Method for simultaneously detecting contents of escherichia coli and salmonella in sample to be detected
阅读说明：本技术 一种同时检测待测样品中大肠杆菌和沙门氏菌的含量的方法 (Method for simultaneously detecting contents of escherichia coli and salmonella in sample to be detected ) 是由 郑金铠 李玉芝 陆畅 周帅帅 高飞 于 2019-10-30 设计创作，主要内容包括：本发明公开了一种同时检测待测样品中大肠杆菌和沙门氏菌的含量的方法。该方法依次包括如下步骤：(1)向待测样品中加入过量的大肠杆菌捕获探针和过量的沙门氏菌捕获探针,孵育；(2)加入过量的大肠杆菌信号探针和过量的沙门氏菌信号探针,孵育；(3)SERS检测拉曼信号强度；(4)将该拉曼信号强度代入根据标准溶液中大肠杆菌O157:H7和沙门氏菌的浓度的Lg值和相应拉曼信号强度绘制标准曲线,得到待测样品中大肠杆菌O157:H7和沙门氏菌的含量。实验证明,该方法可以同时检测待测食品中大肠杆菌O157:H7和沙门氏菌的含量且具有较高的准确性。本发明具有重要的应用价值。(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.)
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 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 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.
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 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 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.
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:
(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) comprises 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) comprises 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.
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:
(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 is shown as the 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 is shown as the 21 st to 60 th positions from the 5' end of SEQ ID NO. 2;
the step (2) comprises the following steps:
(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) comprises the following steps:
(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.
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:
(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 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 is shown as the 21 st to 62 nd positions from the 5' end of SEQ ID NO. 1;
the step (g) comprises 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) comprises 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.
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:
(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 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 is shown as the 21 st to 60 th positions from the 5' end of SEQ ID NO. 2;
the step (p) comprises 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) includes 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.
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
the
the
in each reagent A for detecting bacteria, the bacterial antibody, the
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 single-stranded DNA molecule sequentially comprises a
the
the
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
In any of the
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
the
the
(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
the
the nucleotide sequence of the
(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
the
the nucleotide sequence of the
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 single-stranded DNA molecule A sequentially comprises a
the
the nucleotide sequence of the
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 single-stranded DNA molecule B sequentially comprises a
the
the nucleotide sequence of the
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
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
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
FIG. 3 is a TEM characterization of Au NR, Tag1 and
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.