CN102010865A

CN102010865A - Nucleic acid aptamer capable of specifically recognizing Listeria monocytogenes, screening method and application thereof

Info

Publication number: CN102010865A
Application number: CN 201010295766
Authority: CN
Inventors: 王周平; 丁晓莹
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2010-09-29
Filing date: 2010-09-29
Publication date: 2011-04-13
Anticipated expiration: 2030-09-29
Also published as: CN102010865B

Abstract

The invention discloses a nucleic acid aptamer capable of specifically recognizing Listeria monocytogenes, a screening method and application thereof. By using the SELEX (Systematic Evolution of Ligands By Exponential Enrichment) technology, a single-chain DNA oligonucleotide aptamer capable of specifically recognizing Listeria monocytogenes is obtained, and the aptamer can be transformed into a reporting aptamer by means of fluorescein labels and the like to be used for detecting Listeria monocytogenes. The sequence of the aptamer has wide application prospects in the aspect of accurately and fast detecting Listeria monocytogenes in food.

Description

A kind of nucleic acid aptamer of specific recognition Listeria monocytogenes and screening method thereof and application

Technical field

The present invention relates to biological technical field, specially refer to the SELEX technology of utilizing in the Protocols in Molecular Biology (the Fas lignand system evolution technology of index concentration) and prepare nucleic acid aptamer a kind of and Listeria monocytogenes high specific and high-affinity, for the application of this nucleic acid aptamer in detecting Listeria monocytogenes provides scientific basis and theoretical basis.

Background technology

Listeria monocytogenes (Listeria monocytogenes) is a kind of common, important infecting both domestic animals and human cause of disease bacterium.It can cause the listeriosis of people, animal, mainly show as behind the severe infections be short of breath, vomiting, hemorrhagic fash, purulent conjunctivitis, heating, tic, stupor, spontaneous abortion, meningitis, septicemia be until death.It extensively is present in occurring in nature, and the Listeria monocytogenes that exists in the food has danger to the mankind's safety, but this bacterium growth and breeding still in 4 ℃ of environment is one of refrigerated food The main pathogenic fungi of threatening human health.Therefore, how fast, accurately detect Listeria monocytogenes and have the important research meaning.

The method of traditional detection pathogenic bacteria needs first bacterial isolate microorganism often, by microorganism culturing, identifies with classic methods more then.Consuming time, insensitive is the ubiquitous problems of these methods.Therefore development technology quick, the Sensitive Detection pathogenic micro-organism is very necessary.Though utilize the antibody can the special recognition pathogen bacterium, can be rapidly, accurately sample to be checked is made evaluation, this technology is subjected to specific antibody to prepare the restriction of difficulty.Because according to the outstanding criteria for classification of uncle, the essentially identical bacterial population 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 fine distinction, and finding these difference and preparing corresponding specific antibody obviously is a consuming time and difficult task.

In the last few years, oligonucleotide aptamer substituted molecule as the prospect of antibody molecule, and its research is comparatively noticeable.Oligonucleotide aptamer be by SELEX process screening with target material specificity bonded cluster small molecule DNA or RNA fragment.SELEX technology (Systematic Evolution of Ligands by Exponential Enrichment, phyletic evolution index concentration technology) being a kind of new combinatorial chemistry technique of early 1990s development, is a kind of effective ways of studying nucleic acid construct and function.Its ultimate principle is one of external chemosynthesis single stranded oligonucleotide storehouse at random, mix with the target material with it, form target material-nucleic acid complexes, flush away not with target material bonded nucleic acid molecule, separate and target material bonded nucleic acid molecule, with this nucleic acid molecule is that template is carried out pcr amplification, carries out the screening process of lower whorl again.Repeat screening and amplification by the number wheel, obtain the oligonucleotide aptamer of high-affinity and high specific at last, 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, adaptive son has more obvious superiority, as not relying on zooblast, not limited by immune condition and immunogenicity, the screening of adaptive son is carried out external fully, has time, quality and quantitative selection elasticity, can glue really, fix a point, arbitrarily connect other functional groups and molecule when synthetic; Adaptive sub-sex change and renaturation is reversible and speed is fast, use repeatedly, prolonged preservation and room temperature transportation; Target molecule is wider, outside isolating protein, the Nucleotide macromole, also have small molecules (as dyestuff, Cocaine, caffeine and theophylline etc.), somatomedin, peptide chain, steroid, carbohydrate, cofactor (as FMN etc.), even can be used for complete cell, virus, spore etc.; Combine with target molecule and to have stronger specificity and avidity, do not organized or sample in the interference of non-target protein, can under target character condition of unknown, filter out its corresponding adaptive son; Adaptive son makes disease controlled by occupying target material epi-position, and as the treatment of clinical medicine, the vascular endothelial growth factor when having manifested potential, thrombus generate the effect of the factor, some toxin proteins etc., to reach therapeutic purpose.Aspect microorganism detection, particularly to some pathogenic bacterias or viral research, though do not know the epi-position of its internal structure, function and these materials, but with it as the target material, screen the adaptive son corresponding by the SELEX process with it, detect the target material, focus is explored in the research that has become this field.

