CN102808022B - Application of oligonucleotide aptamer capable of identifying salmonella specifically - Google Patents
Application of oligonucleotide aptamer capable of identifying salmonella specifically Download PDFInfo
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Abstract
The invention relates to application of an oligonucleotide aptamer capable of identifying salmonella specifically. The oligonucleotide aptamer is a single-stranded deoxyribonucleic acid (DNA) which has 78 bases in length, and is applied to the detection of the salmonella 08. Food-borne salmonella 08 is used as a target molecule, and escherichia coli 086: K61 and salmonella choleraesuis are used as reverse screening bacteria, so that a specific aptamer of the salmonella 08 is obtained. The method has the advantages of short detection time, short development period, stable quality, simplicity of operation and the like, and is widely applied to the detection of food and sanitary safety.
Description
Invention field
The invention belongs to biology, food and health detection technique field, be specifically related to the purposes of the oligonucleotide aptamer of specific recognition Salmonellas (serotype O8, hereinafter to be referred as Salmonellas O8).
Background technology
At present, both at home and abroad when Salmonella in Food detects conventional detection method because of its detection means, identifying object each tool relative merits of having nothing in common with each other.The method of traditional detection Salmonellas needs first separation to cultivate again, then by classical method, identifies, consuming time, insensitive is the ubiquitous problems of these methods.Immunological method is simple, convenient, rapid, specificity is better, but still has the weak points such as cross reaction is relatively more serious, false positive is many, sensitivity is on the low side.Polymerase chain reaction (PCR) is though that technology has is accurate, sensitive, feature fast, but its operation when detecting the more sample of number is more numerous and diverse, therefore on the basis of polymerase chain reaction (PCR) technology, derive again a lot of novel polymeric polymerase chain reaction (PCR) technology, as multiple PCR technique, though yet multiplex PCR has been simplified the operation of PCR experiment, but because this technology need severally increase to primer simultaneously, be easy to produce non-specific band or false positive, affect detected result.
The oligonucleotide sequence that phyletic evolution (Systematic evolution of ligands by exponential enrichment, SELEX) technology screening by index concentration part obtains is called aptamer (aptamer).Its principle is utilized Protocols in Molecular Biology exactly, build the strand random oligonucleotide library of synthetic, its stochastic sequence length is 20-100 base left and right, random oligonucleotide library and target molecule are interacted, the oligonucleotide aglucon that retains combination, through repeated amplification, screen several circulations, can make to obtain enrichment with the oligonucleotide sequence of this target specific combination, finally obtain the special oligonucleotide aglucon of target molecule.This technology has the advantages such as storage capacity is large, target molecule scope is wide, avidity is high, high specificity.Aspect clinical detection, the particularly research to some unknown pathogenic bacterias or virus, although do not know the epi-position of its internal structure, function and these materials, but using it as target material, by SELEX technology screening, arrive its corresponding aptamer, to detect target material, become the study hotspot in this field.
Utilize pattern and the protein antibodies of the aptamer identification molecule that SELEX technology screening obtains similar, but compare with protein antibody, nucleic acid aglucon has more superiority, if not limited by immune condition and immunogenicity, can external synthetic, sex change and renaturation are reversible, can modify and be conducive to long-term preservation and room temperature transportation etc.The more important thing is, the target molecule of aptamer is more extensive, comprises metal ion, organic dye, amino acid, cytokine, cofactor, aminoglycoside, microbiotic, base analogue, Nucleotide and polypeptide etc.Wherein protein-based target molecule is maximum, comprises enzyme, somatomedin, antibody, transcription factor, cell adhesion molecule and selection element etc.Complete virion and bacterial pathogens, even complete cell also can or be subdued the oligonucleotide aglucon that SELEX technology screening goes out high-affinity by compound target SELEX technology.Aptamer has higher specificity and accurate recognition capability than antibody, even can identify the undistinguishable protein molecule of monoclonal antibody.These characteristics make aptamer be used widely in biological medicine and food sanitation research field, become indispensable powerful.
Summary of the invention
In order to solve the problems such as the sense cycle existing in existing Salmonellas detection is long, sensitivity is low, false positive is many, the invention provides the application of the oligonucleotide aptamer of a kind of specific recognition Salmonellas.
