Rapid detection method of salmonella based on lamp-crispr / cas12a system

Abstract

The application provides a rapid, efficient, low-cost and visual detection method for detecting Salmonella based on a LAMP-CRISPR / Cas12a system, and relates to the technical field of pathogenic microorganism detection.The sgRNA specific for detecting Salmonella and the Cas12a assembled complex are used to recognize and combine LAMP amplification products, the collateral cleavage activity of Cas12a is activated, the fluorescently labeled DNA is enzymatically cut, and the fluorescent signal can indicate whether Salmonella is contained in the detection sample.The application provides a high-sensitivity, high-specificity, low-cost, visual and convenient and fast detection method for detecting Salmonella.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pathogenic microorganism detection, and in particular to a rapid detection method for Salmonella based on a LAMP-CRISPR / Cas12a system. BACKGROUND

[0002] Salmonella is a major foodborne pathogenic bacteria. According to the Centers for Disease Control and Prevention (CDC), it causes about 1.35 million people to be infected with Salmonella in the United States every year, and more than 100 million people worldwide every year. After people eat food contaminated with Salmonella, they will have symptoms such as diarrhea, vomiting, dizziness, and high fever, not only acute gastroenteritis, but also systemic infection, which can cause the human body to have decreased immunity and repeated infections. At the same time, Salmonella can also cause livestock and poultry to become ill, and Salmonella can also contaminate livestock and poultry products to cause human poisoning and a series of diseases. As a very harmful foodborne bacteria, the detection result of Salmonella has become an important indicator of whether food safety detection is qualified. Therefore, in order to ensure food safety and efficient production of the livestock industry, it is necessary to establish a rapid, accurate and efficient method for detecting Salmonella.

[0003] The national standard GB 29921-2021 "National Food Safety Standard Limit of Pathogenic Bacteria in Prepackaged Food" stipulates that the content of Salmonella in meat products, aquatic products, instant egg products and other foods is 0. The detection steps of the national standard GB 4789.4-2016 "National Food Safety Standard Food Microbiological Examination Salmonella Examination" are complex, in addition, there are bacterial culture identification, gene detection, immunological detection and other methods. However, these detection methods cannot obtain the detection results in a timely manner. Nowadays, the commonly used methods for detecting Salmonella are qPCR and Insulated Isothermal PCR (iiPCR). qPCR uses fluorescence signals to monitor the PCR process in real time, and finally, the unknown template is quantitatively analyzed by a standard curve. iiPCR is based on the Rayleigh-Benard convection principle, and by constantly heating the liquid at the bottom of the reaction tube, the temperature difference between the upper and lower liquids is generated, the rate of heat dissipation and the time of convection are controlled, and a stable temperature gradient is formed in the reaction tube, thereby providing the reaction conditions required for PCR amplification. Awang et al. used electrochemical sensing to detect Salmonella. This system combines immunological techniques, molecular techniques, mass spectrometry, spectroscopy, optical phenotype methods, and biosensor methods. Although these research methods have improved the detection rate, they are time-consuming, costly, and require precise instruments, and cannot be truly popularized, which is not suitable for emergency situations.

[0004] LAMP was invented by Japanese scholar Notomi, under the action of DNA polymerase, with 4-6 pairs of primers, at 60-65℃, the target gene is amplified, and then the amplification results are tested by agarose gel electrophoresis. Compared with traditional PCR, the reaction rate is fast, which can be completed in 45-60 min. The method is low in cost and easy to use, which can be completed without special technical personnel and expensive instruments. The method has high detection sensitivity, compared with ordinary PCR, the sensitivity can be improved by 10000 times. The latest technology based on LAMP reaction-cyclic medium thermal amplification, through the use of DNA polymerase, under isothermal conditions, high specificity, high efficiency of DNA amplification. Suitable for on-site detection in emergency situations. Usually, the detection methods of LAMP amplification products include agarose gel electrophoresis, turbidity detection method, color determination method. Color determination method. The commonly used color developing agent has two categories: metal ion indicator (calcein, hydroxyl naphthol blue and combination of the two) and nucleic acid dye indicator (SYBR Green I, GeneFinder and iodinated propidium). Under the condition of limited detection site, it is the most ideal to be independent of expensive instruments or not to need any instruments. At the same time, the naked eye can accurately determine the result. But the primer design of LAMP, the amount of primer added in the system, reaction temperature, reaction time, enzyme, buffer and other factors are not in the optimal condition, which may lead to false positive, and the above method can not exclude the existence of false positive, which has a negative impact on the accuracy of the test results.

[0005] The CRISPR / Cas system is an acquired immune system developed by bacteria during evolution to defend against the transfer of exogenous plasmids and the invasion of bacteriophages. In 2015, scientist Feng Zhang discovered a CRISPR effector protein, Cas12a. This protein is an RNA-guided DNA-cutting nuclease that, after binding to target DNA, can cis-cleave the target DNA and trans-cleave non-target single-stranded DNA. This system exhibits high sensitivity and specificity and is widely used for the detection of double-stranded or single-stranded DNA. CRISPR / Cas12a-based platforms can be used for rapid and visualized nucleic acid detection and are widely applied in the field of nucleic acid molecular diagnostics. The CRISPR / Cas12a system can detect the presence of pathogenic microorganisms, such as Salmonella, Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes. After diluting the DNA of various pathogens tenfold and performing PCR amplification, the reaction products were used as targets in the Cas12a fluorescent detection system. Results showed that the detection sensitivity could reach up to 10 copies / µL, and each primer and DNA could only specifically participate in the detection reaction of the target bacteria. The CRISPR / Cas12a system has limited inherent detection sensitivity and typically requires combination with nucleic acid amplifiers, PCR, or isothermal amplification techniques. LAMP, with its very high sensitivity, can enhance the detection sensitivity of the CRISPR / Cas12a system when combined with it. Furthermore, secondary detection via Cas12a digestion can eliminate false positives associated with LAMP. Combining these two methods allows for a high level of sensitivity and specificity in Salmonella detection. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] To address the shortcomings of existing technologies, this invention provides a novel, efficient, rapid, and field-applicable method for detecting Salmonella based on a LAMP-based CRISPR / Cas12a system.

[0008] (II) Technical Solution

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] This invention provides a rapid detection method for Salmonella based on the LAMP-CRISPR / Cas12a system, the method comprising the following steps:

[0011] (1) Extract genomic DNA from the sample to be tested;

[0012] (2) Design sgRNA and PCR primers containing sgRNA, and screen for target sgRNA by PCR combined with Cas12a restriction enzyme digestion method;

[0013] (3) Design LAMP primers, use the genomic DNA extracted in step (1) as a template to perform LAMP amplification;

[0014] (4) Use the genomic DNA extracted in step (1) as a template, and the PCR primers designed in step (2) to amplify the specific pathogenic gene SipB, construct it into the pMD-19T cloning vector, and construct the pMD19T-SipB plasmid; dilute the plasmid by ten times as a template for LAMP amplification;

[0015] (5) Fluorescent visual detection, mix the LAMP amplification product, Cas12a, fluorescent probe, target sgRNA, and NEB Buffer 2.1, incubate for 10-30 min, and visually detect the sample under blue light or ultraviolet light to qualitatively detect Salmonella.

[0016] Further, the sample genome extraction in step (1) is a heat lysis method, which does not require additional kits and takes 17-27 min to obtain DNA.

[0017] Further, the sgRNA designed in step (2) is designed for the pathogenic gene SipB sequence and the PAM sequence (TTTN) of Salmonella, and 10 sgRNAs are designed; the nucleotide sequence of the sgRNA is shown in SEQ ID NO. 1-10, the sgRNA is obtained by in vitro transcription and purification or direct synthesis, and the sgRNA is obtained by the method including but not limited to the above method. For the target sequence containing 10 sgRNAs, design PCR primers, PCR combined with Cas12a enzyme cutting, select the target sgRNA, and the nucleotide sequence of the target sgRNA is shown in SEQ ID NO. 6; the nucleotide sequence of the primer is shown in SEQ ID NO. 11 and 12.

