Single-tube dual-mode visual nucleic acid detection method

Abstract

The invention discloses a single-tube dual-mode visual nucleic acid detection method. By designing an asymmetric V-type PCR nucleic acid amplification scheme, a large number of target single chains are generated in a short time, Cas12a-mediated cleavage reaction gets rid of the limitation of PAM sites in the excitation stage through single chain activation, and meanwhile, the single chains can generate stronger fluorescence signals compared with traditional double-chain product activation. In addition, the detection time is shortened by 50%, the visual sensitivity of the detected naked eye color of the bacteria can reach 10 CFU / mL, and the visual sensitivity under fluorescence excitation can reach 1 CFU / mL. Paraffin oil is adopted for layering, and the whole reaction is integrated in a single tube. The experimental steps are simplified, and aerosol pollution in the traditional transfer process is effectively avoided. Meanwhile, visual detection of two modes of naked eye color and fluorescence is realized in a single tube, the detection result is consistent with that of a qPCR standard method, and further advantages are achieved.

Description

Technical Field

[0001] The invention belongs to the field of biochemical analysis, and in particular relates to a single-tube dual-mode visualized nucleic acid detection method. Background Art

[0002] At present, various diseases caused by viruses and bacteria continue to break out, seriously endangering human life and health. In addition, commonly reported cases of foodborne diseases, such as Salmonella infection, Escherichia coli infection, Staphylococcus aureus infection, etc. Therefore, it is necessary to identify and detect bacteria and viruses more accurately and timely, and take corresponding measures and prevention and control measures in time to ensure the food safety and life safety of the public. Nucleic acid amplification technology is a common method for rapid detection of bacteria and viruses, such as polymerase chain amplification reaction (PCR) real-time fluorescence quantitative PCR (qPCR), which can quickly amplify the DNA or RNA of pathogens, thereby realizing rapid identification of targets. These methods have high sensitivity and specificity and are the gold standard for detecting pathogens. However, the traditional PCR-based method requires uncapped gel running to determine the band results, which is easy to cause aerosol contamination, and the qPCR method relies on a dedicated fluorescence monitoring instrument to observe the fluorescence curve, which cannot meet the needs of convenient, simple and intuitive detection. At present, the CRISPR / Cas system, as an emerging gene editing technology, has been used for rapid detection of pathogens. For example, CRISPR-Cas13a can be used to detect viral RNA, while CRISPR-Cas12a can be used to detect bacterial DNA. These methods generally design specific guide RNA (crRNA) to identify the genetic material of the target pathogen, activate the side-cutting effect of the CRISPR protein to cut the single-stranded fluorescent probe, and thus achieve the purpose of fluorescent visualization detection. The advantages of this method are rapid, sensitive, and relatively low cost, making it suitable for rapid on-site detection.

[0003] Although the CRISPR system has great potential in the field of molecular diagnosis, it is limited by PAM (protospacer short palindromic repeat sequence adjacent site). The PAM sequence is a necessary condition for the Cas enzyme and crRNA complex to recognize and bind to the target DNA and activate the cutting activity. Different Cas recognize different PAM sequences. For example, the commonly used Cas9 requires an NGG PAM sequence, the PAM sequence of Cas12a is TTTN, and the PAM site of Cas12b is TTN, which limits the scope of its targeted detection in genomic nucleic acids and makes the site design of crRNA targeting inflexible. In order to overcome the PAM site limitation, some researchers have modified the enzyme by protein engineering to expand the recognition range of its PAM site, thereby increasing the designable range of crRNA. However, these methods often require more professional personnel, and the modification process is also relatively complicated, which is not suitable for the needs of the field of rapid nucleic acid detection. In addition, common CRISPR detection methods generally need to be used in combination with nucleic acid amplification technology to achieve sufficient sensitivity. After obtaining a large amount of amplified products through nucleic acid amplification methods such as PCR, the product is opened and transferred to the CRISPR cutting system that has been incubated in advance. Under the guidance of crRNA, the Cas protein will specifically target the target amplified product with a PAM site and be activated by the nucleic acid target for its trans-cutting activity, and the single-stranded probe in the cutting system realizes signal reporting. However, this two-step detection must be opened after amplification, which is very prone to aerosol contamination and false positives, so it has high requirements for the experimental operation environment. And in the process of transferring the amplified product, only part of the product can often be added to the cutting system, and the amplified product of the previous step cannot be fully utilized. Therefore, it is necessary to develop a new type of PCR-CRISPR nucleic acid detection method that is fast, simple, sensitive and intuitive, so as to realize closed-tube intuitive visual detection without PAM sites. Summary of the invention

