CN112708681B - Primer pair and probe for detecting chicken-derived components, and kit and application thereof - Google Patents
Primer pair and probe for detecting chicken-derived components, and kit and application thereof Download PDFInfo
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Abstract
The invention discloses a primer pair and a probe for detecting chicken-derived components, and discloses a kit designed according to the primer pair and the probe, wherein the kit comprises a RAA reaction system and an LFD test strip; the RAA reaction system comprises RAA reaction general dry powder, tris-HCl buffer solution, an upstream primer JI-F, a downstream primer JI-R, a probe JI-PRO, a sample DNA extracting solution, mgAcO and ddH 2 O; the LFD test strip has an avidin-colloid Jin Teyi antibody on a quality control line and a biotin antibody and a fluorescent group antibody on a detection line. The invention provides a new chicken-origin component adulterated on-site detection method based on a recombinase-mediated isothermal nucleic acid amplification technology lateral flow immunity technology, which can realize visual detection of naked eye results.
Description
Technical Field
The invention relates to a primer pair and a probe for detecting chicken-origin components, and a kit and application thereof, belongs to the field of biotechnology and food safety rapid detection, and in particular relates to a primer pair and a probe for detecting chicken-origin components based on a recombinase-mediated isothermal nucleic acid amplification technology-a lateral flow immunological technology, and a kit and application thereof.
Background
Meat and meat products are considered to be an important source of human proteins and have evolved into a basic dietary ingredient as its enjoyed taste and flavor are widely consumed throughout the world. An unscrupulous producer may attempt to replace beef, mutton, and other expensive meats with chicken due to the temptation of economic profit (adulteration of economic incentives, EMA). Meat adulteration events occur worldwide. The horse-meat smell occurring in the united kingdom and europe in 2013 (the so-called "Ma Men" smell) is one of the most well-known food fraud and pollution events, which demonstrates the vulnerability inherent in the currently complex international food supply chain. Meat adulteration directly threatens the health of consumers, weakens the credit of processing enterprises, and damages trade order and fair competition; in addition, it may violate dietary contraindications in certain countries or nations. Therefore, in order to protect the rights of consumers and avoid unfair market competition, a reliable, efficient, quick and accurate method is provided, the animal species can be accurately identified from meat products, and the condition of meat smell change is prevented.
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 methods are 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 skill, expensive equipment and lengthy procedures, and these conditions or resource limitations limit the detection applications of food meats.
In recent years, there have been developed isothermal amplification techniques of nucleic acids, mainly nucleic acid-dependent amplification detection techniques (Nuclear acid sequence-based amplification, NASBA), loop-mediated isothermal amplification techniques (loop-mediated isothermal amplification, LAMP), helicase-dependent DNA isothermal amplification techniques (Helicase-dependent Isothermal DNA Amplification, HDA), rolling circle nucleic acid amplification techniques (rolling circle amplification, RCA), recombinase polymerase amplification techniques (Recombinase Polymerase Amplification, RPA), recombinase-mediated isothermal nucleic acid amplification techniques (Recombinase Aided Amplification, RAA), and the like. The basic principle is to imitate in vivo nucleic acid replication mechanism, and to participate in the reaction by various proteases to help DNA polymerase replicate DNA so as to realize isothermal amplification of DNA. Compared with PCR, the isothermal amplification technology enables nucleic acid to be amplified under the constant temperature condition, gets 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 is realized.
Recombinase-assisted amplification is combined with lateral flow immunoassay (Recombinase Assisted Amplification-Lateral Flow Device, RAA-LFD), i.e., a combination of recombinase-assisted amplification (RAA) and lateral flow immunoassay (LFD). The method is suitable for rapidly detecting the adulteration of the meat on the detection site (supermarket, meat market and the like) based on the RAA-LFD technology.
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 chicken-derived components.
The invention also aims to provide a kit for detecting chicken-derived components based on a recombinase-mediated isothermal nucleic acid amplification technology-lateral flow immunoassay technology according to the amplification primer pair and the probe.
Another object of the invention is to provide the application of the kit for detecting chicken-derived components based on the recombinase-mediated isothermal nucleic acid amplification technology-lateral flow immunoassay technology.
