A 3D printed multifunctional pork detection device and a portable multifunctional sensor detection method

Abstract

The embodiment of the present invention discloses a 3D printed multifunctional pork detection device and a portable multifunctional sensor detection method, which belongs to the field of meat detection technology. A one-tube RPA-CRISPR / Cas12a sensor is constructed to realize the rapid detection of pork components in food. First, the standard gene in the chromosome of the pig is screened out, and then the primer is designed and the RPA amplification is used for verification and screening to complete the acquisition, identification and amplification of the signal; then the RPA amplification is combined with the CRISPR-Cas12a cutting to convert the nucleic acid signal into a fluorescent signal to complete the signal conversion and output; with the assistance of the 3D printed multifunctional device, the closed tube reaction of RPA-CRISPR / Cas12a is realized by adding paraffin oil, and integrated into a one-tube RPA-CRISPR / Cas12a sensor, and then the performance of the sensor is optimized and the sensitivity verification and actual sample detection are performed.

Description

Technical Field

[0001] The embodiments of the present invention relate to the technical field of meat detection, and in particular to a 3D printed multifunctional pork detection device and a portable multifunctional sensor detection method. Background Art

[0002] Adulteration of animal-derived foods is the most common problem in food adulteration incidents, among which meat adulteration has become one of the focuses of global attention. In recent years, meat adulteration incidents have occurred frequently both at home and abroad. Therefore, building an effective rapid detection method for meat adulteration is one of the important means to curb the occurrence of such incidents.

[0003] At present, the main international detection methods for meat adulteration include morphological detection, metabolite-based detection, immunological detection and molecular biology detection. Morphological detection mainly uses an optical microscope to directly observe the morphology of sample particles to determine the composition of meat products or different animal tissues. This method has a low false positive rate and a low detection limit, but because it is observed by naked eyes by the inspectors, the results are somewhat subjective; metabolite-based detection uses the difference in metabolites between different species of animals to construct sensors such as electronic noses and electronic tongues for detection. The detection method has good repeatability, high sensitivity and simple operation, but it has high requirements for instruments; immunological detection can accurately determine and carry out quantitative analysis, with high sensitivity and good specificity for mixed samples, but due to the disadvantage of protein denaturation, once denatured, the sensitivity of this method is significantly reduced, and this method has high requirements for antibody specificity. If the antibody preparation specificity does not meet the standards, false positive results are likely to occur; molecular biology methods overcome the shortcomings of long time period, cumbersome operation, and difficult to distinguish experimental results, and have become the mainstream technology for meat adulteration detection. Especially since the CRISPR-Cas9 gene scissors won the Nobel Prize in 2020, nucleic acid detection based on gene editing has shown great application prospects. Different nucleic acid amplification technologies can generate different nucleic acid targets, and different nucleic acid amplification technologies combined with the CRISPR-Cas system can also achieve different detection effects.

[0004] The premise and basis of nucleic acid molecule-based detection technology is the need for species-specific gene fragments, that is, the internal standard gene of the species. At present, most of the meat adulteration detection standards and technologies at home and abroad use mitochondrial genes as the internal standard genes of the species to design specific primers for nucleic acid detection. However, due to the high conservatism of mitochondrial genes, the similarity between closely related species is very high, making it difficult to effectively distinguish and identify them. For this reason, screening low-copy number genes on species chromosomes as new internal standard genes is extremely important for accurate detection. Summary of the invention

[0005] To this end, the embodiments of the present invention provide a 3D printed multifunctional pork detection device and a portable multifunctional sensor detection method to solve the problem in the prior art that it is difficult to effectively distinguish and identify due to the high conservatism of mitochondrial genes and the high degree of similarity between closely related species.

