CN104818277A - A group of oligonucleotide aptamers specifically recognizing staphylococcus aureus subsp.aureus - Google Patents
A group of oligonucleotide aptamers specifically recognizing staphylococcus aureus subsp.aureus Download PDFInfo
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- CN104818277A CN104818277A CN201510100548.8A CN201510100548A CN104818277A CN 104818277 A CN104818277 A CN 104818277A CN 201510100548 A CN201510100548 A CN 201510100548A CN 104818277 A CN104818277 A CN 104818277A
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Abstract
The present invention discloses a group of oligonucleotide aptamers (Sp15 and Sp25) specifically recognizing staphylococcus aureus subsp.aureus, and belongs to the field of food hygiene and clinical detection. According to the present invention, the competitive Cell-SELEX technology is adopted, the complete active staphylococcus aureus subsp.aureus cells are adopted as the target, 12 cycles of incubation, washing, dissociation, amplification and restriction enzyme digestion are performed so as to screen the oligonucleotide aptamers capable of specifically combining with the staphylococcus aureus subsp.aureus from the random single-stranded oligonucleotide library, and then sequencing and affinity specificity analysis are performed to obtain the aptamers with the best effect; and the screened aptamers have characteristics of high affinity, specificity, stable character, convenient in vitro synthesis, low cost, and the like, can be used for detection of the staphylococcus aureus subsp.aureus in food and clinical blood samples through the labeling function group or fluorescent dye, and provides the new selection for the current detection method relying on antibody.
Description
Technical field
The present invention relates to biological technical field of food safety, be related specifically to and utilize the SELEX technology in Protocols in Molecular Biology (the Fas lignand system evolution technology of index concentration) to screen a kind of nucleotide aptamer be combined with streptococcus aureus gold subspecies high specific and high-affinity, for the application of this nucleic acid aptamer in inspection streptococcus aureus gold subspecies provides scientific basis and theoretical basis.
Background technology
Staphylococcus (Staphylococcus) is the one in gram-positive cocci, is distributed widely in air, water, soil, feed and people, the body surface of animal, pharynx nasalis and enteron aisle, belongs to infecting both domestic animals and human germ.Wherein the virulence of streptococcus aureus (Staphylococcus aureus) is the strongest, often causes the suppurative inflammation of skin, tissue and organ, and the food poisoning caused by Staphylococcus aureus enterotoxin is a worldwide hygienic issues.In recent years, the food poisoning caused by streptococcus aureus gets more and more, report according to the Center for Disease Control, the food poisoning caused by streptococcus aureus occupies second, account for 33% of whole bacterial food poisoning, Canada is then up to 45%, China's this type of poisoning of annual generation is also very many, streptococcus aureus is as a kind of important food-borne pathogens, it also exists potential harm to food-processing industry and health of people, is therefore badly in need of setting up a kind of quick, sensitive, detection method accurately.
The detection of streptococcus aureus comprises Biological Detection, molecular biosciences detects, immunological detection method.Traditional microbial culture and biochemical identification consuming time, the low and complex operation of sensitivity; Molecular Biological Detection such as PCR method although fast, accurately, interfering factors is many, be subject to operational condition impact, poor repeatability is the ubiquitous problem of the method; Immunology detection has high specificity, accurate, sensitive feature, but this technology prepares the restriction of difficulty by specific antibody, preparation process complexity consuming time, and antibody itself is subject to the impact of the environmental factorss such as temperature, therefore develop quick, sensitive, detection of pathogens technology is very necessary accurately.
In recent years, comparatively noticeable to the research of oligonucleotide aptamer.Oligonucleotide aptamer is by SELEX (Systematic Evolution of Ligands by Exponential Enrichment, the Fas lignand system evolution technology of index concentration) technology screens the cluster small molecule DNA or RNA fragment that obtain from the random single chain oligonucleotide library of external synthesis, and can with the combination of specific structure and target high-affinity, high specific.As the promising alternative molecule of antibody molecule, oligonucleotide aptamer is compared antibody molecule and is shown great advantage: specificity is high, avidity is strong, external synthesis is convenient and cost is low, some functional groups of easy mark and reporter molecules, good stability, the reversible and speed of aptamer Denaturation and Renaturation is fast, can Reusability, preserve for a long time.SELEX technology is a kind of new combinatorial chemistry technique of early 1990s development, is a kind of effective ways studying nucleic acid construct and function, this technology come out at the beginning of the people such as Tuerk just adopt SELEX technology screening to go out the aptamer of phage T4DNA polysaccharase.Nowadays, aptamer has been widely used in target detection, enzyme level, the various field such as regulation and useful for drug delivery.Constantly perfect along with SELEX technology, the target of aptamer has expanded to small-molecule substance (organic dye, metal, medicine, carbohydrate, amino acid, Nucleotide and peptide etc.), protein (comprise enzyme, antibody, the gene regulating factor, and Sugar receptors), tumour cell, virus and pathogenic bacterium etc.Cell-SELEX screening take whole bacterial cells as the screening method of target material, in recent years, adopt Cell-SELEX technology successfully to filter out the aptamer of the bacteriums such as intestinal bacteria, Salmonellas, Listeria monocytogenes, Vibrio parahemolyticus, become the study hotspot in this field based on Cell-SELEX technology screening cause of disease pathogenic bacterium, the isocellular aptamer of tumour cell.
The present invention is can cause the streptococcus aureus gold subspecies of the various diseases such as skin soft-tissue infection, septicemia and catheter associated infection for target, competition Cell-SELEX technology screening is adopted to go out the high specific of streptococcus aureus gold subspecies, the aptamer of high-affinity, this aptamer stability is high, can externally synthesize, easily mark function group and reporter molecules, will be widely used in the rapid detection of food, clinical middle streptococcus aureus gold subspecies.
Summary of the invention
The object of the invention is to provide a kind of microbial molecular biological detection method, particularly a kind of method utilizing quick, the accurate detection streptococcus aureus of aptamer technology.
The phyletic evolution technology (SELEX technology) of the inventive method utilization index level enrichment part, with the complete active bacteria cell of streptococcus aureus gold subspecies for target, aptamer Sp15, Sp25 of screening acquisition 2 and target cell high-affinity, high specific combination, the aptamer of acquisition can be transferred to report aptamer by fluorophor marking method, for the detection of clinical blood sample, Staphylococcus aureus in food, reach object that is quick, Accurate Diagnosis.
Advantage of the present invention:
(1) compared with antibody, single stranded oligonucleotide is more stable, and aptamer can screen in vitro, and the screening cycle is short, and synthesis is convenient, and easily the various functional group of mark and reporter molecules, can preserve use for a long time.
(2) this group sequence be from structure significantly, to combine the avidity that has and select 8 adaptor sequence of different avidity and all stronger one group of adaptor sequence of specificity from streptococcus aureus gold subspecies, can specifically identifying staphylococcus aureus gold subspecies.
Accompanying drawing explanation
Fig. 1 is the secondary structure collection of illustrative plates of streptococcus aureus gold subspecies 8 aptamer family representative series.
Fig. 2 is the saturated binding curve of streptococcus aureus gold subspecies aptamer Sp11, Sp15, Sp25, Sp27 avidity.
Fig. 3 is the specificity figure of streptococcus aureus gold subspecies aptamer Sp11, Sp15, Sp25, Sp27.
Table 1 is streptococcus aureus gold subspecies aptamer dissociation constant Kd value.
Embodiment:
Here is the application by the high special affine streptococcus aureus gold subspecies nucleic acid aptamer of cell-selex technology screening and Rapid Detection streptococcus aureus gold subspecies aspect thereof.
1, external synthesis random single chain DNA (ssDNA) library and primer (Integrated Device Technology, Inc.'s synthesis)
Random single-stranded DNA banks: 5 '-AGCAGCACAGAGGTCAGATG-N40-CCTATGCGTGCTACCGTGAA-3 ', constructs the ssDNA pool that length is 80nt, and two ends are immobilized primer sequence, and centre is the stochastic sequence of 40 bases, and storage capacity reaches 10
14above; Upstream primer: 5 '-AGCAGCACAGAGGTCAGATG-3 '; 5 ' end phosphorylate downstream primer: 5 '-(phosphate)-TTCACGGTAGCACGCATAGG-3 ', all becomes 100 μm of ol/L stock solutions-20 DEG C storage for subsequent use with TE buffer with two kinds of primers ssDNA pool.
2, acquisition and the process of streptococcus aureus gold subspecies used is screened
LB liquid nutrient medium cultivates streptococcus aureus gold subspecies, and 37 DEG C of shaking tables are cultured to logarithmic phase (1.0 × 10
8cfu/mL).The streptococcus aureus gold subspecies bacterium liquid 1mL taken the logarithm vegetative period, at 4 DEG C, under 5000rpm, centrifugal 5min, abandons supernatant, with binding buffer liquid BB (50nM Tric-HCl, 5nM KCl, 100nM NaCl, 1nM MgCl
2, PH=7.4) and rinse twice, wash away medium component, it is for subsequent use to put 4 DEG C of ambient storage.
3, Cell-SELEX technology screening aptamer
During first round screening, reaction system is 600 μ L, and the ssDNA pool got after 2nmol amplification adds BB damping fluid in 95 DEG C of sex change 10min, immediately ice bath 10min, is added the mycetocyte (1 × 10 handled well
8individual) centrifuge tube in.For reducing in conjunction with background, add 10 times of 0.5%BSA solution to ssDNA pool mole number and tRNA in system again, in 37 DEG C of slow oscillation incubation 1h.Hatch rear replacing centrifuge tube, to remove the ssDNA be combined with centrifugal tube wall, then at 4 DEG C, the centrifugal 5min of 5000rpm, abandons supernatant, removes and not to combine or in conjunction with untight ssDNA.Clean 2 times with BB binding buffer liquid subsequently, be resuspended in 100 μ L 1 × PCR damping fluids, at 95 DEG C of sex change 10min, ice bath 10min immediately, then through 4 DEG C, the centrifugal 5min of 5000rpm, draw the ssDNA aptamer that supernatant is first round screening gained, and carry out pcr amplification as template.
50 μ L PCR system comprise ssDNA masterplate 1 μ L, upstream primer (20 μm of ol/L) 1 μ L, downstream primer (10 μm of ol/L) 1 μ L, dNTP (5mmol/L) 0.5 μ L, Taq archaeal dna polymerase (5U/ μ L) 0.5 μ L, 1 × PCR Buffer 5 μ L, ultrapure water 41 μ L.PCR thermal circulation parameters is 94 DEG C of denaturation 5min, 94 DEG C of sex change 45s, 58 DEG C of annealing 45s, and 72 DEG C extend 45s, carry out 20 and take turns circulation, and then 72 DEG C extend 1min, last 12 DEG C of cooling 2min.PCR primer 8% polyacrylamide gel electrophoresis checking expanding effect, verifies under being then placed on UV-light with Gelred dyeing that PCR primer dsDNA size is, after 80bp, adopt phenol-chloroform method to purify.Then with the standby strand time storehouse as next round SELEX screening of Lambda exonuclease digestion legal system, enzyme is cut at 37 DEG C of reaction 30min, go out at 75 DEG C enzyme 10min, after urea-denatured 8%PAGE electrophoresis checking enzymic digestion effect, enzyme being cut single stranded DNA adopts phenol-chloroform method to purify, then measure the concentration of ssDNA with Thermo NanoDrop2000 ultramicrospectrophotometer, calculate the volume dropped in next round library on this basis.
Second takes turns that to take turns reaction system to the 12 be 350 μ L, and wherein ssDNA pool is 100pmol.Screening often increases by one and takes turns, and the mole number increasing adding BSA and tRNA solution is twice, until increase to 8 times.For improving the specificity of screening aptamer, adopt Salmonella typhimurium, false Staphylococcus intermedius every three-wheel, cause let out intestinal bacteria, bacillus cereus, shigella dysenteriae, deactivation streptococcus aureus gold subspecies are counter sieves.
4, Cloning and sequencing
Take turns pcr amplification product that screening obtains ssDNA aptamer to serve Hai Shenggong biotechnology company limited carry out determined dna sequence by the 12, obtain 30 adaptor sequence.
5, adaptor sequence structural analysis
DNAMAN and RNA Structure 4.6 software is adopted to analyze homology information and the secondary structure (shown in Figure of description 1) of 30 sequences respectively.In conjunction with two kinds of software analysis results, sequence is divided into 8 families, 1 Stability Analysis of Structures is selected from each family, the sequence totally 8 that energy level is lower, is synthesized the aptamer of 5 ' end flag F AM, for avidity and specificity analyses by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
6, aptamer and streptococcus aureus gold subspecies avidity and specific assay
6.1 aptamer avidity are analyzed
8 aptamer TE damping fluids of synthesis are joined dilution and is mixed with 10 μm of ol/L solution, for subsequent use at being stored in-20 DEG C.BD FACSCalibur stream type cell analyzer is adopted to analyze the affinity of 8 aptamers.It is different concentration gradients (20,50,75,100,150,200,250,300nmol/L) that the 10 μm of ol/L aptamers getting different volumes add dilution in 500 μ L BB binding buffer liquid, in 95 DEG C of sex change 10min, and 0 DEG C of cooling 10min immediately.Aptamer solution is added handle well 1 × 10
8in individual streptococcus aureus gold subspecies cell, at 37 DEG C, change centrifuge tube, by new centrifuge tube in 4 DEG C after slow oscillation incubation 1h, 3000rpm, centrifugal 5min, abandons supernatant, with BB buffer solution for cleaning once, carry out flow cytometry after being resuspended in 500 μ L BB damping fluids.The fluorescence intensity of blank sample (not adding aptamer) is first regulated, then the forward scatter of working sample under identical parameters, lateral scattering and fluorescence intensity in flow cytometry.The fluorescence intensity per-cent of sample characterizes affinity size (survey and average for three times), utilize the dissociation constant Kd value of each aptamer of GraphPad Prism5 computed in software, as shown in table 1 below, and draw its saturated binding curve (shown in Figure of description 2).
Table 1
6.2 aptamer specificity analyses
Choose and carry out specificity analyses with streptococcus aureus gold subspecies cellular affinity 4 aptamers that more namely Kd value is less, sequence is respectively Sp11, Sp15, Sp25, Sp27 sequence, Kd value difference 49.88 ± 5.87,26.23 ± 1.96,28.36 ± 3.98,62.09 ± 9.76nmol/L.By 50pmol Sp11, Sp15, Sp25, Sp27 aptamer solution respectively with Salmonella typhimurium, false Staphylococcus intermedius, cause and let out intestinal bacteria, bacillus cereus, shigella dysenteriae, deactivation streptococcus aureus gold subspecies in 500 μ L 1 × BB damping fluids in 37 DEG C of slow oscillation incubation 1h, to change after centrifuge tube 4 DEG C, centrifugal 5min under 3000rpm, abandon supernatant, clean once with BB binding buffer liquid, be resuspended in 500 μ L BB damping fluids, after lucifuge mixing, carry out flow cytometry.The combination rate of result display Sp11 and streptococcus aureus gold subspecies only reaches more than 40%, combination rate is starkly lower than Sp15, Sp25, Sp27 tri-aptamers more than 70% combination rate, and repeatability is poor, and Sp27 lets out colibacillary combination rate and has exceeded 40% with causing, with Salmonella typhimurium, the combination rate of the target bacterium of bacillus cereus and deactivation is also relatively high, comparatively speaking, Sp15, Sp25 two aptamers are respectively at false Staphylococcus intermedius, cause let out intestinal bacteria and deactivation target bacterium hatch after combination rate greatly reduce, be about 20%, can high specific combination streptococcus aureus gold subspecies.The aptamer of the streptococcus aureus gold subspecies therefore gone out by SELEX technology screening has high-affinity and high specific, for streptococcus aureus gold subspecies fast, accurately, Sensitive Detection provides important foundation, be with a wide range of applications.
Claims (4)
1. the oligonucleotide aptamer of group-specific identification streptococcus aureus gold subspecies, its nucleotides sequence is classified as the nucleotide sequence shown in sequence table 1,2.
2. oligonucleotide aptamer as described in claim 1, is characterized in that its length is 80 bases longs, and nucleotide sequence has same function with the sequence formed through replacing, lacking or insert one or more Nucleotide.
3. oligonucleotide aptamer as described in claim 1, it is characterized in that its 5 ' end or 3 ' end can flag F AM, mercapto groups, HEX, FITC, vitamin H, FAM, digoxin etc., be further characterized in that the analyzing and testing being used alone or in combination and modifying or do not modify aptamers and all can be used for streptococcus aureus gold subspecies.
4. oligonucleotide aptamer detects the application in streptococcus aureus gold subspecies in food and clinical medicine as described in claim 1.
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| CN113624819A (en) * | 2021-08-09 | 2021-11-09 | 大连工业大学 | Electrochemical aptamer sensor based on exonuclease-assisted amplification |
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Non-Patent Citations (2)
| Title |
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| CAO,X.X. ET AL.: "Combining use of a panel of ssDNA aptamers in the detection of Staphylococcus aureus", 《NUCLEIC ACIDS RESEAR》 * |
| 汪江波等: "SELEX技术筛选耐甲氧西林金黄色葡萄球菌适配子的研究", 《中华医院感染学杂志》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113624819A (en) * | 2021-08-09 | 2021-11-09 | 大连工业大学 | Electrochemical aptamer sensor based on exonuclease-assisted amplification |
| CN113624819B (en) * | 2021-08-09 | 2023-09-19 | 大连工业大学 | Photoelectrochemical aptamer sensor based on exonuclease auxiliary amplification |
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Application publication date: 20150805 |