CN112941200A - Primer pair and probe for detecting duck-origin components, kit and application thereof - Google Patents
Primer pair and probe for detecting duck-origin components, kit and application thereof Download PDFInfo
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Abstract
The invention discloses a primer pair and a probe for detecting duck-origin 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 YA-F, downstream primer YA-R, probe YA-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 duck-origin component adulteration field detection method based on recombinase-mediated isothermal nucleic acid amplification technology side flow immunity technology, can realize visual detection of naked eye results, has high detection sensitivity compared with other detection methods,the 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 duck-origin 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 duck-origin components based on recombinase-mediated isothermal nucleic acid amplification technology-lateral flow immunity technology, a kit and application thereof.
Background
The method is reliable, efficient, rapid and accurate, can accurately identify animal species from meat products and prevent meat from becoming stale, and is imperative for protecting the rights and interests of consumers and avoiding unfair market competition.
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 operating 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 enables nucleic acid to be amplified under a constant temperature condition, expensive thermal cycling experimental instruments are eliminated, and exponential amplification of target fragments can be realized in a short time, so that nucleic acid amplification detection under various non-laboratory conditions is realized.
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 duck-origin components.
The invention also aims to provide a kit for detecting duck-derived components based on 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 duck-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 on-site detection of duck-origin components, have low technical requirements on operators and equipment requirements, are suitable for on-site rapid detection, 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 duck-origin components, wherein the sequences are respectively as follows:
the upstream primer YA-F: TCCTTCCCACAGTATCAATC, as shown in SEQ ID NO: 1;
the downstream primer YA-R: FITC-CGGCTAGCAGGATAGATGAGTT as shown in SEQ ID NO. 2;
probe YA-PRO:
BIOTIN-CCTAAGCCTGTACTTACAAGAAAACATCTA- (H) -TGGCACACCAAGCAC-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 duck-origin 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 fluorescent group 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 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 is a quality control line close to the handheld end liquid absorption area. 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 YA-F is 10 μ M; the concentration of the downstream primer YA-R is 10 mu M; the concentration of the probe YA-PRO was 10. mu.M.
Further, the concentration of MgAcO is 280mM, and the addition amount is 3% 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 of a target region synthesized according to the sequence of ATP6/COX3 genes of ducks.
Still further, the preparation of the positive control comprises the steps of:
according to the sequence of ATP6/COX3 gene of duck in NCBI, performing target sequence cloning sequencing on upstream primer YA-F and downstream primer YA-R by using primers for duck-derived component detection, extracting positive cloning plasmid DNA by using a plasmid DNA miniprep kit, and performing ultraviolet spectrophotometryThe OD of the plasmid DNA was determined260Value according to the formula OD260Calculating the copy number of plasmid DNA by the molecular weight of the dsDNA with the OD of 50 mu g/mL and the recombinant plasmid corresponding to the specific amplification fragment of the duck-derived component, and freezing and storing the obtained plasmid DNA solution with different copy numbers at the temperature of-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 duck-origin components, which is used for detecting duck-origin 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 solution, 4.2% upstream primer YA-F, 1.2% probe YA-PRO, 4.2% downstream primer YA-R, 4% sample DNA extracting solution 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 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 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 duck-origin component exists in the sample or the copy number of the sample is lower than the lowest detection limit of the kit; two lines are formed, one line is positioned on the detection line, and the other line is positioned on the quality control line, so that duck-origin 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 step C, the MgAcO is added in an amount of 3% 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 immediately after the MgAcO is added to prevent the aerosol from diffusing.
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 method is based on a recombinase-mediated isothermal nucleic acid amplification technology lateral flow immunization (RAA-LFD) technology, is a combined application of the RAA technology and the LFD technology, provides a novel duck-origin component adulteration field detection method, and can realize visual detection of naked eye 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, convenient 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 kit 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 the probes and primers for detecting duck-origin components 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 testing duck-origin component nucleic acid in the test 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 strip for detecting duck-derived component nucleic acid in effect test example 2 for the precision and repeatability of amplification;
FIG. 9 shows the results of evaluation of different MgAcO concentrations in RAA-LFD detection 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 evaluation of 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 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, and the color of the meat eye is different according to the particle size of the colloidal gold and the difference of 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 of duck-origin component
Downloading a complete sequence of a mitochondrial genome of a duck (NC-009684.1), taking a sequence of an ATP6/COX3 gene of the duck as a target gene, analyzing sequence characteristics of the duck and comparing the sequence characteristics with sequences of common adulterated meat species, wherein the sequences comprise a pig (NC-000845.1), a cow (NC-006853.1), a sheep (NC-001941.1) and a chicken (NC-040902.1), and a Primer Premier 5.0 software is used for designing a species-specific amplification Primer. The probe sequence is located in the middle section of the amplification primer and is modified by tetrahydrofuran. The primer design follows the following principle:
(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 Duck origin component on-site 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
The preparation of the RAA reaction system (single sample/reaction) was performed according to table 2. 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 YA-F, probe YA-PRO, downstream primer YA-R, sample DNA extracting solution or positive control, ddH2O, MgAcO, performing isothermal in vitro amplification of nucleic acids. 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. MgAcO is added into the inner wall of the reaction tube cap at last, and the next sample RAA is carried out only by buckling the upper tube cap after the MgAcO is addedAnd (4) sample adding operation of the reaction system. Quality control sample ddH with negative sample adding sequence of experimental group and control group2And O, samples to be detected (duck-origin component recombinant plasmids or duck-origin component to-be-detected sample cracking liquid in gradient dilution) are added, and a pipe cover is required to be buckled 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 YA-F (10. mu.M) | 2.1 |
| Probe YA-PRO (10. mu.M) | 0.6 |
| Downstream primer YA-R (10. mu.M) | 2.1 |
| Plasmid or |
2 |
| MgAcO(280mM) | 1.5 |
| 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 duck-origin component exists in the sample or the copy number of the duck-origin component is lower than the lowest detection limit of the kit. Two lines appear, one is located at the detection line (T), and the other is located at the quality control line (C), which indicates that the duck-origin components exist 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:
according to the sequence of ATP6/COX3 gene of duck in NCBI, a plasmid of the target region is synthesized. After cloning and sequencing a target sequence by using a primer for duck-derived component detection, extracting positive clone plasmid DNA by using a plasmid DNA miniprep kit, and then measuring OD of the plasmid DNA by using an ultraviolet spectrophotometer260Value according to the formula OD260Calculating the copy number of the 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 amplification fragment of the duck-derived component, and freezing the obtained plasmid DNA solution with different copy numbers at the temperature of-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/μ L dilution is used as the subsequent amplification template and stored at-20 ℃ for later 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 the initial template of 1 × 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, the test paper strips show negative detection under the condition that beef, chicken, pork plasmid and ultrapure water are used as negative controls. The result is shown in FIG. 6The amplification initial templates of the negative test strips No. 1-3 are beef DNA, chicken DNA and pork DNA respectively, and the amplification initial templates of the positive test strips No. 4-10 are duck-origin 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 the duck-origin component.
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). As shown in FIG. 8, the assay 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 duck-origin 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 duck-derived components. The reaction temperature at which we evaluated the range of MgAcO concentration was 39 ℃ and the reaction time was 20 min. The results are shown in fig. 9, identification of duck-derived components can be realized by 5 concentrations of MgAcO, and the DNA band and brightness of the experimental line change with the change of MgAcO concentration, which indicates that MgAcO concentration is important for RAA amplification efficiency. The intensity and brightness test line of the DNA band showed an optimal 280mM MgAcO addition volume of 1.5. mu.L.
And evaluating different incubation times (15-25 min) of RAA-LFD detection by using transverse flow test paper and electrophoresis, and investigating the influence of the culture time on the detection specificity of the duck-derived component in an RAA reaction system 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 RAA-LFD detection are evaluated by using a transverse flow test paper and electrophoresis, and the incubation temperature is evaluated by using the optimal MgAcO concentration and 20 minutes of incubation time so as to evaluate the influence of the culture temperature on the detection specificity of duck-derived ingredients in an RAA reaction system. As shown in FIG. 11, no difference in the test line was observed when RAA-LFD detection was carried out at 37, 39 and 42 ℃ and there were clear detection lines, indicating that the RAA reaction was suitably carried out 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 duck-origin components, kit and application thereof
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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cctaagcctg tacttacaag aaaacatcta tggcacacca agcac 45
Claims (10)
1. A primer pair and a probe for detecting duck-origin components are characterized in that the sequences are respectively as follows:
the upstream primer YA-F: TCCTTCCCACAGTATCAATC, as shown in SEQ ID NO: 1;
the downstream primer YA-R: FITC-CGGCTAGCAGGATAGATGAGTT as shown in SEQ ID NO. 2;
probe YA-PRO:
BIOTIN-CCTAAGCCTGTACTTACAAGAAAACATCTA- (H) -TGGCACACCAAGCAC-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 duck-origin components 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 3% 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 of a target region synthesized according to the sequence of ATP6/COX3 genes of ducks.
8. The kit of claim 7, wherein the preparation of the positive control comprises the steps of:
according to the sequence of the ATP6/COX3 gene of the duck in NCBI, performing target sequence cloning sequencing on an upstream primer YA-F and a downstream primer YA-R by using a primer for duck-derived component detection, extracting positive cloning plasmid DNA, and taking obtained plasmid DNA solutions 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 application of the kit for detecting duck-origin components as claimed in any one of claims 2 to 8, which is used for detecting duck-origin 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% of Tris-HCl buffer solution, 4.2% of upstream primer YA-F, 1.2% of probe YA-PRO, 4.2% of downstream primer YA-R, 4% of sample DNA extracting solution or positive control or negative quality control sample, 21.8-25.4% of 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 completed20, 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.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114686600A (en) * | 2022-02-24 | 2022-07-01 | 宁波大学 | Primer group and method for meat detection based on seven-fold PCR technology |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103757097A (en) * | 2013-12-10 | 2014-04-30 | 天津出入境检验检疫局动植物与食品检测中心 | Lateral flow test paper strip detection kit for detection of duck source component in food and feed, and application thereof |
| CN110205407A (en) * | 2019-06-21 | 2019-09-06 | 北京市水产技术推广站 | Quickly the RAA amplimer and probe of detection carp edema virus and detection kit and application method |
| CN111139302A (en) * | 2020-01-03 | 2020-05-12 | 华中农业大学 | On-site rapid detection method and detection test strip for bovine-derived and duck-derived components |
| CN111876505A (en) * | 2020-08-06 | 2020-11-03 | 上海海洋大学 | Staphylococcus aureus amplification primer based on RAA-LF technology and application thereof |
-
2021
- 2021-02-08 CN CN202110171460.0A patent/CN112941200B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103757097A (en) * | 2013-12-10 | 2014-04-30 | 天津出入境检验检疫局动植物与食品检测中心 | Lateral flow test paper strip detection kit for detection of duck source component in food and feed, and application thereof |
| CN110205407A (en) * | 2019-06-21 | 2019-09-06 | 北京市水产技术推广站 | Quickly the RAA amplimer and probe of detection carp edema virus and detection kit and application method |
| CN111139302A (en) * | 2020-01-03 | 2020-05-12 | 华中农业大学 | On-site rapid detection method and detection test strip for bovine-derived and duck-derived components |
| CN111876505A (en) * | 2020-08-06 | 2020-11-03 | 上海海洋大学 | Staphylococcus aureus amplification primer based on RAA-LF technology and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| TWISTDX: "TwistAmp® DNA Amplifcation Kits Assay Design Manual", 《TWISTDX.CO.UK》 * |
| 林彦星等: "实时荧光定量PCR检测畜禽肉制品中鸭源性成分", 《动物医学进展》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114686600A (en) * | 2022-02-24 | 2022-07-01 | 宁波大学 | Primer group and method for meat detection based on seven-fold PCR technology |
| CN114686600B (en) * | 2022-02-24 | 2023-12-12 | 宁波大学 | Primer group and method for meat detection based on seven-fold PCR technology |
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