CN109207567A

CN109207567A - It is a kind of based on aptamers and strand displacement amplification reaction to the measuring method of staphylococcus aureus

Info

Publication number: CN109207567A
Application number: CN201811121430.3A
Authority: CN
Inventors: 周楠迪; 蔡蓉凤; 尹凡; 田亚平
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2018-09-26
Filing date: 2018-09-26
Publication date: 2019-01-15
Anticipated expiration: 2038-09-26
Also published as: CN109207567B

Abstract

A determination method for Staphylococcus aureus based on aptamer and strand displacement amplification reaction belongs to the technical field of analytical chemistry, food safety and fermentation analysis. In the present invention, a target recognition system is first constructed, and through the high affinity between Staphylococcus aureus and the aptamer, the cDNA can be competitively released from the double strand; then the cDNA is used as a template, and a trigger primer sequence is added to the solution to amplify a large number of Single-stranded DNA with a specific sequence; after adding a specific hairpin structure, the single-stranded DNA and the hairpin structure self-assemble in solution to form a specific hexagonal structure; finally, a fluorescent dye is added, and the dye binds specifically to the DNA double-strand and releases the fluorescence signal. The content of Staphylococcus aureus in the sample was calculated using the linear relationship between the fluorescence signal and the number of Staphylococcus aureus. According to the principle of fluorescence resonance energy transfer, the invention correlates the fluorescence signal with the quantity of Staphylococcus aureus, and realizes the direct determination of the content of Staphylococcus aureus in food samples such as milk. The established detection method has the characteristics of short detection period, high sensitivity, low cost and strong specificity.

Description

A kind of measurement based on aptamers and strand displacement amplification reaction to staphylococcus aureus Method

Technical field

The present invention relates to a kind of based on aptamers and strand displacement amplification reaction to the measuring method of staphylococcus aureus, can To carry out highly sensitive measurement to staphylococcus aureus content in food and fermented sample, belong to analytical chemistry and food safety With Analysis offermehtations technical field.

Background technique

Staphylococcus aureus is a kind of common human pathogen, is more common in raw milk and raw meat, can produce a variety of Endotoxin causes various infection and food origin disease, such as septicemia, infectious endocarditis (IE) and osteoarthritis, skin Skin and soft tissue, pleura lung etc. seriously threaten human health, about 30% S. aureus carrier in crowd.Together When staphylococcus aureus be also often contaminated bacteria in fermentation process.Therefore research and development are to the highly sensitive, quasi- of staphylococcus aureus True detection method, for more caused by food safety monitoring and Analysis offermehtations, clinical prevention and treatment staphylococcus aureus Kind infection has great importance.

Currently, the method for detection staphylococcus aureus includes tradition culture, biochemistry detection (such as chromogenic culture medium) is immunized Method (such as Enzyme-Linked Immunospot), molecular biology method (such as multiple PCR technique).Wherein, tradition culture and biochemistry Detection method detection time is long, is easy to be influenced by miscellaneous bacteria colonial morphology and quantity and mutation staphylococcus aureus, out Existing false negative result；The immunological method test period is long, complex steps, and various non related antigens easily cause cross reaction, food Enterotoxin Protein agglutination generated in heating process etc. will cause the false positive or false negative of testing result；Molecular biology method It is same to be easy to be influenced by factors such as sample compositions.When these commonly detect methods of staphylococcus aureuses or need long Between processing, skilled worker and valuableness laboratory equipment, or have unstable, sensitivity is low, accuracy is poor etc. to lack Point.Therefore sensitive efficient detection methods of staphylococcus aureus is established, food security supervision and fermentation process are monitored etc. All have important meaning.

