Escherichia coli non-amplification type rapid detection method based on deoxyribozyme probe

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to an escherichia coli non-amplification type rapid detection method based on a deoxyribozyme probe. The method comprises the following steps: detecting Escherichia coli by adopting a fluorescent chip, mixing Escherichia coli lysate and RFD-EC recognition molecules for reaction, dripping into the fluorescent chip for reaction, and detecting the fluorescence intensity after the reaction; the fluorescent chip takes paper as a substrate and is coated with d-DNA, the nucleotide sequence of the d-DNA is shown as SEQ ID NO.3, and the 5'end of the d-DNA is marked with biotin. Fluorescent DNA molecules are enriched through a fluorescent chip micro-fluidic technology, and detection can be completed within 0.5-1 hour without enrichment culture in combination with multi-channel fluorescence detection. The detection lower limit of the method is as low as 100 CFU, and the method has the advantages of rapidness, sensitivity and low cost, is suitable for on-site rapid detection of Escherichia coli in clinical samples and environmental samples, and has important application value in the fields of pollution control and the like.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a non-amplified high-throughput rapid detection method for Escherichia coli based on deoxyribonuclease probes and its application. Background Technology

[0002] Currently, pathogenic bacteria have become a significant threat to public health, and achieving high-throughput, rapid detection of bacteria is crucial for effective control. Developing rapid, high-throughput detection technologies for pathogenic bacteria is of great importance for pollution control, preventing antibiotic overuse, curbing the formation of multidrug-resistant bacteria, and guiding clinical treatment.

[0003] In bacterial detection technologies, culture methods and gene detection methods are two representative approaches, detecting bacteria from the perspectives of growth characteristics and genetic information, respectively, and have significant application value in clinical, research, and public health fields. While culture methods are considered the "gold standard," possessing irreplaceable advantages in accuracy, applicability, and cost, they suffer from drawbacks such as long detection cycles (1 day to several weeks), limited sensitivity, the need for strict aseptic operation, and the inability to detect non-viable bacteria, making them difficult to meet the needs of rapid clinical testing. In recent years, gene detection-based bacterial analysis technologies have integrated nucleic acid amplification technologies (such as the FilmArrayBCID multiplex PCR platform, which can detect 24 pathogens within 1 hour), gene editing detection technologies (such as the CRISPR-Cas system, which achieves high-specificity diagnosis through targeted nucleic acid recognition), high-throughput sequencing technologies (such as Oxford Nanopore MinION nanopore sequencing, which supports rapid on-site metagenomic analysis), and biosensing technologies (such as electrochemical sensors and nanofluorescent probes for real-time detection of biomolecules). Although these technologies have shortened the detection cycle of traditional culture methods from several days to several hours, and some technologies, such as next-generation sequencing, can simultaneously analyze dozens of bacteria, they still have the following limitations: for viable bacterial concentrations below 10... 3 For samples with CFU / mL, traditional culture enrichment methods require 12-48 hours. Direct nucleic acid detection is prone to false negatives due to insufficient extraction efficiency and the presence of PCR inhibitors in the sample. Furthermore, equipment such as nanopore sequencers and flow cytometers are expensive, and nucleic acid extraction methods such as magnetic bead extraction and column extraction are complex and require professional personnel. They may also lead to decreased sensitivity due to incomplete bacterial lysis or nucleic acid loss during purification (theoretical loss rate 10%-30%). Therefore, developing culture-free, single-molecule-level ultrasensitive detection technologies (such as digital PCR and single-cell sequencing) combined with microfluidic chips to achieve fully automated "sample in - result out" analysis is a key technological direction to overcome the current technological bottlenecks.

[0004] In the exploration of technologies to improve detection sensitivity and automation, the functional design of nucleic acid molecules has provided new insights for bacterial detection. Deoxyribonucleases (DNAzymes) are single-stranded DNA molecules with catalytic functions obtained through systematic evolution of ligands by exponential enrichment (SELEX) screening. Functional nucleic acid molecules obtained through SELEX technology also include: 1. Aptamers have the ability to specifically recognize target substances such as proteins and small molecules. Their biological recognition function is similar to that of antibodies, and they have the advantages of a wide target range, high stability, and ease of chemical modification and synthesis. 2. Aptazymes integrate both target recognition domains (aptamers) and catalytic domains (ribozymes), and can trigger catalytic reactions through target binding.

