Triple fluorescent PCR primer probe set, kit and detection method for detecting livestock-derived ingredients

Abstract

The present invention discloses a triple fluorescent PCR primer probe set for detecting livestock-derived components, including a triple primer probe set A for cattle, buffalo, and yak, and a triple primer probe set B for sheep, goats, and antelopes, as shown in Seq.ID No.1 to Seq.ID No.14. The present invention belongs to the field of gene detection technology, and each primer and probe will not cause mutual interference, and can only amplify and excite fluorescent signals for specific target sequences, and has no amplification and fluorescent signals for non-target sequences, and can achieve accurate detection of the above 6 livestock-derived components, and has the advantages of good specificity, small standard error, short detection time, and saving reagent costs, and is suitable for authenticity detection of livestock products.

Description

Technical Field

[0001] The invention belongs to the technical field of gene detection, and particularly relates to a triple fluorescent PCR primer probe set, a kit and a detection method for detecting livestock-derived components. Background Art

[0002] At present, the adulteration of animal products occurs frequently, and there are many reports of this kind at home and abroad. For example, in 2013, many EU countries had beef adulteration incidents of horse meat passing off as beef. In recent years, my country has also continued to expose many cases of animal product adulteration, including various cases seized by local food and drug supervision, quality inspection, and industrial and commercial bureaus and announced by the news media: duck meat passing off as mutton, pork and chicken passing off as beef, cat meat passing off as rabbit meat, and other counterfeiting methods.

[0003] The product characteristics of domestic cattle, buffalo, yaks, sheep, goats, antelopes and other species are extremely similar, but the price difference is large. Many specialty meat products on the market use ingredients derived from buffalo, yaks, sheep and goats as raw materials and as product selling points, but consumers are basically unable to distinguish their source ingredients through sensory evaluation during consumption and eating. In addition, yaks and antelopes also involve the supervision and protection of rare wild animals and the artificial breeding of specialty economic livestock.

[0004] In the study of methods for identifying the authenticity and adulteration of animal products using molecular biology techniques, amplifying and detecting target animal-derived genes through PCR technology is the current mainstream scientific basis and technical means, and has been widely used in recent years. This technology is mainly divided into two parts: ① Extracting sample DNA; ② PCR detection of DNA.

[0005] The extraction of DNA from livestock products can be divided into automated extraction, such as the self-test workstation that is not yet popular; semi-automated extraction, such as the nucleic acid extraction instrument that has gradually become popular; manual extraction, such as the currently common commercial kits, homemade reagents, etc. The DNA loss of unprocessed and semi-processed foods is small and relatively complete, and the above three methods are all applicable. Usually, the DNA purity is OD 260 / OD 280 The pH value is 1.6 to 1.8, and the concentration is at the ng / μL level. It can be slightly diluted or used directly for PCR detection.

[0006] At present, the focus and difficulty lies in the deep processing of livestock products, such as fried foods, refined foods, etc. The degree of DNA damage in these samples is extremely high. If the sample size is extracted according to the nucleic acid extractor or extraction kit, which is usually less than 1g, it is almost impossible to obtain the amount of DNA that can reach the detection limit; if the sample size is increased to more than 10g, it will be difficult to operate because the operating specifications of the nucleic acid extractor or extraction kit cannot be applied. At present, various testing units usually increase the sampling volume and use homemade reagents for manual extraction to obtain DNA. These methods are of various types, very different, and the results are also uneven.

[0007] The first generation of PCR technology, commonly known as conventional PCR technology, first designs primers for species-specific genes, then mixes the sample DNA, primers and amplification reagents, and repeatedly amplifies the target gene through a PCR instrument. The amplified product is then identified by electrophoresis to determine whether it contains species-derived components.

[0008] The second generation of PCR technology is real-time quantitative fluorescent PCR, usually referred to as fluorescent PCR. In addition to designing primers for species-specific genes, a fluorescent probe is designed. After the sample DNA, primers, probes and amplification reagents are mixed, the target gene is repeatedly amplified through a real-time quantitative fluorescent PCR instrument (usually referred to as a fluorescent PCR instrument). The accumulation of the probe's fluorescent signal is synchronized with the gene amplification. Compared with ordinary PCR, it not only improves the specificity of amplification, but also obtains the test results immediately after the amplification is completed.

