CN112708682A - Primer pair and probe for detecting bovine-derived components, kit and application thereof - Google Patents
Primer pair and probe for detecting bovine-derived components, kit and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses a primer pair and a probe for detecting bovine-derived components, and discloses a kit designed according to the primer pair and the probe, wherein the kit comprises an RAA reaction system and an LFD test strip; the RAA reaction system comprises RAA reaction general dry powder, Tris-HCl buffer solution, upstream primer NIU-F, downstream primer NIU-R, probe NIU-PRO, sample DNA extracting solution, MgAcO and ddH2O; the quality control line of the LFD test strip is provided with an avidin-colloidal gold specific antibody, and the detection line is provided with a biotin antibody and a fluorophore antibody. The invention provides a novel bovine-derived component adulteration field detection method based on recombinase-mediated isothermal nucleic acid amplification technology lateral flow immunity technology, can realize visual detection of naked eye results, and has high detection sensitivity compared with other detection methodsThe method is simple, convenient and quick to operate, does not need special equipment, has accurate results, can meet the detection requirements of field samples, and is particularly suitable for field detection of farmer markets, supermarkets, wholesale markets and the like.
Description
Technical Field
The invention relates to a primer pair and a probe for detecting bovine-derived components, a kit and application thereof, belongs to the field of biotechnology and food safety rapid detection, and particularly relates to a primer pair and a probe for detecting bovine-derived components based on recombinase-mediated isothermal nucleic acid amplification technology-lateral flow immunization technology, a kit and application thereof.
Background
Meat and meat products are considered an important source of human protein and have evolved as a basic dietary ingredient, being widely consumed throughout the world with an enjoyable taste and flavor. Because of the temptation of economic profit (adulteration of economic incentive, EMA), unscrupulous producers may try to replace or adulterate beef with other inexpensive meats. Meat adulteration has occurred worldwide. Horse meat rumours (so-called "horse gate" rumours), which occurred in the uk and europe in 2013, are among the most well-known food fraud and contamination events, proving the vulnerability inherent in the current complex international food supply chain. Meat adulteration directly threatens the health of consumers, weakens the credit of processing enterprises, and destroys trade order and fair competition; in addition, it may also violate dietary contraindications in certain countries or nations. Therefore, in order to protect the rights and interests of consumers and avoid unfair market competition, a reliable, efficient, rapid and accurate method is provided, animal species can be accurately identified from meat products, and the situation of meat flavor deterioration is avoided, which is imperative.
Currently, several analytical methods have been validated and developed for screening and monitoring meat adulteration, such as spectroscopic analysis, electrophoresis, enzyme-linked immunosorbent assay (ELISA), chromatographic analysis, and the like. The most widely used method is the Polymerase Chain Reaction (PCR), including conventional PCR, real-time PCR, primer-multiplex PCR, PCR-rflp, High Resolution Melting (HRM) analysis, PCR-sequencing, and the like. However, the prior art requires considerable operational skill, expensive equipment and lengthy procedures, and these conditions or resource limitations limit the application of meat detection.
Recently developed Isothermal nucleic acid Amplification technologies include nucleic acid-dependent Amplification detection (NASBA), loop-mediated Isothermal Amplification (LAMP), Helicase-dependent Isothermal Amplification (HDA), Rolling Circle Amplification (RCA), Recombinase Polymerase Amplification (RPA), and Recombinase-mediated Isothermal Amplification (RAA). The basic principle is to imitate the in vivo nucleic acid replication mechanism, and various proteases participate in the reaction to help DNA polymerase to replicate DNA so as to realize isothermal amplification of the DNA. Compared with PCR, the isothermal amplification technology can amplify nucleic acid under constant temperature, get rid of expensive thermal cycling experimental instruments, and can realize exponential amplification of target fragments in a short time, so that nucleic acid amplification detection under various non-laboratory conditions becomes practical.
