CN110632302A

CN110632302A - Method for simultaneously detecting contents of escherichia coli and salmonella in sample to be detected

Info

Publication number: CN110632302A
Application number: CN201911043414.1A
Authority: CN
Inventors: 郑金铠; 李玉芝; 陆畅; 周帅帅; 高飞
Original assignee: Institute of Food Science and Technology of CAAS
Current assignee: Institute of Food Science and Technology of CAAS
Priority date: 2019-10-30
Filing date: 2019-10-30
Publication date: 2019-12-31
Anticipated expiration: 2039-10-30
Also published as: CN110632302B

Abstract

The invention discloses a method for simultaneously detecting the contents of escherichia coli and salmonella in a sample to be detected. The method sequentially comprises the following steps: (1) adding excessive escherichia coli capture probes and excessive salmonella capture probes into a sample to be tested, and incubating; (2) adding excessive escherichia coli signal probes and excessive salmonella signal probes, and incubating; (3) SERS detects the intensity of Raman signal; (4) and substituting the Raman signal intensity into an Lg value and a corresponding Raman signal intensity according to the concentration of Escherichia coli O157: H7 and salmonella in the standard solution to draw a standard curve, so as to obtain the contents of Escherichia coli O157: H7 and salmonella in the sample to be detected. Experiments prove that the method can simultaneously detect the contents of Escherichia coli O157, H7 and salmonella in the food to be detected and has higher accuracy. The invention has important application value.

Description

Method for simultaneously detecting contents of escherichia coli and salmonella in sample to be detected

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for simultaneously detecting contents of escherichia coli and salmonella in a sample to be detected.

Background

Food-borne pathogenic bacteria refer to microorganisms present in food products that can cause illness in humans or animals. Escherichia coli and salmonella are common food-borne pathogenic bacteria and are easy to pollute food such as vegetables, fruits, eggs, milk and the like. Food poisoning events caused by food-borne pathogenic bacteria occur frequently, causing huge losses.

The effective detection of food-borne pathogenic bacteria is one of the important means for avoiding food poisoning. At present, conventional detection technologies for food-borne pathogenic bacteria mainly include a separation culture method, an immunological detection method (such as an enzyme-linked immunosorbent assay) and a molecular biological detection method (such as a PCR method), and although conventional detection requirements for food-borne pathogenic bacteria can be met, certain disadvantages still exist, for example, the separation culture method is time-consuming, the enzyme-linked immunosorbent assay and the PCR method are easy to generate false positive, and the requirements for simultaneous, rapid and highly sensitive on-site detection of various pathogenic bacteria in a food sample cannot be met. With the development of biotechnology, engineering technology and material technology, biosensor detection methods (such as optical biosensors) have the characteristics of good selectivity, high sensitivity, high speed, low cost and the like, and become an important method for detecting food-borne pathogenic bacteria.

Disclosure of Invention

The invention aims to detect the bacterial content in food to be detected.

The invention firstly protects a kit A for detecting different bacteria contents in a sample to be detected, which can comprise a plurality of reagents A for detecting different bacteria;

each reagent A for detecting bacteria can comprise a bacterial antibody, a single-chain DNA molecule and a Raman signal reporter;

the single-stranded DNA molecule sequentially comprises a DNA segment 1 and a DNA segment 2 from a 5 'end to a 3' end;

the DNA fragment 1 can be composed of N T, C or A, wherein N is a natural number of more than 16;

the DNA fragment 2 may be an aptamer DNA of the bacterium;

in each reagent A for detecting bacteria, the bacterial antibody, the DNA fragment 2 and the Raman signal reporter are different and are used for detecting different bacteria.

Each reagent a for detecting bacteria may specifically be composed of the bacterial antibody, the single-stranded DNA molecule, and the raman signal reporter.

The kit A can be composed of a plurality of reagents A for detecting different bacteria.

In any one of the kit A, a plurality of reagents A for detecting different bacteria have no cross reaction.

The kit A can also comprise a compound B, a compound A capable of being combined with the compound B and gold nano-materials.

Any one of the kit A can be composed of a plurality of reagents A for detecting different bacteria, a compound B, a compound A capable of being combined with the compound B and a gold nano material.

Any one of the kit A can also comprise nano microspheres.

Any one of the kit A can be composed of a plurality of reagents for detecting different bacteria, a compound B, a compound A capable of being combined with the compound B, a gold nano-material and a nano-microsphere.

The invention also discloses a kit B for detecting different bacteria contents in a sample to be detected, which can comprise a plurality of reagents B for detecting different bacteria;

each reagent B for detecting bacteria can comprise a bacterial antibody modified by a compound B, a nano microsphere modified by a compound A and a signal probe, wherein the nano microsphere is combined with the compound B; the signal probe can be a gold nano material jointly marked by a single-stranded DNA molecule and a Raman signal reporter;

the DNA fragment 2 may be an aptamer DNA of the bacterium;

in each reagent B for detecting bacteria, the bacterial antibody modified by the compound B, the nano-microsphere modified by the compound A and capable of being combined with the compound B and the signal probe are different and are used for detecting different bacteria.

In the kit b, the preparation method of the signal probe may be: mixing the gold nano material, the single-stranded DNA molecule and the Raman signal reporter, and reacting in a dark place; and then adding chloroauric acid and hydroxylamine hydrochloride, reacting, and obtaining a precipitate as a signal probe.

Each reagent B for detecting bacteria specifically can be composed of a bacterial antibody modified by the compound B, a nano-microsphere modified by the compound A and capable of being combined with the compound B, and the signal probe.

The kit B can be composed of a plurality of reagents B for detecting different bacteria.

In any one of the kit B, a plurality of reagents B for detecting different bacteria have no cross reaction.

Any of the above single-stranded DNA molecules may specifically consist of the above DNA fragment 1 and the above DNA fragment 2.

In any of the above DNA fragments 1, N may specifically be 20.

The invention also provides a method for detecting different bacteria content in a sample to be detected, which comprises the following steps of (a), step (b), step (c) and step (d):

the step (a) may include the steps of:

(a-1) subjecting the bacterial antibody to compound B modification to obtain a compound B-modified bacterial antibody; carrying out compound A modification on the nano-microsphere which can be combined with the compound B to obtain a compound A modified nano-microsphere; connecting the bacterial antibody modified by the compound B with the nano-microsphere modified by the compound A to obtain a capture probe;

(a-2) preparing different capture probes according to the method of step (a-1); different capture probes form a capture probe group; each capture probe in the set of capture probes is for capturing one bacterium;

(a-3) mixing the gold nano material, the single-stranded DNA molecule and the Raman signal reporter, and carrying out a light-shielding reaction; then adding chloroauric acid and hydroxylamine hydrochloride, reacting, and collecting precipitate; the sediment is a signal probe;

the single-stranded DNA molecule sequentially comprises a DNA fragment 1 and a DNA fragment 2 from a 5 'end to a 3' end;

the DNA fragment 1 consists of N T, C or A, and N is a natural number of more than 16;

the DNA fragment 2 is aptamer DNA of bacteria;

(a-4) preparing different signal probes according to the method of step (a-3); different signal probes form a signal probe group; each signaling probe in the signaling probe set is used for detecting a bacterium;

the step (b) may include the steps of:

(b-1) adding an excessive amount of capture probe sets to a sample to be tested, and incubating;

(b-2) adding an excess of the signal probe set after the step (b-1) is completed, and incubating;

(b-3) after the step (b-2) is completed, SERS detects the intensity of the Raman signal;

the step (c) may include the steps of:

(c-1) adding an excess of capture probe set to the bacterial standard solution, and incubating;

(c-2) adding an excess of the signal probe set after the step (c-1) is completed, and incubating;

(c-3) after the step (c-2) is completed, SERS detects the intensity of the Raman signal;

the step (d): drawing a standard curve according to the concentration of each bacterium in the bacterium standard solution and the corresponding Raman signal intensity, and substituting the Raman signal intensity obtained in the step (b-3) into the standard curve to obtain the content of each bacterium in the sample to be detected.

The invention also provides a method for detecting the contents of Escherichia coli O157: H7 and salmonella in a sample to be detected, which comprises the following steps (1), (2), (3) and (4):

the step (1) may include the steps of:

(1-1) carrying out compound B modification on the Escherichia coli O157: H7 monoclonal antibody to obtain a compound B modified Escherichia coli O157: H7 monoclonal antibody; carrying out compound A modification on the nano-microsphere which can be combined with the compound B to obtain a compound A modified nano-microsphere; connecting the Escherichia coli O157: H7 monoclonal antibody modified by the compound B with the nanometer microsphere modified by the compound A to obtain an Escherichia coli capture probe;

(1-2) modifying the salmonella monoclonal antibody with a compound B to obtain the salmonella monoclonal antibody modified by the compound B; connecting the salmonella monoclonal antibody modified by the compound B with the nano-microsphere modified by the compound A to obtain a salmonella capture probe;

(1-3) mixing the gold nano material, the single-stranded DNA molecule A and the Raman signal reporter A, and reacting in a dark place; then adding chloroauric acid and hydroxylamine hydrochloride, reacting, and collecting precipitate; the precipitate is an escherichia coli signal probe;

the single-stranded DNA molecule A sequentially comprises a DNA fragment 3 and a DNA fragment 4 from the 5 'end to the 3' end;

the DNA fragment 3 consists of N T, C or A, and N is a natural number of more than 16;

the nucleotide sequence of the DNA segment 4 can be shown as 21 st to 62 nd positions from the 5' end of SEQ ID NO. 1;

(1-4) mixing the gold nano material, the single-stranded DNA molecule B and the Raman signal reporter B, and reacting in a dark place; then adding chloroauric acid and hydroxylamine hydrochloride, reacting, and collecting precipitate; the sediment is the salmonella signal probe;

the single-stranded DNA molecule B sequentially comprises a DNA fragment 5 and a DNA fragment 6 from the 5 'end to the 3' end;

the DNA fragment 5 consists of N T, C or A, wherein N is a natural number of more than 16;

the nucleotide sequence of the DNA segment 6 can be shown as 21 st to 60 th positions from the 5' end of SEQ ID NO. 2;

the step (2) may include the steps of:

(2-1) adding excessive escherichia coli capture probes and excessive salmonella capture probes into a sample to be tested, and incubating;

(2-2) after the step (2-1) is finished, adding an excessive escherichia coli signal probe and an excessive salmonella signal probe, and incubating;

(2-3) after the step (2-2) is completed, SERS detects the intensity of the Raman signal;

the step (3) may include the steps of:

(3-1) adding an excess of an Escherichia coli capture probe and an excess of a Salmonella capture probe to a standard solution of Escherichia coli O157: H7 and Salmonella, and incubating;

(3-2) after the step (3-1) is finished, adding an excessive escherichia coli signal probe and an excessive salmonella signal probe, and incubating;

(3-3) after the step (3-2) is completed, SERS detects the intensity of the Raman signal;

the step (4): and (3) drawing a standard curve according to the concentrations of the Escherichia coli O157: H7 and the salmonella in the standard solution and the corresponding Raman signal intensity, and substituting the Raman signal intensity obtained in the step (2-3) into the standard curve to obtain the contents of the Escherichia coli O157: H7 and the salmonella in the sample to be detected.

The invention also provides a method for detecting the content of Escherichia coli O157: H7 in a sample to be detected, which comprises the following steps (f), (g), (H) and (i):

the step (f) may include the steps of:

(f-1) carrying out compound B modification on the Escherichia coli O157: H7 monoclonal antibody to obtain a compound B modified Escherichia coli O157: H7 monoclonal antibody; carrying out compound A modification on the nano-microsphere which can be combined with the compound B to obtain a compound A modified nano-microsphere; connecting the Escherichia coli O157: H7 monoclonal antibody modified by the compound B with the nanometer microsphere modified by the compound A to obtain an Escherichia coli capture probe;

(f-2) mixing the gold nano material, the single-stranded DNA molecule A and the Raman signal reporter A, and reacting in a dark place; then adding chloroauric acid and hydroxylamine hydrochloride, reacting, and collecting precipitate; the precipitate is an escherichia coli signal probe;

the nucleotide sequence of the DNA segment 4 is shown as the 21 st to 62 nd positions from the 5' end of SEQ ID NO. 1;

the step (g) may include the steps of:

(g-1) adding an excessive amount of an escherichia coli capture probe into a sample to be tested, and incubating;

(g-2) after the step (g-1) is completed, adding an excessive amount of an escherichia coli signal probe, and incubating;

(g-3) after the step (g-2) is completed, SERS detects the intensity of the Raman signal;

the step (h) may include the steps of:

(H-1) adding an excess amount of an Escherichia coli capture probe to an Escherichia coli O157: H7 standard solution, and incubating;

(h-2) after the step (h-1) is completed, adding an excessive amount of an escherichia coli signal probe, and incubating;

(h-3) after the step (h-2) is completed, SERS detects the intensity of the Raman signal;

the step (i): and (5) drawing a standard curve according to the concentration of the Escherichia coli O157: H7 in the Escherichia coli O157: H7 standard solution and the corresponding Raman signal intensity, and substituting the Raman signal intensity obtained in the step (g-3) into the standard curve to obtain the content of the Escherichia coli O157: H7 in the sample to be detected.

The invention also provides a method for detecting the content of salmonella in a sample to be detected, which comprises the following steps of (o), step (p), step (q) and step (r):

the step (o) may include the steps of:

(o-1) carrying out compound B modification on the salmonella monoclonal antibody to obtain a compound B modified salmonella monoclonal antibody; carrying out compound A modification on the nano-microsphere which can be combined with the compound B to obtain a compound A modified nano-microsphere; connecting the salmonella monoclonal antibody modified by the compound B with the nano-microsphere modified by the compound A to obtain a salmonella capture probe;

(o-2) mixing the gold nano-material, the single-stranded DNA molecule B and the Raman signal reporter B, and reacting in a dark place; then adding chloroauric acid and hydroxylamine hydrochloride, reacting, and collecting precipitate; the sediment is the salmonella signal probe;

the nucleotide sequence of the DNA segment 6 is shown as the 21 st to 60 th positions from the 5' end of SEQ ID NO. 2;

the step (p) may include the steps of:

(p-1) adding an excessive amount of salmonella capture probes into a sample to be tested, and incubating;

(p-2) after the step (p-1) is completed, adding an excessive amount of salmonella signaling probe, and incubating;

(p-3) after the step (p-2) is completed, SERS detects the intensity of the Raman signal;

the step (q) may include the steps of:

(q-1) adding an excess amount of the salmonella capture probe to the salmonella standard solution, and incubating;

(q-2) after the step (q-1) is completed, adding an excessive salmonella signaling probe, and incubating;

(q-3) after the step (q-2) is completed, SERS detects the Raman signal intensity;

the step (r): and (4) drawing a standard curve according to the concentration of the salmonella in the salmonella standard solution and the corresponding Raman signal intensity, and substituting the Raman signal intensity obtained in the step (p-3) into the standard curve to obtain the content of the salmonella in the sample to be detected.

Any one of the above steps of mixing the gold nano material, the single-stranded DNA molecule and the Raman signal reporter, and carrying out a light-shielding reaction; then adding chloroauric acid and hydroxylamine hydrochloride, reacting, collecting precipitate, mixing gold nano material and single-stranded DNA molecule, and reacting in dark place; adding a Raman signal reporter, mixing and reacting in a dark place; then adding chloroauric acid and hydroxylamine hydrochloride, reacting, and collecting precipitate.

Any one of the above steps of mixing the gold nano material, the single-stranded DNA molecule and the Raman signal reporter, and carrying out a light-shielding reaction; then adding chloroauric acid and hydroxylamine hydrochloride solution, reacting, and collecting the precipitate ", wherein the volume ratio of the gold nanomaterial, single-stranded DNA molecule, Raman signal reporter, chloroauric acid and hydroxylamine hydrochloride solution may be 180:6:2:2:5, wherein the concentration of the DNA solution may be 80-100. mu.M (e.g., 80-90. mu.M, 90-100. mu.M, 80. mu.M, 90. mu.M or 100. mu.M), the concentration of the Raman signal reporter may be 0.5-2mM (e.g., 0.5-1mM, 1-2mM, 0.5mM, 1mM or 2mM), the concentration of the hydroxylamine hydrochloride solution may be 0.35-0.45M (e.g., 0.35-0.40M, 0.40-0.45M, 0.35M, 0.40M or 0.45M), and the concentration of the Raman signal reporter may be 0.5-2mM, 0.0.5 mM, 1mM or 2 mM).

Any one of the above steps of mixing the gold nano material, the single-stranded DNA molecule and the Raman signal reporter, and carrying out a light-shielding reaction; then adding chloroauric acid and hydroxylamine hydrochloride, reacting, and collecting precipitate, wherein the specific steps of:

(K1) uniformly mixing the gold nano material aqueous solution, the single-stranded DNA molecule aqueous solution and the Raman signal reporter aqueous solution, and standing for reaction at room temperature in a dark place;

(K2) after completion of the step (K1), an aqueous chloroauric acid solution and a hydroxylamine hydrochloride solution are added, and the mixture is subjected to shaking reaction at 35 to 39 ℃ (e.g., 35 to 37 ℃, 37 to 39 ℃, 35 ℃, 37 ℃ or 39 ℃) to collect a precipitate.

In the step (K1), the concentration of the gold nanomaterial aqueous solution may be 0.1 nmol/L. The addition amount of the gold nanomaterial aqueous solution may be 90 μ L. The concentration of the aqueous solution of the single-stranded DNA molecules may be 100. mu.M. The amount of the aqueous solution of single-stranded DNA molecules added may be 2. mu.L. The concentration of the aqueous raman signal reporter solution may be 1 mM. The amount of the aqueous raman signal reporter solution added may be 0.5 μ L.

In the step (K1), the reaction time may be 0.5-2h (e.g., 0.5-1h, 1-2h, 0.5h, 1h or 2 h).

The step (K1) can be specifically that the gold nano material aqueous solution and the single-stranded DNA molecule aqueous solution are mixed uniformly, and the mixture is kept standing for reaction for 0.5 to 1 hour (such as 0.5 hour or 1 hour) at room temperature in a dark place; then adding the Raman signal reporter aqueous solution, mixing uniformly, standing for reaction for 0.5-1h (such as 0.5h or 1h) at room temperature in a dark place.

In the step (K1), the single-stranded DNA molecule may be a single-stranded DNA molecule a or a single-stranded DNA molecule b.

In the step (K1), when the single-stranded DNA molecule is a single-stranded DNA molecule a, the raman signal reporter may be a raman signal reporter a. When the single-stranded DNA molecule is the single-stranded DNA molecule b, the raman signal reporter may be the raman signal reporter b.

In the step (2), the concentration of the chloroauric acid aqueous solution can be 1 mM. The amount of the aqueous chloroauric acid solution added may be 1.5. mu.L. The concentration of hydroxylamine hydrochloride solution may be 0.4M. The hydroxylamine hydrochloride solution may be added in an amount of 0.25. mu.L.

In the step (2), the shaking reaction may specifically be 150-250rpm (e.g., 150-200rpm, 200-250rpm, 150rpm, 200rpm or 250rpm) for 1-2h (e.g., 1-1.5h, 1.5-2h, 1h, 1.5hhuo 2 h).

The incubation in the step (b-1), the step (c-1), the step (2-1), the step (3-1), the step (g-1), the step (h-1), the step (p-1), the step (q-1), the step (b-2), the step (c-2), the step (2-2), the step (3-2), the step (g-2), the step (h-2), the step (p-2) and the step (q-2) may be specifically 35 to 39 ℃ (such as 35 to 37 ℃, 37 to 39 ℃, 35 ℃, 37 ℃ or 39 ℃), 10 to 20rpm (10 to 15rpm, 15 to 20rpm, 10rpm, 15rpm or 20rpm), and the mixture may be mixed for 40 to 50min (such as 40 to 45min, or 40 to 50min, 45-50min, 40min, 45min or 50 min).

In the step (b-1), the step (c-1), the step (2-1), the step (3-1), the step (g-1), the step (h-1), the step (p-1), the step (q-1), the step (b-2), the step (c-2), the step (2-2), the step (3-2), the step (g-2), the step (h-2), the step (p-2) and the step (q-2), a step of obtaining a capture target may be further included after incubation; the purpose of obtaining the capture target is to remove impurities and realize concentration. The method for capturing the target object can be specifically separating the nano microspheres. The separation of the nano microspheres can be realized by natural precipitation or centrifugation. When the nano-microspheres are magnetic ferroferric oxide nano-microspheres, the magnetic ferroferric oxide nano-microspheres can be separated through magnetic separation.

Any of the SERS detection parameters described above are as follows: the light source of the instrument is set to be 633nm, the laser intensity is 20W, the signal acquisition time is 10s, and 2 cycles are carried out.

Any one of the compounds B described above may be biotin.

Any one of the compounds A may be avidin. The avidin may be streptavidin.

Any one of the nano-microspheres can be a magnetic ferroferric oxide nano-microsphere.

Any one of the single-stranded DNA molecules A may specifically consist of the DNA fragment 3 and the DNA fragment 4.

The nucleotide sequence of any one of the single-stranded DNA molecules A can be shown as SEQ ID NO. 1.

Any one of the above single-stranded DNA molecules B may specifically consist of the above DNA fragment 5 and the above DNA fragment 6.

The nucleotide sequence of any one of the single-stranded DNA molecules B can be shown as SEQ ID NO. 2.

Any of the raman signal reporter agents described above may be DTNB.

Any one of the raman signal reporter b described above may be MBA.

Any of the above bacterial antibodies may be a bacterial monoclonal antibody or a bacterial polyclonal antibody.

In the above, any of the samples to be tested may be pre-treated to obtain a sample treatment solution, and then the sample treatment solution is tested.

If the sample to be detected is a solid, the pretreatment steps can be as follows: adding PBS buffer solution with equal mass into a sample to be detected, mixing, grinding, filtering, and collecting filtrate; the filtrate is the treatment fluid of the sample to be detected. The purpose of the filtration may be to remove particles larger than 1mm in diameter.

If the sample to be tested is a liquid, the pretreatment steps can be as follows: and adding PBS buffer solution with equal mass into the sample to be detected, and mixing to obtain the sample treatment solution to be detected.

Any one of the samples to be tested can be food to be tested. The food to be tested can be cucumber, chicken, beverage, etc.

In the above, the drawing of the standard curve according to the standard solution and the corresponding raman signal intensity may specifically be drawing of the standard curve according to the Lg value of the standard solution and the corresponding raman signal intensity.

Raman signal reporter herein refers to a substance that can generate a Raman spectrum signal, such as DTNB, MBA.

Any one of the gold nanomaterials can be specifically gold nanomaterials with different shapes, such as gold nanorods, gold nanoparticles, gold nanocages, gold nanoshells, gold triangular plates, gold nanostars and gold nanochains.

The method provided by the invention can be used for simultaneously detecting the contents of different bacteria in the sample to be detected and has higher accuracy. In one embodiment of the invention, the method provided by the invention can be used for simultaneously detecting the contents of Escherichia coli O157: H7 and salmonella in the food to be detected, and has higher accuracy. Compared with a plate culture method, the method provided by the invention also has the following advantages: 1. the method is rapid and does not need long-time pre-culture; 2. the sensitivity is high, and the lowest detection limit reaches 3 cfu/mL; 3. multiple bacteria in the system can be detected simultaneously, and the plate method cannot distinguish the multiple bacteria rapidly. The invention has important application value.

Drawings

FIG. 1 is an SRES assay of the signaling probe prepared in step two of example 1.

FIG. 2 shows UV-vis characterization of Au NR, Tag1 and Tag 2.

FIG. 3 is a TEM characterization of Au NR, Tag1 and Tag 2.

FIG. 4 is the preparation of SERS spectra for detecting Escherichia coli O157: H7 and Salmonella, and a standard curve for detecting Escherichia coli O157: H7 and a standard curve for detecting Salmonella in the third step of example 1.

FIG. 5 is a standard curve for detection of E.coli O157: H7 in step four of example 1 and a standard curve for detection of Salmonella in step five of example 1.

FIG. 6 shows the results of the specificity test in example 2.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

The magnetic ferroferric oxide nano-microspheres modified by streptomycin are products of Ocean nanotech company. DTNB and MBA are both products of Sigma. The monoclonal antibody of Escherichia coli O157: H7 and the monoclonal antibody of Salmonella are both products of the Meridian Life Science company, and the catalog numbers are B65001R and C86309M, respectively. 1 XPSBSbuffer is a product of Solambio. The long-arm biotin modification kit is a product of Elapscience Biotechnology company.

Example 1 detection of the content of Escherichia coli O157: H7 and/or Salmonella in a sample to be tested

First, preparation of Capture Probe

1. Preparation of Escherichia coli Capture Probe

(1) The monoclonal antibody of Escherichia coli O157: H7 was subjected to biotin modification using a long-armed biotin modification kit to obtain a biotin-modified Escherichia coli antibody at a concentration of 1 mg/mL.

(2) After the step (1) is finished, taking 30 mu L of streptomycin modified magnetic ferroferric oxide nano microspheres with the concentration of 1mg/mL, and fully washing the microspheres with 1 multiplied by PBS buffer; and then adding 0.6 mu L of biotin-modified Escherichia coli antibody, uniformly mixing at 37 ℃ and 15rpm for 45min, and separating and removing the unbound antibody to obtain the Escherichia coli monoclonal antibody-modified magnetic ferroferric oxide nano-microsphere, namely the Escherichia coli capture probe (capture probe 1 for short).

2. Preparation of salmonella capture probe

(1) And performing biotin modification on the salmonella monoclonal antibody by using a long-arm biotin modification kit to obtain the biotin-modified salmonella antibody with the concentration of 1 mg/mL.

(2) After the step (1) is finished, taking 30 mu L of streptomycin modified magnetic ferroferric oxide nano microspheres with the concentration of 1mg/mL, and fully washing the microspheres with 1 multiplied by PBS buffer; then 0.6 mu L of biotin-modified salmonella antibody is added, mixed uniformly at 37 ℃ and 15rpm and reacted for 45min, and the unbound antibody is removed by centrifugation, thus obtaining the magnetic ferroferric oxide nano-microsphere modified by the salmonella monoclonal antibody, namely the salmonella capture probe (called capture probe 2 for short).

Preparation and characterization of Signal probes

The nucleotide sequence of the single-stranded DNA molecule A is as follows:

(SEQ ID NO. 1). In the single-stranded DNA molecule A, the 20T's shown at positions 1 to 20 from the 5' end function as: (1) regulating and controlling regrowth of the gold nanorods to obtain a dumbbell-shaped signal probe, (2) reducing steric hindrance in the aptamer recognition process; the DNA molecules shown at positions 21 to 62 are E.coli aptamer DNA.

The nucleotide sequence of the single-stranded DNA molecule B is as follows:

(SEQ ID NO. 2). In the single-stranded DNA molecule B, the 20C shown at the 1 st to 20 th positions from the 5' end functions as: (1) regulating and controlling regrowth of the gold nanorods to obtain a signal probe with a stabbing shape around the body, (2) reducing steric hindrance in the process of aptamer recognition; the DNA molecules shown at positions 21 to 60 are Salmonella aptamer DNA.

