Universal molecular beacon kit for various animal-derived components

Abstract

The invention discloses a universal molecular beacon kit for various animal-derived components, and belongs to the technical field of molecular biological detection. Aiming at gene segments with specific structural characteristics in 11 common livestock and poultry animal genomes, the invention designs a composition of a universal primer and a molecular beacon fluorescent probe. The combination can be used for carrying out real-time fluorescent PCR detection and melting curve analysis by using the kit and the method by taking genome DNA of a sample to be detected as a template. By comparing the shape of a melting peak of a detected sample and the characteristic melting temperature (Tm value) of the detected sample, 11 animal-derived components of livestock and poultry can be quickly and accurately identified under the condition of single-tube detection. Technical innovation is achieved in the field of molecular biological detection, an efficient and reliable solution is provided for animal-derived component detection, and the method has important practical value and popularization potential.

Description

Technical Field

[0001] This invention relates to a universal molecular beacon kit for various animal-derived components, belonging to the field of molecular biology detection technology. Background Technology

[0002] In the crucial field of food authenticity verification, current research has formed a technological system centered on molecular biology, proteomics, and chromatographic analysis. Among these, PCR technology, with its high sensitivity and specificity, has become the cornerstone of species identification; protein analysis techniques (such as mass spectrometry and immunoassay) are widely used in targeted identification; and chromatography provides an effective means for the qualitative and quantitative analysis of specific components. However, existing technologies still have significant limitations when dealing with complex adulteration scenarios. Traditional PCR techniques are mostly designed for single targets, making it difficult to achieve simultaneous detection of multiple species. Detection of multi-source components in complex food matrices often requires cumbersome pretreatment and multiple trials, significantly increasing detection costs and time. Protein analysis techniques are susceptible to protein denaturation during processing and face challenges in antibody development and cross-reactivity when screening multiple targets. While chromatography is precise, its complex pretreatment and expensive equipment make it difficult to meet the demands of high-throughput rapid detection. Especially when dealing with food matrices that are deeply processed and contain multiple components, the above technologies face bottlenecks in terms of detection throughput, timeliness, cost, and anti-interference ability. Furthermore, chromatographic methods have high technical requirements for experimental equipment and operators, making it difficult to meet the needs of grassroots testing institutions.

[0003] Modern detection technologies are evolving towards higher sensitivity, higher specificity, multiplexing, and rapid screening. Emerging technologies such as molecular beacon technology, digital PCR, and high-throughput sequencing are gradually overcoming the limitations of traditional methods, providing better solutions for the detection of animal-derived components. In particular, the technical approach combining melting curve analysis with multiplex PCR shows great potential for achieving multiplexing while maintaining high specificity.

[0004] Therefore, developing a technical solution that can achieve simultaneous, rapid, and accurate identification of multiple targets has become a direction that the industry urgently needs to break through. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides a universal molecular beacon kit for various animal-derived components. The purpose is to overcome the technical problem that existing identification technologies cannot simultaneously and rapidly screen for multiple animal-derived components. This kit can be used to rapidly identify animal-derived components from 11 livestock and poultry species, including pigs, cattle, sheep, chickens, ducks, geese, pigeons, sika deer, camels, donkeys, and foxes, in food. The kit is simple to operate and has specificity.

[0006] The first technical solution provided by the present invention is a composition for detecting animal-derived components, the composition comprising a universal primer pair and a molecular beacon probe, the nucleotide sequences of the universal primers being shown in SEQ ID NO.1 and EQID NO.2, respectively, and the nucleotide sequence of the molecular beacon probe being shown in SEQ ID NO.3.

[0007] The universal upstream primer shown in SEQ ID NO.1 for amplifying the target gene nucleotide sequence is: 5'-CCAAACTGGGATTAGATACC-3'; The universal downstream primer shown in SEQ ID NO.2 is: 5'-GAACAGGCTCCTCTAGGT-3'; The molecular beacon probe used to detect the target gene, SEQ ID NO.3, is: 5'-CGTCGAGCCCTAAACCCAGATGGTTATCGACG-3'.

[0008] In some embodiments, the 5' and 3' ends of the molecular beacon probe are connected to functional groups or molecules.

[0009] Optionally, the functional group or molecule may be a fluorescent marker, a chemiluminescent marker, a bioluminescent marker, a biotin, an affinity ligand, and / or a thiol group.

[0010] Furthermore, the molecular beacon probe has a FAM fluorescence generating group attached to its 5' end and a DABCYL quenching group attached to its 3' end.

[0011] The second technical solution provided by the present invention is a detection system for universal screening of multiple animal-derived components, wherein the detection system contains the composition for detecting animal-derived components described in the first technical solution.

[0012] In some embodiments, the animal-derived ingredients are derived from pigs, cattle, sheep, chickens, ducks, geese, pigeons, sika deer, camels, donkeys, and foxes.

[0013] In some embodiments, the final concentration of the universal upstream and downstream primers in the detection system is 0.1 μM-1.0 μM, and the final concentration of the molecular beacon in the reaction system is 0.05 μM-1.0 μM.

[0014] In some embodiments, the detection system further includes a positive control, qPCR reaction reagents, ROX reference dye, and ultrapure water.

[0015] In some embodiments, the positive control is the animal-derived component from pigs, cattle, sheep, chickens, ducks, geese, pigeons, sika deer, camels, donkeys, and foxes, with a positive control concentration of 100 ng / µL.

[0016] In some embodiments, the test sample is a DNA extract, and the amount of the test sample added to the detection system is 1~100 ng / µL.

[0017] The third technical solution provided by the present invention is a universal screening kit for multiple animal-derived components, wherein the kit contains the composition for detecting animal-derived components described in the first technical solution or the detection system described in the second technical solution.

[0018] In some embodiments, the kit also contains positive controls of pigs, cattle, sheep, chickens, ducks, geese, pigeons, sika deer, camels, donkeys, and foxes. The fourth technical solution provided by this invention is a method for detecting animal-derived components based on molecular beacon melting curve analysis technology. This method is simple to operate, efficient, and has good applicability. Specifically, it includes the following steps: (1) Extract genomic DNA from the unknown sample to be tested and obtain DNA extract solution; (2) The extracted DNA sample was amplified by real-time fluorescence PCR and the melting curve was detected using the composition described in the first technical solution; (3) Perform melting curve analysis on the amplified products, establish a species melting curve Tm value model, and use the peak value of the melting curve to identify animal-derived components in the sample.

[0019] In some embodiments, the animal-derived ingredient may be derived from pigs, cattle, sheep, chickens, ducks, geese, pigeons, sika deer, camels, donkeys, and foxes.

[0020] In some embodiments, in step (2), the concentration of the molecular beacon probe used is 5-20 pmol; the concentration of the universal upstream primer and the universal downstream primer used is 10-30 pmol. More preferably, the concentration of the molecular beacon probe is 10 pmol; and the concentration of the universal upstream primer and universal downstream primer is 20 pmol.

[0021] In some embodiments, in step (2), the reaction system for the fluorescent PCR amplification is as follows: 1-100 ng / μL DNA extraction solution is used as a template, and universal upstream and downstream primers with a final concentration of 0.2 μM, molecular beacon probe with a final concentration of 0.2 μM, qPCR reaction reagents and ROX reference dye (selected according to the qPCR model) are added, and ultrapure water is added to a final volume of 20 μL. The working solution concentration is 5 ng / μL (the final detection concentration is 0.25 ng / μL).

[0022] Furthermore, the qPCR reaction reagents include PCR buffer (containing [Mg2+]), dNTPs and Taq enzyme, or a 2×qPCR premix made of ultrapure water.

[0023] In some embodiments, in step (2), the reaction system for the fluorescent PCR amplification is 1 μL of DNA extract, 10 μL of 2×qPCR premix, 0.4 μL each of universal upstream and downstream primers, 0.4 μL of molecular beacon probe, 0.2 μL of 50×ROX reference dye, and ultrapure water to a final volume of 20 μL.

[0024] In some implementations, in step (2), the amplification program of real-time fluorescence PCR is 95°C pre-denaturation for 30s, 95°C denaturation for 15s, 60°C annealing for 20s and acquisition of fluorescence signal, and 74°C extension for 10s, for a total of 45 cycles.

[0025] In some embodiments, in step (3), the melting curve analysis procedure is as follows: after the PCR amplification reaction is completed, the reaction system is heated to 95°C for 1 min, cooled to 40°C for 5 min, and then slowly increased to 80°C at a rate of 0.05°C per step, while collecting fluorescence simultaneously; the melting curve is plotted with the first negative derivative of fluorescence signal intensity with respect to temperature as the ordinate (-Rn') and temperature as the abscissa.

[0026] In this invention, DNA extraction can be easily performed by those skilled in the art by referring to existing publicly available technical methods or using commercially available kits.

[0027] In this invention, the DNA extraction sample can be fresh meat or processed meat products.

[0028] The fifth technical solution provided by the present invention is the application of the composition described in the first technical solution, the detection system described in the second technical solution, the reagent kit described in the third technical solution, or the method described in the fourth technical solution in the detection of animal-derived components.

[0029] In some embodiments, the animal-derived ingredients are derived from pigs, cattle, sheep, chickens, ducks, geese, pigeons, sika deer, camels, donkeys, and foxes.

[0030] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a rapid detection method based on a multi-species universal primer and molecular beacon probe composition. By combining real-time fluorescence PCR technology with melting curve analysis, it achieves efficient identification of 11 common animal-derived components in samples. The innovation of this technology is mainly reflected in two aspects: the design of universal primers and the optimization of the mismatch resolution capability of molecular beacon probes.

[0031] In the mitochondrial genome, the 12S rRNA gene, due to its sequence conservation and species-specific sites, is an ideal detection target. This invention systematically analyzes the 12S rRNA gene sequences of 11 target species and carefully designs universal primers and specific molecular beacon probes with broad-spectrum amplification capabilities. To ensure detection reliability, this method specifically constructs a highly efficient internal standard system, which can effectively eliminate interference from PCR inhibitors in the sample and significantly improve the accuracy of the detection results. This detection system includes a TaqMan fluorescent probe for monitoring the amplification process, an internal standard probe using a ROX fluorescent reporter group, and universal primers optimized to a length of 100-150 bp to ensure amplification efficiency. Technically, the significant differences in Tm values ​​exhibited after hybridization of different species-specific molecular beacon probes with the target sequence form characteristic melting peaks in the melting curve spectrum, thereby achieving accurate species identification.

[0032] In summary, the advantages of this invention are as follows: 1. A single pair of universal primers can cover the 12S rDNA sequences of all 11 target species, greatly simplifying experimental procedures and significantly improving detection throughput.

[0033] 2. Molecular beacon probe technology has excellent detection sensitivity and specificity, and combined with single-tube closed operation, it effectively avoids exogenous contamination.

[0034] 3. The detection process of this invention is simple and rapid, cost-effective, and easily automated, making it particularly suitable for high-throughput screening of large-scale samples. It can accurately detect the source of ingredients in various animal-derived foods, including processed foods such as meat products and dairy products. Attached Figure Description

[0035] Figure 1 The primers and probes designed for this invention were analyzed using electrophoresis, qPCR, and melting curve results. Figure 1 A represents the primer electrophoresis result. Figure 1 B represents the probe detection result. Figure 1 C represents the probe melting curve.

[0036] Figure 2 The results show the optimized detection system of the reagent kit designed in this invention. Figure 2 A represents the effect of different temperatures on the probe and primers. Figure 2 B represents the effect of probe concentration on the kit. Figure 2 C represents the effect of primer concentration on the reagent kit.

[0037] Figure 3 Results of real-time fluorescence PCR amplification and melting curve analysis of serially diluted sheep, chicken, cattle, pig, and fox DNA templates. Figure 3The components are, in order: A: sheep-derived components, B: chicken-derived components, C: bovine-derived components, D: pig-derived components, and E: fox-derived components.

[0038] Figure 4 The results of melting curve analysis for different types of matrices. Figure 4 A represents the melting curves of goose-derived components from different matrices. Figure 4 B represents the melting curves of bovine-derived components from different matrices. Figure 4 C represents the melting curves of duck-derived components in different matrices. Figure 4 D represents the melting curves of porcine components in different matrices.

[0039] Figure 5 This is a graph showing the results of the melting curve analysis of the mixed sample.

[0040] The attached image includes both Chinese and English text. Derivative Reporter (-Rn'): The value of the negative first derivative; Temperature; Cycle: Number of iterations; Melt Curve Plot: Melting curve; Amplification Plot: Amplification curve. Detailed Implementation

[0041] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0042] Materials used in the examples: 1. The samples used in the following examples include fresh pork, beef, mutton, chicken, duck, goose, pigeon, donkey meat, and fox, sika deer, and camel standard products purchased from the national quality control platform. The samples were sourced from supermarkets, online platforms, and the national quality control platform, and all underwent identification and sequence alignment analysis using standard methods such as "Real-time Fluorescent PCR Method for Detection of Common Animal-Derived Components" (GB / T38164-2019), ensuring a clear species origin.

[0043] 2. The food genome extraction kits and probe-based qPCR detection reagents used in the following examples are all commercially available universal kits; the fluorescence quantitative PCR instrument used is the AB ViiA™ 7 device manufactured by Thermo Fisher Scientific.

[0044] 3. The universal primers and molecular beacon probe combinations used in the following examples were synthesized by a professional biotechnology company on commission.

[0045] Example 1: Design and Optimization of Universal Primer and Molecular Beacon Compositions 1. Mitochondrial genome sequences of 11 livestock and poultry species identifiable by the kit described in this invention were downloaded from the National Center for Biotechnology Information (NCBI) Genome Database. These sequences serve as a reference for universal primer, molecular beacon probe design, and sample species identification, specifically including the following species and their corresponding sequence numbers: pig (… Sus scrofa KP126954), scalpers ( Bos taurus DQ124399), sheep ( Ovis aries KU575248), Chicken ( Gallus gallus KM433666), Duck ( Anas platyrhynchos EU755252), Goose ( Anser sp. MK102803), Pigeon ( Columba livia KP258178), Sika deer ( Cervus nippon JN389443), Camel ( Camelus bactrianus MH109974), donkey ( Equus asinus MG931481) and the fox ( Vulpes vulpes KF387633).

[0046] 2. Based on the collected mitochondrial gene sequences of 11 target species, target gene fragments (100-150 bp) with typical "conserved region-hypervariable region-conserved region" characteristics were screened through multiple sequence alignment. These fragments exhibited high conservation within species (>95%), while also showing significant specific differences between species (containing ≥2 SNP sites). Universal primers were designed using professional software such as Primer Premier, and their specificity was verified using NCBI BLAST. Molecular beacon probes were designed using Mfold and UNAFold software for secondary structure prediction and Tm value analysis, optimizing probe length (15-30 bp) and fluorescent label combinations (FAM / HEX / CY5) to ensure that the Tm value difference of probes for common livestock and poultry species was ≥1℃, and the Tm value difference of probes for special species was ≥0.5℃. The final primer-probe combinations obtained through experiments are as follows: The main types of universal primers and molecular beacon probes designed for upstream and downstream base pair similarity fragments of various species, and those that meet the requirements of target gene sequences, are as follows: The target sequence is the nt610-nt787 fragment (reference sequence AF492351): Upstream primer (FP): 5'-CCAAACTGGGATTAGATACC-3'; Downstream primer (RP): 5'-GAACAGGCTCCTCTAGGT-3'; Probe: 5'-CGTCGAGCCCTAAACCCAGATGGTTATCGACG-3', with the 5' end labeled as a FAM fluorescent generating group and the 3' end labeled as a DABCYL quenching group; The hairpin structure and dimer content of the probe were predicted and evaluated using the online programs Mfold and UNAFold to detect its effectiveness and optimize it.

[0047] (3) Preferred universal primer and molecular beacon probe composition Species DNA samples were serially diluted and then tested according to the real-time quantitative PCR system and reaction conditions specified in "Real-time Fluorescent PCR Method for Detection of Common Animal Derivative Components in Livestock and Poultry" (GB / T 38164-2019). After detection, standard curves were plotted with the logarithm of the initial template copy number as the x-axis and the corresponding Ct value as the y-axis. The correlation coefficient (R²) of the standard curves was calculated. 2 The linear relationship is evaluated using the value, and the formula E=

[10] is used to determine the linear relationship. ( 1 / K) 1]×100%, calculate the amplification efficiency (E). The results are shown in Table 1.

[0048] Table 1. Standard curves and amplification efficiencies of universal primers for each species

[0049] Electrophoretic amplification and qPCR results are as follows Figure 1 As shown in Figure A, the universal primers designed in this invention exhibited stable amplification efficiencies (85.7%-93.4%) in 11 species, including cattle, sheep, pigs, chickens, ducks, geese, pigeons, deer, camels, donkeys, and foxes. The R² values ​​of the standard curves for each species were all above 0.988, indicating good linearity in amplification. Specifically, the amplification efficiencies for cattle, sheep, pigs, deer, camels, donkeys, and foxes all exceeded 90%, while the efficiencies for poultry (chickens, ducks, geese, and pigeons) were slightly lower but still better than 85%, meeting the requirements of qPCR technology. Electrophoresis and qPCR results further validated the balanced recognition ability of the universal primers for target genes, achieving the design expectations and making them suitable for simultaneous detection in multiple species.

[0050] Molecular beacon probes were designed based on the target sequences of the primer pairs. The predicted Tm values ​​after bioinformatics analysis are shown in Table 2. Table 2 Predictions Tm value

[0051] To achieve effective identification of 11 livestock and poultry species, the hybridization molecules between the molecular beacon probe and the genomic DNA of different livestock and poultry species should have significantly different Tm values, and these differences in Tm values ​​should meet the resolution requirements of conventional fluorescent PCR instruments. As shown in Table 2, after the probe hybridizes with the target genes of different species, the Tm values ​​of various products differ, demonstrating discriminative power and making them suitable as universal molecular beacon probes.

[0052] Based on the above analysis results, it was confirmed that the upstream primer FP and downstream primer RP (nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively), as well as the probe (nucleotide sequence is shown in SEQ ID NO.3), met the expectations and were therefore selected as the detection composition for the kit.

[0053] Animal-derived components were detected using a combination of screened universal primer pairs and molecular beacon probes. Real-time fluorescence PCR combined with melting curve analysis was used to detect 11 kinds of fresh meat samples from livestock and poultry.

[0054] The specific testing steps are as follows: (1) Sample DNA extraction: The sample to be tested was ground using a homogenizer, and 200 mg of the well-mixed sample was taken to extract DNA using a commercial food gene extraction kit. After extraction, the concentration and purity of the extracted DNA sample were determined using a micro-ultraviolet spectrophotometer to ensure the accuracy of subsequent experiments.

[0055] (2) Real-time fluorescence PCR: Take 1 μL of the extracted DNA solution for detection. The composition of the reaction system is shown in Table 3: Table 3 Reaction System

[0056] The reaction conditions for real-time fluorescence PCR detection were: 95℃ pre-denaturation for 30s, 95℃ denaturation for 15s, 60℃ annealing for 20s and acquisition of fluorescence signal, and 74℃ extension for 10s, for a total of 45 cycles.

[0057] (3) Melting curve analysis: After PCR amplification, the temperature was raised to 95℃ and held for 1 minute, then lowered to 40℃ and held for 5 minutes. The temperature was then slowly increased to 80℃ at a rate of 0.05℃ per step, while simultaneously collecting fluorescence signals. The melting curve was plotted with the first negative derivative of the fluorescence signal intensity with respect to temperature (-Rn') as the ordinate and temperature as the abscissa. The highest temperature corresponding to the peak of the melting curve is the Tm value of the sample.

[0058] Test results as follows Figure 1As shown, real-time fluorescent PCR was used to detect the source components of 11 livestock and poultry species using molecular beacon probes. The results showed that all 11 livestock and poultry species exhibited clear amplification curves and Ct values ​​< 35 (see [link to study]. Figure 1 (B) demonstrates the effectiveness of the designed molecular beacon probe in the amplification reaction. However, due to the large number of target species and the limited design length of the molecular beacon probe, the specificity between species is slightly inferior to that of a single probe. Further analysis of the melting curves of the molecular beacon probe revealed that each sample exhibited a single and independent melting peak (see [link to analysis]). Figure 1 C) shows that the probe has a certain ability to distinguish between different species.

[0059] The measured melting temperatures (Tm values) for each species were as follows: pig (51.8℃), cattle (54.7℃), sheep (49.6℃), chicken (61.5℃), duck (60.4℃), goose (58.1℃), fox (55.9℃), pigeon (58.8℃), deer (56.6℃), camel (55.1℃), and donkey (57.7℃). Although there are some differences between the measured results and the predicted values, the differences in Tm values ​​among different species meet the resolution requirements of PCR detection, thus enabling effective identification of 11 livestock and poultry species.

[0060] Example 2: Optimization Experiment of Reagent Kit Detection Conditions and Dosage To improve the detection sensitivity, specificity and stability of the kit, the core components and parameters detected in Example 1 were systematically optimized, including the reaction system and reaction conditions.

[0061] For each component, the following conditions were set for optimization: primer / probe concentrations: 0.1, 0.2, 0.4, 0.8 μL; annealing temperatures: 50°C, 55°C, 60°C, 65°C. Different combinations of these conditions were used in the experiments.

[0062] The optimized result is as follows Figure 2 As shown in Table 4, the kit performed optimally when the primer and probe concentrations were 0.4 μL and the temperature was 60℃. This combination also yielded the best results in terms of both cost and effectiveness, meeting experimental expectations. The kit's detection system conditions and dosages are shown in Table 4. Table 4 Reaction System Conditions

[0063] Example 3: Sensitivity and Specificity Experiment This embodiment selects sheep ( Ovis aries ),chicken( Gallus gallus ),ox( Bos taurus ),pig( Sus scrofa domesticus ) and fox ( Vulpes vulpes The source component was the target for detection. A series of concentration standards were prepared using a tenfold serial dilution method, with a concentration gradient range of 10. 1 -10 5 copies / μL (corresponding to a dilution of 10) -1 -10 -5 ).

[0064] The total volume of the reaction system was 20 μL. The real-time fluorescence PCR detection reaction conditions were: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 15 s, 60℃ annealing for 20 s and acquisition of fluorescence signal, and 74℃ extension for 10 s, for a total of 45 cycles.

[0065] Test results as follows Figure 3 As shown, the amplification curves of the sheep, chicken, cattle, pig, and fox DNA templates all exhibited good linearity (R² > 0.98), indicating that the universal primer and molecular beacon probe combination of this invention has excellent quantitative performance over a wide concentration range. The Ct values ​​of all detected species were stably distributed in the range of 17–32 (standard deviation SD < 1.5), meeting the requirements of the detection technology.

[0066] When the Ct value of the real-time fluorescence PCR amplification curve exceeds 35, it may reflect one of the following situations: the sample does not contain any of the 11 animal-derived components, the target DNA content is extremely low, or there is a problem with the quality of sample DNA extraction. In this case, further validation experiments are needed to confirm the results. Although there is interspecies variation in fluorescence intensity due to differences in probe binding sites among different species (ΔRFU = 15–20%), all detection signals are significantly higher than the background value. P <0.01), meeting the testing requirements.

[0067] Sensitivity and stability verification, such as Figure 3 As shown in A, 3C, and 3D, the detection results for sheep, cattle, and pig components indicate that under gradient dilution conditions, the probe sensitivity is not affected, and the amplification efficiency is consistently above 90%, meeting international standards. The limit of detection (LOD) reaches 10 copies / μL, demonstrating high sensitivity. The intra- and inter-batch coefficients of variation (CV) are both <5%, proving the method has good repeatability and stability. These data fully demonstrate that the molecular beacon probe system of this invention possesses: 1. Excellent concentration dependence (R² > 0.98); 2. High detection sensitivity (≤10 copies / μL); 3. Good stability (CV < 5%).

[0068] Example 4: Detection of animal-derived components by different processing methods This embodiment selects five representative samples from three categories: fresh animal tissues (muscle tissues of pigs, cattle, geese, and ducks), processed food products (canned meat, sausages, meat floss, etc.), and animal blood products (plasma protein powder, blood tofu, etc.). To comprehensively evaluate the applicability of the detection system, samples were subjected to different treatments to simulate actual processing conditions, including 121℃ moist heat sterilization for 30 minutes, soaking in pH 2.5 acidic solution for 24 hours, and high-speed homogenization at 10,000 rpm for 5 minutes. Sample DNA was extracted using a commercially available food gene extraction kit, and DNA quality was strictly controlled using ultraviolet spectrophotometry (A260 / A280 ratio 1.7-2.0). Real-time fluorescence PCR detection and melting curve analysis were performed. The detection aims to evaluate the identification effect of the universal primers and molecular beacon probes in this invention on animal-derived components under different processing conditions. The experiment was repeated three times to ensure good repeatability and reliability of the results.

[0069] Real-time fluorescence PCR and melting curve analysis results showed that the molecular beacon probe system designed in this invention exhibits significant stability. Figure 4 As shown, all positive samples from different matrix-derived components exhibited a single characteristic melting peak, with Tm values ​​remaining consistently within a similar range. The peaks were sharp and the half-maximum width was less than 1.5℃. Repeatability verification showed that the CV value for intra-batch repeatability (n=3) was less than 3%, the CV value for inter-batch repeatability (n=3) was less than 5%, and the standard deviation of Tm values ​​was less than 0.3℃, fully demonstrating the reliability of the detection method. This result indicates that the molecular beacon probe of this invention has good adaptability to animal-derived components under complex processing conditions, and its detection sensitivity and specificity are not significantly reduced due to DNA degradation. Furthermore, the probe can accurately distinguish animal-derived components processed by different techniques such as high-temperature treatment and pickling, showing its potential for widespread application in commercially available foods. These characteristics make this technology particularly suitable for applications such as the identification of animal-derived components in processed foods, identification of food adulteration, halal food certification, and import / export food quarantine, providing strong technical support for food safety supervision.

[0070] Example 5: Detection of Mixed Samples Fresh meat samples containing common animal-derived components, such as beef and duck, were selected. DNA was extracted from each sample, and its species origin was determined through sequence identification and comparative analysis. Subsequently, the extracted DNA was mixed in different proportions to simulate the coexistence of multiple animal-derived components commonly found in food. The mixing ratios were set to the actual content ranges in common foods (e.g., 90:10, 80:20, 30:70, 60:40, etc.). To further investigate the impact of processing technology on the detection results, the mixed samples were subjected to high-temperature cooking with the addition of common additives (nitrite, carmine), salt, and monosodium glutamate to simulate common meat product processing procedures. After freezing the mixed samples in liquid nitrogen, they were ground, and the samples were homogenized after thorough mixing.

[0071] DNA was extracted from the mixed samples using a food genomics extraction kit, ensuring the extracted DNA concentration and purity met experimental requirements. A 20 μL real-time fluorescence PCR reaction system was prepared, consisting of universal primers, molecular beacon probes, and an appropriate amount of mixed sample DNA template. To ensure the reliability of the experimental results, a positive control (single-component sample) and a negative control (without DNA template) were included during the detection process. In real-time fluorescence PCR detection, fluorescence amplification curve data of the mixed samples were acquired, and the Ct value corresponding to each animal-derived component was recorded. By comparing the melting peaks and Tm values ​​of the mixed samples and single-component samples, the detection characteristics and distinguishing effects of different animal-derived components in the mixed state were analyzed, providing data support for the component identification of complex foods.

[0072] Depend on Figure 5 It is evident that the identification results of animal-derived components may deviate somewhat from those of single-component detection. However, the temperature differences in the melting peaks of different species are significant, exceeding the potential overlap in detection intervals. This difference is sufficient to ensure accurate identification of each species in the mixed sample.

[0073] Example 6: Blind Sample Simulation Detection Thirty samples were selected from supermarkets, farmers' markets, and online shopping, and qPCR tests were performed on the selected samples to confirm the animal-derived components. The samples were then numbered. The samples were pre-processed into powder form, and a sampling method was used to select samples, which were then numbered A1-A10. The true composition is unknown.

[0074] Weigh 50 mg of blind samples (A1-A10) into a 1.8 mL centrifuge tube for DNA extraction. The real-time fluorescence PCR reaction conditions were as follows: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 15 s, 60℃ annealing for 20 s with fluorescence signal acquisition, and 74℃ extension for 10 s, for a total of 45 cycles. After PCR amplification, the temperature was increased to 95℃ and held for 1 minute, then decreased to 40℃ and held for 5 minutes. The temperature was then slowly increased to 80℃ at a rate of 0.05℃ per step, with simultaneous fluorescence signal acquisition. A melting curve was plotted with the first negative derivative of the fluorescence signal intensity with respect to temperature (-Rn') as the ordinate and temperature as the abscissa. The highest temperature corresponding to the peak of the melting curve is the Tm value of the sample.

[0075] Table 5 Blind Sample Test Results

[0076] The results are shown in Table 5. The kit achieved an accuracy of up to 90.0% in detecting unknown samples. However, other components present in small amounts in the samples could not be effectively identified because the melting curves did not show obvious peaks. Even with complex processing conditions used in the blind sample selection, the kit was still able to effectively identify the components in the sample.

[0077] This indicates that the molecular beacon probe of the present invention can effectively identify different animal-derived components in mixed samples, and its sensitivity and specificity remain good even when multiple components coexist. Since the difference in melting temperature mainly depends on the base pairing stability between the probe and the target DNA fragment, the melting curves of each species have good reproducibility, providing a clear basis for species identification in mixed sample detection.

[0078] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A composition for detecting animal-derived components, characterized in that, The composition comprises a universal primer pair and a molecular beacon probe, the nucleotide sequences of the universal primers being shown in SEQ ID NO.1 and EQ ID NO.2, respectively, and the nucleotide sequence of the molecular beacon probe being shown in SEQ ID NO.

3.

2. The composition according to claim 1, characterized in that, The molecular beacon probe has functional groups or molecules attached to its 5' and 3' ends. These functional groups or molecules may be fluorescent markers, chemiluminescent markers, bioluminescent markers, biotin, affinity ligands, and / or thiol groups.

3. The composition according to claim 1 or 2, characterized in that, The molecular beacon probe has a FAM fluorescence generating group attached to its 5' end and a DABCYL quenching group attached to its 3' end.

4. A detection system for universal screening of multiple animal-derived components, characterized in that, The detection system contains the composition for detecting animal-derived components as described in any one of claims 1 to 3, wherein the animal-derived components are derived from pigs, cattle, sheep, chickens, ducks, geese, pigeons, sika deer, camels, donkeys, and foxes.

5. The detection system according to claim 4, characterized in that, The final concentrations of the universal upstream and downstream primers in the detection system are 0.1 μM-1.0 μM, and the final concentrations of the molecular beacon in the reaction system are 0.05 μM-1.0 μM.

6. The detection system according to claim 4, characterized in that, The detection system also includes a positive control, qPCR reaction reagent, ROX reference dye and ultrapure water. The positive control is the animal-derived component from pigs, cattle, sheep, chickens, ducks, geese, pigeons, sika deer, camels, donkeys and foxes, with a positive control concentration of 100 ng / µL.

7. A universal screening kit for multiple animal-derived components, characterized in that, The kit contains the composition for detecting animal-derived components as described in any one of claims 1 to 3 or the detection system as described in any one of claims 4 to 6, wherein the animal-derived components may be derived from pigs, cattle, sheep, chickens, ducks, geese, pigeons, sika deer, camels, donkeys, and foxes.

8. A method for detecting animal-derived components based on molecular beacon melting curve analysis technology, characterized in that, Includes the following steps: (1) Extract genomic DNA from the unknown sample to be tested and obtain DNA extract solution; (2) Real-time fluorescence PCR amplification and melting curve detection of the extracted DNA sample using the composition according to any one of claims 1 to 3; (3) Perform melting curve analysis on the amplification products, establish a species melting curve Tm value model, and use the melting curve peak value to identify animal-derived components in the sample; The animal-derived components may be derived from pigs, cattle, sheep, chickens, ducks, geese, pigeons, sika deer, camels, donkeys, and foxes.

9. The method according to claim 8, characterized in that, In step (2), the concentration of the molecular beacon probe used is 5-20 pmol; the concentration of the universal upstream primer and the universal downstream primer used is 10-30 pmol; the amplification program of real-time fluorescence PCR is 95℃ pre-denaturation for 30s, 95℃ denaturation for 15s, 60℃ annealing for 20s and collection of fluorescence signal, 74℃ extension for 10s, for a total of 45 cycles; In step (3), the melting curve analysis procedure is as follows: after the PCR amplification reaction is completed, the reaction system is heated to 95℃ for 1 min, cooled to 40℃ for 5 min, and then slowly increased to 80℃ at a rate of 0.05℃ per step, while collecting fluorescence simultaneously. Plot the melting curve with the first negative derivative of fluorescence signal intensity with respect to temperature as the ordinate (-Rn') and temperature as the abscissa.

10. The application of the composition according to any one of claims 1 to 3, or the detection system according to any one of claims 4 to 6, or the kit according to claim 7, or the method according to any one of claims 8 to 9 in the detection of animal-derived components, characterized in that, The animal-derived ingredients are derived from pigs, cattle, sheep, chickens, ducks, geese, pigeons, sika deer, camels, donkeys, and foxes.

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