[0006] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions:

[0007] According to a first aspect of an embodiment of the present invention, the present invention provides a 3D printed multifunctional pork testing device, the 3D printed multifunctional pork testing device comprising a base, an auxiliary bracket, a heating device, a rotor system and a darkroom;

[0008] The auxiliary bracket includes a large bracket and a detachable small bracket, and the small bracket is used to fix the PCR tube containing the sample to be tested; the main body of the heating device is a heatable water tank, which is used to heat the sample to be tested, and a heating rod and a temperature sensor are arranged inside the water tank, and a temperature control panel is arranged outside the water tank, and the temperature control panel is connected to the heating rod and the temperature sensor. A crossbeam is arranged above the water tank for fixing the small bracket, and a turbine is arranged below the center of the crossbeam; the rotor system is used for centrifugation of the sample to be tested, and includes a rotor motor and a rotor fixing buckle, and the rotor fixing buckle is used to fix the auxiliary bracket;

[0009] A rotating arm is connected to the base, and the other end of the rotating arm is connected to a rotor motor. In a heating state, the other end of the rotor motor is connected to the turbine; in a centrifugal state, the other end of the rotor motor is connected to the rotor fixing buckle; the dark chamber is detachably fixed to the base for fluorescence photography of the sample to be tested.

[0010] Furthermore, a circuit system is provided inside the base, and two grooves are provided on the top of the base for equipping PCR tubes and charging cables.

[0011] Furthermore, the outer shell of the darkroom is a light-proof plate, a detachable back panel is provided on the darkroom, a card slot is provided on the back panel for clamping a small bracket, and an ultraviolet lamp, a heat sink and a camera are provided inside the darkroom.

[0012] According to a second aspect of an embodiment of the present invention, the present invention provides a portable multifunctional sensor detection method based on the above-mentioned 3D printed multifunctional pork detection device, and the portable multifunctional sensor detection method comprises the following steps:

[0013] S1: Extract genomic DNA from the sample to be tested, add RPA amplification system, paraffin oil and CRISPR / Cas12a cutting system into the PCR tube in sequence, separate the two systems with paraffin oil, then insert the PCR tube into the small bracket and insert it into the heating water tank of the 3D printing multifunctional device, and turn on the heating and water tank turbine;

[0014] S2: Assemble the small bracket containing the PCR tube onto the large bracket and fix it with the rotor fixing buckle of the rotor system, centrifuge, so that the upper CRISPR / Cas12a system in the PCR tube moves downward and mixes with the RPA amplification system;

[0015] S3: Insert the small bracket into the heating water tank again, and turn on the heating and water tank turbine;

[0016] S4: Take out the small bracket and fix it to the back plate of the darkroom shell, assemble the darkroom to the base to form a closed darkroom, connect the USB data cable to the smart device, turn on the camera software and UV light, and complete the shooting.

[0017] Furthermore, the RPA amplification system includes an upstream primer, a downstream primer, 2×Reaction Buffer, a dNTPs solution, 10×Basic E-mix, 20×Core Reaction Mix, a MgOAc solution and a template DNA; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2;

[0018] The CRISPR / Cas12a system includes: crRNA, Cas12a, a cleavage probe, ddH2O and 10×ToloBuffer; the nucleotide sequence of the crRNA is shown in SEQ ID NO.3, the Cas12a is selected from LbCas12a, and the cleavage probe is selected from FAM-TTTTTT-BHQ1.

[0019] Furthermore, the RPA amplification system is established based on the porcine internal reference gene LOC110262058, and its nucleotide sequence is shown in SEQ ID NO.4.

[0020] Further, the concentration of the upstream primer is 10 μmol / L, the concentration of the downstream primer is 10 μmol / L, the concentration of Cas12a is 50 nmol / L, and the concentration of the cleavage probe is 600 nmol / L.

[0021] Furthermore, in step S1, the amount of RPA amplification system is 10 μL, the amount of CRISPR / Cas12a cutting system is 70 μL, and the amount of paraffin oil is 20 μL. The heating temperature is 36-38°C, and the heating is maintained for 10-60 min; in step S3, the heating temperature is 36-38°C, and the heating is maintained for 20-35 min.

[0022] The embodiments of the present invention have the following advantages:

[0023] 1) Screen out the standard genes in the chromosomes of pigs, and establish the highly specific standard gene LOC110262058 in the chromosomes through intraspecific analysis of the pig genome and interspecific comparison of the genomes of other species, combined with BLASTN and PCR amplification analysis;

[0024] 2) Establish the RPA-CRISPR / Cas12 detection technology based on the standard gene in pork, determine the primer "pig-1" for RPA amplification, the minimum reaction system of 10μL and the shortest reaction time of 10min, determine the optimal system for CRISPR / Cas12a cutting, the most suitable Cas12a concentration of 50nmol / L, the optimal probe addition amount of 600nmol / L, the optimal qualitative detection time of 10min, and the optimal quantitative detection time of 20min.

[0025] 3) A one-tube RPA-CRISPR / Cas12a sensor was established, and a 3D-printed multifunctional device was independently designed, integrating heating, centrifugation, and darkroom imaging functions to assist RPA-CRISPR / Cas12 in achieving closed-tube detection. The sensor has good performance specificity and a sensitivity of 0.01% (w / w), and can be used for the detection of actual samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0027] Figure 1 Specific verification of the LOC110262058 gene provided in Test Example 1 of the present invention (A) BLAST comparison result; (B) PCR verification result (target product 484 bp);

[0028] Figure 2The establishment and optimization results of the RPA reaction provided for Test Example 2 of the present invention (A) Unpurified RPA amplification product; (B) Purified RPA amplification product; (C) Specificity verification of pig-1 primers; (D) Optimization of RPA reaction time; (E) Optimization of RPA reaction volume;

[0029] Figure 3 Establishment and optimization results of the RPA-CRISPR / Cas12a cleavage system provided for Test Example 2 of the present invention (A) Schematic diagram of crRNA / dsDNA complex; (B) Feasibility verification of RPA-CRISPR / Cas12a reaction; (C) Optimization of Cas12a addition; (D) Optimization of cleavage probe addition; (E) Optimization of cleavage time; (F) Fluorescence photography under ultraviolet (310nm), (c) Optimization of Cas12a addition; (d) Optimization of cleavage probe addition; (e) Optimization of cleavage time;

[0030] Figure 4 The 3D printing multifunctional device provided in Test Example 3 of the present invention (A) is a schematic diagram of five parts; (B) is a schematic diagram of the assembly during heating; (C) is a schematic diagram of the assembly during centrifugation; (D) is a schematic diagram of the assembly for photographing and analysis;

[0031] Figure 5 A schematic diagram of the detection and analysis principle of a one-tube RPA-CRISPR / Cas12a sensor provided in Test Example 3 of the present invention;

[0032] Figure 6 Performance test diagram of a one-tube RPA-CRISPR / Cas12a sensor provided for Test Example 3 of the present invention (A) One-tube detection feasibility verification; (B) specificity verification; (C) sensitivity verification: different pork content; (D) Linear relationship between relative gray value and pork content;

[0033] Figure 4 Middle: 1-base; 2-auxiliary bracket; 3-heating device; 4-rotor system; 5-darkroom; 6-heating water tank; 7-crossbeam; 8-temperature sensor; 9-heating rod; 10-temperature control panel; 11-turbine; 12-rotating arm; 13-rotor fixing buckle; 14-motor; 15-small bracket; 16-large bracket; 17-charging cable storage slot; 18-PCR tube storage slot; 19-mechanical lens; 20-UV lamp and radiator; 21-housing; 22-back panel; 23-circuit system. DETAILED DESCRIPTION

[0034] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Test Example 1

[0036] Screening and verification of standard genes in pig chromosomes

[0037] 1. Download 35 sets of pig whole genomes from NCBI, perform intraspecific sequence comparison analysis, and screen out intraspecific conserved gene fragments;

[0038] 2. Compare and analyze the genomes of pigs and other species to screen out the common gene fragments between species;

[0039] 3. Compare and analyze the conserved gene fragments within the species with the shared gene fragments between species, and subtract the interspecies genes from the intraspecies genes to obtain the species-specific gene fragments;

[0040] 4. The species-specific genes were classified and sorted, and the sequence information of 17,947 protein-coding genes and 2,040 pseudogenes were obtained;

