CN116287351A - Probe combination and detection method of staphylococcus aureus - Google Patents
Probe combination and detection method of staphylococcus aureus Download PDFInfo
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- CN116287351A CN116287351A CN202310477173.1A CN202310477173A CN116287351A CN 116287351 A CN116287351 A CN 116287351A CN 202310477173 A CN202310477173 A CN 202310477173A CN 116287351 A CN116287351 A CN 116287351A
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Abstract
The invention relates to the technical field of staphylococcus aureus chromogenic detection, in particular to a probe combination and a staphylococcus aureus detection method. The probe combinations include allosteric probes, template sequences, and hairpin probes. Wherein, the allosteric probe integrates a staphylococcus aureus surface specific protein A aptamer and a connecting sequence which is partially complementary with the protein A aptamer. In the assembled state, the allosteric probe is in a hairpin structure. The aptamer sequence in the allosteric probe can realize specific target recognition, and the connection sequence mediates subsequent dual-signal amplification and color reaction. A series of experimental results prove that the probe combination can specifically identify staphylococcus aureus and has higher specificity and repeatability for detecting staphylococcus aureus. The detection limit of the method for detecting staphylococcus aureus by using the probe combination provided by the invention can reach 100 cfu/mL.
Description
Technical Field
The invention relates to the technical field of pathogen detection, in particular to a probe combination and application thereof, and a detection method of staphylococcus aureus.
Background
The detection of pathogenic bacteria mainly comprises counting and qualitative detection of pathogenic bacteria. Currently, colony culture and counting methods are gold standard in terms of pathogen counts and are widely used in clinic. However, the colony culture and counting method has the problems of low specificity, complex operation and the like caused by long operation period and incapability of qualitatively analyzing the colonies, and influences the efficiency of clinical detection and diagnosis work. In terms of qualitative analysis of pathogenic bacteria, immunoassay is still the most traditional qualitative technology of pathogenic bacteria, and is considered as a gold standard for clinical trials. However, immunoassays are often criticized for their low sensitivity, labor intensity, and high cost. An aptamer is widely used to construct aptamer sensors as a small oligonucleotide sequence that specifically recognizes and binds to biomolecules. The aptamer sensor has the advantages of low cost, simple transformation, rapid response and the like, and can be applied to various experimental and clinical scenes such as disease diagnosis, drug delivery and the like. In recent years, a variety of pathogen detection aptamer sensors have been developed, mainly using an aptamer to convert staphylococcus aureus signals into nucleic acid signals, and amplifying the signals in combination with isothermal nucleic acid signal amplification strategies. The sensors can be simply classified into fluorescence type, electrochemical type, colorimetric type, and other types according to the sensor. Among them, the fluorescent biosensor is attracting attention because of its advantages of stability, programmability, easy operation, and the like. For example, researchers have developed a new method based on the circulating signal amplification cascade technology of CRISPR-Cas12a, enabling sensitive and accurate detection of staphylococcus aureus. The method represents the general idea of pathogen detection based on the aptamer technology at present, namely, the pathogen is specifically identified through the aptamer, and the subsequent signal amplification technology is integrated to ensure higher sensitivity. Although fluorescence biosensors have made remarkable progress on the basis of the traditional staphylococcus aureus detection method, there are two disadvantages: i) False transcription of the oligonucleotide sequence during signal amplification is unavoidable and may interfere with the calculation of the result; ii) the need for fluorescent detection instrumentation to read results is limited in use in some less developed areas. Therefore, developing a high-fidelity signal amplification strategy and integrating the strategy, and accurately and sensitively detecting staphylococcus aureus in a chromogenic mode has important significance.
Disclosure of Invention
The invention aims to provide a probe combination which realizes macroscopic analysis of staphylococcus aureus by a chromogenic technology.
Another object of the present invention is to provide a method for detecting staphylococcus aureus using the aforementioned probe combination, which can accurately detect staphylococcus aureus in a sensitive manner.
Still another object of the present invention is to provide an application of the probe combination in preparing a kit for detecting staphylococcus aureus.
The invention solves the technical problems by adopting the following technical scheme.