The present invention is a target with the common clinically Listeria monocytogenes of food neutralization, utilize the SELEX technology to obtain and Listeria monocytogenes specificity bonded nucleic acid aptamer sequence, this sequence can fast, accurately detect Listeria monocytogenes, because the adaptive sub-stable performance of single strand dna oligonucleotide, can be directly used in fluorescence or chemoluminescence after synthetic convenient and cheap, modified, chromophoric method detects target bacteria, and is therefore simple to operate, direct.This invention can be used widely in fields such as food safety detection, clinical medicine.

Summary of the invention

The object of the invention is to provide a kind of microorganism molecular Biological Detection method, particularly a kind of method of utilizing adaptive sub-technology fast, accurately to detect Listeria monocytogenes.

The phyletic evolution technology (SELEX technology) of the inventive method utilization index level enrichment part, with the complete mycetocyte of Listeria monocytogenes is target, screening obtains the adaptive son with target cell high-affinity specific combination, the adaptive son that will obtain by Fluoresceincarboxylic acid (FAM) marking method transfers the adaptive son of report to, be used for detecting corresponding target bacteria the purpose of reach fast, accurately diagnosing from clinical blood, food culture supernatant.

Advantage of the present invention:

(1) compare with the antibody of protein, single stranded oligonucleotide is more stable; Aptamer can directly externally synthesize, mark, does not 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) this sequence is from structure significantly, have avidity and all the strongest adaptive subsequence of specificity that selects 8 adaptive subsequences of different avidity with target bacteria, can the specific recognition Listeria monocytogenes.

Description of drawings

Fig. 1 is synthetic ssDNA library 2% agarose gel electrophoretogram

Fig. 2 is PCR product 2% agarose gel electrophoretogram of part SELEX screening

Fig. 3 is the adaptive sub-F1-F8 secondary structure collection of illustrative plates of Listeria monocytogenes

Fig. 4 is the saturated binding curve of the adaptive sub-F1-F8 avidity of Listeria monocytogenes

Table 1 is the combination rate of the adaptive son of F2 and nine kinds of contrast bacteriums

Embodiment:

Be nucleic acid aptamer preparation method by SELEX technology screening specific combination Listeria monocytogenes and the application that detects the Listeria monocytogenes aspect fast thereof below.

1, synthetic random single-stranded DNA banks and primer (IDT company is synthetic)

Random single chain DNA (ssDNA) library: 5 '-GGGAGCTCAGAATAAACGCTCAA (N35) TTCGACATGAGGCCCGGATC-3 ', having made up length is the library of ssDNA at random of 78nt, two ends are the immobilized primer sequence, and the centre is the stochastic sequence of 35 bases, and storage capacity is 10 ¹⁴More than; Primer I: 5 '-GGGAGCTCAGAATAAACGCTCAA-3 '; Primer I I:5 '-GATCCGGGCCTCATGTCG AA-3 ', will be at random ssDNA library and two kinds of primers all to be mixed with ℃ storage of 100 μ mol/L stock solutions-20 with the TE damping fluid standby.

2, the pcr amplification in double-stranded DNA (dsDNA) and single stranded DNA (ssDNA) library

The every wheel before the screening is the dsDNA library with the ssDNA amplified library earlier, and is the ssDNA library that template amplification goes out the next round screening with the dsDNA library, for stable condition, do not change reaction system and response procedures.The PCR reaction system is: ssDNA template 2 μ L at random, each 2 μ L (20 μ mol/L) of primer I and primer I I contain Mg ²⁺DNTP mixture 2 μ L (25mmol/L), 10 * PCR damping fluid, 5 μ L, Taq polysaccharase 0.5 μ L adds ultrapure water to 50 μ L; The PCR response procedures is: 97 ℃ of pre-sex change 5min, circulate 96 ℃ of sex change 40s, 59 ℃ of annealing 40s then, 72 ℃ are extended 40s, circulate 30 times, and last 72 ℃ are extended 9min, 12 ℃ of cooling 5min, the PCR product detects with 2% agarose gel electrophoresis, and it is standby to put 4 ℃ of ambient storage.

