Synchronous live and dead bacterium detection sensor based on Cas12-Cas13 combination, detection method and application

Abstract

The invention discloses a synchronous live and dead bacterium detection sensor based on Cas12-Cas13 combination, a detection method and application, the synchronous live and dead bacterium detection sensor comprises a liposome I-DNA reporter gene-96 pore plate-RNA reporter gene-liposome II compound, a Cas12 system and a Cas13 system, in the liposome I-DNA reporter gene-96 pore plate-RNA reporter gene-liposome II compound, the Cas12 system and the Cas13 system are combined, and the Cas12 system and the Cas13 system are combined. A signal molecule I is embedded in the liposome I, and a signal molecule II is embedded in the liposome II; the Cas12 system comprises Cas12 protein and crRNA, and is used for generating a liposome I modified with a DNA cleavage fragment; the Cas13 system comprises Cas13 protein and gRNA, and is used for generating a liposome II modified with an RNA cleavage fragment; after the liposome I modified with the DNA cutting fragment and the liposome II modified with the RNA cutting fragment are collected and crushed, signal molecules I and signal molecules II are released, and the live bacteria and the dead bacteria are synchronously and quantitatively analyzed by detecting the signal intensity of the signal molecules I and the signal intensity of the signal molecules II.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and specifically to a sensor, method and application for synchronous detection of live and dead bacteria based on the combination of Cas12-Cas13. Background Art

[0002] In recent years, diseases caused by pathogenic bacteria have been seriously endangering people's health and causing serious medical and economic burdens worldwide. Among these pathogens, Cronobacter mainly harms newborns and people with weakened immunity. It may cause serious infections such as meningitis and sepsis, especially in premature infants. Although the harm of Cronobacter (also known as Enterobacter sakazakii) is not as great as other common pathogens (such as Salmonella, Staphylococcus aureus, etc.), its severity in neonatal infections has attracted attention. At present, Cronobacter infection is usually related to improperly handled infant formula milk powder. Therefore, rapid detection of Cronobacter that may be present in milk powder is of great significance for the early prevention and later treatment of infant infection.

[0003] For example, the application publication number CN118389716A discloses a MIRA-CRISPRCas12a-based pathogenic Cronobacter typing detection method. The constructed detection method is simple to operate, fast and convenient, highly sensitive, and suitable for on-site detection. It can identify whether there is Cronobacter contamination in the sample to be tested in a relatively short time, and type and determine the type of pathogenic bacteria, which is of great significance for food safety risk assessment caused by Cronobacter. However, this scheme still requires a large amount of MIRA amplification for identification and detection, and the detection efficiency needs to be improved. In addition, there is often a problem of aerosol contamination during the amplification process, which may cause the final test results to be inaccurate.

[0004] In addition to the above-mentioned CRISPR detection methods, there are currently many other methods for bacterial detection, but most of these detection methods can only detect live and dead bacteria indiscriminately, making it difficult to detect live and dead bacteria simultaneously, and it is difficult to determine the ratio of live and dead bacteria in the sample. In actual testing, if live and dead bacteria can be detected at the same time, this can provide a more comprehensive understanding of the infection status, including the pathogenicity of live bacteria and the immune response of dead bacteria, and in food testing, the ratio of live to dead bacteria can reveal the microbial quality of the product and its safety. Therefore, developing a method that can quickly and simultaneously detect live and dead bacteria (such as Cronobacter) in food is crucial to the health of infants and young children. Summary of the invention

[0005] The object of the present invention is to provide a live-dead bacteria synchronous detection sensor, detection method and application based on the combination of Cas12 and Cas13, so as to solve the problems put forward in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A live-dead bacteria synchronous detection sensor based on the combination of Cas12 and Cas13, comprising: a liposome I-DNA reporter gene-96-well plate-RNA reporter gene-liposome II complex, wherein a signal molecule I is embedded in the liposome I, and a signal molecule II is embedded in the liposome II;

[0008] The Cas12 system, which includes Cas12 protein and crRNA. The crRNA is used to complementarily bind to the target DNA of bacteria and guide the Cas12 protein to cleave the DNA reporter gene in the liposome I-DNA reporter gene-96-well plate-RNA reporter gene-liposome II complex, generating liposome I modified with DNA cleavage fragments;

[0009] The Cas13 system, which includes Cas13 protein and gRNA. The gRNA is used to complementarily bind to the target RNA of bacteria and guide the Cas13 protein to cleave the RNA reporter gene in the liposome I-DNA reporter gene-96-well plate-RNA reporter gene-liposome II complex, generating liposome II modified with RNA cleavage fragments;

[0010] After the liposome I modified with DNA cleavage fragments and the liposome II modified with RNA cleavage fragments are collected and broken, the signal molecule I and the signal molecule II are released, and the live bacteria and dead bacteria are synchronously quantitatively analyzed by detecting the signal intensities of the signal molecule I and the signal molecule II.

[0011] As a further solution of the present invention, the liposome I-DNA reporter gene-96-well plate-RNA reporter gene-liposome II complex includes:

[0012] The DNA reporter gene modified with biotin;

[0013] The RNA reporter gene modified with biotin;

[0014] A 96-well plate containing bovine serum albumin (BSA);

[0015] The liposome I modified with biotin, and the biotin between the liposome I and the biotin of the DNA reporter gene is connected by streptavidin to form a liposome I-DNA reporter gene complex. The end of the DNA reporter gene far from the liposome I is connected with NH for binding to bovine serum albumin (BSA) in the 96-well plate 2;

[0016] Biotinylated liposome II, the biotin of liposome II is linked to the biotin of the RNA reporter gene through streptavidin, liposome II-RNA reporter gene complex, and an NH for binding to bovine serum albumin (BSA) in a 96-well plate is linked to one end of the RNA reporter gene far from liposome II 2 .

[0017] As a further aspect of the present invention, it further includes a SERS substrate material (specifically gold-coated silver nanoparticles) for enhancing the SERS effect of signal molecule I and signal molecule II;

[0018] The signal molecule I is Nile blue A perchlorate (NBA), and the signal molecule II is 4-aminothiophenol (4-ATP).

[0019] A method for synchronous detection of live and dead bacteria based on the combined use of Cas12 and Cas13, including the above-mentioned sensor for synchronous detection of live and dead bacteria based on the combined use of Cas12 and Cas13, comprising the following steps:

[0020] S1. Mix the sample containing the bacteria to be detected evenly with the one-step nucleic acid extraction solution, and heat and lyse at a high temperature to obtain a solution containing the nucleic acid (DNA / RNA) of the bacteria to be detected;

[0021] S2. Add the solution containing the nucleic acid (DNA / RNA) of the bacteria to be detected, the Cas12 system, and the Cas13 system to the liposome I-DNA reporter gene-96-well plate-RNA reporter gene-liposome II complex, and incubate at 37 °C to fully shear the DNA reporter gene and the RNA reporter gene;

[0022] S3. Take the supernatant of the 96-well plate solution, and add phosphate buffered saline with Tween (PBST) to break liposome I and liposome II;

[0023] S4. Mix the solution after the breaking treatment with the SERS substrate material, and measure the SERS signal by a portable Raman spectrometer to perform synchronous quantitative analysis on the content of live and dead bacteria in the sample.

[0024] As a further aspect of the present invention, the one-step nucleic acid extraction solution in step S1 includes magnesium chloride, EDTA, Tris-HCl, triethanolamine dodecyl sulfate, and potassium chloride, wherein the concentration of magnesium chloride is 20-80 mM, the concentration of EDTA is 5-20 mM, the concentration of Tris-HCl is 10-50 mM, the mass-volume fraction of triethanolamine dodecyl sulfate is 2%-20%, and the concentration of potassium chloride is 2.0-10.0 mol / L.

[0025] As a further solution of the present invention, the preparation method of the liposome I-DNA reporter gene-96-well plate-RNA reporter gene-liposome II complex in step S2 is as follows:

[0026] A1. Take 75 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) with a concentration of 0.4 M and 75 μL of N-hydroxysulfosuccinimide (Sulfo-NHS) with a concentration of 0.2 M and add them to the 96-well plate, and incubate at room temperature for 2 h.

[0027] A2. Take 300 μL of 5% bovine serum albumin (BSA) (in excess) (dissolved in PBS) and add it to the 96-well plate, incubate in a thermostatic shaker at 37 °C and 100 rpm for 1.5 h, and then wash three times with deionized water.

[0028] A3. Take 100 μL of the liposome I-DNA reporter gene complex with a concentration of 10 μM and 100 μL of the liposome II-RNA reporter gene complex with a concentration of 10 μM and add them to the 96-well plate, incubate at 37 °C for 3 h to generate the liposome I-DNA reporter gene-96-well plate-RNA reporter gene-liposome II complex, and rinse the 96-well plate three times with deionized water for standby.

[0029] As a further solution of the present invention, the preparation method of the liposome I-DNA reporter gene complex is as follows:

[0030] Disperse 5 mg of L-α-phosphatidylcholine, cholesterol and PE-PEG-2000-biotin mixed in a molar ratio of 2:2:1 in 1 mL of chloroform, and rotary evaporate until a thin film is formed; then, resuspend with 5 mL of a signal molecule I solution with a concentration of 4 mM to obtain a liposome I solution coated with signal molecule I, and the surface of the liposome I in the liposome I solution is modified with biotin; among them, the molar number of L-α-phosphatidylcholine is 6.6 μM;

[0031] Put the liposome I solution coated with signal molecule I in a dialysis bag and dialyze it 3-5 times in ultrapure water, and each dialysis lasts for 30-60 min;

[0032] Mix and react the liposome I solution coated with signal molecule I after dialysis, streptavidin and the DNA reporter gene modified with biotin and -NH2 at both ends to form a liposome I-DNA reporter gene complex;

[0033] The preparation method of the liposome II-RNA reporter gene complex is as follows:

[0034] Disperse 5 mg of L-α-phosphatidylcholine, cholesterol, and PE-PEG-2000-biotin mixed in a molar ratio of 2:2:1 in 1 mL of chloroform and rotary evaporate to form a thin film; subsequently, resuspend with 5 mL of a 4 mM solution of signal molecule two to obtain a liposome two solution coated with signal molecule two, and the outer surface of the liposomes in the liposome two solution is modified with biotin; wherein, the molar amount of L-α-phosphatidylcholine is 6.6 μM;

[0035] Place the liposome two solution coated with signal molecule two in a dialysis bag and dialyze it 3-5 times in ultrapure water, with each dialysis lasting 30-60 min;

[0036] Mix and react the liposome two solution coated with signal molecule two after dialysis, streptavidin, and an RNA reporter gene modified with biotin and -NH2 at both ends to form a liposome two-RNA reporter gene complex.

[0037] As a further aspect of the present invention, in step S2, the dosage of the solution containing the nucleic acid (DNA / RNA) of the bacteria to be detected is 1-5 μL, and the final concentrations of Cas12 protein and Cas13 protein are both 50-100 nM; the final concentrations of crRNA and gRNA are both 5-100 nM; the final concentrations of the DNA reporter gene and RNA reporter gene are 5-100 μM; the final concentration of streptavidin is 1-10 mM, and the final concentration of the sulfate Tween buffer (PBST) is 5-10×; the SERS substrate material uses gold-coated silver nanoparticles, and its dosage is 50-500 μL; incubate at 37 °C for an incubation time of 10-30 min; the SERS spectral measurement conditions of the portable Raman spectrometer are 785 nm and 100 mW.

[0038] As a further aspect of the present invention, the bacteria to be detected are specifically Cronobacter, and the crRNA sequence for detecting Cronobacter is shown in SEQ ID NO.1;

[0039] The gRNA sequence for detecting Cronobacter is shown in SEQ ID NO.2;

[0040] The corresponding DNA reporter gene is shown in SEQ ID NO.3;

[0041] The corresponding RNA reporter gene is shown in SEQ ID NO.4.

[0042] An application of a live-dead bacteria synchronous detection sensor based on the combination of Cas12 and Cas13 for simultaneously quantitatively detecting live and dead Cronobacter in food samples.

[0043] Compared with the prior art, the beneficial effects of the present invention are:

[0044] 1. Compared with traditional live or dead bacteria detection methods, the detection method of the present invention can detect live and dead bacteria simultaneously, avoiding the greater harm caused by misestimating the severity of bacterial infection due to the inability to distinguish between live and dead bacteria.

[0045] 2. The detection method of the present invention directly identifies nucleic acids in a non-amplified manner, avoiding the problem of aerosol contamination existing in the traditional amplification process.

[0046] 3. The present invention uses liposomes to encapsulate signal molecules, which has good sealing properties. After fragmentation, the signal molecules are released, and a nano-substrate material is used to enhance the signal. The background signal is reduced and the positive signal is increased throughout the process, resulting in an excellent signal-to-noise ratio.

[0047] 4. After direct identification by the detection method of the present invention, shearing can be carried out immediately. After the separated liposomes are mixed with the nano-substrate material, the SERS signal is directly measured. The whole process takes a short time, has good repeatability, and high sensitivity.

[0048] 5. In addition, after the present invention uses crRNA / gRNA targeting highly conserved DNA / RNA fragments in the Cronobacter genome for identification, the shearing function of the corresponding Cas protein is activated, thereby non-specifically shearing the DNA / RNA reporter gene. After directly aspirating the upper solution of the 96-well plate, the liposomes are separated, and the SERS signal is measured, improving the specificity of the method. Brief Description of the Drawings

[0049] Figure 1 It is a schematic flow chart of a method for simultaneous detection of live and dead bacteria based on the combination of Cas12 and Cas13.

[0050] Figure 2 It is the feasibility analysis data of the present invention; (A) SERS spectra of live bacteria, dead bacteria, and blank groups; (B) Peak data graphs of SERS spectra of live bacteria, dead bacteria, and blank groups at characteristic peaks (593 cm -1 and 1079 cm -1 ).

[0051] Figure 3 is It is the sensitivity analysis data of the present invention; (A-C) are respectively the SERS spectra of live Cronobacter at different concentrations, the peak data graphs at characteristic peaks (593 cm -1 and 1079 cm -1 ), and the standard curve graph; (D-F) are the SERS spectra of dead Cronobacter at different concentrations, the peak data graphs at characteristic peaks (593 cm -1 and 1079 cm -1 ), and the standard curve graph.

[0052] Figure 4 Specific verification data for the present invention. Detailed implementation manners

[0053] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0054] Example 1: A live-dead bacteria synchronous detection sensor based on the combined use of Cas12 and Cas13, comprising a liposome I-DNA reporter gene-96-well plate-RNA reporter gene-liposome II complex, a Cas12 system, and a Cas13 system. In the liposome I-DNA reporter gene-96-well plate-RNA reporter gene-liposome II complex, a signal molecule I is embedded in the liposome I, and a signal molecule II is embedded in the liposome II;

[0055] Among them, the Cas12 system includes a Cas12 protein and a crRNA. The crRNA is used to complement the target DNA of the bacteria and guide the Cas12 protein to cleave the DNA reporter gene in the liposome I-DNA reporter gene-96-well plate-RNA reporter gene-liposome II complex to generate a liposome I modified with a DNA cleavage fragment;

[0056] Among them, the Cas13 system includes a Cas13 protein and a gRNA. The gRNA is used to complement the target RNA of the bacteria and guide the Cas13 protein to cleave the RNA reporter gene in the liposome I-DNA reporter gene-96-well plate-RNA reporter gene-liposome II complex to generate a liposome II modified with an RNA cleavage fragment;

[0057] After the liposome I modified with the DNA cleavage fragment and the liposome II modified with the RNA cleavage fragment are collected and broken, the signal molecule I and the signal molecule II are released, and the live bacteria and dead bacteria are synchronously quantitatively analyzed by detecting the signal intensities of the signal molecule I and the signal molecule II.

[0058] In the embodiment of the present invention, the liposome I-DNA reporter gene-96-well plate-RNA reporter gene-liposome II complex includes:

[0059] A DNA reporter gene modified with biotin;

[0060] An RNA reporter gene modified with biotin;

[0061] A 96-well plate containing bovine serum albumin (BSA);

[0062] Biotinylated liposome I, the biotin of liposome I is linked to the biotin of the DNA reporter gene through streptavidin to form a liposome I-DNA reporter gene complex. At the end of the DNA reporter gene far from liposome I, there is an NH for binding to bovine serum albumin (BSA) in a 96-well plate 2 ;

[0063] Biotinylated liposome II, the biotin of liposome II is linked to the biotin of the RNA reporter gene through streptavidin, forming a liposome II-RNA reporter gene complex. At the end of the RNA reporter gene far from liposome II, there is an NH for binding to bovine serum albumin (BSA) in a 96-well plate 2 。

[0064] In the embodiments of the present invention, it further includes a SERS substrate material for enhancing the SERS effects of signal molecule I and signal molecule II, specifically silver-coated gold nanoparticles; signal molecule I is specifically Nile blue A perchlorate (NBA), and signal molecule II is specifically 4-aminothiophenol (4-ATP).

[0065] The particle size of the silver-coated gold nanoparticles is 20 - 50 nm. The preparation method of the silver-coated gold nanoparticles includes: adding 100 μL of ascorbic acid to 1 mL of gold nanoparticles with a particle size of 20 - 40 nm, quickly vortexing and mixing evenly, then adding 300 μL of silver nitrate and vigorously vortexing at room temperature for 2 - 5 min, continuing to incubate at room temperature for 20 min, vortexing every few minutes during this period to make the system react fully, and storing for standby at 4°C.

[0066] In the specific experiment for feasibility verification, please refer to Figure 2 , a blank control group is set up. The target of the blank control group does not contain Cronobacter, while the target strains are live and dead Cronobacter. After being treated by the method of the present invention, there are no corresponding Raman signal characteristic peaks in the blank control group, which is negative; while the target strains show corresponding Raman characteristic peaks. By the presence or absence and signal intensity of the corresponding Raman characteristic peaks, the number of live bacteria and total colonies of Cronobacter contained in the sample can be calculated.

[0067] In the specific experiment for sensitivity analysis, please refer to Figure 3, Dilute the concentration of Cronobacter to different gradients (from 10 7 CFU / mL diluted to 10 0 CFU / mL), and set up a blank control group. After different concentrations of bacteria are treated by the method of the present invention, corresponding Raman characteristic peaks can be shown according to the high or low bacteria concentration. This method can detect DNA down to 10 1 CFU / mL, and can detect RNA down to 102 CFU / mL, and all have good linear relationships, enabling precise quantification of viable Cronobacter and total colony counts.

[0068] In the specific experiments for specificity verification, please refer to Figure 4 . After treating the Cronobacter group, other non-target bacterial species groups, and the mixed group of Cronobacter and other non-target bacterial species with the method of the present invention and measuring the SERS signals, it can be seen that only the target bacterial species show corresponding Raman characteristic peaks, and when the target bacterial species coexist with non-target bacterial species, the non-target bacterial species do not interfere with the signal intensity of the target bacterial species. Thus, it can be concluded that this method has excellent specificity and can clearly distinguish between target bacterial species and non-target bacterial species.

[0069] Example 2: Please refer to Figure 1 . A method for synchronous detection of viable and dead bacteria based on the combined use of Cas12 and Cas13, including the above-mentioned sensor for synchronous detection of viable and dead bacteria based on the combined use of Cas12 and Cas13, comprising the following steps:

[0070] S1. Mix the sample containing the bacteria to be tested evenly with the one-step nucleic acid extraction solution, heat and lyse at high temperature to obtain a solution containing the nucleic acid (DNA / RNA) of the bacteria to be tested;

[0071] S2. Add the solution containing the nucleic acid (DNA / RNA) of the bacteria to be tested, the Cas12 system, and the Cas13 system to the complex of liposome I - DNA reporter gene - 96-well plate - RNA reporter gene - liposome II, and incubate at 37 °C to fully shear the DNA reporter gene and the RNA reporter gene;

[0072] S3. Take the supernatant of the 96-well plate solution and add phosphate buffered saline with Tween (PBST) to break the liposome I and liposome II;

[0073] S4. Mix the solution after the breaking treatment with the SERS substrate material, and measure the SERS signal by a portable Raman spectrometer to perform synchronous quantitative analysis of the content of viable and dead bacteria in the sample.

[0074] In the embodiment of the present invention, the one-step nucleic acid extraction solution in step S1 includes magnesium chloride, EDTA, Tris-HCl, triethanolamine dodecyl sulfate, and potassium chloride. Among them, the concentration of magnesium chloride is 20 - 80 mM, the concentration of EDTA is 5 - 20 mM, the concentration of Tris-HCl is 10 - 50 mM, the mass volume fraction of triethanolamine dodecyl sulfate is 2% - 20%, and the concentration of potassium chloride is 2.0 - 10.0 mol / L.

[0075] The specific implementation method of the one-step rapid extraction method is as follows: Take 2 - 10 μL of food sample and mix it with 50 - 100 μL of one-step nucleic acid extraction solution (extraction), incubate at 90 °C for 1 - 5 min, and a solution containing the nucleic acid (DNA / RNA) of the bacteria to be detected is obtained.

[0076] In the embodiment of the present invention, the preparation method of the liposome I - DNA reporter gene - 96 - well plate - RNA reporter gene - liposome II complex in step S2 is as follows:

[0077] A1. Take 75 μL of 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide (EDC) with a concentration of 0.4 M and 75 μL of N - hydroxysulfosuccinimide (Sulfo - NHS) with a concentration of 0.2 M and add them to a 96 - well plate, and incubate at room temperature for 2 h.

[0078] A2. Take 300 μL of 5% bovine serum albumin (BSA) (in excess) (dissolved in PBS) and add it to the 96 - well plate, incubate in a constant - temperature shaker at 37 °C and 100 rpm for 1.5 h, and then wash three times with deionized water.

[0079] A3. Take 100 μL of liposome I - DNA reporter gene complex with a concentration of 10 μM and 100 μL of liposome II - RNA reporter gene complex with a concentration of 10 μM and add them to the 96 - well plate, incubate at 37 °C for 3 h to generate the liposome I - DNA reporter gene - 96 - well plate - RNA reporter gene - liposome II complex, and wash the 96 - well plate three times with deionized water for standby.

[0080] In the embodiment of the present invention, the preparation method of the liposome I - DNA reporter gene complex is as follows:

[0081] Disperse 5 mg of L - α - phosphatidylcholine, cholesterol, and PE - PEG - 2000 - biotin mixed in a molar ratio of 2:2:1 in 1 mL of chloroform, and rotary evaporate until a thin film is formed; then, resuspend with 5 mL of signal molecule I solution with a concentration of 4 mM to obtain a liposome I solution coated with signal molecule I, and the surface of liposome I in the liposome I solution is modified with biotin; among them, the molar amount of L - α - phosphatidylcholine is 6.6 μM;

[0082] Put the liposome I solution coated with signal molecule I in a dialysis bag and dialyze it 3 - 5 times in ultrapure water, with each dialysis for 30 - 60 min;

[0083] Mix and react the liposome I solution coated with signal molecule I after dialysis, streptavidin, and a DNA reporter gene modified with biotin and - NH2 at both ends to form a liposome I - DNA reporter gene complex;

[0084] The preparation method of the liposome di-RNA reporter gene complex is as follows:

[0085] Disperse 5 mg of L-α-phosphatidylcholine, cholesterol, and PE-PEG-2000-biotin mixed in a molar ratio of 2:2:1 in 1 mL of chloroform, and rotary evaporate until a thin film is formed; subsequently, resuspend with 5 mL of a 4 mM solution of signal molecule two to obtain a liposome di-solution coated with signal molecule two, and the surface of the liposome di in the liposome di-solution is modified with biotin; among them, the molar amount of L-α-phosphatidylcholine is 6.6 μM;

[0086] Put the liposome di-solution coated with signal molecule two into a dialysis bag and dialyze it 3-5 times in ultrapure water, with each dialysis for 30-60 min;

[0087] Mix and react the liposome di-solution coated with signal molecule two after dialysis, streptavidin, and the RNA reporter gene modified with biotin and -NH2 at both ends to form a liposome di-RNA reporter gene complex.

[0088] In the embodiment of the present invention, in step S2, the dosage of the solution containing the nucleic acid (DNA / RNA) of the bacteria to be detected is 1-5 μL, and the final concentrations of Cas12 protein and Cas13 protein are both 50-100 nM; the final concentrations of crRNA and gRNA are both 5-100 nM; the final concentrations of the DNA reporter gene and the RNA reporter gene are 5-100 μM; the final concentration of streptavidin is 1-10 mM, and the final concentration of the sulfate Tween buffer (PBST) is 5-10×; the SERS substrate material uses gold-coated silver nanoparticles, and its dosage is 50-500 μL; incubate at 37°C for an incubation time of 10-30 min; the SERS spectral measurement conditions of the portable Raman spectrometer are 785 nm and 100 mW.

[0089] In the embodiment of the present invention, the bacteria to be detected are specifically Cronobacter, and the crRNA sequence for detecting Cronobacter is shown in SEQ ID NO.1;

[0090] The gRNA sequence for detecting Cronobacter is shown in SEQ ID NO.2;

[0091] The corresponding DNA reporter gene is shown in SEQ ID NO.3;

[0092] The corresponding RNA reporter gene is shown in SEQ ID NO.4, as shown in the following table.

[0093] Table 1: Gene sequences involved in the present invention

[0094]

[0095] Example 3: Application of a live-dead bacteria synchronous detection sensor based on the combined use of Cas12 and Cas13, which is used to simultaneously and quantitatively detect live and dead Cronobacter in food samples.

[0096] According to the present invention, when live and dead Cronobacter coexist, the RNA of dead Cronobacter is usually degraded. The DNA is recognized by crRNA, and the RNA is recognized by gRNA. Subsequently, the corresponding reporter gene is cleaved, the liposome is broken, and the SERS signal is enhanced and measured.

[0097] Due to the different contents of Cronobacter DNA / RNA, the resulting amounts of liposomes are different, leading to different SERS signal values. The relationship between the levels of different SERS signal values and the content of Cronobacter DNA / RNA is established, thereby quantitatively detecting the content of Cronobacter DNA / RNA and achieving the purpose of simultaneously and quantitatively detecting live and dead Cronobacter. This method has good specificity, high sensitivity, good repeatability, simple operation, strong adaptability, high accuracy, and can complete the detection in a short time.

[0098] This method does not require special expensive instruments and only requires an ordinary small portable Raman spectrometer to simultaneously detect live and dead Cronobacter, which is suitable for carrying out relevant rapid detection work in areas with scarce detection resources.

Claims

1. A sensor for synchronous detection of live and dead bacteria based on the combination of Cas12-Cas13, characterized in that: include: Liposome 1-DNA reporter gene-96-well plate-RNA reporter gene-liposome 2 complex, wherein the liposome 1 is embedded with a signal molecule 1, and the liposome 2 is embedded with a signal molecule 2; Cas12 system, Cas12 system includes Cas12 protein and crRNA, crRNA is used to complement the bacterial target DNA, and guide Cas12 protein to cut the DNA reporter gene in the liposome one-DNA reporter gene-96-well plate-RNA reporter gene-liposome two complex, to generate liposome one modified with DNA cleavage fragments; Cas13 system, the Cas13 system includes Cas13 protein and gRNA, the gRNA is used to complement the bacterial target RNA, and guide the Cas13 protein to cut the RNA reporter gene in the liposome one-DNA reporter gene-96-well plate-RNA reporter gene-liposome two complex to generate liposome two modified with RNA cleavage fragments; After being collected and broken, liposome 1 modified with DNA cleavage fragments and liposome 2 modified with RNA cleavage fragments release signal molecules 1 and 2. By detecting the signal intensities of signal molecules 1 and 2, live bacteria and dead bacteria are simultaneously quantitatively analyzed.

2. A sensor for synchronous detection of live and dead bacteria based on the combination of Cas12-Cas13 according to claim 1, characterized in that: The liposome-DNA reporter gene-96-well plate-RNA reporter gene-liposome two-complex comprises: DNA reporter gene modified with biotin; RNA reporter modified with biotin; 96-well plates containing bovine serum albumin; Liposome 1 is modified with biotin, and the biotin of liposome 1 is connected with the biotin of DNA reporter gene through streptavidin to form a liposome 1-DNA reporter gene complex, and the end of the DNA reporter gene away from liposome 1 is connected with NH2 for binding to bovine serum albumin in a 96-well plate; Liposome II is modified with biotin, and the biotin of liposome II is connected to the biotin of RNA reporter gene through streptavidin to form a liposome II-RNA reporter gene complex. The end of the RNA reporter gene away from liposome II is connected with NH2 for binding to bovine serum albumin in a 96-well plate.

3. A sensor for synchronous detection of live and dead bacteria based on the combination of Cas12-Cas13 according to claim 1, characterized in that: Also included is a SERS substrate material for enhancing the SERS effect of the signal molecule one and the signal molecule two; The first signal molecule is Nile blue A perchlorate, and the second signal molecule is 4-aminothiophenol.

4. A method for synchronous detection of live and dead bacteria based on the combination of Cas12-Cas13, characterized in that: A sensor for synchronous detection of live and dead bacteria based on the combination of Cas12-Cas13 as described in any one of claims 1-3, comprising the following steps: S1. Evenly mix the sample containing the bacteria to be tested with the one-step nucleic acid extraction solution, and heat and lyse them at high temperature to obtain a solution containing the nucleic acid of the bacteria to be tested; S2, adding the solution containing the bacterial nucleic acid to be tested, the Cas12 system and the Cas13 system to the liposome-DNA reporter gene-96-well plate-RNA reporter gene-liposome two complexes, and incubating at 37°C to allow the DNA reporter gene and the RNA reporter gene to be fully sheared; S3, taking the supernatant of the 96-well plate solution, adding sulfate Tween buffer to break up liposome 1 and liposome 2; S4. The crushed solution is mixed with the SERS substrate material, and the SERS signal is measured by a portable Raman spectrometer to perform simultaneous quantitative analysis on the content of live and dead bacteria in the sample.

5. A method for synchronous detection of live and dead bacteria based on the combination of Cas12-Cas13 according to claim 4, characterized in that: The one-step nucleic acid extraction solution in step S1 comprises magnesium chloride, EDTA, Tris-HCl, triethanolamine dodecyl sulfate and potassium chloride, wherein the concentration of magnesium chloride is 20-80 mM, the concentration of EDTA is 5-20 mM, the concentration of Tris-HCl is 10-50 mM, the mass volume fraction of triethanolamine dodecyl sulfate is 2%-20%, and the concentration of potassium chloride is 2.0-10.0 mol / L.

6. A method for synchronous detection of live and dead bacteria based on the combination of Cas12-Cas13 according to claim 4, characterized in that: The preparation method of the liposome-DNA reporter gene-96-well plate-RNA reporter gene-liposome complex in step S2 is: A1. Take 75 μL of 0.4 M 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 75 μL of 0.2 M N-hydroxysulfosuccinimide and add them to a 96-well plate and incubate at room temperature for 2 h. A2. Add 300 μL of 5% bovine serum albumin to a 96-well plate, incubate in a constant temperature shaker at 37°C and 100 rpm for 1.5 h, and then wash three times with deionized water; A3. Take 100 μL of 10 μM liposome-DNA reporter gene complex and 100 μL of 10 μM liposome-RNA reporter gene complex and add them to a 96-well plate. Incubate at 37°C for 3 h to generate liposome-DNA reporter gene-96-well plate-RNA reporter gene-liposome-II complex. Rinse the 96-well plate three times with deionized water and set aside.

7. A method for synchronous detection of live and dead bacteria based on the combination of Cas12-Cas13 according to claim 6, characterized in that: The preparation method of the liposome-DNA reporter gene complex is: 5 mg of L-α-phosphatidylcholine, cholesterol and PE-PEG-2000-biotin mixed in a molar ratio of 2:2:1 were dispersed in 1 mL of chloroform and rotary evaporated to produce a thin film; then, the mixture was resuspended in 5 mL of a signal molecule solution with a concentration of 4 mM to obtain a liposome solution coated with a signal molecule, wherein the surface of the liposome in the liposome solution was modified with biotin; wherein the molar number of L-α-phosphatidylcholine was 6.6 μM; The liposome solution coated with the signal molecule is placed in a dialysis bag and dialyzed in ultrapure water for 3-5 times, each dialysis lasting 30-60 minutes; The dialyzed liposome solution coated with the signal molecule, streptavidin and a DNA reporter gene with biotin and -NH2 modified at both ends are mixed to form a liposome-DNA reporter gene complex; The preparation method of the liposome-RNA reporter gene complex is: 5 mg of L-α-phosphatidylcholine, cholesterol and PE-PEG-2000-biotin mixed in a molar ratio of 2:2:1 were dispersed in 1 mL of chloroform and rotary evaporated until a thin film was produced; then, the mixture was resuspended with 5 mL of a 4 mM signal molecule II solution to obtain a liposome II solution coated with a signal molecule II, wherein the surface of the liposome II in the liposome II solution was modified with biotin; wherein the molar number of L-α-phosphatidylcholine was 6.6 μM; The liposome II solution coated with the signal molecule II is placed in a dialysis bag and dialyzed in ultrapure water for 3-5 times, each dialysis for 30-60 minutes; The dialyzed liposome II solution coated with the signal molecule II, streptavidin and the RNA reporter gene with biotin and -NH2 modified at both ends are mixed to form a liposome II-RNA reporter gene complex.

8. A method for synchronous detection of live and dead bacteria based on the combination of Cas12-Cas13 according to claim 4, characterized in that: In step S2, the amount of the solution containing the bacterial nucleic acid to be tested is 1-5 μL, the final concentrations of the Cas12 protein and the Cas13 protein are both 50-100 nM; the final concentrations of the crRNA and the gRNA are both 5-100 nM; the final concentrations of the DNA reporter gene and the RNA reporter gene are 5-100 μM; the final concentration of the streptavidin is 1-10 mM, and the final concentration of the sulfate Tween buffer is 5-10×; the SERS substrate material uses silver-coated gold nanoparticles, and the amount thereof is 50-500 μL; the incubation time is 10-30 min at 37°C; the SERS spectrum measurement conditions of the portable Raman spectrometer are 785 nm, 100 mW.

9. A method for synchronous detection of live and dead bacteria based on the combination of Cas12-Cas13 according to claim 4, characterized in that: The bacteria to be tested are specifically Cronobacter, and the cr RNA sequence for Cronobacter detection is shown in SEQ ID NO.1; The gRNA sequence used for Cronobacter detection is shown in SEQ ID NO.2; The corresponding DNA reporter gene is shown in SEQ ID NO.3; The corresponding RNA reporter gene is shown in SEQ ID NO.

4.

10. Application of a sensor for synchronous detection of live and dead bacteria based on the combination of Cas12-Cas13 according to any one of claims 1-3, characterized in that: Used for the simultaneous quantitative detection of live and dead Cronobacter in food samples.

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