CN101665821B - Oligonucleotide aptamer group for specifically identifying staphylococcus aureus and use thereof - Google Patents
Oligonucleotide aptamer group for specifically identifying staphylococcus aureus and use thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biotechnology and relates to the sequences of five oligonucleotide aptamers for specifically identifying staphylococcus aurei and use thereof. The five singe-chain DNA oligonucleotide aptamers for specifically identifying the staphylococcus aureus are obtained by a bacterial subtraction SELEX technology. After FITC is marked, the five aptamers are proved by fluorescent staining to be capable of specifically identifying the strain of the staphylococcus aurei. After being used in combination, the five ligands can improve sensibility in identification of target bacteria and the ratios of detected different staphylococcus aureus strains and the detected staphylococcus aurei in different growth states. Therefore, the sequences of the five aptamers have wide application prospects in quick detection of the staphylococcus aurei in samples including suspected terrorist attack biological agents and identification of the staphylococcus aurei and other gram-positive cocci.
Description
Invention field:
The present invention relates to the acquisition and the application of these oligonucleotide aptamers in differentiating streptococcus aureus of the oligonucleotide aptamer of one group of specifically identifying staphylococcus aureus.
Background technology:
Bacterium is the common paathogenic factor of clinical disease, the serious human health that threatening.How quick, sensitive type, the character of differentiating important pathogenic microorganism is that the key subjects that a large amount of manpower and materials are studied are dropped in countries in the world.The method of traditional detection pathogenic bacteria needs first bacterial isolate mikrobe often, through microorganism culturing, identifies with classic methods more then.Consuming time, insensitive is the ubiquitous problems of these methods.Development is quick, the technology of Sensitive Detection pathogenic micro-organism is very necessary, and is particularly more apparent urgent aspect prevention bio-terrorism attack.Though utilize the antibody can the special recognition pathogen bacterium, can be rapidly, accurately sample to be checked is made evaluation, this technology receives specific antibody to prepare the restriction of difficulty.Because according to the outstanding criteria for classification of uncle, the essentially identical colony of biology shape constitutes a bacterial classification, and the close some bacterial classifications of proterties closeness relation are formed a Pseudomonas.In essence, the contained surface antigen overwhelming majority of same Pseudomonas is identical, has only hairline.Finding these difference and preparing specific antibody obviously is a consuming time and difficult task.
In the last few years, oligonucleotide aptamer substituted molecule as the prospect property of antibody molecule, and its research is comparatively noticeable.Oligonucleotide aptamer is obtained by SELEX technology (Systematic evolution of ligands byexponential enrichment) biological library technology screening; This technological principle is utilized Protocols in Molecular Biology exactly; Make up the strand random oligonucleotide library of synthetic, its stochastic sequence length is about 20-100 base.Utilize the flexible and changeable characteristic of single stranded oligonucleotide molecular configurations; Random oligonucleotide library and target molecule are interacted; Reservation is with space structure picture and target molecule bonded oligonucleotide, through increasing, screen several circulations repeatedly, can make the oligonucleotide sequence with this target specific combination obtain enrichment; The final special oligonucleotide aptamer that obtains multiple target molecule, i.e. aptamer.Utilize the pattern and the protein antibodies of the aptamer identification molecule that the SELEX technology screening obtains similar; But compare with protein antibody; Nucleic acid class aglucon has more meliority, limits as not receiving immune condition and immunogenicity, can external synthetic; Sex change and renaturation are reversible, can modify and help prolonged preservation and room temperature transportation etc.The more important thing is that aptamer has higher specificity than antibody, even can discern the undistinguishable protein molecule of monoclonal antibody.And the target molecule of aptamer is very extensive, and is little of dye molecule, big to complete virion and bacterial pathogens, even complete cell also can be through subduing the oligonucleotide aptamer that the SELEX technology screening goes out high-affinity.
With the SELEX compared with techniques of classics, an outstanding feature of subduing the SELEX technology is the characteristic that need not clear and definite target composition before the screening, like differentiation sign (Wang C, et al.J Biotechnol; 2003:102:15-22), therefore,, also might obtain the oligonucleotide aptamer of this bacterium specific antigen composition even if under bacterium specific antigen condition of unknown.Truly have some researchists to utilize the SELEX technology screening to arrive and bacterium specificity bonded aptamer in recent years, explain that specific aptamer has the potential using value aspect the treatment of the detection of mikrobe and pathogenic microorganism.But because same bacterium is easy to morph under different state; The omission (false negative) that utilizes the unique identification thing to identify in this case usually can to cause to this bacterium; And utilize one group of aptamer to detect to different target molecules on the same bacterium, can improve susceptibility and the specificity that target bacteria detects greatly in theory.
