A rainbow trout-derived component primer, detection system, and method based on RPA-CRISPR

Abstract

The present invention discloses a rainbow trout-derived component primer and detection system and method based on RPA-CRISPR. The RPA primer and crRNA of the present invention have good specificity, the constructed identification method shows dual specificity, has no specific recognition reaction to other species, and the detection results are accurate and reliable. At the same time, the rainbow trout-derived component identification method of the present invention is simple to operate, less time-consuming, and has accurate results. It is suitable for on-site rapid detection of rainbow trout-derived components in salmon products by relevant supervisory departments. In addition, the detection method of the present invention is combined with a rapid alkaline lysis method to extract sample nucleic acid. Without relying on large-scale instruments, the detection limit of rainbow trout-derived components adulterated in salmon products can reach 1% (w / w), and the detection sensitivity of rainbow trout genomic DNA is 10 ‑2 ng / μL, suitable for the identification and detection of rainbow trout-derived ingredients in a variety of salmon processed products with high reliability.

Description

Technical Field

[0001] The present invention belongs to the field of food detection technology. More specifically, it relates to a rainbow trout-derived ingredient primer based on RPA-CRISPR and a detection system and method. Background Art

[0002] Currently available methods for identifying species in salmon products are primarily categorized into two main categories: protein-based and nucleic acid-based detection methods. Nucleic acid-based detection methods are highly sensitive and specific. Compared to protein-based methods, they are more stable and accurate, making them suitable for testing complex processed meat products. Therefore, they are widely used for meat species identification. Common nucleic acid-based species identification methods include PCR, real-time fluorescence PCR, DNA barcoding, and PCR-RFLP analysis. Real-time fluorescence PCR is the gold standard for meat species identification in China. Despite their high sensitivity, these methods rely on expensive and cumbersome temperature-variable instruments, are cumbersome and time-consuming, and require a high level of expertise, making them unsuitable for rapid on-site inspections by relevant supervisory authorities.

[0003] In recent years, many DNA isothermal amplification technologies, such as LAMP, RCA, and RPA, have emerged to meet demand, providing the possibility of rapid on-site nucleic acid testing. Currently, DNA isothermal amplification technology has been widely reported to be used for species identification of meat. For example, Li et al. (2022) developed a LAMP amplification combined with a pH indicator colorimetric signal readout method for identifying Atlantic salmon, rainbow trout, and salmon. The results showed that it has good applicability in controlling adulteration and mislabeling in the salmon market. However, LAMP technology requires the design of multiple primers, which can easily cause cross-reactions and produce non-specific amplification results, and the detection results lack stability. In contrast, RPA technology only requires a pair of primers and has a short amplification time. The reaction can be initiated at room temperature and has excellent amplification effect. However, most RPA-based detection methods still require the combination of nucleic acid electrophoresis to interpret the results after amplification, which is complicated and not suitable for rapid on-site detection. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing technologies and provide an RPA primer and a detection system and method that can quickly identify rainbow trout-derived components in salmon products.

[0005] The first object of the present invention is to provide an RPA primer for identifying rainbow trout-derived components.

[0006] The second object of the present invention is to provide the use of the RPA primers in identifying rainbow trout-derived components or preparing products for identifying rainbow trout-derived components.

[0007] A third object of the present invention is to provide a CRISPR / Cas12a detection system for identifying rainbow trout-derived components.

[0008] The fourth object of the present invention is to provide the use of the detection system in identifying rainbow trout-derived components or preparing products for identifying rainbow trout-derived components.

[0009] A fifth object of the present invention is to provide a method for rapidly identifying rainbow trout-derived components in salmon or salmon products.

[0010] A sixth object of the present invention is to provide a kit for identifying rainbow trout-derived components.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention uses rainbow trout mitochondrial DNA as a target. After homology comparison analysis of mitochondrial DNA from different rainbow trout species and mitochondrial DNA from four other common animal species, a pair of highly specific RPA primers for identifying rainbow trout-derived ingredients was designed and screened. These RPA primers exhibit excellent target species specificity and produce small amplified fragments, making them suitable for detecting and identifying rainbow trout-derived ingredients in highly processed salmon products.

