CN113481135A - Group induction system-based detection method of streptococcus mutans and application thereof - Google Patents
Group induction system-based detection method of streptococcus mutans and application thereof Download PDFInfo
- Publication number
- CN113481135A CN113481135A CN202110690715.4A CN202110690715A CN113481135A CN 113481135 A CN113481135 A CN 113481135A CN 202110690715 A CN202110690715 A CN 202110690715A CN 113481135 A CN113481135 A CN 113481135A
- Authority
- CN
- China
- Prior art keywords
- streptococcus mutans
- nlmc
- come
- sfgfp
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/245—Escherichia (G)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a detection method of streptococcus mutans based on a quorum sensing system and application thereof, belonging to the technical field of genetic engineering, wherein the detection method is established based on the quorum sensing system, elements of the quorum sensing system comprise a gene PelB-comD compounded by a signal peptide of escherichia coli and a histidine kinase receptor membrane protein gene, an intracellular response regulatory protein gene comE, and a gene nlmC-sfGFP of a promoter nlmC compounded green fluorescent signal reporter gene sfGFP regulated and activated by the comE; the detection method carries out specific recognition response on a signal molecule CSP secreted and expressed by the streptococcus mutans through the induction system, and further detects the streptococcus mutans. The detection method can specifically identify and detect the streptococcus mutans, has the characteristics of convenience and quickness in operation and low detection cost, and can be applied to caries detection so as to evaluate the health condition of teeth.
Description
Technical Field
The invention relates to the technical field of genetic engineering, and particularly belongs to a streptococcus mutans detection method based on a quorum sensing system and application thereof.
Background
Streptococcus mutans (Streptococcus mutans), a bacterium that grows in the oral cavity, has the characteristic of secreting acidic substances to corrode dental enamel, is one of the main cariogenic bacteria in the oral cavity, and is often found in dental plaque. Streptococcus mutans can corrode by acidic substances produced by metabolism, leading to the occurrence of dental caries, and has been widely studied in the field of oral medicine.
However, the current method for detecting streptococcus mutans is single, and requires expensive instruments and high-precision operation, for example, the current PCR technology mainly used for detecting streptococcus mutans is complex to operate, and the required equipment is complex and expensive, and the detection cost is high. Therefore, it is highly desirable to develop a method for detecting Streptococcus mutans under ordinary culture conditions, which does not require the use of too many complicated and expensive instruments, is relatively simple to operate, and is inexpensive to detect.
In addition, although the PCR technique has the characteristics of high detection sensitivity and high specificity, it also amplifies detection errors, and thus inaccurate results such as false positives are likely to occur, so that it is also necessary to supplement other detection techniques to ensure the accuracy of detection results.
The quorum sensing system is a relatively economic and simple detection method, but is not applied to the detection of streptococcus mutans at present, no suitable quorum sensing system element is found, and how to construct the quorum sensing system is still a mystery group, and the implementation is difficult. Thus, no effective method has been found to date which can be used to detect S.mutans in a complementary manner.
Disclosure of Invention
Aiming at the defects and shortcomings in the background technology, the invention provides the detection method of the streptococcus mutans based on the quorum sensing system, the method can specifically identify and detect the streptococcus mutans, and has the characteristics of convenience in operation and low detection cost.
The invention also aims to apply the detection method of the streptococcus mutans based on the quorum sensing system to the detection of the decayed teeth so as to evaluate the health condition of the teeth.
In order to realize the purpose, the invention adopts the following technical scheme to realize the purpose:
a method for detecting streptococcus mutans based on a quorum sensing system is established based on the quorum sensing system, wherein elements of the quorum sensing system comprise a gene PelB-comD of gene recombination of a signal peptide and a histidine kinase receptor membrane protein of escherichia coli, a gene comE of intracellular response regulatory protein, and a gene nlmC-sfGFP of a promoter nlmC combined with a green fluorescence signal reporter gene sfGFP, wherein the promoter nlmC-sfGFP is regulated and activated by the comE;
the detection method carries out specific recognition response on a signal molecule CSP secreted and expressed by the streptococcus mutans through the induction system, and further detects the streptococcus mutans.
Further measures taken are: the induction system recognizes concentration changes in response to the CSP through the genes comD, comE and nlmC.
Further measures taken are: the nucleotide sequences of the PelB-comD, comE and nlmC-sfGFP are respectively shown in SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5, respectively.
Further measures taken are: the amino acid sequence of the PelB-comD is shown as SEQ ID NO: 2, respectively.
Further measures taken are: the amino acid sequence of the comE is shown as SEQ ID NO: 4, respectively.
Further measures taken are: the method also comprises the construction of an expression vector, and the induction system is constructed on the expression vector.