The recombinant enzyme Assisted Amplification is combined with Lateral Flow immunization (RAA-LFD), namely a combined technology of the recombinant enzyme Assisted Amplification (RAA) and the Lateral Flow immunization (LFD). Based on the RAA-LFD technology, a method suitable for rapidly detecting adulteration of meat products in detection sites (supermarkets, meat markets and the like) is constructed.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide an amplification primer pair and a probe for detecting bovine-derived components.
The invention also aims to provide a kit for detecting bovine-derived components based on the recombinase-mediated isothermal nucleic acid amplification technology-lateral flow immunization technology according to the amplification primer pair and the probe.
The invention also aims to provide application of the kit for detecting the bovine-derived components based on the recombinase-mediated isothermal nucleic acid amplification technology-lateral flow immunization technology.
The kit and the detection method thereof are simple, convenient and rapid, are suitable for detecting bovine-derived components on site, have low technical requirements on operators and equipment requirements, are suitable for rapid detection on site, and have wide application prospects in rapid detection of food safety.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an amplification primer pair and a probe for detecting bovine-derived components, wherein the sequences are respectively as follows:
an upstream primer NIU-F: ACATCGCCTAGCTCCATACA, as shown in SEQ ID NO: 1;
a downstream primer NIU-R: FITC-AGTGAAGAGGCCATAGGGA as shown in SEQ ID NO. 2;
probe NIU-PRO:
BIOTIN-GGCCTGATCAAACTATATTTCCTCTCCTTC- (H) -TAATCACAATCCTTA-PHO, shown in SEQ ID NO: 3;
wherein FITC is a fluorescent group; BIOTIN is BIOTIN; h is a tetrahydrofuran site; PHO is a phosphorylation modification.
The dSpacer Tetrahydrofuran (THF) is inserted into the probe sequence to simulate a basic site for modification, and the THF modified nucleotide can efficiently realize the extension of a DNA chain under the action of DNA polymerase.
The invention provides a kit designed according to the amplification primer pair and the probe for detecting bovine-derived components, which comprises an RAA reaction system and an LFD test strip, wherein a detection line of the LFD test strip is provided with a biotin antibody and a fluorophore antibody, and a quality control line is provided with an avidin-colloidal gold specific antibody.
Further, the fluorophore antibody is a nanogold particle of the FAM antibody.
And carrying out amplification reaction on the primer and the probe which are labeled by biotin and 6-carboxyl fluorescence hormone FAM and the target nucleic acid, wherein the product is an amplicon which is labeled by both biotin and FAM. The front end of the LFD test strip is a detection line close to the immersion liquid area, and the back end of the LFD test strip close to the handheld end is a quality control line. The detection line at the front end of the LFD test strip is provided with nanogold particles of FAM antibodies, the detection line is also provided with biotin antibodies, an amplification product is dripped onto the test strip, FAM groups on amplicons react with the FAM antibodies, after the biotin antibodies at the detection line are combined with the biotin on the amplicons, a display strip is displayed on the detection line, and uncaptured products are combined with the avidin-colloidal gold specific antibodies at the quality control line to form the display strip.
The FAM marker may also be used by selecting an appropriate other marker, such as digoxigenin marker, as desired.
The colors of the detection line and the quality control line are changed according to the selection of the colloidal gold particle size and the reducing agent, the color can be changed by naked eyes, the detection result is not influenced, and the appropriate colloidal gold particle size and the reducing agent can be selected according to the requirement.
Further, the RAA reaction system comprises the following components added into the RAA reaction general dry powder in volume ratio:
furthermore, in the RAA reaction system, the addition amount of the RAA reaction general dry powder is 1 mug/10-30 mug; the RAA reaction universal dry powder comprises: recombinase, Bst DNA polymerase, SSB protein, repair enzyme and dNTPs.
The RAA reaction general dry powder is RAA basic general reaction reagent freeze-dried powder. For example, the product of Jiangsu Qitian gene biology technology Co Ltd, 2 μ g of freeze-dried powder is added in each tube, the reaction specification is 50 μ L, namely 2 μ g of RAA basic general reaction reagent freeze-dried powder is added in each 50 μ L reaction system. One skilled in the art can select an appropriate RAA-based universal reagent product for use as desired.
Further, the Tris-HCl buffer concentration was 300mM, pH 8.0.
Further, the concentration of the upstream primer NIU-F is 10 mu M; the concentration of the downstream primer NIU-R is 10 mu M; the concentration of the probe NIU-PRO was 10. mu.M.
Still further, the MgAcO concentration is 280mM and the addition amount is 2% by volume.
Furthermore, the reaction condition of the kit is that the reaction is carried out for 15-25 min at 37-42 ℃.
Still further, the kit further comprises a positive control; the positive control is a plasmid for synthesizing a target region according to the sequence of the ND5/ND6 gene of cattle.
Still further, the preparation of the positive control comprises the steps of:
according to the sequence of ND5/ND6 gene of cattle in NCBI, using primer for cattle source component detection to clone and sequence an upstream primer NIU-F and a downstream primer NIU-R, using a plasmid DNA miniprep kit to extract positive clone plasmid DNA, and using an ultraviolet spectrophotometer to measure OD of the plasmid DNA260Value according to the formula OD260Calculating the copy number of plasmid DNA according to the molecular weight of the dsDNA with the OD of 50 mu g/mL and the recombinant plasmid corresponding to the specific amplified fragment of the bovine-derived component, and freezing and storing the obtained plasmid DNA solution with different copy numbers at-20 ℃ for later use;
the calculation formula of the plasmid DNA copy number is as follows:
plasmid copy number-plasmid concentration × 6.02 × 1023V (660X total length of plasmid);
the copy number includes, but is not limited to: 1X 100Copy/. mu.L, 1X 101Copy/. mu.L, 1X 102Copy/. mu.L, 1X 103Copy/. mu.L, 1X 104Copies/. mu.L and 1X 105Copy/. mu.L, 1X 106Copy/. mu.L, 1X 108Copies/. mu.L.
The invention provides application of the kit for detecting bovine-derived components, which is used for detecting bovine-derived components and comprises the following steps:
A. taking a sample: reserving a sample to be detected to a sterile centrifuge tube by using sterile scissors for later use;
B. DNA extraction: extracting DNA of a meat sample to be detected by adopting 5% Chelex-100 to obtain a sample DNA extracting solution;
C. RAA reaction system: each sample was formulated in the following volume percentages: 59% Tris-HCl buffer, 4.2% upstream primerNIU-F, 1.2% of probe NIU-PRO, 4.2% of downstream primer NIU-R, 4% of sample DNA extract or positive control or negative quality control sample, 21.8-25.4% ddH2Sequentially adding O into a reaction tube filled with RAA reaction general dry powder, and adding 2-5.6% of MgAcO for reaction; placing the reaction tube at 37-42 ℃ for reaction for 15-25 min to obtain an amplification product;
D. absorbing the amplification product into a new reaction tube, and diluting by 50-300 times;
E. and (3) LFD test strip detection: inserting the immersion area end of the LFD test strip into the diluted reaction tube, after the interpretation area is completely immersed, keeping the test strip flat for 1-3min, and waiting for the appearance of a strip;
F. directly reading a detection result according to the color development condition of the LFD test strip: only one line appears on the quality control line, which indicates that no bovine-derived component exists in the sample or the copy number of the bovine-derived component is lower than the lowest detection limit of the kit; two lines appear, one line is positioned on a detection line, and the other line is positioned on a quality control line, which indicates that bovine-derived components exist in the sample; no strip appears in the quality control line, which indicates that the nucleic acid test strip is invalid.
Further, in the step C, the adding amount of the MgAcO is 2 percent by volume.
Further, in the step C, the MgAcO is finally added to the inner wall of the reaction tube cap, and the tube cap is buckled after the addition to perform the sample adding operation of the next sample RAA reaction system. Mainly because the reaction is started after the MgAcO is added to prevent the dispersion of aerosol.
Further, in step C, the sample adding sequence between the experimental group and the control group is to complete the negative quality control sample ddH of the control group first20, preparing an RAA reaction system, and then preparing an experimental group sample DNA extracting solution and an RAA reaction system of a positive control group.
Further, the results were observed in LFD test strips within 10min, and interpretation was invalid after 10 min.
Compared with the prior art, the invention has the following beneficial effects:
the patent is based on a recombinase-mediated isothermal nucleic acid amplification technology lateral flow immunization (RAA-LFD) technology, is the combined application of the RAA technology and the LFD technology, and provides a method for preparing a recombinant nucleic acidThe novel method for detecting the bovine-derived component adulteration on site can realize visual detection of macroscopic results. Compared with other detection methods, the method has high detection sensitivity, and can stably detect the initial template as low as 1 × 101The copied/mu L RAA amplification product is simple and quick to operate, the whole detection process can be finished in 20-30 minutes, special equipment is not needed, the result is accurate, the detection of a field sample can be met, and the method is particularly suitable for field detection of farmer markets, supermarkets, wholesale markets and the like.
Drawings
FIG. 1 is a diagram illustrating the procedure of lateral flow immunization by recombinase-mediated isothermal nucleic acid amplification;
FIG. 2 is a schematic diagram of the lateral flow immunization technique of recombinase-mediated isothermal nucleic acid amplification technique according to the present invention;
in the figure: panel A shows bidirectional primer recombinase polymerase amplification; panel B shows an amplification reaction between a probe and a target nucleic acid; panel C is a lateral flow immunoassay test;
FIG. 3 is a schematic diagram showing the positions of a probe and a primer for detecting a bovine-derived component in the example;
FIG. 4 is a schematic structural diagram of the disposable nucleic acid detection test strip in the embodiment;
FIG. 5 is a schematic diagram illustrating the interpretation of the test strip results of the disposable nucleic acid test strip in the example;
FIG. 6 shows the results of the test on the sensitivity and specificity of the test strip for bovine-derived component nucleic acid in example 1;
FIG. 7 shows the results of the RAA-LFD detection method and the evaluation of primer specificity in effect test example 1;
FIG. 8 shows the results of the test paper for bovine-derived component nucleic acid test strip in the effect test example 2 on the amplification precision and repeatability;
FIG. 9 shows the results of evaluation of different MgAcO concentrations in RAA-LFD assay using lateral flow test paper (left) and electrophoresis (right) in Effect test example 4;
FIG. 10 shows the results of evaluation of different incubation times in RAA-LFD assay using lateral flow test strips (left) and electrophoresis (right) in Effect test example 4;
FIG. 11 shows the results of evaluating different amplification temperatures in RAA-LFD detection using lateral flow strips (left) and electrophoresis (right) in Effect test example 4.
Detailed Description
The detection program of the recombinase-mediated isothermal nucleic acid amplification technology lateral flow immunity technology is shown in figure 1, and the specific working principle is as follows:
the Recombinase-mediated isothermal nucleic acid Amplification technology lateral flow immunization (RAA-LFD) is a combination technology combining Recombinase-mediated isothermal nucleic acid Amplification technology (RAA) with lateral flow immunoassay (LFD), and the principle is shown in fig. 2: and carrying out amplification reaction on the target nucleic acid by using a primer and a probe which are labeled by biotin and 6-carboxyfluorescein (FAM), and finally forming an amplicon which is simultaneously labeled by the FAM group and the biotin. The front end of the LFD test strip is a detection line close to the immersion liquid area, and the back end of the LFD test strip close to the handheld end is a quality control line. The detection line at the front end of the LFD test strip is provided with nanogold particles of FAM antibodies, the detection line is also provided with biotin antibodies, an amplification product is dripped onto the test strip, FAM groups on amplicons react with the FAM antibodies on the detection line, the biotin antibodies on the detection line are combined with the biotin on the amplicons, red strips are displayed on the detection line, and products which are not captured are combined with the avidin-colloidal gold specific antibodies on the quality control line to display blue strips. The colors of the detection line and the quality control line are different according to the particle size of the colloidal gold and the reducing agent.
The invention will now be further described with reference to the accompanying drawings and specific embodiments.
Examples
1. Sample taking
And (5) reserving the sample to be detected to a sterile centrifuge tube by using sterile scissors for later use.
2. DNA extraction
And 5% Chelex-100 is adopted to extract the DNA of the meat sample to be detected.
3、RAA-LFD
3.1 specific sequence amplification primer and Probe design for bovine derived Components
Downloading a complete mitochondrial genome sequence of a cattle (NC-006853.1), taking a ND5/ND6 gene sequence of the cattle as a target gene, analyzing the characteristics of the cattle sequence and comparing the cattle sequence with common adulterated meat species sequences, wherein the cattle sequence comprises duck (NC-009684.1), pig (NC-000845.1), sheep (NC-001941.1) and chicken (NC-040902.1), and a Primer Premier 5.0 software is used for designing species-specific amplification primers. The probe sequence is located in the middle section of the amplification primer and is modified by tetrahydrofuran. Primer design follows the following principles:
(1) the PCR annealing temperatures of the primers among species are consistent, and the amplification product is 150-350 bp;
(2) the primer has species specificity, and can not perform non-specific amplification under a non-species template;
(3) the primers can be used universally in PCR and RAA;
(4) the length of the probe is about 45-60 bp; the primer amplification segment sequences were aligned using the BLAST function of the National Center for Biotechnology Information (NCBI) to complete the preliminary identification of primer amplification specificity.
A dSpacer Tetrahydrofuran (THF) was inserted into the probe sequence to mimic a basic site.
The identified primers are synthesized by Hangzhou Ongke catalpi and Xi biotechnology limited company, and the purity level is HPLC. The specific sequences of the primers, the labeling conditions of the probes and the primers downstream of the amplification, and the sizes of the amplified fragments are shown in Table 1 and FIG. 3 in detail.
TABLE 1 bovine-derived component in-situ detection kit RAA amplification primer sequence
Wherein, THF: tetrahydrofuran; FITC: fluorescein isothiocyanate; BIOTIN: labeling with biotin; PHO: phosphorylation modification.
3.2 RAA reaction System and reaction time
RAA reaction system was conducted as shown in Table 2Formulation (single sample/reaction). The system design is optimized through experiments. And sequentially adding 50 mu L of each component of the RAA reaction system into the RAA reaction universal dry powder tube: Tris-HCl buffer solution, upstream primer NIU-F, probe NIU-PRO, downstream primer NIU-R, sample DNA extract or positive control, ddH2O, MgAcO, isothermal in vitro amplification of nucleic acids was performed. The RAA reaction universal dry powder comprises: recombinase, Bst DNA polymerase, SSB protein, repair enzyme and dNTPs; purchased from Jiangsu Qitian gene biotechnology limited, 2 μ gRAA per tube of the general lyophilized powder for reaction, the reaction specification was 50 μ L. The concentration of Tris-HCl buffer was 300mM, pH 8.0. And finally adding MgAcO into the inner wall of the reaction tube cap, and then buckling the upper tube cap to perform the sample adding operation of the next sample RAA reaction system. Quality control sample ddH with negative sample adding sequence of experimental group and control group2And O, samples to be detected (bovine-derived component recombinant plasmids or bovine-derived component lysate to be detected in gradient dilution) are added, and a tube cover is required to be fastened immediately after each sample is added, so that aerosol pollution is avoided. The negative quality control sample is added firstly, so that aerosol pollution can be avoided to cause false positive of the quality control sample. Finally, the reaction tube was left to react at 39 ℃ for 25 min.
TABLE 2 RAA reaction System
| Components | Dosage (mu L) |
| Tris-HCl buffer | 29.5 |
| Upstream primer NIU-F (10. mu.M) | 2.1 |
| Probe NIU-PRO (10. mu.M) | 0.6 |
| Downstream primer NIU-R (10. mu.M) | 2.1 |
| Plasmid or |
2 |
| MgAcO(280mM) | 1 |
| Make up the volume with ddH2O to | 50 |
3.3 LFD operation
And after the RAA reaction is finished, opening the Eppendorf tube, sucking the amplified product into a new Eppendorf tube, marking, diluting by 50 times, and immediately carrying out test strip detection. The structure of the LFD test strip is shown in figure 4, nano gold particles of FAM antibody are arranged on a detection line at the front end of the LFD test strip, a biotin antibody is also arranged on the detection line, and an avidin-colloidal gold specific antibody is arranged on a quality control line.
The LFD operation steps are as follows: inserting the immersion area end (marked with blue arrow upwards) of the LFD test strip into an Eppendorf tube, ensuring that the liquid level does not exceed the MAX indicating line of the immersion area, completely immersing the area to be read (about 30-60sec), flatly placing the test strip for 1-3min, and waiting for a red strip to appear. And directly reading the detection result according to the color development condition of the test strip. The method can generate banding within 1min, the result is observed within 10min, and interpretation is invalid after 10 min.
The experimental judgment standard is shown in figure 5, and each test sample at least has one quality control line and has or does not have a detection line. Negative controls showed only one blue line (at quality control line C). Two lines appear in the positive control, one is located at the detection line (T) and is red, and the other is located at the quality control line (C) and is blue. Only one blue line appears on the quality control line, which indicates that no bovine-derived component exists in the sample or the copy number of the bovine-derived component is lower than the lowest detection limit of the kit. Two lines appear, one at the detection line (T) and the other at the quality control line (C), indicating the presence of bovine-derived components in the sample. No blue strip appeared in the quality control line, indicating that the nucleic acid test strip is ineffective.
Effect test example 1 sensitivity and specificity test
Construction of a standard plasmid:
plasmids of the target region were synthesized based on the sequence of the bovine ND5/ND6 gene in NCBI. After cloning and sequencing a target sequence by using the primers for detecting bovine-derived components, extracting positive clone plasmid DNA by using a plasmid DNA miniprep kit, and then measuring the OD of the plasmid DNA by using an ultraviolet spectrophotometer260Value according to the formula OD260The copy number of plasmid DNA is calculated according to the molecular weight of the recombinant plasmid corresponding to the dsDNA with the OD of 50 mu g/mL and the specific amplified fragment of the bovine-derived component, and the obtained plasmid DNA solution with different copy numbers is frozen at-20 ℃ for later use.
The copy number of the plasmid was obtained according to the following formula:
plasmid copy number-plasmid concentration × 6.02 × 1023/(660X Total plasmid length)
Copy number was calculated and diluted to 1X 108Copies/. mu.L were stored at-20 ℃ until use.
At 1 × 106Copy/. mu.L as control disc stock, run 10-1X comparison disc stock solution sample, 10-2X comparison disc stock solution sample, 10-3X comparison disc stock solution sample, 10-4X comparison disc stock solution sample, 10-5X comparison disc stock solution and 10-6And (5) comparing the disc stock solution sample by gradient dilution. Diluting the recombinant plasmid with a determined value to 1 × 105Copy/. mu.L, further 10-fold serial dilution to obtain 1X 100Copy/. mu.L, 1X 101Copy/. mu.L, 1X 102Copy/. mu.L, 1X 103Copy/. mu.L, 1X 104Copies/. mu.L and 1X 105Copy/. mu.L of the dilution was used as the template for subsequent amplification and stored at-20 ℃ until use.
RAA amplification and test strip detection are carried out by using the kit, and the lower detection limit of the obtained product is 1 multiplied by 102Copies/. mu.L. At an initial template of 1X 103～1×106In the case of copying/microliter, the test strip has a fast color development speed, and the test strip is inserted into a PCR reaction tube until the strip develops color for no more than 3 minutes. When the initial template copy number is 1 × 101～1×102In the case of copy/. mu.L, the T-line color development time of the positive sample is consistent with the high copy number, and the band is only slightly light, so that the sensitivity of the detection system can stably detect the initial template as low as 1X 101The entire detection process can be completed in 3 minutes for each μ L of RAA amplification product. Meanwhile, under the condition that pig, chicken, duck plasmids and ultrapure water are used as negative controls, the detection of the test strip shows negative. The results are shown in FIG. 6, the amplification initial templates of the negative test strips No. 1-3 are pork DNA, chicken DNA and duck DNA, respectively, and the amplification initial templates of the positive test strips No. 4-10 are bovine-derived component nucleic acid copy number of 100～106Copies/. mu.L.
FIG. 7 shows the results of the specificity evaluation of the RAA-LFD detection method (left) and the primer (right), and it can be seen that the RAA-LFD detection method provided by the present invention can successfully complete the specificity detection of bovine-derived components.
Effect test example 2 precision and repeatability test
Ultrapure water as a negative control, 1X 100Copy/. mu.L, 1X 101Copy/. mu.L, 1X 102Copies/. mu.L of the recombinant plasmid were used as template for 3 replicates (A, B and C). The results are shown in FIG. 8, and the detection system can stably detect the initial template as low as 1X 101Copying/mu L of RAA amplification product, ensuring that negative control has no false positive, and ensuring that the detection result is stable and reliable.
Effect test example 3 comparative verification example
The results of experimental verification using the RAA-LFD combination of the present invention in comparison with multiplex PCR are shown in Table 3 below.
TABLE 3 RAA-LFD combination and multiplex PCR assay results
As can be seen from the detection examples in Table 3, the RAA-LFD detection method provided by the invention can specifically detect bovine-derived components, has the advantages of visual detection of naked eye results, high sensitivity, simplicity and rapidness in operation, and can be completed in 20-30 minutes in the whole detection process.
Effect test example 4 RAA reaction System optimization evaluation
Different MgAcO concentrations in the RAA-LFD detection are evaluated by using a lateral flow test paper and electrophoresis, and the optimal RAA-LFD condition is optimized by adjusting the concentration of magnesium salt. First, experiments were performed on MgAcO (280 mM MgAcO stock solution of 1. mu.L, 1.5. mu.L, 2.0. mu.L, 2.5. mu.L, 2.8. mu.L) at 5 concentrations to explore the optimal concentration of Mg ions in RAA reaction to identify bovine-derived components. The reaction temperature at which we evaluated the range of MgAcO concentration was 39 ℃ and the reaction time was 20 min. As a result, as shown in FIG. 9, 5 concentrations of MgAcO can realize the identification of bovine-derived components, and the DNA bands and brightness of the experimental line change with the change of the concentration of MgAcO, which indicates that the concentration of MgAcO is important for the RAA amplification efficiency. The intensity and brightness test line of the DNA band showed an optimal 280mM MgAcO addition volume of 1. mu.L.
Different incubation times (15 min-25 min) of RAA-LFD detection are evaluated by using transverse flow test paper and electrophoresis, and the influence of the culture time on the detection specificity of the bovine-derived component in the RAA reaction system is examined at 39 ℃ and the optimal MgAcO concentration. As shown in FIG. 10, it was found from FIG. 10 that clear bands were observed at the amplification times of 15min to 25min, and that a clear test signal was observed when the amplification time was increased to 20min, so that 20min was selected as the optimum incubation time.
Different amplification temperatures (37 ℃ -42 ℃) of the RAA-LFD assay were evaluated using lateral flow dipstick and electrophoresis, and the incubation temperature was evaluated using the optimal MgAcO concentration and 20min incubation time to evaluate the effect of the incubation temperature on the specificity of detection of bovine-derived components in the RAA reaction system. As shown in FIG. 11, the RAA-LFD assays performed at 37, 39 and 42 ℃ all had distinct detection lines, indicating that the RAA reaction is suitable for use in this temperature range.
The present invention is not limited to the above-described embodiments, and various changes and modifications of the present invention are intended to be included within the scope of the claims and the equivalent technology of the present invention if they do not depart from the spirit and scope of the present invention.
Sequence listing
<110> Han mountain teaching college
<120> primer pair and probe for detecting bovine-derived components, kit and application thereof
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Claims (10)
1. A primer pair and a probe for detecting bovine-derived components are characterized in that the sequences are respectively as follows:
an upstream primer NIU-F: ACATCGCCTAGCTCCATACA, as shown in SEQ ID NO: 1;
a downstream primer NIU-R: FITC-AGTGAAGAGGCCATAGGGA as shown in SEQ ID NO. 2;
probe NIU-PRO:
BIOTIN-GGCCTGATCAAACTATATTTCCTCTCCTTC- (H) -TAATCACAATCCTTA-PHO, shown in SEQ ID NO: 3;
wherein FITC is a fluorescent group; BIOTIN is BIOTIN; h is a tetrahydrofuran site; PHO is a phosphorylation modification.
2. The kit for designing the primer pair and the probe for detecting the bovine-derived component according to claim 1, wherein the kit comprises an RAA reaction system and an LFD test strip; the detection line of the LFD test strip is provided with a biotin antibody and a fluorophore antibody, and the quality control line is provided with an avidin-colloidal gold specific antibody.
3. The kit according to claim 2, wherein the RAA reaction system comprises the following components by volume ratio added to RAA reaction general dry powder:
4. the kit according to claim 3, wherein the RAA reaction system is added with the RAA reaction general dry powder in an amount of 1 μ g/10-30 μ L; the RAA reaction universal dry powder comprises: recombinase, Bst DNA polymerase, SSB protein, repair enzyme and dNTPs.
5. The kit according to claim 3, wherein the MgAcO is present at a concentration of 280mM and is added in an amount of 2% by volume.
6. The kit of claim 3, wherein: the reaction condition of the kit is that the reaction is carried out for 15-25 min at 37-42 ℃.
7. The kit of claim 3, wherein the kit further comprises a positive control; the positive control is a plasmid for synthesizing a target region according to the sequence of the ND5/ND6 gene of cattle.
8. The kit of claim 7, wherein the preparation of the positive control comprises the steps of:
according to the sequence of ND5/ND6 gene of cattle in NCBI, after cloning and sequencing an upstream primer NIU-F and a downstream primer NIU-R by using a primer for detecting cattle-derived components, extracting positive clone plasmid DNA, and taking obtained plasmid DNA solution with different copy numbers as positive control;
the copy number includes, but is not limited to: 1X 100Copy/. mu.L, 1X 101Copy/. mu.L, 1X 102Copy/. mu.L, 1X 103Copy/. mu.L, 1X 104Copies/. mu.L and 1X 105Copy/. mu.L, 1X 106Copy/. mu.L, 1X 108Copies/. mu.L.
9. The use of the kit for detecting bovine-derived components according to any one of claims 2 to 8, wherein the kit is used for detecting bovine-derived components, and the detection comprises the following steps:
A. taking a sample: reserving a sample to be detected to a sterile centrifuge tube by using sterile scissors for later use;
B. DNA extraction: extracting DNA of a meat sample to be detected to obtain a sample DNA extracting solution;
C. RAA reaction system: each sample was formulated in the following volume percentages: 59% Tris-HCl buffer solution, 4.2% upstream primer NIU-F, 1.2% probe NIU-PRO, 4.2% downstream primer NIU-R, 4% sample DNA extracting solution or positive control or negative quality control sample, 21.8-25.4% ddH2Adding O into a reaction tube filled with RAA reaction general dry powder, and finally adding 2-5.6% of MgAcO for reaction; placing the reaction tube at 37-42 ℃ for reaction for 15-25 min to obtain an amplification product;
D. absorbing the amplification product into a new reaction tube, and diluting by 50-300 times;
E. and (3) LFD test strip detection: inserting the LFD test strip into the diluted reaction tube, and waiting for the strip to appear;
F. and directly reading the detection result according to the color development condition of the LFD test strip.
10. Use according to claim 9, characterized in that: in step C, the sample adding sequence between the experimental group and the control group is that the negative quality control sample ddH of the control group is firstly completed2And (3) preparing an RAA reaction system of O, and then preparing a DNA extracting solution of a sample of an experimental group and an RAA reaction system of a positive control group.
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