[0013] The present invention obtains a highly specific crRNA for the amplified target gene by designing and screening in the amplified target interval, which is highly specific to rainbow trout mitochondrial DNA fragments.

[0014] Based on the above-mentioned RPA primers and crRNA, the present invention combines an FQ-ssDNA signal reporter probe labeled with a FAM reporter group to construct a CRISPR / Cas12a detection system for identifying rainbow trout-derived components, and establishes a method for rapidly identifying rainbow trout-derived components in salmon products. Because the RPA primers and crRNA designed in the present invention have both high specificity and high sensitivity, the alkaline lysis crude extract can be directly used as the template DNA for the amplification reaction, eliminating the need for complex sample pretreatment and DNA extraction and purification steps, ultimately achieving rapid fluorescent visualization of rainbow trout-derived components in salmon products.

[0015] The present invention provides an RPA primer O5-F / O5-R for identifying rainbow trout-derived components, and the primer sequences are shown in SEQ ID NOs. 1-2.

[0016] The RPA primers O5-F / O5-R described in the present invention have the characteristics of high specificity and high sensitivity, and can be used to identify rainbow trout-derived ingredients in salmon products.

[0017] Therefore, the present invention applies to protect the use of the RPA primers O5-F / O5-R in identifying rainbow trout-derived ingredients or preparing products for identifying rainbow trout-derived ingredients as shown in SEQ ID NOs. 1-2.

[0018] The present invention also provides an RPA-CRISPR / Cas12a detection system for identifying rainbow trout-derived components, which includes RPA primers O5-F / O5-R, crRNA, CRISPR / Cas12a protein and FQ-ssDNA signal reporter probe; the sequence of the crRNA is shown in SEQ ID NO.3, and the sequence of the FQ-ssDNA signal reporter probe is shown in SEQ ID NO.4.

[0019] The present invention also applies to protect the use of the above detection system in identifying rainbow trout-derived ingredients or preparing products for identifying rainbow trout-derived ingredients.

[0020] Preferably, the FQ-ssDNA carries a FAM reporter group and a BHQ quencher group, as shown in Example 2.

[0021] The present invention also provides a method for rapidly identifying rainbow trout-derived components in salmon products, comprising the following steps:

[0022] 1. Extract the DNA of the sample to be tested or directly take the sample and the supernatant of the lysate;

[0023] 2. Using the DNA obtained in step 1 or the supernatant of the sample lysate as a DNA template, perform nucleic acid isothermal amplification using the RPA primers O5-F / O5-R;

[0024] 3. Prepare a detection system using the crRNA, FQ-ssDNA signal reporter probe, and CRISPR / Cas12a protein in the detection system, and add the amplified product obtained in step 2 to react; if the blank control has no fluorescence under blue light irradiation, and the positive control and the tested sample produce green fluorescence, the tested sample contains rainbow trout-derived components.

[0025] When the positive control has no green fluorescence or the blank control has green fluorescence, it indicates that there is an operational error or the reagent is contaminated.

[0026] Preferably, in step 1, the DNA of the sample to be tested is extracted by the SDS method or the sample lysate supernatant is obtained by the alkaline lysis method, see Example 4.

[0027] The alkaline lysis method is Alkaline lysis.

[0028] Preferably, in the reaction system of the nucleic acid isothermal amplification reaction in step 2, the final concentration of primers O5-F / O5-R is 0.3 μM, see Example 1.

[0029] Preferably, the final concentration of dNTPs is 2 mM, see Example 1.

[0030] Specifically, the reaction system of the nucleic acid isothermal amplification reaction in step 2 is: 4 μL C buffer, 1 μL L buffer, 2.4 μL P-core, 0.5 μL dNTPs (10 mM each), primer O5-F / O5-R with a final concentration of 0.3 μM, 0.5 μL B buffer, 0.5 μL DNA template, and a total system of 10 μL.