[0004] In view of the deficiencies of the above-mentioned prior art, the present invention provides a single-tube dual-mode visual nucleic acid detection method for ultra-fast asymmetric amplification without PAM enhanced activation of CRISPR cutting, which aims to solve the problem that the enzyme of the CRISPR detection method needs to be modified to expand the recognition range of its PAM site, and the modification process is also relatively complicated and is not suitable for the needs of the field of rapid nucleic acid detection; and the technical problem that aerosol pollution is very likely to be generated during the combined use of CRISPR detection method and nucleic acid amplification technology, false positives are prone to occur, and the experimental operation environment has high requirements.

[0005] The first technical solution provided by the present invention is a closed-tube reaction system for detecting a target object, wherein the reaction system includes NEB buffer 2.1 (10X), crRNA, Cas12a protein, a single-stranded fluorescent reporter probe, primers, and reagents for VPCR amplification reaction.

[0006] In certain embodiments, the length of the crRNA is 22 nt to 24 nt.

[0007] In certain embodiments, the Cas protein is LbCas12a.

[0008] In certain embodiments, the primers are an upstream and downstream primer set with unequal concentration ratios.

[0009] In certain embodiments, one end of the single-stranded fluorescent reporter probe carries a fluorescent reporter group, and the other end carries a fluorescent quencher group.

[0010] In certain embodiments, the fluorescent reporter group is selected from FAM, HEX, JOE, CY3, and CY5, and the quencher group is selected from DABCYL, TAMRA, and BHQ.

[0011] In certain embodiments, the amplification reagents include Taq DNA polymerase, dNTPs, and ddH2O.

[0012] In certain embodiments, the cleavage reagent includes Cas12a protein, crRNA, NEB buffer 2.1, and ddH2O.

[0013] In certain embodiments, the dilution ratio of the single-side primer is 1:1-1:40.

[0014] In certain embodiments, the ratio of crRNA to Cas12a is 1:0.25-1:2.

[0015] In certain embodiments, the concentration of the Cas12a and crRNA complex is 50nM-400nM.

[0016] In certain embodiments, the final concentration of the single-stranded fluorescent reporter probe is 1 μM-10 μM.

[0017] In some embodiments, the upstream primer and the downstream primer constitute an asymmetric primer pair, and asymmetric v-type PCR amplification is performed with the target as a template. The generated single-stranded activator is recognized by the Cas12a / crRNA complex, activates the side-cutting activity of the Cas protein, and the high-concentration fluorescent signal probe in the side-cutting system can produce naked eye color visualization and fluorescent visualization signals after cleavage.

[0018] In certain embodiments, the target is the invA gene in Salmonella typhimurium.

[0019] In certain embodiments, the nucleotide sequence of the crRNA is shown in any one of SEQ ID NO.1 to 3.

[0020] In certain embodiments, the nucleotide sequence of the asymmetric primer pair is selected from any one of the groups (a) to (c):

[0021] (a) SEQ ID NO.4 and SEQ ID NO.5;

[0022] (a) SEQ ID NO.6 and SEQ ID NO.7;

[0023] (a) SEQ ID NO.8 and SEQ ID NO.9.

[0024] In certain embodiments, the target is Salmonella typhimurium.

[0025] In certain embodiments, the nucleotide sequence of the crRNA is as shown in SEQ ID NO.1.

[0026] In certain embodiments, the nucleotide sequence of the asymmetric primer pair is SEQ ID NO.10 and SEQ ID NO.11.

[0027] In certain embodiments, the nucleotide sequence of the single-stranded fluorescent reporter probe is CCCCCC.

[0028] In certain embodiments, the 5' end of the single-stranded fluorescent reporter probe is modified with a fluorescent reporter group FAM, and the 3' end of the probe is modified with a fluorescent reporter group BHQ.

[0029] The second technical solution provided by the present invention is a method for detecting target nucleic acid in a single tube, wherein the method uses the reaction system described in the first technical solution to detect the target.