[0018] Further, the LAMP primers designed in step (3) contain the target sgRNA in the target sequence of LAMP, which includes the inner primer FIP, the nucleotide sequence of which is shown in SEQ ID NO. 13; the inner primer BIP, the nucleotide sequence of which is shown in SEQ ID NO. 14; the outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO. 15; the outer primer B3, the nucleotide sequence of which is shown in SEQ ID NO. 16; the loop primer LF, the nucleotide sequence of which is shown in SEQ ID NO. 17; and the loop primer LB, the nucleotide sequence of which is shown in SEQ ID NO. 18.

[0019] Further, the LAMP amplification system designed in step (3) is a 25 μL system, which includes 4.4×10 -144.4 ng template DNA, 0.64 mM inner primer FIP and inner primer BIP, 0.08 mM outer primer F3 and outer primer B3, 0.16 mM loop primer LF and loop primer LB, 8 U of Bst 3.0 enzyme, 14 mM of dNTPs, 8 mM of MgSO4 and 1x Bst buffer; the amplification condition is 65℃ incubation for 20-50 min.

[0020] Further, the method for constructing the pMD19T-SipB vector in step (4) is as follows: using the genomic DNA extracted in step (1) as a template and the PCR primers designed in step (2) for amplification, recovering the PCR product by agarose gel electrophoresis, connecting the pMD-19T cloning vector, transforming into the competent cells of Escherichia coli, picking the positive single clone liquid for sequencing, expanding the culture of the bacterial liquid with completely correct sequence to obtain the pMD19T-SipB vector as a template for subsequent sensitivity detection; the annealing temperature of the PCR amplification is preferably 59℃.

[0021] Further, the fluorescent visual detection system in step (5) is a 20 μL system, including 2 μL of LAMP amplification product, 10 nM Cas12a, 132 ng of target sgRNA, 0.7 μM JOE probe, 1x NEB 2.1 buffer and DEPC water; the reaction condition is 37℃ incubation for 10-30 min, and the preferred reaction time is 15 min.

[0022] Further, in step (5), if the LAMP product contains the Salmonella-specific amplification gene, the Cas12a-sgRNA binds to the characteristic sequence, activates the collateral cleavage activity of Cas12a, cuts the single-stranded DNA fluorescent reporter probe with a fluorescent group and a quenching group at both ends, releases the fluorescent group to generate a fluorescent signal, and the solution emits fluorescence; if the LAMP product does not contain the Salmonella-specific amplification gene, the collateral cleavage activity of Cas12a cannot be activated, the fluorescent reporter probe remains intact, and the solution does not emit fluorescence.

[0023] Further, the fluorescent probe sequence used in step (5) is a random 12-base sequence, the 5' end of the probe is modified with JOE, and the 3' end is modified with BHQ1.

[0024] Further, the fluorescent detection condition in step (5) is naked-eye observation under blue light or ultraviolet light; the fluorescent visual detection in step (5) can also use a microplate reader or any fluorescent detection device that can excite and detect fluorescence in the JOE fluorescent channel, and the fluorescent detection condition is to excite fluorescence with excitation light at a wavelength of 520 nm and detect the fluorescence intensity at a wavelength of 548 nm.

[0025] The present application shows through multiple experiments of negative controls that if the agarose gel result of the LAMP amplification product presents a positive result, the Cas12a enzyme cutting secondary detection of the sample will not produce fluorescence, and the secondary detection result can effectively avoid the false positive produced by LAMP amplification.

[0026] The time consumption of each step of the sample detection of the present application has been optimized, which is respectively: 17 min for processing the sample and extracting DNA, 20 min for LAMP amplification, 15 min for Cas12a enzyme cutting, and the total time consumption is 52 min.

[0027] (Three) beneficial effects

[0028] The present application provides a rapid detection method of Salmonella based on LAMP-CRISPR / Cas12a system, which utilizes the high specificity of sgRNA and the high sensitivity of LAMP reaction to effectively ensure the high accuracy of bacterial characteristic sequence nucleic acid detection, and the secondary detection of Cas12a enzyme cutting effectively avoids the false positive produced by LAMP reaction. In the condition of controllable cost instrument, it can be determined within 1 hour whether the pathogenic bacteria exist in the sample to be tested, compared with the traditional PCR detection method, the specificity and sensitivity are greatly improved, and the quality requirement of sample nucleic acid is lower than that of PCR, and the cost is low. The present application is suitable for detection in slaughterhouses and other places, only a constant temperature device and a simple fluorescence detection device are needed to quickly obtain the result, and the result can be determined by naked eye, without the need for special technical personnel, further reducing the cost of on-site detection. The present application has the characteristics of low cost, high sensitivity, strong specificity, short time consumption, high efficiency, accuracy and suitability for on-site detection. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 PCR amplification was carried out on the bacterial DNA extracted by thermal lysis method, and sgRNA transcribed and purified in vitro was used for Cas12a enzyme cutting, and the detection results of the enzyme cutting product in blue light, ultraviolet light and gel imaging system; wherein numbers 1-10 are sgRNA1-sgRNA10, and 11 is a negative control.

[0030] Figure 2 The constructed pMD19T-SipB plasmid was diluted by multiple times, which was used as the template for LAMP amplification, and the detection results of the LAMP amplification product combined with Cas12a enzyme cutting in blue light, ultraviolet light and gel imaging system; wherein numbers 1-15 are plasmid templates diluted by multiple times, and the concentrations are 5.5, 5.5x10 -1 , 5.5x10 -2 , 5.5x10 -3 , 5.5x10 -4 , 5.5x10 -5 , 5.5x10-6 , 5.5 x 10 -7 , 5.5 x 10 -8 , 5.5 x 10 -9 , 5.5 x 10 -10 , 5.5 x 10 -11 , 5.5 x 10 -12 , 5.5 x 10 -13 , 5.5 x 10 -14 ng / μL, 16 is negative control.

[0031] Figure 3 Detection results of LAMP amplification product binding Cas12a enzyme cutting in blue light, ultraviolet light and gel imaging system, wherein ST is Salmonella, SH is Shigella sonnei, LM is Listeria monocytogenes, Sau is Staphylococcus aureus, Eco is Escherichia coli, and NC is negative control.

[0032] Figure 4 Detection results of LAMP reaction combined with Cas12a enzyme cutting in blue light, ultraviolet light and gel imaging system after 11 fresh pork samples were extracted by heat lysis method, wherein 1-11 are different pork samples, and 12 is negative control. DETAILED DESCRIPTION

[0033] To make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. EMBODIMENT

[0034] 1. Design primers, sgRNA in vitro transcription and purification

[0035] In the embodiments of the present application, the specific pathogenic gene SipB of Salmonella is taken as the target gene (sequence number: NC_003197.2), sgRNA is designed according to the PAM sequence (TTTN), and a total of 10 sgRNAs are designed in this embodiment, which are numbered 1-10, for selecting the target sgRNA; the nucleotide sequence of the sgRNA is shown in SEQ ID NO. 1-10; the sequence containing all sgRNAs is taken as the target sequence, and PCR primers are designed, and the primer information is shown in SEQ ID NO. 11, 12; the sequence containing the target sgRNA is taken as the target sequence, and LAMP primers are designed, and the sequence information is shown in SEQ ID NO. 13, 14, 15, 16, 17, 18.

[0036] sgRNA in vitro transcription method: 1 μg Template DNA, 4 μL T7-RNA-polymerase-Mix, 2 μL 10 x Reaction buffer, 100 mM GTP, 100 mM ATP, 100 mM UTP, 100 mM CTP, each 2 μL, add nuclease-free water to 40 μL.