The nucleotide sequence of the oligonucleotide aptamer of specific recognition Salmonellas is as follows: 5'-GATCCGGGCCTCATGTCGAACACCCCCCAACTAAAACAACAAAACACCACCGC CATTGAGCGTTTATTCTGAGCTCCCA-3', the oligonucleotide aptamer of specific recognition Salmonellas is the single stranded DNA that 78 base is long.The present invention, by the screening process of SELEX technology, obtains the oligonucleotide aptamer of high-affinity specific recognition Salmonellas, for fast, accurately detecting Salmonellas O8.
The present invention be take Foodborne salmonella O8 as object target molecule, take intestinal bacteria O86:K61 and Salmonella choleraesuls simultaneously as anti-sieve bacterium, has obtained an aptamer that Salmonellas O8 is special.Present method has that detection time is short, the R&D cycle is short, steady quality, simple operation and other advantages, in food and hygienic safety detect, is used widely.
Accompanying drawing explanation
Fig. 1 is the measurement result figure of single stranded oligonucleotide (ssDNA) enrichment storehouse and Salmonellas O8 combination rate.
Fig. 2 A is No. 10, oligonucleotide aptamer and Salmonellas O8 sensitivity determination result figure.
Fig. 2 B is No. 9, oligonucleotide aptamer and Salmonellas O8 sensitivity determination result figure.
Fig. 3 A is that fluorescence microscope fluorophor labeled oligonucleotide aptamer is combined comparison diagram for No. 10 with intestinal bacteria.
Fig. 3 B is that fluorescence microscope fluorophor labeled oligonucleotide aptamer is combined comparison diagram for No. 10 with Salmonella choleraesuls.
Fig. 3 C is that fluorescence microscope fluorophor labeled oligonucleotide aptamer is combined comparison diagram for No. 10 with Salmonellas O8.
Fig. 3 D is blank figure after fluorescence microscope fluorophor labeled oligonucleotide aptamer is combined wash-out with distilled water No. 10.
Embodiment
The nucleotide sequence of the oligonucleotide aptamer of specific recognition Salmonellas is as follows: 5'-GATCCGGGCCTCATGTCGAACACCCCCCAACTAAAACAACAAAACACCACCGC CATTGAGCGTTTATTCTGAGCTCCCA-3'; The oligonucleotide aptamer of specific recognition Salmonellas is the single stranded DNA that 78 base is long.The present invention, by the screening process of SELEX technology, obtains the oligonucleotide aptamer of high-affinity specific recognition Salmonellas, for fast, accurately detecting Salmonellas O8.
Below by SELEX screening, obtain the special oligonucleotide aptamer of Salmonellas O8, and the oligonucleotide aptamer that wherein avidity is the highest is described further Salmonellas O8 identification and evaluation.
1. random single-stranded DNA banks and primer are synthetic by Shanghai biotechnology company limited
Random single-stranded DNA banks: 5 '-GGGAGCTCAGAATAAACGCTCAA-
35nt-TTCGACATGAGGCCCGGATC-3 ' (note: nt represent any one in A, T, C, G base)
Primer I: 5 ’ – GGGAGCTCAGAATAAACGCTCAA-3 '
Primer II: 5 '-GATCCGGGCCTCATGTCGAA-3 '
Primer III: 5 '-Di Gao Xin – GGGAGCTCAGAATAAACGCTCAA-3 '
Primer IV: 5 '-vitamin H-GATCCGGGCCTCATGTCGAA-3 '.
2. Salmonellas O8(
salmonella O8) preparation
Salmonellas O8(
salmonella O8,purchased from Chinese industrial microbial strains preservation management center C ICC) be inoculated in 37 ℃ of nutrient agar plates and cultivate 15～18 hours, scraping lawn is in 0.9% stroke-physiological saline solution test tube, centrifugal 10 min of 6000 4 ℃ of rpm, washing thalline 3 times, abandon supernatant, be resuspended in 0.9% stroke-physiological saline solution, using and adjusting turbidity than turbid instrument is 0.5, is approximately equivalent to 1.5 * 108/ml Salmonellas O8 bacteria suspension.
