Primer pair, kit and detection method for detecting horse meat component in food

Abstract

The application belongs to the technical field of detecting meat components based on nucleic acid technology, and particularly relates to a primer pair, a kit and a detection method for detecting horse meat components in food. The sequences of the primer pair are shown in SEQ ID NO. 1 and SEQ ID NO. 2. The application further discloses a kit for detecting horse meat components in food and a method for detecting horse meat components. The detection method comprises a pair of primers labeled with 6-FAM fluorescent groups and biotin, can realize high-sensitivity target detection, and can detect as low as 0.1% horse meat in adulterated meat samples. Compared with recombinant enzyme polymerase isothermal amplification real-time fluorescence detection, there is no need to design and screen RPA fluorescent probes. In addition, the application has good application prospect and can be successfully used for detecting horse meat components in donkey meat products, beef products, mutton products and venison products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of detecting meat components based on nucleic acid technology, and particularly relates to a primer pair, a kit and a detection method for detecting horse meat components in food. BACKGROUND

[0002] With the rapid growth of meat consumption, meat adulteration and meat product fraud have become a major problem in the food industry. It is a common practice to replace expensive meat with cheap or unpopular meat. Therefore, in order to effectively avoid the occurrence of adulteration events, maintain the interests of consumers and market integrity, it is necessary to establish a simple, accurate and efficient method to identify horse meat components in adulterated meat.

[0003] Currently, nucleic acid-based detection technology is the mainstream method for detecting animal-derived components. This technology can amplify specific nucleic acid sequences, thereby achieving detection of target sequences. Nucleic acids are widely present in all animal and plant cells and microorganisms, and have relative stability. Polymerase chain reaction (PCR) detection method is the gold standard due to its high sensitivity and specificity, but its disadvantage is time-consuming procedure and requires complex instruments. In contrast, isothermal amplification techniques are promising alternatives. These techniques are simple to operate and do not require a temperature-shifting system for amplification. Among these techniques, recombinase polymerase amplification (RPA) detection is particularly attractive. RPA utilizes three core enzymes: recombinase, single-stranded DNA binding protein (SSB), and strand displacement polymerase. The optimal working temperature of RPA is 37-42℃, and the amplification time is short, making it an ideal choice for rapid identification of species.

[0004] Many studies have explored the application of RPA technology, particularly in meat adulteration detection, providing sufficient data support for its development and application. For example, Zhou et al. developed a duck-specific RPA-based detection method for rapid detection of duck components in animal-derived food. They successfully developed real-time RPA and RPA combined with lateral flow strips (RPA-LFS) detection methods, which can rapidly detect duck meat at temperatures of 39℃ and 40℃ within 20 min, respectively. The minimum detectable duck component in duck and sheep meat powder was 0.1% (weight ratio). Liu et al. developed two RPA-based rapid molecular detection methods that can detect pork and horse meat in 6 to 11 min. These detection methods have high specificity and can identify as little as 0.1% of the target DNA in meat mixtures. In addition, Liu et al. also developed an RPA-CRISPR-Cas12a detection method that can detect the target gene by fluorescence intensity. The RPA-Cas12a-FS system can specifically detect as little as 10 copies of the target gene in 45 min at 37℃. Cao et al. developed a RPA combined with SYBR Green I visual identification method for meat adulteration. This technology can visually detect specific meat in 30 min at 37℃, with a minimum detection rate of 1% for pork adulteration. This technology can be used for samples that are boiled, microwaved, high- pressure or fried. Although these techniques are effective, there are still some drawbacks. Gel electrophoresis requires complex instruments and operations, which prolongs the detection time. Visual detection is subjective and prone to false positive results. LFS and real-time fluorescence detection require special probes, which usually require a lot of synthesis and screening, and are costly and time-consuming. Real-time fluorescence detection also relies on special instruments for real-time monitoring of fluorescence. Therefore, RPA can be quantified by end-point fluorescence detection.

[0005] Magnetic beads (MB) have the advantages of fast and simple magnetic separation, high surface-to-volume ratio, and the ability to bind multiple functional groups, and have been widely used in the separation and detection of nucleic acids (including amplicons). Streptavidin-coated magnetic beads (MB@SA) can effectively bind biotinylated capture probes, making it easy to capture DNA directly from crude samples for detection by real-time quantitative PCR (qPCR). Similarly, MB can also capture the generated amplicons after hybridization with sDNA probes immobilized on magnetic beads. In addition, amplicons can also be detected using biotin-labeled primers. MB@SA is commonly used to enrich, capture and detect biotinylated nucleic acid amplicons generated by various amplification techniques.

[0006] Therefore, there is an urgent need to provide a simple method for detecting horse meat in food. SUMMARY

[0007] In view of the defects and deficiencies in the prior art, the present application provides a primer pair, a kit and a detection method for detecting horse meat components in food.

[0008] To achieve the above technical effects, the present application adopts the following technical solutions.

[0009] The present application provides a primer pair for detecting horse meat components in food, the sequence of the primer pair is shown in SEQ ID NO. 1 and SEQ ID NO. 2.

[0010] In some technical solutions of the present application, the 5' end of the primer shown in SEQ ID NO. 1 is labeled with a 6-FAM fluorescent group; and the 5' end of the primer shown in SEQ ID NO. 2 is labeled with biotin.

[0011] In the present application, the primer pair is designed according to the ATP6-8 gene of equine mitochondrial DNA.

[0012] The present application also provides a kit for detecting horse meat components in food, comprising the primer pair.

[0013] The present application also provides a method for detecting horse meat components in food, comprising: using the primer pair or the kit.

[0014] In some technical solutions of the present application, the method comprises:

[0015] S1, extracting DNA in a sample to be tested to obtain sample genomic DNA;

[0016] S2, using the primer pair to perform RPA amplification on the sample genomic DNA to obtain an amplification product;

[0017] S3, using streptavidin-modified magnetic beads to separate and purify the amplification product, and then using a high-throughput fluorescence detection system to identify and quantify the amplification product; if the fluorescence signal value exceeds a threshold value, it indicates that the amplification product contains horse meat components.

[0018] In some technical solutions of the present application, in S1, after the sample to be tested is lysed by a lysis buffer, the extraction device is placed in the lysis buffer containing the sample, and then the extraction device is washed to remove attached proteins and other substances; after washing, the extraction device is placed in an elution buffer to release the extracted sample genomic DNA;

[0019] The extraction device comprises a paraformaldehyde plate on which a plurality of UiO-66SPME devices are arranged, and each two of the UiO-66SPME devices are parallel to each other.

[0020] The UiO-66SPME device comprises an extraction rod, and a nitrocellulose membrane coated with UiO-66-NH2 is adhered to the extraction rod.

[0021] In some technical solutions of the present application, in S1, a 96-well high-throughput DNA extraction system is used, which is composed of 96 UiO-66SPME devices and a paraformaldehyde plate, and DNA is separated through three steps: lysis, washing, and elution, and the extraction time of each sample is less than 7.2 seconds.

[0022] In some technical solutions of the present application, the lysis solution comprises: 95-105 mM Tris-HCl, 4.5-5.5 mM EDTA, 195-205 mM NaCl, and 0.8-1.2 % SDS, with a pH of 7.5-8.5.

[0023] In some technical solutions of the present application, the elution solution is 8-12 mM Tris.

[0024] In some technical solutions of the present application, in S2, the RPA amplification comprises: adding primer-free rehydration buffer, forward primer and reverse primer, sample genomic DNA, and ddH2O into a reaction tube in which recombinase and polymerase freeze-dried powder are premixed, mixing uniformly, then adding magnesium acetate, mixing uniformly, and then amplifying at a temperature of 37-41 DEG C for 15-20 min.

[0025] The addition amount of the forward primer and the reverse primer is 2.8-3.2 muL respectively, and the addition amount of the magnesium acetate is 2.0-2.6 muL.

[0026] In some technical solutions of the present application, in S3, the threshold value is 834, and when the fluorescence signal value is greater than 834, it indicates that the amplification product contains horse meat components.

[0027] In some technical solutions of the present application, in S3, the volume ratio of the streptavidin-modified magnetic beads to the amplification product is 2-3:1-2.

[0028] In S3, the detection step of magnetic separation and fluorescence detection comprises:

[0029] First, 100 muL of MB@SA solution is taken, and washed with 1xTBS buffer three times. After magnetic separation (MS) and careful decanting of the supernatant, the washed beads are dispersed in 200 muL of 2xBinding&Washing (B&W) buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2 M NaCl, 0.01 % Tween-20) and stored at -4 DEG C for standby.

[0030] Then, 15 muL of amplification product and 10 muL of dispersed MB@SA mixture are placed in a centrifuge tube, the mixture is vortexed and incubated at room temperature for 5 min. The centrifuge tube is separated on a magnetic stand, the supernatant is removed, and then washed with 1xB&W buffer three times. Finally, the washed magnetic bead complex is resuspended in 200 muL of ultrapure water.

[0031] The mixed solution was directly transferred to a polystyrene black 96-well enzyme-coated plate. End-point fluorescence detection (FD) was performed by detecting the fluorescence signal through a multifunctional enzyme marker (TECAN Infinite200 PRO).

[0032] In the present application, beef products, donkey meat products, deer meat products and mutton products were purchased respectively, and the above-mentioned RPA-MS-FD system was used for qualitative and quantitative analysis of 21 kinds of processed meat products. Then the accuracy of the results was verified by using the Chinese industry standard "Identification of Livestock Components in Foods and Feeds Part 5: Detection of Horse Meat Components-Real-time PCR Method" (SN / T3730.5-2013).

[0033] Compared with the prior art, the present application has the following beneficial effects:

[0034] (1) The present application only needs a pair of labeled primers to perform RPA amplification in the reaction system to detect fluorescence. Compared with RPA real-time fluorescence detection, the design and screening process of special fluorescent probes is omitted.

[0035] (2) The present application adopts a 96-well high-throughput DNA extraction method for DNA separation and purification. Then the separated DNA is amplified by RPA technology, and the amplicon is separated by magnetic separation (MS) strategy. This detection method only needs a pair of primers labeled with 6-FAM fluorescent groups and biotin. Finally, the fluorescence intensity is measured by an enzyme marker to qualitatively and quantitatively analyze the DNA amplicon, which provides a high-throughput, rapid and accurate method for detecting horse meat adulteration. The DNA separation method provided by the present application can purify DNA for subsequent multiplex PCR analysis. In addition, a high-throughput DNA separation method based on magnetic ionic liquid is also provided, and then real-time PCR analysis is performed. These methods save the time-consuming and laborious sample preparation steps, thereby saving the time of analysts and improving the overall analysis efficiency of researchers.

[0036] (3) At present, high-throughput sample preparation strategies have been widely used in clinical, pharmaceutical, food and environmental science fields. These strategies improve sample throughput and preparation efficiency, which helps to quickly analyze complex samples. In the present application, based on the newly developed 96-SPME extraction device, the extraction time of each sample is less than 7.2 seconds using the extraction device.

[0037] (4) High efficiency. The present application is combined with a 96 high-throughput DNA separation system, which can analyze 96 meat samples in 31.5 min. This includes 11.5 min of rapid DNA extraction, 15 min of RPA amplification and 5 min of fluorescence detection. The time-consuming and laborious sample preparation steps are omitted, thereby saving the time of analysts and improving the overall analysis efficiency of researchers.

[0038] (5) High sensitivity. The present application can detect as little as 0.1% horse meat in adulterated meat samples.

[0039] (6) High specificity. The present application can highly specifically recognize horse meat, and no positive amplification was observed in the DNA of other 11 kinds of meat.