[0037] sgRNA purification method: add 2 μL DNase I to 20 μL in vitro transcription product, incubate at 37°C for 15 min; mix 160 μL nuclease-free water with the in vitro transcription product treated in the previous step and 20 μL 3M sodium acetate, extract twice with 200 μL 1:1 phenol chloroform isoamyl alcohol mixture, take the supernatant, add two volumes of pre-cooled anhydrous ethanol, mix well, precipitate RNA at -20°C for 30 min, centrifuge and discard the supernatant, rinse the precipitate twice with pre-cooled 500 μL 70% ethanol, dissolve the precipitated RNA with 20-40 μL DEPC water.

[0038] 2. Screening target sgRNA

[0039] Bacterial genomic DNA was extracted by heat lysis method as template, PCR primers containing target sequence of sgRNA were amplified, the PCR product was purified, and then Cas12a enzyme cutting was performed, the system was: 10 nM Cas12a, 0.7 μM probe, 2 μL 2.1 Buffer, 2 μL PCR purified product, 132 ng sgRNA, add DEPC water to 20 μL. Incubate at 37°C for 10-30 min. Determine the results on the blue light instrument or ultraviolet instrument respectively, the results are shown in Figure 1 Figure 1 No. 1-10 is sgRNA1-sgRNA10, No. 11 is negative control; the results show that the No. 6 sgRNA is the brightest, and the No. 6 sgRNA is finally selected as the target sgRNA, and the nucleotide sequence of the target sgRNA is shown in SEQ ID NO. 6.

[0040] 3. Construction of pMD19T-SipB vector

[0041] Bacterial genomic DNA was used as template, and designed PCR primers were amplified. The amplified product was purified and ligated with pMD-19T cloning vector, and the competent cells of E. coli were transformed. Single colony bacteria were picked for bacterial liquid PCR, and Sanger sequencing was performed. The bacterial liquid with completely correct sequence was expanded and cultured, and the plasmid was extracted. The plasmid was diluted by ten times as a sensitivity detection template.

[0042] 4. Sensitivity detection​

[0043] LAMP amplification was performed using pMD-SipB plasmid diluted by ten times as a template, and the LAMP amplification system was as follows: 0.8 μL of plasmid DNA diluted by ten times, 1.6 μM of inner primer 1 and inner primer 2, 0.2 μM of outer primer 1 and outer primer 2, 0.2 μM of loop primer 1 and loop primer 2, 8 U of Bst 3.0 enzyme, 14 mM of dNTPs, 150 mM of MgSO4, and 1×Bst buffer; the amplification condition was 65℃ incubation for 20-50 min. Then 2 μL of LAMP amplification product, 20 nM Cas12a, 0.7 μM probe, 2 μL 2.1 Buffer, 132 ng sgRNA, and DEPC water were added to make up to 20 μL. 37℃ incubation for 10-30 min. The concentrations of the plasmid template diluted by ten times were 5.5, 5.5×10 -1 , 5.5×10 -2 , 5.5×10 -3 , 5.5×10 -4 , 5.5×10 -5 , 5.5×10 -6 , 5.5×10 -7 , 5.5×10 -8 , 5.5×10 -9 , 5.5×10 -10 , 5.5×10 -11 , 5.5×10 -12 , 5.5×10 -13 , 5.5×10 -14 ng / μL, a total of fifteen, corresponding to numbers 1-15 in Figure 2 , and number 16 was a negative control group. According to the detection results of blue light, ultraviolet light, and gel imaging system, it was preliminarily judged that the detection lower limit of LAMP combined with Cas12a enzyme cutting was 5.5×10 -14 ng / μL, as shown in Figure 2 .

[0044] The same pMD-SipB plasmid diluted by the same ratio as above was used as a template for fluorescence quantitative PCR, and the plasmid copy number was converted by an absolute quantitative method. The fluorescence quantitative PCR system: 7.5 μL 2x SYBR-Green Mix, 0.5 μL of forward and reverse PCR primers, 5 μL of plasmid DNA diluted by the ratio, and 15 μL of nuclease-free water. Quantitative detection was performed in an ABI7500 fast RealTime PCR System, and the reaction program was 95°C pre-denaturation for 10 min, 95°C denaturation for 15 s, 60°C annealing for 1 min, and fluorescence signal collection at 60°C, for a total of 45 cycles. The equation of the standard curve is: Y=-4.782x+9.316, R2=0.991. According to the standard curve, the conversion of the plasmid copy number showed that the detection limit of LAMP combined with Cas12a enzyme digestion was 1.37x10 -4 copies / μL, indicating that the method described in the application has high sensitivity for detection of Salmonella.

[0045] 5. Specific detection

[0046] Five kinds of bacterial DNA of Salmonella (ST), Shigella sonnei (SH), Listeria monocytogenes (LM), Staphylococcus aureus (Sau), and Escherichia coli (Eco) and a negative control (NC) were used as templates for LAMP amplification, and the sample system was: 0.8 μL of bacterial DNA, 0.64 μM of inner primer 1 and inner primer 2, 0.08 μM of outer primer 1 and outer primer 2, 0.16 μM of loop primer 1 and loop primer 2, 8 U of Bst3.0 enzyme, 14 mM of dNTPs, 8 mM of MgSO4, and 1x Bst buffer; the amplification condition was 65°C incubation for 20-50 min. Then 2 μL of LAMP amplification product, 20 nM Cas12a, 0.7 μM probe, 2 μL 2.1 Buffer, 132 ng sgRNA, and DEPC water were added to make up to 20 μL. Incubation at 37°C for 10-30 min. According to the detection results of blue light, ultraviolet light and gel imaging system, only Salmonella strain emitted fluorescence, and the rest of the strains did not emit light, indicating that the method described in the application has high specificity for detection of Salmonella, and the results are shown in Figure 3 . Example

[0047] Detection of Salmonella in fresh pork samples

[0048] Fresh pork samples were purchased from different markets, 11 samples in total, 10 g of each pork sample was added into 10 mL PBS, and then gently beaten for 5 min. The solution obtained by beating was taken into a centrifuge tube and centrifuged at 1000 rpm / min for 1 min. The supernatant was used to extract bacterial DNA by heat lysis. The extracted bacterial DNA was used as a template for LAMP amplification. The system was as follows: 0.8 μL of bacterial DNA, 0.64 μM of inner primer 1 and inner primer 2, 0.08 μM of outer primer 1 and outer primer 2, 0.16 μM of loop primer 1 and loop primer 2, 8 U of Bst 3.0 enzyme, 14 mM of dNTPs, 8 mM of MgSO4 and 1 × Bst buffer; the amplification condition was 65℃ incubation for 20-50 min. Then 2 μL of LAMP amplification product, 10 nM Cas12a, 0.7 μM probe, 2 μL 2.1 Buffer, 132 ng sgRNA, and DEPC water were added to make up to 20 μL. 37℃ incubation for 10-30 min. According to the detection results of blue light, ultraviolet light and gel imaging system, it can be known that No. 2, 4, 5, 6, 7, 8, 9 and 10 pork samples were contaminated with Salmonella, and the results are shown in Figure 4

[0049] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.​

Claims

1. A rapid detection method for Salmonella based on the LAMP-CRISPR / Cas12a system for non-diagnostic purposes, characterized in that, Includes the following steps: (1) Extract genomic DNA from the sample to be tested; (2) Design sgRNA and PCR primers containing sgRNA, and screen for target sgRNA by PCR combined with Cas12a enzyme digestion method; the sgRNA is designed to target the pathogenic gene SipB sequence and PAM sequence of Salmonella, and its nucleotide sequence is shown in SEQ ID NO.6; The nucleotide sequences of the PCR primers are shown in SEQ ID NO.11 and 12, and they are used for screening the target sgRNA by combining conventional PCR with Cas12a restriction enzyme digestion. (3) Design LAMP primers and use the genomic DNA extracted in step (1) as a template for LAMP amplification; the LAMP primer sequences are shown in SEQ ID NO.13-18, and the target sequence of LAMP contains the target sgRNA, including the outer primer F3, whose nucleotide sequence is shown in SEQ ID NO.13; the outer primer B3, whose nucleotide sequence is shown in SEQ ID NO.14; the inner primer FIP, whose nucleotide sequence is shown in SEQ ID NO.15; the inner primer BIP, whose nucleotide sequence is shown in SEQ ID NO.16; the loop primer LF, whose nucleotide sequence is shown in SEQ ID NO.17; and the loop primer LB, whose nucleotide sequence is shown in SEQ ID NO.

18. (4) Using the genomic DNA extracted in step (1) as a template, the PCR primers designed in step (2) were used to amplify the specific pathogenic gene SipB, which was then constructed into the pMD-19T cloning vector to construct the pMD19T-SipB plasmid; the plasmid was serially diluted tenfold as a template for LAMP amplification. (5) Fluorescent visual detection: Mix LAMP amplification product, Cas12a, fluorescent probe, target sgRNA, and NEB Buffer 2.1, incubate for 10-30 min, and then visually detect Salmonella in the sample under blue or ultraviolet light.

2. The rapid detection method for Salmonella based on the LAMP-CRISPR / Cas12a system according to claim 1, characterized in that, The LAMP amplification system designed in step (3) is a 25 μL system, including 5.5 × 10⁻⁶ LAMP. -14 ~5.5 ng template DNA, 0.64 μM inner primer FIP and inner primer BIP, 0.08 μM outer primer F3 and outer primer B3, 0.16 μM circular primer LF and circular primer LB, 8 U Bst3.0 enzyme, 14 mM dNTPs, 8 mM MgSO4 and 1× Bst buffer; amplification conditions: incubation at 65 °C for 20–50 min.

3. The rapid detection method for Salmonella based on the LAMP-CRISPR / Cas12a system according to claim 1, characterized in that, The method for constructing the pMD19T-SipB vector in step (4) is as follows: using the genomic DNA extracted in step (1) as a template, the PCR primers designed in step (2) are used for amplification, the PCR products are recovered by agarose gel electrophoresis, and the pMD-19T cloning vector is ligated to obtain the pMD19T-SipB vector, which is used as a template for sensitivity detection.

4. The rapid detection method for Salmonella based on the LAMP-CRISPR / Cas12a system according to claim 1, characterized in that, The fluorescent probe sequence used in step (5) is a random 12-base sequence. The 5' end of the probe is modified with JOE and the 3' end is modified with BHQ1. Its specific structure is 5'-JOE-12bp-BHQI-3'.

5. The rapid detection method for Salmonella based on the LAMP-CRISPR / Cas12a system according to claim 1, characterized in that, In step (5), the fluorescence visual detection system is a 20 μL system, including 2 μL of LAMP amplification product, 10 nM Cas12a, 132 ng of target sgRNA, 0.7 μM JOE probe, 1×NEB buffer 2.1 and DEPC water; the reaction conditions are incubation at 37℃ for 10-30 min.

6. The rapid detection method for Salmonella based on the LAMP-CRISPR / Cas12a system according to claim 1, characterized in that, The fluorescence detection conditions in step (5) are blue light or ultraviolet light naked-eye observation.

7. The rapid detection method for Salmonella based on the LAMP-CRISPR / Cas12a system according to claim 1, characterized in that, The fluorescence visualization detection in step (5) uses an ELISA reader or any fluorescence detection device that can excite and detect fluorescence on the JOE fluorescence channel. The fluorescence detection conditions are to excite fluorescence with excitation light at a wavelength of 520 nm and detect fluorescence intensity at a wavelength of 548 nm.

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