The kit and the detection method thereof are simple, convenient and quick, are suitable for detecting chicken-derived components on site, have lower technical requirements and equipment requirements on operators, are suitable for on-site quick detection, and have wide application prospects in quick detection of food safety.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides an amplification primer pair and a probe for detecting chicken-derived components, which have the following sequences:
the upstream primer JI-F: CCAGCAAATTATAGACCCAC, as shown in SEQ ID NO. 1;
the downstream primer JI-R: FITC-GATTGTTGGTAGGATGATCT, shown as SEQ ID NO. 2;
probe JI-PRO:
BIOTIN-CCTCTCAATCTGACCAGTAGAAAACCAAAC- (H) -CCATCATTCGCCCTT-PHO, as shown in SEQ ID NO. 3;
wherein FITC is a fluorescent group; BIOTIN is BIOTIN; h is tetrahydrofuran locus; PHO is a phosphorylation modification.
A 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 an amplification primer pair and a probe for detecting chicken-derived components, which comprises a 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-colloid Jin Teyi antibody.
Further, the fluorescent group antibody is a gold nanoparticle of a FAM antibody.
The primers and probes with biotin marks and 6-carboxyl fluorescence hormone FAM marks are subjected to amplification reaction with target nucleic acid, and the product is an amplicon with both biotin and FAM marks. The front end of the LFD test strip is a detection line close to the immersion area, and the rear end close to the liquid absorption area at the hand-held end is a quality control line. The detection line at the front end of the LFD test strip is provided with nano gold particles of FAM antibody, the detection line is also provided with biotin antibody, amplified products are dripped on the test strip, FAM groups on the amplicon react with the FAM antibody, after the biotin antibody on the detection line is combined with biotin on the amplicon, the detection line displays a strip, and the uncaptured products are combined with avidin-colloid Jin Teyi antibody on the quality control line to display the strip.
FAM markers may also be used by selecting other markers as appropriate, such as digoxin markers.
The detection line and the quality control line are colored, the color seen by naked eyes is changed according to the selection change of the colloid Jin Lijing and the reducing agent, the detection result is not affected, and the proper colloid Jin Lijing and the reducing agent can be selected according to the requirement.
Further, the RAA reaction system comprises the following components in volume ratio added into the RAA reaction general dry powder:
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 general dry powder comprises the following components: recombinase, bst DNA polymerase, SSB protein, repair enzyme, dNTPs.
The RAA reaction general dry powder is RAA basic general reaction reagent freeze-dried powder. For example, 2 mug of lyophilized powder per tube of Jiangsu Qiyan gene biotechnology Co., ltd, the reaction specification is 50 mug, i.e., 2 mug of RAA-based universal reagent lyophilized powder is added per 50 mug of reaction system. The skilled artisan can select the 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 JI-F is 10. Mu.M; the concentration of the downstream primer JI-R was 10. Mu.M; the concentration of the probe JI-PRO was 10. Mu.M.
Further, the concentration of MgAcO is 280mM, and the addition amount is 3% by volume.
Further, the reaction condition of the kit is that the reaction is carried out for 15-25 min at 37-42 ℃.
Further, the kit also comprises a positive control; the positive control is a plasmid which synthesizes a target region according to the sequence of the ND4L gene of the chicken.
Still further, the preparation of the positive control comprises the steps of:
according to the sequence of ND4L gene of chicken in NCBI, using the primer for detecting chicken origin component to clone and sequence the target sequence of upstream primer JI-F and downstream primer JI-R, using plasmid DNA small quantity extraction kit to extract positive clone plasmid DNA, using ultraviolet spectrophotometer to measure OD of plasmid DNA 260 Value according to formula OD 260 1 OD=50 mug/mL dsDNA, and molecular weight of recombinant plasmid corresponding to specificity amplified fragment of chicken origin component, calculating plasmid DNA copy number, and freezing the obtained plasmid DNA solution with different copy number at-20 deg.C for standby;
the calculation formula of the plasmid DNA copy number is as follows:
plasmid copy number = plasmid concentration x 6.02 x 10 23 /(660. Times. Plasmid total length);
the copy number includes, but is not limited to: 1X 10 0 Copy/. Mu.L, 1X 10 1 Copy/. Mu.L, 1X 10 2 Copy/. Mu.L, 1X 10 3 Copy/. Mu.L, 1X 10 4 Copy/. Mu.L and 1X 10 5 Copy/. Mu.L, 1X 10 6 Copy/. Mu.L, 1X 10 8 Copy/. Mu.L.