[0040] (7) Good practical application. The present application is successfully used to analyze 21 kinds of commercial meat products. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 Figure 1 is a diagram of agarose gel electrophoresis results of RPA amplification of 12 species of meat product genomic DNA by 5 pairs of primers, wherein a-e represent electrophoresis results of target fragments amplified by primer sets with sizes of 114bp, 166bp, 170bp, 152bp, and 276bp, respectively, and lanes 1-11 are genomic DNA of horse, chicken, duck, goose, turkey, pig, cow, sheep, camel, rabbit, and no template control (NTC), respectively. Figure 1 Figure 1 is a diagram of agarose gel electrophoresis results of RPA amplification of 12 species of meat product genomic DNA by 5 pairs of primers, wherein a-e represent electrophoresis results of target fragments amplified by primer sets with sizes of 114bp, 166bp, 170bp, 152bp, and 276bp, respectively, and lanes 1-11 are genomic DNA of horse, chicken, duck, goose, turkey, pig, cow, sheep, camel, rabbit, and no template control (NTC), respectively. Figure 1 Figure 1 is a diagram of agarose gel electrophoresis results of RPA amplification of 12 species of meat product genomic DNA by 5 pairs of primers, wherein a-e represent electrophoresis results of target fragments amplified by primer sets with sizes of 114bp, 166bp, 170bp, 152bp, and 276bp, respectively, and lanes 1-11 are genomic DNA of horse, chicken, duck, goose, turkey, pig, cow, sheep, camel, rabbit, and no template control (NTC), respectively.

[0042] Figure 2 Figure 1 is a diagram of agarose gel electrophoresis results of RPA amplification of 12 species of meat product genomic DNA by 5 pairs of primers, wherein a-e represent electrophoresis results of target fragments amplified by primer sets with sizes of 114bp, 166bp, 170bp, 152bp, and 276bp, respectively, and lanes 1-11 are genomic DNA of horse, chicken, duck, goose, turkey, pig, cow, sheep, camel, rabbit, and no template control (NTC), respectively.

[0043] Figure 3 Figure 1 is a diagram of agarose gel electrophoresis results of RPA amplification of 12 species of meat product genomic DNA by 5 pairs of primers, wherein a-e represent electrophoresis results of target fragments amplified by primer sets with sizes of 114bp, 166bp, 170bp, 152bp, and 276bp, respectively, and lanes 1-11 are genomic DNA of horse, chicken, duck, goose, turkey, pig, cow, sheep, camel, rabbit, and no template control (NTC), respectively.

[0044] Figure 4 Figure 1 is a diagram of agarose gel electrophoresis results of RPA amplification of 12 species of meat product genomic DNA by 5 pairs of primers, wherein a-e represent electrophoresis results of target fragments amplified by primer sets with sizes of 114bp, 166bp, 170bp, 152bp, and 276bp, respectively, and lanes 1-11 are genomic DNA of horse, chicken, duck, goose, turkey, pig, cow, sheep, camel, rabbit, and no template control (NTC), respectively.

[0045] Figure 5 Figure 1 is a diagram of agarose gel electrophoresis results of RPA amplification of 12 species of meat product genomic DNA by 5 pairs of primers, wherein a-e represent electrophoresis results of target fragments amplified by primer sets with sizes of 114bp, 166bp, 170bp, 152bp, and 276bp, respectively, and lanes 1-11 are genomic DNA of horse, chicken, duck, goose, turkey, pig, cow, sheep, camel, rabbit, and no template control (NTC), respectively. Figure 5 Figure 1 is a diagram of agarose gel electrophoresis results of RPA amplification of 12 species of meat product genomic DNA by 5 pairs of primers, wherein a-e represent electrophoresis results of target fragments amplified by primer sets with sizes of 114bp, 166bp, 170bp, 152bp, and 276bp, respectively, and lanes 1-11 are genomic DNA of horse, chicken, duck, goose, turkey, pig, cow, sheep, camel, rabbit, and no template control (NTC), respectively. Figure 5 Figure 1 is a diagram of agarose gel electrophoresis results of RPA amplification of 12 species of meat product genomic DNA by 5 pairs of primers, wherein a-e represent electrophoresis results of target fragments amplified by primer sets with sizes of 114bp, 166bp, 170bp, 152bp, and 276bp, respectively, and lanes 1-11 are genomic DNA of horse, chicken, duck, goose, turkey, pig, cow, sheep, camel, rabbit, and no template control (NTC), respectively. Figure 5 Figure 1 is a diagram of agarose gel electrophoresis results of RPA amplification of 12 species of meat product genomic DNA by 5 pairs of primers, wherein a-e represent electrophoresis results of target fragments amplified by primer sets with sizes of 114bp, 166bp, 170bp, 152bp, and 276bp, respectively, and lanes 1-11 are genomic DNA of horse, chicken, duck, goose, turkey, pig, cow, sheep, camel, rabbit, and no template control (NTC), respectively.

[0046] Figure 6 Figure 1 is a diagram of agarose gel electrophoresis results of RPA amplification of 12 species of meat product genomic DNA by 5 pairs of primers, wherein a-e represent electrophoresis results of target fragments amplified by primer sets with sizes of 114bp, 166bp, 170bp, 152bp, and 276bp, respectively, and lanes 1-11 are genomic DNA of horse, chicken, duck, goose, turkey, pig, cow, sheep, camel, rabbit, and no template control (NTC), respectively.Figure 6 In this context, 'a' represents the fluorescence intensity of the RPA amplification product without MS. Figure 6 In this context, 'b' represents the fluorescence intensity of the RPA amplification product with MS.

[0047] Figure 7 This is a histogram showing the normal distribution of fluorescence intensity in the blank sample.

[0048] Figure 8 The image shows the morphological characterization results of MB@SA and the MB@SA-DNA complex; among them, Figure 8 In this context, 'a' represents the Fourier transform infrared spectrum of MB@SA, MB@SA-DNA, DNA, and SA. Figure 8 In the diagram, 'b' represents the UV-Vis absorption profile of MB@SA, MB@SA-DNA, DNA, and SA at wavelengths of 230-350 nm.

[0049] Figure 9 Scanning electron microscope (SEM) images and elemental spectra of MB@SA and MB@SA-DNA are shown; among them, Figure 9 In this context, 'a' represents the MB@SA scanning electron microscope image; Figure 9 In this text, 'b' represents a scanning electron microscope image of MB@SA-DNA; Figure 9 c represents the elemental spectrum of MB@SA; Figure 9 In this context, 'd' represents the elemental spectrum of MB@SA-DNA.