Further measures taken are: the expression vector comprises a vector pET28a (+) -PelB-comD-comE-nlmC-sfGFP formed by PelB-comD, comE, nlmC and sfGFP.
Further measures taken are: the construction of genetic engineering bacteria is also included, and the induction system is connected to a pET28a (+) vector and is introduced into the genetic engineering bacteria; the genetic engineering bacteria enable a sensing system to respond and activate the expression of a downstream green fluorescent reporter gene by the recognition and combination of a signal molecule CSP which responds to the secretion and expression of the streptococcus mutans.
Further measures taken are: the induction system is activated when the CSP concentration reaches above 0.5mg/mL, and the nlmC promoter is started to up-regulate the expression of the up-regulated fluorescent gene sfGFP.
The detection method of the streptococcus mutans based on the quorum sensing system is applied to caries detection to evaluate the health condition of teeth.
The invention establishes the detection method of the streptococcus mutans based on the group induction system and provides the elements of the induction system, thereby facilitating the detection of the streptococcus mutans, and the detection is carried out through the response of the group induction system.
According to the invention, streptococcus mutans can be used for synthesizing a secretion signal molecule CSP, a system capable of identifying and responding the CSP signal molecule in the environment is constructed, namely, a set of detection induction system capable of identifying and responding the CSP concentration change can be formed through the genes comD, comE and nlmC, when the CSP concentration is lower, a receptor can not be activated, and green fluorescent protein at the downstream of a promoter is not expressed; when the CSP reaches a certain concentration threshold, the signal molecule CSP is combined with receptor membrane protein to activate the receptor, the activated receptor is contacted with intracellular response regulatory protein to activate the intracellular response regulatory protein, the activated intracellular response regulatory protein can be combined with the nlmC promoter to start the expression of the fluorescent reporter gene, thereby realizing the detection of the CSP concentration, judging the concentration and the quantity of the streptococcus mutans and realizing the detection of the dental caries.
Specifically, PelB-comD and comE genes of streptococcus mutans are constitutively expressed in a genetic engineering strain, wherein PelB-comD as a membrane receptor protein can be identified and combined with a signal molecule CSP secreted and expressed from streptococcus mutans, when the signal molecule CSP reaches a certain concentration, the signal molecule CSP can identify and combine a membrane receptor and activate the receptor, the activated receptor can be contacted with an intracellular response regulatory protein, so that the intracellular response regulatory protein is subjected to autophosphorylation activation, the intracellular response regulatory protein activated by two molecules can be combined with an nlmC promoter and starts the expression of a fluorescence reporter gene sfGFP at the downstream of the promoter, and the signal peptide gene PelB is favorable for assisting the comD to form a membrane protein.
Wherein the detection system cannot activate the detection system to up-regulate the expression of the fluorescent gene sfGFP in the absence or low concentration of CSP signal molecules; when the CSP concentration reaches above 0.5mg/mL, the upregulation of expression of the nlmC promoter can only be initiated, as shown by the results in FIG. 5.
The invention really establishes a detection method of the streptococcus mutans based on a quorum sensing system, and provides a gene element sequence, a construction method of each component, a method for transferring engineering bacteria, a working way and specific application of the detection method of the streptococcus mutans; the engineering bacteria containing the streptococcus mutans quorum sensing system are introduced to realize the specific identification response of the signal molecule CSP of the streptococcus mutans, and a CSP concentration gradient response experiment is carried out, so that the specific detection application of the streptococcus mutans is realized; and a set of detection system constructed in the engineering bacteria has the function of specifically identifying and detecting the streptococcus mutans, simplifies the detection steps, and ensures simple detection operation and low detection cost.
Through the technical scheme, compared with the prior art, the invention has the following beneficial effects:
1. the invention can detect the characteristic of CSP concentration by using a streptococcus mutans quorum sensing system to detect whether streptococcus mutans exists or not, so as to predict the concentration and the quantity of the streptococcus mutans, and the streptococcus mutans is a main pathogenic bacterium of dental caries, so that the constructed detection method can be applied to dental caries detection to realize the evaluation of the health condition of teeth.
2. The substance CSP detected by the induction system is a signal molecule specific to the streptococcus mutans, so that the streptococcus mutans can be specifically identified and detected, the detection accuracy can be effectively improved, false positive is avoided, the result accuracy is ensured, the transient amplification of detection errors is avoided, and the induction system can be used as an effective supplementary detection means for the streptococcus mutans.
3. The invention firstly constructs a detection method based on a streptococcus mutans quorum sensing system in the field, provides specific implementation technologies such as a gene element sequence and the like, and fills the blank of the industry.
