Salmonella detection system and method and application thereof

Abstract

The invention provides a novel salmonella molecular detection method based on an EXPAR-CRISPR / Cas12a detection system, the method specifically amplifies a yfiR gene of salmonella through an EXPAR isothermal technology, and uses a CRISPR / Cas12a system for cutting and detection, and has the advantages of rapidness, sensitivity, strong specificity and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial detection, and in particular to a Salmonella detection method based on the EXPAR-CRISPR / Cas12a system. Background Art

[0002] Salmonella is one of the main pathogens of bacterial foodborne diseases, causing about 78 million cases of infection and 59,000 deaths worldwide each year, posing a severe challenge to public health security. Infected people usually show non-specific symptoms such as abdominal pain, diarrhea, and fever, so rapid and accurate laboratory testing is the key to disease prevention and control. Although traditional detection methods (such as isolation and culture, enzyme-linked immunosorbent assay ELISA, chemiluminescent immunoassay, fluorescent immunoassay, and PCR nucleic acid amplification technology) have been widely used, they generally have limitations such as high equipment dependence, long detection cycle (several hours to several days), complex operation, and insufficient sensitivity and specificity, making it difficult to meet the needs of point-of-care diagnosis (POCT). The development of detection technologies that are both highly specific and sensitive, simple and fast, has become a core demand for improving public health emergency response capabilities.

[0003] In recent years, isothermal DNA amplification technology has emerged in the field of molecular diagnosis due to its simple operation, strong portability, short detection time (usually <1 hour) and cost-effectiveness. Representative technologies include strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA) and exponential isothermal amplification (EXPAR). Among them, EXPAR technology can achieve 10% amplification of target nucleic acid within 30 minutes through DNA template, deoxynucleoside triphosphates (dNTPs) and dual enzyme catalysis system. 6 The CRISPR / Cas system, with its ultra-high specificity of nuclease targeted cleavage, can be used in conjunction with isothermal amplification technology (such as CRISPR-Cas12a / LAMP, CRISPR-Cas13a / RPA) to construct an "amplification-recognition" cascade reaction, and achieve visual detection through fluorescent signals or lateral flow chromatography test strips, further breaking through the bottlenecks of traditional methods in sensitivity and specificity. This type of technological integration provides innovative solutions for on-site rapid screening and precise traceability of Salmonella, and is expected to reshape the paradigm of foodborne pathogen detection. Summary of the invention

[0004] The present invention provides a new method for molecular detection of Salmonella based on the EXPAR-CRISPR / Cas12a detection system. The method specifically amplifies the yfiR gene of Salmonella through the EXPAR isothermal technology and uses the CRISPR / Cas12a system for detection, which has the advantages of being rapid, sensitive, and highly specific.

[0005] The present invention first provides a detection system for Salmonella, which includes an isothermal amplification system and a CRISPR / Cas12a detection system. The isothermal amplification system recognizes a target gene and amplifies to produce an EXPAR product of single-stranded DNA, and the CRISPR / Cas12a detection system can recognize and cleave the EXPAR product to generate detectable fluorescence:

[0006] Wherein, the isothermal amplification system contains a hairpin probe and an Nt-type nicking enzyme. The hairpin probe contains a complementary hybridization sequence to the target gene and a specific sequence that cooperates with the Nt-type nicking enzyme; the target gene is the Salmonella yfiR gene.

[0007] In one embodiment according to the present invention, the nucleotide sequence of the target gene is SEQ ID NO:1.

[0008] In one embodiment according to the present invention, the isothermal amplification system contains: an Nt-type nicking enzyme, a DNA polymerase, dNTPs, a penetrant, a buffer; preferably, the Nt-type nicking enzyme is Nt.BstNBI, the nucleotide sequence of the hairpin probe is SEQ ID NO:2; preferably, the penetrant is betaine; preferably, the DNA polymerase is the large fragment of Bst DNA polymerase.

[0009] In one embodiment according to the present invention, the CRISPR / Cas12a detection system contains a Cas12a enzyme, a crRNA, and an ssDNA. The nucleotide sequence of the crRNA is SEQ ID NO:3, and / or, the ssDNA is SEQ ID NO:4.

[0010] The present invention also provides a method for detecting Salmonella, which includes:

[0011] 1) Amplifying a detection sample using the isothermal amplification system of the above detection system to obtain an EXPAR product;

[0012] 2) Using the CRISPR / Cas12a detection system of the above detection system to recognize and cleave the EXPAR product, detecting and recording the fluorescence signal;

[0013] 3) Analyzing the fluorescence signal.

[0014] In one embodiment according to the present invention, the reaction system of the isothermal amplification system comprises: 8 - 12 U large fragment of Bst DNA polymerase, 10 - 15 U Nt.BstNBI, 10 - 15 nM hairpin probe, 20 - 30 μM betaine, dNTPs and buffer; preferably, the concentration of dNTPs is 200 - 300 μM; preferably, the buffer is 1×NEBuffer 3.1;

[0015] In one embodiment according to the present invention, the amplification reaction conditions of the isothermal amplification system are carried out at 42 °C for 60 minutes.

[0016] In one embodiment according to the present invention, the cleavage reaction of the CRISPR / Cas12a system comprises: 20 μL EXPAR product, 500 nM LbCas12a, 500 nM crRNA and 1000 nM ssDNA;

[0017] Preferably, the reaction conditions of the cleavage reaction are: carried out at 37 °C for 60 minutes; preferably, the fluorescence signal is recorded every 5 minutes by using a qPCR instrument.

[0018] The present invention further provides the above detection system, and / or the application of the detection method in detecting Salmonella in biological samples, environmental samples and / or clinical samples. The biological samples are selected from one or more of meat, poultry products, eggs, dairy products, aquatic products, vegetables, feeds, cosmetics and ready-to-eat foods; and / or, the environmental samples are selected from one or more of water sources, soil, sewage, farm excreta and air dust; the clinical samples are selected from one or more of feces, blood, vomitus, urine, bile and tissue biopsy specimens. Brief Description of the Drawings

[0019] Figure 1 It is a schematic diagram of the working principle of the EXPAR-CRISPR / Cas12a detection system;

[0020] Figure 2 It is a map for evaluating the identification of the yfiR gene of Salmonella by the EXPAR-CRISPR / Cas12a detection system. Among them, A is the real-time fluorescence measurement in 7 different EXPAR-CRISPR / Cas12a reactions. 1: Hairpin probe, 2: Target DNA, 3: Hairpin probe bound to the target DNA, 4: Complete EXPAR components excluding DNA polymerase, 5: Lack of Nt.BstNBI nicking endonuclease, 6: Excluding target DNA, 7: Completely assembled EXPAR components. B is the fluorescence detection photo of 7 reactions. C is the photo of agarose gel electrophoresis analysis of 7 reactions. M: DNA molecular weight marker (range 25 bp - 500 bp).

[0021] Figure 3 Optimization comparison spectra of reaction conditions. Among them, A is the fluorescence curve of the EXPAR reaction containing hairpin probes with different concentrations. B is the fluorescence and S / N ratio of the EXPAR reaction containing hairpin probes with different concentrations. C is the fluorescence curve of EXPAR under different reaction temperature conditions. D is the fluorescence and S / N ratio of EXPAR under different reaction temperature conditions. Error bars represent standard deviation, n = 3.

[0022] Figure 4 Sensitivity and linear analysis spectra of the EXPAR reaction. Among them, A is the fluorescence curve of the EXPAR reaction containing different concentrations of yfiR gene fragments. B is the fluorescence intensity of the EXPAR reaction containing different concentrations of yfiR gene fragments. Error bars represent standard deviation, n = 3. C is the linear relationship between the average fluorescence intensity at 60 minutes and the logarithm of the target DNA concentration. D is the fluorescence intensity of the EXPAR-CRISPR / Cas12a detection of different concentrations of yfiR gene fragments at 60 minutes. Error bars represent standard deviation, n = 3.

[0023] Figure 5 Detection specificity analysis spectra. Among them, A is the fluorescence curve of the EXPAR-CRISPR / Cas12a system for detecting different bacteria (including Salmonella, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Acinetobacter baumannii; NC is the negative control). B is the fluorescence intensity at the end point of the reaction. Error bars represent standard deviation, n = 3.

[0024] Figure 6 Interference experiment determination and specificity analysis spectra. Among them, A is the fluorescence intensity at the end point of the reaction detected by the EXPAR-CRISPR / Cas12a system (including target DNA, target DNA + R1, target DNA + R2, target DNA + R3). B is the fluorescence intensity at the end point of the reaction detected by the EXPAR-CRISPR / Cas12a system (including target DNA, NC_001472.1, NC_002016.1, NC_004718.3). Detailed implementation mode

[0025] To make the technical problems, technical solutions, and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0026] For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0027] Reagent materials:

[0028] A mixture of Nicking endonuclease (Nt.BstNBI), large fragment of Bst DNA polymerase, dNTP, LbCas12a and 10×NEBuffer3.1 was purchased from New England Biolabs.

[0029] The yfiR gene fragment (SEQ ID NO:1), crRNA (SEQ ID NO:3), hairpin probe (SEQID NO:2), ssDNA fluorescent reporter probe (SEQ ID NO:4), random sequence 1 (SEQ ID NO:5), random sequence 2 (SEQ ID NO:6), random sequence 3 (SEQ ID NO:7), NC_001472.1 (SEQ ID NO:8), NC_002016.1 (SEQ ID NO:9), NC_004718.3 (SEQ ID NO:10) were synthesized by Shanghai Sangon.

[0030] All bacterial strains used in this invention were from BeNa Culture Collection Center (BNCC, Shanghai, China) or isolated from clinical specimens of the First Affiliated Hospital of Guangdong Medical University in Dongguan.

[0031] Other chemicals were purchased from reputable commercial suppliers and were of analytical purity.

[0032] All buffer solutions applied in this invention were prepared with ultrapure water in our laboratory.

[0033] A comprehensive list of the sequences used in this invention can be found in Table 1. The hairpin probe was heated to 95°C for 15 minutes and then gradually cooled to room temperature before use.

[0034] Table 1 Sequences of synthetic oligonucleotide chains

[0035]

[0036] Example 1 EXPAR-CRISPR / 12a assay procedure

[0037] Figure 1 The schematic diagram of the detection system is shown as follows, and the operation steps are as follows:

[0038] 1) EXPAR amplification Each EXPAR reaction consisted of 1×NE buffer 3.1, 250 μM dNTP, 8 U large fragment of Bst DNA polymerase, 10 U Nt.BstNBI, 25 μM betaine, 10 nM hairpin probe, 10 nM target DNA and DEPC water, with a final volume of 20 μL. The amplification was carried out at 42°C for 60 minutes to obtain the EXPAR amplification product.

[0039] 2) The CRISPR / Cas12a fluorescence analysis reaction system was 30 μL, including 20 μL of EXPAR amplification product, 500 nM LbCas12a, 500 nM crRNA, 1000 nM ssDNA fluorescence reporter probe, and 1×NEB r2.1 buffer. The reaction was carried out at 37 °C for 60 minutes using a Gentier96E qPCR instrument (Tianlong, Xi'an, China), and fluorescence measurements were recorded every 5 minutes. The fluorescence signal was analyzed using ultraviolet light detection (Tano MINI Space 3000).

[0040] 3) Gel electrophoresis

[0041] The EXPAR reaction products were subjected to gel electrophoresis using a 4% agarose gel (composed of 3% NuSeive GTG agarose and 1% agarose). The electrophoresis was carried out at a constant voltage of 65 V for approximately 75 minutes. After staining with ethidium bromide, the electrophoresis gel was scanned using a gel imaging system (Tano MINI Space 3000).

[0042] Figure 2 The ability of the EXPAR-CRISPR / Cas12a detection system to identify the Salmonella yfiR gene was demonstrated. Figure 2 A Real-time fluorescence measurements were performed in 7 different EXPAR-CRISPR / Cas12a reactions. Among them, 1 was the hairpin probe, 2 was the target DNA, 3 was the hairpin probe bound to the target DNA, 4 was the complete EXPAR component without DNA polymerase, 5 lacked the Nt.BstNBI nicking endonuclease, 6 did not include the target DNA, and 7 was the fully assembled EXPAR component. Figure 2 B Fluorescence detection photos of the 7 reactions. Only the 7th EP tube with the fully assembled EXPAR component detected a fluorescence reaction.

[0043] Figure 2 C Photos of the agarose gel electrophoresis analysis of the 7 reactions. Among them, M was the DNA molecular weight marker (range 25 bp - 500 bp). The detection results showed that only the amplification product was clearly detected in the 7th lane.

[0044] Therefore, based on Figure 2 the detection results, it can be seen that only the fully assembled components can produce fluorescence signals and amplification products.

[0045] Example 2 Optimization of reaction conditions

[0046] To obtain the optimal reaction conditions, comparative tests were carried out on different concentrations of hairpin probes (5 nM, 10 nM, and 20 nM) and different reaction temperatures (42 °C, 50 °C, 60 °C).

[0047] The reaction system is as follows: 1×NEBuffer 3.1, 250 μM dNTP, 8 U large fragment of Bst DNA polymerase, 10 U Nt.BstNBI, 25 μM betaine, and DEPC water

[0048] A Fluorescence curves of EXPAR reactions containing hairpin probes at different concentrations (5 nM, 10 nM, and 20 nM) (reaction temperature was 42 °C, reaction time was 60 minutes).

[0049] B Fluorescence and S / N ratios of EXPAR reactions containing hairpin probes at different concentrations (5 nM, 10 nM, and 20 nM) (reaction temperature was 42 °C, reaction time was 60 minutes).

[0050] C Fluorescence curves of EXPAR at different reaction temperatures (42 °C, 50 °C, 60 °C) (hairpin probe concentration was 10 nM, reaction time was 60 minutes).

[0051] D Fluorescence and S / N ratios of EXPAR at different reaction temperatures (42 °C, 50 °C, 60 °C). Error bars represent standard deviation, n = 3 (hairpin probe concentration was 10 nM, reaction time was 60 minutes).

[0052] As Figure 3 shown, the optimal reaction conditions were finally determined as follows: hairpin probe concentration was 10 nM, reaction temperature was 42 °C.

[0053] Example 3 Determination of the sensitivity of the reaction system

[0054] Effect of the concentration of the yfiR gene fragment on the EXPAR reaction Different concentrations of the yfiR gene fragment were added to the EXPAR amplification system, and the concentrations were set to 0, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, and 100 nM respectively.

[0055] The total volume of the reaction system was 30 μL. In addition to the yfiR gene fragment, the following components were also included: 1×NEBuffer 3.1, 250 μM dNTP, 8 U large fragment of Bst DNA polymerase, 10 U Nt.BstNBI, 25 μM betaine, 10 nM hairpin probe, and DEPC water. The reaction system was incubated at 42 °C for 60 minutes, and the fluorescence intensity of the reaction was measured using CRISPR / Cas12a.

[0056] The fluorescence curves of the EXPAR reactions containing different concentrations of the yfiR gene fragment are as shown in Figure 4 A of

[0057] The fluorescence intensities of the EXPAR reactions containing different concentrations of the yfiR gene fragment are as shown in Figure 4 B of

[0058] The linear relationship between the average fluorescence intensity at 60 minutes and the logarithm of the target DNA concentration is as shown in Figure 4 C below.

[0059] The fluorescence intensities of different concentrations of Salmonella genomic DNA detected by EXPAR-CRISPR / Cas12a at 60 minutes are as shown in Figure 4 D below.

[0060] The experimental results show that the detection limit of the EXPAR-CRISPR / Cas12a detection system is 100 nM, and there is a linear relationship between the target DNA concentration in the range of 10 fM to 100 nM.

[0061] Example 4 Determination of the specificity of the reaction system

[0062] Different types of bacterial genomic DNA (20 ng / μL) were added to the EXPAR amplification system

[0063] Salmonella, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Acinetobacter baumannii were used as experimental groups respectively; DEPC water was used as the negative control group (NC group).

[0064] The total volume of the reaction system was 30 μL. In addition to different gene fragments, it also included the following components: 1×NEBuffer3.1, 250 μM dNTP, 8 U large fragment of Bst DNA polymerase, 10 U Nt.BstNBI, 25 μM betaine, 10 nM hairpin probe, and DEPC water. The added volume of the yfiR gene fragment was 20 μL. The reaction system was incubated at 42 °C for 60 minutes, and the fluorescence intensity of the reaction was measured using CRISPR / Cas12a.

[0065] The results are as shown in Figure 5 shown Figure 5 A below shows the fluorescence curves of the EXPAR-CRISPR / Cas12a system when detecting different bacteria. Among them, a fluorescence curve was detected for Salmonella, while Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Acinetobacter baumannii, and NC all showed no detection. Figure 5 B below shows the fluorescence intensities of each experimental group and NC at the end point of the reaction.

[0066] According to the detection results, it can be determined that the detection method provided by the present invention has significant specificity, can effectively reduce false positives caused by other strains, and significantly improves the accuracy and reliability of the detection results.

[0067] Example 5 Determination of Interference and Specificity of the Reaction System

[0068] In the EXPAR amplification system, random sequence 1 (SEQ ID NO: 5), random sequence 2 (SEQ ID NO: 6), and random sequence 3 (SEQ ID NO: 7) were added as experimental groups for the interference experiment. Different types of DNA were added to the EXPAR amplification system, and the experimental groups were respectively NC_001472.1 (SEQ ID NO: 8), NC_002016.1 (SEQ ID NO: 9), NC_004718.3 (SEQ ID NO: 10), and target DNA (SEQ ID NO: 1), and DEPC water was used as the negative control group (NC group).

[0069] The total volume of the reaction system was 30 μL. In addition to different DNA fragments, the following components were included: 1×NEBuffer3.1, 250 μM dNTP, 8 U large fragment of Bst DNA polymerase, 10 U Nt.BstNBI, 25 μM betaine, 10 nM hairpin probe, and DEPC water. The reaction system was incubated at 42 °C for 60 minutes, and the fluorescence intensity of the reaction was measured using CRISPR / Cas12a.

[0070] The results are as Figure 6 shown, Figure 6 As shown in A, the fluorescence intensities of the target DNA group, target DNA + R1, target DNA + R2, and target DNA + R3 groups detected by the EXPAR-CRISPR / Cas12a system are shown. Among them, obvious fluorescence was generated in all experimental groups. Figure 6 As shown in B, the statistical chart of the fluorescence intensities of different types of DNA detected by the EXPAR-CRISPR / Cas12a system is shown. Among them, obvious fluorescence was generated by the target DNA, and no obvious fluorescence was generated in the NC_001472.1, NC_002016.1, NC_004718.3, and NC groups.

[0071] The above is the preferred implementation manner of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A detection system for Salmonella, comprising an isothermal amplification system and a CRISPR / Cas12a detection system, wherein the isothermal amplification system recognizes a target gene and amplifies an EXPAR product that produces single-stranded DNA, and the CRISPR / Cas12a detection system can recognize and cut the EXPAR product to produce detectable fluorescence; in, The isothermal amplification system comprises a hairpin probe and an Nt-type nicking enzyme, wherein the hairpin probe comprises a hybridization sequence complementary to the target gene and a specific sequence that cooperates with the Nt-type nicking enzyme; The target gene is the Salmonella yfiR gene.

2. The detection system according to claim 1, characterized in that: The nucleotide sequence of the target gene is SEQ ID NO:

1.

3. The detection system according to claim 1 or 2, characterized in that: The isothermal amplification system comprises: Nt-type nicking enzyme, DNA polymerase, dNTPs, permeabilizing agent, and buffer; preferably, the Nt-type nicking enzyme is Nt.BstNBI, and the nucleotide sequence of the hairpin probe is SEQ ID NO: 2; preferably, the permeabilizing agent is betaine; preferably, the DNA polymerase is BstDNA polymerase large fragment.

4. The detection system according to any one of claims 1 to 3, characterized in that: The CRISPR / Cas12a detection system comprises a Cas12a enzyme, crRNA and ssDNA, wherein the nucleotide sequence of the crRNA is SEQ ID NO: 3, and / or the ssDNA is SEQ ID NO:

4.

5. A method for detecting Salmonella, comprising: 1) amplifying the test sample using the isothermal amplification system of the detection system according to any one of claims 1 to 4 to obtain an EXPAR product; 2) using the CRISPR / Cas12a detection system of any one of claims 1 to 4 to identify and cut the EXPAR product, and detect and record the fluorescence signal; 3) Analyze the fluorescence signal.

6. The detection method according to claim 5, characterized in that The reaction system of the isothermal amplification system comprises: 8-12U Bst DNA polymerase large fragment, 10-15U Nt.BstNBI, 10-15nM hairpin probe, 20-30μM betaine, dNTPs and buffer; preferably, the concentration of dNTPs is 200-300μM; preferably, the buffer is 1×NEBuffer 3.

1.

7. The detection method according to claim 6, characterized in that The amplification reaction condition of the isothermal amplification system is 42° C. for 60 minutes.

8. The detection method according to any one of claims 5 to 7, characterized in that: The cleavage reaction of the CRISPR / Cas12a system comprises: 20 μL EXPAR product, 500 nM LbCas12a, 500 nM crRNA and 1000 nM ssDNA; preferably, the reaction conditions of the cleavage reaction are: at 37° C. for 60 minutes; preferably, the fluorescence signal is recorded every 5 minutes using a qPCR instrument.

9. Use of the detection system according to any one of claims 1 to 4, and / or the detection method according to any one of claims 5 to 8 in detecting Salmonella in daily life samples, environmental samples and / or clinical samples.

10. The use according to claim 9, characterized in that The living samples are selected from one or more of meat, poultry products, eggs, dairy products, aquatic products, vegetables, feed, cosmetics and ready-to-eat foods; and / or, the environmental samples are selected from one or more of water sources, soil, sewage, farm excrement and air dust; and the clinical samples are selected from one or more of feces, blood, vomitus, urine, bile and tissue biopsy specimens.

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