[0050] Figure 10 The figure shows the optimization results for primer and magnesium acetate addition amounts; among them... Figure 10 In this context, A and B represent the optimized primer addition amount. Figure 10 In B, lanes 1-5 were 0.5 μL, 1.3 μL, 2.1 μL, 2.9 μL and 3.7 μL respectively, and M was 2K Marker for all lanes. Figure 10 In the figure, C and D represent the optimized results of magnesium acetate addition. Figure 10 In D, lanes 1-5 were 1.5 μL, 2.0 μL, 2.5 μL, 3.0 μL and 3.5 μL respectively, and M was 2K Marker for all lanes.

[0051] Figure 11 The figure shows the optimization results of amplification time and amplification temperature; among them, Figure 11 Figures A and B represent the optimized amplification time results. Figure 11 In lane B, lanes 1-5 were 5 min, 10 min, 15 min, 20 min, and 25 min respectively, and lane M was 2K Marker; Figure 11 In the figure, C and D represent the optimized amplification temperature. Figure 11D in the figure, lanes 1-5 are 35℃, 37℃, 39℃, 41℃ and 43℃ respectively, and M is 2K Marker.

[0052] Figure 12 Figure for the optimization results of RPA-MS-FD detection conditions; Figure 12 a in the figure indicates the optimization ratio of RPA amplicon volume to resuspended MB@SA volume; Figure 12 b in the figure indicates the optimization incubation time; Figure 12 c in the figure indicates the optimization number of washing times.

[0053] Figure 13 Figure for the specificity test results of RPA-MS-FD detection, gel electrophoresis 1-13 lanes are respectively horse, donkey, deer, chicken, duck, goose, pig, cow, sheep, camel, turkey, rabbit genomic DNA and NTC; M is 2K Marker.

[0054] Figure 14 Figure for the verification results of RPA-MS-FD detection method; wherein, Figure 14 a in the figure indicates the detection limit and calibration curve of RPA-FS-FD detection method; Figure 14 b in the figure indicates the corresponding gel electrophoresis image of meat samples, lanes 1-11 respectively show 100%, 80%, 60%, 40%, 20%, 10%, 1%, 0.5%, 0.1% and 0.01% adulterated horse beef samples and NTC; M is 2K Marker.

[0055] Figure 15 Figure for the results of RPA-MS-FD and RT-PCR two detection methods to identify the adulteration of commercial meat products; wherein, Figure 15 a in the figure indicates the quantitative detection results of commercial meat adulteration by RPA-MS-FD method; Figure 15 b in the figure indicates the quantitative detection results of commercial meat adulteration by RT-PCR method; wherein 19 indicates the identification results of the 19th sample. DETAILED DESCRIPTION

[0056] The present application will be described in detail below with specific examples, but should not be understood as limiting the present application. If not specially stated, the technical means used in the following examples are the conventional means familiar to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specially stated, can be obtained from commercial channels.

[0057] 1. Experimental materials and reagents

[0058] TwistAmp Basic kit was purchased from TwistDX (Cambridge, UK). Streptavidin (SA)-coated magnetic beads (MB@SA) were purchased from Biyun Tian Biotechnology Co., Ltd. (Shanghai, China). 1xTBS buffer, RNase-free double distilled water (ddH2O) and 2xTaqMan Fast PCR Master Mix were purchased from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). Sodium chloride (NaCl), ethylenediaminetetraacetic acid (EDTA), tris-hydroxymethyl aminomethane hydrochloride (Tris-HCl), sodium dodecyl sulfate (SDS) and agarose were purchased from Macklin Biochemical Technology Co., Ltd. (Shanghai, China). SA and Tween 20 were purchased from Beijing Solabio Technology Co., Ltd. (Beijing, China). Loading buffers were purchased from Beijing Zhenxi Gold Biotechnology Co., Ltd. (Beijing, China). Gelstain Red nucleic acid dye and DL 2000 DNA Marker were purchased from Suzhou Youyilan Di Biological Technology Co., Ltd. (Suzhou, China). All chemicals were of analytical grade.

[0059] Example 1: Primer screening

[0060] 1. Primer design

[0061] Three complete mitochondrial gene sequences of horse (MN187574.1, NC_009154.3, NC_001640.1) were downloaded from the National Center for Biotechnology Information (NCBI https: / / www.ncbi.nlm.nih.gov / ). RPA primers were designed using Primer Premier 5 according to TwistDxTM primer design guidelines. The designed primer sets were preliminarily verified using "BLAST" (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast), and according to the verification results, five pairs of primer sets were selected for synthesis by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). The DNA of horse, chicken, duck, goose, turkey, pig, cow, sheep, camel and rabbit was used as a template for RPA amplification, and the specificity of the five primer sets was verified by agarose gel electrophoresis, and the most suitable primer was selected.

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[0063] Table 1 Amplification primer

[0064]

[0065] Example 2: A method for detecting horse meat components in food

[0066] 1. A method for detecting horse meat components in food, which uses a 96-well high-throughput DNA extraction method for DNA separation and purification. Then the separated DNA is amplified by RPA technology, and the amplified product is separated by magnetic separation (MS) strategy, which is named as RPA-MS-FD method, which specifically includes the following steps:

[0067] (1) High-throughput DNA extraction

[0068] As Figure 5DNA was extracted using a high-throughput DNA extraction system developed by the inventors, designated as 96-SPME extraction device, which consists of 96 M-UiO-66-NH2 coated DNA extraction devices and a polyformaldehyde plate. The 96 M-UiO-66-NH2 coated DNA extraction devices are evenly attached to one side of the polyformaldehyde plate, with each two M-UiO-66-NH2 coated DNA extraction devices being parallel to each other. The M-UiO-66-NH2 coated DNA extraction devices were prepared as follows: zirconium tetrachloride (320 mg), terephthalic acid (125 mg), 2-amino terephthalic acid (125 mg), acetic acid (9.864 g), hydrochloric acid (270 mg), and N-N'-dimethylformamide (DMF) (50 mL) were mixed and then poured into a hydrothermal reactor, which was heated in a muffle furnace at 120°C for 24 h. After cooling to room temperature, the solid particles were separated by centrifugation and washed with DMF and ethanol several times until the supernatant was completely transparent. Finally, the white powder was dried in a vacuum drying oven at 150°C for 12 h; then the white powder was placed in a muffle furnace and heated at 350°C for 120 min. After cooling to room temperature, the adsorbent material M-UIO-66-NH2 was obtained (for detailed synthesis process, please refer to the invention patent with application number 202210626821.0). Then, 10 mg of M-UiO-66-NH2 was added to 6 mL of methanol and ultrasonically treated for 2 h, and 300 mg of polyvinylpyrrolidone was added under continuous stirring, and the nitrocellulose membrane was immersed in the mixture for 20 s and dried at room temperature to make the M-UIO-66-NH2 coated membrane. Then, the improved membrane was attached to a stainless steel rod. This simple M-UiO-66-NH2 coated DNA extraction device is called "extraction rod". The extraction rod separates DNA through three steps: lysis, washing, and elution (less than 7.2 seconds per sample). The purity and concentration of the DNA were determined by ultraviolet-visible spectrophotometry (Nano300, Allsheng, Hangzhou, China), and the 260 / 280 nm ratio of the DNA template was between 1.8 and 2.0.

[0069] The specific extraction method is as follows:

[0070] 1) 30 mg of meat sample was added to each well of the 96-well plate, followed by the addition of 600 μL of lysis buffer to each well. Then, the 96-SPME device was immersed in the lysis solution for 60 seconds.

[0071] The composition of the lysis buffer is as follows: 100 mM Tris-HCl (pH = 8.0), 5 mM EDTA, 200 mM NaCl, 1% SDS. The volume of the lysis buffer and the lysis time were optimized. The results are shown in Table 1. Figure 3a and b, the recovery rate increased as the volume of lysis buffer increased from 300 μL to 600 μL. However, a larger volume of lysis buffer did not further promote the isolation of DNA, which might be due to the dynamic balance between the extraction stage and the sample volume. Considering the trade-off between the obtained DNA concentration and the consumed organic solvent, a volume of 600 μL of lysis buffer was chosen as the optimal volume for further study; when the lysis time reached 60 seconds, the amount of extracted DNA could reach a maximum.

[0072] 2) Transfer the 96-SPME extraction device to another 96-well plate and rinse each extraction rod with deionized water for 30 seconds to remove attached proteins and other substances.

[0073] 3) Place the 96-SPME extraction device treated in (2) into a 96-well plate containing elution buffer for 10 min to release the extracted DNA.

[0074] The composition of the elution buffer: 300 μL of 10 mM Tris (pH = 7.0). The volume of the elution buffer and the elution time were optimized. The volume of the elution buffer is a necessary parameter for the quantitative recovery of target DNA, as shown in Figure 4 a, as the volume of the elution buffer increased, the DNA concentration was diluted, and the recovery rate decreased. According to the research results, the experiment selected a volume of 300 μL of elution buffer as the optimal volume. A long enough elution time not only ensures that the DNA is completely released from the coating, improving the extraction sensitivity, but also avoids the influence of DNA residues on subsequent experiments. The time curve of eluting DNA from the SPME extraction device is shown in Figure 4 b. It takes about 10 min to elute most of the DNA from the SPME coating. After 10 min, due to the dynamic balance between the solid coating phase and the liquid phase, the extraction device may absorb DNA from the elution buffer, resulting in a decrease in DNA concentration. Therefore, elution for 10 min was used for the next step of research.

[0075] 2, RPA amplification of horse DNA

[0076] As shown in Figure 5 b, the RPA reaction was performed using the TwistAmp Basic kit. The RPA reaction mechanism is that the labeled primer undergoes exponential amplification under the action of recombinase, strand displacement DNA polymerase and single-stranded DNA binding protein, producing amplified products labeled at both ends.

[0077] The reaction system of RPA amplification was 50 μL: 29.5 μL primer-free rehydration buffer, 2.9 μL labeled forward primer (10 μM L -1 ), 2.9 μL labeled reverse primer (10 μM L -1 ), 2.0 μL (1) DNA template obtained by high-throughput extraction (10 ng μL -1 ), and 10.2 μL ddH2O were added to the reaction tube pre-mixed with recombinase and polymerase freeze-dried powder to make the total volume 47.5 μL. After mixing well, 2.5 μL 280 mM magnesium acetate (MgOAc) was added to the cover of the reaction tube, and mixed by inverting up and down for 8-10 times. The mixed reaction was incubated at 39 °C for 15 min in a 96-well T100 thermal cycler. The amplification product was detected by 2% agarose gel stained with Gelstain Red, and analyzed by Gel Doc TM XR system.

[0078] 3. Magnetic separation (MS) and fluorescence detection of RPA amplification product

[0079] The magnetic separation and fluorescence detection of RPA amplification product are as shown in c of Figure 5 .

[0080] First, 100 μL of MB@SA solution was taken and washed three times with 1 x TBS buffer. After magnetic separation (MS) and careful pouring of the supernatant, the washed beads were dispersed in 200 μL of 2 x Binding & Washing (B&W) buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2 M NaCl, 0.01% Tween-20) and stored at -4 °C for later use. Then, 10 μL of washed streptavidin magnetic beads (MB@SA) were introduced into 15 μL of RPA amplification product obtained from step 2 and incubated at room temperature in the dark for 5 min. During the incubation, streptavidin (tetrameric protein) non-covalently binds to four biotin molecules in the liquid phase at the same time, thereby forming a complex of MB@SA and amplification product (MB@SA-DNA). After the incubation was completed, the MB@SA-DNA complex was separated from the solution under the action of an external magnetic field, the supernatant was poured to remove excess labeled primers and other substances, and then the MB@SA-DNA complex was washed three times with 1 x B&W buffer. Finally, the washed magnetic bead complex was resuspended in 200 μL of ultrapure water; the mixture containing the MB@SA-DNA complex was directly transferred to a polystyrene black 96-well enzyme plate, and the multifunctional enzyme plate instrument was parameter set to excitation wavelength (λex) 484 nm, emission wavelength (λem) 529 nm, and manual gain setting to 150. Then end-point fluorescence detection (FD) was performed, and the fluorescence intensity value was obtained. In the FD method, the 6-FAM fluorescent group has a unique excitation and emission wavelength, which can be recognized and determined by the multifunctional enzyme plate instrument. Finally, the fluorescence intensity of the fluorescent group-labeled amplification product was detected for qualitative and quantitative analysis (e.g. Figure 5

[0081] Therefore, the RPA-based detection is realized by combining it with magnetic separation (MS) and fluorescence detection (FD) (RPA-MS-FD).

[0082] The provided detection method was also analyzed in this example:

[0083] (1) Verify the feasibility of MS

[0084] The feasibility of MS was evaluated by comparing the fluorescence intensity measurements of RPA amplification products with and without MS. When detecting RPA amplicons without MS, 15 μL of the amplicons were diluted to 200 μL with ultrapure water. The mixture was directly transferred to a black polystyrene 96-well enzyme plate, and the fluorescence intensity was detected using a multifunctional enzyme plate instrument. MS of RPA amplicons was performed according to the method described in step (3). As shown in Figure 6 ​As shown in a and b, when MS was used, the fluorescence signal ratio F / F0 (F and F0 are the fluorescence intensities with and without horse DNA templates, respectively) increased from 2.09 to 10.68, indicating that residual primers were successfully removed and interference from subsequent FD was minimized. This explains the basic principle of RPA-MS-FD and verifies the effectiveness of MS in improving detection performance.

[0085] (2) Determine the fluorescence threshold for a positive result.

[0086] Following the developed RPA-MS-FD method, 500 fluorescence intensity measurements were performed on blank (template-free control, NTC) samples to determine the threshold for distinguishing between negative and positive results. Data analyzed using SPSS statistical software showed a normal distribution (e.g., ...). Figure 7 (As shown). The average value was calculated through statistical analysis. The value was 505.7, and the standard deviation (SD) was 109.6. According to the three-standard-deviation rule, data points exceeding this range are generally considered outliers. Therefore, [the following was chosen]. Using the corresponding threshold signal value as the standard for a positive signal, the positive fluorescence threshold was determined to be 834. When the measured fluorescence signal value is greater than the threshold of 834, it indicates that the sample contains horse meat.

[0087] (3) Characterization of MB@SA and MB@SA-DNA

[0088] To further investigate the adsorption of DNA on MB@SA, we used FI-IR and UV-VIS techniques to analyze MB@SA, MB@SA-DNA, DNA, and SA. Figure 8 As shown in a, in MB@SA, Fe-O (621 cm⁻¹) was observed. -1 ) and C=O stretch (1637cm) -1 Corresponding characteristic peaks. Interestingly, MB@SA-DNA and pure DNA exhibit similar functional groups, including CN extension (1101 cm⁻¹). -1 ) and CH stretch (2888cm) -1 This indicates that DNA was successfully adsorbed onto the MB@SA surface. Furthermore, as... Figure 8 As described in section b, SA and MB@SA exhibited maximum absorbance at a wavelength of 284 nm, while DNA and MB@SA-DNA exhibited maximum absorbance at a wavelength of 262 nm, which confirms the effective binding of DNA to MB@SA.

[0089] Morphological characterization of MB@SA and MB@SA-DNA complex by scanning electron microscope (SEM) showed that MB@SA formed clusters due to their magnetism, while in MB@SA-DNA complex, magnetic beads gathered together, which might be due to their being wrapped in DNA and protein and other substances (as shown in a-b of Figure 9 In addition, the elemental mapping of MB@SA and MB@SA-DNA complex after incubation confirmed that the weight ratio of each element in MB@SA-DNA complex after incubation increased significantly (as shown in c-d of Figure 9 and Table 2).

[0090] In summary, MB@SA showed good binding properties with DNA, which was mainly attributed to the non-covalent coupling of streptavidin on magnetic beads, which facilitated the binding with biotin on the amplification product.

[0091] Table 2 Weight ratio of each element in MB@SA and MB@SA-DNA

[0092] Element Weight ratio of MB@SA (%) Weight ratio of MB@SA-DNA (%) Fe 44.7 44.7 C 16.5 43.6 O 36.4 46.5 N 1.2 3.0 P 1.1 2.9 S 0.1 0.2 Cl 0.0 1.9

[0093] (4) Optimization of RPA-MS-FD conditions

[0094] In order to obtain the best RPA reaction conditions, the primer addition amount, MgOAc addition amount, amplification temperature and amplification time were optimized. The optimization of all variables was based on the maximum fluorescence difference (△F, △F = F-F0, where F and F0 represent the fluorescence intensity when DNA template exists and does not exist, respectively).

[0095] The primer addition amount selected in this experiment was 0.5 μL, 1.3 μL, 2.1 μL, 2.9 μL and 3.7 μL, respectively. As shown in A of Figure 10 , the increase of primer addition amount led to the steady rise of positive fluorescence signal. However, the more the amount of primer, the more conducive to the formation of primer dimers, resulting in the gradual increase of negative control fluorescence. The largest fluorescence difference was observed when 2.9 μL of primer was added, which was consistent with the gel electrophoresis results (as shown in B of Figure 10 ).

[0096] We tested 1.5 μL, 2.0 μL, 2.5 μL, 3.0 μL and 3.5 μL of MgOAc addition amount. As shown in C and D of Figure 10 , with the increase of MgOAc addition amount, the positive fluorescence signal began to increase and then decrease. According to the brightness of the band and the maximum fluorescence difference, the optimal addition amount was 2.5 μL.

[0097] The amplification time of 5 min, 10 min, 15 min, 20 min and 25 min was tested, respectively. As shown in Figure 11As shown in Figures A and B, the longer the amplification time, the brighter the observed bands and the stronger the positive fluorescence signal. The fluorescence signal tends to stabilize when the amplification time is 15 minutes. Therefore, we selected 15 minutes as the optimal amplification time.

[0098] We adjusted the amplification temperature from 35℃ to 43℃ in 2℃ increments. The results are as follows... Figure 11 As shown in C and D, the fluorescence signal initially increases with increasing amplification temperature, then decreases. Although no significant change in band brightness was observed in gel electrophoresis, the optimal amplification temperature was 39 °C.

[0099] The analytical performance of the RPA-MS-FD method is affected by the ratio of RPA amplicon volume to resuspended MB@SA volume. Ratios of 1:2, 2:3, 1:1, 3:2, and 2:1 were tested. Figure 12 As shown in a, the higher the ratio, the more pronounced the fluorescence signal. At the optimal ratio of 3:2, the positive fluorescence signal tends to stabilize, indicating that MB@SA binds sufficiently to the amplification product.

[0100] The incubation times for the tests were 2 min, 5 min, 10 min, 15 min, and 20 min. The fluorescence signal tended to stabilize after 5 min (e.g., ...). Figure 12 As shown in b) indicates that 5 minutes is the optimal incubation time. Furthermore, we tested washing cycles of 1, 2, 3, 4, and 5 times, and observed that the fluorescence signal gradually decreased with increasing washing cycles. This is likely because during washing, in addition to removing excess primers, the MB@SA-DNA complex is also lost, leading to a gradual weakening of the fluorescence signal. Optimal results were obtained after 3 washes (as shown in b). Figure 12 (as shown in c).

[0101] Example 3: Specificity test of the method for determining horse meat components in food provided in Example 2

[0102] The specificity of the method was tested using horse (Equus caballus) and other 11 animal meats, including pig (Sus scrofa), cow (Bos taurus), goat (Capra hircus), chicken (Gallus gallus), duck (Anas platyrhynchos), goose (Anser cygnoides orientalis), camel (Camelus Linnaeus), rabbit (Oryctolagus cuniculus), duck (Anas platyrhynchos), goose (Anser cygnoides orientalis), camel (Camelus Linnaeus), rabbit (Oryctolagus cuniculus), turkey (Meleagris gallopavo), donkey (Equus asinus) and deer (Cervus nippon). Genomic DNA extracted from these 12 kinds of meat was used as template. The reaction was carried out under optimal conditions. All samples were repeated three times, and ddH2O was used as no-template control (NTC).

[0103] In the specificity detection, DNA from horse, donkey, deer, chicken, duck, goose, pig, cow, goat, camel, turkey and rabbit of different sources was used as template. As shown in Figure 13 only the reaction containing horse meat DNA showed a positive signal, while other samples (including NTC) did not show amplification signal. This highlights the high specificity of the RPA-MS-FD method for horse meat components. Similar results were also obtained by gel electrophoresis detection. It is worth noting that the detection time of RPA-MS-FD method is only 5 min / 96 samples, which is much shorter than the 45 min / sample required by gel electrophoresis method, which highlights the high efficiency of RPA-MS-FD method.

[0104] Example 4: Sensitivity test

[0105] Horse and beef were respectively minced to make a series of binary mixed meat samples, with horse meat content of 100%, 80%, 60%, 40%, 20%, 10%, 1%, 0.5%, 0.1% and 0.01%, to detect the limit of detection (LOD) of the method. DNA extracted from these different binary meat mixtures was used as template. All samples were repeated three times, and ddH2O was used as no-template control.

[0106] The sensitivity and practicability of RPA-MS-FD method were evaluated by analyzing beef samples containing different proportions of horse meat (100%, 80%, 60%, 40%, 20%, 10%, 1%, 0.5%, 0.1% and 0.01%). As shown in Figure 14The fluorescence results shown in figure a indicate that this method successfully detected horse meat content as low as 0.1% (by weight) in beef, with the fluorescence signal decreasing as the adulteration ratio decreased. The detection limit results are consistent with the gel electrophoresis results (e.g., Figure 14 (As shown in b) although the latter is qualitative rather than quantitative. It is worth noting that detecting adulteration percentages below 5% is uneconomical. These results demonstrate that the RPA-MS-FD method has high sensitivity and is suitable for identifying adulteration in meat products on the market. Furthermore, this method shows a good linear relationship between the fluorescence signal and the adulteration percentage, with the linear equation being y = 797.28x + 1710.7, R0 2 The value is 0.9904.

[0107] Example 5: Quantitative Analysis of Commercial Meat

[0108] Processed meat products (including 10 beef products, 7 donkey meat products, 2 venison products, and 2 mutton products) were purchased from local markets and online stores. All meat samples were cut into small pieces, labeled with numbers 1-21, and immediately stored at -20°C until use.

[0109] The method described in Example 2 was used for qualitative and quantitative analysis of 21 processed meat products and a control sample (water, sample number 22). The accuracy of the results was then verified using the Chinese industry standard "Identification of Livestock Components in Food and Feed Part 5: Detection of Horse Meat Components - Real-time PCR" (SN / T3730.5-2013). The sequences of the primers and fluorescent probes, as shown in SEQ ID NO. 3-5 in Table 3, were synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). The detection was performed using a LightCycler 96 real-time PCR system (Roche Diagnostics, Basel, Switzerland). The solution used consisted of 12.5 μL of 2×TaqMan Fast PCR MasterMix, 1 μL of primers and probes (10 nmol L⁻¹), 2 μL of template DNA (50 ng), and supplemental ddH₂O, for a total volume of 25 μL. Reaction conditions: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 5 s, 60℃ annealing for 30 s, fluorescence collection at 60℃, 40 cycles.

[0110] Table 3 Primers and fluorescent probes used in real-time fluorescence PCR

[0111]

[0112] like Figure 15The results of the RPA-MS-FD method are shown in Table 4. In the 21 commercial samples tested, the RPA-MS-FD identified 1 positive sample and 20 negative samples, which were consistent with the results of the RT-PCR method. The positive sample was sample No. 19, which was a donkey meat product. The quantitative adulteration percentage of the positive sample is shown in Table 4, which confirms the accuracy and practicability of the technology.

[0113] Table 4 Positive results of RPA-MS-FD strategy for commercial products

[0114]

[0115]

[0116] Note: (-) indicates a negative sample detected by the RPA-MS-FD method.

[0117] Example 6: Comparison with other DNA amplification methods for detecting horse meat

[0118] The RPA-MS-FD method was compared with other DNA-based amplification methods for horse meat detection, and the results are shown in Table 5. PCR has accurate and sensitive detection capabilities. For example, Panzhu Qin et al. (Qin, P., Qiao, D., Gao, Y., Yao, L., Lu, J., Xu, J., & Chen, W. (2019). Self-signal-on fluorescent colorimetric protocol for rapid authentication of horse meat adulterated beef samples with functional designed probes. International Journal of Food Science and Technology, 54(5), 1752-1759. https: / / doi.org / 10.1111 / ijfs.14068) developed a new self-signal fluorescent detection PCR method that uses unique hairpin primers as functional probes without the need for expensive instruments or additional fluorescent probes. This method can quickly detect adulterated horse meat in beef samples based on fluorescent signals. Maria et al. (Magiati, M., Myridaki, V. M., Christopoulos, T. K., & Kalogianni, D. P. (2019). Lateral flow test for meat authentication with visual detection. Food Chemistry, 274, 803-807. https: / / doi.org / 10.1016 / j.foodchem.2018.09.063) demonstrated a method for identifying horse meat by visual inspection using PCR-LFS. This detection method can be completed within 25-30 min after amplification and can detect as little as 0.01% horse meat in a binary mixture. However, these PCR-based methods still have limitations, such as reliance on variable temperature amplification instruments and long time consumption.

[0119] Compared to the salted round amplification method (SRCA), the RPA-MS-FD fluorescent detection method has a milder amplification temperature and shorter amplification time. In addition, our method was also evaluated with RT-ERA, ERA-LFS, and RT-RPA methods, highlighting its advantages. Our RPA-MS-FD method only uses a pair of 6-FAM and biotin-labeled primers without the need for additional fluorescent probes. Effective primers and probes usually require a lot of synthesis and screening, which is costly and time-consuming, while our method simplifies this process.

[0120] The RPA-MS-FD method provided by the application is comparable to the performance of all three methods in detecting horse meat as low as 0.1% (wt%). In addition, the detection time (96 samples 31.5 min) of the RPA-MS-FD method is shorter than that of the three methods, while the detection time of the RT-RPA method is only 15 min. Compared with the extraction of DNA using a commercial kit, the high-throughput DNA extraction method of our application greatly shortens the sample pretreatment time. The use of 96 high-throughput DNA extraction method is conducive to the separation and purification of DNA, and the concentration and purity of the extracted DNA are comparable to those of traditional commercial kits. In summary, compared with existing horse meat detection methods, our method has the advantages of being fast, convenient and simple.

[0121] Table 5 Comparison of RPA-MS-FD method with other DNA-based horse meat detection methods

[0122]

[0123] Note: SRCA: salt rolling circle amplification; RT: real-time; ERA: enzymatic recombinase amplification; "-" means no such item.

[0124] In summary, the application develops a RPA-MS-FD method for qualitative and quantitative detection of horse meat. For 96 samples, the entire process from DNA extraction to detection can be completed within 31.5 min (including 11.5 min of DNA extraction, 15 min of RPA amplification and 5 min of fluorescence detection). This method does not rely on a thermal cycler. The method is accurate and does not produce cross-reactions with other animal-derived nucleic acids. The sensitivity is sufficient for adulteration detection, and 0.1% (wt%) of horse meat component in adulterated meat can be detected. The method uses MB@SA to specifically bind to the RPA amplification product, without the need to design and screen RPA probes. It solves the problem of real-time fluorescence detection of RPA dependence on portable fluorescence constant temperature amplification instrument and false positive of fluorescent dye. The RPA-MS-FD method can simultaneously qualitatively, quantitatively and high-throughput detect multiple DNA targets. The method is suitable for detecting commercial samples and provides a new choice for rapid detection of food safety.

[0125] Although preferred embodiments of the application have been described, those skilled in the art, once aware of the basic inventive concept, can make additional changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the application.

[0126] Obviously, many modifications and variations of the present application are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims and their equivalents, the application can be practiced otherwise than as specifically described.

Claims

1. A method for detecting horse meat components in food, characterized in that, include: S1, Extract DNA from the sample to be tested to obtain genomic DNA of the sample; S2, using primer pairs with sequences as shown in SEQ ID NO. 1 and SEQ ID NO. 2, RPA amplification of the sample genomic DNA is performed to obtain amplification products; wherein, the 5′ end of the primer shown in SEQ ID NO. 1 is labeled with a 6-FAM fluorescent group; and the 5′ end of the primer shown in SEQ ID NO. 2 is labeled with biotin. S3. The amplification products were separated and purified using streptavidin-modified magnetic beads, and then identified and quantified using a high-throughput fluorescence detection system. A fluorescence signal value exceeding the threshold indicated a positive result, meaning that the amplification product contained horse meat. In S1, after the sample to be tested is lysed with lysis buffer, the extraction device is placed in the lysis buffer containing the sample for 55-65 seconds, and then the extraction device is washed to remove attached proteins and other substances; after washing, the extraction device is placed in elution buffer to release the extracted genomic DNA of the sample. The extraction device includes: a polyoxymethylene board on which a plurality of UiO-66 SPME devices are arranged, with every two UiO-66 SPME devices in parallel; The UiO-66 SPME device includes an extraction rod on which a nitrocellulose membrane coated with UiO-66-NH2 is adhered.

2. The method as described in claim 1, characterized in that, The lysis buffer comprises: 95-105 mM Tris-HCl with a pH of 7.5-8.5, 4.5-5.5 mM EDTA, 195-205 mM NaCl, and 0.8%-1.2% SDS by mass; the eluent is 8-12 mM Tris.

3. The method as described in claim 1, characterized in that, In S2, RPA amplification includes: adding primer-free rehydration buffer, forward and reverse primers, sample genomic DNA, and ddH2O to a reaction tube premixed with recombinase and polymerase lyophilized powder, mixing thoroughly, adding magnesium acetate, mixing well, and amplifying at 37℃-41℃ for 15 min-20 min. The amounts of the forward and reverse primers added were 2.8 μL-3.2 μL, and the amount of magnesium acetate added was 2.0 μL-2.6 μL.

4. The method as described in claim 1, characterized in that, In S3, the threshold is 834. When the fluorescence signal value is greater than 834, it indicates a positive result, meaning that the amplification product contains horse meat.

5. The method as described in claim 1, characterized in that, In S3, the volume ratio of the streptavidin-modified magnetic beads to the amplification product is 2-3:1-2.

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