Drawings
FIG. 1 is a schematic diagram of the construction process of the quorum sensing system expression plasmid of the present invention;
FIG. 2 shows the detection result of the target gene PelB-comD in the vector construction process of the present invention;
FIG. 3 shows the detection result of the target gene nlmC-sfGFP in the vector construction process of the present invention;
FIG. 4 shows the result of detection of the target gene comE in the process of constructing the vector of the present invention;
FIG. 5 is a graph showing the trend of real-time monitoring of the change in fluorescence intensity of different concentrations of CSP;
FIG. 6 shows the detection results of fluorescence control test of engineering bacteria.
Detailed Description
In order to clearly understand the technical solutions adopted by the present invention, the following description is made on the preferred embodiments of the present invention, and it should be understood that the embodiments described herein are only used for illustrating and explaining the present invention, and are not used to limit the present invention.
Example (b): a method for detecting streptococcus mutans based on a quorum sensing system is established based on the quorum sensing system, wherein elements of the quorum sensing system comprise a gene PelB-comD of gene recombination of a signal peptide and a histidine kinase receptor membrane protein of escherichia coli, a gene comE of intracellular response regulatory protein, and a gene nlmC-sfGFP of a promoter nlmC combined with a green fluorescence signal reporter gene sfGFP, wherein the promoter nlmC-sfGFP is regulated and activated by the comE;
the detection method carries out specific recognition response on a signal molecule CSP secreted and expressed by the streptococcus mutans through the induction system, and further detects the streptococcus mutans.
The induction system recognizes concentration changes in response to the CSP through the genes comD, comE and nlmC.
The nucleotide sequences of the PelB-comD, comE and nlmC-sfGFP are respectively shown in SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5, respectively.
The amino acid sequence of PelB-comD can also be shown as SEQ ID NO: 2, respectively.
The amino acid sequence of the comE can also be as set forth in SEQ ID NO: 4, respectively.
The method also comprises the construction of an expression vector, and the induction system is constructed on the expression vector. By constructing the induction system on an expression vector, the induction system is convenient to replicate in a microorganism and the expression plays a role.
The expression vector comprises a vector pET28a (+) -PelB-comD-comE-nlmC-sfGFP formed by PelB-comD, comE, nlmC and sfGFP.
The construction of genetic engineering bacteria is also included, and the induction system is connected to a pET28a (+) vector and is introduced into the genetic engineering bacteria; the genetic engineering bacteria enable a sensing system to respond and activate the expression of a downstream green fluorescent reporter gene by the recognition and combination of a signal molecule CSP which responds to the secretion and expression of the streptococcus mutans.
The induction system is activated when the CSP concentration reaches above 0.5mg/mL, and the nlmC promoter is started to up-regulate the expression of the up-regulated fluorescent gene sfGFP.
The detection method of the streptococcus mutans based on the quorum sensing system can be applied to the detection of the decayed tooth to evaluate the health condition of the tooth.
In order to verify the detection effect of the present example, the following test was performed.
1. Preparing engineering bacteria, expression vector, enzyme and other reagents
Wherein the engineering bacteria is escherichia coli, the escherichia coli (e.coli BL21) strain is purchased from limited biotechnology of hanfenghui in the south of lake, and the expression vector pET28a (+) is purchased from limited biotechnology of hanfenghui in the south of lake.
Enzymes and other reagents used during the assay: kanamycin sulfate, 1kb DNA Ladder and a plasmid small-amount extraction kit are purchased from Beijing Sorley Tech Co., Ltd;
the super-fidelity 2X pre-mixed liquid,
golden Gate Assembly kit (
v2) from NEB (beijing) ltd; LB medium powder was purchased from Biotechnology engineering (Shanghai) GmbH.
2. Main instrument equipment and source used in verification process
Table 1: main equipment and manufacturer
3. Preparation of culture Medium
Lb (luria broth) liquid medium: 10g/L tryptone, 5g/L yeast extract, 10g/L NaCl, pH 7.0; sterilizing with high pressure steam at 121 deg.C for 20 min.
LB solid medium: adding 2% agar powder on the basis of LB (Luria broth) liquid culture medium; sterilizing with high pressure steam at 121 deg.C for 20 min.
4. Cloning of genes of Streptococcus mutans PelB-comD, comE, nlmC-sfGFP
(1) PelB-comD, comE, nlmC-sfGFP genes were synthesized by Kinsrui Biotechnology GmbH.