Based on above consideration; The present invention is the purpose target bacteria with common clinically streptococcus aureus; Simultaneously with suis and epidermis staphylococcus for subduing target bacteria; Utilize bacterium to subdue the SELEX technology and obtained five aptamer that streptococcus aureus is special, Combination application can be rapidly, responsive, special detect streptococcus aureus.Because the adaptive sub-stable performance of single strand dna oligonucleotide, synthetic convenient and cheap, after modifying, can directly be used for fluorescence or chemoluminescence, chromophoric method detects target bacteria, and is so simple to operate, direct.This invention can be used widely in clinical medicine, military medicine and emergent protection.
Summary of the invention:
The object of the invention is to propose the sequence and the application thereof of five oligonucleotide aptamers of specifically identifying staphylococcus aureus; Comprise the streptococcus aureus that detects fast in the sample, differentiate the application of streptococcus aureus and other gram-positive cocci aspect.。
Advantage of the present invention:
1) compare with the antibody of protein, the oligonucleotide of strand is more stable; Aptamer can directly externally synthesize, mark, does not therefore need two of mark to resist, and makes operation more simple, rapid; The synthetic cost of aptamer is low than the Antibody Preparation cost, and the cycle is short.
2) these five sequences all can be distinguished the different targets in specifically identifying staphylococcus aureus surface.
3) compare with unique sequence, these five sequence associations are used, and can improve susceptibility and specificity to streptococcus aureus, improve the recall rate to target bacteria.
Description of drawings:
The copolymerization close-burning of five oligonucleotide aptamer specific recognition of Fig. 1 streptococcus aureus is really schemed.
The copolymerization close-burning that five oligonucleotide aptamer Combination application of Fig. 2 are identified streptococcus aureus is really schemed.
The streaming result that five oligonucleotide aptamer Combination application of Fig. 3 are identified streptococcus aureus.
Five oligonucleotide aptamer Combination application of Fig. 4 are to the streaming result of different staphylococcus aureus strains recall rates.
Five oligonucleotide aptamer Combination application of Fig. 5 are to the streaming result of the streptococcus aureus recall rate under the different growth conditions.
Embodiment:
Obtain the special oligonucleotide aptamer of streptococcus aureus and five special oligonucleotide aptamers are wherein relatively further described the present invention to streptococcus aureus identification and recall rate through subduing SELEX screening below.
1. synthetic random single-stranded DNA banks and primer ((completion of Invitrogen company).
Library GP45:5 '-GCAATGGTACGGTACTTCC (45N) CAAAAGTGCACGCTACTTTGCTAA-3 '
5 ' primer (Plong-1): 5 '-GCAATGGTACGGTACTTCC-3 '
3 ' primer (P11): 5 '-TTAGCAAAGTAGCGTGCACTTTTG-3 '
3 ' primer (Pstem-loop3): 5 '-GCTAAGCGGGTGGGACTTCCTAGTCCCACCCGCTTAGCAAAGTAG CGTGCACTTTTG-3 '
2. the bacterium target of screening usefulness obtaining and handling.
TSB culture medium culturing streptococcus aureus, suis, staphylococcus epidermidis, 37 ℃ of equal shaking tables are cultured to the early stage (OD of logarithmic growth
600Be about 0.2).Collect bacterium then respectively, get 100 μ l bacterium liquid, dilution 10
5The coated plate counting.The centrifugal supernatant that goes of all the other bacterium liquid utilizes 1 * PBS to wash, the medium component that the place to go is unnecessary.90% methyl alcohol-20 is 10min ℃ fixedly, and then fixes with 100% methyl alcohol, places-20 ℃ of preservations, and is subsequent use.
3. subdue the SELEX screening and obtain the special oligonucleotide aptamer of streptococcus aureus
1) subdue subdue (the negative screening) of bacterium to the library:
A. get the ready bacterium 1 * 10 of subduing
6, place 1ml PCR pipe;
B. with the ssDNA library in 100 ℃ of sex change 5min; Place immediately on ice fully cooling (the 1st take turns screening input amount be 1500pmol; Later on every input amount of taking turns screening is that 100pmol, 50pmol successively decrease, for the acquisition of aglucon, the 1st, 2 take turns do not subdue screening);
C. with the ssDNA library with subdue bacterium mixing (later every take turns the volume of hatching altogether that screening uses be 100 μ l) in 200 μ l confining liquid damping fluids, hatch 1hr altogether in 37 ℃, ssDNA library and cell are fully acted on;
D. separate not with the centrifugal 5min of 10000r/m under the room temperature and subdue bacterium bonded ssDNA library (subtractive library) 2) screening (positive-selecting) of streptococcus aureus 8325-4 specific combination ssDNA aglucon:
A. get 1 * 10
7The streptococcus aureus 8325-4 that gets ready places 1.5ml PCR pipe;
B. with above-mentioned subtractive library and streptococcus aureus 8325-4 in the confining liquid damping fluid mixing (1 μ g/ μ l yeast tRNA 1%BSA), is hatched 1hr in 37 ℃ altogether, and ssDNA library and cell are fully acted on;
C.0.05%Tween-20, PBS damping fluid washing 3 times, 1ml/ time.
3) oligonucleotide aptamer of PCR enrichment and streptococcus aureus 8325-4 specific combination:
Every take turns behind positive-selecting, obtain obtain enrichment with streptococcus aureus bonded oligonucleotide aptamer library through pcr amplification.Be about to contain the bacterial precipitation that combines aglucon and be dissolved in an amount of tri-distilled water, move into new PCR pipe, conventional pcr amplification.
A. produce the single stranded product of two different lengthss through 5 ' primer (Plong-1) and 3 ' primer (Pstem-loop3) amplification; Two products of urea PAGE electrophoretic separation; Cut glue and reclaim the single stranded product (low strap, normal chain) that 5 ' primer (Plong-1) extends, be used for the next round screening.
B. increase through 5 ' primer (Plong-1) and 3 ' primer (P11) and produce double-stranded DNA, reclaim the back and connect the T carrier, picking cloning and sequencing, the sequence information of the oligonucleotide aptamer of acquisition enrichment.
The c.PCR amplification condition:
Strand: 94 ℃ of 5min, 94 ℃ of 1min, 37 ℃ of 1min20sec, 58 ℃ of 40sec, through suitable cycle number, 58 ℃ of 2min.
Double-stranded: 94 ℃ of 5min, 94 ℃ of 1min, 37 ℃ of 1min20sec, 72 ℃ of 40sec, through suitable cycle number, 72 ℃ of 10min.
4) repeat screening: repeat above-mentioned negative screening, positive-selecting and pcr amplification enrichment process, carry out 6 altogether and take turns screening.
5) the enrichment oligonucleotide is known the sequencing result and the analysis in gamete library
A. last enriched library amplification is two strands, connects the pUC19-T carrier, Transformed E .coliDH5 α, 55 positive colonies of picking carry out determined dna sequence (called after SA1-SA55 respectively) at random from 500 clones.
B. adopt MEME online tool and RNA STRUCTURE analysis software, obtain the higher structure of 55 sequence homology information and each bar sequence through software analysis.
C. combine the analysis and the aglucon higher structure of MEME software, on average select 1-2 bar Stability Analysis of Structures in each family, the sequence that energy level is lower is representative, and totally 11 sequences are carried out next step evaluation.
4. the flow cytometer screening of the special oligonucleotide aptamer of one group of streptococcus aureus focuses on together and identifies
1) send Invitrogen company synthetic and the contrast of above-mentioned 11 sequences and former storehouse at 5 ' end flag F ITC.
FITC-SA1：
5’-GCAATGGTACGGTACTTCCGCTTTGCCGGTTATGGGCTATCGCACGTCAGGTAGTTCGAACCGACAAAAGTGCACGCTACTTTGCTAA-3’
FITC-SA3：
5’-GCAATGGTACGGTACTTCCCCTTACCCGCTTGTATCTTTACCTGCTACCTAGCGTGTAGTGGCACAAAAGTGCACGCTACTTTGCTAA-3’
FITC-SA20：
5’-GCAATGGTACGGTACTTCCGCGCCCTCTCACGTGGCACTCAGAGTGCCGGAAGTTCTGCGTTATCAAAAGTGCACGCTACTTTGCTAA-3’
FITC-SA23：
5’-GCAATGGTACGGTACTTCCGGGCTGGCCAGATCAGACCCCGGATGATCATCCTTGTGAGAACCACAAAAGTGCACGCTACTTTGCTAA-3’
FITC-SA31：
5’-GCAATGGTACGGTACTTCCTCCCACGATCTCATTAGTCTGTGGATAAGCGTGGGACGTCTATGACAAAAGTGCACGCTACTTTGCTAA-3’
FITC-SA32：
5’-GCAATGGTACGGTACTTCCTGTAAGCGAACCTGCTGTTGCCTGTCTGTATTCATGCCCCCATTTCAAAAGTGCACGCTACTTTGCTAA-3’
FITC-SA33：
5’-GCAATGGTACGGTACTTCCGTAGAACCTAGGCGTCGTCTATACATACCACTACGATGAAACACGCAAAAGTGCACGCTACTTTGCTAA-3’
FITC-SA34：
5’-GCAATGGTACGGTACTTCCCACAGTCACTCAGACGGCCGCTATTGTTGCCAGATTGCCTTTGGCCAAAAGTGCACGCTACTTTGCTAA-3’
FITC-SA43：
5’-GCAATGGTACGGTACTTCCTCGGCACGTTCTCAGTAGCGCTCGCTGGTCATCCCACAGCTACGTCAAAAGTGCACGCTACTTTGCTAA-3’
FITC-SA46：
5’-GCAATGGTACGGTACTTCCGTCACTTGGCTGATGGTAGATAGGCGGTGGTTGCGAGGCTTGGTCAAAAGTGCACGCTACTTTGCTAA-3’
FITC-SA50：
5’-GCAATGGTACGGTACTTCCGTGCTTAGACCACCCTAGTGATACGCCGAACTGTGCAGTATTAAGCAAAAGTGCACGCTACTTTGCTAA-3’
FITC-GP45：
5’-GCAATGGTACGGTACTTCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCAAAAGTGCACGCTACTTTGCTAA-3’
Annotate: N represent A, T, G, C any one.
2) utilize flow cytometer further to filter out the aptamer of staphylococcus aureus specific.
A. (the 240nM/ bar is PBS) respectively with 1 * 10 oligonucleotide aptamer and library
6Subdue bacterium and target bacterium and hatch 45min, washing, the centrifugal supernatant of abandoning at 37 ℃;
B. cell heavily is dissolved in 300 μ l PBS, flow cytometer detects (table 1).
3) fluorescence co-focusing is identified five special aptamer that streaming filters out
A. (the 240nM/ bar is PBS) respectively with 1 * 10 five oligonucleotide aptamers and library
6Subdue bacterium and target bacterium and hatch 45min, the centrifugal supernatant of abandoning at 37 ℃;
B. the bacterium deposition is hanged with 10 μ l1 * PBS damping fluid, be coated on the slide glass, it is fixing to overdo.
C. slide glass is placed 1 * PBS damping fluid washing 2-3 time, 2 minutes/inferior.Washed for 3 seconds with sterile distilled water at last.
D. dry, add mountant, deckglass.Fluorescent microscope or Laser Scanning Confocal Microscope are observed combination situation (Fig. 1) down.
5. the Combination application of five special oligonucleotide aptamers
Through Laser Scanning Confocal Microscope and flow cytometer; Five aptamer and single aptamer (is example with best SA43) that comparison combination is used are to the susceptibility of streptococcus aureus evaluation, specificity and to the advantage aspect the streptococcus aureus recall rate under different staphylococcus aureus strains, the different growth conditions.The preparation of bacterium testing sample, with the hatching of adaptive son, combine, confocal fluorescent microscopic examination or flow cytometer detection method be the same.
Experimental result:
Through subduing SELEX screening and flow cytometry analysis; Five oligonucleotide aptamers that streptococcus aureus is special have been obtained; And after five the adaptive sub-Combination application, the susceptibility that can improve streptococcus aureus identification reaches the recall rate to the streptococcus aureus of different staphylococcus aureus strains, different growth conditions.
11 oligonucleotide aptamers of table 1 cells were tested by flow cytometry and streptococcus aureus and suis bonded positive rate (%)
Table 1 listed 11 sequences that flow cytometry analysis identifies respectively with streptococcus aureus and suis bonded positive rate; Can obtain conclusion: in 11 sequences of test; Wherein five sequences (SA20, SA23, SA31, SA34 and SA43) can well be distinguished streptococcus aureus 8325 and suis A5005, can take place specific combination the (representing with * in the table) with streptococcus aureus 8325.
Can obtain conclusion by Fig. 1: five oligonucleotide aptamers are 3 kinds of staphylococcus aureus strains to be measured of specific recognition in various degree all, do not have obvious the combination with suis, GP45 contrast and above-mentioned bacterium debond.
Can obtain conclusion by Fig. 2: can improve the fluorescence intensity that unique sequence detects streptococcus aureus after five combined sequence, not combine and have with suis.
Can be reached a conclusion by Fig. 3: five aglucon Combination application, the fluorescence intensity curves that flow cytometer detects can obviously be separated three kinds of staphylococcus aureus strains and suis, staphylococcus epidermidis and intestinal bacteria.
Can be reached a conclusion by Fig. 4: compare with single aglucon SA43, five aglucon Combination application can improve the detecting of each staphylococcus aureus strains, and the fluorescence intensity peak value moves to right more obvious.
Can be reached a conclusion by Fig. 5: compare with single aglucon SA43, five aglucon Combination application can improve detecting the streptococcus aureus of different growth conditions.
In a word, five the equal ability of oligonucleotide aptamer specifically identifying staphylococcus aureus, the Combination application detection sensitivity is higher, and high specificity has broad application prospects.
Sequence table
< 110>Military Medical Science Institute's basic medical institute
< 120>application of one of specifically identifying staphylococcus aureus group of oligonucleotide aptamer
<130>
<160>5
<210>1
<211>88
<212>DNA
< 213>artificial sequence
<220>
< 223>by subduing the oligonucleotide aptamer sequence that bacterium SELEX screening obtains
<400>1
<210>2
<211>88
<212>DNA
< 213>artificial sequence
<220>
< 223>by subduing the oligonucleotide aptamer sequence that bacterium SELEX screening obtains
<400>2
<210>3
<211>88
<212>DNA
< 213>artificial sequence
<220>
< 223>by subduing the oligonucleotide aptamer sequence that bacterium SELEX screening obtains
<400>3
<210>4
<211>88
<212>DNA
< 213>artificial sequence
<220>
< 223>by subduing the oligonucleotide aptamer sequence that bacterium SELEX screening obtains
<400>4
<210>5
<211>88
<212>DNA
< 213>artificial sequence
<220>
< 223>by subduing the oligonucleotide aptamer sequence that bacterium SELEX screening obtains
<400>5
Claims (3)
- One group of specifically identifying staphylococcus aureus oligonucleotide aptamer, it is characterized in that comprising that five are respectively the long single stranded DNAs of 88 bases shown in the sequence table.
- According to one group of specifically identifying staphylococcus aureus described in the claim 1 oligonucleotide aptamer, it is characterized in that five oligonucleotide aptamers are external chemosynthesis, or through PCR preparation.
- According to one group of specifically identifying staphylococcus aureus described in the claim 1 oligonucleotide aptamer, it is characterized in that, five oligonucleotide aptamers 5 ' end or 3 ' end through FITC, vitamin H or digoxigenin labeled.
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| CN101949929A (en) * | 2010-09-07 | 2011-01-19 | 福建出入境检验检疫局检验检疫技术中心 | Method for using stranded DNA aptamer to detect Lysteria monocytogenes |
| CN102220311B (en) * | 2011-04-27 | 2014-10-01 | 福建出入境检验检疫局检验检疫技术中心 | Screening method for BT-transgene paddy rice Cry1Ab/Ac protein aptamer |
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| CN107525930B (en) * | 2017-08-23 | 2019-02-15 | 湖南科技大学 | Detect the preparation method and application of staphylococcus aureus and enterotoxin B kit |
| CN108982848B (en) * | 2018-08-19 | 2021-08-20 | 潍坊医学院 | Fluorescent detection method for methicillin-resistant staphylococcus aureus based on aptamer |
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| CN116970677B (en) * | 2023-08-03 | 2024-04-30 | 西北大学 | Application of copper cluster nano material based on framework nucleic acid in preparation of pathogenic bacteria detection products |
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Address after: 100850 No. 27 Taiping Road, Beijing, Haidian District Patentee after: Chinese People's Liberation Army Military Medical Research Institute Address before: 100850 No. 27 Taiping Road, Beijing, Haidian District Patentee before: Institute of Basic Medical Sciences |