1. Preparation of Escherichia coli Signal Probe 1

(1) Taking a flask, adding 5mL of an aqueous solution of LCTAB (the concentration is 0.2mol/L) and 5mL of an aqueous solution of chloroauric acid (the concentration is 0.5mM) and uniformly mixing; then 600 mul of sodium borohydride aqueous solution (the concentration is 10mmol/L) is added, mixed evenly and kept stand for reaction for 2h at room temperature, and seed solution is obtained.

(2) After the step (1) is finished, taking another round-bottom flask, adding 10mL of aqueous solution of LCTAB (concentration is 0.2mol/L), adding 300 μ L of aqueous solution of silver nitrate (concentration is 4mmol/L), 10mL of aqueous solution of chloroauric acid (concentration is 1mM), 140 μ L of aqueous solution of ascorbic acid AA (concentration is 78.8mmol/L) and 24 μ L of seed solution, mixing uniformly, and standing at 30 ℃ for reaction for 2h to obtain the gold nanorods.

(3) And (3) taking the system which finishes the step (2), centrifuging, collecting precipitates, and dissolving the precipitates in ultrapure water to obtain a gold nanorod solution (hereinafter referred to as Au NR) with the concentration of 0.1 nmol/L.

(4) Adding 2 mu L of single-stranded DNA molecule A aqueous solution (the concentration is 100 mu M) and 0.5 mu L of LDTNB aqueous solution (the concentration is 1mM) into 90 mu L of gold nanorod solution, uniformly mixing, and standing for reaction for 1h at room temperature in a dark place; then, 1.5. mu.L of an aqueous chloroauric acid solution (concentration: 1mM) and 0.25. mu.L of a hydroxylamine hydrochloride solution (concentration: 0.4M) were added thereto, and the mixture was shaken at 37 ℃ and 200rpm for 1.5 hours, centrifuged, and the precipitate was collected and dissolved in ultrapure water to obtain an E.coli signal probe 1 (hereinafter referred to as Tag1(Au NR-apt1-DTNB) or Tag1) having a concentration of 0.1 nmol/L.

The purpose of performing step (4) is to: and (4) regrowing the Au NR obtained in the step (3).

2. Preparation of Salmonella Signaling Probe 2

(1) The same as step 1 (1).

(2) The same as step (2) in step 1.

(3) The same as step (3) in step 1.

(4) Adding 2 mu L of single-stranded DNA molecule B aqueous solution (the concentration is 100 mu M) and 0.5 mu L of LMBA aqueous solution (the concentration is 1mM) into 90 mu L of gold nanorod solution, uniformly mixing, and standing for reaction for 1h at room temperature in a dark place; then, 1.5. mu.L of an aqueous chloroauric acid solution (concentration: 1mM) and 0.25. mu.L of a hydroxylamine hydrochloride solution (concentration: 0.4M) were added thereto, and the mixture was shaken at 37 ℃ and 200rpm for 1.5 hours, centrifuged, and the precipitate was collected and dissolved in ultrapure water to obtain a Salmonella signaling probe 2 (hereinafter referred to as Tag2(Au NR-apt2-MBA) or Tag2) having a concentration of 0.1 nmol/L.

3. Preparation of Escherichia coli contrast signal probe

(1) The same as step 1 (1).

(2) The same as step (2) in step 1.

(3) The same as step (3) in step 1.

(4) Adding 2 mu L of single-stranded DNA molecule A aqueous solution (the concentration is 100 mu M) and 0.5 mu L of LDTNB aqueous solution (the concentration is 1mM) into 90 mu L of gold nanorod solution, uniformly mixing, and standing for reaction for 1h at room temperature in a dark place; centrifuging, collecting precipitate, and dissolving in ultrapure water to obtain Escherichia coli contrast signal probe (Au NR-DTNB) with concentration of 0.1 nmol/L.

4. Preparation of salmonella contrast signal probe

(1) The same as step 1 (1).

(2) The same as step (2) in step 1.

(3) The same as step (3) in step 1.

(4) Adding 2 mu L of single-stranded DNA molecule B aqueous solution (the concentration is 100 mu M) and 0.5 mu L of LMBA aqueous solution (the concentration is 1mM) into 90 mu L of gold nanorod solution, uniformly mixing, and standing for reaction for 1h at room temperature in a dark place; centrifuging, collecting precipitate, and dissolving in ultrapure water to obtain Salmonella contrast signal probe (Au NR-MBA) with concentration of 0.1 nmol/L.

5. SRES detection

A small amount of DTNB aqueous solution (concentration 1mM), MBA aqueous solution (concentration 1mM), Tag1, Tag2, Au NR-DTNB or Au NR-MBA was drawn up by a capillary and placed under a laser confocal Raman microscope. The light source of the instrument is set to be 633nm, the laser intensity is 20W, the signal acquisition time is 10s, and 2 cycles are carried out. After the light source is focused on the capillary, the visible light is turned off, the laser is turned on, and signal acquisition is carried out.

The results are shown in FIG. 1(DTNB is an aqueous DTNB solution, and MBA is an aqueous MBA solution). The results show that the Raman signal intensity values of the DTNB aqueous solution and the MBA aqueous solution are both less than 10, the Raman signal intensity values of the Au NR-DTNB and the Au NR-MBA are both about 100, and the Raman signal intensity values of the Tag1 and the Tag2 are both about 400. The results show that DTNB in Tag1 is better wrapped and adsorbed compared with Au NR-DTNB; compared with Au NR-MBA, MBA in the Tag2 is better wrapped and adsorbed; tag1 and Tag2 are more beneficial to formation of Raman hot spots in the SERS detection process, and larger Raman signal intensity is obtained.

6. Characterization of

(1) UV-vis characterization

Taking a micro cuvette, soaking the cuvette in aqua regia, cleaning the cuvette with ultrapure water, and drying the cuvette; au NR, Tag1, or Tag2 (see fig. 2 (b)) was then added to the microcuvette and a spectrum scan was performed using an ultraviolet-visible spectrophotometer. The scanning range is set to be 220-800nm, and the step length is 0.1 nm/s.

The results of the spectral scan are shown in fig. 2 (a). The result shows that the Au NR has two absorption peaks which are 520nm and 650nm respectively; the absorption peak of Tag1 is red-shifted, and the two absorption peaks are 523nm and 670nm respectively; the absorption peak of Tag2 is red-shifted, and the two absorption peaks are 536nm and 678nm respectively. Therefore, after Au NR regrows, the long diameter and the short diameter of the gold nanorod are increased. There are two distinct absorption peaks, indicating that the synthesized Tag1 and Tag2 are uniform in shape.

(2) TEM characterization

Dripping 10 μ LAu NR, Tag1(Au NR-apt1-DTNB) or Tag2(Au NR-apt2-MBA) on the copper net, and sucking off the excessive solution with filter paper after 2 min; the copper mesh was then placed in a clean environment until dry. And observing the appearance of the sample by using a transmission electron microscope.

The results are shown in FIG. 3((a) is Au NR, (b) is Tag1(Au NR-apt1-DTNB), (c) is Tag2(Au NR-apt 2-MBA)). The result shows that the short diameter of the Au NR is 15-20nm, and the long diameter is 55-60 nm; the Tag1(Au NR-apt1-DTNB) mediates regrowth, the shape is dumbbell-shaped, the short diameter is 20-25nm, and the long diameter is 70-75 nm; the Tag2(Au NR-apt2-MBA) mediates regrowth, the appearance is a stabbing gold rod on the whole body, the short diameter is 20-25nm, and the long diameter is 75-80 nm. Therefore, the single-stranded DNA molecule A and the single-stranded DNA molecule B can mediate the growth of the gold nanorods, but different irregular sharp shapes are obtained.

Based on the above results, Tag1 and Tag2 were selected for subsequent experiments.

Third, detecting the contents of Escherichia coli O157: H7 and salmonella in the sample to be detected

A. The detection steps of the contents of Escherichia coli O157, H7 and salmonella in the sample to be detected are as follows:

1. preparation of standard curve for detecting Escherichia coli O157H 7 and standard curve for detecting salmonella

(1) Taking Escherichia coli O157: H7, and culturing with LB liquid medium to obtain Escherichia coli liquid. And (3) culturing the salmonella by using an LB liquid culture medium to obtain salmonella bacteria liquid.

(2) After completion of step (1), Escherichia coli is subjected toMixing the bacterial liquid and salmonella bacterial liquid to obtain Escherichia coli O157H 7 with Salmonella concentration of 10¹cfu/mL of solution 1, Escherichia coli O157H 7 and Salmonella all at 10²cfu/mL of solution 2, Escherichia coli O157H 7 and Salmonella all at 10³cfu/mL solution 3, Escherichia coli O157H 7 and Salmonella concentration were all 10⁴cfu/mL of solution 4, Escherichia coli O157H 7 and Salmonella all at 10⁵cfu/mL of solution 5 and E.coli O157H 7 and Salmonella concentrations were all 10⁶cfu/mL of solution 6.

(3) And (3) after the step (2) is finished, taking 0.5mL of solution 1, solution 2, solution 3, solution 4, solution 5 or solution 6, adding excessive capture probe 1 (specifically adding 30 mu L of capture probe 1 with the concentration of 1 mg/mL) and excessive capture probe 2 (specifically adding 30 mu L of capture probe 2 with the concentration of 1 mg/mL), uniformly mixing at 37 ℃ and 15rpm for 45min, carrying out magnetic separation, and collecting enriched bacterial liquid.

(4) And (3) adding 3 times of volume of an escherichia coli signal probe 1 (specifically adding 180 mu L of escherichia coli signal probe 1 with the concentration of 0.4 nmol/L) and 3 times of volume of a salmonella signal probe 2 (specifically adding 180 mu L of salmonella signal probe 2 with the concentration of 0.4 nmol/L) into the enriched bacterial liquid collected in the step (3), uniformly mixing at 37 ℃ and 15rpm for 45min, carrying out magnetic separation, and collecting the enriched bacterial liquid.

(5) And (4) sucking a small amount of the enriched bacterial liquid collected in the step (4) by using a capillary tube, and placing under a laser confocal Raman microscope. The light source of the instrument is set to be 633nm, the laser intensity is 20W, the signal acquisition time is 10s, and 2 cycles are carried out. After the light source is focused on the capillary, the visible light is turned off, the laser is turned on, and signal acquisition is carried out.

The detection result is shown in (a) (10) in FIG. 4¹cfu/mL is solution 1, 10²cfu/mL is solution 2, 10³cfu/mL is solution 3, 10⁴cfu/mL is solution 4, 10⁵cfu/mL is solution 5, 10⁶cfu/mL is solution 6; 1331cm-¹The peak at (A) was produced by DTNB on Tag1 bound to E.coli O157: H7, 1074cm-¹And 1586cm-¹Peak at (MBA) was MBA production on Tag2 bound to salmonella).

Using Escherichia coli O157H 7The lg value of (A) is shown on the abscissa, the corresponding Raman signal intensity is shown on the ordinate, and a standard curve for detecting Escherichia coli O157: H7 is drawn. The standard curve for detecting Escherichia coli O157: H7 is shown in FIG. 4 (b), and the linear relationship is: 91.0X +185.7, R²＝0.9914。

And drawing a standard curve for detecting the salmonella by taking the lg value of the salmonella as an abscissa and the corresponding Raman signal intensity as an ordinate. The standard curve for detecting salmonella is shown in fig. 4 (c), and the linear relationship is: y is 64.2X +99.7, R²＝0.9800。

The lowest quantitative concentration was 3 × the lowest detected concentration. The lowest quantitative concentrations of Escherichia coli O157: H7 and Salmonella were 10cfu/mL, and therefore the lowest detection limits of Escherichia coli O157: H7 and Salmonella were 3 cfu/mL.

2. Detecting the contents of Escherichia coli O157H 7 and salmonella in a sample to be detected

(1) And (4) taking a sample to be detected, and carrying out pretreatment to obtain a sample treatment solution to be detected.

If the sample to be detected is solid, the pretreatment method comprises the following steps: an equal mass of 1 x PBS buffer was added to the sample to be tested, mixed, milled, and then filtered (to remove particles larger than 1mm in diameter), and the filtrate was collected. The filtrate is the treatment fluid of the sample to be detected.

If the sample to be detected is liquid, the pretreatment method comprises the following steps: and adding 1 XPBS buffer with equal mass into the sample to be detected, and mixing to obtain the sample treatment solution to be detected.

(2) And (3) taking 0.5mL of the sample treatment solution to be detected obtained in the step (1), adding the excessive capture probe 1 and the excessive capture probe 2, uniformly mixing at 37 ℃ and 15rpm for 45min, carrying out magnetic separation, and collecting the enriched solution.

(3) And (3) adding the enrichment liquid collected in the step (2) into an escherichia coli signal probe 1 with the volume being 3 times of that of the enrichment liquid and a salmonella signal probe 2 with the volume being 3 times of that of the enrichment liquid, uniformly mixing for 45min at 37 ℃ and 15rpm, carrying out magnetic separation, and collecting the enrichment liquid.

(4) And (4) sucking a small amount of the concentrated solution collected in the step (3) by using a capillary tube, and placing under a laser confocal Raman microscope. The light source of the instrument is set to be 633nm, the laser intensity is 20W, the signal acquisition time is 10s, and 2 cycles are carried out. And after the light source is focused on the capillary tube, the visible light is turned off, the laser is turned on, and the signal acquisition is carried out to obtain the Raman signal intensity of the sample treatment solution to be detected.

(5) According to the standard curve for detecting the Escherichia coli O157: H7 and the standard curve for detecting the salmonella, the contents of the Escherichia coli O157: H7 and the salmonella in the treatment fluid of the sample to be detected can be calculated, and the contents of the Escherichia coli O157: H7 and the salmonella in the sample to be detected are further obtained.

B. Accuracy detection

1. Taking a sample to be detected (tap water, commercially available cucumber or commercially available chicken), carrying out autoclaving at 121 ℃ for 15min, and then adding Escherichia coli O157: H7 and salmonella to obtain the sample to be detected containing Escherichia coli O157: H7 and salmonella. In a sample to be detected containing Escherichia coli O157H 7 and salmonella, the concentration of Escherichia coli O157H 7 is 3.5 multiplied by 10²cfu/mL, Salmonella concentration 2.0X 10²cfu/mL。

2. After the step 1 is finished, the method in the step A is adopted to detect the contents of Escherichia coli O157H 7 and salmonella in a sample to be detected containing Escherichia coli O157H 7 and salmonella. The recovery of both bacteria was then calculated separately, with a (%) recovery of ═ N_SERS/N_{Adding into}X 100% where N_SERSNumber of bacteria detected for SERS method, N_{Adding into}The number of bacteria added.

3. After completion of step 1, the total content of Escherichia coli O157, H7 and Salmonella in the test sample containing Escherichia coli O157, H7 and Salmonella was determined by plate culture (described in: Biosensors and Bioelectronics 74(2015) 504-511).

4. After

steps

2 and 3 are completed, the deviation is calculated, and the deviation (%) is (N)_SERS-N_{Flat plate})/N_{Flat plate}X 100% where N_SERSNumber of bacteria detected for SERS method, N_{Flat plate}The number of bacteria was determined by plating.

The results are shown in Table 1. The method provided by the invention can be used for simultaneously detecting the contents of Escherichia coli O157, H7 and salmonella in a sample to be detected, and has higher accuracy. Meanwhile, compared with a plate culture method, the method provided by the invention also has the following advantages: 1. the method is rapid and does not need long-time pre-culture; 2. the sensitivity is high, and the lowest detection limit reaches 3 cfu/mL; 3. multiple bacteria in the system can be detected simultaneously, and the plate method cannot distinguish the multiple bacteria rapidly.

TABLE 1

Fourthly, detecting the content of Escherichia coli O157H 7 in the sample to be detected

The detection steps of the content of Escherichia coli O157H 7 in the sample to be detected are as follows:

1. preparation of Standard Curve for detecting Escherichia coli O157H 7

(1) Taking Escherichia coli O157H 7, culturing with LB liquid medium to obtain Escherichia coli O157H 7 with concentration of 10¹cfu/mL solution 1, E.coli O157H 7 concentration of 10²cfu/mL solution 2, E.coli O157H 7 concentration of 10³cfu/mL solution 3, E.coli O157H 7 concentration of 10⁴cfu/mL solution 4, E.coli O157H 7 concentration of 10⁵cfu/mL of solution 5 and E.coli O157H 7 concentration of 10⁶cfu/mL of solution 6.

(2) And (3) after the step (1) is finished, taking 0.5mL of solution 1, solution 2, solution 3, solution 4, solution 5 or solution 6, adding excessive capture probes 1, uniformly mixing at 37 ℃ and 15rpm for 45min, carrying out magnetic separation, and collecting enriched bacterial liquid.

(3) And (3) adding the enriched bacterial liquid collected in the step (2) into an escherichia coli signal probe 1 with the volume being 3 times, uniformly mixing at 37 ℃ and 15rpm, reacting for 45min, carrying out magnetic separation, and collecting the enriched bacterial liquid.

(4) And (4) sucking a small amount of the enriched bacterial liquid collected in the step (3) by using a capillary tube, and placing under a laser confocal Raman microscope. The light source of the instrument is set to be 633nm, the laser intensity is 20W, the signal acquisition time is 10s, and 2 cycles are carried out. After the light source is focused on the capillary, the visible light is turned off, the laser is turned on, and signal acquisition is carried out.

And drawing a standard curve for detecting the Escherichia coli O157: H7 by taking the lg value of the Escherichia coli O157: H7 as an abscissa and the corresponding Raman signal intensity as an ordinate. The standard curve for detecting Escherichia coli O157: H7 is shown in FIG. 5 (a), and the linear relationship is: Y105.4X +254.7, R²＝0.9942。

The lowest quantitative concentration was 3 × the lowest detected concentration. The lowest quantitative concentration of Escherichia coli O157: H7 was 10cfu/mL, and therefore, the lowest detection limit of Escherichia coli O157: H7 was 3 cfu/mL.

2. Detection of content of Escherichia coli O157H 7 in sample to be detected

(1) The same as step (1) in the third step 2.

(2) And (2) taking 0.5mL of the sample treatment solution to be detected obtained in the step (1), adding excessive capture probes 1, uniformly mixing at 37 ℃ and 15rpm for 45min, carrying out magnetic separation, and collecting the enrichment solution.

(3) And (3) adding the enrichment liquid collected in the step (2) into an escherichia coli signal probe 1 with the volume being 3 times of that of the enrichment liquid, uniformly mixing at 37 ℃ and 15rpm for 45min, carrying out magnetic separation, and collecting the enrichment liquid.

(5) According to the standard curve for detecting the Escherichia coli O157: H7, the content of the Escherichia coli O157: H7 in the treatment fluid of the sample to be detected can be calculated, and the content of the Escherichia coli O157: H7 in the sample to be detected is further obtained.

Fifthly, detecting the content of salmonella in the sample to be detected

The detection steps of the content of the salmonella in the sample to be detected are as follows:

1. preparation of Standard Curve for detecting Salmonella

(1) Culturing Salmonella with LB liquid culture medium to obtain Salmonella concentration 10¹cfu/mL solution 1, Salmonella concentration 10²cfu/mL ofSolution 2, Salmonella concentration 10³cfu/mL solution 3, Salmonella concentration 10⁴cfu/mL solution 4, Salmonella concentration 10⁵ Solution 5 cfu/mL and Salmonella concentration 10⁶cfu/mL of solution 6.

(2) And (3) after the step (1) is finished, taking 0.5mL of solution 1, solution 2, solution 3, solution 4, solution 5 or solution 6, adding excessive capture probes 2, uniformly mixing at 37 ℃ and 15rpm for 45min, carrying out magnetic separation, and collecting enriched bacterial liquid.

(3) And (3) adding the enriched bacterial liquid collected in the step (2) into a salmonella signal probe 2 with the volume being 3 times of that of the enriched bacterial liquid, uniformly mixing at 37 ℃ and 15rpm for reaction for 45min, carrying out magnetic separation, and collecting the enriched bacterial liquid.

And drawing a standard curve for detecting the salmonella by taking the lg value of the salmonella as an abscissa and the corresponding Raman signal intensity as an ordinate. The standard curve for detecting salmonella is shown in fig. 5 (b), and the linear relationship is: y is 87.6X +59.5, R²＝0.9955。

The lowest quantitative concentration was 3 × the lowest detected concentration. The lowest quantitative concentration of Salmonella was 10cfu/mL, and therefore the lowest detection limit for Salmonella was 3 cfu/mL.

2. Detection of salmonella content in sample to be detected

(1) The same as step (1) in the third step 2.

(2) And (2) taking 0.5mL of the sample treatment solution to be detected obtained in the step (1), adding excessive capture probe 2, uniformly mixing at 37 ℃ and 15rpm for 45min, carrying out magnetic separation, and collecting the enrichment solution.

(3) And (3) adding the enrichment liquid collected in the step (2) into a salmonella signal probe 2 with the volume being 3 times of that of the enrichment liquid, uniformly mixing the enrichment liquid at 37 ℃ and 15rpm for 45min, carrying out magnetic separation, and collecting the enrichment liquid.

(5) And calculating the content of the salmonella in the treatment fluid of the sample to be detected according to the standard curve for detecting the salmonella, and further obtaining the content of the salmonella in the sample to be detected.

Example 2 specificity detection

1. Preparation of samples to be tested

The sample to be detected is a sample I to be detected, a sample II to be detected, a sample III to be detected, a sample IV to be detected, a sample V to be detected and a sample VI to be detected.

The first sample to be detected is a mixed bacteria solution. In the mixed bacteria solution, the concentrations of Escherichia coli DH5 alpha, staphylococcus aureus and vibrio parahaemolyticus are all 10⁵The concentration of cfu/mL, Escherichia coli O157H 7 and Salmonella were all 10³cfu/mL。

The concentration of the second sample to be detected is 10⁵cfu/mL of Escherichia coli DH 5. alpha. bacterial liquid.

The concentration of the sample III to be detected is 10⁵cfu/mL Staphylococcus aureus liquid.

The concentration of the sample four to be detected is 10⁵cfu/mL of Vibrio parahaemolyticus liquid.

The concentration of the sample to be detected is five and is 10³cfu/mL of Escherichia coli O157H 7 bacterial liquid.

The concentration of the sample six to be detected is 10³cfu/mL salmonella bacterial liquid.

2. And (3) after the step 1 is finished, respectively adding 1 XPBS buffer with equal mass into the sample to be detected, and mixing to sequentially obtain the sample treatment solution to be detected.

3. Respectively taking 0.5mL of sample treatment solution to be detected, adding excessive capture probe 1 and excessive capture probe 2, uniformly mixing at 37 ℃ and 15rpm for 45min, carrying out magnetic separation, and collecting the enriched solution.

4. And (3) adding the enrichment liquid collected in the step (3) into an escherichia coli signal probe 1 with the volume being 3 times and a salmonella signal probe 2 with the volume being 3 times, uniformly mixing for 45min at 37 ℃ and 15rpm, carrying out magnetic separation, and collecting the enrichment liquid.

5. And (4) sucking a small amount of the enrichment liquid collected in the step (4) by using a capillary tube, and placing under a laser confocal Raman microscope. The light source of the instrument is set to be 633nm, the laser intensity is 20W, the signal acquisition time is 10s, and 2 cycles are carried out. And after the light source is focused on the capillary tube, the visible light is turned off, the laser is turned on, and the signal acquisition is carried out to obtain the Raman signal intensity of the sample treatment solution to be detected.

The detection result is shown in FIG. 6(Mixture is a first sample to be detected, Escherichia coli is a second sample to be detected, Staphylococcus aureus is a third sample to be detected, Vibrio parahaemolyticus is a fourth sample to be detected, Escherichia coli O157: H7 is a fifth sample to be detected, and Salmonella is a sixth sample to be detected). The result shows that the method provided by the invention can specifically detect the characteristic signals of Escherichia coli O157: H7 and salmonella, and has no obvious signal response to other bacteria (such as Escherichia coli DH5 alpha, staphylococcus aureus and Vibrio parahaemolyticus).

<110> institute for agricultural product processing of Chinese academy of agricultural sciences

<120> method for simultaneously detecting contents of escherichia coli and salmonella in sample to be detected

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 62

<212> DNA

<213> Artificial sequence

<400> 1

tttttttttt tttttttttt ccggacgctt atgccttgcc atctacagag caggtgtgac 60

gg 62

<210> 2

<211> 60

<212> DNA

<213> Artificial sequence

<400> 2

cccccccccc cccccccccc tatggcggcg tcacccgacg gggacttgac attatgacag 60

Claims

1. A kit A for detecting the content of different bacteria in a sample to be detected comprises a plurality of reagents A for detecting different bacteria;

each reagent A for detecting bacteria comprises a bacterial antibody, a single-chain DNA molecule and a Raman signal reporter;

the DNA fragment 2 is an aptamer DNA of the bacterium;

2. The kit a of claim 1, wherein: the kit A also comprises a compound B, a compound A capable of being combined with the compound B and gold nano-materials.

3. A kit B for detecting the content of different bacteria in a sample to be detected comprises a plurality of reagents B for detecting different bacteria;

each reagent B for detecting bacteria comprises a bacterial antibody modified by a compound B, a nano microsphere modified by a compound A and capable of being combined with the compound B, and a signal probe; the signal probe is a gold nano material jointly marked by a single-stranded DNA molecule and a Raman signal reporter;

the DNA fragment 2 is an aptamer DNA of the bacterium;

4. The kit b of claim 3, wherein: the preparation method of the signal probe comprises the following steps: mixing the gold nano material, the single-stranded DNA molecule and the Raman signal reporter, and reacting in a dark place; and then adding chloroauric acid and hydroxylamine hydrochloride, reacting, and obtaining a precipitate as a signal probe.

5. The kit A according to claim 1 or 2, or the kit B according to claim 3 or 4, wherein: the bacterial antibody is a monoclonal antibody of bacteria or a polyclonal antibody of bacteria.

6. The kit A according to claim 1, 2 or 5, or the kit B according to any one of claims 3 to 5, wherein: the compound B is biotin; the compound A is avidin.

7. A method for detecting the content of different bacteria in a sample to be detected comprises the following steps (a), (b), (c) and (d):

the step (a) includes the steps of:

the DNA fragment 2 is aptamer DNA of bacteria;

the step (b) comprises the steps of:

the step (c) comprises the steps of:

8. A method for detecting contents of Escherichia coli O157: H7 and salmonella in a sample to be detected comprises the following steps (1), (2), (3) and (4):

the step (1) comprises the following steps:

the step (2) comprises the following steps:

the step (3) comprises the following steps:

9. A method for detecting the content of Escherichia coli O157H 7 in a sample to be detected comprises the following steps (f), (g), (H) and (i):

the step (f) includes the steps of:

the step (g) comprises the steps of:

the step (h) comprises the steps of:

10. A method for detecting the content of salmonella in a sample to be detected comprises the following steps of (o), step (p), step (q) and step (r):

the step (o) includes the steps of:

the step (p) comprises the steps of:

the step (q) includes the steps of: