CN117230069B - Nucleic acid aptamer capable of specifically recognizing Cronobacter and application thereof - Google Patents
Nucleic acid aptamer capable of specifically recognizing Cronobacter and application thereof Download PDFInfo
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Abstract
The invention discloses a nucleic acid aptamer for specifically recognizing Cronobacter and application thereof, belonging to the technical field of biological detection. According to the invention, cronobacter is used as a target, on the basis of an original aptamer sequence obtained by SELEX screening, an aptamer with high affinity and high specificity combined with a target cell is obtained by adopting truncated optimization, an optimal aptamer is obtained by further truncated optimization, and a round two-chromatographic test shows that the optimal aptamer is more compact and stable than a secondary structure stem region of the original aptamer. On the basis, the aptamer sequence is combined with the magnetic nano particles, so that the Cronobacter in the enriched sample can be specifically separated. The aptamer is a novel recognition element of Cronobacter, has the advantages of high sensitivity, low cost, easiness in preparation and modification and labeling, and can be applied to construction of various detection methods.
Description
Technical Field
The invention relates to a nucleic acid aptamer for specifically recognizing Cronobacter and application thereof, belonging to the technical field of biological detection.
Background
Cronobacter as a common rod-shaped food-borne pathogenic bacterium is commonly found in environmental and infant formulas. It has low nutrition requirement, osmotic pressure resistance and dryness resistance, can survive for a long time in infant formula milk powder, can cause infants and immunocompromised adults or the elderly to be infected with diseases with extremely high mortality, and seriously threatens public health safety and human health. Enterobacter sakazakii and salmonella are listed together as group A pathogenic bacteria of infant formula. It is considered that enterobacter sakazakii, salmonella and the like in the infant formula are main causes of infection, disease and death of infants, and especially have the most killing power for infants with dysplasia and poor immune function. FDA data shows that the infection dose is similar to that of E.coli O157: H7. Recent studies have shown that cronobacter is divided into 7 species, cronobacter sakazakii, cronobacter malonate, cronobacter zurich, mo Jinsi, cronobacter Kang Dimeng, cronobacter You Niwo and cronobacter dublinii, cronobacter sakazakii are species of this genus, and all seven species were detected in foods. Therefore, the detection of the Cronobacter in the food is realized accurately, rapidly and sensitively, and the method has extremely important significance for preventing the infection of the Cronobacter, guaranteeing the food safety and protecting the human health.
Common methods for detecting Cronobacter include traditional plate separation culture and physiological and biochemical identification methods, ELISA methods, PCR methods, fluorescent quantitative PCR methods, immunological methods and the like. These strategies have attractive features such as rapid detection and excellent sensitivity; however, they also have unsatisfactory drawbacks, including laborious and complex sample pretreatment, expensive instrumentation and reagents, and the need for sufficient expertise, and non-specific binding and stability of antibodies to the analog will severely impact detection performance. Therefore, a novel sensor with efficient identification elements, ultrasensitive, selective and rapid detection of cronobacter to control food safety, and to supervise the entry and exit inspection and quarantine work is highly desirable.
Nucleic acid aptamers (aptamers) are oligonucleotide sequences with high specificity and affinity for target substances (such as cells, viruses, toxins, proteins, vitamins, etc.) obtained by screening random oligonucleotide libraries based on the SELEX technology. Compared with the traditional identification component antibody, the aptamer has the advantages of easy in-vitro synthesis, good repeatability, high stability and low cost, and can be applied to various biosensors, thereby showing great potential in cell biology, biomedicine and food safety detection. In recent years, many biosensors based on aptamer detection of cronobacter have been developed. These methods of detection increase the efficiency of detection of Cronobacter in food products, but also have great limitations, since most of these methods are based on the detection of model species, which cannot identify all species of Cronobacter at one time.