[0009] Multiplex real-time quantitative fluorescence PCR (hereinafter referred to as multiplex fluorescence PCR) is a type of fluorescence PCR. For multiple species-specific genes, primers and probes are designed one by one, and different probes are labeled with fluorescent groups of different wavelengths. After the sample DNA, primers, probes and amplification reagents are mixed, multiple target genes are repeatedly amplified through a multi-channel fluorescence PCR instrument, and the requirement of simultaneously detecting multiple target genes is achieved by monitoring multiple fluorescence signals. Compared with ordinary fluorescence PCR, each additional set of primers and probes can double the detection efficiency and halve the cost of other reagents.

[0010] The prior art discloses multiplex fluorescence PCR methods for identifying animal-derived ingredients, such as 20151012723.2 "Taqman-LNA multiplex fluorescence quantitative PCR method, primers, probes and kits for simultaneous detection of bovine and porcine ingredients in meat and meat products", 201610272771.5 "A nested fluorescence PCR detection primer, probe, kit, detection method and application of donkey, horse, bovine and porcine ingredients in donkey-hide gelatin", 201510527762.1 "Primer, probe composition, kit and multiplex fluorescence PCR detection method for identifying donkey, horse and bovine ingredients in cosmetics", etc. However, none of the above methods involve how to distinguish the six species of cattle, buffalo, yak, sheep, goat and antelope through technical means. Summary of the invention

[0011] In order to solve the problems existing in the prior art, the present invention sets specific primer probe sets for the genomes of six large cattle and sheep in my country, namely cattle (Bos taurus), buffalo (Bubalusbubalis), yak (Bos grunniens), sheep (Ovis aries), goat (Capra aegagrus), and antelope (Saigatatarica). The primers and probes will not cause mutual interference, and can only amplify specific target sequences and excite fluorescent signals, and there is no amplification and fluorescent signals for non-target sequences. The present invention can achieve accurate detection of cattle, buffalo, yak, sheep, goat, and antelope-derived components. The universal primer pairs of sheep and goats do not use degenerate bases, have the advantages of good specificity, small standard error, short detection time, saving reagent costs, etc., and are conducive to the full detection of livestock-derived components of different strains. At the same time, the present invention optimizes the sample pretreatment method to address the problems of low efficiency and poor quality in DNA extraction of deep-processed livestock products in livestock-derived fluorescent PCR detection. The method has the characteristics of high extraction efficiency and good extraction quality, and can meet the requirements of existing DNA extraction methods or extraction kits. In subsequent fluorescent PCR detection, the Ct value can basically reach 25-35, which is within the threshold period where most standards do not require retesting.

[0012] There are many difficulties in the DNA extraction process of deeply processed livestock foods, such as fried foods and refined foods. First, the degree of damage to the sample DNA is extremely high. If the sample is extracted according to the sampling volume of the nucleic acid extractor or extraction kit, which is usually less than 1g, it is almost impossible to obtain the amount of DNA that can reach the detection limit; if the sampling volume is increased to more than 10g, it will be difficult to operate because the operating specifications of the nucleic acid extractor or extraction kit cannot be applied. This method first cleans the surface of the sample with n-hexane and then dries it at 60°C. The amount of the cleaned sample can be selected within the range of 20 to 200g according to actual needs or experience, ensuring sufficient sample DNA content.

[0013] Secondly, samples of deeply processed animal food are usually in the form of blocks, strips, or powders, in which the cell components are usually homogenized and contain a large number of indigestible or difficult-to-degrade carbonized or inert components. These carbonized or inert components usually exist in the form of a large number of particles, powders, and precipitates. During the DNA extraction process, the DNA solution is usually adsorbed on them. Even if it is centrifuged, it will be quickly and massively absorbed by the particles / powders / precipitates within a few seconds after centrifugation and cannot be effectively separated. This method adds an equal volume of 3d H 2 O Extract overnight on a shaker at 37°C and 200 rpm to ensure that the sample DNA is dissolved into the water phase as much as possible; then separate most of the liquid phase through a manual or electric squeezer, avoiding the disadvantage that centrifugal means cannot effectively separate. Finally, centrifuge at 10,000 rpm for 10 minutes to obtain the water phase, which is concentrated to less than 1 mL by a vacuum concentrator. Conventional manual methods, commercial kits, DNA extractors, etc. can be used for DNA purification.