[0041] 5. Select the three sequences with the highest scores in "coding protein" and "pseudogene" respectively, and verify them through BLASTN and PCR experiments in the NCBI database. Reliable endogenous reference genes should show high specificity in different species. Through BLASTN analysis, six genes showed high specificity in pig species and had no homology with other related species. However, through PCR experiment verification, only LOC110262058 gene was highly specific (gene sequence and primer sequence are shown in Table 1). Figure 1As shown in B, lane M: DNAladderDL2000; lane 1: negative control; lane 2: fish; lane 3: snake; lane 4: dog; lane 5: rabbit; lane 6: rat; lane 7: goose; lane 8: duck; lane 9: chicken; lane 10: deer; lane 11: donkey; lane 12: horse; lane 13: goat; lane 14: sheep; lane 15: yak; lane 16: cattle; lane 17: pig. Only the pig LOC110262058 gene showed a positive reaction, and no amplification products appeared in other species. The LOC110262058 (GeneBank: NC_010450.4) gene is located on chromosome 8, with the start and end positions between 11084067 and 11084627, which is related to the formation of the uncharacterized protein LOC110262058. Therefore, the LOC110262058 gene on chromosome was selected as the internal standard gene for pigs.

[0042] Table 1

[0043]

[0044]

[0045] Test Example 2

[0046] Establishment and optimization of RPA-CRISPR / Cas12a technology

[0047] 1. Primer and crRNA design

[0048] Two sets of RPA primers were designed based on the pig endogenous standard gene LOC110262058, and the crRNA sequence was designed based on the product sequence. The specific information is shown in Table 2.

[0049] Table 2

[0050]

[0051] 2. Establishment and optimization of RPA reaction

[0052] The RPA amplification system includes: 2×ReactionBuffer 5μL, dNTPs solution 1.8μL, 10×Basic E-mix 1μL, upstream primer 0.5μL with a concentration of 10μmol / L, downstream primer 0.5μL with a concentration of 10μmol / L, 20×CoreReactionMix 0.5μL, MgOAc solution 0.5μL, and template DNA 0.2μL.

[0053] The TwistAmpTM LiquidBasic kit was used for RPA amplification, and the results of 2% agarose gel electrophoresis of the amplified product were as follows: Figure 2As shown in A. It can be seen that the amplification verification results of the two sets of primers both have bands, and it can be seen that the primers and genomic DNA undergo RPA amplification. Figure 2 In A, lane M: DNAladderDL2000; lane 1: negative control; lanes 2-3: pig-1, primer set; lanes 4-5: pig-2, primer set. Figure 2 The band in A is more diffuse, and the size of the band is larger than the target fragment (297bp). The reason may be that the RPA reaction system is more complex, containing recombinase, polymerase and SSB proteins. If the amplified DNA fragment is attached with impurities such as proteins, it will slow down its migration speed in the agarose gel, making the amplification result appear larger. The amplified product was purified using the Tiangen General DNA Product Purification Kit and then subjected to 2% agarose gel electrophoresis. The results are as follows: Figure 2 As shown in B, lane M: DNAladderDL2000; lane 1: negative control; lane 2-3: pig-1, primer group; lane 4-5: pig-2, primer group, the product is consistent with the target fragment size. Combining 2A and 2B, the pig-1 primer amplification effect is better, and further specificity verification is needed. The results are as follows Figure 2 As shown in C, lane M: DNAladderDL2000; lane 1: pig; lane 2: cattle; lane 3: sheep; lane 4: chicken; lane 5: duck; lane 6: goose. Beef, mutton, chicken, duck, and goose meat all have no target fragment bands, while pork has obvious target fragment bands, which shows that the primers have good specificity and can be used for subsequent experiments.

[0054] Considering the timeliness of the detection, the RPA reaction time was optimized. The products after 10min, 20min, 30min, 40min, 50min, and 60min of RPA reaction were subjected to 2% agarose gel electrophoresis. The results are shown in Figure 2 As shown in D, lane M: DNAladder250 II; lane 1: negative control; lanes 2-3: 10min; lanes 4-5: 20min; lanes 6-7: 30min; lanes 8-9: 40min; lanes 10-11: 50min; lanes 12-13: 60min. In the RPA reaction, a relatively obvious target product band can be seen after 10min, so the RPA reaction is determined to be 10min. Considering the cost of detection, the amount of reagents used in the RPA reaction is optimized. 10μL, 20μL, 30μL, and 40μL of RPA reaction systems were prepared respectively, and the products were tested by 2% agarose gel electrophoresis after the reaction. The results are shown in Figure 2As shown in E, lane M: DNA ladder DL2000; lane 1-2: 40 μL; lane 3-4: 30 μL; lane 5-6: 20 μL; lane 7-8: 10 μL. The target product band can already appear in the 10 μL system, so the RPA reaction system was reduced to 10 μL for subsequent experiments.