In recent years, with the development of combinatorial chemistry and biosensor technique, a variety of biosensor quilts based on aptamers For detecting pathogenic microorganism.These biosensors are made of detecting element and recognition component, are detected by signal conversion Target substance such as electrochemical sensor, fluorescent optical sensor, colorimetric sensor etc..Aptamers are obtained by SELEX screening technique, are There is the DNA or RNA molecule of high-affinity and specificity to particular target.Compared with antibody, aptamers are more easily-synthesized and store, To widen their application.Importantly, many method for amplifying signal based on nucleic acid molecules have further been widened and have been fitted Detection range with body sensor, such as rolling circle amplification (RCA), strand displacement amplification (SDA) and hybridization chain reaction (HCR) etc., these technologies are more and more widely used in the external qualitative detection of DNA/RNA.Biosensor has The features such as quickly, specificity is high, and sensitivity is strong for detection, reusable, application range is also increasingly extensive.

Summary of the invention

The purpose of the present invention is overcoming above-mentioned shortcoming, one kind is established based on aptamers and strand displacement amplification reaction to gold Staphylococcus aureus carries out highly sensitive, high specific, accurate measuring method.

Technical solution of the present invention, key technology of the present invention include cDNA in conjunction with aptamers after, staphylococcus aureus With the affine race problem of aptamers；The building of Nb.bpu10I nicking restriction endonuclease and Bsm archaeal dna polymerase dual-enzyme coupling system, Expand specific dna sequence；It is designed, DNA is made to be self-assembly of specific space structure and is characterized using DNA sequence dna；It utilizes Fluorescent dye converts the content of staphylococcus aureus to the variation of fluorescence signal.Include content are as follows: first design with it is golden yellow The cDNA of color staphylococcus aptamers partial complementarity is allowed to form part heteroduplex with aptamers, and is fixed on magnetic bead surfaces, When, there are when staphylococcus aureus, due to its affinity between aptamers, cDNA being made to be replaced and discharge in solution；Secondly In the presence of triggering primer, the cDNA of release is in conjunction with triggering primer, there are Nb.bpu10I restriction endonuclease, Bsm polymerase and When free dNTPs, a large amount of single stranded DNA is generated by the collective effect iterative cycles amplification of two kinds of enzymes；Then toward detection system Middle hairpin structure sequence of the addition through designing, the single stranded DNA generated with amplification are incubated for by certain time, form specific six side Shape DNA self-assembled structures；Finally using DNA self-assembled structures as template, SYBR Green I fluorescent dye is added, it is total by fluorescence Energy transfer principles of shaking emit fluorescence signal, utilize the linear relationship of the fluorescence intensity and staphylococcus aureus content that detect To calculate Gold Samples staphylococcus aureus content (Fig. 1).

It is described based on aptamers and strand displacement amplification reaction to the measuring method of staphylococcus aureus, first building target Identification system, the sequence cDNA of partial complementarity forms double-strand by the staphylococcus aureus aptamers that are fixed on magnetic bead and therewith Structure, by the high affinity between staphylococcus aureus and aptamers, the competitor release effect cDNA from double-strand；Then with CDNA is template, triggering primer sequence is added in the solution, using the Nb.bpu10I restriction enzyme with special cleavage site Enzyme and Bsm archaeal dna polymerase with strong strand displacement effect carry out coupling reaction, amplify a large amount of single-stranded with particular sequence DNA；After specific hairpin structure is added, single stranded DNA and hairpin structure are self-assembly of specific hexagonal structure in the solution；Most SYBR Green I fluorescent dye is added afterwards, dyestuff and DNA double chain are specifically bound, and discharge fluorescence signal.Utilize fluorescence signal The staphylococcus aureus content in sample is calculated with the linear relationship of staphylococcus aureus quantity.