[0005] By binding aptamers or aptamer ribozymes to fluorescent groups, molecular beacon sensors based on conformational changes can be constructed, which induce fluorescence signal release through target recognition. Deoxyribozymes, on the other hand, typically trigger fluorescence signal amplification by catalyzing substrates (such as cutting RNA / DNA). All of the above functional nucleic acid molecules have shown application potential in the field of biosensing and can be widely used for the detection of nucleic acids, proteins, bioactive molecules, and disease biomarkers.

[0006] In the innovation process of biosensing technology, functional nucleic acid molecules (including deoxyribozymes, aptamers, and aptamer ribozymes) have provided a new technical path for solving existing problems in the field of pathogen detection due to their unique properties. Currently, the detection of pathogens such as Escherichia coli still relies on traditional methods in some scenarios: culture methods are a classic confirmatory method, but their detection cycle is as long as 24-48 hours, making it difficult to meet the immediate needs of emergency infection diagnosis or on-site food screening; traditional PCR technology can achieve quantitative detection within 6 hours, but it suffers from high equipment costs and requires professional personnel for sample preparation and data analysis. In the detection of clinical samples (such as stool from patients with diarrhea) and environmental samples (such as fresh food and drinking water), point-of-care testing (POCT) has become an important development direction because it can shorten the diagnostic cycle and reduce laboratory dependence. However, existing POCT technologies (such as immunogold test strips) have insufficient sensitivity (the detection limit is usually 10). 4 -10 5The detection of E. coli suffers from limitations such as limited CFU / mL or high throughput (single-sample detection). Therefore, developing highly sensitive detection methods based on novel biorecognition elements (such as aptamers and antibodies), such as nanofluorescent probe technology, and combining them with microfluidic chip technology to achieve automated sample processing and multiplexing, is of significant technical importance and application value in improving the rapid on-site response capability and high-throughput screening efficiency of E. coli detection. Summary of the Invention

[0007] Aptamer ribozymes, as single-molecule nucleic acid elements that combine target recognition (aptamer domain) and catalytic function (ribozyme domain), have biological recognition functions of aptamer domains that are very similar to those of antibodies. They also have advantages such as a wide target range, high stability, and ease of chemical modification and synthesis.

[0008] The aptamer ribozyme probe (RFD-EC) screened in this invention is a complex formed by base complementarity pairing of a catalytic DNA strand (5'short) and a substrate strand (FS28), the structure of which is shown in [Figure number missing]. Figure 1 The FS28 chain has a fluorescent group (F) and a quenching group (Q) bonded to its two ends. In the absence of the E. coli-specific secretory protein S, the 5' short binds to FS28 to form a stem-loop structure, bringing F and Q closer together and quenching the fluorescence signal. However, in the presence of the secretory protein S, the 5' short acts as a deoxyribonuclease to specifically cleave FS28, causing F and Q to separate and significantly enhancing the fluorescence signal. Previous experiments have verified the recognition ability of this probe for E. coli, but its effectiveness is limited by the sensitivity of the detection method (the limit of detection is 10). 3 For samples with a concentration of CFU / mL, enrichment culture for 2-4 hours is required to meet the detection threshold.

[0009] To address these shortcomings, this invention provides a non-amplification rapid detection method for Escherichia coli based on aptamer ribozyme probes. By integrating paper chip microfluidic technology with a high-sensitivity fluorescence detection module, it achieves efficient concentration and signal amplification of bacteria in the sample, and can complete the detection within 0.5-1 hours without enrichment culture.

[0010] The present invention adopts the following technical solution: This invention provides a method for detecting Escherichia coli, which uses a fluorescent chip for detection. E. coli lysate and RFD-EC recognition molecules are mixed and reacted, then placed on the fluorescent chip for reaction. The fluorescence intensity is detected after the reaction. The fluorescent chip uses paper as a substrate and is coated with… d -DNA, the d The nucleic acid sequence of the DNA is shown in SEQ ID NO.3. d - The 5' end of the DNA is labeled with biotin for fixation. d-DNA.