[0030] In certain embodiments, the method comprises the following specific steps:

[0031] (1) Assembling Cas12a and crRNA in the reaction system at 37° C. for 20 min;

[0032] (2) Add the sample to be tested, primers and reagents for VPCR amplification reaction into the reaction tube, and finally add paraffin oil;

[0033] (3) adding the Cas12a / crRNA complex incubated in (1) to the single-stranded fluorescent reporter probe, mixing well and adding to the reaction tube cap in (2) for VPCR amplification reaction for 30 to 35 minutes;

[0034] (4) Centrifuge for 10 to 30 seconds;

[0035] (5) Incubate at 37°C for 5 min and then determine the result.

[0036] In certain embodiments, the target nucleic acid is obtained after nucleic acid extraction of the sample to be tested.

[0037] In certain embodiments, the target nucleic acid is single-stranded DNA.

[0038] In certain embodiments, after the reaction is completed, dual-mode monitoring of naked eye color visualization and fluorescence visualization is performed.

[0039] The third technical solution provided by the present invention is a kit, which contains the reaction system described in the first technical solution.

[0040] The fourth technical solution provided by the present invention is the use of the reaction system described in the first technical solution, or the method described in the second technical solution, or the kit described in the third technical solution in detecting foodborne pathogens.

[0041] In certain embodiments, the foodborne pathogenic bacteria include Salmonella, Escherichia coli, Staphylococcus aureus. In certain embodiments, the foodborne pathogenic bacteria is Salmonella typhimurium.

[0042] The technical effects of the present invention are as follows:

[0043] (1) By designing an asymmetric V-type PCR nucleic acid amplification scheme, a large number of target single strands are generated in a short time, so that the Cas12a-mediated cleavage reaction is freed from the limitation of the PAM site in the excitation stage, which is more conducive to the flexible design of nucleic acid amplification primers and the flexible design of crRNA targeting intervals, increasing the designable intervals, and even intervals without the optimal PAM site can be detected using the CRISPR system. At the same time, the results show that the single-stranded product system activates the cleavage activity of Cas12a more efficiently than the double-stranded product activation, and can produce a stronger fluorescent signal. In addition, asymmetric V-type PCR nucleic acid amplification is faster than traditional PCR (time is shortened by 50%), and the naked eye color visualization sensitivity of bacteria can reach 10CFU / mL, and the visualization sensitivity under fluorescence excitation can reach 1CFU / mL.

[0044] (2) Compared with the traditional method, we used spatial separation operation, paraffin oil stratification, and regulation of the distribution of the aqueous phase system to integrate the entire reaction into a single tube. This simplified the experimental steps and effectively avoided aerosol contamination in the traditional transfer process. At the same time, two modes of visual detection, naked eye color and fluorescence, were achieved in a single tube. The entire reaction was completed within 40 minutes, and the test results were consistent with the standard qPCR method. In comparison, it has the advantages of being faster, simpler, more sensitive and intuitive, and getting rid of the fluorescence monitoring instrument. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a schematic diagram of the mechanism of the present invention.

[0046] Figure 2 To verify that single-stranded DNA can waive the limitation of PAM site to activate Cas12a trans-cutting activity.

[0047] Figure 3 This is the establishment of an asymmetric v-type PCR-CRISPR / nucleic acid detection basic reaction system; the positive color visualized by the naked eye is green, and the negative color is purple; the positive color visualized by fluorescence is green, and the negative color is black.

[0048] Figure 4 Optimize the amplification system conditions (dNTP, Taq polymerase, annealing temperature, asymmetric primer dilution multiple).

[0049] Figure 5 Optimize the ratio of Cas12a to crRNA.

[0050] Figure 6 Optimize the dosage of Cas12a / crRNA complex.

[0051] Figure 7 Optimize concentration for probe visualization.

[0052] Figure 8 This is a sensitivity experiment of the asymmetric v-type PCR-CRISPR method, taking Salmonella typhimurium as an example.

[0053] Fig. 9 Evaluation of the specificity of the asymmetric v-PCR-CRISPR method.

[0054] Fig.10 The sensitivity of the standard qPCR method for detecting Salmonella Typhimurium.

[0055] Fig.11 Comparison of the results of standard qPCR and asymmetric v-PCR-CRISPR methods in detecting Salmonella in actual samples. DETAILED DESCRIPTION

[0056] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0057] Taking the detection of foodborne pathogens as an example, the detection principle of the present invention is as follows:

[0058] This principle is shown in the schematic diagram ( Figure 1) is described. The detection strategy of traditional PCR-CRISPR / Cas12a usually relies on time-consuming ordinary PCR amplification, and it is necessary to transfer the amplified product part to the CRISPR cutting system assembled in advance. Subsequently, crRNA will specifically target the target product containing PAM sites, thereby activating the trans-cleavage activity of Cas12a to cleave the fluorescent probe. For traditional methods, its biggest shortcoming is the limitation of PAM sites. Due to the inherent properties of Cas12a protein, a PAM site with a TTTN sequence must be recognized by the Cas12a / crRNA complex, and some of the best primer amplification intervals do not necessarily contain the best PAM site, so the design of amplification primers and the screening difficulty of the targeting interval of crRNA are greatly improved in practical applications. And the method of transforming Cas protein to make it free from the limitation of PAM sites is too complicated and professional, so the present invention focuses on the improved design of nucleic acid amplification methods. By adopting the asymmetric v-type PCR amplification method, a large number of single-stranded targets in the conserved gene interval of foodborne pathogens can be amplified in a short time. The Cas protein is not restricted by the PAM site when recognizing single-stranded targets, and the signal of single-stranded activation is stronger than that of double-stranded activation. The CRISPR system is activated by a large number of single-stranded amplification products to produce a stronger trans-cleavage activity than double-stranded product activation, thereby cleaving high-concentration fluorescent probes and achieving dual visualization of naked eye color and fluorescence. The entire reaction is integrated into a single tube. The amplification system is placed at the bottom of the tube in a spatially separated manner and liquid-sealed with paraffin oil. Due to the difference in water and oil density, the water phase can be isolated from the outside world, and evaporation during thermal cycling can be prevented. Losses caused by loss. After the Cas12a / crRNA cutting system is assembled in advance, it is placed above the tube cap. Due to the surface tension of water droplets, the cutting will not drip into the tube. After the ultra-fast asymmetric V-type PCR amplification of the target DNA of the genome of foodborne pathogens produces a large number of single strands, the reaction tube is taken out and centrifuged briefly to fully mix the cutting system with the amplified product. The reaction is then kept at 37°C for 5 minutes to determine the presence or absence of the pathogen nucleic acid target through the naked eye color and fluorescence excitation dual visualization mode. When the foodborne pathogen nucleic acid target is present, the naked eye color is green and the fluorescence is visible green. When the nucleic acid target is not present, the naked eye color is purple and the fluorescence is invisible.

[0059] The raw materials used in the embodiment:

[0060] 1. Cas12a protein was expressed in the laboratory according to the reference (Liu Ru, Li Xiaolong, Zhang Xiaoqian, et al. Prokaryotic expression and activity detection of LbCas12a protein [J]. Chinese Journal of Animal Husbandry and Veterinary Medicine, 2022, 49(05): 1621-1629. DOI: 10.16431 / j.cnki.1671-7236.2022.05.003). The plasmid was designed using SNAPEGENE software, and the plasmid was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0061] 2. The RNA, DNA, and FQ used in the method were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0062] 3. Taq DNA Polymerase was purchased from Nanjing Novezan Biotechnology Co., Ltd., with the catalog number P101-01.

[0063] 4. dNTP Mixture (2.5 mM each) was purchased from Biotime Biotechnology Co., Ltd. with the catalog number D7371.

[0064] 5. NEB buffer 2.1 was purchased from the official website of NEB Life Sciences Reagents, product number B6002V.

[0065] 6. Paraffin oil was purchased from Sangon Biotechnology Co., Ltd.

[0066] 7. Nucleic acid extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0067] 8. Salmonella Typhimurium ATCC 14028, Escherichia coli ATCC43888, Staphylococcus aureus ATCC 29213, Listeria monocytogenes ATCC 19115, and Vibrio parahaemolyticus ATCC 17802 were purchased from ATCC.