The invention provides application of the kit for detecting chicken-derived ingredients, which is used for detecting chicken-derived ingredients and comprises the following steps of:
A. sample retention: reserving a sample to be detected by using sterile scissors to a sterile centrifuge tube 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 JI-F, 1.2% probe JI-PRO, 4.2% downstream primer JI-R, 4% sample DNA extract or positive control or negative quality control sample, 21.8-25.4% ddH 2 O is added into a reaction tube of the split RAA reaction general dry powder in sequence, and finally 2 to 5 is added.6% MgAcO for reaction; placing the reaction tube at 37-42 ℃ to react for 15-25 min to obtain an amplification product;
D. sucking the amplified product into a new reaction tube, and diluting 50-300 times;
E. LFD test strip detection: inserting the immersion area end of the LFD test strip into the diluted reaction tube, soaking the area to be interpreted, flatly placing the test strip for 1-3min, and waiting for the strip to appear;
F. and 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 chicken-derived component exists in the sample, or the copy number of the chicken-derived component is lower than the lowest detection limit of the kit; two lines appear, one is positioned on the detection line, and the other is positioned on the quality control line, so that the existence of chicken-origin components in the sample is indicated; no band appears on the quality control line, indicating that the nucleic acid test strip is invalid.
Further, in the step C, the addition amount of the MgAcO is 3% by volume.
In step C, mgAcO is finally added into the inner wall of the reaction tube cover, and then the tube cover cap is buckled after the MgAcO is added, so that the sample adding operation of the RAA reaction system of the next sample is performed. Mainly because MgAcO is added to start the reaction to prevent the dispersion of aerosol.
Further, 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 finished first 2 0, preparing the RAA reaction system, and preparing the RAA reaction system of the experimental group sample DNA extracting solution and the positive control group.
Further, the LFD test strip was observed within 10min, and after 10min the reading was not valid.
Compared with the prior art, the invention has the following beneficial effects:
the patent provides a new chicken-origin component adulteration on-site detection method based on a recombinase-mediated isothermal nucleic acid amplification (RAA-LFD) technology, which is the combined application of the RAA technology and the LFD technology, 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 1×10 1 Copy/. Mu.L of RAA amplification productThe method is simple and rapid to operate, the whole detection process can be completed within 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 test procedure for a side-stream immunological technique of the recombinase-mediated isothermal nucleic acid amplification technique of the invention;
FIG. 2 is a schematic diagram of a side-stream immunological technique of the recombinase-mediated isothermal nucleic acid amplification technique of the present invention;
in the figure: FIG. A is a two-way primer recombinase polymerase amplification; panel B shows the amplification reaction of a probe with a target nucleic acid; panel C is a lateral flow immunological detection;
FIG. 3 is a schematic diagram showing the positions of probes and primers for detecting a chicken-derived component in the examples;
FIG. 4 is a schematic diagram of a disposable nucleic acid test strip according to an embodiment;
FIG. 5 is a schematic diagram showing the result interpretation of a disposable nucleic acid test strip in the examples;
FIG. 6 shows the sensitivity and specificity test results of the chicken-derived component nucleic acid test strip detection system in effect test example 1;
FIG. 7 shows the results of RAA-LFD test method and primer specificity evaluation in effect test example 1;
FIG. 8 shows the results of amplification accuracy and repeatability of the chicken-derived component nucleic acid test strip detection system of effect test example 2;
FIG. 9 is a graph showing the results of evaluation of different MgAcO concentrations in RAA-LFD test using lateral flow dipsticks (left) and electrophoresis (right) in effect test example 4;
FIG. 10 shows the results of evaluation of different incubation times in RAA-LFD test using lateral flow dipsticks (left) and electrophoresis (right) in effect test example 4;
FIG. 11 shows the results of evaluation of different amplification temperatures in RAA-LFD test using lateral flow dipsticks (left) and electrophoresis (right) in effect test example 4.
Detailed Description
The detection procedure of the side-stream immunological technology of the recombinase-mediated isothermal nucleic acid amplification technology is shown in fig. 1, and the specific working principle is as follows:
the side-stream immunological technique (Recombinase Aided Amplification-lateral flow device, RAA-LFD) of the recombinase-mediated isothermal nucleic acid amplification technique refers to a combined technique of combining the recombinase-mediated isothermal nucleic acid amplification technique (RAA) with a lateral flow immunological test strip (LFD), and the principle of the combined technique is shown in figure 2: the primers and probes marked by FAM with biotin marks and 6-carboxyl fluorescence hormone are used for carrying out amplification reaction with the target nucleic acid, and finally the amplicon with FAM groups and biotin marks is formed. The front end of the LFD test strip is a detection line close to the immersion area, and the rear end close to the liquid absorption area at the hand-held end is a quality control line. The detection line at the front end of the LFD test strip is provided with nano gold particles of FAM antibodies, the detection line is also provided with biotin antibodies, amplified products are dripped on the test strip, FAM groups on the amplicon react with the FAM antibodies on the detection line, the biotin antibodies at the detection line are combined with biotin on the amplicon, a red band is displayed on the detection line, and the uncaptured products are combined with avidin-colloid Jin Teyi antibodies at the quality control line to display a blue band. The detection line and the quality control line are colored, and the colors seen by naked eyes are different according to the difference of the colloid Jin Lijing and the reducing agent.
The invention will now be further described with reference to the drawings and specific examples.
Examples
1. Sample retention
And (5) reserving the sample to be detected by using sterile scissors to a sterile centrifuge tube for later use.
2. DNA extraction
The DNA of the meat sample to be tested was extracted using 5% Chelex-100.
3、RAA-LFD
3.1 design of specific sequence amplification primers and probes for chicken-derived Components
Downloading complete mitochondrial genome sequence of chicken (NC_ 040902.1), taking the sequence of chicken ND4L gene as a target gene, analyzing the characteristics of chicken sequence and comparing with common adulterated meat species sequences, including duck (NC_ 009684.1), cow (NC_ 006853.1), sheep (NC_ 001941.1) and pig (NC_ 000845.1), and designing a species-specific amplification Primer by using Primer Premier 5.0 software. The probe sequence is positioned in the middle section of the amplification primer and is modified by tetrahydrofuran. Primer design follows the following principle:
(1) The PCR annealing temperature of the inter-species primers is consistent, and the amplified product is 150-350 bp;
(2) The primer has species specificity, and non-specific amplification can not occur under a non-native species template;
(3) Primers can be used commonly in PCR and RAA;
(4) The probe length is about 45-60 bp; the initial identification of primer amplification specificity was accomplished by alignment of primer amplification segment sequences using BLAST function of the National Center for Biotechnology Information (NCBI).
A dSpacer Tetrahydrofuran (THF) was inserted into the probe sequence to mimic a basic site.
The identified primer is synthesized by the catalpa ovata biological technology limited company in Qingzhou, and the purity grade is HPLC. The specific sequences of the primers, the probes and the labeling conditions of the amplified downstream primers and the sizes of the amplified fragments are shown in Table 1 and FIG. 3 in detail.
TABLE 1 on-site detection kit RAA amplification primer sequences for chicken-derived components
Wherein, THF: tetrahydrofuran; FITC: fluorescein isothiocyanate fluorescein isothiocyanate; BIOTIN: labeling with biotin; PHO: phosphor ylation 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. Sequentially adding 50 mu L of each component of the RAA reaction system into a RAA reaction general dry powder tube: tris-HCl buffer, upstream primer JI-F, probe JI-PRO, downstream primer JI-R, sampleDNA extract or positive control, ddH 2 O, mgAcO, isothermal in vitro amplification of nucleic acids was performed. The RAA reaction general dry powder comprises: recombinase, bst DNA polymerase, SSB protein, repair enzyme, dNTPs; 2. Mu.g of RAA reaction universal lyophilized powder per tube purchased from Jiangsu Qiyan Gene biotechnology Co., ltd, was reacted at a specification of 50. Mu.L. Tris-HCl buffer concentration was 300mM, pH=8.0. And finally, mgAcO is added into the inner wall of the reaction tube cover, and then the tube cover cap is buckled after the MgAcO is added, so that the sample adding operation of the RAA reaction system of the next sample is performed. The sample adding sequence of the experimental group and the control group is negative quality control sample ddH 2 O, the sample to be detected (the gradient diluted chicken-origin component recombinant plasmid or chicken-origin component sample lysate) needs to be buckled with a tube cover immediately after each sample is added, so that aerosol pollution is avoided. The negative quality control sample can be added first to avoid aerosol pollution and cause false positive of the quality control sample. Finally, the reaction tube is placed at 39 ℃ for reaction for 25min.
TABLE 2RAA reaction System
| Component (A) | Dosage (mu L) |
| Tris-HCl buffer | 29.5 |
| The upstream primer JI-F (10. Mu.M) | 2.1 |
| Probe JI-PRO (10. Mu.M) | 0.6 |
| Downstream primer JI-R (10. Mu.M) | 2.1 |
| Plasmid or sample lysate | 2 |
| MgAcO(280mM) | 1.5 |
| With ddH 2 O complements the volume to | 50 |
3.3 LFD operations
After the RAA reaction is finished, the Eppendorf tube is opened, the amplified product is sucked into a new Eppendorf tube, marked and diluted by 50 times, and the test strip detection is immediately carried out. The structure of the LFD test strip is shown in figure 4, the front end detection line of the LFD test strip is provided with nanometer gold particles of FAM antibody, the detection line is also provided with biotin antibody, and the quality control line is provided with avidin-colloid Jin Teyi antibody.
The LFD operation steps are as follows: inserting the end of the immersion area (marked with blue arrow upwards) of the LFD test strip into the Eppendorf tube, wherein the liquid level cannot exceed the MAX indication line of the immersion area, and keeping the test strip flat for 1-3min after the whole immersion area is immersed (about 30-60sec is needed), and waiting for the appearance of red stripes. And directly reading the detection result according to the color development condition of the test strip. The method can generally generate a strip within about 1min, the result is observed within 10min, and the interpretation is invalid after 10 min.
The experimental judgment standard is shown in fig. 5, and at least one quality control line, with or without detection lines, appears in each test sample. Only one blue line appears for the negative control (at quality control line C). Two lines appear in the positive control, one is located in the detection line (T) and is red, and the other is located in the quality control line (C) and is blue. Only one blue line appears on the quality control line, which indicates that the sample has no chicken-derived component or the copy number is lower than the lowest detection limit of the kit. Two lines appear, one located in the detection line (T) and the other located in the quality control line (C), indicating the presence of chicken-derived components in the sample. The quality control line showed no blue band, indicating failure of the nucleic acid test strip.
Effect test example 1 sensitivity and specificity test
Standard plasmid construction:
plasmids of the target region were synthesized based on the sequence of the ND4L gene of chickens in NCBI. After clone sequencing of target sequence by using primers for detecting chicken-derived components, extracting positive clone plasmid DNA by using a plasmid DNA small-quantity extraction kit, and measuring OD of the plasmid DNA by using an ultraviolet spectrophotometer 260 Value according to formula OD 260 1 OD=50 mug/mL dsDNA, and molecular weight of recombinant plasmid corresponding to specific amplified fragment of chicken origin component, calculating plasmid DNA copy number, and freezing the obtained plasmid DNA solution with different copy numbers at-20 deg.C for standby.
The copy number of the plasmid was obtained according to the following formula:
plasmid copy number = plasmid concentration x 6.02 x 10 23 /(660X total length of plasmid)
Calculate copy number and dilute to 1×10 8 Copy/. Mu.L, store at-20deg.C for later use.
At 1X 10 6 Copy/. Mu.L as the master for the control disk 10 -1 Sample of stock solution of X comparison disk, 10 -2 Sample of stock solution of X comparison disk, 10 -3 Sample of stock solution of X comparison disk, 10 -4 Sample of stock solution of X comparison disk, 10 -5 X comparison of disc stock solution and 10 -6 The original liquid sample of the X comparison disc is subjected to gradient dilution and comparison. Diluting the recombinant plasmid with a fixed value to 1X 10 5 Copy/. Mu.L, serial 10-fold dilution to obtain 1X 10 0 Copy/. Mu.L, 1X 10 1 Copy/. Mu.L, 1X 10 2 Copy/. Mu.L, 1X 10 3 Copy/. Mu.L, 1X 10 4 Copy/. Mu.L and 1X 10 5 The copy/. Mu.L of the diluent is used as a follow-up amplification template, and the template is preserved at-20 ℃ for standby.