[0011] In the above technical solution, the RFD-EC recognition molecule further comprises the catalytic chain 5'short shown in SEQ ID NO.1 and the substrate chain FS28 shown in SEQ ID NO.2; wherein, the substrate chain FS28 contains an RNA base A, with T bases on both sides of which are connected to a fluorescent group F and a quenching group Q, respectively. The catalytic chain 5'short is partially complementary to the substrate chain FS28 and has recognition, catalytic, and cleavage functions. After binding to the E. coli-specific secretory protein S, it can cleave the SEQ ID NO.2 chain to break it into a short chain, thereby promoting the separation of the fluorescent group F and the quenching group Q to release a fluorescent signal.

[0012] In the above technical solution, the fluorescent group F includes any one of FAM, FITC, Cy3, and Cy5, and the quenching group Q includes Dabcyl.

[0013] In the above technical solution, further, after the catalytic chain 5'short binds to the E. coli-specific secretory protein S, it catalytically cleaves the substrate chain FS28, breaking it into short chains, resulting in the separation of the fluorescent group F and the quenching group Q, releasing a fluorescent signal; the fluorescent DNA molecules generated by the reaction f -DNA is specifically recognized by fluorescent chips. d -DNA enrichment, the d - The DNA pairs complementary to specific sequence bases of the substrate chain FS28.

[0014] In the above technical solution, the detection method further includes the following steps: (1) Preparation and lysis of bacterial culture: Escherichia coli was cultured and the bacterial concentration was diluted to 10. 4 ~10 6 CFU / mL, centrifuged and resuspended in a buffer reaction solution containing HEPES, NaCl and BaCl2, and ultrasonically oscillated to obtain a bacterial mixed lysate; (2) The catalytic chain 5'short is mixed with the substrate chain FS28 to form an RFD-EC recognition molecule; (3) Bacterial mixed lysis buffer, RFD-EC recognition molecules and buffer, reacted at room temperature in the dark and then dropped onto the fluorescent chip area, washed and enriched. f -DNA, detected by a fluorescence spectrometer.

[0015] In the above technical solution, further, in step (1), after culturing Escherichia coli in a shaker at 37 ℃ and 150~200 rpm, the bacterial concentration is diluted to 10. 4 ~10 6 The reaction solution contained 20-60 mmol / L HEPES, 120-160 mmol / L NaCl, and 5-20 mmol / L BaCl2. The centrifugation conditions were 4 ℃, 8000 rpm for 10 min, and ultrasonic oscillation for 1-5 min.

[0016] In the above technical solution, further, in step (2), the concentration of the catalytic chain 5'short is 80~120 μmol / L and the volume is 2.5 μL, and the concentration of the substrate chain FS28 is 8~12 μmol / L and the volume is 0.5 μL.

[0017] In the above technical solution, further, the reaction system in step (3) is 25 μL mixed lysis buffer, 3 μL LRFD-EC recognition molecule and 100 μL buffer. The reaction is carried out at room temperature in the dark for 5~30 min. The reaction is performed by inserting a fluorescence instrument or a handheld fluorescence instrument. Different fluorescence excitation and detection channels are used for detection. The fluorescence detection uses blue, green and red channels, and the corresponding excitation wavelength and emission wavelength are as follows: blue channel, excitation wavelength Ex: 415~480 nm, emission wavelength Em: 495~520 nm; green channel, excitation wavelength Ex: 530~550 nm, emission wavelength Em: 560~610 nm; red channel, excitation wavelength Ex: 615~645 nm, emission wavelength Em: 655~720 nm.