3, the used target bacterium of screening obtaining and handling

The LB liquid nutrient medium is cultivated Listeria monocytogenes, and 37 ℃ of shaking tables are cultured to logarithmic phase (OD ₆₀₀Be about 0.3), stop to cultivate, collect OD ₆₀₀Be about 0.3 bacterium liquid 1mL, 4 ℃, the centrifugal 10min of 8000rpm abandons supernatant, and (it is standby to put 4 ℃ of ambient storage for 1 * BB) flushing twice, the medium component that flush away is unnecessary with binding buffer liquid.

4, the SELEX technology screening obtains the nucleic acid aptamer of specific recognition Listeria monocytogenes

The first round, reaction system was 600 μ L when screening, and the library of dsDNA at random of getting after 2nmol increases adds 1 an amount of * BB in 95 ℃ of sex change 5min, ice bath 10min immediately.The mycetocyte (1 * 10 that its adding is handled well ⁸Individual) in the centrifuge tube, add 5 times again to the 5%BSA solution and the yeast tRNA of ssDNA library mole number at random, to reduce, in 37 ℃ of vibration hatching 1h in conjunction with background.Need to change centrifuge tube after the hatching, to remove and centrifugal tube wall bonded ssDNA, with new centrifuge tube in 4 ℃, the centrifugal 10min of 6000rpm, abandon supernatant, remove not in conjunction with or in conjunction with untight ssDNA at random library, subsequently with the 1 * BB that contains 0.05%BSA by resuspension and centrifugal elutriation 2 times, add 100 μ L, 1 * PCR damping fluid at last, in 95 ℃ of sex change 5min, 0 ℃ is cooled off 10min immediately, through 4 ℃, the centrifugal 10min of 8000rpm draws supernatant to another clean centrifuge tube, is the adaptive son of first round screening gained.As the template pcr amplification is the dsDNA library, is used for second and takes turns screening.Second takes turns that to take turns reaction system to the 8th be 350 μ L, and wherein the ssDNA library is 100pmol at random, whenever takes turns screening and need repeat to screen the aptamers storehouse that obtains Listeria monocytogenes for 8 times with fresh bacterium liquid.Every screening PCR product of taking turns is detected with 2% agarose gel electrophoresis.

(1 * BB) is 50mMTris-HCl (pH 7.4) to above-mentioned screening binding buffer liquid, 5mM KCl, 100mM NaCl, 1mMMgCl ₂Dcq buffer liquid is to contain 1 * BB of 0.05%; Elution buffer is 1 * PCR damping fluid.

5, clone and order-checking

With the 8th ssDNA library of taking turns enrichment, pcr amplification is double-stranded, serves Hai Boshang Bioisystech Co., Ltd and carries out determined dna sequence, obtains 20 adaptive subsequences.

6, adaptive subsequence structural analysis

Adopt DNAMAN software and RNA Structure 4.2 softwares respectively adaptive subsequence to be carried out primary structure and secondary structure analysis, obtain the homology information and the secondary structure collection of illustrative plates of 20 sequences.In conjunction with primary structure homology and secondary structure sequence is divided into 8 families, from each family, select 1 Stability Analysis of Structures, the sequence that energy level is lower is that next step evaluation is carried out in representative, totally 8, as shown in Figure 3, loop-stem structure and the hairpin structure in every adaptive son may be adaptive son and target bacteria bonded basis

7, adaptive son and Listeria monocytogenes avidity and specific assay

7.1 adaptive sub-avidity analysis

With above-mentioned 8 adaptive subsequences 5 ' end flag F AM, synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, be used for avidity and measure.

