CN113355330B - ssDNA aptamer for specifically recognizing Weissella viridescens and screening method and application thereof - Google Patents
ssDNA aptamer for specifically recognizing Weissella viridescens and screening method and application thereof Download PDFInfo
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Abstract
The invention relates to a ssDNA aptamer for specifically recognizing Weissella viridescens and a screening method and application thereof, wherein the screening method comprises the following steps: constructing a random library and a primer pair, taking Weissella viridescens as a positive screening strain, taking Lactobacillus plantarum, Lactobacillus brevis, Leuconostoc mesenteroides, Pediococcus pentosaceus and Staphylococcus aureus as a counter screening strain, excluding sequences with lower specificity, excluding sequences with non-specific combination by bovine serum albumin and tRNA, and screening to obtain a ssDNA library; sequencing the ssDNA library obtained by screening, selecting a sequence with stable structure and lower free energy, and screening a sequence with high affinity and high specificity to Weissella viridis to obtain the ssDNA aptamer for specifically recognizing Weissella viridis. The ssDNA aptamer has potential application prospect in the aspect of accurate, rapid and sensitive detection of Weissella viridis.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a ssDNA aptamer for specifically recognizing Weissella viridis, a screening method and application thereof.
Background
Weissella viridis (Weissellaviridiscens) is a common dominant contaminating microorganism in low temperature meat product processing, and is also a typical spoilage lactic acid bacterium in ultra-high pressure low temperature meat products. The Weissella viridis has the capability of resisting a plurality of external adverse conditions, and can not be completely killed by other adverse conditions such as organic acid treatment and heat besides the capability of resisting ultrahigh pressure treatment. When exposed to oxygen, some meat products, such as sausages, vacuum packed meats, smoked meats, etc., turn green. Weissella viridis has become an important putrefying bacterium threatening the quality and safety of meat products, and causes huge economic loss to the meat processing industry. Therefore, it is necessary to develop a method for rapidly and accurately detecting Weissella viridis in food.
The traditional Weissella viridescens detection method comprises the steps of enrichment culture, separation and purification, microscopic examination, biochemical identification and the like, and is long in time consumption, complex in operation and low in detection efficiency. The aptamer is single-stranded DNA or RNA with the length ranging from 10 to 100 nucleotides obtained by SELEX screening. The aptamer sequence can be folded through interaction of base complementary pairing and the like to form a unique secondary structure and a unique tertiary structure, and the aptamer sequence is specifically combined with a target. The Cell-SELEX technology uses complete cells, bacteria or spores and other macromolecular substances as targets, and obtains aptamers capable of being combined with the targets in a high specificity mode through a centrifugal precipitation method. The method can screen the aptamer corresponding to the target under the condition of unknown target property, and is not interfered by other non-target proteins in tissues or samples. However, at present, no aptamer related to Weissella viridis has been reported.
Disclosure of Invention
In order to solve the technical problems, the invention provides the ssDNA aptamer for specifically recognizing Weissella viridis, has the characteristics of high specificity, high stability, convenience in synthesis, easiness in labeling functional groups and the like, and can be widely applied to convenient detection of Weissella viridis in food.
The nucleotide sequence of the ssDNA aptamer for specifically recognizing Weissella viridescens is a sequence shown in SEQ ID NO.1, and the specific sequence is as follows:
5’-AGCAGCACAGAGGTCAGATGGCTTTAGGGTGCCTTATTTATTGCATCT AGATTATAGTGACCTATGCGTGCTACCGTGAA-3’。
further, the 3 'end or 5' end of the ssDNA aptamer is modified with a functional group or molecule.
Further, the functional group or molecule is an isotope, an electrochemical label, an enzyme label, a fluorophore, biotin, an affinity ligand or a thiol group. The functional groups or molecules are used to increase the stability of the aptamer, provide a detectable signal, or to link the aptamer with other substances to form a composition.
The invention claims the application of the ssDNA aptamer in detecting Weissella viridis.
The Weissella viridescens detection kit comprises the ssDNA aptamer.