[0031] Preferably, the reaction conditions of the nucleic acid isothermal amplification reaction in step 2 are 37° C., 30 min, as shown in Example 1.

[0032] Preferably, in the detection system described in step 3, the final concentration of Cas12a is 125 nM, the final concentration of crRNA is 187.5 nM, and the final concentration of FQ-ssDNA fluorescent signal reporter probe is 500 nM, as shown in Example 2.

[0033] Specifically, the CRISPR / Cas12a fluorescence detection system described in step 3 is: CRISPR / Cas12a detection system: 2.5 μL 10×NEBuffer 2.1, 2.5 μL Cas12a (1 μM), 1.875 μL crRNA (2 μM), 1 μL FQ-ssDNA (10 μM), total system 10 μL.

[0034] Preferably, the reaction conditions of step 3 are 37° C., 15 min, as shown in Example 2.

[0035] Specifically, the salmon products described in the present invention include but are not limited to fresh salmon, dried salmon, frozen salmon, salmon balls, cooked salmon meat, salmon powder, etc.

[0036] The present invention also provides a kit for identifying rainbow trout-derived components, wherein the kit contains RPA primers O5-F / O5-R, CRISPR / Cas12a protein, crRNA and FQ-ssDNA fluorescent signal reporter probe.

[0037] The present invention has the following beneficial effects:

[0038] (1) The RPA primers and crRNA described in the present invention are both highly specific and only specifically amplify rainbow trout mitochondrial target DNA fragments, and do not perform non-specific amplification and identification detection on other species. The rainbow trout-derived component identification method constructed based on the RPA primers and crRNA has dual specificity for the identification and detection of rainbow trout-derived components, and is suitable for the detection and identification of rainbow trout-derived components in salmon products, with accurate results.

[0039] (2) The method for identifying rainbow trout-derived components constructed by the present invention can realize the rapid identification and detection of rainbow trout-derived components in salmon products without relying on the laboratory environment. It is easy to operate and the results are easy to observe. It is suitable for on-site rapid detection of rainbow trout-derived components, and is particularly suitable for on-site rapid spot checks by relevant supervisory departments. It has strong practicality. The present invention optimizes the reaction conditions and reaction procedures of RPA constant temperature amplification and RPA-CRISPR / Cas12a reaction. Under relatively low temperature (37°C), amplification and detection can be completed. The RPA amplification process only takes 30 minutes, and the CRISPR / Cas12a detection process only takes 15 minutes. In addition, combined with the direct lysis method, sample pretreatment and DNA extraction can be achieved within 35 minutes. The identification of rainbow trout-derived components can be achieved by taking the sample lysate supernatant. The whole process can be shortened to 80 minutes, with high detection efficiency and reduced detection cost.

[0040] (3) The present invention constructs an Atlantic salmon adulteration model with different rainbow trout doping ratios and finds that by extracting sample DNA using the SDS method or a commercial kit, the detection limit of rainbow trout-derived components in salmon products reaches 1% (w / w), meeting the detection requirements for adulterated rainbow trout components in salmon products.

[0041] (4) The RPA-CRISPR / Cas12a detection system and identification method for identifying rainbow trout-derived components established in the present invention are suitable for the identification and testing of rainbow trout-derived components in various salmon products such as fresh salmon, dried salmon, frozen salmon, salmon balls, cooked salmon meat, and salmon powder. It has a wide range of applications and is completely consistent with the detection results of the real-time fluorescence PCR method established by Zhou Lu et al. (2019). It has high reliability and is suitable for the rapid detection of rainbow trout-derived components in salmon products on the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The results of specificity detection of rainbow trout RPA primers are shown. Lane M is a 5000 bp DNA marker. Lanes 1 to 5 correspond to species such as rainbow trout, Atlantic salmon, Chinook salmon, Coho salmon, and Humpback salmon, respectively. Lane 6 is a negative control (ddH2O).