The invention provides a probe combination, which comprises an allosteric probe, a template sequence c and a hairpin probe, wherein the allosteric probe and the hairpin probe are of hairpin structures in an assembled state; the allosteric probe is provided with a connecting sequence b and a nucleic acid aptamer sequence a for specifically recognizing staphylococcus aureus; the 3' end of the hairpin probe is a primer sequence d which is used for mediating the chain extension assisted by DNA polymerase; the 5' end of the hairpin probe consists of a sequence g and a sequence f, wherein the sequence g is partially complementary to the b sequence of the allosteric probe, and the sequence f is complementary to the restriction endonuclease cleavage site; the template sequence consists of two parts, wherein one part is a C-rich sequence for transcribing the G-tetrad, and the other part is partially complementary to the b sequence of the allosteric probe; the g sequence of the hairpin probe is partially complementary to the b sequence of the allosteric probe; the 3' -end sequence of the template probe is partially complementary with the b sequence of the allosteric probe; a sequence f' complementary to the sequence f is used to mediate hybridization of the allosteric probe with the hairpin probe; one end of the template sequence c may be complementary to a partial sequence c 'on the linker sequence b, the sequence c' being used to mediate hybridization of the template sequence c with the allosteric probe, the sequence f 'being located downstream of the sequence c'; the primer sequence d is used for mediating the extension of a sequence N capable of forming a G-tetramer by taking the template sequence c as a template, and the sequence N is connected with the primer d through a sequence of a recognition site of endonuclease.
The invention also provides a detection method of staphylococcus aureus, which comprises the following steps: mixing and culturing the assembled allosteric probe and the sample to be detected for 20-30 min, adding the template sequence and the assembled hairpin probe, mixing and culturing for 20-30 min, adding the DNA ligase for culturing for 20-30 min, adding the phi29 DNA polymerase and the endonuclease for culturing for 50-70 min, finally adding potassium ions and heme to obtain a product solution, and mixing the product solution with TMB-H 2 O 2 Mixing substrate solutions, wherein if the mixed solution is blue, the presence of staphylococcus aureus in a sample to be detected is indicated, otherwise, the presence of staphylococcus aureus is not indicated.
The invention also provides application of the probe combination in detection of staphylococcus aureus and preparation of a kit for detecting staphylococcus aureus.
The allosteric probe in the probe combination provided by the invention recognizes the surface protein A of staphylococcus aureus through an aptamer sequence, and causes the allosteric probe to become allosteric and releases a connecting sequence; the two ends of the connecting sequence are respectively complementary with the hairpin probe and the template sequence, the 5 'end of the hairpin probe and the 3' end of the template sequence are pulled up, and the hairpin probe and the template sequence are assembled into a whole under the action of ligase; synthesizing a G-rich strand complementary to the template sequence by using the 3' -end of the hairpin probe as a primer under the action of DNA polymerase, and releasing and recognizing the other hairpin probe and the template sequence complex to mediate signal circulation during the synthesis of the allosteric probe-staphylococcus aureus complex in the G-rich strand (namely, the strand extension process); the endonuclease recognition site at the 5' -end of the generated G-rich sequence can be recognized and cleaved by the endonuclease and a nick is generated. Under the combined action of endonucleases and polymerases, a large number of G-rich sequences are produced. The G-rich sequence is assembled to form a quadruplet under the action of potassium ions and is combined with heme to form a two-dimensional DNA structure with a catalytic effect to catalyze an ATBS-mediated color reaction. Meanwhile, experiments show that the specific target recognition and signal amplification based on the nucleic acid aptamer are combined, so that the detection performance is good, staphylococcus aureus can be specifically recognized, and the detection of staphylococcus aureus is high in specificity and repeatability. The detection limit of the method for detecting staphylococcus aureus by using the probe combination provided by the invention can reach 100 cfu/mL.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the operation of an allosteric probe and hairpin probe provided by the invention;
FIG. 2 shows fluorescence intensity of FAM-labeled allosteric probes before and after allosteric;
FIG. 3 shows the detection results of the method provided by the invention for detecting Staphylococcus aureus in the presence or absence of DNA polymerase, nb.BbvCI and heme;
FIG. 4 is a graph showing the F/F0 values obtained by the method of the invention for detecting Staphylococcus aureus at different concentrations;
FIG. 5 is a linear relationship between F/F0 and Staphylococcus aureus concentration.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The present invention will be described in detail with reference to specific examples.
The invention provides a probe combination, which comprises an allosteric probe, a template sequence c and a hairpin probe, wherein the allosteric probe and the hairpin probe are of hairpin structures in an assembled state; the allosteric probe is provided with a connecting sequence b and a nucleic acid aptamer sequence a for specifically recognizing staphylococcus aureus; the 3' end of the hairpin probe is a primer sequence d which is used for mediating the chain extension assisted by DNA polymerase; the 5' end of the hairpin probe consists of a sequence g and a sequence f, wherein the sequence g is partially complementary to the b sequence of the allosteric probe, and the sequence f is complementary to the restriction endonuclease cleavage site; the template sequence consists of two parts, wherein one part is a C-rich sequence for transcribing the G-tetrad, and the other part is partially complementary to the b sequence of the allosteric probe; the g sequence of the hairpin probe is partially complementary to the b sequence of the allosteric probe; the 3' -end sequence of the template probe is partially complementary with the b sequence of the allosteric probe; a sequence f' complementary to the sequence f is used to mediate hybridization of the allosteric probe with the hairpin probe; one end of the template sequence c may be complementary to a partial sequence c 'on the linker sequence b, the sequence c' being used to mediate hybridization of the template sequence c with the allosteric probe, the sequence f 'being located downstream of the sequence c'; the primer sequence d is used for mediating the extension of a sequence N capable of forming a G-tetramer by taking the template sequence c as a template, and the sequence N is connected with the primer d through a sequence of a recognition site of endonuclease.
Further, the nucleotide sequence of the nucleic acid aptamer in the probe combination is shown in positions 1 to 36 in SEQ ID No. 1.
Further, the nucleic acid nicking endonuclease in the above-described probe combination is nb.
Further, the sequence f in the above probe combination is shown in positions 31 to 36 of SEQ ID No. 2.
Further, the nucleotide sequence of the allosteric probe in the probe combination is a DNA molecule shown as SEQ ID No. 1.
Further, the nucleotide sequence of the hairpin probe in the probe combination is a DNA molecule shown as SEQ ID No. 2.
Further, the template sequence in the probe combination is a DNA molecule shown as SEQ ID No. 3.
The invention also provides a detection method of staphylococcus aureus, which comprises the following steps: mixing and culturing the assembled allosteric probe and the sample to be detected for 20-30 min, adding the template sequence and the assembled hairpin probe, mixing and culturing for 20-30 min, adding the DNA ligase for culturing for 20-30 min, adding the phi29 DNA polymerase and the endonuclease for culturing for 50-70 min, finally adding potassium ions and heme to obtain a product solution, and mixing the product solution with TMB-H 2 O 2 Mixing substrate solutions, wherein if the mixed solution is blue, the presence of staphylococcus aureus in a sample to be detected is indicated, otherwise, the presence of staphylococcus aureus is not indicated.
Further, the allosteric probe in the assembled state and the hairpin probe in the assembled state in the above detection method are obtained by the following operations: and respectively heating the synthesized allosteric probe and hairpin probe to 90 ℃ and incubating for 8-12 min, and then cooling to room temperature according to 0.5 ℃/min to form the allosteric probe in an assembled state and the hairpin probe in an assembled state.
The invention also provides application of the probe combination in preparation of a kit for detecting staphylococcus aureus.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
The allosteric probes and hairpin probes in the following examples were synthesized by the biological engineering company (Shanghai). The addition of the fluorescent and quenching groups of the allosteric probes in the following examples was also accomplished by the company Biotechnology Co.
dNTP mix solutions were also purchased from Biotechnology Inc. (Shanghai, china). KF and nb.bvci are from New England Biolabs (Ipswich, MA, USA). phi29 enzyme (DNA polymerase), heme, CEA, PCT, urease and pepsin were purchased from Takara Biotech (inc., da, china). All chemical reagents were analytical grade, and Rnase-free water was used throughout the process.
Example 1
The object of the present embodiment is to provide a probe assembly.
The probe combinations provided in this example include an allosteric probe and a hairpin probe, the structures of which are respectively as follows.
1. Allosteric probe
The allosteric probe has a hairpin structure, the structure of which is shown as the allosteric probe in figure 1, and the sequence of which is shown as SEQ ID No. 1. In SEQ ID No.1 (5 '-3'), the nucleotides 1 to 36 are the sequence a, and the sequence a is used for recognizing the surface-specific protein A of staphylococcus aureus, and the nucleotides 37 to 60 are the sequence b in SEQ ID No. 1. In SEQ ID No.1, nucleotides 3 to 17 are complementary to the bases of nucleotides 46 to 60, and nucleotides 18 to 45 are non-complementary sequences, which can be folded into a loop. The sequences shown for the allosteric probes were synthesized by the biological engineering company (Shanghai).
SEQ ID No.1：ATA TAC ACC CCA CCT CGC TCC CGT GAC ACT AAT GCT ATT TTT TTT GCG AGG TGG GGT GTA
2. Hairpin probe
The hairpin probe has a hairpin structure, the structure of which is shown as the hairpin probe in figure 1, and the sequence of which is shown as SEQ ID No. 2. In SEQ ID No.2 (5 '-3'), nucleotide numbers 43 to 51 are sequence d, nucleotide numbers 23 to 30 are sequence e, nucleotide numbers 12 to 19 are sequence f, and nucleotide numbers 1 to 9 are sequence g; the nucleotides d and e are base-complementary paired and the remaining nucleotides are non-complementary sequences. Wherein the sequence shown in g is partially complementary with 53 th to 60 th bases of SEQ ID No. 1. The sequences shown for hairpin probes were synthesized by the biological engineering company (Shanghai).
SEQ ID No.2：P-CCT CGC AAA CCA CCT CAG CAC CGA TAA AAA CGT GAC ACA GCT ATT TTT TAT
3. Template sequence
The template sequence is shown as SEQ ID No.3 (5 '-3'). Wherein, the 37 th to 45 th bases are complementary to the 52 th to 60 th bases of SEQ ID No. 1. The 1 st to 27 th bases are responsible for transcription of the G-rich sequence. The sequences shown in the template sequence were synthesized by the biological engineering company (Shanghai).
SEQ ID No.3：TAC CTC AGC ATC CCT ATC CCT ATC CCT ACC CCA CCA TAC ACC CCA
The working mechanism of the probe assembly provided in this embodiment is shown in fig. 1.
(1) In the assembled state, both the allosteric probe and the hairpin probe are hairpin structures, which prevents hybridization of the allosteric probe to the hairpin probe.
(2) When pathogenic bacteria are present in the sensing system, the aptamer a on the allosteric probe (i.e., the nucleotide sequences shown in SEQ ID No.1 at positions 1 to 36) will specifically bind to pathogenic bacteria (Staphylococcus aureus surface-specific protein A), causing the allosteric probe to become allosteric, thereby breaking down the hairpin structure into a linear state, forming an allosteric probe-pathogenic bacteria complex, and exposing the linker sequence b (i.e., the nucleotide sequences shown in SEQ ID No.1 at positions 37 to 60).
(3) One end of the exposed linker sequence b (bases 53 to 60 of SEQ ID No. 1) can be complementarily paired with sequences g and f on the hairpin probe (sequences shown as nucleotides 31 to 62 of SEQ ID No. 2), and the other end (bases 46 to 52 of SEQ ID No. 1) can be complementarily paired with the template sequence (bases 1 to 7 of SEQ ID No. 3), mediating hybridization of the hairpin probe with the allosteric probe-Staphylococcus aureus complex.
(4) Because the two ends of the connecting sequence b are respectively complementary and paired with one chain of the beacon stem region of the hairpin probe and the template sequence, the connecting sequence b is used as a template under the action of DNA ligase to synthesize a complete template chain M (connecting the template sequence c and the sequence g and comprising the sequence c and the sequence g) complementary with the connecting sequence b, so as to form the hairpin probe-allosteric probe-pathogenic bacteria complex.
(5) In the synthesis of the G-rich strand (i.e., the strand extension process), under the action of phi29 enzyme (DNA polymerase), the double helix structure of the template strand M and the connecting sequence b is opened, and the allosteric probe-staphylococcus aureus complex originally connected with the template strand M is released; meanwhile, the 3' end (sequence d) of the other strand of the beacon stem region of the hairpin probe is used as a primer to synthesize a sequence F complementary to the sequence F and a G-rich strand complementary to the template strand M (namely, a sequence shown as N in FIG. 2), so that the sequence N and the primer d are connected through a nucleic acid nicking endonuclease recognition sequence F; the released allosteric probe-staphylococcus aureus complex recognizes another set of hairpin probe and template sequence complexes, mediating signal circulation;
(6) The endonuclease recognition site (i.e., the sequence shown as F in FIG. 2) at the 5' end of the resulting G-rich sequence is capable of being cleaved by the endonuclease recognition and is nicked so that the G-rich sequence is released from the allosteric probe. Under the combined action of endonucleases and polymerases, a large number of G-rich sequences are produced.