RAA amplification and test strip detection are carried out by using the kit, and the detection lower limit of the obtained product is 1 multiplied by 10 2 Copy/. Mu.L. At the initial template of 1×10 3 ～1×10 6 In the case of copy/. Mu.L, the color development speed of the test strip is very high, and the color development time of the test strip is not more than 3 minutes from the time of inserting the test strip into the PCR reaction tube. When the initial template copy number is 1×10 1 ～1×10 2 Copy/. Mu.LUnder the condition that the T line color development time of the positive sample is consistent with the high copy number, the strip is only slightly shallow, thus the sensitivity of the detection system can stably detect the initial template as low as 1X 10 1 The whole detection process can be completed in 3 minutes by copying/mu L of RAA amplification product. Meanwhile, under the condition that plasmids of pigs, cattle and ducks and ultrapure water are used as negative controls, the detection of the test paper strip is negative. As shown in FIG. 6, the amplification initial templates of the negative test strips 1-3 are pork DNA, beef DNA and duck DNA respectively, and the amplification initial templates of the positive test strips 4-10 are chicken-derived component nucleic acid with copy number of 10 respectively 0 ～10 6 Copy/. Mu.L.
FIG. 7 shows the results of specific evaluation of RAA-LFD detection method (left) and primer (right), and it can be seen that the RAA-LFD detection method provided by the invention can successfully complete the specific detection of chicken-derived components.
Effect test example 2 precision and repeatability test
With ultrapure water as negative control, 1X 10 0 Copy/. Mu.L, 1X 10 1 Copy/. Mu.L, 1X 10 2 Copy/. Mu.L of recombinant plasmid was used as template and 3 replicates were performed (A, B and C). As shown in FIG. 8, the detection system can stably detect the initial template as low as 1×10 1 Copy/. Mu.L of RAA amplification product, and ensure that the negative control has no false positive, and the detection result is stable and reliable.
Effect test example 3 comparative verification example
Experimental verification of comparison with multiplex PCR using the RAA-LFD technique of the present invention, the results are shown in Table 3 below.
TABLE 3RAA-LFD combination technique and multiplex PCR detection results
As can be seen from the detection examples in Table 3, the RAA-LFD detection method provided by the invention can specifically detect chicken-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.
Effect test example 4 RAA reaction System optimization evaluation
Different MgAcO concentrations in RAA-LFD assays were evaluated using lateral flow test paper and electrophoresis, with magnesium salt concentrations adjusted to optimize the best RAA-LFD conditions. First, experiments were performed on 5 concentrations of MgAcO (1. Mu.L, 1.5. Mu.L, 2.0. Mu.L, 2.5. Mu.L, 2.8. Mu.L of 280mM MgAcO stock solution) to search for the optimal concentration of Mg ions in the RAA reaction to identify chicken-derived components. We assessed the reaction temperature in the MgAcO concentration range as 39℃and the reaction time as 20min. As shown in FIG. 9, the identification of chicken-derived components can be realized by 5 concentrations of MgAcO, and the DNA band and the brightness of the experimental line change along with the change of the concentration of MgAcO, which shows that the concentration of MgAcO is critical to the amplification efficiency of RAA. The intensity and brightness test line of the DNA bands showed an optimal 280mM MgAcO addition volume of 1.5. Mu.L. Therefore, mgAcO (280 mM) was added to a volume of 1.5. Mu.L, which was the most specific for detection of chicken-derived components, in a 50. Mu.L RAA reaction system.
Different incubation times (15 min-25 min) of RAA-LFD detection were evaluated using lateral flow test paper and electrophoresis, and the effect of incubation time on chicken-derived component detection specificity in the RAA reaction system was examined at 39℃and optimal MgAcO concentration. As a result, as shown in FIG. 10, we found that clear bands were seen at the amplification times of 15min to 25min, and that a clear test signal was seen at the amplification time increased to 20min, so we selected 20min as the optimal incubation time.
Different amplification temperatures (37 ℃ C. -42 ℃ C.) for RAA-LFD detection were assessed using lateral flow test paper and electrophoresis, and the incubation temperature was assessed using optimal MgAcO concentrations and incubation times of 20 minutes to assess the effect of incubation temperature on chicken-derived component detection specificity in the RAA reaction system. As shown in FIG. 11, no difference in the test lines was observed when RAA-LFD was performed at 37, 39 and 42℃and there was a clear detection line, indicating that it is appropriate to perform the RAA reaction in this temperature range.