[0014] When designing specific primers and probes for cattle, buffalo, yaks, sheep, and goats, their taxonomic status must be clarified first. Domestic cattle Bo.taurus domestica and goats C.aegagrus hircus are domesticated species of wild cattle Bo.taurus and wild goats C.aegagrus, respectively; there is no scientific name difference between the domesticated species of buffalo Bu.bubalis, yak Bo.grunniens, and sheep O.aries and the original species. According to relevant research on molecular taxonomy, the genetic differences between wild subspecies of higher species are very small, usually only a few genetic differences, especially for domesticated subspecies, whose genomes are almost indistinguishable from the original wild species. In addition, in each genomic library, the species source notes of the relevant sequences are generally only to the genus and species; those that are accurate to subspecies, variants, serotypes, and mutant strains are mostly certain pathogenic bacteria and viruses. Therefore, when searching for target genes, the present invention uses the related sequences of Bo. taurus, Bu. bubalis, Bo. grunniens, O. aries, and C. aegagrus as references, without distinguishing between original species and domesticated species.

[0015] When designing specific primers and probes for antelopes, their taxonomic status should be clarified first. According to literature reports, in taxonomy, antelopes do not refer to a specific family or genus; there are a total of 86 species of antelopes, belonging to 11 tribes and 32 genera. Among these genera and species, most are rare and wild species; the present invention selects the Saiga antelope S. tatarica, which has significant medicinal value and is under strict monitoring and protection, as the identification object.

[0016] After searching NCBI's Genbank, dozens of mitochondrial-related sequences of Bo.taurus, Bu.bubalis, Bo.grunniens, O.aries, C.aegagrus, and S.tatarica were obtained. The conserved sequences were first aligned using the MegAlign software in Lasergene v7.1.0, and then screened after verification by Oligo v7.0.1 and NCBI's Primer-Blast. The inventors found that according to the specific sites of their glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes, a pair of universal primers and two specific probes for Bo.taurus and Bu.bubalis, a pair of universal primers and two specific probes for O.aries and C.aegagrus, exclusive primers and specific probes for Bo.grunniens, and exclusive primers and specific probes for S.tatarica can be designed. The above probes respectively use NFQ-MGB fluorescence quenching groups, which can more effectively avoid nonspecific amplification caused by single-base differences compared to TAMRA, BHQ, etc.

[0017] Compared with the existing methods, the primers and probes provided by the present invention are 34bp in the probe of SN / T 4397-2015 "Real-time fluorescence PCR method for detecting yak-derived ingredients in exported food", while the probe of the present invention is only 20-22bp, which reduces the probability of non-specific matching in the middle of the sequence; compared with SN / T 2051-2008 "Real-time PCR method for detecting cattle, sheep and pig-derived ingredients in food, cosmetics and feed", the present invention refines the classification of cattle and sheep, which is closer to daily life and actual detection; compared with SN / T2980-2011 "Triple real-time fluorescence PCR detection method for cattle, goat and sheep-derived ingredients in animal products", the universal primer pair for sheep and goats does not use degenerate bases, which improves the specificity; compared with SN / T 3730.7-2013 "Methods for identification of common livestock species in food and feed Part 7 Real-time fluorescence PCR method for detection of buffalo components" and SN / T Compared with SN / T 2051-2008, SN / T3730.7-2013 and SN / T 4397-2015, the detection method provided by the present invention solves the detection problem of deep-processed samples and is easier to operate.

[0018] Examples of primer probe set detection sites for Bo.taurus, Bu.bubalis, Bo.grunniens, O.aries, C.aegagrus, and S.tatarica are as follows:

[0019] (1) The GAPDH gene of Bo. taurus, the detection site is 2437 bp to 2510 bp on the NC007303.6 sequence in NCBI.

[0020] (2) The GAPDH gene of Bu. bubalis, the detection site is 2416 bp to 2490 bp on the NW005785176.1 sequence in NCBI.

[0021] (3) GAPDH gene of Bo. grunniens, the detection site is 102 bp to 191 bp on the EU195062.1 sequence in NCBI.

[0022] (4) Mitochondrial gene of O. aries, the detection site is 146 bp to 269 bp on the KF469290.1 ​​sequence in NCBI.

[0023] (5) Mitochondrial gene of C. aegagrus, the detection site is 775bp to 880bp on the GQ240309.1 sequence in NCBI.

[0024] (6) Mitochondrial gene of S. tatarica, the detection site is 6484 bp to 6585 bp on the KY829450.1 sequence in NCBI.

[0025] The triple fluorescent PCR primer probe set for detecting six kinds of animal-derived components provided by the present invention includes a triple primer probe set A for Bo. taurus, Bu. bubalis, and Bo. grunniens, and a triple primer probe set B for O. aries, C. aegagrus, and S. tatarica, as shown in Table 1. Preferably, the triple primer probe set A and the triple primer probe set B can be selected for simultaneous detection to improve efficiency, or the triple primer probe set A or the triple primer probe set B can be selected separately for identification to save operation and reagents according to the actual detection needs of the sample.

[0026] Table 1

[0027]

[0028] The present invention also provides a triple fluorescent PCR kit for detecting six kinds of animal-derived components, including the above-mentioned primer probe set, fluorescent PCR reagent, positive control, negative control and blank control. In the selection of supporting reagents, for multiple fluorescent PCR reagents, a model with a thermosensitive Taq antibody is selected to inhibit nonspecific annealing of primers or nonspecific amplification caused by primer dimers under low temperature conditions, extend the shelf life of the kit, and allow more freeze-thaw times; the fluorescent PCR reagent includes ROX dye, which can improve the detection accuracy for multiple fluorescent PCR instruments that require ROX dye. More preferably, the blank control is 3dH 2 O.

[0029] Preferably, the concentrations of the universal upstream primer and downstream primer of Bo.taurus and Bu.bubalis are 20 μmol / L, respectively, and the concentrations of the universal upstream primer and downstream primer of O.aries and C.aegagrus are 20 μmol / L, respectively; the concentrations of the Bo.grunniens upstream primer, Bo.grunniens downstream primer, S.tatarica upstream primer, and S.tatarica downstream primer are 10 μmol / L, respectively, and the concentrations of the 6 probes described in Table 1 are all 10 μmol / L. Preferably, the positive control is a mixed DNA fragment or genome that is amplified by the primer set included in the above primer probe set and detected positive by the probe set included in the above primer probe set, with a concentration of 10 5 copies / μL level.

[0030] Preferably, the negative control is non-animal-derived DNA, and the blank control is ultrapure water.

[0031] In addition, the present invention also provides a triple fluorescence PCR method for detecting six kinds of animal-derived components, comprising the following steps: 1) performing sample pretreatment and extracting sample DNA; 2) establishing a multiplex fluorescence PCR reaction system including the primer probe set described in claim 1 or 2, and the reaction conditions are: 95°C 20s-2min or 10-15min; 95°C 5s-1min, 60°C 20s-2min; 40 cycles and collecting fluorescence signals to judge the detection results. When it is a biological thermosensitive antibody, 95°C 20s-2min or 10-15min; when it is a chemical thermosensitive antibody, 95°C 10-15min.

[0032] Preferably, the sample is a deeply processed animal food, and the sample pretreatment includes: cleaning the sample surface with n-hexane and drying at 60°C, adding an equal volume of 3d H 2Overnight extraction was carried out on a shaker at 37 °C and 200 rpm. After separating the liquid phase by a press, the aqueous phase was obtained by centrifugation at 10,000 rpm for 10 min and concentrated to less than 1 mL by a vacuum concentrator.

[0033] Preferably, the method for judging the detection result includes:

[0034] ① Quality control standard: In the PCR reaction systems of the triple primer-probe sets A and B of the positive control, there is a logarithmic increase in fluorescence for FAM, VIC, and NED, and the Ct value ≤ 30.0. There is no fluorescence signal and logarithmic increase in the negative control and the blank control, and the Ct value ≥ 40.0. Then, the judgment of the detection result of the livestock-derived components in ② is carried out;