The original aptamer obtained by SELEX contains 60-100 nucleotides, however, not all bases participate in the recognition process of the aptamer and the target, and redundant bases not only increase the cost of synthesis, but may also enhance the flexibility of the aptamer structure and form steric hindrance and the like to reduce the affinity of the aptamer to the target. The truncated optimized aptamer has fewer bases, can be used for researching an aptamer-target binding mechanism more efficiently, and is designed into a more sensitive biosensor.
Disclosure of Invention
In order to solve the problems, the invention provides an optimized aptamer sequence for specifically recognizing all species of Cronobacter cloacae. The invention uses Cronobacter as a target, performs truncation optimization on the basis of an original sequence A9P of an 80-nucleotide-long Cronobacter cloonobacter cloacae DSM 18703 aptamer obtained by SELEX screening, obtains an optimized aptamer sequence capable of recognizing all species bacteria combined with the Cronobacter cloacae with high affinity and specificity, and uses the optimal aptamer for separation and enrichment of Cronobacter cloacae in a sample.
It is a first object of the present invention to provide a nucleic acid aptamer specifically recognizing cronobacter, said nucleic acid aptamer being selected from any one of the following:
(1) A nucleotide sequence shown as SEQ ID NO. 4;
(2) One of the optimized sequences is truncated by the nucleotide sequence shown in SEQ ID NO. 4.
Further, the sequence of the nucleotide sequence shown in SEQ ID NO.4 after truncated optimization is shown in SEQ ID NO. 5.
Further, the 5 'end or the 3' end of the nucleic acid aptamer is modified with a functional group or a molecule.
Further, the functional group or molecule is selected from at least one of an isotope, an electrochemical label, an enzyme label, a fluorescent group, biotin, an affinity ligand, a thiol, and an amino group.
The synthesis, treatment and characterization operations of the aptamer are as follows:
1. Synthesis of aptamers
The aptamer marked by the FAM group at the 5' end is synthesized by Shanghai Biotechnology engineering services Inc. for affinity and specificity analysis, the unmarked aptamer sequence is synthesized for round dichroism analysis, and the biotinylated aptamer is synthesized for capturing Cronobacter in a sample.
2. Treatment of bacterial species
1ML (1X 10 8) of the bacterial liquid in the logarithmic phase (OD 600 =0.3) was taken out in a centrifuge tube, the supernatant was discarded after refrigerated centrifugation at 5000r/min at 4℃for 5min, and the supernatant was washed twice with 1 Xbinding buffer (1 XBB) (50 mmol/L Tris-HCl (pH 7.4), 5mmol/L KCl,100mmol/L NaCl,1mmol/L MgCl 2) at room temperature to remove the excess medium components, resuspended in 1 XBB and stored at 4℃for further use.
3. Characterization of aptamer sequences
(1) Aptamer affinity assay
The synthesized aptamer was diluted with 1 XBB buffer to prepare a 10. Mu.M solution, which was stored at-4℃for further use. The affinity of the aptamer was analyzed using a flow cytometer. After dissolving the aptamer marked by the FAM group at the 5' end in 1 XBB and pre-deforming at 95 ℃ to restore the room temperature, preparing aptamer solutions (25, 50, 100, 150, 200, 300 nM) with different concentrations, and then incubating the aptamer solutions with the treated Cronobacter cloacae for 45min at 25 ℃ in a dark place. 5000r/min, freeze centrifugation at 4℃for 5min, discarding supernatant, and re-suspending in 500. Mu.L of 1 XBB buffer for flow cytometry, and comparing with the bacteria solution without aptamer. The fluorescence intensity of the blank sample (no aptamer added) was adjusted first, and then the forward scatter, side scatter and fluorescence intensity of the sample were measured under the same parameters. The dissociation constant K d values for each aptamer were fitted using GRAPHPAD PRISM 9.0.0 software and the saturated binding curves were plotted.