FAM-F1

5′-GGGAGCTCAGAATAAACGCTCAAGGGGGGCCTAGACTAGGGGGAGAGGGTGGGACGGTTTCGACATGAGGCCCGGATC-3′

FAM-F2

5′-GGGAGCTCAGAATAAACGCTCAATACTATCGCGGAGACAGCGCGGGAGGCACCGGGGATTCGACATGAGGCCCGGATC-3′

FAM-F3

5′-GGGAGCTCAGAATAAACGCTCAAGGGGCGGCGGCGGTGGTACGGGGTTGGGAGCGGGCT?TCGACATGAGGCCCGGATC-3′

FAM-F4

5′-GGGAGCTCAGAATAAACGCTCAAGGCGTATGCGCAGCGAGGGCGGCCGGGCGACGTCGTTCGACATGAGGCCCGGATC-3′

FAM-F5

5′-GGGAGCTCAGAATAAACGCTCAACCACGGGAACAACATCGTGGCAGGGACGAGCGTCCTTTCGACATGAGGCCCGGATC-3′

FAM-F6

5′-GGGAGCTCAGAATAAACGCTCAAGCGCGCTGCCGACGCGGGGGGGCTGATTAGCGTGGTTCGACATGAGGCCCGGATC-3′

FAM-F7

5′-GGGAGCTCAGAATAAACGCTCAAACTGAGGGGCGGCGGACGGGATGGGAAATGTAGGTTCGACATGAGGCCCGGATC-3′

FAM-F8

5′-GGGAGCTCAGAAFAAACGCTCAATAGCGTGGGTAACCGTGTTGGGGGGTGCCACGGTCTTCGACATGAGGCCCGGATC-3′

With eight adaptive sons use respectively 1 * BB dilution for different concentration gradients (10,20,50,100,150,200,250,300nmol/L), with 1 * 10 ⁸Individual Listeria monocytogenes at 37 ℃ of incubations in conjunction with 1h, 4 ℃, 6000rpm, centrifugal 10min, abandon supernatant, with 1 * BB flushing twice, add 100 μ L, 1 * BB, the lucifuge mixing, with FL-7000 fluorescent spectrophotometer assay fluorescent value (survey and average for three times), utilize the dissociation constant Kd value of each adaptive son of Sigma Plot11.0 computed in software, and draw its saturated binding curve, as shown in Figure 4.

7.2 specificity analyses

The adaptive subsequence minimum with the strongest Kd of the being value of Listeria monocytogenes cellular affinity is the F2 sequence, the Kd value is 58.85nmol/L, with the adaptive son (100nmol/L of 10pmol F2,1 * BB) respectively with streptococcus aureus, Salmonella typhimurium, Shigellae, intestinal bacteria, Vibrio parahemolyticus, subtilis, suis, Pseudomonas aeruginosa, Lactobacterium acidophilum in 37 ℃ of warm the region between the heart and the diaphragms in conjunction with 1h, 4 ℃, 6000rpm, centrifugal 10min, abandon supernatant, with twice of 1 * BB flushing, mycetocyte heavily is dissolved in 100 μ L, 1 * BB, with FL-7000 fluorescent spectrophotometer assay fluorescent value (survey and average for three times).The result shows that the combination rate of the adaptive son of F2 and nine kinds of contrast bacterium is no more than 5%, as shown in table 1, the specificity that shows adaptive son of F2 and Listeria monocytogenes is good, therefore, utilize the adaptive son of Listeria monocytogenes high-affinity specific nucleic acid of SELEX technology screening to detect Listeria monocytogenes, be with a wide range of applications.

The combination rate (%) of the table adaptive son of 1F2 and nine kinds of contrast bacteriums

Claims

1. the nucleic acid aptamer of a specific recognition Listeria monocytogenes and screening method thereof and application is characterized in that: the nucleotide sequence of described oligonucleotide aptamer is shown in sequence 1-8 in the sequence table.

2. the preparation method of the oligonucleotide aptamer of target Listeria monocytogenes according to claim 1, carry out according to the following step step:

(1) structure and the primer in random single chain DNA (ssDNA) library

Random single chain DNA (ssDNA) library:

5′-GGGAGCTCAGAATAAACGCTCAA(N35)TTCGACATGAGGCCCGGATC-3′；

Primer I: 5 '-GGGAGCTCAGAATAAACGCTCAA-3 ';

Primer I I:5 '-GATCCGGGCCTCATGTCG AA-3 ';

(2) PCR prepares ssDNA library at random;

(3) nucleic acid aptamer of SELEX screening Listeria monocytogenes;

(4) dna clone and order-checking;

(5) adaptive subsequence primary structure and secondary structure analysis;

(6) adaptive sub-avidity and specificity are identified.

3. the application of the oligonucleotide aptamer of target Listeria monocytogenes according to claim 1 aspect the detection Listeria monocytogenes, it is characterized in that described oligonucleotide aptamer is external chemosynthesis, or prepare by PCR or other molecular biology methods.

4. according to the application described in the claim 1, it is characterized in that 5 of described oligonucleotide aptamer ' end or 3 ' end can pass through marks such as FITC (FAM), vitamin H, digoxin.