly and collect by centrifugation. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator.

[0215] 1×10 of the product amplified by the upstream and downstream primers of each primer set 6 copies / μL positive plasmid dilution as template, use The nucleic acid amplification kit (strip method) (JY0204) is used to prepare a dual RAA reaction system of porcine and bovine components (single sample / reaction), and the system is as follows:

[0216]

[0217] Set up negative control samples (1×10 6 The recombinant plasmid solution was replaced with the same volume of ultrapure water (1×10 copies / μL), and the order of sample addition was negative control sample, 1×10 6 Copies / μL of the same concentration of porcine and bovine components mixed plasmid solution, each sample must be immediately capped after adding to avoid aerosol contamination. Mix the above reaction system and add the basic reaction unit. Dissolve the lyophilized powder fully. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator. Open the reaction unit, add 2.5μL280mM MgAc2 to each 0.2mL eppendorf tube, mix thoroughly and collect by centrifugation. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator.

[0218] 1×10 of the product amplified by the upstream and downstream primers of each primer set 6 Copies / μL of plasmid dilution were used as templates. The nucleic acid amplification kit (strip method) (JY0204) is used to prepare a dual RAA reaction system of duck-derived components and sheep-derived components (single sample / reaction), and the system is as follows:

[0219]

[0220] Set up negative control samples (1×10 6 The recombinant plasmid solution was replaced with the same volume of ultrapure water (1×10 copies / μL), and the order of sample addition was negative control sample, 1×10 6 Copies / μL of the same concentration of duck-derived components and sheep-derived components mixed plasmid solution. After each sample is added, the tube cap must be fastened immediately to avoid aerosol contamination. Mix the above reaction system and add the basic reaction unit. Dissolve the lyophilized powder fully. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator. Open the reaction unit and add 2.5μL280mM MgAc2 to each 0.2mL eppendorf tube, mix thoroughly and collect by centrifugation. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator.

[0221] 1×10 of the product amplified by the upstream and downstream primers of each primer set 6 Copies / μL of plasmid dilution were used as templates. Nucleic Acid Amplification Kit (Test Strip Method) (JY0204) is used to prepare a dual RAA reaction system of porcine and sheep components (single sample / reaction), the system is as follows:

[0222]

[0223] Set up negative control samples (1×10 6 The recombinant plasmid solution was replaced with the same volume of ultrapure water (1×10 copies / μL), and the order of sample addition was negative control sample, 1×10 6 Copies / μL of the same concentration of porcine and sheep-derived components mixed plasmid solution, each sample must be immediately capped after adding to avoid aerosol contamination. Mix the above reaction system and add the basic reaction unit. Dissolve the lyophilized powder fully. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator. Open the reaction unit, add 2.5μL280mM MgAc2 to each 0.2mL eppendorf tube, mix thoroughly and collect by centrifugation. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator.

[0224] The reaction tube was placed at 39°C for 16 minutes. After the RAA reaction was completed, the eppendorf tube was opened, the amplified product was transferred to a new eppendorf tube, marked, and diluted 20 to 50 times.

[0225] The schematic diagram of the dual-target disposable nucleic acid test strip (JY0209) is as follows Fig.18 As shown, insert the immersion area of ​​the test strip (marked with a blue arrow pointing upward) into the eppendorf tube. The liquid level must not exceed the MAX indicator line of the immersion area. Wait until the judgment area is completely soaked (about 30 to 60 seconds), lay the test strip flat for 1 minute, and wait for the red strip to appear. Read the test result directly according to the color development of the test strip. Observe the result within 10 minutes, and the judgment after 10 minutes is invalid.

[0226] From the results of the double RAA-LFS detection of the positive recombinant plasmid and the negative control, it can be seen that the mix4 ( Fig.19 ), a mix of the porcine component detection primer set H and the bovine component detection primer set I ( Fig. 20 ), mix3 of duck-derived component detection primer set A and sheep-derived component detection primer set M ( Fig.21 ), mix4 of primer set H for detecting porcine components and primer set M for detecting sheep components ( Fig. 22 ) was consistent with the expected color development results, where the negative control amplification product was diluted 20 times and the test strip test result was negative, and the positive plasmid amplification product was diluted 20 times and the test strip test result was positive, which can be used for the subsequent dual RAA-LFS detection of meat. Experimental Example 10: Drawing of absolute quantitative standard curves for duck-derived component detection primer set 2, pig-derived component detection primer set 5, cattle-derived component detection primer set 11 and sheep-derived component detection primer set 13

[0227] Real-time qPCR was performed using the comparison plate in Example 2, and the forward primers and reverse primers in duck-derived component detection primer set 2, pig-derived component detection primer set 5, bovine-derived component detection primer set 11, and sheep-derived component detection primer set 13 were used to verify the sensitivity of the primers, and the copy number was absolutely quantified.

