Microfluidic detection kit for detecting animal-derived components in beef and beef products

Abstract

The invention discloses a microfluidic detection kit for detecting animal-derived components in beef and beef products, and belongs to the field of molecular biology. The microfluidic detection kit provided by the invention is based on a CarryOn P1000Q platform, integrates DNA extraction and purification by a magnetic bead method and real-time fluorescent PCR detection, and comprises a lysis solution, an eluent, a magnetic bead solution and a PCR reaction solution. The microfluidic detection kit provided by the invention is used for detecting animal-derived components in beef and beef products, is simple to operate, does not need professional technicians and PCR reaction laboratory environment, can realize a rapid nucleic acid detection process, is simple, convenient and safe, and is consistent in accuracy rate in a laboratory. The invention provides an effective, rapid and accurate detection means for import and export inspection and quarantine and food safety detection, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and particularly to a microfluidic detection kit for detecting animal-derived components in beef and beef products. Background Art

[0002] The safety traceability and species identification of meat products have gradually attracted the attention of government departments, food production enterprises, and consumers in various countries. In order to ensure the quality and safety of meat products, protect the legitimate rights and interests of consumers, and avoid the occurrence of unfair competition events in the market, it is urgent to establish a method that can quickly and accurately identify and detect animal-derived components.

[0003] Currently, the identification methods for meat varieties in China mainly include two categories: protein identification and nucleic acid identification. Protein identification has been successfully applied to the identification of fresh meat species. However, when meat undergoes deep processing such as chopping, mixing, steaming, and roasting, the structure and stability of proteins in the meat are damaged, and species-specific proteins or antigenic determinants are also destroyed. Therefore, the reliability and stability of using protein technology to identify meat species are poor, and it cannot meet the requirements of modern meat adulteration detection. In contrast, due to the relative stability of DNA, nucleic acid technology has great advantages in the detection of meat species in deeply processed products, and detection can be achieved as long as the specific genes of animal species are known.

[0004] However, although conventional nucleic acid detection methods have certain specificity and sensitivity, the meat to be detected needs to undergo a series of operations such as sample treatment, nucleic acid extraction, and PCR amplification before the final result can be obtained. The whole process is not only cumbersome, complex, and time-consuming, but also has high requirements for site, personnel, instruments, reagents, etc., and there is also a possibility of false positives and false negatives.

[0005] In addition, whether it is protein detection or traditional nucleic acid detection, usually only one or a few animal species can be identified. In actual detection, due to the variety and complexity of meat products, it is more necessary to simultaneously detect and identify multiple animal-derived components.

[0006] Therefore, there is an urgent need to establish a detection method that is simple to operate, accurate in results, and capable of simultaneously detecting multiple animal-derived components. Summary of the Invention

[0007] The object of the present invention is to provide a microfluidic detection kit for detecting animal-derived components in beef and beef products, so as to solve the problems existing in the above-mentioned prior art. Based on the CarryOn P1000Q platform, the present invention provides a product and method for detecting animal-derived components in beef and beef products. By adopting the principle of a microfluidic chip, the magnetic bead method for nucleic acid extraction and purification and the real-time fluorescence PCR standard detection process are integrated. Its equipment is simple to operate, does not require professional technical personnel to operate, nor does it require a PCR laboratory environment, realizing a rapid nucleic acid detection process of "sample in, result out". The entire detection process can be completed in only 50 minutes.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention also provides a microfluidic detection kit, which includes a lysis solution, an elution solution, a magnetic bead solution, and a PCR reaction solution.

[0010] Preferably, the lysis solution includes 4-6M guanidine hydrochloride, 0.1-1M sodium acetate, 1wt%-4wt% Triton X-100, and the pH value is 3.5-4.5.

[0011] Preferably, the elution solution includes 10-50mM tris(hydroxymethyl)aminomethane, and the pH value is 7.5-8.5.

[0012] Preferably, the magnetic bead solution includes 0.1-1M trehalose and 10-50mg / mL magnetic beads.

[0013] Preferably, the magnetic beads are composed of Fe3O4 and SiO2;

[0014] The surface modification group of the magnetic beads is a hydroxyl group; the particle size of the magnetic beads is 100nm-800nm.