3.SELEX screening obtains the special oligonucleotide aptamer of Salmonellas O8
1) SELEX screening process:
A. first run screening, gets synthetic random single chain DNA 1/3 OD (10 μ g) and joins 400ul 1 * binding buffer liquid, and 95 degree sex change 5min, are then placed in rapidly 10min on ice;
B. add 1mL bacteria suspension 1.5 * 10
8, in 37 ° of C shaking table 100 rpm, in conjunction with 1 hour (can reduce binding time) later, single-stranded DNA banks and bacterium are fully acted on;
C. change centrifuge tube, to remove the single stranded DNA of being combined with tube wall, the single-stranded DNA banks that under room temperature, the centrifugal 10min separation of centrifugal 10 000 rpm is not combined with bacterium;
D. abandon supernatant, add 600 μ L 1 * dcq buffer liquid, the centrifugal 10min of centrifugal 10 000 rpm, repeats this process 4 times, and object is to wash away the nucleic acid fragment of not being combined with bacterium;
E. connect step and remove supernatant after centrifugal, add 99 ° of C heating 3min of 100 μ L deionized water, the centrifugal 15min of high speed centrifugation 18 000 rpm abandons precipitation, and replace tubes stays supernatant (nucleic acid fragment is stored in supernatant), and-20 ° of C save backup.
2) oligonucleotide aptamer of PCR enrichment and Salmonellas O8 specific combination:
Increased and obtained enrichment by PCR in every oligonucleotide aptamer library with Salmonellas O8 specific combination obtaining after screening of taking turns;
A. take above-mentioned supernatant as template, by primer I and the amplification of primer IV, produce the biotin labeled double-stranded DNA of one end band;
B. pcr amplification condition: 95 ° of C denaturation 3min, then carry out 95 ° of C sex change 30s of 30 circulation, 60 ° of C annealing 30s, 72 ° of C extend 30s, and last 72 ° of C extend 10min;
C. after PCR product purification, band biotin labeled double-stranded DNA in one end is combined by the effect magnetic bead crosslinked with streptavidin between vitamin H-streptavidin, through 1 * connection (the standard Binding and Washing Buffer, B & W) damping fluid washing is 3 times, 37 ° of C sex change 30 min of fresh NaOH with 100mM, make not to be with the single stranded DNA of vitamin H to elute from the crosslinked magnetic bead of streptavidin, measure the concentration of its single stranded DNA, as the enrichment storehouse of next round screening.
3) repeat screening: repeat above-mentioned SELEX screening process and pcr amplification enrichment process, carry out altogether 9 and take turns screening, wherein the 5th, 8 take turns respectively with intestinal bacteria, Salmonella choleraesuls are counter sieves.
4) mensuration of enrichment oligonucleotide aptamer and bacterium combination rate:
A. the 1st, 3,5,7,9,11 take turns the products that SELEX screens, with the primer III of mark digoxin and the primer IV of mark vitamin H, carry out pcr amplification, the PCR product of purifying fully reacts and unwinds with NaOH with the magnetic bead of streptavidin mark, and the single stranded DNA of digoxigenin labeled is free in supernatant;
B. press single stranded DNA: Salmonellas O8=300pmol:1.5 * 10
8combination in 1 * binding buffer liquid, centrifugal, abandon supernatant, alkaline phosphatase enzyme reaction 15 min that add anti digoxin antibody mark, repeated washing, washes away the alkaline phosphatase of the anti digoxin antibody combination of not being combined with the upper single stranded DNA-digoxin of Salmonellas O8, with 5-bromo-4-chloro-3-indolylphosphate salt/NBT (BCIP/NBT), develops the color, after termination reaction, with enzyme connection instrument, in 405 nm, measure absorbance A value, see Fig. 1.
5) sequencing result in enrichment oligonucleotide aptamer library and analysis:
A. by last enriched library amplification, be two strands, connect pEGM-T carrier, transform
e.colidH5 α, random 22 positive colonies of picking carry out determined dna sequence, and wherein 19 is available sequences, in Table 1;
B. adopt the homology of Clustal W, RNA STRUCTURE and 19 sequence primary structures of DNAMAN software analysis and carry out the prediction of secondary structure;
C. binding affinity height and aglucon higher structure, select the sequence (No. 10, oligonucleotide aptamer, No. 9, oligonucleotide aptamer) of two oligonucleotide aptamers that avidity is the highest, energy level is lower, carries out next step evaluation.