[0228] from Fig.23 It can be seen that the qPCR melting curve analysis of duck-derived component detection primer group 2, pig-derived component detection primer group 5, bovine-derived component detection primer group 11 and sheep-derived component detection primer group 13 are all sharp single peaks, indicating that the duck-derived component detection primer group, pig-derived component detection primer group, bovine-derived component detection primer group and sheep-derived component detection primer group screened in this embodiment have high amplification specificity and amplification efficiency.

[0229] Fig.24 The results show that the above-mentioned duck-derived component detection primer group 2, pig-derived component detection primer group 5, and bovine-derived component detection primer group 11 (can successfully detect 10 1 ~10 6The initial template of the copy) and the sheep-derived component detection primer set 13 (can successfully detect 10 2 ~10 6 The dynamic detection range spanned more than 5 logarithmic values, and the Cq value difference of the template dilution gradient with a 10-fold difference was similar, showing a good detection effect.

[0230] Duck-derived component detection primer set 2 Cq value and logarithm-converted copy number (y = -5.5095x + 46.385, R 2 >0.993, 5Cq value of the porcine component detection primer set and the logarithmic-converted copy number (y=-5.0431x+37.514, R 2 >0.9965, Cq value of primer set 11 for detection of bovine components and the copy number after logarithmic transformation (y=-3.9078x+33.246, R 2 >0.9974, Cq value of the primer set 13 for detecting sheep-derived components and the copy number after logarithmic transformation (y=-5.47x+51.056, R 2 >0.9975), indicating that the primers designed in this example have good amplification specificity and sensitivity, and can be used for RT-PCR and RAA analysis of duck-derived components, pig-derived components, bovine-derived components, and sheep-derived components.

[0231] Experimental Example 11: Dual RAA-LFS sensitivity detection of duck-derived components, pig-derived components, cattle-derived components, and sheep-derived components by primer sets

[0232] Using the duck-derived component detection primer group A, pig-derived component detection primer group H, bovine-derived component detection primer group I and sheep-derived component detection primer group M verified in Example 10, the corresponding plasmids of the corresponding amplified fragments were used as templates, and the RAA amplification system in Experimental Example 9 was used to perform 1×10 3 copies / μL, 1×10 2 copies / μL, 1×10 1 Copies / μL of duck-derived and bovine-derived components at the same concentration, 1×10 3 copies / μL, 1×10 2 copies / μL, 1×10 1 Copies / μL of the same concentration of porcine and bovine components, 1×10 3 copies / μL, 1×10 2 copies / μL, 1×10 1 Copies / μL of duck-derived and sheep-derived components at the same concentration, 1×10 3 copies / μL, 1×10 2 copies / μL, 1×10 1The double RAA amplification of the positive mixed plasmid dilutions of duck-derived components and sheep-derived components at the same concentration was performed, and the amplification products were diluted 20 times and then tested on test strips to determine the detection sensitivity of the primer combination.

[0233] See also Figure 25-28 The sensitivity test results of dual RAA-LFS with different primer combinations showed that the minimum detection threshold of dual RAA-LFS for simultaneous accurate detection of duck-derived component detection primer group A and bovine-derived component detection primer group I was 10 3 The minimum detection threshold for simultaneous accurate detection of dual RAA-LFS by primer set H for porcine components and primer set I for bovine components is 10 4 The minimum detection threshold for accurate simultaneous detection of dual RAA-LFS by primer set A for duck-derived components and primer set M for sheep-derived components is 10 4 The minimum detection threshold for simultaneous accurate detection of dual RAA-LFS by primer set H for porcine components and primer set M for sheep components is 10 1 copies.

[0234] Experimental Example 12: Optimization of the dual RAA-LFS detection system for duck-derived ingredients, pig-derived ingredients, bovine-derived ingredients, and sheep-derived ingredients

[0235] use The nucleic acid amplification kit (strip method) (JY0204) is used to prepare a double RAA reaction system of duck-derived component detection primer group A and bovine-derived primer group I components (single sample / reaction), and the system is as follows:

[0236]

[0237] Set up negative quality control samples (replace the recombinant plasmid solution with the same volume of ultrapure water), and add samples in the following order: negative quality control samples, 1×10 1 copies / μL, 1×10 2 copies / μL, 1×10 3 Copies / μL of duck-derived and bovine-derived components positive mixed plasmid solution of the same concentration. After each sample is added, the tube cap must be fastened immediately to avoid aerosol contamination. Mix the above reaction system and add the basic reaction unit. Dissolve the lyophilized powder fully. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator. Open the reaction unit and add 2.5μL 280mM MgAc2 to each 0.2mL eppendorf tube, mix thoroughly and collect by centrifugation. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator.

[0238] use The nucleic acid amplification kit (strip method) (JY0204) is used to prepare a dual RAA reaction system of porcine and bovine components (single sample / reaction), and the system is as follows:

[0239]

[0240]

[0241] Set up negative quality control samples (replace the recombinant plasmid solution with the same volume of ultrapure water), and add samples in the following order: negative quality control samples, 1×10 1 copies / μL, 1×10 2 copies / μL, 1×10 3 Copies / μL of the same concentration of the positive mixed plasmid solution of porcine and bovine components. After each sample is added, the tube cap must be fastened immediately to avoid aerosol contamination. Mix the above reaction system and add the basic reaction unit. Dissolve the lyophilized powder fully. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator. Open the reaction unit and add 2.5μL of 280mM MgAc2 to each 0.2mL eppendorf tube, mix thoroughly and collect by centrifugation. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator.

[0242] use The nucleic acid amplification kit (strip method) (JY0204) is used to prepare a dual RAA reaction system of duck-derived components and sheep-derived components (single sample / reaction), and the system is as follows:

[0243]

[0244] Set up negative quality control samples (replace the recombinant plasmid solution with the same volume of ultrapure water), and add samples in the following order: negative quality control samples, 1×10 1 copies / μL, 1×10 2 copies / μL, 1×10 3 Copies / μL of duck-derived and sheep-derived components of the same concentration of positive mixed plasmid solution. After each sample is added, the tube cap must be fastened immediately to avoid aerosol contamination. Mix the above reaction system and add the basic reaction unit. Dissolve the lyophilized powder fully. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator. Open the reaction unit, add 2.5μL 280mM MgAc2 to each 0.2mL eppendorf tube, mix thoroughly and collect by centrifugation. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator.

[0245] use Nucleic Acid Amplification Kit (Test Strip Method) (JY0204) is used to prepare a dual RAA reaction system of porcine and sheep components (single sample / reaction), the system is as follows:

[0246]

[0247] Set up negative quality control samples (replace the recombinant plasmid solution with the same volume of ultrapure water), and add samples in the following order: negative quality control samples, 1×10 1 copies / μL, 1×10 2 copies / μL, 1×10 3 Copies / μL of the same concentration of the positive mixed plasmid solution of porcine and sheep components. After each sample is added, the tube cap must be fastened immediately to avoid aerosol contamination. Mix the above reaction system and add the basic reaction unit. Dissolve the lyophilized powder fully. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator. Open the reaction unit and add 2.5μL of 280mM MgAc2 to each 0.2mL eppendorf tube, mix thoroughly and collect by centrifugation. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator.

[0248] The reaction tube was placed at 39°C for 16 minutes. After the RAA reaction was completed, the eppendorf tube was opened, the amplified product was transferred to a new eppendorf tube, marked, and diluted 20 to 50 times.

[0249] The results of the dual RAA-LFS sensitivity test of the primer set for duck-derived and bovine-derived components are as follows Fig.29 As shown in Figure 2, the sensitivity test results of the dual RAA-LFS primer set for detecting porcine and bovine components are as follows: Fig.30 As shown in the figure, the sensitivity test results of the double RAA-LFS of the primer set for detecting duck-derived components and sheep-derived components are as follows Fig.31 As shown in the figure, the sensitivity test results of the dual RAA-LFS of the primer set for detecting porcine and sheep components are as follows Fig.32 As shown in the figure, except for the detection primer set mix4 for porcine and bovine components, the minimum detection threshold of dual RAA-LFS can reach 10 1 The minimum detection threshold of dual RAA-LFS of the combination mix2 in other dual detection systems can reach 10 1 copies.

[0250] Experimental Example 13: Dual RAA-LFS Detection of Animal-derived Components in Meat and Meat Products after Simple Lysis

[0251] After qPCR identification, meat processed products (including marinated meat processed products) containing duck-derived ingredients, pig-derived ingredients, cattle-derived ingredients, and sheep-derived ingredients were collected. About 20 mg of sample and 100 μL of Cool Flash Nucleic Acid Releaser (BT0068) was mixed, and the sample to be tested was cut into pieces with ophthalmic scissors, centrifuged, and 2 μL of the lysate supernatant was taken for testing.

[0252] The optimized dual RAA amplification system in Experimental Example 11 was used for amplification, and the supernatant of meat and processed meat lysate was used as a template. The nucleic acid amplification kit (strip method) (JY0204) is used to prepare a dual RAA reaction system of duck-derived components and bovine-derived components (single sample / reaction), and the system is as follows:

[0253] Components Dosage (μL) Basal buffer 29.4 DuckDerived-F (5μM) 0.88 DuckDerived-R(5μM) 1.26 DuckDerived-P(1μM) 1.26 BovineDerived-F(5μM) 1.17 BovineDerived-R(5μM) 1.68 BovineDerived-P(1μM) 1.68 Lysate supernatant 2 Make up to volume with water 47.5

[0254] The optimized dual RAA amplification system in Experimental Example 11 was used for amplification, and the supernatant of meat and processed meat lysate was used as a template. The nucleic acid amplification kit (strip method) (JY0204) is used to prepare a dual RAA reaction system of porcine and bovine components (single sample / reaction), and the system is as follows:

[0255]

[0256]

[0257] The dual RAA amplification system in Experimental Example 11 was used for amplification, with the supernatant of meat and processed meat product lysate as the template, and The nucleic acid amplification kit (strip method) (JY0204) is used to prepare a dual RAA reaction system of duck-derived components and sheep-derived components (single sample / reaction), and the system is as follows:

[0258] Components Dosage (μL) Basal buffer 29.4 DuckDerived-F (5μM) 0.58 DuckDerived-R(5μM) 0.84 DuckDerived-P(1μM) 0.84 OvineDerived-F(5μM) 1.03 OvineDerived-R(5μM) 1.48 OvineDerived-P(1μM) 1.48 Lysate supernatant 2 Make up to volume with water 47.5

[0259] The dual RAA amplification system in Experimental Example 11 was used for amplification, with the supernatant of meat and processed meat product lysate as the template, and Nucleic Acid Amplification Kit (Test Strip Method) (JY0204) is used to prepare a dual RAA reaction system of porcine and sheep components (single sample / reaction), the system is as follows:

[0260] Components Dosage (μL) Basal buffer 29.4 PigDerived-F(5μM) 0.51 PigDerived-R(5μM) 0.73 PigDerived-P(1μM) 0.73 OvineDerived-F(5μM) 0.88 OvineDerived-R(5μM) 1.26 OvineDerived-P(1μM) 1.26 Lysate supernatant 2 Make up to volume with water 47.5

[0261] The order of adding samples is negative quality control samples (the supernatant of the lysate is replaced with the same volume of ultrapure water), meat and meat processed product lysate supernatant. After each sample is added, the tube cap must be fastened immediately to avoid aerosol contamination. Mix the above reaction system and add the basic reaction unit. Dissolve the lyophilized powder fully. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator. Open the reaction unit, add 2.5μL 280mM MgAc2 to each 0.2mL eppendorf tube, mix thoroughly and collect by centrifugation. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator.

[0262] The reaction tube was placed at 39°C for 16 minutes. After the RAA reaction was completed, the eppendorf tube was opened, the amplified product was transferred to a new eppendorf tube, marked, and diluted 20 to 50 times.