[0043] Figure 2 Figure 1 shows the specificity of the RPA-CRISPR / Cas12a detection system. Figure A shows the statistical analysis of the fluorescence values ​​for the specificity detection of the RPA-CRISPR / Cas12a detection system. Figure B shows the detection results. Tubes 1 to 5 correspond to species such as rainbow trout, Atlantic salmon, Chinook salmon, Coho salmon, and Humpback salmon, respectively. Lane 6 is the negative control (ddH2O).

[0044] Figure 3 This is the absolute sensitivity test result of the RPA-CRISPR / Cas12a detection system. The amount of rainbow trout genomic DNA tested is 100, 10-1, 10-2, 10-3, 10-4, 10-5, 10-6 ng, and NTC, respectively.

[0045] Figure 4 These are the detection limit results of the RPA-CRISPR / Cas12a detection system. The detected proportions of rainbow trout and Atlantic salmon adulteration are 100%, 10%, 5%, 1%, 0.1%, 0.01%, 0%, and NTC, respectively.

[0046] Figure 5 These are the test results of the RPACRISPR / Cas12a detection method applied to 36 commercially available salmon products (samples 1-62); NTC is the blank control (ddH2O); rainbow trout is the positive control (rainbow trout DNA).

[0047] Figures 6-7 The real-time fluorescence PCR method established by Zhou Lu et al. (2019) was used to confirm the test results; Figure 6 These are the real-time fluorescence PCR test results for rainbow trout-derived components in samples 1 to 18; Figure 7 Real-time fluorescence PCR of rainbow trout-derived components from samples 18 to 36. DETAILED DESCRIPTION

[0048] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0049] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0050] Example 1 Design of RPA primers and optimization of the reaction.

[0051] Design and screening of rainbow trout-specific isothermal amplification primers (RPA primers).

[0052] The present invention downloaded rainbow trout mitochondrial DNA and mitochondrial DNA sequences of four other animal species from the GenBank database. The species information involved is shown in Table 1. SnapGene software was used to perform homology comparison analysis on rainbow trout homologous sequence DNA and the mitochondrial DNA sequences of the other four different animal species.

[0053] Table 1 Species information involved in RPA primer design

[0054]

[0055]

[0056] Through homologous comparison, we selected the regions that are conserved within the mitochondrial DNA of rainbow trout species but have large interspecies differences with the mitochondrial DNA sequences of the other four animal species as targets, and designed rainbow trout-specific isothermal amplification primers.

[0057] The designed primer sequences are shown in the table below, and 7 pairs of primers were obtained through preliminary screening.

[0058]

[0059] The designed primers were screened for specificity using the Primer-BLAST tool on an online website. RPA experiments were then performed using rainbow trout genomic DNA as the amplification template. The amplified products were purified and tested by nucleic acid electrophoresis. Primer pairs with single, bright bands were selected as candidate primers. Finally, RPA amplification was performed using DNA from four related species to verify primer specificity. Ultimately, the primers with single, bright bands and no nonspecific amplification were selected as the optimal primers. After a series of screening tests, the RPA primers ultimately selected were O5-F / O5-R, with the following sequences:

[0060] The sequence of O5-F (SEQ ID NO. 1) is 5'-CCTCGCAGTCCCACTGTGGCTTGCTACAGTAA-3'

[0061] O5-R (SEQ ID NO.2) sequence: 5'-CGTTTTCTTGTAAATAGAGGCTTAGGAGTAAG-3'

[0062] The fragment amplified by the specific primer pair O5-F / O5-R for isothermal amplification of rainbow trout-derived components was 356 bp in size.

[0063] The specificity test results of RPA primers O5-F / O5-R are as follows Figure 1 As shown, lane M is a 5000bp DNA marker; lanes 1 to 5 correspond to species such as rainbow trout, Atlantic salmon, Chinook salmon, Coho salmon, and Humpback salmon; lane 6 is a negative control (ddH2O). Figure 1 It can be seen that the RPA isothermal amplification primers O5-F / O5-R designed and screened by the present invention have high specificity and can be used for the detection of rainbow trout-derived components.

[0064] In order to further improve the detection effect, the present invention optimized the reaction conditions of RPA isothermal amplification. The specific optimized parameters are shown in Table 2.