(2) Aptamer-specific assay
1X 10 8 other 6 bacteria (E.coli, listeria monocytogenes, salmonella, shigella flexneri, staphylococcus aureus, vibrio parahaemolyticus) and 5' -terminal FAM group-labeled aptamer were incubated at 25℃for 45min in the dark, then washed with 1 XBB buffer, resuspended in 500. Mu.L of 1 XBB buffer, and subjected to flow cytometry after mixing in the dark.
(3) Round two chromatography
After denaturation of the aptamer solution (10. Mu.M) at 95℃for 5min, it was allowed to stand at room temperature for 30min, so that the aptamer formed a stable spatial structure. The measurement of the circular dichroism was carried out on a CHIRASCAN V CD spectrometer, using a quartz cuvette with a path length of 1mm, setting a spectral scan range of 220-320 nm. The data were fit analyzed using Pro-DATA VIEWER software.
4. Visual observation of aptamer binding to target
Binding of FAM-labeled aptamer to bacteria was observed under confocal microscopy. After incubating 500 μl of the fluorescent-labeled aptamer with the pretreated bacteria at 25 ℃ for 45min, unbound aptamer was removed using 1×bb buffer, the mixture was resuspended in 100 μl of dnase and rnase free water, the bacterial suspension was dropped onto a glass bottom slide, and images were acquired using a laser confocal microscope.
A second object of the present invention is to provide the use of the above-mentioned nucleic acid aptamer in detection of Cronobacter, such as preparation of detection products including, but not limited to, kits, test papers, etc.
Further, the Cronobacter comprises one or more of Cronobacter sakazakii, cronobacter malonate, cronobacter zurich, cronobacter Mo Jinsi, cronobacter Kang Dimeng, cronobacter You Niwo and Cronobacter dublin.
It is a third object of the present invention to provide the use of the above-mentioned nucleic acid aptamer in food detection or medical detection.
It is a fourth object of the present invention to provide a product for detecting Cronobacter, comprising the above-mentioned aptamer.
Further, the product may be a biosensor.
Further, the product for detecting cronobacter further comprises a magnetic separation medium, such as Magnetic Nanoparticles (MNPs), attached to the nucleic acid aptamer.
The invention has the beneficial effects that:
(1) Compared with the antibody, the aptamer has the advantages of capability of screening in vitro, short screening period, convenience in synthesis, easiness in labeling various functional groups and reporter molecules, stable property, capability of long-term storage and use and the like;
(2) The sequence is an aptamer sequence with strong affinity, specificity and stability, which is obtained by cutting based on the secondary structure of the original sequence, and can specifically identify all species bacteria of the Cronobacter;
(3) Compared with the original Cronobacter oxydans aptamer obtained by screening, the sequence has ideal affinity, good specificity and lower synthesis cost, and the Cronobacter oxydans in the environment and food can be detected and separated more sensitively based on a detection method constructed.
(4) The aptamer is a novel recognition element of Cronobacter, has the advantages of high sensitivity, low cost, easiness in preparation and modification and marking, and can be applied to construction of various detection methods.
(5) The aptamer provided by the invention can be combined with the magnetic nanoparticle to achieve the purpose of quantitatively detecting and enriching the Cronobacter. The method comprises the following steps: biotin modification is carried out on the 5' -end of the aptamer to obtain a biotinylation aptamer; then, the aptamer is combined to the surface of the magnetic nanoparticle by utilizing the specific combination of biotin and avidin, so as to obtain the aptamer functionalized magnetic nanoparticle; and then, utilizing the specific combination of the aptamer and the Cronobacter, separating and enriching the Cronobacter in the sample under the action of an external magnetic field, so as to realize the qualitative or quantitative detection of the Cronobacter.
Drawings
FIG. 1 is a flow chart of aptamer sequence truncation optimization of the invention.
FIG. 2 is a saturated binding curve of aptamer A9P-43 fitted using GRAPHPADPRISM software according to the present invention.
FIG. 3 shows the results of the specificity of the aptamer A9P-43 obtained by flow cytometry according to the present invention.