(7) The G-rich sequences assemble to form G-tetrahedra, mediating color development: the G-rich sequence is assembled to form a quadruplet under the action of potassium ions and is combined with heme to form a two-dimensional DNA structure with a catalytic effect to catalyze an ATBS-mediated color reaction.
Example 2
The present example was directed to investigate the assembly effect of the allosteric probe provided in example 1.
To investigate the assembly effect of the allosteric probe, fluorescent dye FAM and corresponding quenching dye BHQ were labeled at both ends of the allosteric probe provided in example 1, and whether it was assembled into a stem-loop structure from linearity (the state of the allosteric probe was linear before unassembled after synthesis) was evaluated by detecting fluorescence change before and after assembly of the allosteric probe. The allosteric probe used in this example was the allosteric probe provided in example 1, and was labeled with a FAM group at the 5 'end and a BHQ group (labeled by the company of biotechnology, ltd) at the 3' end.
Preparing an allosteric probe solution: the aforementioned fluorogenic labeled allosteric probe solution of FAM group and BHQ group labeled by Biotechnology Co., ltd was prepared as 10. Mu.M fluorogenic labeled allosteric probe solution with DEPC water, which is abbreviated as allosteric probe solution in this example.
The systems to be detected in each group are prepared as follows:
(1) Linear group (referred to as linear in fig. 2): taking 10 mu L of 10 mu M of the allosteric probe solution, namely the solution to be detected containing the allosteric probe in a linear state.
(2) Hairpin structure group (referred to as hairpin structure in fig. 2): 10 mu M of an allosteric probe solution (10 mu M) was taken, heated to 90 ℃ and incubated for 10min, and then the solution was gradually cooled to room temperature to obtain a solution to be detected (10 mu M) containing an allosteric probe with a hairpin structure.
(3) Cp+sa group: preparation of an allosteric Probe solution (10. Mu.M) containing hairpin structures according to the hairpin Structure group, 10. Mu.L of the allosteric Probe solution containing hairpin structures was taken with a concentration of 10. Mu.L of 2. Mu.L 4 After mixed incubation of cfu/mL of Staphylococcus aureus Solution (SA) for 30 minutes, the solutions to be tested of the CP+SA group were obtained.
(4) Cp+cea group: preparation of an allosteric probe solution (10. Mu.M) containing hairpin structure according to hairpin structure group, mixing and culturing 10. Mu.L of allosteric probe solution containing hairpin structure with 2. Mu.L of carcinoembryonic antigen CEA solution (purchased from Takara Biotech) for 30min to obtain the solution to be detected of CP+CEA group.
(5) Cp+pct group: preparation of an allosteric probe solution containing hairpin structure according to hairpin structure group (10. Mu.M), mixing and culturing 10. Mu.L of allosteric probe solution containing hairpin structure with 2. Mu.L of procalcitonin PCT solution (purchased from Takara Biotech) for 30 minutes to obtain the solution to be detected of CP+PCT group.
The fluorescence signal intensity (a.u.) in the three groups of samples was detected by using a fluorescence spectrophotometer, and the result is shown in fig. 2, and the fluorescence intensity of the linear probe group is higher than that of the hairpin structure group, so that after the allosteric probe provided in example 1 is assembled into the hairpin structure by high-temperature annealing and renaturation, the 5 '-end-labeled FAM group is quenched due to the closer distance between the FAM group and the 3' -end-labeled BHQ group, resulting in lower fluorescence intensity of the hairpin structure group. Meanwhile, the fluorescence intensity of the CP+SA group is higher than that of the CP+CEA group and the CP+PCT group, which shows that the assembled allosteric probe can be specifically combined with staphylococcus aureus in a system, so that the assembled allosteric probe becomes linear, and stronger fluorescence intensity is detected, and the CP+CEA group and the CP+PCT group cannot be identified by the allosteric probe because CEA and PCT cannot cause the allosteric probe to become allosteric to excite a fluorescence signal. Therefore, the allosteric probe provided in the embodiment 1 is incubated at 90 ℃ for 10min and gradually cooled to room temperature, so that the card starting structure can be assembled well, and the allosteric probe has higher specificity to staphylococcus aureus.
Example 3
The purpose of this example is to verify that DNA polymerase, nicking endonuclease (Nb.BbvCI) and heme are necessary to detect Staphylococcus aureus using the method provided by the invention, i.e., to provide a method of detecting Staphylococcus aureus.
Preparing an allosteric probe solution: the allosteric probe provided in example 1 was prepared into a 2 μm solution of the allosteric probe with DEPC water, and the solution was incubated at 90 ℃ for 10min and gradually cooled to room temperature to obtain an assembled allosteric probe, which was abbreviated as an allosteric probe solution in the following.
Hairpin probe solution preparation: the hairpin probe provided in example 1 was prepared into a 2. Mu.M hairpin probe solution with DEPC water, and the assembled hairpin probe was obtained by incubating at 90℃for 10min and gradually cooling to room temperature, followed by abbreviated as an allosteric probe solution.
Template sequence solution: the template sequence provided in example 1 was formulated with DEPC water as a 2. Mu.M template sequence solution.
The systems to be detected in each group are prepared as follows:
(1) The DNA polymerase + endonuclease + Heme group (i.e. the groups are all present in fig. 3): after 10. Mu.L of the allosteric probe (assembled state) was mixed with 2. Mu.L of the target (Staphylococcus aureus solution) and incubated for 20 minutes, 10. Mu.L of the hairpin probe solution (assembled state) and the template sequence solution were added to the system and mixed and incubated for 20 minutes. Then 10. Mu.L of T4 DNA ligase was added, and the DNA ligase was inactivated by heating to 65℃after 30 minutes of incubation at room temperature. Then, 2. Mu.L of phi29 DNA polymerase and 2. Mu.L of Nb.BbvCI endonuclease were added to the above solution and incubated for 60 minutes, and then 2. Mu.L of potassium ion and 2. Mu.L of heme (heme) were added to the solution to obtain a product solution. 180. Mu.L TMB-H 2 O 2 Mixing the substrate solution with 20 mu L of the product solution, if oxidation-reduction reaction occurs, the mixed solution is yellow, otherwise, the mixed solution does not change color, and the final solution is obtained after reaction for 5 min. The blue solution was subjected to ultraviolet-visible (UV-vis) spectroscopic analysis using an ultraviolet spectrophotometer. The final solution of the experimental group (staphylococcus aureus present) was counted as F at a value of absorbance at 480 nm.
(2) The DNA free polymerase group (i.e., the polymerase (-) group in FIG. 3): the difference from the DNA polymerase+endonuclease+heme group is that 2. Mu.L of phi29 DNA polymerase was not added, and the rest of the procedure was the same as the DNA polymerase+endonuclease+heme group.
(3) No endonuclease group (i.e., endonuclease (-) group in FIG. 3): the difference from the DNA polymerase+endonuclease+heme group is that 2. Mu.L of Nb.BbvCI endonuclease was not added, and the rest of the operations were the same as the DNA polymerase+endonuclease+heme group.
(4) Heme-free group (i.e., heme (-) group in fig. 3): the difference from the DNA polymerase+endonuclease+heme group is that 2. Mu.L of Heme (Heme) was not added, and the rest of the procedure was the same as the DNA polymerase+endonuclease+heme group.
(5) Control group: the difference from the DNA polymerase+endonuclease+heme group is that 2. Mu.L of Staphylococcus aureus was not added, and the rest of the procedures were the same as the DNA polymerase+endonuclease+heme group. The absorbance value at 480nm of the final solution (yellow solution) obtained in the control group was F0.
As shown in FIG. 3, the absorbance ratio F/F0 of the DNA polymerase+endonuclease+heme group is higher than that of the DNA polymerase group, the endonuclease group and the Heme group, indicating that ABTS exists in the final solution of the DNA polymerase+endonuclease+heme group - The content of (C) is higher than that of the DNA-free polymerase group, the endonuclease-free group and the heme-free group. Thus, the detection method provided in this example requires participation of DNA Polymerase (Polymerase), endonuclease Nb.BbvCI and heme to detect a stronger fluorescent signal.
Example 4
The purpose of this example is to optimize the method provided in example 3.
The staphylococcus aureus source used in this example was the same as in example 2.
Nb.bvci endonuclease solutions of different concentrations: the Nb.BbvCI endoenzyme was formulated with RNase-free water in 5 concentration gradients of 2U/L, 3U/L, 4U/L, 5U/L, 6U/L, 7U/L.