(2) Amplification of PelB-comD, comE, nlmC-sfGFP Gene sequences
Design of a primer, shown as SEQ ID NO: 6. SEQ ID NO: 7 (amplified PelB-comD) and SEQ ID NO: 8. SEQ ID NO: 9 (amplified comE), SEQ ID NO: 10. SEQ ID NO: 11 (amplified nlmC-sfGFP).
b: the amplification system was as follows:
table 2: PCR amplification system
C: PCR amplification reaction:
table 3: PCR amplification reaction
(3) Purification and recovery of PCR amplification product
(4) Enzyme digestion ligation reaction of recovered fragment and vector
Table 4: enzyme digestion ligation reaction system
Table 5: enzyme digestion ligation reaction program
Wherein the nucleotide sequences of PelB-comD, comE and nlmC-sfGFP are respectively shown in SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5, respectively.
The amino acid sequences of PelB-comD and comE are respectively shown in SEQ ID NO: 2. SEQ ID NO: 4, respectively.
5. Construction of expression vector based on Streptococcus mutans quorum sensing system
The specific construction scheme of the quorum sensing system expression plasmid is shown in figure 1. The sequence of the joining of the target genes PelB-comD, comE and nlmC-sfGFP to the vector pET28a (+) is shown.
Linearized pET28a (+), PelB-comD, comE and nlmC-sfGFP sequences are obtained by PCR amplification, and the fragments are synchronously digested and connected to pET28a (+) vector by Golden Gate kit to construct expression vector pET28a (+) -PelB-comD-comE-nlmC-sfGFP of detection system based on streptococcus mutans quorum sensing system.
The vector construction process is shown in FIGS. 2-4. As can be seen from the results in the figure, the size of PelB-comD is 886bp, the size of comE is 468bp, and the size of nlmC-sfGFP is 1003bp, which is consistent with the expected size, thereby realizing the successful construction of the expression vector.
6. Engineering strain construction in streptococcus mutans detection method
The transgenic receptor selected in the example is Escherichia coli (Escherichia coli BL21) from limited biotechnology of hunan fenghui, and the operation process is as follows.
A single colony of the host bacteria of the Escherichia coli is picked from the plate, inoculated into a test tube of 3mL LB liquid culture medium and kept at 37 ℃ for 200r/min overnight (16-18 h). Inoculating 1mL of bacterial liquid into a conical flask containing 100mL of LB culture medium, shaking and culturing for 2-3 h at 37 ℃, measuring an OD600 value every 20min after culturing for 2h to detect the growth condition of the culture, immediately carrying out ice bath on the conical flask for 15min when the OD600 value reaches 0.5-0.6, and cooling the culture to 0 ℃. Bacteria were transferred aseptically to a sterile, ice-precooled 50mL centrifuge tube. The cells were recovered by centrifugation at 4000r/min for 10min at 4 ℃. The culture solution was poured out, and the centrifuge tube was placed on a filter paper for 1min to drain the last residual culture solution. The cells were resuspended in approximately 20mL (10 mL per tube) of ice-chilled 0.1mol/L CaCl2 solution (the resuspension procedure was light) and incubated in ice for 30 min. The cells were recovered by centrifugation at 4000r/min for 10min at 4 ℃. The supernatant was discarded and placed on filter paper for 1min to drain the last remaining liquid. Then, 4mL of 0.1mol/L CaCl2 solution precooled with ice was added to the suspension and the cells were resuspended by gentle shaking. And (3) subpackaging the competent cells into Eppendorf tubes, adding glycerol preservation solution with the volume of 30% into each 200 mu L, and placing the tubes in a refrigerator at the temperature of-80 ℃ for freezing and storing for later use.
Transformation of the expression vector plasmid into E.coli (Escherichia coli BL21)
200. mu.L of the prepared competent cells were taken in a sterile Eppendorf tube. Add 10. mu.L of plasmid to be transformed (about 30-50 ng plasmid) per tube, mix plasmid and competent cells gently with a pipette tip, and ice-wash for 30 min. The ice bath mixture was heat shocked in a 42 ℃ water bath for 2min without shaking the centrifuge tube. Taking out and cooling in ice bath for 2 min. 800. mu.L of Kana-free LB medium preheated to 37 ℃ was added to each tube, and gentle shaking was carried out at 37 ℃ and 150r/min for 45 min. And (3) coating 0.2mL of bacterial liquid on an LBK plate containing Kana, and performing inverted culture at 37 ℃ for 12-16 h. A negative control (plasmid was replaced by distilled water) was also performed. The expression vector plasmid containing the streptococcus mutans quorum sensing system is transformed into escherichia coli, a set of functional detection system with the function of specifically recognizing and detecting the streptococcus mutans is constructed in engineering bacteria of the escherichia coli, the specific recognition response of a signal molecule CSP of the streptococcus mutans is realized, a CSP concentration gradient response experiment is carried out, and the specific detection application of the streptococcus mutans is realized.
7. Function verification of the detection method of the embodiment
Real-time monitoring of fluorescence intensity changes
While culturing the strain, CSP with different concentrations is added, and whether the fluorescence intensity changes or not is monitored in real time, so that whether the detection system can work or not is judged.
