A method for imaging bacterial single-base mutations

Abstract

The present invention discloses a single-base mutation imaging method based on in situ ARMS-PCR and Cas labeling. This method belongs to the field of single-cell analysis. By adding base mismatch sites to PCR primers, effective amplification and specific identification of single nucleotide variation information within bacteria are achieved. At the same time, the amplified products are labeled by modified gRNA / Cas12a. This method can also accurately quantify Salmonella resistant bacteria in complex environments and obtain cellular heterogeneity and spatial location information.

Description

Technical Field

[0001] The present invention belongs to a method for imaging single-base mutations in cells, and relates to a method for improving the specificity of single-base mutation recognition by adding mismatch sites in PCR primers, and using CRISPR / Cas12a to illuminate single cells, thereby achieving high-contrast imaging of single nucleotide variation information of pathogens. Background Art

[0002] Overuse of antibiotics and environmental changes can induce single-base mutations in foodborne pathogens, conferring drug resistance. In situ imaging of single-nucleotide variants in single cells can capture heterogeneity within a population of drug-resistant bacteria. Currently, traditional methods for detecting drug resistance in foodborne pathogens primarily include PCR, sequencing (NGS), and fluorescence in situ hybridization (FISH). PCR- and sequencing-based detection methods are unable to visualize single-nucleotide variant information in images, fail to capture cellular heterogeneity and population spatial information, and obscure the characteristics of a few cells. FISH often requires multiple fluorescent probes to be positioned at the target sequence for imaging to enhance the fluorescent signal, but this is not conducive to the detection of single-nucleotide variants. Using a single probe to detect single nucleotides can result in false positives and nonspecific binding. Consequently, FISH methods are unable to detect shorter target sequences, let alone specifically identify single-nucleotide variants.

[0003] Therefore, the present invention uses in situ ARMS-PCR to specifically identify single nucleotide variants and uses CRISPR / Cas12a to label target sequences, thereby enabling the detection of pathogenic genes. Furthermore, the method provided by this patent not only adds mismatch sites to PCR primers to improve the specificity of ARMS-PCR, but also optimizes the gRNA structure. Compared to FISH, only a single single-stranded DNA reporter molecule is used to image the target gene, thereby obtaining high-contrast imaging of cellular heterogeneity and spatial location information. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of existing pathogen gene detection technologies by developing a method for visualizing pathogen gene detection based on in situ ARMS-PCR and Cas12a labeling. This method increases the specificity of single-base mutation recognition by adding base mismatch sites to PCR primers. The target gene sequence is amplified using in situ ARMS-PCR, and Cas12a and a DNA reporter molecule are then combined with the amplified product to illuminate single cells. This method improves single-nucleotide resolution within cells, enabling high-signal, low-background in situ imaging, simplifying analysis steps, and reducing costs. Furthermore, this method can accurately quantify drug-resistant Salmonella in co-culture systems with varying ratios of drug-resistant and drug-sensitive Salmonella. The competitive survival ability of drug-resistant and drug-sensitive Salmonella strains under stress with different concentrations of antimicrobial peptides (0% and 0.05%) was studied.

[0005] The specific steps of the single-cell in situ ARMS-PCR-Cas12a detection method described in the present invention are as follows:

[0006] (1) Pretreatment of bacteria. First, culture the bacteria at 37°C and 180 rpm for 12 h. Centrifuge the enriched bacteria at 6000 rpm for 5-10 min, and remove the supernatant. Then, add 4% paraformaldehyde solution, resuspend the bacterial pellet, and let it stand at room temperature for 1-2 h. Add 1× PBS buffer, shake thoroughly, wash the bacterial pellet, and centrifuge under the same conditions to remove the supernatant. Then, add 50 μg / mL proteinase K, incubate the bacteria at 37°C for 10-30 minutes, and centrifuge to remove the supernatant. Add 5‰ Triton X-100, let it stand at room temperature for 5-10 min, and finally wash with 1× PBS buffer and centrifuge to remove the supernatant. Keep the bacterial pellet.

[0007] (2) In situ bacterial amplification and imaging. The pretreated bacteria were first amplified by PCR. A certain volume of buffer, primers (100 μM), H2O, and pretreated bacteria were added. The PCR-amplified bacteria were centrifuged and the supernatant was removed. The bacterial pellet was washed twice with 1× PBS and centrifuged. The supernatant was removed and the bacterial pellet was retained.

[0008] The Cas12a reaction system was added to the amplified bacterial pellet. First, premix the gRNA and DNA reporter. Add a certain volume of gRNA (20 μM), DNA reporter (20 μM), buffer, and H2O, and premix at 75°C for 3-10 minutes to obtain a premix. Then, add Cas12a (10 μM), buffer, premix, and water to the PCR-amplified bacterial pellet and incubate at 37°C for 1-2 hours. Then, wash by centrifugation, remove the supernatant, and resuspend the bacterial pellet in a certain volume of 1× PBS buffer. After mixing, a certain volume of the bacterial solution was added to a slide modified with polylysine (100 μg / mL) and the bacteria were imaged in situ using a fluorescence microscope (Nikon Ts2R-FL).

[0009] (3) Growth and competition of Salmonella under antimicrobial peptide stress. LB medium was prepared to contain different concentrations of colistin sulfate (0%, 0.0125%, 0.025%, 0.05%, 0.1%, 0.2%, and 0.3%). Salmonella was inoculated at a ratio of 500:1 and incubated at 37°C for 10-12 h. The OD600 value of the bacterial solution was measured every 2 h using a UV spectrophotometer to plot the Salmonella growth curve.

[0010] Resistant and susceptible bacteria were added to LB medium containing an optimal concentration of colistin sulfate solution in a proportional manner and incubated at 37°C for 12 hours. Simultaneously, bacterial cultures were aspirated every 12 hours for in situ imaging and plate counts. Plate counts were performed on standard XLT4 plates and resistant XLT4 plates, with 8 μg / mL ciprofloxacin solution added to the resistant plates. Measurements were taken at six consecutive time points. The inoculated plates were incubated in a biological incubator at 37°C for 24-30 hours before counting.

[0011] The principle of the in situ ARMS-PCR-Cas12a technology-based pathogen gene detection method of the present invention is as follows: Figure 1 As shown. We artificially added base mismatch sites in the PCR primers to improve the specificity of the method for identifying single-base variations, resulting in different amplification efficiencies between drug-resistant bacteria and drug-sensitive bacteria. After the amplification is completed, we use CRISPR / Cas12a to identify the amplified products within the bacteria and light up the drug-resistant bacteria, thereby achieving the effect of signal output. At the same time, this method can be used to accurately quantify drug-resistant bacteria in a co-culture system (Salmonella resistant bacteria and drug-sensitive bacteria). We also studied the competitive growth ability of Salmonella resistant bacteria and drug-sensitive bacteria under the stress of a certain concentration of the antimicrobial peptide colistin sulfate, and obtained cell heterogeneity information and spatial location information. During the experiment, we optimized the mismatch sites of the ARMS-PCR primers, as shown in the following figure. Figure 2As shown. Ensure that the primer has one base mismatch with drug-resistant bacteria and two base mismatches with drug-sensitive bacteria. Therefore, this results in that this method can only specifically identify and amplify the target sequence in drug-resistant bacteria, and drug-sensitive bacteria cannot be effectively amplified. By extracting the DNA sequences of drug-resistant and drug-sensitive Salmonella bacteria, the qPCR amplification efficiency under different primers was determined. The results showed that the F3 primer (the fourth mismatch site at the 3' end of the primer) maximized the difference in amplification efficiency between drug-resistant and drug-sensitive bacteria and was the optimal primer. Primer sequence information is shown in Figure 7 shown.

[0012] In order to further improve the fluorescence intensity, we also modified the structure of gRNA. Different gRNA binding sites and structural changes will affect the recognition ability of Cas12a effector protein. Four different gRNA binding sites were selected according to the target sequence, and a sequence was randomly extended at the 5' or 3' end of the gRNA. This sequence will bind to the DNA reporter molecule for in situ imaging. Figure 3 As shown, by selecting a sequence extension at the 3' end of the gRNA and binding to the PAM 2 site, drug-resistant bacteria produced a more pronounced green fluorescence signal than drug-susceptible bacteria. The overall fluorescence intensity of the drug-susceptible group was low, almost identical to the background, achieving the desired effect. Furthermore, we achieved the highest signal-to-noise ratio (resistant bacteria / drug-susceptible bacteria) of 471.1%, significantly higher than the signal-to-noise ratios of other sites (81.9%-256.2%).

[0013] To further evaluate the feasibility of this method, we used the CRISPR / Cas12a system to identify ARMS-PCR amplification products. When both Cas12a protein and DNA reporter molecules were present, the drug-resistant bacteria group produced a significant fluorescent signal. In contrast, the signal produced by the drug-sensitive bacteria group was close to that of the low-fluorescence control group, indicating that no target product of the drug-sensitive bacteria could be recognized. Therefore, the drug-resistant bacteria can be illuminated and produce a high fluorescence signal, with an average fluorescence intensity 19 times that of the control group. However, the control group produced only weak fluorescence, which may be because the reaction lacks Cas12a effector protein or DNA reporter molecules, such as Figure 4 The above experimental results prove that pathogen genes can be illuminated and visualized through in situ ARMS-PCR and CRISPR / Cas12a strategies.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) This method provides a visualization detection method based on in situ ARMS-PCR-Cas to illuminate pathogen genes, allowing imaging of single-base mutations and accurately obtaining cellular heterogeneity information and spatial location information.

[0016] (2) This method explores the feasibility of in situ ARMS-PCR for intracellular amplification and detection of single-base mutation genes in Salmonella, achieving specific identification of single-base mutations at the single-molecule level. Furthermore, modified gRNA and single-stranded DNA reporter molecules are used to illuminate drug-resistant bacteria. This strategy can significantly increase the fluorescence intensity of drug-resistant bacteria, reduce background signals, and obtain high-contrast in situ imaging.

[0017] (3) The CRISPR / Cas12a pathogen-lighting strategy provided by this method can avoid the complex probe design steps in traditional fluorescence in situ hybridization technology, improve the signal-to-background ratio, and reduce false positives.

[0018] (4) This method can be used to accurately quantify drug-resistant bacteria in co-culture systems and explore the competitive survival advantages of drug-resistant and drug-sensitive Salmonella under antimicrobial peptide stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of a visualization detection method for identifying single-base mutations based on in situ ARMS-PCR-Cas provided by the present invention;

[0020] Figure 2 For the optimization of ARMS-PCR primer mismatch sites;

[0021] Figure 3 For the modification and verification of gRNA structure;

[0022] Figure 4 To verify the signal output of gRNA and DNA reporter molecules on ARMS-PCR amplification products;

[0023] Figure 5 To identify drug-resistant and drug-sensitive bacteria based on in situ ARMS-PCR-Cas;

[0024] Figure 6 To study the competitive ability of Salmonella under antimicrobial peptide stress based on in situ ARMS-PCR-Cas;

[0025] Figure 7 The sequence information of ARMS-PCR primers and gRNA is shown in Figure 2. DETAILED DESCRIPTION

[0026] The following is a further description of the visualization detection method for identifying pathogen genes based on in situ ARMS-PCR-Cas provided by the present invention with reference to examples and drawings.

[0027] Example 1: In situ imaging distinguishes between drug-resistant and sensitive bacteria mixed in different proportions

[0028] In this example, the method proposed in this invention can be used to differentiate mixed drug-resistant and drug-sensitive bacteria by in situ imaging. The specific steps are as follows: We selected five co-culture systems with different ratios, adding 10%, 20%, 40%, 60%, and 100% of drug-resistant bacteria. The co-culture system was centrifuged at 6000-8000 rpm for 5-10 minutes, and the supernatant was removed. The precipitate was washed with PBS buffer and centrifuged and the supernatant was removed. A certain volume of 4% paraformaldehyde was then added for fixation for 1-2 hours. Next, the bacteria were treated with 50µg / mL proteinase K and incubated at 37°C for 5-10 minutes and 5‰ Triton X-100 and allowed to stand at 37°C for 5-10 minutes. Next, a certain volume of gRNA (100nM), Cas12a (100nM), and DNA reporter (1μM) were added to the sample to be tested and mixed. After a reaction time of 45-60 minutes, in situ imaging was performed using a fluorescence microscope. The results showed that this method can accurately quantify drug-resistant bacteria in the co-culture system.

[0029] Example 2: Studying the competitive growth of Salmonella under antimicrobial peptide stress

[0030] In this example, the competitive growth of drug-resistant and drug-susceptible Salmonella under stress conditions with a certain concentration of antimicrobial peptide was investigated. The specific steps are as follows: First, the growth curves of drug-susceptible Salmonella were studied under different concentrations of colistin sulfate to select the optimal colistin sulfate concentration. Colonies of drug-resistant and drug-susceptible Salmonella were counted using a preliminary plate count and added to LB medium containing the optimal colistin sulfate concentration and standard LB medium, respectively, in proportion. Every 12 hours, the plate was diluted into fresh medium and plated onto standard XLT4 plates and resistant XLT4 plates (containing 8 μg / mL ciprofloxacin), respectively. The plates were incubated at 37°C for 12 hours, and colonies were counted. In this example, the culture was incubated for a total of 72 hours, with six consecutive measurements. Simultaneously, the culture medium was imaged in situ at different time periods using the method described in this invention, and drug-resistant bacteria were counted. The results showed that the statistical results for drug-resistant bacteria obtained by this patented research method were consistent with those obtained by the plate count method, demonstrating the feasibility and accuracy of this method. At the same time, the counting results showed that under the pressure of antimicrobial peptides, drug-resistant bacteria have greater survival competitive advantages than drug-sensitive bacteria.

Claims

1. A single-base mutation imaging method for non-disease diagnosis or treatment purposes, characterized in that: Intracellular ARMS-PCR was used to specifically amplify the mutation target, and the amplified product was labeled based on Cas12a / gRNA and a reporter probe to achieve visual detection of single-base mutations in drug-resistant genes in single Salmonella cells; the primer sequences are shown in SEQ ID NO.4 and SEQ ID NO.6; the gRNA sequence is shown in SEQ ID NO.10; the 5' end of the reporter probe was modified with a fluorescent group, and the sequence was GACTGGATCTCATGGTTCGA.

2. The single-base mutation imaging method for non-disease diagnosis or treatment purposes according to claim 1, characterized in that: The method includes two reaction systems: ARMS-PCR and CRISPR / Cas12a. The ARMS-PCR reaction system includes 100 μM primers, pretreated bacteria, buffer, and H2O; the CRISPR / Cas12a reaction system includes 20 μM gRNA, 20 μM reporter probe, 10 μM Cas12a, buffer, and H2O. The reaction is carried out for 1.5-2 hours, and in situ imaging is performed using a fluorescence microscope.

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