Specific step is as follows:

(1) target identifies system:

A, the formation of aptamers and cDNA duplex structure: the aptamers sequence provided according to document, it will be on the label of the end aptamers 5' Biotin, and design the cDNA sequence with aptamers partial complementarity；By 0.25 μm of olL^-1CDNA and 0.25 μm of olL^-1It is suitable 0.1molL is added in ligand^-1It is mixed in 7.4 system of PBS, pH, 37 DEG C is slowly cooled to after 95 DEG C of denaturation 5min, be incubated for 120min makes aptamers and cDNA form duplex structure by base pair complementarity principle；

Wherein aptamers sequence is as shown in SEQ ID NO.1；With the cDNA sequence such as SEQ ID NO.2 institute of aptamers partial complementarity Show；

The document is specially Moon J, Kim G, Park S B, et al. Comparison of Whole-Cell SELEX Methods for the Identification of Staphylococcus Aureus-Specific DNA Aptamers[J]. Sensors, 2015, 15(4):8884-8897。

B, magnetic bead cleaning and modification: the suspension containing magnetic beads that Streptavidin is modified are mixed with vortex mixer, take 100 μ L magnetic beads outstanding Liquid, Magnetic Isolation removes PBS buffer solution after being cleaned magnetic bead 3 times using 1mL PBS buffer solution, and it is resulting mixed that 100 μ L step a are added Liquid is closed, 37 DEG C of shaking tables, 220rmin are placed in^-1It is incubated for 45min, double-strand is passed through into the affinity between Streptavidin and biotin Double-strand is fixed to magnetic bead surfaces；

C, the competitive binding of staphylococcus aureus and separation: by the sample containing staphylococcus aureus in 3000rmin^-1 It is centrifuged 5min, is used PBS buffer solution cleaning precipitating 3 times；The resulting suspension containing magnetic beads of step b clean magnetic bead 3 with 1mL PBS buffer solution It is secondary, it is finally resuspended in the PBS buffer solution that 100 μ L contain staphylococcus aureus, is mixed, in 37 DEG C of shaking tables, 220 r min^-1It is incubated for 45 min；Magnetic bead is removed into system using Beads enrichment system, retains the supernatant containing free cDNA；

(2) strand displacement amplification system

D, cDNA and triggering primer form double-strand: by 0.0125 μm of olL^-1Primer is triggered as shown in SEQ ID NO.3,95 It is added in step c supernatant after DEG C high-temperature denaturation and slowly cools to 37 DEG C, be incubated for 120min, make to trigger in primer and supernatant CDNA passes through base pair complementarity；

E, the amplified reaction of strand displacement shearing: 7.5U Nb.bpu10I nicking restriction endonuclease being added into 3 μ L above-mentioned steps d solution, 3U Bsm archaeal dna polymerase and 250 μm of olL^-1Free deoxyribonucleoside triphosphate, 10 mmolL^-1 Tris- HCl, 10 mmolL^-1 MgCl₂, 100 mmolL^-1KCl, 0.1 mgmL^-1BSA is incubated for after mixing at 37 DEG C 100min obtains the single stranded DNA of massive amplification；

(3) DNA self assembly: 0.715 μm of olL^-1Step (2) are added after 95 DEG C of denaturation 5min and complete strand displacement for hairpin structure The reaction solution of amplification slowly cools to 37 DEG C of incubation 120min；

(4) fluorescence signal detection and Specification Curve of Increasing: SYBR Green I fluorescent dye is diluted 10000 times, 30 μ L is taken to add Enter in above-mentioned steps (3) solution, dye 120min, 96 hole elisa Plates are added, read blank using multi-function microplate reader and contains There is the fluorescence signal of staphylococcus aureus solution to change, for the excitation wavelength used for 497 nm, measurement launch wavelength is 520 The fluorescence signal of nm；By shaking flask culture staphylococcus aureus solution, by diluting again, according to sensor fluorescent value and golden yellow Relationship between staphylococcic concentration draws out corresponding linear relationship curve；

(5) actual sample detects: by the sample containing staphylococcus aureus with step (1) ~ (4) described operation, determining phase The fluorescent value answered calculates corresponding bacteria concentration from standard curve.

Step (1) described magnetic bead is the magnetic bead that Streptavidin modification is commercialized, and is purchased from the Tianjin good limited duty of science and technology of benefit Ren company.