100. mu.L of a bacterial suspension containing the adjusted bacterial cell OD600 of 0.8 was inoculated into a 96-well plate, and CSP was added to the sample wells so that the concentrations of CSP in the sample wells became 1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL and 0mg/mL, respectively, in the presence of excitation light: 515nm, emission light: the reaction was monitored at 570nm for 24 hours in real time, and the results are shown in FIG. 5. Wherein Blank is shown in the figure, and Control is a negative Control added with sterile water, namely CSP concentration is 0.
As is apparent from the results in the figure, when the concentration of CSP reached 0.5mg/ml, the comD-CSP complex bound the phosphorylation of comE, the phosphorylated comE and the nlmC promoter, thereby initiating the expression of the downstream fluorescent gene GFP. The fluorescence intensity gradually increased with the time of incubation, and after 24 hours, there was a significant difference in fluorescence intensity. The result shows that the streptococcus mutans detection method based on the quorum sensing system is successfully established, plays a detection function, can be used for detecting streptococcus mutans, and is activated when the CSP concentration reaches above 0.5mg/mL, and promotes the nlmC promoter to up-regulate the expression of the up-regulated fluorescent gene sfGFP.
In order to obviously express and excite fluorescence under an ultraviolet lamp, engineering bacteria fluorescence contrast test is also carried out: the engineering strain and the CSP of the embodiment are respectively added into four test tubes as test samples, the other two test tubes are empty bacteria, the same amount of CSP is added as a control sample, and then the six samples are subjected to fluorescence detection.
The detection results are shown in FIG. 6, wherein the first four are test samples of the engineering strain and the second two are empty bacteria control samples from left to right in the figure. The results in the figure clearly show that the engineering bacteria are obviously expressed under an ultraviolet lamp and excited to emit fluorescence; the engineering bacteria containing the streptococcus mutans quorum sensing system can realize specific identification response to the CSP, so that specific detection to the streptococcus mutans is realized.
By combining the embodiment, the detection result and the comparison test, the detection method can specifically identify and detect the streptococcus mutans by detecting the characteristic of the CSP concentration, so as to predict the concentration and the quantity of the streptococcus mutans, avoid generating false positive, ensure the accuracy of the result, have simple and convenient operation and low detection cost, are suitable for being applied to the detection of the decayed tooth, and realize the evaluation of the health condition of the tooth.
The above description is only for the purpose of illustrating the embodiments of the present invention and not for the purpose of limiting the same, and equivalent modifications and variations of the embodiments of the present invention will be apparent to those skilled in the art without departing from the overall spirit of the invention.
Sequence listing
<110> Shenzhen profession and technology college
<120> detection method of streptococcus mutans based on quorum sensing system and application thereof
<130> 2021.01.01
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 804
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 1
atgaagtacc tgctgccgac cgcggcggcg ggtctgctgc tgctggcggc gcagccggcg 60
atggcgatga acgcgatgag caacggcacc tataccgtta gcaaagtgag caacgttacc 120
agcaagaaaa ccagcagcaa catggcggtg accatggtta acgtgaacta cgcgtatgcg 180
agctacaacc gtaacagcag caactatggt gtggcgagca gcgaccgtcg tgcggatggt 240
accggcgtta tgggtagcag caccacctac atggcgggcg cgagctatag cgttaacgtg 300
gacggccgta aggatagcac caagatgaaa gttaagaaac gtatgaacac catgtactat 360
gtttacgtga gctataacgt gaccaagcgt aaagtggttg tgtacagcag ctataccaag 420
aaagttaaca tggcgaaggc gcgtaacacc tacagcagct ataaagaccg tagccgtcac 480
gattacaaca ccagccgtgg taacaaagac gcgagcaagt accacaaaac cggtcacgat 540
acccgttata acggccacgc gaacaacgac gcggtgaagg gcagcgcgaa agcgaacaag 600
aaagcggtta acgttgtgag cagcaagatg gacacgagct gcgataacgc ggcggcgagc 660
aacgcgaaga aaaacggtag cgtgaacagc accaagaaag atgttagcaa gaaaaactac 720
agcaccaaag gcagcaaccg tggtggcgcg aaggtgaacc accactataa aaccagcacc 780
agcaaccacc accacaagaa ataa 804
<210> 2
<211> 245
<212> PRT
<213> Artificial Synthesis (Artificial Sequence)
<400> 2
Met Asn Ala Met Ser Asn Gly Thr Tyr Thr Val Ser Lys Val Ser Asn
1 5 10 15
Val Thr Ser Lys Lys Thr Ser Ser Asn Met Ala Val Thr Met Val Asn
20 25 30
Val Asn Tyr Ala Tyr Ala Ser Tyr Asn Arg Asn Ser Ser Asn Tyr Gly
35 40 45
Val Ala Ser Ser Asp Arg Arg Ala Asp Gly Thr Gly Val Met Gly Ser
50 55 60
Ser Thr Thr Tyr Met Ala Gly Ala Ser Tyr Ser Val Asn Val Asp Gly
65 70 75 80
Arg Lys Asp Ser Thr Lys Met Lys Val Lys Lys Arg Met Asn Thr Met
85 90 95
Tyr Tyr Val Tyr Val Ser Tyr Asn Val Thr Lys Arg Lys Val Val Val
100 105 110
Tyr Ser Ser Tyr Thr Lys Lys Val Asn Met Ala Lys Ala Arg Asn Thr
115 120 125
Tyr Ser Ser Tyr Lys Asp Arg Ser Arg His Asp Tyr Asn Thr Ser Arg
130 135 140
Gly Asn Lys Asp Ala Ser Lys Tyr His Lys Thr Gly His Asp Thr Arg
145 150 155 160
Tyr Asn Gly His Ala Asn Asn Asp Ala Val Lys Gly Ser Ala Lys Ala
165 170 175
Asn Lys Lys Ala Val Asn Val Val Ser Ser Lys Met Asp Thr Ser Cys
180 185 190
Asp Asn Ala Ala Ala Ser Asn Ala Lys Lys Asn Gly Ser Val Asn Ser
195 200 205
Thr Lys Lys Asp Val Ser Lys Lys Asn Tyr Ser Thr Lys Gly Ser Asn
210 215 220
Arg Gly Gly Ala Lys Val Asn His His Tyr Lys Thr Ser Thr Ser Asn
225 230 235 240
His His His Lys Lys
245
<210> 3
<211> 435
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 3
atgagcgttg acgatggtcg taccaccgcg gcgatgaaga aaaactggag ctacaagacc 60
ggtaaagacg cgaagggcaa ccacgataag aaaaagaaag gcgttgcgaa ccgtcacaac 120
agcgcggtgg ttgtgaccac ccacagcatg acctatgtga gcgcggacga taaaagcaac 180
agccaccgta gcgcgtacta tgcgatgaac agcaagaacg gtagccacag cagcaccgtt 240
gcgtacacca gcagcaccgc gcacaaatgc tacacctatg accgttatgg cagcatgacc 300
gacgttaaaa tggataagcg ttgccaccgt agcgtgaacg cgaacacccg tgaccgtaag 360
aaacgtgcgt accgtaacaa caaaagctgt agccgtacca agaccaaacg tgcggtggcg 420
gatcgtcgtg cgaag 435
<210> 4
<211> 145
<212> PRT
<213> Artificial Synthesis (Artificial Sequence)
<400> 4
Met Ser Val Asp Asp Gly Arg Thr Thr Ala Ala Met Lys Lys Asn Trp
1 5 10 15
Ser Tyr Lys Thr Gly Lys Asp Ala Lys Gly Asn His Asp Lys Lys Lys
20 25 30
Lys Gly Val Ala Asn Arg His Asn Ser Ala Val Val Val Thr Thr His
35 40 45
Ser Met Thr Tyr Val Ser Ala Asp Asp Lys Ser Asn Ser His Arg Ser
50 55 60
Ala Tyr Tyr Ala Met Asn Ser Lys Asn Gly Ser His Ser Ser Thr Val
65 70 75 80
Ala Tyr Thr Ser Ser Thr Ala His Lys Cys Tyr Thr Tyr Asp Arg Tyr
85 90 95
Gly Ser Met Thr Asp Val Lys Met Asp Lys Arg Cys His Arg Ser Val
100 105 110
Asn Ala Asn Thr Arg Asp Arg Lys Lys Arg Ala Tyr Arg Asn Asn Lys
115 120 125
Ser Cys Ser Arg Thr Lys Thr Lys Arg Ala Val Ala Asp Arg Arg Ala
130 135 140
Lys
145
<210> 5
<211> 857
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 5
atcaaaaatg accgtttagg acaaaatagc taccatttag gatattttgc tccattttga 60
aaataaattg ttatactaga gatgtcggtt gcgcagccag acaaaaaact aaaaatgggg 120
aaggggtatt tatcatgaag gagatgcgta aaggcgagga actgttcacc ggtgtggttc 180
cgatcctggt ggaactggac ggcgatgtta acggtcacaa atttagcgtg cgtggtgagg 240
gcgaaggtga cgcgaccaac ggcaagctga ccctgaaatt catttgcacc accggtaaac 300
tgccggtgcc gtggccgacc ctggttacca ccctgaccta cggcgtgcag tgctttgcgc 360
gttatccgga ccacatgaag caacacgatt tctttaaaag cgcgatgccg gagggctacg 420
ttcaggaacg taccatcagc ttcaaggacg atggtaccta taaaacccgt gcggaagtga 480
agtttgaagg cgacaccctg gttaaccgta tcgagctgaa gggtattgac ttcaaagaag 540
atggcaacat cctgggtcac aaactggagt acaactttaa cagccacaac gtgtatatta 600
ccgcggataa gcagaaaaac ggcatcaagg cgaacttcaa aattcgtcac aacgtggaag 660
acggtagcgt tcaactggcg gatcactacc agcaaaacac cccgattggt gatggtccgg 720
tgctgctgcc ggataaccac tatctgagca cccaaagcgt tctgagcaag gacccgaacg 780
agaaacgtga tcacatggtg ctgctggaat ttgttaccgc ggcgggcatt acccacggta 840
tggatgagct gtacaag 857
<210> 6
<211> 35
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 6
ggctacggtc tcgactcact aaagtgaagg agatg 35
<210> 7
<211> 48
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 7
ggctacggtc tcctcatttt tgatgctagc gctagcgcaa aaaacccc 48
<210> 8
<211> 35
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 8
ggctacggtc tccatgaccg tttaggacaa aatag 35
<210> 9
<211> 31
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 9
ggctacggtc tcgcaaaaaa cccctcaaga c 31
<210> 10
<211> 53
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 10
ggctacggtc tcgtttgcgg atccggatcc taatacgact cactaaagtg aag 53
<210> 11
<211> 47
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 11
ggctacggtc tccttccttt cgggctttgg tcgaccttcg cacgacg 47
Claims (10)
1. A detection method of streptococcus mutans based on a quorum sensing system is characterized by comprising the following steps: the detection method is established based on a quorum sensing system, and elements of the sensing system comprise a gene PelB-comD compounded by a signal peptide of escherichia coli and a histidine kinase receptor membrane protein gene, an intracellular response regulatory protein gene comE, and a gene nlmC-sfGFP of a promoter nlmC compounded with a green fluorescent signal reporter gene sfGFP under the regulation and activation of the comE;
the detection method carries out specific recognition response on a signal molecule CSP secreted and expressed by the streptococcus mutans through the induction system, and further detects the streptococcus mutans.
2. The method according to claim 1, wherein the method comprises the following steps: the induction system recognizes concentration changes in response to the CSP through the genes comD, comE and nlmC.
3. The method according to claim 1, wherein the method comprises the following steps: the nucleotide sequences of the PelB-comD, comE and nlmC-sfGFP are respectively shown in SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5, respectively.
4. The method according to claim 1, wherein the method comprises the following steps: the amino acid sequence of the PelB-comD is shown as SEQ ID NO: 2, respectively.
5. The method according to claim 1, wherein the method comprises the following steps: the amino acid sequence of the comE is shown as SEQ ID NO: 4, respectively.
6. The method according to claim 1, wherein the method comprises the following steps: the method also comprises the construction of an expression vector, and the induction system is constructed on the expression vector.
7. The method according to claim 6, wherein the method comprises the following steps: the expression vector comprises a vector pET28a (+) -PelB-comD-comE-nlmC-sfGFP formed by PelB-comD, comE, nlmC and sfGFP.
8. The method according to claim 7, wherein the method comprises the following steps: the construction of genetic engineering bacteria is also included, and the induction system is connected to a pET28a (+) vector and is introduced into the genetic engineering bacteria; the genetic engineering bacteria enable a sensing system to respond and activate the expression of a downstream green fluorescent reporter gene by the recognition and combination of a signal molecule CSP which responds to the secretion and expression of the streptococcus mutans.
9. The method according to claim 1, wherein the method comprises the following steps: the induction system is activated when the CSP concentration reaches above 0.5mg/mL, and the nlmC promoter is started to up-regulate the expression of the up-regulated fluorescent gene sfGFP.
10. The use of a quorum sensing system-based streptococcus mutans detection method according to any one of claims 1-9 in caries detection for assessing dental health.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110690715.4A CN113481135B (en) | 2021-06-22 | 2021-06-22 | Method for detecting streptococcus mutans based on quorum sensing system and application of method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110690715.4A CN113481135B (en) | 2021-06-22 | 2021-06-22 | Method for detecting streptococcus mutans based on quorum sensing system and application of method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113481135A true CN113481135A (en) | 2021-10-08 |
| CN113481135B CN113481135B (en) | 2023-06-30 |
Family
ID=77935727
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110690715.4A Active CN113481135B (en) | 2021-06-22 | 2021-06-22 | Method for detecting streptococcus mutans based on quorum sensing system and application of method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113481135B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2332733A1 (en) * | 2000-04-10 | 2001-10-10 | Dennis G. Cvitkovitch | Signal peptides, nucleic acid molecules and methods for treatment of caries |
| JP2003038167A (en) * | 2001-07-25 | 2003-02-12 | Eiken Chem Co Ltd | Chromogenic medium for detecting cariogenic bacterium |
| JP2009085619A (en) * | 2007-09-27 | 2009-04-23 | Tdk Corp | Biosensor |
| TW202018088A (en) * | 2018-10-31 | 2020-05-16 | 國立中正大學 | Test piece, detection method and detection device of dental caries factors |
-
2021
- 2021-06-22 CN CN202110690715.4A patent/CN113481135B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2332733A1 (en) * | 2000-04-10 | 2001-10-10 | Dennis G. Cvitkovitch | Signal peptides, nucleic acid molecules and methods for treatment of caries |
| JP2003038167A (en) * | 2001-07-25 | 2003-02-12 | Eiken Chem Co Ltd | Chromogenic medium for detecting cariogenic bacterium |
| JP2009085619A (en) * | 2007-09-27 | 2009-04-23 | Tdk Corp | Biosensor |
| TW202018088A (en) * | 2018-10-31 | 2020-05-16 | 國立中正大學 | Test piece, detection method and detection device of dental caries factors |
Non-Patent Citations (3)
| Title |
|---|
| 季晓黎等: "变异链球菌二元信号传导系统的研究进展", 《牙体牙髓牙周病学杂志》 * |
| 徐蓉蓉等: "口腔链球菌密度感应信号系统comE基因及luxS基因的检测分析", 《华西口腔医学杂志》 * |
| 欧美珍等: "牙菌斑生物膜密度感应信号的研究进展", 《国际口腔医学杂志》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113481135B (en) | 2023-06-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2670847B1 (en) | Methods and materials for enhancing functional protein expression in bacteria | |
| CN113943373A (en) | Beta coronavirus polymer antigen, preparation method and application thereof | |
| CN111150833B (en) | Application of LTX-315 in preparation of product for inhibiting coronavirus | |
| CN108796040B (en) | Bioluminescence detection probe based on transcription activator-like effector and construction method and application thereof | |
| CN112941237A (en) | CRISPR nucleic acid detection kit for specifically detecting H7N9 avian influenza A virus | |
| CN107828769A (en) | A kind of heat-resisting lyases MMPpgh and the polynucleotides for encoding this enzyme | |
| CN111087453A (en) | Preparation method and application method of chlamydia pneumoniae recombinant antigen | |
| CN113481135A (en) | Group induction system-based detection method of streptococcus mutans and application thereof | |
| CN116445448B (en) | Acinetobacter baumannii PLPFP recombinant protein, preparation method and application | |
| CN110746496B (en) | PAL recombinant protein of Acinetobacter baumannii, encoding gene thereof and application of PAL recombinant protein and encoding gene | |
| CN111197098A (en) | Method for detecting mycobacterium tuberculosis from sputum | |
| TW201237170A (en) | Method of examining Riemerella anatipestifer and its kit | |
| CN115028743A (en) | Fluorescent sensor for detecting D-2-hydroxyglutaric acid and construction method and application thereof | |
| CN111808177B (en) | Signal peptide for improving protein expression quantity and application thereof | |
| Kim et al. | Homologous expression and T3SS-dependent secretion of TAP-tagged Xo2276 in Xanthomonas oryzae pv. oryzae induced by rice leaf extract and its direct in vitro recognition of putative target DNA sequence | |
| CN113583141A (en) | Swine epidemic diarrhea virus Nsp9 protein, fusion protein containing Nsp9 protein, and preparation method and application thereof | |
| CN101633917A (en) | Firefly luciferase and preparation method thereof | |
| CN114199848B (en) | High-throughput protein expression detection method based on protein ligase | |
| CN113088505B (en) | Application of polysaccharide lyase coding gene 04147 in preparation of recombinant peach gum polysaccharide hydrolase | |
| CN116462743B (en) | Acinetobacter baumannii translation elongation factor recombinant protein, preparation method and application | |
| CN114133439B (en) | Mutant xSUMO, mutant xE1 and xSUMO-xE1 combined mutant and related products and application thereof | |
| CN114958933B (en) | Method for preparing sulforaphane by using myrosinase Emyr | |
| CN114717362A (en) | Method for detecting respiratory syncytial virus by using Cas13a protein and application thereof | |
| CN107988188A (en) | A kind of D- alanyls-D-alanine carboxypeptidase and preparation method thereof | |
| CN113528412B (en) | Explosive visual biosensor based on escherichia coli cell surface display technology and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |