CN110632300A

CN110632300A - Aptamer-based biosensor for detecting salmonella and preparation method and application thereof

Info

Publication number: CN110632300A
Application number: CN201910890363.XA
Authority: CN
Inventors: 王玉; 李莎莎; 刘素; 黄加栋; 张儒峰; 赵一菡; 瞿晓南; 张雪; 宋晓蕾; 王海旺; 王敬锋
Original assignee: University of Jinan
Current assignee: University of Jinan
Priority date: 2019-09-20
Filing date: 2019-09-20
Publication date: 2019-12-31
Anticipated expiration: 2039-09-20
Also published as: CN110632300B

Abstract

The invention relates to the technical field of biosensors, in particular to a biosensor for detecting salmonella based on a nucleic acid aptamer, which is characterized in that based on the specific recognition of the nucleic acid aptamer and a target object, a hairpin probe HAP is opened, S1 is replaced from a composite probe S by utilizing a fulcrum-mediated strand displacement reaction, the replaced S can expose a sequence for forming a G-tetrad in a catalytic hairpin self-assembly (CHA) amplification mode, and the G-tetrad/heme DNase is formed in the presence of heme. The catalytic performance of G-tetrad/heme horseradish peroxidase is used for oxidizing cysteine into cystine, and charge transfer between cysteine and silver clusters through gold-sulfur bonds cannot be realized, so that fluorescence signal conduction is regulated and controlled, and an aptamer biosensor is constructed.

Description

Aptamer-based biosensor for detecting salmonella and preparation method and application thereof

Technical Field

The invention relates to the technical field of biosensors, in particular to a biosensor for detecting salmonella based on a nucleic acid aptamer, and also relates to a preparation method thereof.

Background

Salmonella is a common food-borne pathogenic bacterium, and is a gram-negative intestinal bacterium parasitized in cells. The bacterium is widely existed in nature, not only can cause acute, chronic or recessive infection of livestock, poultry and other animals, but also can cause food poisoning of people by polluting food, thereby causing great threat to human beings. According to statistics, the food poisoning caused by salmonella is often listed as the top in various countries in the world of bacterial food poisoning. Salmonella causes 70 million deaths each year. If no action is taken to stop this global threat, it is expected that 1000 million people will die globally by the year 2050. Salmonella is the most common pathogenic bacterium in food poisoning in China, and accounts for the first place of food poisoning.

The currently reported methods for detecting salmonella include traditional culture methods, enzyme-linked immunosorbent assay, PCR technology, etc. The traditional salmonella detection methods have long detection period, complicated procedures and expensive equipment, and can not meet the requirements far away. Therefore, a rapid, accurate, simple and trace analysis method is urgently needed to detect salmonella in food. In recent years, DNA biosensing detection technology has gained wide attention by virtue of its high sensitivity and specificity. The fluorescence technology has the remarkable advantages of high sensitivity, strong specificity, low price, no need of sample pretreatment and the like, and is more and more emphasized by people in the fields of biology, medicine and the like.

Disclosure of Invention

In order to solve the problems of low specificity and sensitivity, high cost and long detection period of the method for detecting the salmonella in the prior art, the invention provides the biosensor for detecting the salmonella, which has high specificity and sensitivity, low cost and high detection speed and is based on enzyme-free fluorescence signal conduction.

Another object of the present invention is to provide a method and use of the above biosensor for detecting Salmonella.

In order to achieve the purpose, the invention adopts the following technical scheme.

A biosensor for detecting salmonella comprises a hairpin probe HAP, a composite probe S, a nucleic acid strand S2, a nucleic acid strand C, a hairpin probe H1, a hairpin probe H2, a hairpin probe H3, cysteine, heme, salmonella and a buffer solution;

the HAP base series is shown as SEQ No. 1;

the base series of S0 is shown as SEQ No. 2;

the base series of S1 is shown as SEQ No. 3;

the base series of S2 is shown as SEQ No. 4;

the H1 base series is shown as SEQ No. 5;

the H2 base series is shown as SEQ No. 6;

the H3 base series is shown as SEQ No. 7;

the C base series is shown as SEQ No. 8.

The detection of the salmonella is realized in a homogeneous solution, and the amplification of a signal is realized in an isothermal amplification mode without enzyme assistance, so that the high-sensitivity detection of the salmonella is realized, and a lower detection lower limit is obtained. In the homogeneous reaction, the reaction conditions were 37 ℃ and the reaction time was 90 min.

The preparation method of the biosensor for detecting the salmonella comprises the following steps:

(1) constructing a composite probe S;

(2) synthesis of DNA silver nanoclusters (AgNCs-DNA);

(3) homogeneous reaction: adding salmonella and hairpin probe HAP into the homogeneous phase, simultaneously adding composite probe S, nucleic acid chain S2, H1, H2, H3, AgNCs-DNA cysteine, heme and buffer solution, mixing uniformly and then incubating;

(4) the fluorometer detects the intensity of the fluorescence.

The construction steps of the composite probe S in the step (1) are as follows:

sterile water, 10 XPB, S0 probe and S1 probe were added to pre-prepared sterile EP tubes, shaken for 30S, incubated at 95 ℃ for 5min, slowly cooled to room temperature to hybridize as probes, stored at-20 ℃ until use.

The step (2) of synthesizing the DNA silver nanoclusters (AgNCs-DNA) comprises the following operation steps:

mu.L of 100. mu.M nucleic acid strand C and 73. mu.L of 20 mM PB (pH 7.0) buffer were added to an EP tube wrapped in tinfoil, followed by 6. mu.L of a 1.5 mM AgNO3 solution (ensuring Ag)⁺Mixing with H3 at a ratio of 6: 1), shaking for 1 min, and standing at 4 deg.C for 30 min;

after 30 min, continuously adding 6 μ L of 1.5 mM NaBH4 into the EP tube, shaking for 1 min, and standing in the dark at 4 deg.C for more than 6h to obtain the final product.

The homogeneous reaction operation in the step (3) comprises the following steps:

3 μ L of Salmonella (5.0X 10)⁵cfu/mL), hairpin probe HAP (1.5. mu.L, 500 nM), composite probe S (3. mu.L, 1. mu.M), nucleic acid strand S2 (3. mu.L, 1. mu.M), H1 (3. mu.L, 1. mu.M), H2 (3. mu.L, 1. mu.M), H3 (3. mu.L, 1. mu.M), AgNCs-DNA (14. mu.L, 4. mu.M), cysteine 1. mu.M, heme (3. mu.L, 1. mu.M), and buffer were added to the centrifuge tubes, shaken for 30S, and water-washed at 37 ℃ for 90 min.

And (4) setting the excitation wavelength of the fluorometer to 570 nm.

The biosensor is used for detecting salmonella in food and water.

The sequences used in this application are:

HAP: 5’- AGTAATGCCCGGTAGTTATTCAAAGATGAGTAGGAAAAGATTGGCATTACTAT

GGGTCTCACTATG-3’

S0: 5’-AAAAGAACCCATAGTGAGACCCATGAACCCATAGTGAGACCCATAGTAATG

CC-3’

S1: 5’- ATGGGTCTCACTATGGGTTCAACG-3’

S2: 5’- GTCTCACTATGGGTTCATGGGTCTCACTATGG-3’

H1: 5’-TGGGTAGGGCGGGTCGTTGAACCCATAGTGAGACCCATATGGGTCAAGA

CATGGGTCTCACTATGGGT-3’

H2: 5’-TGGGTAGGGCGGGTAGTGAGACCCATGTCTTGACCCATATGGGTTCAAC

GATGGGTCAAGACATGGGT-3’

H3: 5’-TGGGTAGGGCGGGTGTCTTGACCCATCGTTGAACCCATATGGGTCTCACT

ATGGGTTCAACGATGGGT-3’

C: 5’-CCCCCCCCCCCC-3’

the black italic part of hairpin probe HAP is the aptamer sequence of salmonella, and the black bold font of hairpin probe H1, H2 and H3 is the split G tetrad sequence. Hybridizing the S0 probe and the S1 probe in a ratio of 1:2 to synthesize a composite probe S, wherein when a target exists, the target is specifically combined with the aptamer, thereby opening the hairpin structure of the HAP, the opened HAP can bind to the terminal toehold region of the composite probe S, replace S1 from the composite probe S, while the second foothold region is exposed, the nucleic acid strand S2 hybridizes thereto through the exposed foothold region, the other strand S1 and the opened HAP are further displaced, the displaced opened HAP is subjected to the next round of circulation, and then obtaining a large amount of S1, the replaced S1 can open H1, the opened H1 can continue to open H2, the opened H2 can further open H3, the opened H3 can replace S1, the catalytic hairpin self-assembly amplification (CHA) is realized, at the same time, the sequence forming the G-tetrad is exposed, and G-tetrad/heme DNA enzyme is formed in the presence of heme. The catalytic performance of G-tetrad/heme horseradish peroxidase is used for oxidizing cysteine into cystine, and charge transfer between cysteine and silver clusters through gold-sulfur bonds cannot be realized, so that fluorescent signal conduction is regulated and controlled, and quantitative detection of salmonella is realized.

The detection mode of the invention is fluorescence detection, and a great amount of G-quadruplet DNA enzyme is generated by utilizing the chain displacement cyclic amplification and CHA amplification functions mediated by the fulcrum region, so that the oxidation of the cysteine is realized, and the fluorescence intensity is obviously changed. The detection of the target is performed by detecting the fluorescence intensity of the solution.

The invention utilizes the special identification of the aptamer to realize the high specificity detection of the target salmonella; using the chain displacement circulation amplification mediated by the fulcrum region to generate a large amount of secondary products S1; by utilizing the CHA amplification function, G-quadruplet DNA enzyme is continuously generated, the oxidation of cysteine is realized, the detection signal is amplified, the detection sensitivity is improved, and the ultra-sensitivity detection of the target salmonella is realized; the sensor has mild reaction conditions and high reaction speed; compared with other optical detection means, the detection method is simple and convenient to operate and short in detection period; the detection process is realized in a homogeneous phase, so that the complexity of operation is reduced; the process cost of the biosensor is effectively reduced without enzyme participation, and the method is suitable for the requirements of low cost in industrialization.

The invention has the beneficial effects that:

1. detection limit is low

The specific recognition of the aptamer is utilized, and the combination of the aptamer and salmonella is utilized to realize the high-specificity detection of the target object; the chain displacement cyclic amplification mediated by the fulcrum region is utilized, the cyclic utilization of HAP is realized, a large amount of secondary products S1 are generated, the detection signal is amplified, and the detection sensitivity is improved; the CHA amplification function is utilized to continuously generate G-quadruplet DNA enzyme, so that the sensitivity of the sensor is effectively improved; the detection line can reach 0.45.

2. Simple method and stable performance

The construction of the sensor only needs one step, thereby effectively avoiding the pollution possibly caused by adding samples in multiple steps and having the advantages of simple and convenient operation, high reaction speed and the like; the main processes of the detection principle are realized in a homogeneous phase, so that the reaction speed is improved, the complexity of operation is reduced, and the rapid, simple and sensitive detection of the target object is realized.

3. Detection of salmonella in food and water

The process for manufacturing the biosensor has low cost and is suitable for the requirement of low price in industrialization. Is suitable for food safety, the detection of salmonella in water and the practical application of biosensor industrialization.

Drawings

FIG. 1 is a schematic diagram of the experiment;

FIG. 2 is a graph showing the results of concentration optimization of H1 in example 1;

FIG. 3 is a graph showing the optimized detection results of hemoglobin concentration in example 2

FIG. 4 is a graph showing the results of the reaction time optimization assay in example 3.

FIG. 5 is a standard curve for the detection of Salmonella in example 4.

Detailed Description

The present invention is further illustrated by the following specific examples.

Example 1

The preparation method of the fluorescence biosensor comprises the following steps:

the synthetic operation steps of the composite probe S are as follows:

mu.L of sterilized water, 3. mu.L of 10 XPB, 3. mu.L of 100. mu.M S0 probe and 6. mu.L of 100. mu.M S1 probe were added to a pre-prepared sterilized EP tube, shaken for 30S, incubated at 95 ℃ for 5min, slowly cooled to room temperature to hybridize as probes, and stored at-20 ℃ until use.

The operation steps of the synthesis of DNA silver nanoclusters (AgNCs-DNA) are as follows:

mu.L of 100. mu.M nucleic acid strand C and 73. mu.L of 20 mM PB (pH 7.0) buffer were added to an EP tube wrapped in tinfoil, followed by 6. mu.L of 1.5 mM AgNO3 solution (ensuring a ratio of Ag + to H3 of 6: 1), shaking for 1 min, and standing at 4 ℃ for 30 min; after 30 min, continuously adding 6 mu L of 1.5 mM NaBH4 into the EP tube, shaking for 1 min, and placing in the dark at 4 ℃ for more than 6h to obtain the product;

the main steps of the reaction process in the homogeneous solution are as follows:

3 μ L of Salmonella (5.0X 10)⁵cfu/mL), hairpin probe HAP (1.5. mu.L, 500 nM), composite probe S (3. mu.L, 1. mu.M), nucleic acid strand S2 (3. mu.L, 1. mu.M), H1 (final concentrations 0.4. mu.M, 0.6. mu.M, 0.8. mu.M, 1.0. mu.M, 1.2. mu.M, 1.4. mu.M), H2 (3. mu.L, 1. mu.M), H3 (3. mu.L, 1. mu.M), AgNCs-DNA (14. mu.L, 4. mu.M), cysteine 1. mu.M, hemoglobin (3. mu.L, 1. mu.M), and buffer were added to the centrifuge tube, shaken for 30S, and water bath at 37 ℃ for 90 min.

The fluorescence intensity detection by the fluorescence instrument mainly comprises the following steps:

the solution after the homogeneous reaction (30. mu.L) was diluted to 100. mu.L, and the fluorescence peak intensity was detected at 635nm using a fluorometer.

The excitation wavelength of the fluorometer is set to 570nm, the emission wavelength is 635nm, the detection range is 570nm-800nm, the change of a fluorescence signal is read, and the target object is detected.

The results are shown in FIG. 2, from which it can be seen that the peak of the detected fluorescence intensity decreases with increasing concentration of H1, and that the fluorescence intensity tends to stabilize when the concentration exceeds 1.0. mu.M. Therefore, the optimal final concentration of H1 was 1.0. mu.M.

Example 2

the synthetic operation steps of the composite probe S are as follows:

3 μ L of Salmonella (5.0X 10)⁵cfu/mL), hairpin probe HAP (1.5. mu.L, 500 nM), composite probe S (3. mu.L, 1. mu.M), nucleic acid strand S2 (3. mu.L, 1. mu.M), H1 (3. mu.L, 1. mu.M), H2 (3. mu.L, 1. mu.M), H3 (3. mu.L, 1. mu.M), AgNCs-DNA (14. mu.L, 4. mu.M), cysteine 1. mu.M, 3. mu.L of hemoglobin (final concentrations of 0.01. mu.M, 0.05. mu.M, 0.1. mu.M, 0.5. mu.M, 1. mu.M, 5. mu.M, 10. mu.M, respectively), and buffer were added to a centrifuge tube, shaken for 30S, and water bath at 37 ℃ for 90 min.

The results are shown in FIG. 3, from which it can be seen that the peak of the detected fluorescence intensity decreases as the concentration of hemoglobin increases, and that the fluorescence intensity tends to stabilize when the concentration exceeds 1.0. mu.M. Therefore, the optimal final concentration of hemoglobin is 1.0. mu.M.

Example 3

the synthetic operation steps of the composite probe S are as follows:

3 μ L of Salmonella (5.0X 10)⁵cfu/mL), hairpin probe HAP (1.5. mu.L, 500 nM), composite probe S (3. mu.L, 1. mu.M), nucleic acid strand S2 (3. mu.L, 1. mu.M), H1 (3. mu.L, 1. mu.M), H2 (3. mu.L, 1. mu.M), H3 (3. mu.L, 1. mu.M), AgNCs-DNA (14. mu.L, 4. mu.M), cysteine 1. mu.M, heme (3. mu.L, 1. mu.M), buffer were added to the centrifuge tubes, shaken for 30S, bathed in water at 37 ℃ for 30 min, 45min, 60min, 75 min, 90min, 105 min, 120 min.

As a result, as shown in FIG. 4, it can be seen that the peak value of the detected fluorescence intensity decreases with the increase of the reaction time, and the fluorescence intensity tends to be stable when the reaction time exceeds 90 min. The optimum homogeneous reaction time is 90 min.

Example 4

the synthetic operation steps of the composite probe S are as follows:

3 μ L of Salmonella (5.0X 10)⁵，1.0×10⁵，5.0×10⁴，1.0×10⁴，5.0×10³，1.0×10³，5.0×10²，1.0×10²50, 10 cfu/mL), hairpin probe HAP (1.5. mu.L, 500 nM), composite probe S (3. mu.L, 1. mu.M), nucleic acid strand S2 (3. mu.L, 1. mu.M), H1 (3. mu.L, 1. mu.M), H2 (3. mu.L, 1. mu.M), H3 (3. mu.L, 1. mu.M), AgNCs-DNA (14. mu.L, 4. mu.M), cysteine 1. mu.M, heme (3. mu.L, 1. mu.M), and buffer were added to the centrifuge tube, shaken for 30S, and water-washed at 37 ℃ for 90 min.

The results are shown in FIG. 5, from which it can be seen that the peak of the detected fluorescence intensity decreases with increasing Salmonella concentration, when the concentration exceeds 5.0X 10⁵After cfu/mL, the fluorescence intensity tends to be stable. Therefore, the optimal final concentration of Salmonella is 5.0X 10⁵ cfu/mL。

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the embodiments, and any other changes, modifications, combinations, substitutions and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.

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Claims

1. A biosensor for detecting salmonella is characterized by comprising a hairpin probe HAP, a composite probe S, a nucleic acid strand S2, a nucleic acid strand C, a hairpin probe H1, a hairpin probe H2, a hairpin probe H3, cysteine, heme, salmonella and a buffer solution;

the composite probe S is composed of an S0 probe, an S1 probe 1:2 into a double strand;

the HAP base series is shown as SEQ No. 1;

the base series of S0 is shown as SEQ No. 2;

the base series of S1 is shown as SEQ No. 3;

the base series of S2 is shown as SEQ No. 4;

the H1 base series is shown as SEQ No. 5;

the H2 base series is shown as SEQ No. 6;

the H3 base series is shown as SEQ No. 7;

the C base series is shown as SEQ No. 8.

2. The method for preparing a biosensor in accordance with claim 1, comprising the steps of:

(1) constructing a composite probe S;

(2) synthesizing DNA silver nanocluster AgNCs-DNA;

(3) homogeneous reaction: uniformly mixing salmonella, a hairpin probe HAP, a composite probe S, a nucleic acid chain S2, H1, H2, H3, AgNCs-DNA, cysteine, heme and a buffer solution, and then incubating;

(4) the fluorometer detects the intensity of the fluorescence.

3. The method for detecting Salmonella according to claim 2, wherein the composite probe S of step (1) is constructed by the following steps:

the sterile water, 10 XPB, S0 probe and S1 probe are shaken for 30S, incubated at 95 ℃ for 5min, slowly cooled to room temperature, hybridized into probe, and stored at-20 ℃ for standby.

4. The method for detecting salmonella as claimed in claim 2, wherein the step (2) of synthesizing the DNA silver nanocluster AgNCs-DNA comprises the following steps:

mixing the nucleic acid chain C and PB buffer solution, and then adding AgNO₃Shaking the solution for 1 min, and standing at 4 deg.C for 30 min;

adding NaBH₄Shaking for 1 min, and standing at 4 deg.C in dark for more than 6 hr.

5. The method for detecting Salmonella of claim 2, wherein the homogeneous reaction of step (3) is carried out by:

mixing salmonella, hairpin probe HAP, composite probe S, nucleic acid chain S2, H1, H2, H3, AgNCs-DNA, cysteine, heme and buffer solution, shaking for 30S, and water-bathing for 90min at 37 ℃.

6. The method for detecting Salmonella of claim 2, wherein said step (4) fluorometer sets the excitation wavelength to 570 nm.

7. The biosensor of claim 1 for detecting salmonella in food and water.