[0068] Example 1 Comparison of CRIPSR / Cas12a cleavage activity activated by single-stranded DNA and double-stranded DNA

[0069] In this embodiment, taking the invA gene in Salmonella typhimurium as an example, it is verified that single-stranded DNA (ssDNA, as shown in Table 1) can also activate Cas, and has a significant enhancement effect compared to double-stranded DNA (dsDNA, as shown in Table 2). Three different target region simulation chains in the invA interval were synthesized, one of which contained the best PAM site, and the other two target intervals had no PAM site. First, the double-stranded hybridization experimental system is as follows: ddH2O 30μL, Annealing Buffer 10μL, single-stranded DNA upstream chain (ssDNA-UP) (100μM, 5μL), single-stranded DNA downstream chain (ssDNA-DOWN) (100μM, 5μL). Hybridization procedure: (1) 95℃2min; (2) 0.1℃ drop every 8s, about 90min; (3) 4℃ storage. After hybridization is completed, Cas12a is activated with ssDNA-UP in these three intervals and dsDNA after hybridization, and the cutting reaction is carried out at 37℃ and the fluorescence signal is monitored. The CRISPR / Cas12a cutting system is as follows: NEB buffer 2.1 (10X) 2 μL, Cas12a (5 μM) 0.2 μL, crRNA (2.5 μM) 0.4 μL, FQ (10 μM) 1 μL, dsDNA / ssDNA (1 μM) 1 μL, ddH2O 15.4 μL.

[0070] Table 1 The sequences of ssDNA, crRNA and fluorescent probe (FQ) are as follows (5'-3')

[0071]

[0072] Table 2 dsDNA information

[0073] dsDNA-KY KY-ssDNA-UP and KY-ssDNA-DOWN hybridize to form double-stranded DNA dsDNA-1 No PAM1-ssDNA-UP and No PAM1-ssDNA-DOWN hybridize to form double-stranded DNA dsDNA-2 No PAM2-ssDNA-UP and No PAM2-ssDNA-DOWN hybridize to form double-stranded DNA

[0074] The experimental results show that the target region can activate the side cutting activity of Cas12a regardless of whether it contains the optimal PAM site or the ssDNA without PAM site ( Figure 2 ), indicating that the ssDNA target broke the activation restriction of the PAM site, and the overall fluorescence intensity of the ssDNA activation group was higher than that of the dsDNA group, proving that ssDNA activation of CRISPR does have a signal amplification effect.

[0075] Example 2 Establishment of asymmetric V-type PCR-CRISPR / nucleic acid detection basic reaction system

[0076] In this embodiment, taking Salmonella typhimurium as an example, a nucleic acid detection method is established.

[0077] 1 Design asymmetric primer pairs and crRNA for the target (Salmonella typhimurium), as shown in the table below.

[0078] Table 3 Primers and crRNA

[0079]

[0080] 2. Nucleic Acid Extraction

[0081] The genomic DNA of Salmonella typhimurium was extracted using a nucleic acid extraction kit.

[0082] 3 Establishment of asymmetric V-type PCR-CRISPR single-tube detection basic reaction system

[0083] CRISPR cleavage system: NEB Buffer 2.1 (10X) 3 μL, Cas12a (5 μM) 1.8 μL, crRNA (5 μM) 1.8 μL, FQ (100 μM) 2.4 μL, ddH2O 1 μL. First, assemble all components except FQ, incubate in a 37°C oven for 20 min, then add the high-concentration probe and mix well for later use.

[0084] Asymmetric V-type PCR amplification system: dNTP (2.5mM) 0.8μL, Taq polymerase (5U / μL) 0.4μL, TaqBuffer (10X) 2.5μL, upstream primer KY-F (1μM) 3μL, downstream primer KY-R (10μM) 3μL, target nucleic acid DNA 6μL, ddH2O 4.3μL. A total of 20μL of amplification system was mixed evenly and placed at the bottom of the tube, 30μL of paraffin oil was added to the top for separation, 10μL of the incubated cutting system was added to the tube cap, and then the reaction tube was placed in a thermal cycler for asymmetric V-type PCR amplification reaction.

[0085] Table 4 Asymmetric V-type PCR reaction program

[0086]

[0087] After the amplification reaction is completed, centrifuge the reaction tube for 10 to 30 seconds using a mini centrifuge and place it at 37°C for 5 minutes for the cutting reaction. Observe the visual results with the naked eye or under fluorescence excitation. The results of the basic reaction system are as follows Figure 3 As shown, for the naked eye color mode, the positive is green and the negative is purple. For the fluorescent mode, the positive is green and the negative is no fluorescence (black). When there is no Cas12a, crRNA, DNA polymerase, upstream and downstream primers, fluorescent probes and targets in the system, no cleavage reaction occurs, and the color and fluorescent signal are unchanged. Only when all components are present, the whole reaction can proceed normally, resulting in changes in color signal and fluorescent signal, and the experimental results show that the dual-mode visualization system is successfully established.

[0088] Example 3 Reaction system optimization

[0089] 1. Optimization of amplification system conditions

[0090] The reaction system was optimized in order to obtain a reaction system with strong specificity and high sensitivity: the reaction system was the same as that in Example 2.3, and the parameters that needed to be optimized were adjusted under the same conditions.

[0091] 1dNTP concentration optimization

[0092] The reaction system was the same as in Example 2, except that the final dNTP concentrations were adjusted to 100 μM, 150 μM, 200 μM, 250 μM, and 300 μM. Figure 4 As shown in (A), when the dNTP concentration is 100 μM or 250 μM, the end point fluorescence signal value is higher and the intensity is comparable. From the perspective of economic benefits, the optimal dNTP concentration of 100 μM was selected in this experiment to save costs. In addition, low concentrations of dNTP can also reduce nonspecific amplification products during amplification.

[0093] 2Taq DNA polymerase concentration optimization

[0094] The reaction system was the same as in Example 2, except that the final concentration of Taq polymerase was adjusted to 0.05U / μL, 0.1U / μL, 0.15U / μL, 0.2U / μL, 0.25U / μL, and 0.3U / μL. Figure 4 As shown in (B), when the concentration of Taq polymerase is 0.1 U / μL, the end point fluorescence signal value reaches the highest value, so the optimal concentration of Taq polymerase is 0.1 U / μL.

[0095] 3 Annealing temperature optimization

[0096] The reaction system was the same as in Example 2, except that the annealing temperature of the amplification program was adjusted to 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, and 64°C. Figure 4 As shown in (C), when the annealing temperature is 64°C, the endpoint fluorescence signal value reaches the strongest, so the optimal annealing temperature is 64°C.

[0097] 4. Optimization of upstream primer dilution multiple

[0098] The reaction system was the same as in Example 2, except that the dilution ratio of the upstream primer KY-F was adjusted to 1:1, 1:10, 1:20, 1:30, and 1:40. Figure 4 As shown in (D), when KY-F was diluted ten-fold, the endpoint fluorescence signal value reached the highest value, so the optimal KY-F dilution factor was 1:10.

[0099] 2. Optimization of the ratio of Cas12a to crRNA

[0100] The reaction system is the same as in Example 2, only the usage ratio of crRNA and Cas12a is adjusted to 1:0.25, 1:0.5, 1:1, 1:1.25, 1:1.5 and 1:2, and the results are as follows Figure 5 As shown, when the ratio of Cas12a to crRNA is 1:1, the endpoint fluorescence signal value reaches the strongest, so the optimal ratio of Cas12a to crRNA is 1:1.

[0101] 3. Optimization of the dosage of Cas12a-crRNA complex

[0102] The reaction system was the same as in Example 2, except that the final concentrations of crRNA and Cas12a were adjusted to 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM and 400 nM. Figure 6 As shown, when the concentration of Cas12a and crRNA is 300nM, the endpoint fluorescence signal value reaches the strongest, so the optimal concentration of Cas12a and crRNA is 300nM.

[0103] 4. Optimization of probe visualization concentration

[0104] The reaction system was the same as in Example 2, except that the final concentration of the probe was adjusted to 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM. The results were as follows: Figure 7 As shown, when the final concentration of the probe is 10 μM, the endpoint fluorescence signal value reaches the strongest and the visualization effect is the best, so the optimal probe concentration is 10 μM.

[0105] Example 4 Asymmetric V-type PCR-CRISPR method sensitivity experiment

[0106] In order to explore the sensitivity of this method, the gradient diluted Salmonella template was tested using the method established in Example 3. The specific process is as follows: First, the Salmonella template DNA extracted from the kit was gradiently diluted 10 times using ddH2O to obtain 10 6 CFU / mL~10 0 There were seven groups of template DNA with CFU / mL, plus a total of 8 groups with a negative control group. Then, according to the optimal detection system established in Example 3, an asymmetric vPCR amplification reaction was performed first, followed by centrifugation for 10-30s, and finally a Cas12a cleavage reaction at 37°C. Finally, the signal was visualized by fluorescence and color, respectively, and the detection limit of this method for Salmonella was obtained.

[0107] The experimental results show that the detection limit of this method is 10 CFU / mL in naked eye mode and 1 CFU / mL in fluorescence mode. Both modes have high sensitivity. The sensitivity of fluorescence mode is 10 times higher than that of color method. This proves that this method is suitable for multi-scene detection needs, that is, naked eye color visualization meets the needs of ordinary on-site instrument-free initial screening, and fluorescence visualization meets the needs of further accurate verification ( Figure 8 ).

[0108] Example 5 Evaluation of the specificity of the asymmetric V-type PCR-CRISPR method

[0109] In order to further investigate the specificity of the present method, Salmonella Typhimurium ATCC 14028, Escherichia coli ATCC 43888, Staphylococcus aureus ATCC 29213, Listeria monocytogenes ATCC 19115, Vibrio parahaemolyticus ATCC 17802 and the template mixture of these five bacteria were respectively used for detection according to the detection method established in Example 3. According to the fluorescence intensity bar graph and the dual-mode visualization results, only when Salmonella Typhimurium is present can the reaction occur to generate a fluorescent signal, and green ( Fig. 9 ).

[0110] Example 6 Comparison of asymmetric V-type PCR-CRISPR method and standard qPCR method

[0111] Taking Salmonella typhimurium as an example, fluorescence quantitative PCR (qPCR) method was used for detection.

[0112] Table 5 qPCR primers

[0113] Sal-Primer1(5'-3'):GCGGCGTTGGAGAGTGATA Sal-Primer2(5'-3'):AGCAATGGA AAAAGCAGGATG Sal-Siganl(5'-3'):FAM-CATTTCTTAAACGGCGGTGTCTTTCCCT-BHQ1

[0114] Configure the reaction system and set up the reaction program according to the instructions of the industry standard (SN / T 1870-2016).

[0115] The experimental results showed that the sensitivity of the qPCR method was 10 2 CFU / mL( Fig.10 ), the method of the present invention is 100 times more sensitive than it. More importantly, the method does not require expensive qPCR instruments, has the advantages of short detection time (30-40 minutes) and can achieve color fluorescence dual mode visualization.

[0116] Table 6 Comparison between the present invention and standard qPCR method

[0117]

[0118] Example 7 Actual sample detection

[0119] Foodborne pathogens seriously endanger human health, and their spread poses a high food safety risk. Therefore, the feasibility of the detection method established in Example 3 for actual sample detection was evaluated, and 30 meat samples (chicken and pork) from the retail market were collected. At the same time, the standard qPCR method (SN / T1870-2016) was used as a control method. The results show that the present invention and the standard qPCR method have high consistency in detecting Salmonella DNA in food samples, and have higher sensitivity. The present invention detected 7 Salmonella-positive samples and 23 negative samples, while the standard qPCR method detected 6 Salmonella-positive samples and 24 negative samples ( Fig.11 ). In addition, the dual-mode visual signal readout of the present invention makes the determination of the test results more convenient and quick, and has more advantages in practical applications in the field of food safety testing in the future.

[0120] In summary, the present invention designs an ultra-fast V-type PCR amplification method, the principle of which is to avoid the time consumption in the traditional PCR denaturation-annealing-extension process by setting a fast instantaneous temperature rise and fall process, thereby greatly shortening the time of the amplification reaction, and shortening the original 1.5h nucleic acid target preparation process to about 0.5h. At the same time, the concentration ratio of the upstream and downstream primers in the ultra-fast V-type PCR amplification reaction is adjusted, and the bacterial genomic DNA is amplified to produce a large number of target single strands through asymmetric amplification. Since the CRISPR system can activate its cutting activity without the need for a PAM site when identifying a target single strand, the limitation of the traditional double-stranded product PAM site is avoided, making the design of the primer amplification interval more flexible. At the same time, the activation of the CRISPR system by a single-stranded product will produce a stronger and faster fluorescent signal than the activation of the double-stranded product, thereby improving the detection rate and sensitivity. At the same time, in order to avoid aerosol contamination, we have adopted an integrated detection method for spatial separation in a single tube, in which the amplification system is placed at the bottom of a centrifuge tube, and then paraffin oil is added to its surface to isolate and seal the amplification system. The CRISPR cutting system and the high-concentration fluorescent reporter probe are placed in the centrifuge tube cap. Due to the effect of its surface tension, the reagents placed in the tube cap will not drip. The specific implementation method is: first, an ultra-fast asymmetric V-type PCR amplification reaction is carried out in a thermal cycler, which takes about 30 minutes to produce a large number of target single strands that can realize PAM-free enhanced activation of the CRISPR system. Then the centrifuge tube is taken out for a simple centrifugation to fully mix the cutting system and the amplification product, and then reacted at a constant temperature of 37°C for 5 minutes. Due to the presence of high-concentration fluorescent probes, dual-mode signal readout based on naked eye color visualization and fluorescent naked eye can be completed. Therefore, a more time-saving, PAM-free, pollution-free, signal-enhanced integrated ultra-fast single-tube detection is achieved. Since single-stranded products have a higher activation efficiency for the CRISPR system than double-stranded products, the entire method has extremely high sensitivity (1 CFU / mL). The use of naked-eye color visualization avoids complex readout forms such as traditional PCR and qPCR reactions that require gel running and observation of fluorescence curves, gets rid of dependence on expensive instruments, and reduces reagent costs and operating thresholds. It is therefore more conducive to grassroots promotion and use.

[0121] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A reaction system for detecting a target object, characterized in that: The reaction system includes NEB buffer 2.1, crRNA, Cas12a protein, single-stranded fluorescent reporter probe, asymmetric primer pairs and reagents for VPCR amplification reaction; Using the target as a template, VPCR amplification is performed using an asymmetric primer pair. The single-stranded activator produced is recognized by the Cas12a / crRNA complex, which activates the side-cutting activity of the Cas12a protein without the PAM site. The high-concentration fluorescent signal probe in the side-cutting system can produce naked eye color visualization and fluorescent visualization signals after cleavage.

2. The reaction system according to claim 1, characterized in that The target is the invA gene in Salmonella typhimurium; the nucleotide sequence of the crRNA is shown in any one of SEQ ID NOs. 1 to 3; The nucleotide sequence of the asymmetric primer pair is selected from any one of the groups (a) to (c): (a) SEQ ID NO.4 and SEQ ID NO.5; (a) SEQ ID NO.6 and SEQ ID NO.7; (a) SEQ ID NO.8 and SEQ ID NO.

9.

3. The reaction system according to claim 1, characterized in that The target is Salmonella typhimurium; The nucleotide sequence of the crRNA is shown in SEQ ID NO.1; The nucleotide sequences of the asymmetric primer pair are SEQ ID NO.10 and SEQ ID NO.

11.

4. The reaction system according to any one of claims 1 to 3, characterized in that: One end of the single-stranded fluorescent reporter probe carries a fluorescent reporter group, and the other end carries a fluorescent quencher group; the nucleotide sequence of the single-stranded fluorescent reporter probe is CCCCCC.

5. The reaction system according to any one of claims 1 to 4, characterized in that: The dilution ratio of the single-side primer in the asymmetric primer pair is 1:1-1:40; The ratio of crRNA to Cas12a is 1:0.25-1:2; The concentration of the Cas12a and crRNA complex is 50nM-400nM; The final concentration of the reporter probe is 1 μM-10 μM.

6. A method for detecting a target nucleic acid, characterized in that: The method is to detect the target nucleic acid using the reaction system described in any one of claims 1 to 5.

7. The method according to claim 6, characterized in that The specific steps are as follows: (1) Assemble Cas12a protein and crRNA at 37°C for 20 min; (2) adding the sample to be tested, the asymmetric primer pair and the reagents for the VPCR amplification reaction into the reaction tube, and finally adding paraffin oil; (3) adding the Cas12a / crRNA complex incubated in (1) to the single-stranded fluorescent reporter probe, mixing well and adding to the reaction tube cap in (2) for VPCR amplification reaction for 30 to 35 minutes; (4) Centrifuge for 10 to 30 seconds; (5) Incubate at 37°C for 5 min and then determine the result.

8. A kit, characterized in that: The kit contains the reaction system according to any one of claims 1 to 5.

9. Use of the reaction system according to any one of claims 1 to 5, or the method according to claim 6 or 7, or the kit according to claim 8 in detecting foodborne pathogens.

10. The use according to claim 9, characterized in that: The foodborne pathogens include Salmonella, Escherichia coli, and Staphylococcus aureus.

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