A method for detecting staphylococcus aureus based on CRISPR technology

Abstract

The application provides a method for detecting Staphylococcus aureus based on CRISPR technology, and the method comprises the following steps: S1, amplifying a to-be-detected sample to obtain an amplification product; the to-be-detected sample is amplified by using an RPA amplification primer group; S2, contacting the amplification product with Cas protein, sgRNA and single-stranded nucleic acid detector, detecting a detectable signal generated by the Cas protein cutting the single-stranded nucleic acid detector, and thus detecting Staphylococcus aureus; the sequence of the RPA amplification primer group is shown as SEQ ID No. 3 and SEQ ID No. 7, and the sequence of the target region in the sgRNA for hybridizing with the target nucleic acid is shown as SEQ ID No. 9. The method has the advantages of good inclusivity, strong specificity, high sensitivity and the like, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nucleic acid detection, and particularly relates to a method for detecting Staphylococcus aureus based on CRISPR technology. BACKGROUND

[0002] Staphylococcus aureus (S. aureus) is also called "golden staphylococcus", belonging to Staphylococcus, and is a representative of gram-positive bacteria, which is a common foodborne pathogenic microorganism. The optimal growth temperature of the bacteria is 37℃, the pH is 7.4, and the bacteria are resistant to high salt and can grow in an environment with a salt concentration close to 10%. Staphylococcus aureus often parasitizes the skin, nasal cavity, throat, intestines and stomach, abscesses, and suppurative sores of humans and animals, and is ubiquitous in the air, sewage and other environments.

[0003] The present application provides a novel method for detecting Staphylococcus aureus, which is based on RPA-CRISPR technology, especially based on the trans activity of RPA recombinase polymerase amplification technology and Cas12b, and provides a detection method with high specificity and high detection sensitivity. SUMMARY

[0004] Therefore, the main purpose of the present application is to provide a method for detecting Staphylococcus aureus.

[0005] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0006] On the one hand, the present application provides a method for detecting Staphylococcus aureus, which comprises the following steps:

[0007] S1, amplifying a to-be-tested sample to obtain an amplification product;

[0008] The to-be-tested sample is amplified by using an RPA amplification primer set, and the sequence of the RPA amplification primer set is shown in SEQ ID No. 3 and SEQ ID No. 7;

[0009] S2, contacting the amplification product with Cas protein, sgRNA and single-stranded nucleic acid detector, detecting the detectable signal generated by the Cas protein cutting the single-stranded nucleic acid detector, and thereby detecting Staphylococcus aureus, wherein the sgRNA comprises a region combined with the Cas protein and a guide sequence hybridized with the target nucleic acid, the target nucleic acid is a nucleic acid derived from Staphylococcus aureus, and the sequence of the target region in the sgRNA hybridized with the target nucleic acid is shown in SEQ ID No. 9.

[0010] In one embodiment, the method is an RPA-CRISPR detection method.

[0011] In one embodiment, the amplification product is as shown in SEQ ID No. 1 or is a partial sequence of the sequence shown in SEQ ID No. 1.

[0012] In one embodiment, the target nucleic acid is the genomic sequence DQ507377.1 of Staphylococcus aureus.

[0013] In one embodiment, the sequence of the RPA amplification primer set is as shown in any one of SEQ ID No. 2-7, SEQ ID No. 12-13. Preferably, the sequence of the RPA amplification primer set is as shown in SEQ ID No. 3 and SEQ ID No. 7.

[0014] In the present application, the region that binds with the CRISPR / CAS effector protein (Cas protein) is also referred to as the scaffold region, which interacts with the Cas protein and thereby binds with the Cas protein.

[0015] In one embodiment, the gRNA comprises, in order from 5' end to 3' end, a region that binds with the Cas protein and a targeting region that hybridizes with the target nucleic acid.

[0016] In one embodiment, the sequence of the targeting region that hybridizes with the target nucleic acid comprises any one of SEQ ID No. 8-10; preferably, the sequence of the targeting region that hybridizes with the target nucleic acid is as shown in SEQ ID No. 9.

[0017] In one embodiment, the region that binds with the Cas protein is as shown in SEQ ID No. 11.

[0018] In one embodiment, the Cas protein is selected from the V-type Cas protein, for example, Cas12, Cas14 family protein or a mutant thereof.

[0019] In one embodiment, the Cas protein is Cas12b. Preferably, the sequence of the Cas protein is as shown in SEQ ID No. 14.

[0020] Preferably, the sequence of the region that binds with the Cas protein is as shown in SEQ ID No. 11.

[0021] Further, the method further comprises a step of obtaining the target nucleic acid from the sample to be tested; preferably, the target nucleic acid is obtained from the sample to be tested by using an amplification method.

[0022] The amplification is selected from one or any of PCR, nucleic acid sequencing based amplification (NASBA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HDA), or nicking enzyme amplification reaction (NEAR), multiple displacement amplification (MDA), rolling circle amplification (RCA), ligase chain reaction (LCR), or derivative amplification methods (RAM), preferably the amplification is LAMP amplification.

[0023] In the present application, the sample can be a sample from an animal, for example, a human, a cow, a sheep, a pig, a horse, a donkey.

[0024] In one embodiment, the sample can be a blood, excrement (feces, urine), milk, meat, internal organs, fur, abortion product, secretion or bone marrow sample.

[0025] In other embodiments, the sample can also be derived from an environmental sample, for example, air, water, soil, equipment in a farm, etc.

[0026] In the present application, the detectable signal is achieved by the following methods: visual-based detection, sensor-based detection, color detection, gold nanoparticle-based detection, fluorescence polarization, fluorescent signal, colloidal phase transition / dispersion, electrochemical detection and semiconductor-based detection.

[0027] In some embodiments, the method of the present application further comprises the step of measuring the detectable signal produced by the Cas protein. The Cas protein can trigger the cleavage activity of any single-stranded nucleic acid after recognizing or hybridizing with the target nucleic acid, thereby cleaving the single-stranded nucleic acid detector and generating a detectable signal.

[0028] In one embodiment, the detectable signal can also be achieved by the following methods: different labeling molecules are arranged at the 5' end and 3' end of the single-stranded nucleic acid detector, for example, a fluorescent group and a quenching group, the fluorescent group is selected from one or any of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED460; the quenching group is selected from one or any of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.

[0029] In one embodiment, the single-stranded nucleic acid detector is FAM-TTTTTTT-BHQ1.

[0030] In one embodiment, the two ends of the single-stranded nucleic acid detector are arranged with labels that can be detected by colloidal gold.

[0031] In an embodiment, the steps S1 and S2 can be in one reaction system or in different systems.

[0032] In an embodiment, the step S1 is an RPA amplification method; preferably, the amplification system in the step S1 comprises a basic RPA premix, an RPA amplification primer group, magnesium acetate, template DNA, and polyethylene glycol 35000; more preferably, the amplification system in the step S1 comprises 1x basic RPA premix, 37.5nM RPA amplification primer group, 10.5mM magnesium acetate, 20-200ng template DNA, and 0.9% polyethylene glycol 35000, and the RPA reaction parameter is 43℃ / 20min.

[0033] In an embodiment, the step S2 is a CRISPR detection method; preferably, the detection system in the step S2 comprises a CRISPR buffer, a Cas12b protein, an sgRNA, and a single-stranded nucleic acid detector; more preferably, the detection system in the step S2 comprises 1x CRISPR buffer, 0.2μM Cas12b protein, 0.25μM sgRNA, and 3.3μM single-stranded nucleic acid detector; and the CRISPR reaction parameter is 43℃ / 30min.

[0034] In an embodiment, the method has good inclusivity, strong specificity, and high sensitivity. The method can detect Staphylococcus aureus of various sources; and the method can detect nucleic acids at the fg level.

[0035] In another aspect, the present application also provides a system or composition for detecting Staphylococcus aureus, which comprises the above-mentioned RPA amplification primer group, Cas protein, and sgRNA. Further, the system or composition further comprises the above-mentioned single-stranded nucleic acid detector.

[0036] In another aspect, the present application also provides the use of the above-mentioned system or composition in detecting Staphylococcus aureus.

[0037] In another aspect, the present application also provides the use of the above-mentioned system or composition in preparing a reagent or kit for detecting Staphylococcus aureus.