[0055] 3. Establishment and optimization of RPA-CRISPR / Cas12a reaction

[0056] The Cas12 protein used in this experiment is LbCas12a (Cpf1). Cas12a has a RuvC nuclease domain, which can activate its endonuclease and trans-cleavage activity when the crRNA is complementary to the target gene for 15 or more bases. Cas12a with activated trans-cleavage activity can non-specifically cleave fluorescently labeled ssDNA to show fluorescence. Therefore, crRNA can be designed based on the location of the PAM site and its downstream 15-20 bases in the gene fragment amplified by the pig-1 primer. The sequence is as follows Figure 3 As shown in A. To prove the feasibility of the designed crRNA and CRISPR-Cas12a system, it is necessary to verify it. First, the pork genome was amplified by RPA, and the reaction product was taken to construct the CRISPR-Cas12a cutting system according to Table 3. The conditions for the cutting reaction were 37°C for 40 minutes. Take out the system after the reaction, observe whether there is green fluorescence under ultraviolet light (310nm), and use an enzyme reader to scan the spectrum (excitation light 488nm).

[0057] Table 3

[0058]

[0059] The fluorescence of the RPA product after the CRISPR-Cas12a reaction observed under UV light and the spectral image scanned using an ELISA reader are shown in Figure 2. Figure 3 As shown in B. The CRISPR-Cas12a system to which the RPA product was added can observe green fluorescence under ultraviolet light after the reaction, and the spectral image shows that it has a maximum absorption peak at 520nm, while the system to which the RPA product was not added has no green fluorescence, and the spectral image has no absorption peak. The above results prove that the crRNA designed in this experiment can be paired with the target gene amplified by RPA and activate the trans-cleavage activity of Cas12a, cutting the fluorescent quenching probe. Therefore, the CRISPR-Cas12a system constructed based on the pig-1 primer amplification fragment is feasible.

[0060] In order to improve the specificity and sensitivity of the sensor, it is necessary to use RPA products to optimize the performance of the sensor to ensure that the CRISPR-Cas12a gene editing sensor performs result analysis with optimal performance. Specifically, it includes three aspects: the amount of Cas12a added, the amount of probe added, and the cutting time.

[0061] (1) Cas12a is the effector enzyme that cuts the fluorescence quenching probe. When the probe concentration is constant, there should be a maximum concentration of Cas12a that makes the system fluorescence value reach the maximum and no longer change. Figure 3 C and 3Fc show that the Cas12a concentration is 12.5nmol / L, 25nmol / L, 37.5nmol / L, 50nmol / L, 62.5nmol / L, and 70nmol / L, and the 525nm fluorescence intensity of the reaction product scanned by a microplate reader and ultraviolet photography. It can be seen from the figure that after the Cas12a concentration reaches 50nmol / L, its fluorescence intensity is not much different from 62.5nmol / L and 75nmol / L, so the optimal Cas12a concentration of the RPA-CRISPR-Cas12a system is determined to be 50nmol / L.

[0062] (2) The amount of probe added is the variable that has the greatest impact on the fluorescence intensity of the CRISPR-Cas12a system. In order to maximize the sensitivity of the sensor while saving reagents, it is necessary to find the optimal amount of probe added so that the system produces strong fluorescence without consuming too much probe. Figure 3 D and 3Fd show the fluorescence intensity of the reaction product at 525nm scanned by a microplate reader and UV photography when the probe concentration in the CRISPR-Cas12a system is 100nmol / L, 200nmol / L, 300nmol / L, 400nmol / L, 500nmol / L, 600nmol / L, and 700nmol / L. It can be seen from the figure that the fluorescence intensity is significantly different when the probe concentration is 500nmol / L and 600nmol / L, while there is no significant difference in fluorescence intensity when the concentration is 600nmol / L and 700nmol / L. Therefore, the optimal probe addition amount of the RPA-CRISPR-Cas12a system is determined to be 600nmol / L.

[0063] (3) In order to pursue the rapid detection performance of the sensor, it is necessary to further shorten the 40 minutes required for the original CRISPR-Cas12a system reaction while ensuring that the fluorescence intensity does not decrease significantly. Figure 3E and 3Fe show the 525nm fluorescence intensity and UV photography of the products with reaction times of 5min, 10min, 15min, 20min, 25min, 30min, and 35min respectively, scanned by a microplate reader. It can be seen from the figure that the effect of reaction time on fluorescence intensity is not as significant as the amount of Cas12a and the amount of probe. After 10min of reaction, the fluorescence intensity can reach more than 50%. Considering the time cost, the reaction time can be set to 5min only when qualitative analysis is performed. In addition, it can be seen from the figure that there is no obvious difference in fluorescence intensity after 20min of reaction. From the perspective of precise quantification, it is more appropriate to set the reaction time to about 20min.

[0064] Test Example 3

[0065] One-tube RPA-CRISPR / Cas12a-based sensor technology established

[0066] 1. Design of 3D printing multifunctional device

[0067] This device is an integrated instrument with heating, centrifugation and darkroom imaging functions. It has complete functional modules and is compact and practical. This device is divided into 5 modules: base 1, auxiliary bracket 2, heating device 3, rotor system 4, darkroom 5. Figure 4 shown.

[0068] The first part is the base 1, which is the basic part of the instrument. The base is a black box, and the inside of the box is mainly equipped with circuits, including power supplies, circuit boards and other circuit systems 23. Two slots are opened on the edge of the base. The PCR tube storage slot 18 is used to hold PCR tubes, and the charging cable storage slot 17 is used to hold charging cables. The base is also connected to a rotating arm 12, which can be twisted to a suitable position as needed. The rotating shaft of the rotating arm can connect the rotor and the turbine 11 of the heating tank.

[0069] The second part is the auxiliary bracket 2, which has a large bracket 16 and two small brackets 15, and each small bracket 15 can be inserted with 4 PCR reaction tubes. The small bracket 15 is detachable and can be fixed in the water tank beam 7, or can be inserted into the large bracket 16 and fixed on the rotor, or can be stuck on the back plate 22 of the darkroom 5.

[0070] The third part is the heating device 3, the main body of which is a heatable water tank 6, and there is a heating rod 9 in the water tank, which can keep the water in the water tank at the required temperature. At the same time, the water tank is also equipped with a temperature sensor 8, which can monitor the temperature of the water tank and display it in real time on the temperature control panel 10. There is a crossbeam 7 above the water tank, which is not only used to fix the auxiliary bracket 2, but also has a turbine 11 under the center, which can be connected to the rotating arm 12 of the base 1. By rotating the turbine 11, the water temperature in the water tank is consistent, so that the reaction tube is heated more evenly.

[0071] The fourth part is the rotor system 4. There is a rotor fixing buckle 13 under the rotating arm 12, which can fix the auxiliary bracket 2, so that the overall function is the same as that of a centrifuge. During the reaction process, by turning on the rotor motor 14, substances with different densities in the solution can be separated or mixed under the action of centrifugal force.

[0072] The fifth part is the darkroom 5. The darkroom shell is made of opaque plate to facilitate imaging of the fluorescent substance in the reaction tube. The back plate 22 of the shell can be disassembled. There are two card slots on the back plate to clamp the auxiliary small bracket. An ultraviolet lamp is installed inside and equipped with a radiator 20 to make it easier to observe the fluorescent substance. A mechanical lens 19 is also equipped inside, which is connected to the camera software of the computer via a USB data cable to take pictures.

[0073] 2. Establishment of a one-tube RPA-CRISPR / Cas12a sensor

[0074] RPA-CRISPR-Cas12a technology can detect pork components in the sample to be tested. However, when used for actual testing, it cannot be guaranteed that the testing environment has laboratory conditions, and repeated opening of the lid to add reagents can easily cause aerosol contamination. Therefore, the RPA amplification system and the CRISPR / Cas12a system are reacted in the same PCR tube, so that signal recognition and amplification as well as signal conversion and output are completed in the same PCR tube, thereby avoiding repeated opening of the lid and reducing the possibility of aerosol contamination. Since the RPA and CRISPR / Cas12a reactions require the same temperature, in order to prevent the trans-cutting activity of Cas12a from being activated prematurely and directly cutting the template, paraffin oil is used to separate the two systems. The specific detection process is as follows: Figure 5As shown, 10 μL RPA amplification system, 20 μL paraffin oil and 70 μL CRISPR / Cas12a cutting system are added to the PCR tube, and then the PCR tube is inserted into the small bracket and stuck in the heating water tank of the 3D printing multifunctional device, the heating and water tank turbines are turned on, the temperature is controlled to 37±1°C, and the heating is maintained for 10 min (i.e., RPA reaction); then the small bracket with the PCR tube is assembled to the large bracket, and the rotor buckle of the rotor system is used to fix it, and the CRISPR / Cas12a system in the upper layer of the PCR tube moves down and mixes with the RPA amplification system, and then the small bracket is stuck in the 3 heating water tank again, the heating and water tank turbines are turned on, the temperature is controlled to 37±1°C, and the heating is maintained for 10-20 min (i.e., CRISPR / Cas12a cutting reaction); finally, the small bracket is taken out and fixed to the back plate of the dark box shell, the dark box is assembled to the base to form a closed dark room, the USB data cable is connected to smart devices such as mobile phones, tablets, and computers, the camera software and ultraviolet light are turned on, and the shooting is completed. If there is pork in the detection system, a green fluorescence image can be obtained ( Figure 6 A).

[0075] After verifying the feasibility of the one-tube RPA-CRISPR / Cas12a sensor, the specificity of the system needs to be verified to ensure the effectiveness of the sensor. The specificity verification was carried out using pigs, cattle, sheep, chickens, ducks, and geese. Figure 6 In B, tube 1: pig; tube 2: cattle; tube 3: sheep; tube 4: chicken; tube 5: duck; tube 6: goose; tube 7: fish; tube 8: negative control; it can be seen that only the pork group can be observed to have green fluorescence under ultraviolet light, and the other five meats have no obvious fluorescence. Therefore, it is proved that only the RPA amplification product of the pork genome can activate the trans-cleavage activity of Cas12a to cut the fluorescence quenching probe, making the system show green fluorescence, further ensuring the specificity of the sensor.

[0076] The sensitivity of the sensor was then analyzed. 100g of pork samples with different ingredients were prepared respectively, 100g pork (100% pork, w / w), 100g pig / chicken (10% pork, w / w), 100g pig / chicken (1% pork, w / w), 100g pig / chicken (0.1% pork, w / w), 100g pig / chicken (0.01% pork, w / w), 100g pig / chicken (0.001% pork, w / w), 100g pig / chicken (0.0001% pork, w / w). The samples were made into minced meat, followed by genome extraction and analysis using a one-tube RPA-CRISPR / Cas12a sensor. Fluorescent photos were collected by ultraviolet photography, and then the grayscale analysis of the fluorescent image was performed using ImageJ software. The results are as follows. Figure 6 C and 6D. Figure 6In C, tube 1: 100g; tube 2: 10g; tube 3: 1g; tube 4: 0.1g; tube 5: 0.01g; tube 6: 0.001g; tube 7: 0.0001g; tube 8: 0g; From the fluorescence photograph under ultraviolet light, it can be seen that the reaction products of 100% pork, 10% pork and 1% pork have obvious green fluorescence, and the reaction products of 0.1% pork and 0.01% pork can also be observed to have very light green fluorescence, and no green fluorescence can be observed after 0.01% pork. Therefore, the qualitative detection limit by visual observation is 0.01% (w / w). In order to determine the content of pork components more accurately, we used the "color inversion" function of ImageJ software to process the fluorescence image and analyze the relationship between the relative gray value and the pork content in the processed image. The relative gray value is Gp-Gn, where Gp is ​​the gray value of the detection with pork components, and Gn is the gray value of the detection without pork components, which is the negative control. Figure 6 As shown in D, within the range of 104 to 108 ng (per 100 g) of pork content, the relative gray value is linearly related to the pork content, R2 = 0.9691, and the fitted regression equation is y = 26.75x-98.67, where y represents the relative fluorescence intensity and x represents the logarithm of the pork content. Therefore, the quantitative detection line of this sensor is 0.01 g, that is, 0.01% (w / w).

[0077] Test Example 4

[0078] Application of one-tube RPA-CRISPR / Cas12a-based sensor technology in real samples

[0079] 1. The one-tube RPA-CRISPR / Cas12a sensor of the present invention was used to detect actual samples. The test results are shown in Table 4. Among the samples tested, samples containing pork components were successfully detected, proving the effectiveness of the one-tube RPA-CRISPR / Cas12a sensor.

[0080] Table 4

[0081]

[0082]

[0083] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A 3D printed multifunctional pork testing device, characterized in that: The 3D printed multifunctional pork testing device comprises a base, an auxiliary bracket, a heating device, a rotor system and a dark chamber; The auxiliary bracket includes a large bracket and a detachable small bracket, and the small bracket is used to fix the PCR tube; the main body of the heating device is a heatable water tank, the interior of the water tank is provided with a heating rod and a temperature sensor, the exterior of the water tank is provided with a temperature control panel, the temperature control panel is connected to the heating rod and the temperature sensor, a crossbeam is provided above the water tank for fixing the small bracket, and a turbine is provided below the center of the crossbeam; the rotor system includes a rotor motor and a rotor fixing buckle, and the rotor fixing buckle is used to fix the auxiliary bracket; The base is connected with a rotating arm, and the other end of the rotating arm is connected to a rotor motor. In a heating state, the other end of the rotor motor is connected to the turbine; in a centrifugal state, the other end of the rotor motor is connected to the rotor fixing buckle; the darkroom is detachably fixed to the base; The base has a circuit system inside, and two grooves are provided on the top of the base for equipping PCR tubes and charging cables; The outer shell of the darkroom is a light-proof plate. A detachable back plate is arranged on the darkroom. A card slot is arranged on the back plate for clamping a small bracket. An ultraviolet lamp, a radiator and a camera are arranged inside the darkroom.

2. A portable multifunctional sensor detection method based on the 3D printed multifunctional pork detection device according to claim 1, characterized in that: The portable multifunctional sensor detection method comprises the following steps: S1: Extract genomic DNA from the sample to be tested, add RPA amplification system, paraffin oil and CRISPR / Cas12a cutting system into the PCR tube in sequence, separate the two systems with paraffin oil, then insert the PCR tube into the small bracket and insert it into the heating water tank of the 3D printing multifunctional device, and turn on the heating and water tank turbine; S2: Assemble the small bracket containing the PCR tube onto the large bracket and fix it with the rotor fixing buckle of the rotor system, centrifuge, so that the upper CRISPR / Cas12a system in the PCR tube moves downward and mixes with the RPA amplification system; S3: Insert the small bracket into the heating water tank again, and turn on the heating and water tank turbine; S4: Take out the small bracket and fix it to the back plate of the darkroom shell, assemble the darkroom to the base to form a closed darkroom, connect the USB data cable to the smart device, turn on the camera software and UV light, and complete the shooting.

3. The portable multifunctional sensor detection method according to claim 2, characterized in that: The RPA amplification system includes an upstream primer, a downstream primer, 2×Reaction Buffer, a dNTPs solution, 10×Basic E-mix, 20×Core ReactionMix, a MgOAc solution and a template DNA; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2; The CRISPR / Cas12a system includes: crRNA, Cas12a, a cleavage probe, ddH2O and 10×Tolo Buffer; the nucleotide sequence of the crRNA is shown in SEQ ID NO.3, the Cas12a is selected from LbCas12a, and the cleavage probe is selected from FAM-TTTTTT-BHQ1.

4. The portable multifunctional sensor detection method according to claim 2, characterized in that: The RPA amplification system is established based on the porcine internal reference gene LOC110262058, and its nucleotide sequence is shown in SEQ ID NO.

4.

5. The portable multifunctional sensor detection method according to claim 3, characterized in that: The concentration of the upstream primer is 10 μmol / L, the concentration of the downstream primer is 10 μmol / L, the concentration of the Cas12a is 50 nmol / L, and the concentration of the cleavage probe is 600 nmol / L.

6. The portable multifunctional sensor detection method according to claim 2, characterized in that: In step S1, the amount of RPA amplification system is 10 μL, the amount of CRISPR / Cas12a cutting system is 70 μL, and the amount of paraffin oil is 20 μL. The heating temperature is 36-38 ° C, and the heating is maintained for 10-60 min; in step S3, the heating temperature is 36-38 ° C, and the heating is maintained for 20-35 min.

Images