[0263] After simple lysis, the results of the dual RAA-LFS test of animal-derived ingredients in meat and processed meat products are as follows: Fig.33 As shown in the figure, the primer sets for detecting duck-derived components and bovine-derived components were used to detect duck or beef samples, respectively; the primer sets for detecting pig-derived components and bovine-derived components were used to detect pork or beef samples, respectively; the primer sets for detecting duck-derived components and sheep-derived components were used to detect duck or mutton samples, respectively; and the primer sets for detecting pig-derived components and sheep-derived components were used to detect pork or mutton samples, respectively. After the primer sets for detecting animal-derived components of each sample were tested by double RAA-LFS, when the negative control showed negative, the color development results of the amplified products were consistent with the qPCR identification results.

[0264] Experimental Example 14: Rapid detection of homemade adulterated beef products containing duck-derived ingredients, adulterated beef products containing pig-derived ingredients, adulterated beef products containing duck-derived ingredients, and adulterated mutton products containing pig-derived ingredients by dual RAA-LFS

[0265] Duck meat and beef samples were frozen in liquid nitrogen and ground into powder using a mortar. The frozen duck meat powder and beef powder were mixed in a ratio of 1:1, 1:9, 1:99 and 1:999, respectively. 40 mg of each sample was weighed and divided into two equal parts. One of the 20 mg homemade adulterated meat samples was extracted with a rapid universal genomic DNA extraction kit (Xinjing Bio, Hangzhou) for qPCR analysis, Cq value determination, and sample copy number calculation. The other 20 mg sample was The method provided by Cool Flash Nucleic Acid Releaser was used to complete the lysis of the homemade adulterated meat sample, and the dual RAA amplification system in Experimental Example 11 was amplified. The system is as follows:

[0266]

[0267]

[0268] After the reaction solution was prepared, 2.5 μL of 280 mM MgAc2 was added to each 0.2 mL eppendorf tube and mixed thoroughly.

[0269] Pork and beef samples were frozen in liquid nitrogen and ground into powder using a mortar. The frozen pork powder and beef powder were mixed in a ratio of 1:1, 1:9, 1:99 and 1:999, respectively. 40 mg of each sample was weighed and divided into two equal parts. A 20 mg homemade adulterated meat sample was used to extract total DNA using a rapid universal genomic DNA extraction kit (Xinjing Bio, Hangzhou) for qPCR analysis, to determine the Cq value, and to calculate the sample copy number. The other 20 mg sample was used as The method provided by Cool Flash Nucleic Acid Releaser was used to complete the lysis of the homemade adulterated meat sample and amplify the dual RAA amplification system in Experimental Example 11. The system is as follows:

[0270] Components Dosage (μL) Basal buffer 29.4 PigDerived-F(5μM) 0.73 PigDerived-R(5μM) 1.05 PigDerived-P(1μM) 1.05 BovineDerived-F(5μM) 0.44 BovineDerived-R(5μM) 0.63 BovineDerived-P(1μM) 0.63 Meat and processed meat sample lysate 2 Make up to volume with water 47.5

[0271] After the reaction solution was prepared, 2.5 μL of 280 mM MgAc2 was added to each 0.2 mL eppendorf tube and mixed thoroughly.

[0272] Duck and mutton samples were frozen in liquid nitrogen and ground into powder using a mortar. The frozen duck powder and mutton powder were mixed in a ratio of 1:1, 1:9, 1:99 and 1:999. 40 mg of each sample was weighed and divided into two equal parts. A 20 mg homemade adulterated meat sample was used to extract total DNA using a rapid universal genomic DNA extraction kit (Xinjing Bio, Hangzhou) for qPCR analysis, to determine the Cq value, and to calculate the sample copy number. The other 20 mg sample was used as The method provided by Cool Flash Nucleic Acid Releaser was used to complete the lysis of the homemade adulterated meat sample and amplify the dual RAA amplification system in Experimental Example 11. The system is as follows:

[0273] Components Dosage (μL) Basal buffer 29.4 DuckDerived-F (5μM) 0.58 DuckDerived-R(5μM) 0.84 DuckDerived-P(1μM) 0.84 OvineDerived-F(5μM) 1.03 OvineDerived-R(5μM) 1.48 OvineDerived-P(1μM) 1.48 Meat and processed meat sample lysate 2 Make up to volume with water 47.5

[0274] After the reaction solution was prepared, 2.5 μL of 280 mM MgAc2 was added to each 0.2 mL eppendorf tube and mixed thoroughly.

[0275] Pork and mutton samples were frozen in liquid nitrogen and ground into powder using a mortar. The frozen pork powder and mutton powder were mixed in a ratio of 1:1, 1:9, 1:99 and 1:999. 40 mg of each sample was weighed and divided into two equal parts. A 20 mg homemade adulterated meat sample was used to extract total DNA using a rapid universal genomic DNA extraction kit (Xinjing Bio, Hangzhou) for qPCR analysis, to determine the Cq value, and to calculate the sample copy number. The other 20 mg sample was used as The method provided by Cool Flash Nucleic Acid Releaser was used to complete the lysis of the homemade adulterated meat sample, and the dual RAA amplification system in Experimental Example 11 was amplified. The system is as follows:

[0276]

[0277]

[0278] After the reaction solution is prepared, add 2.5 μL of 280 mM MgAc2 to each 0.2 mL eppendorf tube and mix thoroughly; place the 0.2 mL eppendorf tube in a PCR instrument with a heated cover and keep warm at 39°C for 16 minutes. After the RAA reaction is completed, open the eppendorf tube, pipette the amplified product into a new eppendorf tube, mark it, and dilute it 20 times.

[0279] Comparison of RAA-LFS test results of homemade adulterated meat samples of different proportions of animal origin ( Fig.34 ) and qPCR test results show that the RAA-LFS detection system invented by the present invention team can realize the rapid and accurate detection of duck-derived components and bovine-derived components, porcine-derived components and bovine-derived components, duck-derived components and sheep-derived components, and pig-derived components and sheep-derived components through rapid lysis of samples.

[0280] Table 5 qPCR test results of random samples of homemade adulterated meat of animal origin with different proportions

[0281]

[0282]

[0283] In summary, the present application screened out primer sets that can simultaneously detect duck-derived ingredients and bovine-derived ingredients, pig-derived ingredients and bovine-derived ingredients, duck-derived ingredients and sheep-derived ingredients, and pig-derived ingredients and sheep-derived ingredients through a series of embodiments. The primer set can effectively amplify the target gene, has high specificity and sensitivity, has no cross-reaction with other species, and can be used for on-site rapid detection of duck-derived ingredients, pig-derived ingredients, bovine-derived ingredients, and sheep-derived ingredients, which is of great significance for food safety.

[0284] The invention shown and described herein can be implemented in the absence of any element, limitation specifically disclosed herein. The terms and expressions used are used as illustrative terms rather than limiting, and it is not intended to exclude any equivalents of the features shown and described or parts thereof in the use of these terms and expressions, and it should be recognized that various modifications are feasible within the scope of the present invention. It should therefore be understood that, although the present invention is specifically disclosed through various embodiments and optional features, modifications and variations of the concepts described herein can be adopted by those of ordinary skill in the art, and it is believed that these modifications and variations fall within the scope of the present invention as defined in the appended claims.

[0285] The contents of the articles, patents, patent applications, and all other documents and electronically available information described or recorded 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 into this application any and all materials and information from any such article, patent, patent application, or other document.

Claims

1. A RAA detection primer set for detecting duck-derived components, pig-derived components, cattle-derived components and sheep-derived components, characterized in that: The primer set includes duck-derived component detection primers, pig-derived component detection primers, bovine-derived component detection primers and sheep-derived component detection primers, duck-derived component detection probes, pig-derived component detection probes, bovine-derived component detection probes and sheep-derived component detection probes; wherein, The duck-derived component detection primers include a forward primer Duck Derived-F and a reverse primer Duck Derived-R. The sequence of the forward primer Duck Derived-F was 5′-CCACTTCTTAGAGTACTCCCAG-3′, and the sequence of the reverse primer Duck Derived-R was 5′-GGGTTGTAACTGTTGATAGTG-3′; The porcine-derived component detection primers include a forward primer Pig Derived-F and a reverse primer Pig Derived-R. The sequence of the forward primer Pig Derived-F is 5′-CCAATAAGCAATGATCAACC-3′, The sequence of the reverse primer Pig Derived-R was 5′-CTATGGCTACTGAGATGTATCC-3′; The bovine-derived component detection primers include a forward primer Bovine Derived-F and a reverse primer Bovine Derived-R. The sequence of the forward primer Bovine Derived-F is: 5′-CGTATCCCTACCCATCCTTACA-3′, The sequence of the reverse primer, Bovine Derived-R, was: 5′-ACCCGATTCAGACAAGTAGT-3′; The sheep-derived component detection primers include a forward primer Ovine Derived-F and a reverse primer Bovine Derived-R. The sequence of the forward primer Ovine Derived-F is: 5′-CAACACCACACTTCACAGCTTGC-3′, The sequence of the reverse primer Ovine Derived-R is: 5′-GGATCCTGCTAGTGTGTAA-3′; wherein the duck-derived component detection probe Duck Derived-P, Its sequence is 5′-GCACAAGCTCCAACACAACAAATAAAGTCAHCAAGAGCCCTCAACC-3′; The pig-derived component detection probe Pig Derived-P has a sequence of 5′ -CGAATCACCCGTATCATAAATTACTCAATCCHCAAGCCATTAAACTT-3′; The bovine-derived component detection probe Bovine Derived-P has a sequence of 5′- CTCTTCGTGCCTCCCAATTACACCAAATTCHATTAGTAAGGTCAGC-3′; The sheep-derived component detection probe Ovine Derived-P has a sequence of 5′ -CCATATGACTTCTAACCCTAATACTCATAHCTAGCCAACATCATC-3′; The reverse primers Duck Derived-R and Pig Derived-R for detecting duck-derived components and pig-derived components are modified with FITC at their 5′ ends, the detection probes Duck Derived-P and Pig Derived-P are modified with biotin at their 5′ ends, the 3′ ends of Duck Derived-P and Pig Derived-P are phosphorylated, and H is tetrahydrofuran; the reverse primers Bovine Derived-R and Ovine Derived-R for detecting bovine-derived components and sheep-derived components are modified with digoxigenin at their 5′ ends, the detection probes Bovine Derived-P and Ovine Derived-P are modified with rhodamine at their 5′ ends, the 3′ ends of Bovine Derived-P and Ovine Derived-P are phosphorylated, and H is tetrahydrofuran.

2. A kit for detecting whether meat and processed meat contain bovine, ovine, duck and / or porcine components, characterized in that: The kit comprises the primer set according to claim 1.

3. Use of the primer set as claimed in claim 1 in the preparation of a reagent or a kit for detecting whether the beef or mutton meat or processed meat to be tested contains duck-derived ingredients or pig-derived ingredients.

4. Use of the kit as claimed in claim 3 in detecting whether meat and processed meat contain bovine, ovine, duck and / or porcine components.

5. A method for detecting whether the beef or mutton meat and processed meat to be tested contain duck-derived ingredients or pig-derived ingredients by using a kit, characterized in that: The following steps are involved: 1) Extracting the total DNA of the meat and processed meat to be tested, or rapidly lysing the meat and processed meat samples to release nucleic acids; 2) using the total DNA extracted in step 1) or the supernatant of the sample lysate as a template, and using the forward primer and the reverse primer in the kit of claim 2 to perform RAA amplification to obtain an amplified product; 3) detecting the amplification product, and judging whether the amplification product contains duck-derived components and / or bovine-derived components, pig-derived components and / or bovine-derived components, duck-derived components and / or sheep-derived components, pig-derived components and / or sheep-derived components based on the detection result of the amplification product; If the amplified product contains a 287bp DNA fragment, the sample to be tested contains duck-derived components; if the amplified product does not contain the 287bp DNA fragment, the sample to be tested does not contain duck-derived components; if the amplified product contains a 274bp DNA fragment, the sample to be tested contains pig-derived components; if the amplified product does not contain the 274bp DNA fragment, the sample to be tested does not contain pig-derived components; if the amplified product contains a 351bp DNA fragment, the sample to be tested contains bovine-derived components; if the amplified product does not contain the 351bp DNA fragment, the sample to be tested does not contain bovine-derived components; if the amplified product contains a 397bp DNA fragment, the sample to be tested contains sheep-derived components; if the amplified product does not contain the 397bp DNA fragment, the sample to be tested does not contain sheep-derived components.