FIG. 4 is a CD diagram of the aptamer A9P and truncated sequence A9P-43 of the invention.
FIG. 5 is a fluorescent image of the binding of FAM-labeled aptamer A9P-43 of the invention to Cronobacter sakazakii.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
Example 1 truncated optimization of aptamer sequences
The secondary structure of each truncated aptamer predicted by M-fold was determined by sequence affinity using a flow cytometer and the dissociation constant was calculated (K d). Sample and blank groups (no aptamer added) were set, after which detection of forward scatter, side scatter and fluorescence intensity was performed using a flow cytometer. The K d value is an important indicator for characterizing aptamer affinity. The greater the K d value, the weaker the affinity of the aptamer. Conversely, the smaller the K d value, the stronger the affinity of the aptamer.
The aptamer sequence truncation optimization flow is shown in figure 1. Cronobacter sakazakii, dublin Cronobacter sakazakii and Mo Jinsi Cronobacter sakazakii are taken as targets. First, the primer region (A) at both ends of A9P (nucleotide sequence shown in SEQ ID NO. 1) was removed to obtain the sequence A9P-62, wherein the nucleotide sequence is shown in SEQ ID NO.2. The affinity results are shown in table 1, and the reduced affinity of the truncated sequences compared to the original long aptamers suggests that the primer regions are fully or partially involved in the target recognition and binding process, which is critical for aptamer formation into stable target binding conformation. The aptamer A9P-57 obtained by truncating the (B) region, the nucleotide sequence of which is shown in SEQ ID NO.3, does not meet the expectation that truncated aptamers have increased affinity for all four targets. The aptamer A9P-58 is obtained by cutting off the (C) region, the nucleotide sequence of the aptamer is shown as SEQ ID NO.4, and the affinity of the aptamer for three targets is increased. Therefore, on the basis of the aptamer A9P-58, the aptamer A9P-43 (the nucleotide sequence is shown as SEQ ID NO. 5) is obtained by cutting off the (E) region again, and the sequence saturation binding curve is shown in FIG. 2. The K d values are all obviously reduced, the affinity is respectively improved by 1.62,1.63,5.35 times compared with that of the original aptamer A9P, and the base is reduced by 37.
The lower the Gibbs free energy (DeltaG) value of the aptamer is, the more stable the structure of the aptamer is, the DeltaG of each aptamer is predicted by M-fold as shown in Table 1, the original long sequence DeltaG is-10.02 kcal/mol, and the DeltaG of truncated optimized aptamer A9P-43 is-11.75 kcal/mol, which indicates that the optimized sequence has better stability. By analysis of the above results, we successfully obtained truncated aptamer A9P-43, which has significantly better affinity and stability than the original aptamer.
TABLE 1
Example 2 aptamer-specific assay
And carrying out specificity analysis on the aptamer obtained by cutting optimization by using a flow cytometer. As shown in FIG. 3, the specificity measurement results show that the fluorescence intensities of the aptamer A9P-43 and Mo Jinsi Cronobacter sakazakii after being combined with Dublin Cronobacter sakazakii and Cronobacter sakazakii are 66.38%,61.14% and 69.10%, respectively, and the values of the aptamer A9P-43 and the Cronobacter sakazakii for the rest food-borne pathogenic bacteria are not more than 11%, which shows that the aptamer A9P-43 has higher affinity and good specificity for three Cronobacter sakazakii.
EXAMPLE 3 round two chromatography
Round dichroism (Circular dichroism, CD) is an important method to study conformational changes before and after aptamer binding to a target. A CD map of the aptamer A9P and the truncated sequence A9P-43 is shown in FIG. 4. Both the original aptamer A9P and the truncated aptamer A9P-43 have a positive peak at 260-290nm, which is a characteristic peak of base stacking, a negative peak around 240nm is the result of helix force, the position of the positive and negative peaks of the truncated aptamer A9P-43 are offset compared with A9P, the intensity is enhanced, and an additional positive peak is displayed at 210nm, which indicates that the truncated aptamer structure is changed, the aptamer double helix stacking effect and helix force are enhanced, the stem region of the aptamer secondary structure is presumed to become more compact and stable, and the stem-loop structure enhancement may be due to the formation of a new stem-loop structure or an nonstandard base complementary pairing.