The present invention is not limited to the above-described embodiments, but it is intended that the present invention also includes modifications and variations if they fall within the scope of the claims and the equivalents thereof, if they do not depart from the spirit and scope of the present invention.
Sequence listing
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Claims (8)
1. A kit for detecting a primer pair and probe design of chicken-derived components is characterized by comprising a RAA reaction system and an LFD test strip; the detection line of the LFD test strip is provided with a biotin antibody and a fluorescent group antibody, and the quality control line is provided with an avidin-colloid Jin Teyi antibody;
the RAA reaction system contains an amplification primer pair and a probe for detecting chicken-derived components, and the sequences are as follows:
the upstream primer JI-F: CCAGCAAATTATAGACCCAC, as shown in SEQ ID NO. 1;
the downstream primer JI-R: FITC-GATTGTTGGTAGGATGATCT as shown in SEQ ID NO. 2;
probe JI-PRO:
BIOTIN-CCTCTCAATCTGACCAGTAGAAAACCAAAC- (H) -CCATCATTCGCCCTT-PHO, as shown in SEQ ID NO. 3;
wherein FITC is a fluorescent group; BIOTIN is BIOTIN; h is tetrahydrofuran locus; PHO is a phosphorylation modification;
the RAA reaction system comprises the following components in volume ratio added into RAA reaction general dry powder:
Tris-HCl buffer 59%;
the upstream primer JI-F4.2%;
1.2% of the probe JI-PRO;
the downstream primer JI-R4.2%;
4% of sample DNA extract;
MgAcO 2~5.6%；
ddH 2 O 21.8~25.4% 。
2. the kit according to claim 1, wherein the addition amount of the RAA reaction universal dry powder in the RAA reaction system is 1 mug/10-30 mug; the RAA reaction general dry powder comprises the following components: recombinase, bst DNA polymerase, SSB protein, repair enzyme, dNTPs.
3. The kit according to claim 1, wherein the concentration of MgAcO is 280mM and the addition amount is 3% by volume.
4. The kit of claim 1, wherein: the reaction condition of the kit is that the reaction is carried out for 15-25 min at 37-42 ℃.
5. The kit of claim 1, wherein the kit further comprises a positive control; the positive control is a plasmid which synthesizes a target region according to the sequence of the ND4L gene of the chicken.
6. The kit of claim 5, wherein the preparation of the positive control comprises the steps of:
according to the sequence of ND4L gene of chicken in NCBI, cloning and sequencing target sequences by using a primer pair for detecting chicken origin components, namely an upstream primer JI-F and a downstream primer JI-R, extracting positive cloning plasmid DNA, and taking obtained plasmid DNA solutions with different copy numbers as positive control;
the copy number is: 1X 10 0 Copy/. Mu.L, 1X 10 1 Copy/. Mu.L, 1X 10 2 Copy/. Mu.L, 1X 10 3 Copy/. Mu.L, 1X 10 4 Copy/. Mu.L and 1X 10 5 Copy/. Mu.L, 1X 10 6 Copy/. Mu.L, 1X 10 8 Copy/. Mu.L.
7. Use of a kit for detecting a chicken-derived ingredient as claimed in any one of claims 1 to 6, characterized in that it is used for detecting a chicken-derived ingredient, said detection comprising the steps of:
A. sample retention: reserving a sample to be detected by using sterile scissors to a sterile centrifuge tube 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 JI-F, 1.2% probe JI-PRO, 4.2% downstream primer JI-R, 4% sample DNA extract or positive control or negative quality control sample, 21.8-25.4% ddH 2 O is added into a reaction tube of the split RAA reaction general dry powder, and finally 2 to 5.6 percent of MgAcO is added for reaction; placing the reaction tube at 37-42 ℃ to react for 15-25 min to obtain an amplification product;
D. sucking the amplified product into a new reaction tube, and diluting 50-300 times;
E. 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 a detection result according to the color development condition of the LFD test strip.
8. The use according to claim 7, characterized in that: in the 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 finished firstly 2 0, preparing the RAA reaction system, and preparing the RAA reaction system of the experimental group sample DNA extracting solution and the positive control group.
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