6. A method for detecting duck-derived components and / or bovine-derived components, porcine-derived components and / or bovine-derived components, duck-derived components and / or sheep-derived components, porcine-derived components and / or sheep-derived components in meat and processed meat samples based on RAA-lateral flow chromatography technology, characterized in that: The following steps are involved: 1) Rapidly lyse meat and processed meat samples to release nucleic acids, or extract total DNA from biological samples to be tested; 2) using the sample lysate supernatant or the extracted total DNA obtained in step 1) as a template, and performing RAA amplification using the primer set described in claim 1; 3) Use lateral flow chromatography test strips to detect RAA amplification products of duck-derived components and / or bovine-derived components in meat and processed meat samples. If only the quality control zone band of the test strip is colored, and no band is colored in the detection zone, the test result is negative, indicating that the sample does not contain duck-derived components and bovine-derived components; if two bands of the test strip are colored, one of which is located in the quality control zone and the other is located in the detection zone T1, the test result is positive, indicating that the test sample contains bovine-derived components but no duck-derived components; if the test strip has two bands, If the strips are colored, one of which is located in the quality control area and the other is located in the test area T2, the test result is positive, indicating that the test sample contains duck-derived components but no bovine-derived components; if the test strip has three colored strips, two of which are colored, one of which is located in the quality control area, one in the test area T1, and one in the test area T2, the test result is positive, indicating that the sample contains both bovine-derived components and duck-derived components; if the test strip's quality control area and both test areas are not colored, the test is invalid and the test strip needs to be replaced and retested; 4) Use lateral flow chromatography test strips to detect RAA amplification products of porcine and / or bovine components in meat and processed meat samples. If only the quality control zone band of the test strip is colored, and no band is colored in the detection zone, the test result is negative, indicating that the sample does not contain porcine and bovine components; if two bands of the test strip are colored, one of which is located in the quality control zone and the other is located in the detection zone T1, the test result is positive, indicating that the test sample contains bovine components but no porcine components; if the test strip has two bands, If one of the three strips is colored, one is located in the quality control area, and the other is located in the test area T2, the test result is positive, indicating that the test sample contains porcine components but no bovine components; if the test strip has three strips colored, two of which are colored, one is located in the quality control area, one is located in the test area T1, and one is located in the test area T2, the test result is positive, indicating that the sample contains both bovine and porcine components; if the quality control area and the two test areas of the test strip are not colored, the test is invalid and the test strip needs to be replaced and retested; 5) Use lateral flow chromatography test strips to detect RAA amplification products of duck-derived components and / or sheep-derived components in meat and processed meat samples. If only the quality control zone band of the test strip is colored, and no band is colored in the detection zone, the test result is negative, indicating that the sample does not contain duck-derived components and sheep-derived components; if the test strip has two colored bands, one of which is located in the quality control zone and the other is located in the detection zone T1, the test result is positive, indicating that the test sample contains sheep-derived components but does not contain duck-derived components; if the test strip has two colored bands, one of which is located in the quality control zone and the other is located in the detection zone T1, the test result is positive, indicating that the test sample contains sheep-derived components but does not contain duck-derived components; If the test strip has three colored bands, one of which is located in the quality control area and the other in the test area T2, the test result is positive, indicating that the test sample contains duck-derived components but no sheep-derived components; if the test strip has three colored bands, two of which are colored, one of which is located in the quality control area, one in the test area T1, and one in the test area T2, the test result is positive, indicating that the sample contains both sheep-derived components and duck-derived components; if the quality control area and the two test areas of the test strip do not show color, the test is invalid and the test strip needs to be replaced and retested; 6) Use lateral flow chromatography test strips to detect RAA amplification products of porcine and / or goat-derived components in meat and processed meat samples. If only the quality control zone band of the test strip is colored and no band is colored in the detection zone, the test result is negative, indicating that the sample does not contain porcine and goat-derived components; if two bands of the test strip are colored, one of which is located in the quality control zone and the other is located in the detection zone T1, the test result is positive, indicating that the test sample contains goat-derived components but no porcine components; if the test strip has two bands, If one of the strips is colored, and one of them is located in the quality control area, and the other is located in the detection area T2, the test result is positive, indicating that the test sample contains porcine components but no sheep-derived components; if the test strip has three colored strips, and two of them are colored, one of which is located in the quality control area, one is located in the detection area T1, and one is located in the detection area T2, then the test result is positive, indicating that the sample contains both sheep-derived and porcine components; if both the quality control area and the detection area of ​​the test strip are not colored, the test is invalid and the test strip needs to be replaced and retested.

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