Coagulase negative staphylococcus aptamer molecular beacon fluorescent biosensor
The molecular beacon fluorescent biosensor constructed by SELEX screened with coagulase-negative Staphylococcus nucleic acid aptamer solves the time and accuracy of coagulase-negative Staphylococcus detection in peritoneal dialysis-related peritonitis, and achieves fast and low-cost high-sensitivity detection.
Patent Information
- Application Number
- CN202510561650.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-30
- Publication Date
- 2025-08-05
AI Technical Summary
The prior art consumes time and has a high culture negative rate in diagnosing peritonitis related to peritoneal dialysis, making it difficult to achieve early, fast and accurate coagulase-negative staphylococci detection, affecting antibiotic selection and patient prognosis.
The coagulase-negative Staphylococcus nucleic acid aptamer obtained by SELEX screening was constructed to construct a fluorescent biosensor of coagulase-negative Staphylococcus aptamer molecular beacon, and the binding changes of FAM fluorescent groups and ThT fluorescent dyes were used to achieve rapid and low-cost detection.
The rapid, low-cost and ultra-sensitive detection of coagulase-negative staphylococci is achieved, with the detection range of 3×100~3×104CFU/mL, and the detection limit is as low as 0.54CFU/mL, which has industrialization potential.
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Figure CN120424937A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biosensors, in particular to a coagulase-negative staphylococcus aptamer molecular beacon fluorescence biosensor. Background Art
[0002] Coagulase-negative Staphylococci are the most common pathogens causing peritoneal dialysis-associated peritonitis (PD-associated peritonitis). PD-associated peritonitis is a common complication in peritoneal dialysis (PD) patients, occurring in over 40% of patients and a major cause of PD technical failure and peritoneal membrane failure. Currently, clinical diagnosis of the causative bacteria is generally performed through bacterial culture, but this is time-consuming and has a high negative culture rate. Therefore, early, rapid, and accurate identification of the pathogen is crucial for guiding antibiotic selection, avoiding the side effects of broad-spectrum antibiotics, and improving the prognosis of PD-associated peritonitis patients.
[0003] In 1990, aptamers were first screened in vitro by Ellington and Szostak using the systematic evolution of ligands by exponential enrichment (SELEX) technique. Over the following three decades, more and more aptamers have been isolated, capable of binding to target molecules with affinity and specificity. In terms of detection, aptamer biosensors have developed rapidly in recent years. They are able to generate output signals in a target-responsive manner, are stable, and are easy to transport and store. Thioflavin T (ThT) is a small molecule fluorescent dye that can be embedded in specific nucleic acid conformations, such as G-quadruplexes, hairpin structures, or B-DNA duplexes, to produce high fluorescence quantum yields.
[0004] The present invention proposes a coagulase-negative staphylococcus nucleic acid aptamer obtained by SELEX screening, and successfully constructs a coagulase-negative staphylococcus aptamer molecular beacon fluorescent biosensor based on the aptamer, ultimately realizing rapid, low-cost, and ultrasensitive fluorescence detection of coagulase-negative staphylococci. Summary of the Invention
[0005] Based on this, the present invention proposes a coagulase-negative staphylococcus nucleic acid aptamer and successfully constructs a coagulase-negative staphylococcus aptamer molecular beacon fluorescent biosensor.
[0006] In one aspect, the present application provides a coagulase-negative staphylococcal nucleic acid aptamer.
[0007] The coagulase-negative staphylococcal nucleic acid aptamer sequence is: 5'-CTCTGGGTGTTCCCTCTGCTGCCCCGGTGT-3', as shown in SEQ ID NO: 1.
[0008] On the other hand, the present invention provides a coagulase-negative staphylococcus aptamer molecular beacon fluorescent biosensor, characterized by: (1) FAM-labeled sequence of the coagulase-negative staphylococcus nucleic acid aptamer biosensor; (2) coagulase-negative staphylococcus detection;
[0009] The sequence of the coagulase-negative staphylococcal aptamer molecular beacon fluorescent biosensor is: 5'-FAM-CTCTGGGTGTTCCCTCTGCTGCCCCGGTGT-3', as shown in SEQ ID NO: 1, where FAM is a fluorescent group modified at the 5' end;
[0010] The signal reporter molecules of the coagulase-negative staphylococcus aptamer molecular beacon fluorescent biosensor are FAM fluorescent group and ThT fluorescent dye;
[0011] The concentration of ThT fluorescent dye in the solution of the coagulase-negative staphylococcal aptamer molecular beacon fluorescent biosensor is 4 μM;
[0012] The buffer solution of the coagulase-negative staphylococcus molecular aptamer molecular beacon fluorescent biosensor is: 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.76 mM KH2PO4, pH 7.4;
[0013] The detection of coagulase-negative staphylococci is achieved by binding the aptamer to the coagulase-negative staphylococci, which produces a conformational change and, under the action of ThT, a change in the FAM fluorescence signal intensity. Under specific excitation conditions, the FAM fluorescence value exhibits a gradient change with the concentration of coagulase-negative staphylococci in the solution, thereby enabling the detection of coagulase-negative staphylococci.