[0065] Table 2

[0066]

[0067] While keeping other conditions unchanged, the assay was tested under different primer concentrations, dNTP concentrations, amplification times, and reaction temperatures. The fluorescence brightness of the reaction tubes under blue light and the fluorescence values ​​measured in the QPCR instrument were compared. Taking fluorescence intensity and reagent costs into consideration, the optimal RPA reaction parameters for the reaction system were determined. The optimal reaction system included a final primer concentration of 0.3 μM, a final dNTP concentration of 2 mM, an optimal reverse amplification time of 30 minutes, and an optimal amplification temperature of 37°C.

[0068] Example 2 is the construction and optimization of the RPA-RISPR / Cas12a detection system

[0069] The present invention designs and screens the amplification target interval of the RPA primers described in Example 1, and obtains a specific crRNA for the amplified target DNA, whose sequence is 5'-UAAUUUCUACUAAGUGUAGAUUUGCCUGAAGGAACCCCCGUU-3' (SEQ ID NO.3); At the same time, an FQ-ssDNA fluorescent probe is designed, whose sequence is 5'FAM-TTTTTT-3'BHQ (SEQ ID NO.4), thereby establishing an RPA-CRISPR / Cas12a detection system. The FQ-ssDNA fluorescent probe carries a FAM reporter group and a BHQ quencher group.

[0070] Based on the optimized RPA amplification system, the crRNA / Cas12a ratio, Cas12a enzyme concentration, FQ-ssDNA fluorescent probe concentration, and reaction time in the rainbow trout CRISPR / Cas12a reaction system were optimized. The specific optimization conditions are shown in Table 3:

[0071] Table 3

[0072]

[0073] While keeping other conditions unchanged, the detection effects under different Cas12a concentrations, crRNA / Cas12a ratios, and FQ-ssDNA concentrations were tested, and the fluorescence brightness of the reaction tube under blue light and the fluorescence value measured in the QPCR instrument were compared. Taking into account the fluorescence intensity and reagent cost, the optimal CRISPR / Cas12a reaction condition parameters of the reaction system were obtained. Among them, the final concentration of Cas12a in the optimal reaction system should be 125nM, the crRNA / Cas12a ratio should be 1.5:1, the optimal FQ-ssDNA concentration should be 500nM, and the optimal reaction time should be 15

[0074] The present invention utilizes the constructed RPA-CRISPR / Cas12a detection system and the optimized optimal reaction system and reaction time, combined with DNA extraction, to identify rainbow trout-derived components in salmon products.

[0075] (1) Extract the sample DNA using the SDS method, animal genomic DNA extraction kit, or other recognized DNA extraction methods with equivalent effectiveness and store at -20°C until use.

[0076] In this embodiment, the sample DNA was obtained by rapid extraction of AL lysis method. Take about 15mg of salmon chunks or meat powder and place it in a 1.5mL centrifuge tube, add 200μL NaOH solution (0.2mol / L), and grind it with a handheld electric grinder for 1 to 2 minutes. Continue to add 100μL 0.2mol / L NaOH solution to the above grinding liquid, vortex to mix, and lyse in a 75℃ water bath for 30min, mixing every 10min during the period. The obtained lysate was briefly centrifuged, 10μL of the middle layer liquid was drawn into a new 200μL centrifuge tube, 30μL 0.04mol / L Tris-HCl solution was added, vortex to mix, and stored at -20℃. The supernatant of the obtained lysate was used as a DNA template for PCR reaction. (2) After multiple optimizations of the RPA constant temperature amplification system and the CRISPR / Cas12a detection system in the RPA-CRISPR / Cas12a detection system, the final RPA constant temperature amplification system and CRISPR / Cas12a detection system are as follows:

[0077] ①RPA constant temperature amplification system: 4μL C buffer, 1μL L buffer, 2.4μL P-core, 0.5μL dNTPs (10mM each), primer O5-F / O5-R final concentration of 0.3μM, 0.5μL B buffer, DNA template 0.5μL, total system 10μL.

[0078] ②CRISPR / Cas12a detection system: 2 μL 10×NEB Buffer r2.1, 2.5 μL Cas12a (1 μM), 1.875 μL crRNA (2 μM), 1 μL FQ-ssDNA (10 μM), 0.5 μL 0.8 U RNase Inhibitor, total system 10 μL.

[0079] The reaction conditions of the RPA-CRISPR / Cas12a detection system are as follows: the RPA isothermal amplification system is reacted at 37°C for 30 minutes; the prepared CRISPR / Cas12a detection system is added and reacted at 37°C for 15 minutes.

[0080] The RPA-CRISPR / Cas12a test results were observed using a micro blue light gel cutting instrument, and photos were taken with a mobile phone to save the results for result judgment: when both the sample to be tested and the positive control produced green fluorescence, and the blank control had no green fluorescence, the sample to be tested was judged to be positive for rainbow trout-derived components; when the positive control produced green fluorescence, the sample to be tested did not produce green fluorescence, and the blank control had no green fluorescence, it was judged that no rainbow trout-derived components were detected in the sample; if the positive control did not produce green fluorescence or the blank control produced green fluorescence, it indicated that the operation failed or there was reagent contamination, and the experiment needed to be repeated.

[0081] Example 3 Specificity Verification of RPA-CRISPR / Cas12a Detection System

[0082] In order to verify the specificity of the RPA-CRISPR / Cas12a detection system constructed by the present invention, the present invention extracted DNA from rainbow trout, Atlantic salmon, and Chinook salmon as detection objects, and performed specificity verification on the constructed RPA-CRISPR / Cas12a detection system. DNA extraction, RPA-CRISPR / Cas12a detection system and reaction conditions were the same as those in Example 1, and the fluorescence value was determined by real-time fluorescence PCR instrument. The specificity verification results of the RPA-CRISPR / Cas12a detection system are shown in Figure 2. Figure 2 As shown, it can be seen that the detection system of the present invention only produces obvious green fluorescence visible to the naked eye for the DNA sample of rainbow trout, and has no fluorescent reaction for the other four species such as Atlantic salmon and Chinook salmon, indicating that the RPA-CRISPR / Cas12a detection system of the present invention has high specificity.

[0083] Example 4 Sensitivity of RPA-CRISPR / Cas12a Detection Method

[0084] In order to verify the sensitivity of the constructed RPA-CRISPR / Cas12a detection method, the present invention uses rainbow trout genomic DNA as a template and sets up a system with different target DNA contents for detection. The amount of rainbow trout genomic DNA in the system is 100, 10-1, 10-2, 10-3, 10-4, 10-5, and 0 (NTC). The test results are as follows Figure 3 As shown in the figure, it can be seen that the detection limit of the RPA-CRISPR / Cas12a detection method constructed by the present invention for rainbow trout genomic DNA is 10-2ng.

[0085] Example 5 Detection Limit of RPA-CRISPR / Cas12a Detection Method

[0086] To verify the detection limit of the constructed RPA-CRISPR / Cas12a detection method, the present invention prepared a rainbow trout adulteration model by mixing rainbow trout and Atlantic salmon meat powder in different proportions to simulate the situation where rainbow trout is adulterated with Atlantic salmon on the market. The RPA-CRISPR / Cas12a detection system described in the present invention was used for evaluation and verification.

[0087] Table 4 Binary adulteration model of rainbow trout mixed with Atlantic salmon

[0088]

[0089] The detection limit evaluation results are as follows Figure 4 As shown, when Atlantic salmon is mixed with ≥1% rainbow trout components, it can be detected by the RPA-CRISPR / Cas12a system described in the present invention, and the test tube emits obvious green fluorescence visible to the naked eye, showing a positive test result. When the adulteration ratio is less than 1%, the system cannot recognize the rainbow trout target, the test tube does not produce green fluorescence, and the test result is consistent with the NTC and is negative. Therefore, the detection limit of the RPA-CRISPR / Cas12a system constructed by the present invention for rainbow trout-derived components can reach 1%.

[0090] Example 6 Practical Application and Accuracy of RPA-CRISPR / Cas12a Detection Method

[0091] In order to test whether the RPA-CRISPR / Cas12a detection method constructed by the present invention can be applied to actual detection and verify its detection effect. The present invention collected and purchased 36 commercial salmon products, including salmon sashimi, salmon balls, salmon powder, salmon cubes, dried salmon and other salmon products through online shopping platforms and offline restaurants. The sample DNA rapid extraction, RPA-CRISPR / Cas12a detection system and method are the same as those in Example 1. The detection results are as follows Figure 5 As shown in the figure, the test results showed that 9 of the 36 salmon samples were adulterated with rainbow trout, with an adulteration rate of 25%. Twenty-seven salmon products, representing 75%, had no detectable pork components. These results demonstrate that the RPA-CRISPR / Cas12a detection method constructed in this invention can be applied to the rapid identification of rainbow trout-derived ingredients in commercially available salmon products. With its short detection time and low cost, it is particularly suitable for on-site detection of salmon product adulteration.

[0092] In order to prove the accuracy of the RPA-CRISPR / Cas12a detection method of the present invention, the real-time fluorescence PCR method established by Zhou Lu et al. (2019) was used to verify the accuracy of the constructed RPA-CRISPR / Cas12a detection system. The rainbow trout-derived components in 36 different types of salmon samples were detected according to the real-time fluorescence PCR method. The results are as follows Figures 6-7 The results showed that the results of the reference real-time fluorescence PCR detection method were completely consistent with those of the present invention (Table 5), which demonstrated that the RPA-CRISPR / Cas12a detection method of the present invention has high accuracy and can be applied to the rapid detection of rainbow trout-derived components in salmon products on the market.

[0093] Table 5 Comparison of detection results of RPA-CRISPR / Cas12a of the present invention and real-time fluorescence PCR

[0094]

[0095] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An RPA-CRISPR / Cas12a composition for identifying rainbow trout-derived components, characterized in that: The method is composed of RPA primers O5-F / O5-R, crRNA, CRISPR / Cas12a protein and FQ-ssDNA fluorescent probe for rapid identification of rainbow trout-derived components; the sequences of the RPA primers O5-F / O5-R are shown in SEQ ID NOs. 1 to 2, the sequence of the crRNA is shown in SEQ ID NO. 3, and the sequence of the FQ-ssDNA is shown in SEQ ID NO.

4.

2. Use of the composition according to claim 1 in identifying rainbow trout-derived ingredients or preparing products for identifying rainbow trout-derived ingredients.

3. A method for rapidly identifying rainbow trout-derived ingredients in salmon products, characterized in that: The following steps are involved: 1) Extract the DNA of the sample to be tested or directly take the sample and the supernatant of the lysate; 2) Using the DNA obtained in step 1 or the supernatant of the sample lysate as a template, performing a nucleic acid isothermal amplification reaction using the RPA primers O5-F / O5-R described in claim 1; 3) A detection system is prepared using the crRNA, FQ-ssDNA, and CRISPR / Cas12a protein in the composition of claim 1, and the amplification product obtained in step 2 is added to react; the 5' end of the FQ-ssDNA is labeled with a FAM group and the 3' end is labeled with a BHQ group; a portable blue light is used to directly visually determine whether the sample contains rainbow trout-derived components.

4. The method according to claim 3, characterized in that In step 1, the DNA of the sample to be tested is extracted by the SDS method or the sample lysate supernatant is obtained by the alkaline lysis method.

5. The method according to claim 3, characterized in that: In the reaction system of the nucleic acid isothermal amplification reaction in step 2, the final concentration of primers O5-F / O5-R is 0.3 μM.

6. The method according to claim 3, characterized in that: In the detection system described in step 3, the final concentration of Cas12a is 125 nM, the final concentration of crRNA is 187.5 nM, and the final concentration of FQ-ssDNA is 500 nM.

7. A kit for identifying rainbow trout-derived ingredients, characterized in that: Contains the RPA-CRISPR / Cas12a composition of claim 1.

Images