[0035] ② Detection result: In the PCR reaction system of the triple primer-probe set A of the sample, if there is a logarithmic increase in fluorescence for FAM and / or VIC and / or NED, and the Ct value ≤ 30.0, then it contains the corresponding Bos taurus and / or Bubalus bubalis and / or Bos grunniens-derived components; in the PCR reaction system of the triple primer-probe set B of the sample, if there is a logarithmic increase in fluorescence for FAM and / or VIC and / or NED, and the Ct value ≤ 30.0, then it contains the corresponding Ovis aries and / or Capra hircus and / or Gazella subgutturosa-derived components; if there is no signal and logarithmic increase in one or several of the above fluorescences, then it does not contain the corresponding livestock-derived components; if 30.0 < Ct value < 40.0, the template amount is increased for recheck. If the Ct value ≥ 40.0, the detection result is negative. If the Ct value < 40.0, the detection result is positive.

[0036] Compared with the prior art, the beneficial effects of the present invention include:

[0037] (1) The present invention sets primer-probe sets for the specific genes of Bos taurus, Bubalus bubalis, Bos grunniens, Ovis aries, Capra hircus, and Gazella subgutturosa. Each primer-probe will not cause interference with each other, and can only amplify and excite fluorescence signals for specific target sequences, without amplification and fluorescence signals for non-target sequences, with good specificity and high detection sensitivity.

[0038] (2) The detection method provided by the present invention is conducive to the full detection of different strains of livestock-derived components. Compared with the multiplex PCR method, it has smaller standard error (when the initial concentration of the standard 3 kbp plasmid is at the 10 5 level, the quantitative standard error is at the 10 4 level), shorter detection time (the detection result can be obtained within about 1 h after loading), and less generation of toxic and harmful substances (the dosage of the fluorescent dye is at the ng / μL level); compared with the fluorescence PCR method, it has the advantages of simultaneously detecting multiple target genes and saving reagent costs; compared with the ordinary PCR method, it has both the above two advantages.

[0039] (3) The primers, probes, kits and methods provided by the present invention have been applied to the daily detection of this unit (detection limit 0.01% mass fraction), and have also been verified by another testing unit in the same industry. Hundreds of batches of samples have no missed detection or false detection, and the detection results are the same as those of the standard method. Practice has shown that the present invention saves more than 70% of operation time and more than 70% of reagent costs compared with the standard method, and has good practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The test result diagram of Example 3 of the present invention; wherein, 1-6 are the positive controls of the initial test, 15-26 are the negative controls and blank controls of the initial test; 7-12 are the positive controls of the retest, 27-38 are the negative controls and blank controls of the retest, 13 is the bovine-derived fluorescent signal of the initial test, and 14 is the bovine-derived fluorescent signal of the retest. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0042] In the present invention, the reagents and materials involved are conventional commercial products, or can be obtained by conventional technical means in the art.

[0043] Example 1 Construction and verification of a triple fluorescent PCR kit for detecting six animal-derived ingredients

[0044] ① Primer probe set: As shown in Table 1, it can be synthesized by a company with the ability to synthesize primers and probes. In this embodiment, Shanghai Sangon Biotechnology Technology Service Co., Ltd. was selected for synthesis. The primer and probe dry powders were diluted to 100 μmol / L as a stock solution, and prepared into 10 μmol / L as a working solution according to Table 2, and stored at -20°C in a black 1.5 mL centrifuge tube.

[0045] Table 2 Preparation of 100 μmol / L stock solution to 10 μmol / L working solution

[0046]

[0047] ② Fluorescent PCR reagent: Common commercially available fluorescent PCR reagent, this example uses ABI Path-ID TM qPCR Master Mix.

[0048] ③ Positive control: Take all the primer stock solutions and dilute them to 10 μmol / L. Extract 6 kinds of livestock DNA standards, use the corresponding primers and common PCR reagents to amplify on a common PCR instrument, cut the target band and recover it, then transfer it to TaKaRaT-Vector pMD TM20 vector, replicated with competent E. coli cells JM109, extracted pMT-20 plasmid, and diluted to 10 6 copies / μL concentration, take 10μL of each, mix well and then use 3d H 2 O to 100 μL, the final concentration is 10 5 copies / μL level.

[0049] ④Negative control: non-animal DNA.

[0050] ⑤Blank control: ultrapure water.

[0051] ⑥ ABI Quantstudio 5 was used as the multi-channel fluorescence PCR instrument. Sample characteristics were verified as shown in Table 3. The reaction solution was prepared according to Table 4 and the reaction was carried out according to Table 5.

[0052] Table 3 Sample characteristics

[0053]

[0054] Table 4 30μL reaction system (unit: μL)

[0055]

[0056] Table 5 Reaction conditions

[0057]

[0058] Note * : Detect fluorescence signal.

[0059] ⑦The reaction results are shown in Table 6:

[0060] Table 6 Sample test results: measured Ct value and the presence or absence of S-shaped curve

[0061]

[0062]

[0063] ⑧ Verification results: The kit was sent to the microbiology (including molecular biology) testing laboratory of the same industry testing unit in the province, and the results were consistent. Through the setting of positive control, spiked control, negative control, and blank control, it was shown that the components of the kit were effective, with good specificity, small standard error, short detection time, and reagent cost saving.

[0064] Example 2: Detection of samples commissioned by individuals: chicken feet in black bean sauce, spicy duck neck, roast goose, braised beef, buffalo milk, yak jerky, fat lamb rolls, lamb slices, and antelope horn powder

[0065] The samples commissioned by individuals for testing were retrieved, including black bean sauce chicken feet, spicy duck neck, roast goose, braised beef, buffalo milk, yak jerky, fat lamb rolls, lamb slices, and antelope horn powder, numbered as #1, #2, #3, #4, #5, #6, #7, #8, and #9. Solid samples were ball-milled and liquid samples were directly sampled. The DNA of the samples was extracted on a small magnetic rack (Qiagen MagAttract Magnetic Rack) using an externally purchased DNA extraction kit (Qiagen MagAttract Hmw DNA kit). All samples were 3d H 2 O was diluted (or vacuum concentrated) to a concentration of 50 ng / μL. The primer set, positive control, negative control, blank control, etc. were all based on the kit components in Example 1. The purchased fluorescent PCR reagent was Multiplex PCR Kit, tested on an ABI fluorescent PCR instrument (model 7500Fast).

[0066] The test results are shown in Table 7: ① Quality control standard: FAM, VIC, and NED of the positive control system A and system B all have fluorescence logarithmic growth, and the Ct value is ≤30.0; the negative control and blank control have no fluorescence signal and logarithmic growth, and the Ct value is ≥40.0. ② FAM #4, VIC #5, NED #6 in system A and FAM #7, VIC #8, and NED #9 in system B have fluorescence signals and logarithmic growth, and the Ct value is ≤30.0, and the others have no fluorescence signals and logarithmic growth, and the Ct value is ≥40.0, indicating that braised beef, buffalo milk, yak jerky, fat sheep rolls, mutton slices, and antelope horn powder contain cattle, buffalo, yak, sheep, goat, and antelope derived ingredients, respectively, and other samples do not contain the above-mentioned derived ingredients. ③Comparison results: consistent with the results of relevant source component testing of samples in accordance with or with reference to SN / T 2980-2011, SN / T 3730.7-2013, SN / T 4397-2015 and other standards.

[0067] Table 7 Sample test results: Measured Ct value and presence or absence of S-shaped curve

[0068]

[0069]

[0070] Example 3: Testing of the sample commissioned by the enterprise - Detection of the beef strips with light shadow

[0071] Take the sample entrusted by the enterprise for testing, 1 portion of Dengying beef shreds, 200g of sample, set as 10# sample, clean the surface of the sample with n-hexane and dry it at 60℃, add an equal volume of 3d H 2O was extracted overnight at 37 °C and 200 rpm on a shaker. After separating the liquid phase by an electric press, the aqueous phase was obtained by centrifugation at 10,000 rpm for 10 min, concentrated to 1 mL by a vacuum concentrator, and purified and concentrated to 50 μL on a small magnetic stand (Agencourt Spristand 6 - position tube magnet) using a nucleic acid purification kit (Beckman Agencourt AMPure XP). The externally purchased fluorescent PCR reagent selected the environmental sample - optimized reagent ABI TaqMan TM Environmental Master Mix 2.0, and the DNA extract loading volume was selected as 5 μL. The primer probe set, positive control, negative control, blank control, etc. were all in accordance with the kit components in Example 1 and were tested on a machine (ABI fluorescence PCR instrument, model Quantstudio 5).

[0072] The test results are shown in Figure 1 and Table 8: ① Quality control standard: For the positive control, FAM, VIC, and NED in both System A and System B showed logarithmic fluorescence growth, and the Ct value ≤ 30.0; the negative control and the blank control had no fluorescence signal and logarithmic growth, and the Ct value ≥ 40.0. ② In System A, for #10, 30.0 < Ct value < 40.0 for FAM, and there was no fluorescence signal and logarithmic growth for the others and the Ct value ≥ 40.0; after increasing the DNA extract loading volume to 10 μL and retesting, the Ct value < 40.0, and the test result was judged as positive, indicating that it contained bovine - derived components, and the other samples did not contain the above - mentioned derived components. ③ Comparison result: It was consistent with the results of detecting relevant derived components in the samples according to or referring to standards such as SN / T 2980 - 2011, SN / T 3730.7 - 2013, and SN / T 4397 - 2015.

[0073] Table 8 Initial and retest results of samples: Measured Ct values

[0074]

[0075] The above content is a further detailed description of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, and all should be regarded as belonging to the protection scope of the present invention. Sequence Listing <110> Guizhou Institute of Product Quality Supervision and Inspection <120> Triplex fluorescent PCR primer - probe set, kit and detection method for detecting livestock - derived components <160> 14 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial sequence (RenGongXuLie) <400> 1 aggccatcac catcttcca 19 <210> 2 <211> 18 <212> DNA <213> Artificial sequence (RenGongXuLie) <400> 2 cccagtgcct cctccaag 18 <210> 3 <211> 20 <212> DNA <213> Artificial sequence (RenGongXuLie) <400> 3 tgaccccttc attgaccttc 20 <210> 4 <211> 20 <212> DNA <213> Artificial sequence (RenGongXuLie) <400> 4 ttctctgcct tgactgtgcc 20 <210> 5 <211> 20 <212> DNA <213> Artificial sequence (RenGongXuLie) <400> 5 aggcctattc ctagcaatac 20 <210> 6 <211> 20 <212> DNA <213> Artificial sequence (RenGongXuLie) <400> 6 gtttgcgtgt atatatcgga 20 <210> 7 <211> twenty one <212> DNA <213> Artificial sequence (RenGongXuLie) <400> 7 atatatacat gcaaacggag c 21 <210> 8 <211> twenty three <212> DNA <213> Artificial sequence (RenGongXuLie) <400> 8 ttgcgaatag aagaataact cca 23 <210> 9 <211> twenty one <212> DNA <213> Artificial sequence (RenGongXuLie) <400> 9 ttcctgttcc gtctctcaca c 21 <210> 10 <211> twenty one <212> DNA <213> Artificial sequence (RenGongXuLie) <400> 10 tgttctgtct cccacactta c 21 <210> 11 <211> 20 <212> DNA <213> Artificial sequence (RenGongXuLie) <400> 11 tactggaaca tatagaccat 20 <210> 12 <211> twenty one <212> DNA <213> Artificial sequence (RenGongXuLie) <400> 12 cagccatagt ttacgtctcg g 21 <210> 13 <211> twenty one <212> DNA <213> Artificial sequence (RenGongXuLie) <400> 13 cagccataat ttacatctcg a 21 <210> 14 <211> twenty two <212> DNA <213> Artificial sequence (RenGongXuLie) <400> 14 attcatacac gtaggacgag gc 22

Claims

1. Triple fluorescent PCR primer probe set for detecting livestock-derived ingredients, Features: The invention comprises a triple primer probe set A for cattle, buffalo and yak and a triple primer probe set B for sheep, goat and antelope; the triple primer probe set A comprises: a universal upstream primer Seq.ID No.1 for cattle and buffalo, a universal downstream primer Seq.ID No.2 for cattle and buffalo, an upstream primer Seq.ID No.3 for yak, a downstream primer Seq.ID No.4 for yak, a fluorescent probe Seq.ID No.9 for cattle, a fluorescent probe Seq.ID No.10 for buffalo and a fluorescent probe Seq.ID No.11 for yak; the triple primer probe set B comprises: a universal upstream primer Seq.ID No.5 for sheep and goat, a universal downstream primer Seq.ID No.6 for sheep and goat, an upstream primer Seq.ID No.7 for antelope, a downstream primer Seq.ID No.8 for antelope, a fluorescent probe Seq.ID No.12 for sheep, a fluorescent probe Seq.ID No.13 for goat and a fluorescent probe Seq.ID No.14 for antelope. No.14; The 5' end of the cattle fluorescent probe is modified with FAM, the 5' end of the buffalo fluorescent probe is modified with VIC, the 5' end of the yak fluorescent probe is modified with NED, the 5' end of the sheep fluorescent probe is modified with FAM, the 5' end of the goat fluorescent probe is modified with VIC, the 5' end of the antelope fluorescent probe is modified with NED, and the 3' ends of the probes are respectively modified with NFQ-MGB; The antelope is a Saiga antelope.

2. Triple fluorescence PCR kit for detecting livestock-derived ingredients, Features: The method comprises the primer probe set according to claim 1, a fluorescent PCR reagent, a positive control, a negative control, and a blank control.

3. The triple fluorescence PCR kit for detecting livestock-derived components according to claim 2, Features: The concentrations of the universal upstream primer and downstream primer for cattle and buffalo are 20 μmol / L, respectively; the concentrations of the upstream primer and downstream primer for yaks are 10 μmol / L, respectively; the concentrations of the universal upstream primer and downstream primer for sheep and goats are 20 μmol / L, respectively; the concentrations of the upstream primer and downstream primer for antelopes are 10 μmol / L, respectively; and the concentrations of the probes are 10 μmol / L.

4. The triple fluorescence PCR kit for detecting livestock-derived components according to claim 2, Features: The positive control is a mixed DNA fragment or genome that is amplified and tested positive using the triple fluorescent PCR primer probe set of claim 1, with a concentration of 10 5 copies / μL level.

5. The triple fluorescence PCR kit for detecting livestock-derived components according to claim 2, Features: The negative control is non-animal-derived DNA; the blank control is ultrapure water.

6. Triple fluorescence PCR method for detecting livestock-derived ingredients, Features: It includes the following steps: 1) Pretreat the sample to extract the sample DNA; 2) Establish a multiplex fluorescence PCR reaction system including the primer-probe set described in claim 1, and amplify with the primer-probe set described in claim 1. The reaction conditions are: 95°C for 20 s–2 min or 10–15 min; 95°C for 5 s–1 min, 60°C for 20 s–2 min; 40 cycles and collect fluorescence signals to judge the test results.

7. The triple fluorescence PCR method for detecting livestock-derived components according to claim 6, characterized in that: The sample is a deeply processed animal food. The sample pretreatment includes: cleaning the sample surface with n-hexane and drying at 60°C, adding an equal volume of 3d H 2 O was extracted on a shaker at 37°C and 200 rpm overnight. The liquid phase was separated by squeezing, and then the aqueous phase was obtained by centrifugation at 10,000 rpm for 10 min, which was concentrated to less than 1 mL using a vacuum concentrator.

8. The triple fluorescence PCR method for detecting livestock-derived components according to claim 6, characterized in that: The judgment of the test results includes: ① Quality control standard: In the PCR reaction systems of the triple primer-probe set A and the triple primer-probe set B of the positive control, there is a logarithmic increase in fluorescence for FAM, VIC, and NED, and the Ct value ≤ 30.

0. There is no fluorescence signal and logarithmic increase for the negative control and the blank control, and the Ct value ≥ 40.

0. Then, judge the detection results of the livestock-derived components in ②; ② Test results: In the PCR reaction system of the triple primer-probe set A of the sample, if there is a logarithmic increase in fluorescence for FAM and / or VIC and / or NED, and the Ct value ≤ 30.0, it contains the corresponding Bos taurus and / or Bubalus bubalis and / or Bos grunniens-derived components; in the PCR reaction system of the triple primer-probe set B of the sample, if there is a logarithmic increase in fluorescence for FAM and / or VIC and / or NED, and the Ct value ≤ 30.0, it contains the corresponding Ovis aries and / or Capra hircus and / or Gazella subgutturosa-derived components; if there is no signal and logarithmic increase in one or several of the above fluorescences, it does not contain the corresponding livestock-derived components; if 30.0 < Ct value < 40.0, increase the template amount for retesting. If the Ct value ≥ 40.0, the test result is negative. If the Ct value < 40.0, the test result is positive.

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