Experimental group: after 10. Mu.L of the allosteric probe (assembled state, preparation method referred to example 2 or example 3) was mixed with 2. Mu.L of the target (Staphylococcus aureus solution) and incubated for 20 minutes, 10. Mu.L of the hairpin probe solution (assembled state, preparation method referred to example 2 or example 3) and the template sequence solution were added to the system and incubated for 20 minutes. Then 10. Mu.L of T4 DNA ligase was added thereto, and the DNA ligase was inactivated by heating to 65℃after 30 minutes of incubation at room temperature. Then, 2. Mu.L of phi29 DNA polymerase and 2. Mu.L of Nb.BbvCI endonuclease solution (five groups of 2U/L, 3U/L, 4U/L, 5U/L, 6U/L, 7U/L of Nb.BbvCI endonuclease solution were added to the above solution and incubated for 60 minutes, and then 2. Mu.L of potassium ions and 2. Mu.L of heme (heme) were added to the solution to obtain a product solution. 180. Mu.L TMB-H 2 O 2 The substrate solution was mixed with 20. Mu.L of the product solution (if redox reaction occurred, the mixed solution was yellow, otherwise, no discoloration occurred) and reacted for 5 minutes to obtain the final solution. The blue solution was subjected to ultraviolet-visible (UV-vis) spectroscopic analysis using an ultraviolet spectrophotometer. The final solution of the experimental group (staphylococcus aureus present) was counted as F at a value of absorbance at 480 nm.
Control group: the difference from the experimental group is that 2. Mu.L of staphylococcus aureus is not added, the rest of the operations are the same as the experimental group, and the final solution of the control group is not discolored. The final control solution (staphylococcus aureus was not present) was counted as F0 as absorbance at 480 nm.
The color change in the test panel was reflected in the ratio F/F0. And calculating the ratio of the F value of the Nb.BbvCI endonucleases with different concentrations to the F0 of the control group to obtain the F/F0 value of the Nb.BbvCI endonucleases with different concentrations. A linear graph was made using the F values of the Nb.BbvCI endonucleases at different concentrations and the concentrations of the Nb.BbvCI endonucleases at different concentrations, and the results are shown in FIG. 4, wherein the absorbance of the finally obtained blue solution at 480nm gradually increases with the increase of the concentration of the Nb.BbvCI endonucleases. Due to TMB-H 2 O 2 Oxidation of in-system ABTS 2- ABTS gradually oxidized to green - Yellow solution with ABTS - The increase in the content gradually changed to blue, so that the higher the absorbance at 480nm of the finally obtained blue solution, the more the ABTS therein was shown - The higher the content, i.e.the more G-rich sequences are produced.
Example 5
The purpose of this example was to verify the sensitivity of the method provided in example 3.
Preparation of staphylococcus aureus as 10 Using RNase-free Water -6 cfu/ml、10 -5 cfu/ml、10 -4 cfu/ml、10 -3 cfu/ml、10 -2 cfu/ml、10 -1 cfu/ml of staphylococcus aureus solution.
Experimental group: after 10. Mu.L of the allosteric probe solution (assembled state, preparation method referred to example 2 or example 3) was mixed with 2. Mu.L of Staphylococcus aureus at different concentrations for 20 minutes, 10. Mu.L of hairpin probe solution (assembled state, preparation method referred to example 2 or example 3) and template sequence solution were added to the system and mixed for 20 minutes. Then 10. Mu.L of T4 DNA ligase was added, and the DNA ligase was inactivated by heating to 65℃after 30 minutes of incubation at room temperature. Then, phi29 DNA polymerase (2. Mu.L) and Nb.BbvCI endonuclease (2. Mu.L) were added to the above solution and incubated for 60 minutes, and then (2. Mu.L) potassium ion and 2. Mu.L heme (heme) were added to the solution to obtain a product solution. 180. Mu.L TMB-H 2 O 2 The substrate solution was mixed with 20. Mu.L of the product solution and reacted for 5 minutes to obtain a final solution. The blue solution was subjected to ultraviolet-visible (UV-vis) spectroscopic analysis using an ultraviolet spectrophotometer. The final solution of the experimental group (staphylococcus aureus present) was counted as F at a value of absorbance at 480 nm.
Control group: the difference from the experimental group was that 2. Mu.L of Staphylococcus aureus was not added, and the rest of the operations were the same as the experimental group. The absorbance value at 480nm of the final solution (yellow solution) obtained in the control group was F0.
And calculating the ratio of the F value of the staphylococcus aureus with different concentrations to the F0 value of the control group to obtain the F/F0 value of the staphylococcus aureus with different concentrations. A linear graph was made using the F/F0 values of the staphylococcus aureus groups of different concentrations and the concentrations of the staphylococcus aureus groups of different concentrations, and the result is shown in fig. 5, where the correlation equation y=1.900×lgc+13.05 for the line shown in fig. 5, and the correlation coefficient is 0.9985. As can be seen from FIG. 5, the detection method provided by the present application has high sensitivity.
In summary, the probe combination and the detection method of staphylococcus aureus provided by the invention can effectively identify staphylococcus aureus in a system, the whole reaction does not need to use an external complex instrument, and whether staphylococcus aureus exists in the solution can be judged only by whether the final reaction solution turns blue, so that the reaction solution turns blue to indicate that staphylococcus aureus exists in the system. Meanwhile, the content of staphylococcus aureus can be quantified by means of an ultraviolet spectrophotometer, and the whole detection method is simple, convenient and high in sensitivity.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the invention is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Claims (10)
1. A probe combination comprising an allosteric probe, a template sequence c and a hairpin probe, the allosteric probe and hairpin probe being hairpin structures in an assembled state;
the allosteric probe is provided with a connecting sequence b and a nucleic acid aptamer sequence a for specifically recognizing staphylococcus aureus;
the 3' end of the hairpin probe is a primer sequence d which is used for mediating the chain extension assisted by DNA polymerase; the 5' end of the hairpin probe consists of a sequence g and a sequence f, wherein the sequence g is partially complementary to the b sequence of the allosteric probe, and the sequence f is complementary to the restriction endonuclease cleavage site;
the template sequence consists of two parts, wherein one part is a C-rich sequence for transcribing the G-tetrad, and the other part is partially complementary to the b sequence of the allosteric probe;
the g sequence of the hairpin probe is partially complementary to the b sequence of the allosteric probe; the 3' -end sequence of the template probe is partially complementary with the b sequence of the allosteric probe; a sequence f' complementary to the sequence f is used to mediate hybridization of the allosteric probe with the hairpin probe; one end of the template sequence c may be complementary to a partial sequence c 'on the linker sequence b, the sequence c' being used to mediate hybridization of the template sequence c with the allosteric probe, the sequence f 'being located downstream of the sequence c'; the primer sequence d is used for mediating the extension of a sequence N capable of forming a G-tetramer by taking the template sequence c as a template, and the sequence N is connected with the primer d through a sequence of a recognition site of endonuclease.
2. The probe combination of claim 1, wherein the nucleotide sequence of the nucleic acid aptamer is shown at positions 1 to 36 of SEQ ID No. 1.
3. The probe combination of claim 1, wherein the endonuclease is nb.
4. A probe combination according to claim 3 wherein the sequence f is shown at positions 12 to 19 of SEQ ID No. 2.
5. The probe combination of any one of claims 1 to 4, wherein the nucleotide sequence of the allosteric probe is a DNA molecule as shown in SEQ ID No. 1.
6. The probe combination of any one of claims 1 to 4, wherein the nucleotide sequence of the hairpin probe is a DNA molecule as shown in SEQ ID No. 2.
7. The probe combination of any one of claims 1 to 4, wherein the template sequence is a DNA molecule as shown in SEQ ID No. 3.
8. The method for detecting staphylococcus aureus is characterized by comprising the following steps: mixing and culturing the assembled allosteric probe and the sample to be detected for 20-30 min, adding the template sequence and the assembled hairpin probe, mixing and culturing for 20-30 min, adding the DNA ligase for culturing for 20-30 min, adding the phi29 DNA polymerase and the endonuclease for culturing for 50-70 min, finally adding potassium ions and heme to obtain a product solution, and mixing the product solution with TMB-H 2 O 2 Mixing the substrate solution, if the mixed solution is blue,and indicating that staphylococcus aureus exists in the sample to be detected, otherwise, not exists.
9. The method according to claim 8, wherein the assembled allosteric probe and the assembled hairpin probe are obtained by: and respectively heating the synthesized allosteric probe and hairpin probe to 90 ℃ and incubating for 8-12 min, and then cooling to room temperature according to 0.5 ℃/min to form the allosteric probe in an assembled state and the hairpin probe in an assembled state.
10. Use of a probe combination according to any one of claims 1 to 7 for the preparation of a kit for detecting staphylococcus aureus.
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| CN117706088B (en) * | 2024-02-05 | 2024-05-03 | 首都医科大学附属北京康复医院 | Method for detecting methicillin staphylococcus aureus |
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