A multi-site crisper pathogenic gene visual detection method

Abstract

The application discloses a pathogenic bacteria gene visual in-situ detection method based on a multi-site CRISPR / Cas12a technology, and belongs to the in-situ detection field of intracellular genes. The trans-cleavage activity of a gRNA / Cas12a complex on a reporter probe is used to light up a target gene, and a multi-site gRNA design strategy can significantly improve the fluorescence intensity, more quickly recognize the target gene in a complex environment, and does not need to denature and digest the cells. Therefore, the multi-site combined non-repetitive gene imaging method developed by the application can realize in-situ detection of the differentiation of drug-resistant bacteria and drug-sensitive bacteria, and can be adapted to complex environments (such as intestinal slices).

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of intracellular gene in situ detection, and relates to a method for activating CRISPR / Cas12a transcleavage activity in situ in cells, and realizing single-cell visual detection of pathogenic bacteria genes by using multi-site binding of multiple Cas12a, without denaturation or digestion process. BACKGROUND

[0002] Single-cell in situ analysis is a key way to understand cell behavior and function, and can obtain heterogeneity information of cell population. Single-cell visualization technology can improve the time and spatial resolution of gene detection. Traditional gene analysis techniques include real-time quantitative polymerase chain reaction (qPCR) and next-generation sequencing (NGS), etc. The above techniques analyze the nucleic acid extracted from a large number of cells after pretreatment, which may mask the spatial information and heterogeneity information of subpopulations (such as drug-resistant bacteria), and it is difficult to obtain the gene expression characteristics of a small number of key strains. Therefore, people usually use single-cell in situ detection methods to study the information of subpopulations. The traditional in situ detection method of cell and tissue gene expression is in situ hybridization technology, among which the most widely used is fluorescence in situ hybridization (FISH). FISH technology can obtain spatial information and cell heterogeneity information of RNA expression. However, FISH method has low sensitivity, and it is difficult to detect low-concentration RNA in cells, and the technology cannot detect non-repetitive gene information.

[0003] Therefore, the present application provides a multi-site CRISPR / Cas12a technology-based pathogenic bacteria gene visual in situ detection method, which can detect non-repetitive genes in the target, without denaturation or digestion of the target. At the same time, the method provided by the present application has the potential to construct a dynamic research tool for pathogen function and promote single-cell behavior visualization. SUMMARY

[0004] The present application aims to overcome the shortcomings of the existing intracellular gene in situ detection technology, and to explore a multi-site CRISPR / Cas12a technology-based pathogenic bacteria gene visual in situ detection method. By activating CRISPR / Cas12a transcleavage activity in situ in cells, non-repetitive genes in single cells can be directly detected, without digestion and denaturation of the target. The method can simplify the detection and analysis steps, reduce costs, and improve detection efficiency. Through the detection method, imaging of drug-resistant Salmonella typhimurium in intestinal slices can be realized, and the competitive survival ability of drug-resistant strains and drug-sensitive strains under salt stress environmental conditions can be studied.

[0005] The specific steps of the single-cell in situ detection method according to the present application are as follows:

[0006] (1) In situ imaging of bacteria. Centrifuge at 6000-8000 rpm for 3-5 min, remove the supernatant, resuspend in 1x PBS buffer, centrifuge at 6000-8000 rpm for 3-5 min, remove the supernatant. Add a certain volume of 4% paraformaldehyde for 30-60 min. Then, the bacteria are treated with 50 μg / mL proteinase K at 37°C for 5-10 min and 5‰ Triton X-100 at 37°C for 5-10 min. Next, add a certain volume of gRNA (100 nM), Cas 12a (100 nM) and reporter probe (1 μM) to the test mixture. After 30-60 min of reaction, in situ imaging is performed using a fluorescence microscope (Nikon Ts2R-FL). Image J is used for quantitative analysis of fluorescence images.

[0007] (2) Growth and competition of Salmonella under salt stress. LB medium is configured to contain different concentrations of sodium chloride (0%, 2%, 4%, 8%). Salmonella S. (ATCC 14028) is inoculated at a ratio of 1:100, incubated at 37°C for 10-12 h, and the OD600 of the bacterial solution is measured every hour using a UV spectrophotometer to draw a growth curve.

[0008] The drug-resistant strain and the drug-sensitive strain are cultured in LB medium without NaCl to 1x10 8 CFU / mL, and the culture temperature is 37°C. At the same time, culture in LB medium containing different concentrations of NaCl, temperature is 37°C, culture for 10-12 h. Next, the cultured bacterial solution is inoculated into new LB medium at a ratio of 1:100, the bacterial solution is gradiently diluted, and plated on agar plates with or without 8 μg / mL ciprofloxacin XLT4. Then incubate at 37°C under aerobic conditions for 24 hours, count the colonies. Measure 6 time points continuously according to the above method.

[0009] (3) Animal experiments. 12 SPF BALB / c mice were randomly divided into 2 groups, 6 in each group. Each group was placed in an isolator separately. Both groups were fed with the same sterile feed. Two weeks after the start of the experiment, antibiotics were added to the drinking water to eliminate intestinal bacteria (vancomycin 0.5 g / L, neomycin sulfate 0.5 g / L, ampicillin 0.5 g / L). From the 15th to the 17th day, group C was fed with 1x10 8 CFU of Salmonella ATCC 14028, and group T was fed with 1x10 8 CFU of S29. All mice were sacrificed on the 18th day, and the colon and cecum were collected under sterile conditions.

[0010] The principle of the pathogenic bacteria gene visualization in situ detection method based on the multi-site CRISPR / Cas12a technology described in the present application is as follows: Figure 1The CRISPR-Cas12a is a single RNA-guided DNA cleavage nuclease that can cleave non-target single-stranded DNA (ssDNA). When the gRNA / Cas12a complex recognizes the target gene, the reporter probe can be cleaved and the fluorescence can be restored. In the experiment, the Salmonella drug resistance gene oqxB was used as the target. A Salmonella strain (S29) randomly selected from the oqxB positive strain was used as the experimental object to perform the ciprofloxacin minimum inhibitory concentration test and the growth and competition experiment of Salmonella under salt stress. In order to improve the signal-to-noise ratio during imaging, a multiple site binding strategy of Cas12a was used. First, 20 gRNA sites were designed on the oqxB gene, as shown in Figure 6 In vitro fluorescence detection was performed on each site using the extracted DNA. Except for sites 12, 16, 17, and 19, most gRNA sites can effectively activate the trans-cleavage activity of Cas12a. Then, in order to prevent the diffusion of fluorescent signals outside the bacteria, the effect of different reporter probes on in situ imaging was explored. The optimized reporter probe has a more complex structure, which can avoid the diffusion of fluorescence, thereby causing a significant difference between cells and background. The results show that the traditional single-stranded reporter probe is easy to diffuse to the bacteria and is not suitable for single-cell analysis, and may be completely digested, as shown in Figure 2

[0011] Meanwhile, in order to further evaluate whether the multiple binding strategy of Cas12a can enhance the activation of Cas12a and improve the signal-to-background ratio, a single gRNA site and different gRNA sites were used for testing while maintaining the total concentration of Cas12a. The pixel intensity of 65 bacteria was calculated. When recognizing the target oqxB gene, a single gRNA site cannot trigger a significant fluorescence signal. When the combined gRNAs increase from 5 sites to 20 sites, the pixel intensity of the drug-resistant strain increases from 23.07 to 72.06, and the pixel intensity of the sensitive strain increases from 14.53 to 25.60. We use the sensitive strain as the background group and the drug-resistant strain as the signal group. The signal-to-background ratio increases from 1.01 to 2.81. Notably, when using 20 gRNA sites, the pixel intensity increases by 187.55% compared to the background group, while using a single gRNA site, the signal increases by 0.93% (as shown in Figure 3 ). Furthermore, the present application also compared the effect of time on the fluorescence imaging results under different combined gRNA sites. The results show that when the gRNA sites are insufficient, the fluorescence of the bacteria cannot be significantly distinguished from the background fluorescence. With the increase of time, the fluorescence signals of all samples gradually recover. The fluorescence signal of 20 gRNA sites recovers the fastest and reaches the maximum value at 30 min, as shown in Figure 3 ​The above experimental results prove that the multiplex binding strategy combined with Cas12a can activate the trans-cleavage activity of the CRISPR / Cas12a system in situ.

[0012] Compared with the prior art, the present application has the following beneficial effects:

[0013] 1. The method provides a pathogenic gene visualization in situ detection method based on multi-site CRISPR / Cas12a technology, which allows imaging of single cell genes and can provide key information about cell population heterogeneity.

[0014] 2. The method explores the possibility of activating the CRISPR / Cas12a trans-cleavage activity in situ, realizes the in situ detection of Salmonella drug resistance gene oqxB, and uses the trans-cleavage activity of gRNA / Cas12a complex to report probe to light up the target gene. The multi-site gRNA design strategy can significantly improve the fluorescence intensity and more quickly identify the target gene in a complex environment.

[0015] 3. The multiplex binding strategy combined with Cas12a provided by the present application improves the signal-to-background ratio and avoids the denaturation and digestion process of the target in traditional detection.

[0016] 4. The method can be used in complex environments (intestinal sections) and can be used for single cell research of Salmonella typhimurium under salt stress. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Principle diagram of the pathogenic gene visualization in situ detection method based on multi-site CRISPR / Cas12a technology provided by the present application

[0018] Figure 2 Influence of different structures of reporter probes on in situ imaging

[0019] Figure 3 Fluorescence intensity imaging of multi-site drug-resistant strains and drug-sensitive strains and the different effects of time on sites

[0020] Figure 4 Differentiation of Salmonella isolated from poultry farms based on multi-site Cas 12a

[0021] Figure 5 Differentiation of drug-resistant strains and sensitive strains in mouse cecum based on multi-site Cas 12a

[0022] Figure 6 Sequence information of 20 gRNAs DETAILED DESCRIPTION

[0023] The pathogenic bacteria gene visualization in-situ detection method based on the multi-site CRISPR / Cas12a technology provided by the present application is further described below in combination with examples and drawings. Example 1: In-situ imaging to distinguish drug-resistant strains and sensitive strains mixed in different proportions

[0024] In this embodiment, the drug-resistant strains and sensitive strains mixed can be distinguished by the method provided in the present application, and the specific operation steps are as follows: take strains mixed in different proportions, centrifuge at 6000-8000 rpm for 3-5 min, remove the supernatant, resuspend in 1x PBS buffer, centrifuge at 6000-8000 rpm for 3-5 min, remove the supernatant. Add a certain volume of 4% paraformaldehyde to fix for 45-60 min. Then, treat the bacteria with 50 μg / mL proteinase K at 37°C for 5-10 min and 5‰ Triton X-100 at 37°C for 5-10 min. Next, add a certain volume of gRNA (100 nM), Cas12a (100 nM) and optimized reporter probe (1 μM) to the test mixture. After 45-60 min of reaction, in-situ imaging is performed using a fluorescence microscope (Nikon Ts2R-FL).

[0025] Example 2: Detection of Salmonella isolated from poultry farms

[0026] In this example, the method is applied to detect whether the Salmonella isolated from poultry farms contains drug-resistant genes. The operation steps are as follows: first, randomly select 30 strains of clinically isolated Salmonella for in-situ detection, then pretreat the strains (the pretreatment steps mainly refer to Example 1), add Cas12a combined with multiple sites, then perform in-situ imaging, and finally use Image J to quantitatively analyze the fluorescence images. The results show that this method can better distinguish drug-resistant strains from drug-sensitive strains, as shown in Figure 4

[0027] Example 3: Distinguish drug-resistant strains and drug-sensitive strains in mouse cecum

[0028] ​In this embodiment, we performed Salmonella challenge on antibiotic-treated mice. After 7 consecutive days of challenge, we performed intestinal sectioning and imaging using the method of the present application to verify the feasibility of imaging drug-resistant Salmonella in complex environments. The Salmonella ingested by mice mainly parasitizes the intestinal tract, especially the cecum. The complex components of the intestinal tract increase background fluorescence interference. The specific steps of the embodiment are as follows: we randomly divided 12 SPF BALB / c mice into 2 groups, 6 in each group. Each group was individually placed in an isolation room. Both groups were fed with the same sterile feed. After 2 weeks of experiment, antibiotics were added to the drinking water to remove intestinal bacteria (vancomycin 0.5 g / L, neomycin sulfate 0.5 g / L, ampicillin 0.5 g / L). After 2 weeks of antibiotic treatment, the mice were divided into two groups and orally administered with drug-resistant and sensitive strains, respectively. From day 15 to day 17, group C was fed with 1 x 10 8 CFU of Salmonella ATCC 14028, and group T was fed with 1 x 10 8 CFU of S29. All mice were sacrificed on day 18, and the colon and cecum were collected under sterile conditions. Then, the sections were imaged in situ, and the experimental results showed that the present experiment could correctly distinguish between drug-resistant and drug-sensitive strains in the cecum of mice, proving that the binding strategy of multiple sites of Cas12a could enhance the fluorescence signal output.

[0029] Example Four: Study of competitive growth of Salmonella under salt stress conditions

[0030] In this embodiment, the competitive growth of Salmonella under salt stress conditions was studied. The specific operation steps are as follows: Salmonella ATCC 14028 and S29 were cultured in LB medium without NaCl at 37°C to 1 x 10 8 CFU / mL. An equal amount was inoculated into medium containing different concentrations of sodium chloride (0%, 4%), and incubated at 37°C for 10-12 h. The bacterial culture was inoculated at a ratio of 1:100 and the bacterial culture was serially diluted and spread on agar plates. Then, the plates were incubated at 37°C under aerobic conditions for 20-24 hours, and the number of colonies was counted. The above method was continuously measured at 6 time points. SEQUENCE LISTING <110> Sichuan University <120> A method for visualizing detection of pathogenic bacteria with multiple sites of CRISPR <160> 22 <170> SIPOSequenceListing 1.0 <210> 1 <211> 41 <212> RNA <213> Artificial Sequence <400> 1 uaauuucuac uaaguguaga uauuuuuauc accggguuaa u 41 <210> 2 <211> 41 <212> RNA <213> Artificial Sequence <400> 2 uaauuucuac uaaguguaga uaaaacauga uguacaugaa a 41 <210> 3 <211> 41 <212> RNA <213> Artificial Sequence <400> 3 uaauuucuac uaaguguaga ugcuccggug aauaugcgau g 41 <210> 4 <211> 41 <212> RNA <213> Artificial Sequence <400> 4 uaauuucuac uaaguguaga ucugaucucc auuaacgccc a 41 <210> 5 <211> 41 <212> RNA <213> Artificial Sequence <400> 5 uaauuucuac uaaguguaga ucagucaccc ggcgcuaacg c 41 <210> 6 <211> 41 <212> RNA <213> Artificial Sequence <400> 6 uaauuucuac uaaguguaga uguccgcgac uccauccgcg c 41 <210> 7 <211> 41 <212> RNA <213> Artificial Sequence <400> 7 uaauuucuac uaaguguaga uagcauucuc uaucugcugg g 41 <210> 8 <211> 41 <212> RNA <213> Artificial Sequence <400> 8 uaauuucuac uaaguguaga uucuccgggg ucaccggcca g 41 <210> 9 <211> 41 <212> RNA <213> Artificial Sequence <400> 9 uaauuucuac uaaguguaga uuucccaccc aggauaagcu g 41 <210> 10 <211> 41 <212> RNA <213> Artificial Sequence <400> 10 uaauuucuac uaaguguaga uccggggcuu aacgcgcugc a 41 <210> 11 <211> 41 <212> RNA <213> Artificial Sequence <400> 11 uaauuucuac uaaguguaga uccgcuuuuu uaucgacagg c 41 <210> 12 <211> 41 <212> RNA <213> Artificial Sequence <400> 12 uaauuucuac uaaguguaga uccgagaccg uggcgacgcc g 41 <210> 13 <211> 41 <212> RNA <213> Artificial Sequence <400> 13 uaauuucuac uaaguguaga uaggugcaga accgcgucgc g 41 <210> 14 <211> 41 <212> RNA <213> Artificial Sequence <400> 14 uaauuucuac uaaguguaga uucccccggc gggaaguacg a 41 <210> 15 <211> 41 <212> RNA <213> Artificial Sequence <400> 15 uaauuucuac uaaguguaga ugcgccgagc cgcugccgca g 41 <210> 16 <211> 41 <212> RNA <213> Artificial Sequence <400> 16 uaauuucuac uaaguguaga ugcaauauca uucugaaaac g 41 <210> 17 <211> 41 <212> RNA <213> Artificial Sequence <400> 17 uaauuucuac uaaguguaga ucgccgcuug ccgcggcgca u 41 <210> 18 <211> 41 <212> RNA <213> Artificial Sequence <400> 18 uaauuucuac uaaguguaga ucugcgcagc ucgaacggcu a 41 <210> 19 <211> 41 <212> RNA <213> Artificial Sequence <400> 19 uaauuucuac uaaguguaga ugaccagcgc aaacacacgg c 41 <210> 20 <211> 41 <212> RNA <213> Artificial Sequence <400> 20 uaauuucuac uaaguguaga ugcuuuucca uccugccgcc g 41 <210> 21 <211> 12 <212> DNA <213> Artificial Sequence <400> 21 gggttttttg gg 12 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <400> 22 tagctagcgc tagctatttt tt 22

Claims

1. A single-cell visualization method for detecting antibiotic resistance genes in pathogens for purposes other than disease diagnosis or treatment, characterized in that, Twenty gRNAs were designed based on the Salmonella resistance gene oqxB. The gRNA / Cas12a complex binds to the target gene at multiple sites within the bacteria and generates trans-cleavage activity, thereby cleaving the reporter probe and producing specific fluorescence to illuminate the target gene. No denaturation or digestion process is required. The gRNA sequences are shown in SEQ ID NO.1-SEQ ID NO.20; the reporter probe sequence is shown in SEQ ID NO.

22.

2. The method according to claim 1, characterized in that, The reaction system included pretreated Salmonella, Cas12a protein, gRNA, and reporter probe. The concentrations of Cas12a, gRNA, and reporter probe were 100 nM and 1 μM, respectively. After reacting for 45-60 min, in situ imaging was performed using a fluorescence microscope.

Images