Step (1) aptamers are the staphylococcus aureus aptamers of 5 ' terminal modified biotins, are purchased from raw work biology Engineering (Shanghai) limited liability company.

The staphylococcus aureus solution of step (4) described various concentration is specially 7 ~ 7 × 10⁷ CFU/mL。

SEQ ID NO.1 aptamers:

biotin-(CH₂)₆-CACACCGCAGCAGTGGGAACGTTTCAGCCATGCAAGCATCACGCCCGT；

SEQ ID NO.2 partial complementarity DNA (cDNA):

TAATGCCTGATAATGCCTGATAATGCCTGATAATGCCTGATAATGCCTGATAATGCCTGAGCACGGGCGTGA TGCTTGCA；

SEQ ID NO.3 triggers primer: TGCAAGCATC；

SEQ ID NO.4 hairpin structure (Hp) CGACTAATGCCTGAGTCG.

Beneficial effects of the present invention: the present invention constructs the gold based on strand displacement amplification art and DNA self-assembled structures Staphylococcus aureus aptamer sensor.The concentration that critical sequences have been expanded by strand displacement amplification art, is exaggerated fluorescence signal, Expand the sensor detection range, improves detection sensitivity.Tradition side of this method compared to detection staphylococcus aureus Method, high specificity, high sensitivity.

Detailed description of the invention

Fig. 1 is the staphylococcus aureus fluorescence detection schematic diagram based on strand displacement amplification art and DNA self-assembled structures.

Fig. 2 is staphylococcus aureus standard fluorometric detection curve.

Specific embodiment

Application principle of the invention is further described below with reference to specific example.

Nb.bpu10I nicking restriction endonuclease and Bsm archaeal dna polymerase in following embodiment are purchased from the silent winged generation of match, and you are scientific and technological (China) Co., Ltd；SYBR Green I fluorescent dye is purchased from Sangon Biotech (Shanghai) Co., Ltd..

The nucleic acid sequence of aptamers used in the embodiment of the present invention and cDNA etc.:

Table 1

。

The drafting of 1 staphylococcus aureus concentration standard curve of embodiment

Magnetic bead and 100 μ L aptamers-cDNA the compounds 220rmin in 37 DEG C of shaking tables for taking 100 μ L Streptavidins to modify^-1It is incubated for 45min.Magnetic bead is cleaned using PBS buffer solution, after Magnetic Isolation, ten times of gradient dilutions are added in each EP pipe respectively Staphylococcus aureus bacterium solution, concentration range be 7 ~ 7 × 10⁷ CFU·mL^-1.In 37 DEG C of shaking table 220rmin^-1It is incubated for 45min cleans magnetic bead, retains supernatant after Magnetic Isolation.3 μ L supernatants are taken, 0.0125 μm of olL is added^-1Primer is triggered, 0.1 U·μL^-1Bsm archaeal dna polymerase, 0.25 U μ L^-1Nb.bpu10I nicking restriction endonuclease, 250 μm of olL^-1Free Deoxyribonucleoside triphosphate, 10 mmolL^-1Tris-HCl, 10 mmolL^-1 MgCl₂, 100 mmolL^-1KCl, 0.1mg·mL^-1BSA is incubated for 100 min in 37 DEG C of water-baths after mixing, is eventually adding 30 μ L SYBR Green I dyeing 40min measures fluorescent intensity.It is mapped to obtain standard curve with fluorescence intensity to staphylococcus aureus concentration, such as Fig. 2 institute Show, fluorescence intensity increases with the increase of staphylococcus aureus concentration, and equation of linear regression is the x+ of y=26561 12219, R²It is 0.9976, wherein y indicates fluorescence intensity, and x indicates Log (c [staphylococcus aureus]/CFUmL^-1), it should The detection of method is limited to 1.69 CFU mL^-1。

The measurement of staphylococcus aureus content in the practical milk sample of embodiment 2.

In order to further verify accuracy of this method in measuring practical milk sample when staphylococcus aureus content, select Commercially available certain milk, 12000 rmin^-115min is centrifuged with protein precipitation, by 0.22 μm of water phase membrane filtration two of supernatant It is secondary.By 80 μ L staphylococcus aureuses in an aseptic environment with 10²To 10⁶ CFU·mL^-1Be inoculated into 20 by diluted concentration again In the processed milk sample product of μ L.The magnetic bead and 100 μ L aptamers-cDNA compounds for taking 100 μ L Streptavidins to modify are in 37 220 rmin in DEG C shaking table^-1It is incubated for 45 min.Magnetic bead is cleaned, after Magnetic Isolation, pretreated milk actual sample is added, In 37 DEG C of 220 rmin of shaking table^-145 min are incubated for, magnetic bead is cleaned, retains supernatant after Magnetic Isolation.3 μ L supernatants are taken, are added Enter 0.0125 μm of olL^-1Trigger primer, 0.1 U μ L^-1Bsm archaeal dna polymerase, 0.25 U μ L^-1Nb.bpu10I nicking Restriction endonuclease, 250 μm of olL^-1Free deoxyribonucleoside triphosphate, 10 mmolL^-1Tris-HCl, 10 mmolL^-1 MgCl₂, 100 mmolL^-1KCl, 0.1 mgmL^-1BSA is incubated for 100 min in 37 DEG C of water-baths after mixing, is eventually adding 30 μ L SYBR Green I dyes 40 min, measures fluorescent intensity, and staphylococcus aureus can be calculated by substituting into standard curve Concentration, concrete outcome are as shown in table 2.

Table 2

。

Sequence table

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Claims

1. a kind of assay method to Staphylococcus aureus based on aptamer and strand displacement amplification reaction, it is characterized in that the steps are as follows: first build a target recognition system, by being fixed on the Staphylococcus aureus aptamer on magnetic beads and The partially complementary sequence cDNA forms a double-stranded structure. Through the high affinity between Staphylococcus aureus and the aptamer, the cDNA is competitively released from the double-strand; The Nb.bpu10I restriction endonuclease with a special cutting site and the Bsm DNA polymerase with a strong strand displacement effect are coupled to amplify a large number of single-stranded DNA with a specific sequence; The strand DNA and the hairpin structure self-assemble in solution to form a specific hexagonal structure; finally, SYBR Green I fluorescent dye is added, and the dye binds specifically to the DNA double-strand to release a fluorescent signal; the fluorescence signal is used to correlate with the number of Staphylococcus aureus. A linear relationship was used to calculate the Staphylococcus aureus content in the sample.

2. the assay method to Staphylococcus aureus based on aptamer and strand displacement amplification reaction according to claim 1, is characterized in that concrete steps are as follows:

(1) Target recognition system:

a. Formation of aptamer and cDNA double-stranded structure: label the 5' end of the aptamer sequence with biotin, and design a cDNA sequence that is partially complementary to the aptamer; combine 0.25μmol·L ^-1 cDNA with 0.25μmol· L ^-1 aptamer was added to 0.1mol·L ^-1 PBS, pH 7.4 system, mixed well, denatured at 95°C for 5 min, slowly cooled to 37°C, and incubated for 120 min, so that the aptamer and cDNA formed double-stranded pairing based on the principle of complementary base pairing. chain structure;

The aptamer sequence is shown in SEQ ID NO.1; the cDNA sequence complementary to the aptamer part is shown in SEQ ID NO.2;

b. Magnetic bead cleaning and modification: Mix the streptavidin-modified magnetic bead suspension with a mixer, take 100 μL of the magnetic bead suspension, wash the magnetic beads with 1 mL of PBS buffer three times, and remove the PBS buffer by magnetic separation Add 100 μL of the mixture obtained in step a, place it on a shaker at 37°C, incubate at 220 r·min ^-1 for 45 min, and immobilize the double strands on the surface of the magnetic beads through the affinity between streptavidin and biotin;

c. Competitive binding and separation of Staphylococcus aureus: Centrifuge the sample containing Staphylococcus aureus at 3000 r·min ^-1 for 5 min, and wash the precipitate 3 times with PBS buffer; the magnetic bead suspension obtained in step b is buffered with 1 mL of PBS The magnetic beads were washed 3 times with the solution, and finally resuspended in 100 μL of PBS buffer containing Staphylococcus aureus, mixed well, and incubated at 37 °C for 45 min at 220 r·min ^-1 ; the magnetic beads were separated by a magnetic bead separation system. Bead removal system, retaining supernatant containing free cDNA;

(2) Strand displacement amplification system

d. The cDNA and the trigger primer form double-stranded: 0.0125 μmol·L ^-1 trigger primer is denatured at a high temperature of 95°C, then added to the supernatant of step c, slowly cooled to 37°C, and incubated for 120 minutes, so that the trigger primer and the cDNA in the supernatant are combined. by complementary base pairing;

The trigger primer is shown in SEQ ID NO.3;

e. Amplification reaction of strand displacement shearing: add 7.5U Nb.bpu10I nicking endonuclease, 3UBsm DNA polymerase and 250 μmol·L ^-1 free deoxyribonucleoside triphosphate to 3 μL of step d solution, 10 mmol·L ^-1 Tris-HCl, 10 mmol·L ^-1 MgCl ₂ , 100 mmol·L ^-1 KCl, 0.1 mg·mL ^-1 BSA, and incubate at 37°C for 100 min after mixing to obtain a large amount of amplified single-chain DNA;

(3) DNA self-assembly: 0.715 μmol·L ^-1 hairpin structure was denatured at 95°C for 5 minutes, then added to the reaction solution of step (2) to complete the strand displacement amplification, and slowly cooled to 37°C and incubated for 120 minutes;

The hairpin structure is shown in SEQ ID NO.4;

(4) Fluorescence signal detection and standard curve drawing: Dilute SYBR Green I fluorescent dye 10,000 times, add 30 μL to the solution in step (3), stain for 120 minutes, add to 96-well microplate, and use a multi-function microplate reader to read the blank As well as the change of the fluorescence signal of the solution containing Staphylococcus aureus, the excitation wavelength was 497 nm, and the fluorescence signal was measured at the emission wavelength of 520 nm; the Staphylococcus aureus solution was cultured in a shaker flask, and the solution was diluted step by step. The relationship between the concentrations of Staphylococcus aureus, draw the corresponding linear relationship curve;

(5) Actual sample detection: The samples containing Staphylococcus aureus are operated as described in steps (1) to (4), the corresponding fluorescence values are determined, and the corresponding bacterial concentration is calculated from the standard curve.

3. The assay method for Staphylococcus aureus based on aptamer and strand displacement amplification reaction according to claim 2, characterized in that: the different concentrations of Staphylococcus aureus solutions described in step (4) are specifically 7-7 ×10 ⁷ CFU/mL.

4. the assay method to Staphylococcus aureus based on aptamer and strand displacement amplification reaction according to claim 2, is characterized in that:

SEQ ID NO.1 aptamer:

biotin-(CH ₂ ) ₆ -CACACCGCAG CAGTGGGAAC GTTTCAGCCA TGCAAGCATC ACGCCCGT;

SEQ ID NO.2 Partially complementary DNA (cDNA):

TAATGCCTGA TAATGCCTGA TAATGCCTGA TAATGCCTGA TAATGCCTGA TAATGCCTGAGCACGGGCGT GATGCTTGCA;

SEQ ID NO.3 trigger primer: TGCAAGCATC;

SEQ ID NO. 4 Hairpin structure: (Hp) CGACTAATGC CTGAGTCG.