[0018] Furthermore, in the above technical solution, when the S protein secreted by E. coli is present in the system, SEQ ID NO.1 can specifically recognize and catalyze the cleavage of SEQ ID NO.2, generating fragmented fluorescent DNA molecules ( ƒ ƒ-DNA); after the reaction solution is dropped into the fluorescent chip area, ƒ-DNA is specifically recognized by the sequence within the chip. d -DNA is selectively enriched, and then the fluorescence intensity is detected by a fluorometer or a handheld fluorometer. The fluorescence intensity of the blank solution is close to the background signal, while the fluorescence intensity of the sample containing E. coli shows a linear positive correlation with the bacterial content, thus achieving high-throughput detection of E. coli content.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: The method of this invention does not require enrichment culture, and the detection cycle is 0.5 to 1 hour, which has the advantage of rapid response. The detection can be completed in as little as 30 minutes. The method of this invention has high detection sensitivity and detection throughput, with a detection limit as low as 100 Escherichia coli, and can detect simultaneously through three detection channels, for a total of 12 detection pathways.

[0020] The method of this invention has good specificity and can effectively distinguish Escherichia coli from a variety of Gram-negative and Gram-positive bacteria. It has significant recognition specificity, making the sensing results of Escherichia coli more accurate.

[0021] The method of this invention can specifically detect Escherichia coli in actual samples without relying on expensive PCR systems and professional technicians. It is low-cost and enables rapid, high-throughput detection of Escherichia coli. It is especially suitable for rapid on-site detection scenarios and has great significance in pollution control. Attached Figure Description

[0022] The other objectives and technical advantages of the present invention will become clearer through the detailed description of the technical solution of the present invention in conjunction with the accompanying drawings, which will help to fully understand the technical features and innovations of the present invention.

[0023] Figure 1 The diagram shows the structures of SEQ ID NO.1 and SEQ ID NO.2.

[0024] Figure 2 This is a schematic diagram illustrating the principle of RFD-EC recognition of Escherichia coli in this invention.

[0025] Figure 3 This is the specificity of RFD-EC in this invention.

[0026] Figure 4 This is the standard curve obtained from the Escherichia coli detection method of the present invention.

[0027] Figure 5 This is a schematic diagram of the detection steps of the present invention. Detailed Implementation

[0028] To better understand the present invention, it is now described through embodiments. The specific embodiments listed are only for illustrating the present invention and are not intended to limit the present invention.

[0029] Table 1 Nucleic acid sequences used in this invention

[0030] Example 1: Rapid detection of Escherichia coli in river water using RFD-EC recognition molecules This method utilizes RFD-EC to identify molecules for the detection of E. coli, and the procedure is as follows: Figure 5As shown, the specific operation steps are as follows: (1) The test solution includes a blank solution, a standard solution containing a known number of Escherichia coli standards filtered through a 0.45 μm filter membrane, and a pretreated actual water sample filtered through a 0.45 μm filter membrane; (2) Bacterial culture and concentration calibration are as follows: After overnight culture, the concentration of Escherichia coli standard solution is determined by microplate reader. When OD600 = 0.5 (corresponding to a bacterial concentration of approximately 10...), the concentration is determined. 8 When the concentration reaches (CFU / mL), remove the bacterial culture and dilute it to 10 with sterile water. 5 CFU / mL; calculate the gradient concentrations of 100, 1000, and 10000 bacteria in a 10.5 μL bacterial sample and prepare standard solutions; centrifuge the E. coli standard solution sample at 4 ℃ and 8000 rpm for 10 min, discard the supernatant, and resuspend it in the reaction buffer (40 mmol / L HEPES, 140 mmol / L NaCl, 10 mmol / L BaCl2), repeatedly aspirate and mix well, and then sonicate for 3 min to obtain E. coli lysate; (3) The method for preparing the RFD-EC recognition molecule is as follows: the deoxynucleotide chain of SEQ ID NO.2 labeled with fluorescent group F and fluorescent quencher group Q is mixed with the deoxynucleotide chain of SEQ ID NO.1 to form the RFD-EC recognition molecule, wherein the concentration of SEQ ID NO.1 is 100 μmol / L and the volume is 2.5 μL, and the concentration of SEQ ID NO.2 is 10 μmol / L and the volume is 0.5 μL; (4) The fluorescence detection procedure is as follows: E. coli lysis buffer, RFD-EC recognition molecule and buffer are thoroughly mixed and reacted at room temperature in the dark for 20 min to obtain the reaction solution. The reaction solution is dropped onto the fluorescent chip area and washed multiple times to make the specific recognition sequence of the chip area. d - DNA enrichment of fragmented fluorescent DNA molecules (ƒ-DNA: TCGATCCTTCTCA), after 5 min of reaction, insert the paper chip into a fluorometer or handheld fluorometer, use the blue channel (excitation wavelength Ex: 415-480nm, emission wavelength Em: 495-520nm, exposure time 280ms) to detect and record the fluorescence intensity, and complete the E. coli count.

[0031] (5) Testing the actual water sample: The filtered actual water sample is cultured according to the method in step (2) to obtain Escherichia coli lysate, and the fluorescence intensity is detected according to the method in step (4). The fluorescence intensity is substituted into the standard curve to calculate the content of Escherichia coli in the actual water sample.

[0032] (6) The verification experiment was designed as follows: The blank solution, E. coli standard solution, and actual water sample were each divided into 6 portions. Three portions were placed in 5 mL SOB medium and cultured overnight at 37 ℃ and 150 rpm as the verification group. The verification group was counted under a microscope, and the counting results were compared with those of this method. The other three portions were tested in parallel using the test strip method described above. The experimental results are shown in Figure 4. The fluorescence intensity was positively correlated with the actual number of E. coli, verifying the effectiveness of this method.

[0033] Example 2: Specificity of RFD-EC (1) The test solutions used for specific detection in this example include: Escherichia coli standard solution, Klebsiella pneumoniae standard solution, Bacillus subtilis standard solution, Pseudomonas aeruginosa standard solution, and Bacillus gladiolus standard solution; (2) Bacterial culture and concentration calibration: After overnight culture, the E. coli standard solution was used to determine the bacterial concentration using an ELISA reader. When OD600 = 0.5 (corresponding to a bacterial concentration of approximately 10⁸ CFU / mL), the bacterial solution was removed and diluted to 10⁻¹⁰ with sterile water. 5 CFU / mL; (3) Preparation of lysis buffer: The Escherichia coli standard solution sample was centrifuged at 4 ℃ and 8000 rpm for 10 min. After discarding the supernatant, it was resuspended in the reaction buffer (40 mmol / L HEPES, 140 mmol / L NaCl, 10 mmol / L BaCl2). After repeated aspiration and mixing, it was sonicated for 3 min to obtain the Escherichia coli lysis buffer. (4) Following steps (2) and (3) above, Klebsiella pneumoniae standard solution, Bacillus subtilis standard solution, Pseudomonas aeruginosa standard solution and Bacillus gladiolus standard solution are respectively processed into corresponding bacterial lysates; (5) The method for preparing the RFD-EC recognition molecule is as follows: the deoxynucleotide chain of SEQ ID NO.2 labeled with fluorescent group F and fluorescent quencher group Q is mixed with the deoxynucleotide chain of SEQ ID NO.1 to form the RFD-EC recognition molecule, wherein the concentration of SEQ ID NO.1 is 100 μmol / L and the volume is 2.5 μL, and the concentration of SEQ ID NO.2 is 10 μmol / L and the volume is 0.5 μL; (4) The fluorescence detection procedure is as follows: Bacterial lysis buffer (E. coli lysis buffer or other lysis buffers), RFD-EC recognition molecules, and buffer are thoroughly mixed and reacted at room temperature in the dark for 20 min to obtain the reaction solution. The reaction solution is then dropped onto the fluorescent chip area. After multiple washes, the specific recognition sequence d-DNA in the chip area is enriched with fragmented fluorescent DNA molecules (ƒ-DNA: TCGATCCTTCTCA). After reacting for 5 min, the paper chip is inserted into a fluorometer or handheld fluorometer. The fluorescence intensity is detected and recorded using the blue channel (excitation wavelength Ex: 415-480 nm, emission wavelength Em: 495-520 nm, exposure time 280 ms). For other bacterial species, simply replace the E. coli lysis buffer with their respective lysis buffers and follow the above steps, recording the fluorescence intensity.

[0034] (5) Results are as follows Figure 3 As shown, when Escherichia coli lysis buffer was added to the reaction system, the fluorescence detection area showed a strong fluorescence signal; however, when Klebsiella pneumoniae lysis buffer, Bacillus subtilis lysis buffer, Pseudomonas aeruginosa lysis buffer, or Bacillus gladiolus lysis buffer were added to the reaction system, the fluorescence signal intensity in the fluorescence detection area was significantly lower than that of Escherichia coli (close to the background fluorescence level).

Claims

1. A method for detecting Escherichia coli, characterized in that, The detection of Escherichia coli using a fluorescent chip involves mixing E. coli lysate with RFD-EC recognition molecules, reacting the mixture, and then adding it to the fluorescent chip for reaction. The fluorescence intensity is then measured after the reaction. The fluorescent chip uses paper as a substrate and is coated with… d -DNA, the d The nucleic acid sequence of the DNA is shown in SEQ ID NO.

3. d - The 5' end of the DNA is labeled with biotin.

2. The method for detecting Escherichia coli according to claim 1, characterized in that, The RFD-EC recognition molecule comprises the catalytic chain 5'short shown in SEQ ID NO.1 and the substrate chain FS28 shown in SEQ ID NO.2; wherein the substrate chain FS28 contains an RNA base A, with a fluorescent group F and a quenching group Q attached to its two T bases, respectively; the catalytic chain 5'short is partially complementary to the substrate chain FS28 and has recognition, catalytic, and cleavage functions.

3. The method for detecting Escherichia coli according to claim 2, characterized in that, The catalytic chain 5'short, after binding to the E. coli-specific secreted protein S, catalytically cleaves the substrate chain FS28, breaking it into short chains. This causes the fluorescent group F to separate from the quenching group Q, releasing a fluorescent signal; the resulting fluorescent DNA molecules... f -DNA is specifically recognized by fluorescent chips. d -DNA enrichment, the d -Specific sequence bases of DNA and substrate chain FS28 ( f -DNA complementary pairing.

4. The detection method according to claim 1, characterized in that, The fluorescent group F includes any one of FAM, FITC, Cy3, and Cy5, and the quenching group Q includes Dabcyl.

5. The detection method according to claim 1, characterized in that, Includes the following steps: (1) Culture Escherichia coli and dilute the bacterial concentration to 10. 4 ~10 6 CFU / mL, centrifuged and resuspended in a buffer reaction solution containing HEPES, NaCl and BaCl2, and ultrasonically oscillated to obtain a bacterial mixed lysate; (2) The catalytic chain 5'short is mixed with the substrate chain FS28 to form an RFD-EC recognition molecule; (3) Bacterial mixed lysate, RFD-EC recognition molecules and bacteriophages ff After reacting at room temperature in the dark, the sample is dropped onto the fluorescent chip area, washed, and enriched. f -DNA, detected by a fluorescence spectrometer.

6. The detection method according to claim 5, characterized in that, The reaction solution buffer in step (1) consists of 20-60 mmol / L HEPES, 120-160 mmol / L NaCl, and 5-20 mmol / L BaCl2. The centrifugation conditions are 4℃, 8000 rpm for 10 min, and ultrasonic oscillation time is 1-5 min.

7. The detection method according to claim 5, characterized in that, In step (2), the concentration of the catalytic chain 5'short is 80~120 μmol / L and the volume is 2.5 μL, while the concentration of the substrate chain FS28 is 8~12 μmol / L and the volume is 0.5 μL.

8. The detection method according to claim 5, characterized in that, In step (3), the reaction system consists of 25 μL of bacterial mixed lysis buffer, 3 μL of RFD-EC recognition molecule, and 100 μL of bu ff The reaction time was 5–30 min. Fluorescence detection was performed using blue, green, and red channels, with corresponding excitation and emission wavelengths as follows: blue channel Ex 415–480 nm, Em 495–520 nm; green channel Ex 530–550 nm, Em 560–610 nm; red channel Ex 615–645 nm, Em 655–720 nm.

9. The detection method according to claim 5, characterized in that, The fluorescence intensity of the fluorescent chip was detected by a fluorescence meter. The fluorescence intensity of the blank solution was close to the background signal, while the fluorescence intensity of the sample containing E. coli was linearly positively correlated with the bacterial content.

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