The Weissella viridescens detection test paper comprises the ssDNA aptamer.
The Weissella viridescens detection composition comprises the ssDNA aptamer.
The fluorescence biosensor for detecting Weissella viridescens comprises one or more ssDNA aptamers, a fluorescent group and a fluorescence quencher.
Further, the fluorescence quencher is graphene oxide.
The invention relates to a screening method of ssDNA aptamers for specifically recognizing Weissella viridis, which comprises the following steps:
constructing a random library and a primer pair of a nucleotide sequence shown by SEQ ID NO.6-7, taking Weissella viridis as a positive screening strain, taking Lactobacillus plantarum, Lactobacillus brevis, Leuconostoc mesenteroides, Pediococcus pentosaceus and Staphylococcus aureus as a counter screening strain, excluding sequences with lower specificity, excluding sequences with non-specific combination by bovine serum albumin and tRNA, and screening to obtain a ssDNA library;
sequencing the ssDNA library obtained by screening, selecting a sequence with stable structure and lower free energy, and screening out a sequence with high affinity and high specificity to Weissella viridis to obtain a ssDNA aptamer for specifically recognizing Weissella viridis;
the sequences of the random library all structurally conform to the structural features shown in the following general formula: 5 '-AGCAG CACAG AGGTC AGATG-N40-CCTAT GCGTG CTACC GTGAA-3', wherein N represents any one of bases A, T, C, G and N40 represents a random fragment of 40 bases in length;
wherein, the nucleotide sequence of the primer pair is specifically as follows:
an upstream primer: 5'-AGCAGCACAGAGGTCAGATG-3', respectively;
a downstream primer: 5'-TTCACGGTAGCACGCATAGG-3' are provided.
In the screening method, the lactobacillus plantarum, the lactobacillus brevis, the leuconostoc mesenteroides and the pediococcus pentosaceus are common coexisting bacteria in the low-temperature meat product, the staphylococcus aureus is a bacterium which widely exists in the environment, the survival environment of the Weissella viridis under natural conditions can be better simulated by adopting the bacteria for reverse screening, and the practicability of the obtained ssDNA aptamer for specifically recognizing the Weissella viridis in detection is ensured.
Further, the screening method comprises 11 rounds of positive screening and 4 rounds of negative screening, wherein the positive screening is carried out on the positive screening strain, and the negative screening is carried out on the negative screening strain.
Further, the ssDNA library obtained by screening consists of ssDNA obtained by round 15 screening.
Further, the concentration of bovine serum albumin and tRNA increased with the number of screening rounds until it increased to twice the concentration used in the first round of screening to increase the screening pressure.
Further, the excess amount of bovine serum albumin and tRNA in the system relative to the ssDNA sequence reduces non-specific binding of the ssDNA sequence to Weissella viridis.
By the scheme, the invention at least has the following advantages:
the invention provides a high-specificity aptamer sequence which can be screened in vitro, has short screening period, is convenient to synthesize, has good stability, high affinity, is easy to modify and mark, and has high detection sensitivity and strong specificity for the detection of Weissella viridis.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.
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In order that the present disclosure may be more readily and clearly understood, reference will now be made in detail to the present disclosure, examples of which are illustrated in the accompanying drawings.
FIG. 1 is a schematic diagram of the screening method of ssDNA aptamers that specifically recognize Weissella viridae of the present invention;
FIG. 2 is a map of the secondary structure of 5 higher affinity ssDNA aptamers obtained in example 2;
FIG. 3 is a graph of the affinity saturation binding curves for the 5 higher affinity ssDNA aptamers obtained in example 2;
FIG. 4 shows the specific detection results of 5 ssDNA aptamers with higher affinity obtained in example 2.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Example 1
1. Synthesis of random libraries and primers was performed by Biotechnology engineering (Shanghai) Inc.
Random library:
5’-AGC AGC ACA GAG GTC AGA TG-N40-CCT ATG CGT GCT ACC GTG AA-3’；
5' carboxyfluorescein labeled upstream primer: 5 '-FAM-AGC AGC ACA GAG GTC AGA TG-3';
5' phosphorylated downstream primer: 5 '-P-TTC ACG GTA GCA CGC ATA GG-3'.
Random library and primers were made up into 10uM stock in TE buffer and stored at-20 ℃ until use.
2. Cultivation of bacterial species
Inoculating activated Weissella viridescens into MRS broth, culturing at 37 deg.C, transferring bacteria (OD600 ═ 0.6) in exponential phase into centrifuge tube according to growth curve of Weissella viridescens, centrifuging at 5000r/min and 4 deg.C for 5min, discarding supernatant bacterial culture solution, washing white thallus precipitate at bottom of centrifuge tube with binding buffer BB (10mmol/L Tris-HCl, 5mmol/L KCl, 100mmol/L NaCl, 5mmol/L MgCl2, pH7.4), and removing excessive culture medium components.
3. Screening of Weissella viridis ssDNA aptamers
(1) In the first round of screening, the input amount of the random library was 1nmol, and the concentration of Weissella viridis was 108cfu/mL, random library was denatured at 95 ℃ for 5min before incubation, then ice-cooled for 5min, and then incubated with Weissen green at room temperatureAnd (5) incubating the bacteria. Meanwhile, 0.5% Bovine Serum Albumin (BSA) solution and excessive transfer RNA (tRNA) which are 10 times of the mole number of the random library are added into the system, the total volume is 600 mu L, the mixture is uniformly mixed in a binding buffer BB, and the mixture is incubated for 1h at room temperature by shaking, so that the random library and the Weissella viridis are fully bound.
(2) After incubation is finished, the bacterial suspension is placed in a centrifugal machine for centrifugation at 5000r/min at 4 ℃ for 5min, ssDNA combined with Weissella viridescens is centrifugally precipitated to the bottom of a test tube along with the Weissella viridescens, ssDNA not combined with Weissella viridescens is dissociated in supernatant, supernatant is removed, the precipitate is washed twice by using a binding buffer BB, and ssDNA which is nonspecifically adsorbed by thalli or has weak binding capacity with thalli is removed.
(3) Adding 300 mu L of 1 XPCR buffer solution into the composite sediment of the ssDNA and the Weissella viridescens, heating for 10min at 95 ℃, destroying the thallus structure through thermal denaturation, dissociating the combined ssDNA from the thallus, quickly centrifuging for 10min at 8000r/min and 4 ℃ in a precooled centrifuge, obtaining supernatant which is the ssDNA capable of being combined with the Weissella viridescens, and collecting the supernatant as a first round of aptamer library.
(4) PCR amplification was performed using the first round aptamer library as template. The amplification system contained 1. mu.L of ssDNA, 0.5. mu.L of each of the upstream and downstream primers at a concentration of 10. mu.M, 1. mu.L of dNTP mix at a concentration of 5mM, 5. mu.L of 10 XPCR buffer, 0.5. mu.L of Taq DNA polymerase, and 31.5. mu.L of sterile ultrapure water, so that the total volume of PCR was 50. mu.L. The PCR program was set to 95 ℃ denaturation for 5min, 95 ℃ denaturation for 30s, 56 ℃ annealing for 30s, and 72 ℃ extension for 30s, and the cycle was repeated for 14 cycles, then 72 ℃ extension for 1min, and finally the PCR product was stored at 4 ℃ under refrigeration.
(5) And the PCR product is purified by a purification kit, the purified product is subjected to electrophoresis verification by 8% non-denatured polyacrylamide gel, the electrophoresis band is single and bright, and the band is positioned at the position of 80 bp.
(6) Adding 1/10 volumes of lambda exonuclease buffer solution and a proper amount of lambda exonuclease into the PCR purified product, carrying out enzyme digestion for 50min at 37 ℃, heating in a water bath at 75 ℃ for 15min, and inactivating enzyme to terminate enzyme digestion. The enzyme digestion product is electrophoresed by 8% denatured polyacrylamide gel (containing 7M urea), the electrophoretic band is single and bright, and the band and the synthesized library are in the same position. Adding 1/10 volumes of 3mol/L sodium acetate and 2 volumes of absolute ethyl alcohol into the enzyme digestion product which is verified to be single-chain by electrophoresis, mixing evenly, and precipitating in a refrigerator at minus 80 ℃ overnight. The overnight solution was centrifuged at 14000rpm/min for 15min at 4 ℃ and the supernatant removed, washed by adding 100. mu.L of 70% ethanol by volume, subsequently centrifuged at 14000rpm/min for 15min at 4 ℃ and the supernatant removed, dried in an oven at 50 ℃ and added 200. mu.L of binding buffer BB as the library for the next round of screening.
For each additional round of screening, the concentration of added BSA and tRNA was increased by 25% until it increased to 2-fold. Reverse screening was performed in rounds 5, 7, 9 and 12 using Lactobacillus plantarum, Lactobacillus brevis, Leuconostoc mesenteroides, Pediococcus pentosaceus and Staphylococcus aureus.
4. Cloning and sequencing
The ssDNA obtained from the 15 th round of screening was amplified by PCR, and the PCR products were analyzed and sequenced by Biotechnology engineering (Shanghai) GmbH to obtain 29 ssDNA sequences. The sequences were divided into 8 families based on the homology information of 29 sequences, 1 sequence with stable structure and low free energy was selected from each family (8 sequences are respectively marked as L3, L5, L6, L7, L9, L10, L11 and L15), and 5' end labeled FAM aptamers were synthesized by Biotechnology (Shanghai) GmbH for affinity and specificity analysis.
Example 2
(1) Aptamer affinity assay
The 8 ssDNA sequences were dissolved to a concentration of 10. mu.M with binding buffer BB and stored at-20 ℃ until use. The affinity of 8 ssDNA sequences for Weissella viridescens was analyzed by flow cytometry. The concentration gradients of 8 ssDNA were 10, 20, 40, 80, 160 and 320nmol/L, respectively, and after L0min denaturation at 95 ℃ the samples were immediately cooled in ice bath for 10 min. Subsequently, ssDNA solution was added to the Weissella viridans somatic pellet that had been washed 3 times with binding buffer BB and incubated at 25 ℃ for 1h with slow shaking. The Weissella viridescens suspension was then washed with binding buffer BB and the Weissella viridescens-ssDNA complex was finally resuspended in 500. mu.L of binding bufferFlow cytometry analysis was performed in liquid BB. Percent fluorescence intensity of the complexes characterizes the magnitude of the affinity, and dissociation constant K for each ssDNA was calculated using GraphPad Prism5 softwaredThe values are shown in table 1 below, and their saturation binding curves are plotted.
TABLE 1 dissociation constant K of ssDNAdValue of
(2) Aptamer specificity assay
Specific analysis is carried out on 5 aptamers (L3, L5, L6, L9 and L15 with strong affinity to Weissella viridescens, and the nucleotide sequences of the aptamers are respectively shown as SEQ ID NO. 1-5), namely aptamers L3, L5, L6, L9 and L15, the secondary structure maps of the aptamers are shown as figure 2, and K of the 5 aptamersdThe saturation binding curves are shown in FIG. 3 for values of 68, 65, 90, 82 and 69nmol/L, respectively. Aptamers L3, L5, L6, L9 and L15 with the concentration of 160nmol/L are respectively incubated with Weissella viridescens, Lactobacillus plantarum, Lactobacillus brevis, Leuconostoc mesenteroides, Pediococcus pentosaceus and Staphylococcus aureus at 25 ℃ for 1h under oscillation, then the thalli are washed by a binding buffer BB, and are resuspended in the binding buffer BB to remove the nonspecific binding of the aptamers and the thalli, and then the flow cytometry analysis is carried out in a light-shielded state. The results show (fig. 4) that the binding rates of L3, L5, and L15 to weissella viridis are all over 60%, but the binding rates of L5 to pediococcus pentosaceus, leuconostoc mesenteroides are over 20%, and the binding rates of L15 to leuconostoc mesenteroides, staphylococcus aureus are over 20%, so L3 is selected as an aptamer capable of binding to weissella viridis with high specificity.
The aptamer capable of recognizing Weissella viridescens with high affinity and high specificity is obtained by Cell-SELEX technology screening, a sensitive molecular probe is provided for rapid and accurate detection of Weissella viridescens in food, and the aptamer has potential practical application prospects.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.
Sequence listing
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Claims (9)
1. The ssDNA aptamer capable of specifically recognizing Weissella viridescens is characterized in that the nucleotide sequence of the ssDNA aptamer is shown as SEQ ID NO. 1.
2. The ssDNA aptamer of claim 1, wherein: the 3 'end or the 5' end of the ssDNA aptamer is modified with a functional group or molecule.
3. The ssDNA aptamer of claim 2, wherein: the functional group or molecule is isotope, electrochemical marker, enzyme marker, fluorescent group, biotin, affinity ligand or sulfhydryl.
4. Use of the ssDNA aptamer of any of claims 1-3 in the detection of Weissella viridis.
5. A Weissella viridescens detection kit is characterized in that: comprising the ssDNA aptamer of any of claims 1-3.
6. The green Weissella detection test paper is characterized in that: comprising the ssDNA aptamer of any of claims 1-3.
7. A Weissella viridis detection composition is characterized in that: comprising the ssDNA aptamer of any of claims 1-3.
8. A fluorescence biosensor for detecting Weissella viridis, comprising: comprising the ssDNA aptamer of any of claims 1-3, further comprising a fluorophore and a fluorescence quencher.
9. The fluorescence biosensor of claim 8, wherein: the fluorescence quencher is graphene oxide.
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| CN102010865A (en) * | 2010-09-29 | 2011-04-13 | 江南大学 | Nucleic acid aptamer capable of specifically recognizing Listeria monocytogenes, screening method and application thereof |
| CN102010866A (en) * | 2010-09-29 | 2011-04-13 | 江南大学 | Nucleic acid aptamer capable of specifically recognizing shigella, screening method and application thereof |
| CN103031305A (en) * | 2011-09-30 | 2013-04-10 | 江南大学 | Screening and application of oligonucleotide aptamer for specific recognition of Salmonella Typhimurium |
| CN104278036A (en) * | 2014-10-08 | 2015-01-14 | 江南大学 | Set of oligonucleotide aptamers for specifically recognizing Sh.sonnei |
| CN106906296A (en) * | 2017-04-05 | 2017-06-30 | 江南大学 | A kind of label of high frequency zone Wei Si Salmonellas and its application |
| CN112175958A (en) * | 2020-10-09 | 2021-01-05 | 江南大学 | Optimized aptamer sequence for specifically recognizing Listeria monocytogenes and application thereof |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102010865A (en) * | 2010-09-29 | 2011-04-13 | 江南大学 | Nucleic acid aptamer capable of specifically recognizing Listeria monocytogenes, screening method and application thereof |
| CN102010866A (en) * | 2010-09-29 | 2011-04-13 | 江南大学 | Nucleic acid aptamer capable of specifically recognizing shigella, screening method and application thereof |
| CN103031305A (en) * | 2011-09-30 | 2013-04-10 | 江南大学 | Screening and application of oligonucleotide aptamer for specific recognition of Salmonella Typhimurium |
| CN104278036A (en) * | 2014-10-08 | 2015-01-14 | 江南大学 | Set of oligonucleotide aptamers for specifically recognizing Sh.sonnei |
| CN106906296A (en) * | 2017-04-05 | 2017-06-30 | 江南大学 | A kind of label of high frequency zone Wei Si Salmonellas and its application |
| CN112175958A (en) * | 2020-10-09 | 2021-01-05 | 江南大学 | Optimized aptamer sequence for specifically recognizing Listeria monocytogenes and application thereof |
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