Example 4 visualization of aptamer binding to target
The interaction of the truncated optimized aptamer A9P-43 with three Cronobacter sakazakii was observed using a laser confocal microscope. The fluorescence image of binding of FAM-labeled aptamer A9P-43 to Cronobacter crescentus is shown in FIG. 5. In comparison with the blank, under a laser confocal microscope, aptamer A9P-43 containing FAM label showed fluorescent signals after binding to Cronobacter sakazakii, cronobacter dublin and Mo Jinsi Cronobacter sakazakii; in contrast, none of the three Cronobacter bacteria were able to detect fluorescent signals without binding to any of the aptamers. In conclusion, the aptamer A9P-43 obtained through truncation optimization in the study has good binding capacity to Cronobacter sakazakii, cronobacter dublinii and Cronobacter Mo Jinsi. Therefore, we consider that the A9P-43 aptamer has potential for application in aptamer-based assay platforms for detection of cronobacter contamination in food products.
Example 5 application of aptamer functionalized magnetic beads to capture Cronobacter in sample
(1) Synthetic aptamer functionalized magnetic beads
The aminated magnetic beads are dispersed in PBS buffer solution with the avidin concentration of 250 mug/mL, the supernatant containing the free avidin is removed under the action of magnetic separation after shaking for 4 hours at 37 ℃ under 130r/min in a dark place, and the supernatant is resuspended in 5mL PBS buffer solution containing 5% BSA and shaking for 3 hours in a dark place slowly. After the reaction, the reaction mixture was washed with 1 XB & W buffer and finally resuspended in 1 XB & W buffer. Avidin magnetic beads with the volume ratio of 1:1 were incubated with 4. Mu.M biotinylated A9P-43 at 37℃for 2h at 130r/min to synthesize MNPs-A9P-43. After three magnetic washes with 1 XB & W buffer, resuspended in 1 XB & W buffer and allowed to stand overnight to equilibrate.
(2) Enrichment capture detection of Cronobacter
Bacteria were grown to 10 8 cfu/mL, a series of bacterial suspensions were obtained by dilution of the bacterial broth, and incubated with 700. Mu.L of MNPs-A9P-43 at a concentration of 1mg/mL at 25 ℃. The captured bacteria were collected by magnetic separation, the captured cells were washed twice with BB buffer, resuspended in BB buffer, 100. Mu.L was plated on DFI plates, incubated at 37℃for 12h, the number of enriched captured bacteria and the total bacteria in the bacterial suspension were determined by three counts by plate counting, and the capture efficiency was calculated. Capture efficiency = number of captured bacteria/total number of bacteria in bacterial suspension x 100%. Six non-target bacteria food-borne pathogens (E.coli, salmonella, vibrio parahaemolyticus, shigella flexneri, staphylococcus aureus, listeria monocytogenes) were used for specificity verification. The bacterial concentration of 10 3 cfu/mL was selected and the capture rate was determined under the same detection conditions. The experimental results are shown in table 2, and the capturing efficiencies of the aptamer functionalized magnetic beads to Mo Jinsi Cronobacter sakazakii, dublin Cronobacter sakazakii and Cronobacter sakazakii in the samples are respectively 80.38%, 78.75% and 84.86%; the capturing efficiency of other strains is not more than 20%, and the method has good specificity and can effectively separate the enriched Cronobacter.
TABLE 2
(3) Cronobacter analysis of infant formula
In order to verify the enrichment capture performance of the constructed aptamer-based enrichment capture Cronobacter platform for all species of Cronobacter, we extended it to other species of Cronobacter (Cronobacter malonate, kang Dimeng Cronobacter, you Niwo Cronobacter stoniensis, cronobacter zurich). The affinity of the aptamer A9P-43 for four strains was first verified by flow cytometry, and the results are shown in FIG. 2, wherein the aptamer A9P-43 shows good affinity for Cronobacter malonate, cronobacter Kang Dimeng, cronobacter zurich and Cronobacter You Niwo, indicating that the truncated and optimized aptamer has excellent binding performance for the four Cronobacter.
And selecting infant formula milk powder samples for the standard adding and recycling experiment. The total number of bacteria in the infant formula sample before enrichment and the number of bacteria captured by MNPs-A9P-43 after enrichment were determined by using a conventional plate counting method, and the results are summarized in Table 3, and the recovery rate is 66.67% -89.95%. The result not only shows that the truncated optimized aptamer can specifically identify all species bacteria of the enterobacter sakazakii, greatly solves the difficulty of enrichment and capture limitation, but also shows that the constructed platform can be used for enrichment and capture of the Cronobacter sakazakii in actual samples.
TABLE 3 Table 3
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (7)
1. A nucleic acid aptamer specifically recognizing cronobacter, wherein the nucleic acid aptamer is a nucleotide sequence shown as SEQ ID No. 5.
2. The nucleic acid aptamer of claim 1, wherein: the 5 'end or the 3' end of the nucleic acid aptamer is modified with a functional group or a molecule.
3. The nucleic acid aptamer of claim 2, wherein: the functional group or molecule is selected from at least one of isotope, electrochemical marker, enzyme marker, fluorescent group, biotin, affinity ligand, sulfhydryl and amino.
4. Use of a nucleic acid aptamer according to any one of claims 1-3 for the preparation of a product for detecting cronobacter, characterized in that: the Cronobacter is one or more selected from Cronobacter sakazakii, cronobacter malonate, cronobacter zurich, cronobacter Mo Jinsi, cronobacter Kang Dimeng, cronobacter You Niwo and Cronobacter dublin.
5. A product for detecting cronobacter, characterized in that: a nucleic acid aptamer comprising any one of claims 1-3, wherein the cronobacter is selected from one or more of cronobacter sakazakii, cronobacter malonate, cronobacter zurich, mo Jinsi cronobacter, kang Dimeng cronobacter, you Niwo s cronobacter and dublin cronobacter.
6. The product according to claim 5, wherein: the product is a biosensor.
7. The product according to claim 5, wherein: the product for detecting the Cronobacter comprises a magnetic separation medium connected with the nucleic acid aptamer.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109593837A (en) * | 2017-09-29 | 2019-04-09 | 东北农业大学 | A method of the nanogold colorimetric method based on magnetic capture quickly detects the rugged Cronobacter sakazakii of slope |
| CN109825554A (en) * | 2017-11-23 | 2019-05-31 | 东北农业大学 | A method of the quickly detection rugged Cronobacter sakazakii of slope is captured using immune magnetic |
| CN110951898A (en) * | 2019-12-30 | 2020-04-03 | 广东省微生物研究所（广东省微生物分析检测中心） | Specific novel molecular target of 4 species in Cronobacter and rapid detection method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109593837A (en) * | 2017-09-29 | 2019-04-09 | 东北农业大学 | A method of the nanogold colorimetric method based on magnetic capture quickly detects the rugged Cronobacter sakazakii of slope |
| CN109825554A (en) * | 2017-11-23 | 2019-05-31 | 东北农业大学 | A method of the quickly detection rugged Cronobacter sakazakii of slope is captured using immune magnetic |
| CN110951898A (en) * | 2019-12-30 | 2020-04-03 | 广东省微生物研究所（广东省微生物分析检测中心） | Specific novel molecular target of 4 species in Cronobacter and rapid detection method thereof |
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