[0038] Advantages of the present application:

[0039] The present application provides a method for detecting Staphylococcus aureus, which is an RPA-CRISPR detection method, and uses an RPA amplification primer group and sgRNA with high detection efficiency, and has the advantages of good inclusivity, strong specificity, and high sensitivity.

[0040] Sequence information

[0041] Part of the sequence information involved in the present application is provided as follows:

[0042]

[0043]

[0044] The following drawings and examples are only used to illustrate the present application, and are not limited to the scope of the present application. According to the following detailed description of the drawings and preferred embodiments, various purposes and advantages of the present application will become apparent to those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 Schematic diagram of the position of sgRNA targeting sequence (sgRNA-1, sgRNA-2, sgRNA-3) on the target sequence.

[0046] Figure 2 Detection efficiency of sgRNA-1.

[0047] Figure 3 Detection efficiency of sgRNA-2.

[0048] Figure 4 Detection efficiency of sgRNA-3.

[0049] Figure 5 Schematic diagram of the detection efficiency of RPA-F1 / R1 in detecting target nucleic acid.

[0050] Figure 6 Schematic diagram of the detection efficiency of RPA-F1 / R2 in detecting target nucleic acid.

[0051] Figure 7 Schematic diagram of the detection efficiency of RPA-F1 / R3 in detecting target nucleic acid.

[0052] Figure 8 Schematic diagram of the detection efficiency of RPA-F2 / R1 in detecting target nucleic acid.

[0053] Figure 9 Schematic diagram of the detection efficiency of RPA-F2 / R2 in detecting target nucleic acid.

[0054] Figure 10 Schematic diagram of the detection efficiency of RPA-F2 / R3 in detecting target nucleic acid.

[0055] Figure 11 Schematic diagram of the detection efficiency of RPA-F3 / R1 in detecting target nucleic acid.

[0056] Figure 12 Schematic diagram of the detection efficiency of RPA-F3 / R2 in detecting target nucleic acid.

[0057] Figure 13 This diagram illustrates the detection efficiency of RPA-F3 / R3 when detecting target nucleic acids.

[0058] Figure 14 This is the result of the inclusiveness verification of the RPA-CRISPR detection system.

[0059] Figure 15 The results validated the exclusivity of the RPA-CRISPR detection system in 61 common cross-strains.

[0060] Figure 16 Schematic diagram of sensitivity verification results for the RPA-CRISPR detection system Figure 1 .

[0061] Figure 17 Schematic diagram of sensitivity verification results for the RPA-CRISPR detection system Figure 2 .

[0062] Figure 18 Schematic diagram of sensitivity verification results for the RPA-CRISPR detection system Figure 3 .

[0063] Figure 19 Schematic diagram of sensitivity verification results for the RPA-CRISPR detection system Figure 4 .

[0064] Figure 20 Schematic diagram of sensitivity verification results for the RPA-CRISPR detection system Figure 5 .

[0065] Figure 21 Schematic diagram of sensitivity verification results for the RPA-CRISPR detection system Figure 6 . Detailed Implementation

[0066] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0067] The technical scheme of the present application is based on the principle of combining RPA amplification with CRISPR technology, which has the characteristics of rapidness, sensitivity, specificity and high efficiency. Under the premise that specific nucleic acids of target pathogenic bacteria exist in the sample, specific primers bind to the target sequence, and the target sequence is enriched by RPA amplification. Cas enzyme (Cas protein Cas12b, amino acid sequence as shown in SEQ ID No. 14) binds to the amplification product under the guidance of sgRNA, activates the trans cleavage activity of Cas protein, and cleaves the Reporter (one end of the Reporter is connected with a fluorescent group, and the other end is connected with a quencher group) in the system. The Reporter is cleaved by the Cas protein to release fluorescence to present the detection result. In other embodiments, the two ends of the single-stranded nucleic acid detector (Reporter) can also be marked with colloidal gold for detection.

[0068] Example 1, Staphylococcus aureus target-specific sgRNA and RPA primer screening

[0069] In this embodiment, sgRNA design and target nucleic acid amplification are performed on the specific nucleic acid of Staphylococcus aureus.

[0070] According to the genome sequence of Staphylococcus aureus, we set the target sequence on the nuc fragment (DQ507377.1, 1-300), and the nucleic acid sequence is shown as SEQ ID No. 1. sgRNA design and sgRNA performance verification are performed. We first use PCR primers (sequences as shown in SEQ ID No. 12-13) to amplify double-stranded fragments, and the PCR primer sequences are shown in the following table:

[0071]

[0072] The sequences of the designed sgRNAs are shown in the following table (the targeting region sequences of nuc-sgRNA-1-3 are shown as SEQ ID No. 8-10, and the backbone region sequence is shown as SEQ ID No. 11):

[0073]

[0074] Among them, the targeting regions of nuc-sgRNA-1, nuc-sgRNA-2 and nuc-sgRNA-3 are shown as SEQ ID No. 8-10, respectively, and the backbone region sequences of nuc-sgRNA-1, nuc-sgRNA-2 and nuc-sgRNA-3 are shown as SEQ ID No. 11.

[0075] The sgRNA position of Staphylococcus aureus target is as shown in Figure 1 , and the screening result is as shown in Figures 2-4 , and the screening result is as shown inFigures 2-4 In particular, 1 cycle = 1 minute, and the smaller the number of cycles to reach the plateau represents the higher sgRNA cleavage efficiency. According to the results of the above experiment, the sgRNA with the highest cleavage efficiency is selected for the subsequent experiment. Figures 2-4 It can be seen that nuc-sgRNA-1 does not reach the fluorescence plateau within 30 minutes, nuc-sgRNA-2 reaches the fluorescence plateau in 13 cycles, and nuc-sgRNA-3 reaches the fluorescence plateau in 19 cycles. That is, the cleavage efficiency of nuc-sgRNA-2 is higher, and nuc-sgRNA-2 will be used for subsequent experiments.

[0076] On the basis of the higher cleavage efficiency of nuc-sgRNA-2, three RPA upstream primers and three RPA downstream primers were designed for combination (RPA-F1 / R1, RPA-F1 / R2, RPA-F1 / R3, RPA-F2 / R1, RPA-F2 / R2, RPA-F2 / R3, RPA-F3 / R1, RPA-F3 / R2, RPA-F3 / R3) for amplification screening. The sequences of RPA-F1, RPA-F2, RPA-F3, RPA-R1, RPA-R2, RPA-R3 are shown in SEQ ID No. 2-7, and the sequences are shown in the following table:

[0077]

[0078] The results of different combinations of primers are shown in Figures 5-13 From the fluorescence growth rate and the time to reach the platform value, Nuc-RPA-F2 / R3 is the best, so Nuc-RPA-F2 / R3 combination is selected for subsequent testing.

[0079] Example 2, RPA-CRISPR reaction system

[0080] Based on the results of RPA and CRISPR, we finally selected nuc-RPA-F2 / R3 with nuc-sgRNA-2 for subsequent performance verification.

[0081] According to the component final concentration and volume in Table 1 and Table 2, the RPA and CRISPR reaction system is configured.

[0082] Table 1, RPA fluorescence method reaction system

[0083]

[0084] Table 2, CRISPR reaction system

[0085]

[0086] The fluorescence probe (also known as single-stranded nucleic acid detector) is FAM-TTTTTTT-BHQ1.

[0087] Note 1: In order to avoid the reaction from starting too early, when configuring the RPA reaction system, avoid mixing magnesium acetate with other components in advance. It is recommended to add it separately on the tube wall or tube cover. After all components are configured, mix all components.

[0088] Note 2: The CRISPR components should be mixed with the RPA components after the RPA process is completed. It is recommended to add the CRISPR reaction system to a new tube cover after the RPA and CRISPR reaction systems are configured. Be careful to cover it to avoid the CRISPR reaction system falling during the RPA process. After the RPA reaction process is completed, centrifuge thoroughly and mix well, and then place it in a fluorescence PCR instrument for CRISPR reaction. Alternatively, after the RPA process is completed, wait for the RPA system to cool down to room temperature, and then add the CRISPR components.

[0089] Note 3: It is recommended to use commercially available RPA and CRISPR systems for experiments, and adjust the components and final volume of the reaction system according to the instructions for different raw materials, and ensure that the final concentration of the components listed in the table remains unchanged.

[0090] The RPA reaction parameters are: 43°C / 20min;

[0091] The CRISPR reaction parameters are: 43°C / 30min, and the fluorescence signal is collected every minute.

[0092] Example 3, Inclusivity and exclusivity verification of RPA-CRISPR detection of Staphylococcus aureus

[0093] The RPA primers and sgRNA selected in Example 2 were used for inclusivity and exclusivity verification in different sources of Staphylococcus aureus strains and common cross-species bacteria. The inclusivity test strains and experimental results are shown in the following table and Figure 14

[0094]

[0095]

[0096] The exclusivity test strains and experimental results are shown in the following table and Figure 15

[0097]

[0098]

[0099]

[0100] ​​According to the inclusive experimental results, the CRISPR results of 22 strains of S. aureus standard strains and sample isolates are consistent with the traditional biochemical results, and there is no false negative. According to the exclusive experimental results, the CRISPR results of 61 strains of non-S. aureus standard strains and sample isolates are consistent with the traditional biochemical results, and there is no false negative, indicating high specificity.

[0101] Example 4, sensitivity test of RPA-CRISPR detection system

[0102] After diluting the single colony of S. aureus after culture with water, the magnetic bead method was used for extraction, and the final concentration was 10 ng / uL. The template was diluted step by step to determine the minimum concentration of target nucleic acid that could be detected in the sample to be tested by the RPA-CRISPR detection system.

[0103] The RPA amplification system and Cas enzyme cutting detection system refer to Example 3, and the reaction process refers to Example 2. The template with a final concentration of 10 ng / uL was diluted by 10 times, 100 times, 1000 times, 10^4 times, 10^5 times and 10^6 times, respectively, and each dilution gradient was repeated three times. The results are shown in Figures 16-21

[0104] The results show that when the concentration of the template is 10 ng / uL and is diluted by 10 times, 100 times, 1000 times, 10^4 times and 10^5 times, the RPA-CRISPR detection system can stably detect it. When diluted by 10^6 times, the three repeats cannot be detected. According to the above results, 10^5 times dilution of the single colony after culture at 10 ng / uL can be detected, and the RPA-CRISPR detection system in the present application can detect fg-level nucleic acid.

[0105] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings disclosed herein, and these changes are within the scope of protection of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.​

Claims

1. A method for detecting Staphylococcus aureus based on CRISPR technology, which is a method for non-disease diagnosis and treatment purposes, characterized by, The method includes the following steps: S1. Amplify the sample to be tested to obtain the amplification product; The test sample was amplified using an RPA amplification primer set, the sequences of which are shown in SEQ ID No. 3 and SEQ ID No.

7. S2. The amplification product is contacted with Cas protein, sgRNA and a single-stranded nucleic acid detector, and the detectable signal generated by the Cas protein cleaving the single-stranded nucleic acid detector is detected, thereby detecting Staphylococcus aureus. The sgRNA includes a region that binds to the Cas protein and a target region that hybridizes with the target nucleic acid. The target nucleic acid is a nucleic acid derived from Staphylococcus aureus. The sequence of the target region that hybridizes with the target nucleic acid in the sgRNA is shown in SEQ ID No.

9. The Cas protein is Cas12b; The backbone region of the sgRNA is shown in SEQ ID No.

11.

2. The method of claim 1, wherein, The amplification product is shown in SEQ ID No.

1.

3. The method according to claim 1, characterized in that, The detectable signal can be achieved through any of the following methods: vision-based detection, sensor-based detection, gold nanoparticle-based detection, fluorescence polarization, fluorescence signal, colloidal phase transition, electrochemical detection, or semiconductor-based detection.

4. The method according to claim 1, characterized in that, Step S1 is the RPA amplification method.

5. The method according to claim 1, characterized in that, Step S2 is a CRISPR detection method, and the detection system in step S2 includes CRISPR buffer, Cas12b protein, sgRNA and single-stranded nucleic acid detector.

6. A composition for detecting Staphylococcus aureus, the composition comprising the RPA amplification primer set, Cas protein, sgRNA and single-stranded nucleic acid detector as described in claim 1.

7. Use of the composition of claim 6 in the preparation of reagents or kits for the detection of Staphylococcus aureus.

8. The use according to claim 7, characterized in that, The test samples were derived from animals.

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