[0015] Preferably, the PCR reaction solution includes 0.1-1μM bovine-specific primer probe, 0.1-1μM porcine-specific primer probe, 0.1-1μM chicken-specific primer probe, 0.1-1μM duck-specific primer probe, DNA polymerase, dNTP (deoxyribonucleoside triphosphate), PCR reaction buffer, air-drying protectant, and water.

[0016] The present invention also provides an application of the microfluidic detection kit in the preparation of a product for detecting animal-derived components in beef or beef products.

[0017] The present invention also provides a microfluidic chip for detecting animal-derived components in beef or beef products. The chip includes a reagent chamber, a nucleic acid extraction chamber, a PCR reaction chamber, a waste liquid chamber, and the kit according to any one of the above.

[0018] Among them, the lysis solution and the elution solution are stored in the reagent cartridge; the magnetic bead solution is stored in the nucleic acid extraction cartridge; and the PCR reaction solution is stored in the PCR reaction cartridge.

[0019] The present invention also provides an application of the microfluidic detection kit or the microfluidic chip as described above in detecting animal-derived components in beef or beef products.

[0020] The present invention also provides a method for detecting animal-derived components in beef or beef products, including the step of using the microfluidic detection kit or the microfluidic chip as described above to detect the beef or beef products to be tested.

[0021] The present invention discloses the following technical effects:

[0022] The microfluidic detection kit and detection method provided by the present invention optimize the detection technology, cost, and efficiency, and have high specificity and sensitivity, and can quickly and accurately detect specific meat sources. This method adopts the principle of a microfluidic chip, integrates the nucleic acid extraction and purification by the magnetic bead method and the real-time fluorescence PCR standard detection process. Its equipment is easy to operate. Put in the chip, click to run, and all reactions are automatically completed in a fully enclosed chip, without the risk of pathogen diffusion and product contamination. From sample to result, no manual intervention is required, and it only takes about 50 minutes to complete the detection. It does not require professional technical personnel to operate, nor does it require a PCR laboratory environment, but the accuracy is consistent with the laboratory method. It can be tested immediately after sampling, greatly improving the nucleic acid detection efficiency, getting rid of the environment, equipment, personnel, cold chain, etc. required for traditional nucleic acid detection, and having the characteristics of simplicity, rapidity, and safety. This method takes species-specific DNA as the detection target, based on the principle of multiplex PCR, and uses bioinformatics means to select 4 kinds of animal samples such as cattle, pigs, chickens, and ducks to establish a method that can simultaneously detect 4 kinds of animal-derived components, greatly shortening the detection time, and providing an effective, rapid, and accurate detection means for import and export inspection and quarantine and food safety detection. Description of the Drawings

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0024] Figure 1 It is a detection result diagram of a beef sample of the microfluidic chip in Experimental Example 1;

[0025] Figure 2 It is a detection result diagram of a mixed sample of beef and chicken of the microfluidic chip in Experimental Example 1;

[0026] Figure 3 Detection result diagram of beef and duck mixed sample for the microfluidic chip in Experimental Example 1;

[0027] Figure 4 Detection result diagram of beef and pork mixed sample for the microfluidic chip in Experimental Example 1;

[0028] Figure 5 Detection result diagram of goose meat sample for the microfluidic chip in Experimental Example 1;

[0029] Figure 6 Detection result diagram of beef sample for the microfluidic chip prepared based on Example 2 in Experimental Example 2;

[0030] Figure 7 Detection result diagram of beef sample for the microfluidic chip prepared based on Example 3 in Experimental Example 2;

[0031] Figure 8 Detection result diagram of beef sample for the microfluidic chip prepared based on Example 4 in Experimental Example 2;

[0032] Figure 9 Detection result diagram of beef and duck mixed sample for the microfluidic chip prepared based on Example 5 in Experimental Example 3;

[0033] Figure 10 Detection result diagram of beef and chicken mixed sample for the microfluidic chip prepared based on Example 6 in Experimental Example 3;

[0034] Figure 11 Detection result diagram of beef and pork mixed sample for the microfluidic chip prepared based on Example 7 in Experimental Example 3. Detailed implementation manners

[0035] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation to the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0036] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0037] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0038] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the description of this invention, which will be obvious to those skilled in the art. Other embodiments obtained from the description of this invention will be obvious to those skilled in the art. The description and examples of this invention are merely exemplary.

[0039] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0040] Example 1

[0041] This example provides a microfluidic detection kit for detecting animal-derived components in beef and beef products, including:

[0042] The lysis buffer contains 4M guanidine hydrochloride, 0.5M sodium acetate, 2% Triton X-100, and the pH is 3.5;

[0043] The elution buffer contains 30mM Tris-HCl and the pH is 7.5;

[0044] The magnetic bead solution contains 0.1M trehalose and 50mg / mL magnetic beads;

[0045] The magnetic beads are composed of Fe3O4 and SiO2, and the surface is modified with hydroxyl groups, namely silica hydroxyl magnetic beads;

[0046] The particle size of the magnetic beads is 400nm;

[0047] The PCR reaction solution contains bovine specific primer probes, porcine specific primer probes, chicken specific primer probes, duck specific primer probes, DNA polymerase, dNTP (deoxyribonucleoside triphosphate), PCR reaction buffer, air-drying protectant and water. Among them,

[0048] The upstream primer F sequence for detecting beef is: 5'-TAATCCTTCTGCTCAC-3' (SEQ ID NO.1);

[0049] The downstream primer R sequence for detecting beef is: 5'-TCCTCAGAATGATAG-3' (SEQ ID NO.2);

[0050] The probe P sequence for detecting beef is: 5'-FAM-TTTGGCTCTCTCC-BHQ1-3' (SEQ ID NO.3);

[0051] The upstream primer F sequence for detecting pork is: 5'-ATCTACATGATTCATT-3' (SEQ ID NO.4);

[0052] The downstream primer R sequence for detecting pork is: 5'-TGTTTTTTGAGTTTTGA-3' (SEQ ID NO.5);

[0053] The probe P sequence for detecting pork is: 5'-HEM-ATCTCAAACTACTCATA-BHQ1-3' (SEQ ID NO.6);

[0054] The upstream primer F sequence for detecting chicken is: 5'-CCCTCCTCCTTTCATCCT-3' (SEQ ID NO.7);

[0055] The downstream primer R sequence for detecting chicken is: 5'-GTCATAGCGGAACCGTG-3' (SEQ ID NO.8);

[0056] The probe P sequence for detecting chicken is: 5'-ROX-CTATGAATCCGGGCCTC-BHQ2-3' (SEQ ID NO.9);

[0057] The upstream primer F sequence for detecting duck is: 5'-GGCCACACAAATCCTCA-3' (SEQ ID NO.10);

[0058] The downstream primer R sequence for detecting duck is: 5'-TGTGTTGGCTACTGAGG-3' (SEQ ID NO.11);

[0059] The probe P sequence for detecting duck is: 5'-CY5-TACTGGCTATGCACTACACCG-BHQ2-3' (SEQ IDNO.12).

[0060] The main components of the PCR reaction buffer are 0.4M Tris-HCl (Tris-hydroxymethyl aminomethane hydrochloride), 55mM MgCl2, 1M KCl, 1% Tween-20 (polyoxyethylene sorbitan monolaurate), water, and the pH value is 9.06 - 9.1;

[0061] The protective agents are BSA (bovine serum albumin) and trehalose;

[0062] The components and dosages of the PCR amplification reagent and the PCR amplification system where the protective agent is located are shown in the following table.

[0063] Table 1 PCR amplification system

[0064]

[0065]

[0066] Configure according to the system described in Table 1. After mixing evenly, dispense 12.5 μL into the microfluidic chip one by one. Use a blast drying oven to air-dry the chip containing the PCR amplification system. The air-drying conditions adopted in this example are: temperature is 80 °C and time is 20 min.

[0067] After air-drying, detect the water loss rate by weighing. It is required that the water loss rate is greater than 90%. After air-drying, seal the microfluidic chip and combine it with the reagent cartridge containing the purification reagent for standby.

[0068] Experimental example 1

[0069] Prepare the reagent according to the microfluidic detection kit in Example 1, and store the lysis solution and elution solution in the reagent cartridge, the magnetic bead solution in the nucleic acid extraction cartridge, the PCR reaction solution in the PCR reaction cartridge, and the waste liquid cartridge for standby, and make a microfluidic chip.

[0070] The specific detection method is as follows: Take 50 mg of the meat tissue to be tested, add it to the lysis solution, mix well, let it stand at room temperature for 5 min, and inject the lysed liquid into the PCR reaction cartridge of the microfluidic chip. After sealing the lid, insert the microfluidic chip into the CarryOn P1000Q instrument to automatically complete the nucleic acid extraction and PCR amplification processes.

[0071] Sample No. 1 is a beef sample, and its detection result is as Figure 1 shown, and only the specific amplification curve appears in the beef channel, which is consistent with the actual situation.

[0072] Sample No. 2 is a mixture of beef and chicken, and its detection result is as Figure 2 shown, and the specific amplification curves appear in both the beef channel and the chicken channel, which is consistent with the actual situation.

[0073] Sample No. 3 is a mixture of beef and duck, and its detection result is as Figure 3 shown, and the specific amplification curves appear in both the beef channel and the duck channel, which is consistent with the actual situation.

[0074] Sample No. 4 is a mixture of beef and pork, and its detection result is asFigure 4 As shown, specific amplification curves simultaneously appeared in the beef channel and the pork channel, which was consistent with the actual situation.

[0075] Sample No. 5 was a goose meat sample, and its test results were as Figure 5 shown. No specific amplification curve appeared in all channels, which was consistent with the actual situation.

[0076] Example 2

[0077] The difference from Example 1 was only that the lysis buffer composition was: 4M guanidine hydrochloride, 0.7 mM sodium acetate, 2 wt% Triton X-100, pH = 4.

[0078] Example 3

[0079] The difference from Example 1 was only that the lysis buffer composition was: 5M guanidine hydrochloride, 0.5 mM sodium acetate, 4 wt% Triton X-100, pH = 3.7.

[0080] Example 4

[0081] The difference from Example 1 was only that the lysis buffer composition was: 6M guanidine hydrochloride, 0.3 mM sodium acetate, 3 wt% Triton X-100, pH = 4.2.

[0082] Experimental Example 2

[0083] Configure the reagents according to the microfluidic detection kits provided in Examples 2 to 4, fabricate the chip according to the method described in Experimental Example 1, and take the test samples for detection.

[0084] Using the chip prepared according to Example 2 to detect Sample No. 1, the results were as Figure 6 shown. Although the results were consistent with the actual situation, the Ct values obtained were relatively larger than those in Experimental Example 1.

[0085] Using the chip prepared according to Example 3 to detect Sample No. 1, the results were as Figure 7 shown. Although the results were consistent with the actual situation, the Ct values obtained were relatively larger than those in Experimental Example 1.

[0086] Using the chip prepared according to Example 4 to detect Sample No. 1, the results were as Figure 8 shown. Specific amplification curves appeared in the chicken and duck channels, which was inconsistent with the actual situation.

[0087] Example 5

[0088] The difference from Example 1 is only that the components of the PCR reaction solution are as follows: beef upstream primer F 0.5 μM, beef downstream primer R 0.5 μM, beef fluorescent probe P 0.5 μM; pork upstream primer F 0.3 μM, pork downstream primer R 0.3 μM, pork fluorescent probe P 0.5 μM; chicken upstream primer F 0.2 μM, chicken downstream primer R 0.2 μM, chicken fluorescent probe P 0.3 μM; duck upstream primer F 0.3 μM, duck downstream primer R 0.3 μM, duck fluorescent probe P 0.3 μM.

[0089] Example 6

[0090] The difference from Example 1 is only that the components of the PCR reaction solution are as follows: beef upstream primer F 0.2 μM, beef downstream primer R 0.2 μM, beef fluorescent probe P 0.4 μM; pork upstream primer F 0.4 μM, pork downstream primer R 0.4 μM, pork fluorescent probe P 0.4 μM; chicken upstream primer F 0.5 μM, chicken downstream primer R 0.5 μM, chicken fluorescent probe P 0.3 μM; duck upstream primer F 0.8 μM, duck downstream primer R 0.8 μM, duck fluorescent probe P 0.4 μM.

[0091] Example 7

[0092] The difference from Example 1 is only that the components of the PCR reaction solution are as follows: beef upstream primer F 0.3 μM, beef downstream primer R 0.3 μM, beef fluorescent probe P 0.3 μM; pork upstream primer F 0.5 μM, pork downstream primer R 0.5 μM, pork fluorescent probe P 0.5 μM; chicken upstream primer F 0.4 μM, chicken downstream primer R 0.4 μM, chicken fluorescent probe P 0.4 μM; duck upstream primer F 0.6 μM, duck downstream primer R 0.6 μM, duck fluorescent probe P 0.6 μM.

[0093] Experimental Example 3

[0094] Configure the reagents according to the microfluidic detection kit provided in Examples 5 to 7, fabricate the chip as described in Experimental Example 1, and take the test sample for detection.

[0095] Using the chip prepared according to Example 5, sample No. 3 was detected, and the results are as Figure 9 shown. Compared with the results obtained in Experimental Example 1, the Ct value obtained in the duck channel is larger.

[0096] Using the chip prepared according to Example 6, sample No. 2 was detected, and the results are as Figure 10 shown. Compared with the results obtained in Experimental Example 1, the Ct value obtained in the chicken channel is larger.

[0097] The chip prepared according to Example 7 was used to detect Sample No. 4, and the results are as follows Figure 11 shown. Compared with the results obtained in Experimental Example 1, the Ct value obtained from the pork channel is larger.

[0098] Experimental Example 4

[0099] The kit described in Example 1 of the present invention was compared with a commercial kit.

[0100] The operation process of the kit of Example 1 is as described in Experimental Example 1.

[0101] Nucleic acid extraction was performed on the same samples using a commercial nucleic acid extraction kit, and then detection was performed using a commercial beef detection kit, pork detection kit, chicken detection kit, and duck detection kit, respectively.

[0102] The experimental results are shown in Table 2

[0103] Table 2 Control test results of commercial kits

[0104]

[0105]

[0106] Using the same samples, the detection results obtained with the microfluidic detection kit prepared in Example 1 are consistent with those of the commercial kit.

[0107] Conclusion: The microfluidic kit provided by the present invention can efficiently and accurately detect beef-derived components in the sample to be tested.

[0108] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A microfluidic detection kit, characterized in that, It includes a lysis solution, an elution solution, a magnetic bead solution, and a PCR reaction solution.

2. The microfluidic detection kit according to claim 1, wherein The lysis solution includes 4 - 6M guanidine hydrochloride, 0.1 - 1M sodium acetate, 1wt% - 4wt% Triton X-100, and the pH value is 3.5 - 4.

5.

3. The microfluidic detection kit according to claim 1, wherein The elution solution includes 10 - 50mM tris(hydroxymethyl)aminomethane, and the pH value is 7.5 - 8.

5.

4. The microfluidic detection kit according to claim 1, wherein The magnetic bead solution includes 0.1 - 1M trehalose and 10 - 50mg / mL magnetic beads.

5. The microfluidic detection kit according to claim 4, wherein The magnetic beads are composed of Fe3O4 and SiO2; The surface modification group of the magnetic beads is a hydroxyl group; the particle size of the magnetic beads is 100nm - 800nm.

6. The microfluidic detection kit according to claim 1, wherein The PCR reaction solution includes 0.1 - 1μM bovine specific primer probe, 0.1 - 1μM porcine specific primer probe, 0.1 - 1μM chicken specific primer probe, 0.1 - 1μM duck specific primer probe, DNA polymerase, dNTP (deoxyribonucleoside triphosphate), PCR reaction buffer, air-drying protectant, and water.

7. Use of the microfluidic detection kit according to any one of claims 1 - 6 in the preparation of a product for detecting animal-derived components in beef or beef products.

8. A microfluidic chip for detecting animal-derived components in beef or beef products, characterized in that, The chip includes a reagent chamber, a nucleic acid extraction chamber, a PCR reaction chamber, a waste liquid chamber, and the kit according to any one of claims 1 - 6; Among them, the reagent chamber stores the lysis solution and the elution solution; the nucleic acid extraction chamber stores the magnetic bead solution; the PCR reaction chamber stores the PCR reaction solution.

9. Use of the microfluidic detection kit according to any one of claims 1 - 6 or the microfluidic chip according to claim 8 in the detection of animal-derived components in beef or beef products.

10. A method for detecting animal-derived components in beef or beef products, characterized in that, It includes the step of using the microfluidic detection kit according to any one of claims 1 - 6 or the microfluidic chip according to claim 8 to detect the beef or beef products to be tested.

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