4. fluorophor labeled oligonucleotide aptamer (No. 10, oligonucleotide aptamer) is identified with the fluorescent microscope of Salmonellas O8
1) No. 10 sequences of oligonucleotide aptamer being served to marine life Engineering Co., Ltd synthesizes and holds mark hydroxyl fluorescein (FAM) 5 '.
5'-FAM-GATCCGGGCCTCATGTCGAACACCCCCCAACTAAAACAACAAAACACCACCGCCATTGAGCGTTTATTCTGAGCTCCCA-3'；
2) by No. 10, the oligonucleotide aptamer of FAM mark respectively with Salmonellas O8, the combination in 1 * binding buffer liquid of Salmonella choleraesuls and intestinal bacteria, 37 ° of C are hatched 1 hour, the centrifugal supernatant of abandoning, washing for several times, until supernatant does not have fluorescence;
3) bacterium precipitation is resuspended with 10 μ L 1 * lavation buffer solutions, be coated on slide glass, overdo fixing;
4) slide glass is placed in to damping fluid and washs 2-3 time, 2 minutes/time, finally with sterile distilled water, rinsed for 2 seconds;
5) dry, add mountant, cover glass.Fluorescence microscopy Microscopic observation is shown in Fig. 3 in conjunction with situation.
experimental result:
By SELEX, screen with avidity and analyze, No. 10, the special oligonucleotide aptamer of avidity is the highest, energy level is minimum Salmonellas O8 have been obtained, and No. 10 sequences of oligonucleotide aptamer are carried out after fluorophor tag application, and upper microscopes is obviously observed with Salmonellas O8 specific combination is occurred.
As seen from Figure 1, along with the increase of screening wheel number, the bonding force in single stranded DNA enrichment storehouse increases gradually, and the 9th takes turns screening has obtained good avidity, but when the binding site on Salmonellas reaches a state of saturation, the single stranded DNA being adsorbed onto on Salmonellas just no longer increases.
The oligonucleotide aptamer sequence that table 1 obtains for SELEX screening
No. 1, oligonucleotide aptamer:
GATCCGGGCCTCATGTCGAACCCACACCCCACAACCACCCAGCCCCAGCCCGCTATTGAGCGTTTATTCTGAGCTCCC
No. 2, oligonucleotide aptamer:
GATCCGGGCCTCATGTCGAACACAACACCCAGCCAACGACCACAACTCCAACTCATTGAGCGTTTATTCTGAGCTCCC
No. 3, oligonucleotide aptamer:
GATCCGGGCCTCATGTCGAAACCACAACCCAACAACAAGAACCACAAAGAGCCCCTTGAGCGTTTATTCTGAGCTCCC
No. 4, oligonucleotide aptamer:
GATCCGGGCCTCATGTCGAACACACACAGAACCACAACCACTAAACACGAGGGCCTTGAGCGTTTATTCTGAGCTCCC
No. 5, oligonucleotide aptamer:
GATCCGGGCCTCATGTCGAACCAACAAGGCAGAAAGAAACCGACAAACCGCACTGTTGAGCGTTTATTCTGAGCTCCC
No. 6, oligonucleotide aptamer:
GATCCGGGCCTCATGTCGAACCAACAAGGCAGAAAGAAACCGACAAACCGCACTGTTGAGCGTTTATTCTGAGCTCCC
No. 7, oligonucleotide aptamer:
GATCCGGGCCTCATGTCGAACCAACAAGGCAGAAAGAAACCGACAAACCGCACTGTTGAGCGTTTATTCTGAGCTCCC
No. 8, oligonucleotide aptamer:
GATCCGGGCCTCATGTCGAACCGAACGACTCAAAGATCAAGCCAAGCCACGCCCGTTGAGCGTTTATTCTGAGCTCCC
No. 9, oligonucleotide aptamer:
GATCCGGGCCTCATGTCGAACCAACCCAACACCAAAGAGACCACCACCACACGAGTTGAGCGTTTATTCTGAGCTCCC
No. 10, oligonucleotide aptamer:
GATCCGGGCCTCATGTCGAACACCCCCCAACTAAAACAACAAAACACCACCGCCATTGAGCGTTTATTCTGAGCTCCC
No. 11, oligonucleotide aptamer:
GATCCGGGCCTCATGTCGAACACGAGCCACCAACGCACCAAAACCCGTCCTCCACTTGAGCGTTTATTCTGAGCTCCC
No. 12, oligonucleotide aptamer:
GATCCGGGCCTCATGTCGAAGGCACGCGCACCAAAACACAAACTCCCCCCGCACCTTGAGCGTTTATTCTGAGCTCCC
No. 13, oligonucleotide aptamer:
GATCCGGGCCTCATGTCGAAGGGCCACCTAACCACCTCTGCCAATACCCCCCGCGTTGAGCGTTTATTCTGAGCTCCC
No. 14, oligonucleotide aptamer:
GATCCGGGCCTCATGTCGAAGGGAAGTCATGGCATGGATGCGCTCCCCGCCCACCTTGAGCGTTTATTCTGAGCTCCC
No. 15, oligonucleotide aptamer:
GATCCGGGCCTCATGTCGAAGGGAAGTCATGGCATGGATGCGCTCCCCGCCCACCTTGAGCGTTTATTCTGAGCTCCC
No. 16, oligonucleotide aptamer:
GATCCGGGCCTCATGTCGAACGGCGCAGTGACACGGTAGGGAGTCGTGCCGCGGCTTGAGCGTTTATTCTGAGCTCCC
No. 17, oligonucleotide aptamer:
GGGAGCTCAGAATAAACGCTCAAGGTGGGCGGGCGGCGTTGCTGTTCTTTTGCTGGTGTTCGACATGAGGCCCGGATC
No. 18, oligonucleotide aptamer:
GGGAGCTCAGAATAAACGCTCAAGAGGCGGCCTGTTCGCTTGTTGTGGGTCCGCTTGGTTCGACATGAGGCCCGGATC
No. 19, oligonucleotide aptamer:
GGGAGCTCAGAATAAACGCTCAAGGGCACTGGGTGGTGATGTTTTGTTTGTCTGGCGGTTCGACATGAGGCCCGGATC
No. 20, oligonucleotide aptamer:
GGGAGCTCAGAATAAACGCTCAAGGGGGGGGTGGAGGGGTATGCAGTTTCGGTGGCCGTTCGACATGAGGCCCGGATC
Table 1 has been listed 20 sequences, and in general, the secondary structure that free energy is minimum is the most stable.We select No. 10, oligonucleotide aptamer and oligonucleotide aptamer that free energy is lower to measure its avidity No. 9.
As seen from Figure 2, the dissociation constant that No. 10, oligonucleotide aptamer (Kd) is 32.04, and the dissociation constant that No. 9, oligonucleotide aptamer (Kd) is 175.9.Lower according to dissociation constant (Kd) value, fit avidity is just higher, finally we to select oligonucleotide aptamer be optimal fit body No. 10, and do further checking.
By Fig. 3, can obtain conclusion: No. 10 energy specific recognition Salmonellas O8 of fluorophor labeled oligonucleotide aptamer, have stronger fluorescence, and with intestinal bacteria and Salmonella choleraesuls without obvious combination, only there is the fluorescence of minute quantity.
In a word, No. 10 energy specific recognition Salmonellas O8 of oligonucleotide aptamer that screening obtains, susceptibility is high, speed is fast, and this invention can be used for the preparation of detection kit.The present invention is used for the concrete grammar of specific detection Salmonellas O8 as embodiment step 4.
Claims (2)
1. the oligonucleotide aptamer of specific recognition Salmonellas serotype O8, is characterized in that: the nucleotide sequence of described oligonucleotide aptamer is as follows:
5'-
GATCCGGGCCTCATGTCGAACACCCCCCAACTAAAACAACAAAACACCACCGCCATTGAGCGTTTATTCTGAGCTCCCA-3'。
2. the application of oligonucleotide aptamer according to claim 1 in detecting Foodborne salmonella serotype O8.
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