[0014] The specific steps for establishing the standard curve were as follows: different concentrations of coagulase-negative staphylococci were added to PBS buffer solution containing 10 μM aptamer and 4 μM ThT solution. The concentrations of coagulase-negative staphylococci were 3×10 0 , 3×10 1 , 3×10 2 , 3×10 3 , 3×10 4CFU / mL; the fluorescence intensity of FAM before and after the addition of bacterial solution to the solution system was measured using a fluorescence spectrophotometer, with an excitation wavelength of 492 nm and an emission wavelength of 520 nm. A standard curve was drawn based on the difference in fluorescence intensity before and after and the concentration of coagulase-negative Staphylococcus. The results showed that the coagulase-negative Staphylococcus aptamer molecular beacon fluorescence sensor had a high sensitivity and specificity at 3×10 0 ~3×10 4 There was a good linear relationship between the concentration range of CFU / mL coagulase-negative staphylococci (R 2 =0.9992), the linear regression equation was Y=30.93X+20.18, and the detection limit was as low as 0.54 CFU / mL.
[0015] On the other hand, it relates to the application of the biosensor in the development of coagulase-negative staphylococcus detection methods or clinical detection.
[0016] Compared with the prior art, the present invention has the following beneficial effects:
[0017] 1. The present invention obtained a new coagulase-negative Staphylococcus nucleic acid aptamer through SELEX screening, which has good affinity and an affinity constant of 16.87nM;
[0018] 2. The nucleic acid aptamer of the present invention undergoes conformational changes after binding to coagulase-negative staphylococci, and has molecular beacon properties, which can achieve signal conversion and output;
[0019] 3. The coagulase-negative staphylococcal aptamer molecular beacon fluorescent biosensor proposed in this invention is 3×10 0 ~3×10 4 There was a good linear relationship between the concentration range of CFU / mL coagulase-negative staphylococci (R 2 =0.9992), the linear regression equation was Y=30.93X+20.18, and the detection limit was as low as 0.54 CFU / mL;
[0020] 4. The coagulase-negative staphylococcus aptamer molecular beacon fluorescent biosensor proposed in the present invention can achieve rapid, low-cost, stable and ultra-sensitive coagulase-negative staphylococcus detection, and has certain versatility and industrialization potential. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 The secondary structure of the coagulase-negative staphylococcal aptamer.
[0022] Figure 2 The figure shows the results of measuring the affinity constant of nucleic acid aptamer for coagulase-negative staphylococci by flow cytometry.
[0023] Figure 3Validation results of the target-induced allosteric ability of the coagulase-negative staphylococcal aptamer. A. is the full-wavelength scanning result, B. is the dynamic monitoring result.
[0024] Figure 4 Schematic diagram of the coagulase-negative staphylococcal aptamer molecular beacon fluorescent biosensor.
[0025] Figure 5 Optimization results for the coagulase-negative Staphylococcus aureus aptamer molecular beacon fluorescent biosensor. A, B. Optimization results for ThT concentration in the sensor; C, D. Optimization results for buffer type; E, F. Optimization results for aptamer concentration in the sensor.
[0026] Figure 6 Figure 2. Standard curve of the coagulase-negative Staphylococcus aptamer molecular beacon fluorescence biosensor. A. Fluorescence spectra at different target concentrations. B. Standard curve based on fluorescence signal. DETAILED DESCRIPTION
[0027] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.
[0028] Example 1. SELEX screening of coagulase-negative staphylococcal aptamers
[0029] The nucleotide sequences involved in the present invention are shown in Table 1.
[0030] Table 1. Coagulase-negative Staphylococcus aptamer sequences
[0031]
[0032] The present invention provides a method for screening coagulase-negative staphylococcal nucleic acid aptamers, comprising the following steps:
[0033] (1) Random library denaturation
[0034] The random library was placed in a 95°C metal bath for denaturation for 10 minutes, and then immediately placed in an ice bath for 10 minutes;
[0035] (2) Target incubation
[0036] Add target coagulase-negative Staphylococcus to the denatured library and incubate to form a single-stranded DNA-target complex;
[0037] (3) Sequence separation
[0038] First, remove the unadsorbed and weakly bound nucleic acid sequences by centrifugation and washing, and then heat at 95°C for 10 minutes to denature the bound sequences and separate them from the bacterial surface;
[0039] (4) PCR amplification
[0040] The single-stranded DNA bound to the target is used as a template for PCR amplification;
[0041] (5) Enzymatic digestion and purification
[0042] The amplified product of step (4) is digested with Lambda exonuclease to prepare single strands, and the secondary library is obtained by recovery and purification;
[0043] (6) Multiple rounds of screening
[0044] The screening library in step (1) is replaced by the secondary library obtained in step (5), and steps (1) to (5) are repeated for multiple rounds of screening;
[0045] (7) High-throughput sequencing
[0046] After the screening is completed, the latest secondary library is subjected to high-throughput sequencing analysis to obtain candidate coagulase-negative Staphylococcus aureus nucleic acid aptamers to be tested.
[0047] Example 2. Structure prediction and affinity determination of coagulase-negative staphylococcal aptamers
[0048] First, the structure of the coagulase-negative Staphylococcus aureus nucleic acid aptamer (shown in SEQ ID NO: 1) obtained by sequencing was predicted. The secondary structure of the aptamer was predicted using DNAfold WebServer. The results are as follows: Figure 1 As shown, it can be seen that the screened coagulase-negative staphylococcal nucleic acid aptamer contains a stem-loop structure.
[0049] Subsequently, the affinity of the coagulase-negative staphylococcal nucleic acid aptamer (shown in SEQ ID NO: 1) was verified using flow cytometry. The principle is that when coagulase-negative staphylococci are present in the system, the aptamer will specifically bind to the coagulase-negative staphylococci. After separating the unbound aptamer, the binding of the aptamer to the coagulase-negative staphylococci at different concentrations was measured using flow cytometry. The specific experimental steps are as follows:
[0050] The FAM-modified aptamer solution was gradiently diluted to 10 μM and 1 μM dilutions, denatured at 95°C for 5 minutes, and immediately placed in an ice box for 10 minutes. Different volumes of coagulase-negative staphylococcal solution and the same concentration volume were mixed in a brown EP tube to prepare suspensions with final aptamer concentrations of 10 nM, 50 nM, 100 nM, 200 nM, 400 nM, and 600 nM, respectively. After incubation at low temperature in the dark on a shaker at 200 rpm for 45 minutes, the fluorescence value of each sample was measured using a flow cytometer, and the curve was plotted using Graphpad Prism 10 and K was analyzed. d The values are fitted.
[0051] like Figure 2 As shown, the screened coagulase-negative Staphylococcus aureus aptamer (shown in SEQ ID NO: 1) has good affinity for the target, and the average fluorescence value increases with increasing aptamer concentration. Nonlinear fitting of the flow cytometry results revealed that the affinity constant of the coagulase-negative Staphylococcus aureus aptamer (shown in SEQ ID NO: 1) is 16.87 nM.
[0052] Example 3. Optimization of coagulase-negative staphylococcal aptamer molecular beacon fluorescence sensor
[0053] The ThT fluorescent dye was used to verify that the stem-loop structured aptamer undergoes conformational changes after binding to coagulase-negative staphylococci, and has molecular beacon characteristics, which can realize the output of fluorescent signals ( Figure 3 The specific detection principle is as follows: the ThT-nucleic acid aptamer complex formed by the FAM fluorescent group-labeled aptamer and ThT has high fluorescence intensity. When the target coagulase-negative staphylococci exist in the system, they bind to the FAM-labeled aptamer, resulting in a decrease in the fluorescence intensity of the system. The higher the concentration of coagulase-negative staphylococci, the lower the fluorescence intensity of the system. Figure 4 ).
[0054] The ThT concentration, buffer type and FAM-labeled aptamer concentration in the sensor were optimized. Figure 5 As shown, the fluorescence intensity change increases when the ThT concentration increases from 1 μM to 4 μM, but decreases above 4 μM. Therefore, 4 μM was selected as the ThT concentration. The fluorescence intensity change was greatest when PBS buffer (137 mM NaCl, 10 mM Na₂HPO₄, 2.7 mM KCl, 1.76 mM KH₂PO₄, pH 7.4) was used in the sensor. The fluorescence intensity change increased when the final concentration of the FAM-labeled aptamer increased from 0 μM to 2 μM, but the increase in the intensity became slower above 2 μM. Therefore, a PBS buffer with a ThT concentration of 4 μM and a FAM-labeled aptamer concentration of 2.5 μM was selected.
[0055] Example 4. Sensitivity Evaluation of Coagulase-Negative Staphylococcus Aptamer Molecular Beacon Fluorescent Sensor
[0056] The coagulase-negative Staphylococcus aureus nucleic acid aptamer modified with FAM fluorescent group (as shown in SEQ ID NO: 1) was used to detect coagulase-negative Staphylococcus aureus of known concentration, and a standard curve was prepared based on the change of FAM fluorescence value in the solution. Different concentration gradients of bacterial solution (3×10 0 ~3×10 4 CFU / mL) was added to a PBS buffer solution (137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.76 mM KH2PO4, pH 7.4) containing 2.5 μM aptamer and 4 μM ThT solution. The fluorescence intensity of FAM before and after the addition of the bacterial solution was measured using a fluorescence spectrophotometer (excitation wavelength: 492 nm, emission wavelength: 520 nm). A standard curve was plotted based on the difference in fluorescence intensity before and after addition and the concentration of coagulase-negative staphylococci.
[0057] like Figure 6 As shown, the coagulase-negative staphylococcal aptamer molecular beacon fluorescence sensor was detected at 3×10 0 ~3×10 4 The fluorescence signal of the coagulase-negative staphylococcus aptamer molecular beacon fluorescence sensor decreased with the increase of coagulase-negative staphylococcus concentration within the range of CFU / mL, showing a good detection trend. 0 ~3×10 4 There was a good linear relationship between the concentration range of CFU / mL coagulase-negative staphylococci (R 2 =0.9992), the linear regression equation was Y=30.93X+20.18, and the detection limit was as low as 0.54 CFU / mL.
Claims
1. A coagulase-negative staphylococcal nucleic acid aptamer, characterized in that: The nucleic acid aptamer sequence is: 5'-CTCTGGGTGTTCCCTCTGCTGCCCCGGTGT-3', as shown in SEQ ID NO:
1.
2. Use of the aptamer sequence according to claim 1 in the development of a method for detecting coagulase-negative Staphylococci.
3. Use of the aptamer sequence according to claim 1 in a coagulase-negative staphylococcus detection kit.
4. A coagulase-negative staphylococcal aptamer molecular beacon fluorescent biosensor, characterized in that: The fluorescent biosensor comprises: (1) a sequence of a FAM-labeled coagulase-negative staphylococcus nucleic acid aptamer biosensor; (2) detection of coagulase-negative staphylococci; The sequence of the coagulase-negative staphylococcal aptamer molecular beacon fluorescent biosensor is: 5'-FAM-CTCTGGGTGTTCCCTCTGCTGCCCCGGTGT-3', as shown in SEQ ID NO: 1, where FAM is a fluorescent group modified at the 5' end.
5. The coagulase-negative staphylococcal aptamer molecular beacon fluorescent biosensor according to claim 4, characterized in that: The signal reporting molecules of the coagulase-negative staphylococcus aptamer molecular beacon fluorescent biosensor are FAM fluorescent group and thioflavin T fluorescent dye.
6. The coagulase-negative staphylococcal aptamer molecular beacon fluorescent biosensor according to claim 4, characterized in that: The concentration of Thioflavin T fluorescent dye in the solution of the biosensor is 4 μM.
7. The coagulase-negative staphylococcal aptamer molecular beacon fluorescent biosensor according to claim 4, characterized in that: The buffer solution of the biosensor is: 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.76 mM KH2PO4, pH 7.
4.
8. The method for quantitatively detecting coagulase-negative staphylococci using the coagulase-negative staphylococcus aptamer molecular beacon fluorescent biosensor according to claim 4, characterized in that: Establishment of standard curve: Bacterial solutions with different concentration gradients were added to a buffer solution containing the aptamer and thioflavin T solution. The fluorescence intensity of FAM before and after the addition of the bacterial solution was measured using a fluorescence spectrophotometer with an excitation wavelength of 492 nm and an emission wavelength of 520 nm. A standard curve was drawn based on the difference in fluorescence intensity before and after addition and the concentration of coagulase-negative staphylococci.
9. Use of the label-free biosensor according to any one of claims 4 to 7 or the method according to claim 8 in the development of a coagulase-negative staphylococcus detection method or clinical detection.
10. Use of the label-free biosensor according to any one of claims 4 to 7 or the method according to claim 8 in the development of a clinical detection kit for coagulase-negative Staphylococcus.