CN105603070B - Detection method and detection kit for aflatoxin B1 - Google Patents

Detection method and detection kit for aflatoxin B1 Download PDF

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CN105603070B
CN105603070B CN201610041767.8A CN201610041767A CN105603070B CN 105603070 B CN105603070 B CN 105603070B CN 201610041767 A CN201610041767 A CN 201610041767A CN 105603070 B CN105603070 B CN 105603070B
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CN105603070A (en
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夏晓东
陈佳鑫
田彩霞
黄昊文
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Hunan University of Science and Technology
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Abstract

The invention relates to a method and a kit for detecting aflatoxin B1, wherein the method comprises the following steps: (A) hybridizing an aflatoxin B1 aptamer Apt with a single-stranded signal probe ssDNA to form a hybrid chain; (B) the hybrid chain is contacted with a sample to be detected, and when aflatoxin B1 exists in the sample to be detected, the hybrid chain reacts with aflatoxin B1 to release ssDNA; (C) using DNA amplification to make the hybrid chain into double-stranded DNA, then using exonuclease to hydrolyze the double-stranded DNA into mononucleotide to remove the double-stranded DNA, and at this time, leaving ssDNA in the system; (D) under the induction of ssDNA, silver ions are reduced to generate near-infrared fluorescent silver nanoclusters; detecting the fluorescence intensity of the system, thereby determining the content of aflatoxin B1 in the sample to be detected. The method has the characteristics of high sensitivity, simple operation, low cost and the like.

Description

Detection method and detection kit for aflatoxin B1
Technical Field
The invention relates to the field of nano biosensing and biological detection, and particularly provides a detection method and a detection kit for aflatoxin B1.
Background
Aflatoxin B1 has strong toxicity to humans and several animals, is the most carcinogenic of known chemicals, and is extremely harmful to humans and animals. Aflatoxins are metabolites produced by aspergillus flavus, aspergillus parasiticus, and the like. Aflatoxins are widely found in soil, soybeans, rice, corn, macaroni, seasonings, milk and its products, edible oils, meat (fish) products, peanuts and walnuts and other animals and plants and various nuts, especially peanuts and walnuts. When people ingest food polluted by aflatoxin B1, acute poisoning such as acute hepatitis, hemorrhagic necrosis and the like can occur, even death occurs; when ingested in a micro amount, chronic poisoning, growth disorder, carcinogenesis, teratogenesis, etc. may be caused. Aflatoxin is almost unavoidable in agricultural products, aflatoxin B1 is the most common in natural food and has the strongest hazard, and the national quality control Bureau stipulates that aflatoxin B1 is one of the necessary inspection items of most foods. The food hygiene standard in China stipulates that: the aflatoxin in the corn, peanut oil, peanut and products thereof can not be more than 20 mug/kg; the rice and other edible oil can not be more than 10 mug/kg; other grains, beans and fermented food can not be more than 5 mug/kg; the infant milk replacer can not detect aflatoxin. The contents of aflatoxin B1 in grains, peanuts and products thereof are specified in 2002 by EU and other countries, the content of aflatoxin B1 in the peanuts directly used by human beings is required to be less than or equal to 2 mug/kg, and the content of peanut aflatoxin B1 imported as a food raw material is required to be less than or equal to 8 mug/kg. If the content of aflatoxin in food exceeds a certain standard, people can directly threaten the health of people. Therefore, it is very valuable to establish a detection method of aflatoxin B1 with high selectivity and high sensitivity.
At present, the domestic and foreign researches mainly comprise Thin Layer Chromatography (TLC), High Performance Liquid Chromatography (HPLC), immunoaffinity column method, enzyme linked immunosorbent assay and the like. The TLC method has poor specificity and sensitivity, and the required standard substance has high concentration and high potential pollution. The HPLC method needs personnel with special operation technology to carry out detection, and the technical requirement is high; the purification column consumes more, and the detection cost is higher, and immunoaffinity column specificity is better, still needs special technical personnel can be competent at the detection work, and the detection technology requires height. The immunoassay method has many advantages of simple and convenient operation, rapidness, less pollution, higher sensitivity and the like, but generally needs expensive biological reagents such as enzyme labeling reagents, enzymes, antigens, antibodies and the like, and the biological reagents are easy to inactivate.
Disclosure of Invention
The invention provides a method for detecting aflatoxin B1, which comprises the following steps:
(A) hybridizing an aflatoxin B1 aptamer Apt with a single-stranded signal probe ssDNA to form a hybrid chain;
(B) adding a sample solution to be detected into the hybrid chain solution, and selectively reacting the hybrid chain with aflatoxin B1 to release a single-chain signal probe ssDNA when the aflatoxin B1 exists in the sample to be detected;
(C) eliminating the interference of impurity cross-linking, utilizing DNA amplification to make the hybrid chain into double-stranded DNA, then using exonuclease to hydrolyze the double-stranded DNA into mononucleotide to remove the double-stranded DNA and leave single-stranded signal probe ssDNA;
(D) the aflatoxin B1 aptamer-aflatoxin combination is utilized to not induce silver ions to be reduced into near-infrared fluorescent silver nanoclusters, only the single-chain signal probe ssDNA can induce the silver ions to be reduced into the near-infrared fluorescent silver nanoclusters, and the fluorescence intensity of the system is detected in a silver ion reduction detection system, so that the aflatoxin B1 content in a sample to be detected is determined.
The silver ion reduction detection system comprises silver ions and a borohydride reducing agent, such as sodium borohydride, which are added in sequence and enable the silver ions to be reduced into near-infrared fluorescent silver nano-clusters. And (D) detecting, for example, sequentially adding a silver ion solution and a sodium borohydride solution into the system in sequence, and reacting to generate the near-infrared fluorescent silver nanocluster.
The aflatoxin B1 aptamer is 5' -AAAAAGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3’。
The ssDNA of the single-stranded signal probe is 5' -CCCCCCACACCCGATCCCCCCTGTGGGCCTAGCG-3’。
The detection principle of the invention is as follows: apt was first hybridized to ssDNA (underlined); when aflatoxin B1 exists, the hybrid chain reacts with aflatoxin B1 to release ssDNA; Apt-ssDNA (residual), Apt-aflatoxin B1 and ssDNA are present in the system. Apt-aflatoxin B1 can not induce silver ions to be reduced into near-infrared fluorescent silver nanoclusters, and the presence of Apt-aflatoxin B1 has no interference to the determination; the hybrid strand may interfere; in order to eliminate the interference of impurity cross-linking: a. using DNA amplification to make the hybrid strand double-stranded DNA, b. using exonuclease to hydrolyze the double-stranded DNA to single nucleotides and remove the double-stranded DNA. At the moment, only ssDNA capable of inducing the generation of the near-infrared fluorescent silver nanocluster is left in the system, and the content of aflatoxin B1 can be determined by detecting the fluorescence intensity of the system. Because the interference of background fluorescence in the system is eliminated, the sensitivity and the precision of detection are improved.
The invention also provides a kit for detecting aflatoxin B1, which at least comprises: the kit comprises an aflatoxin B1 aptamer, a single-stranded signal probe ssDNA, a DNA amplification system, an exonuclease and a silver ion reduction detection system.
The aflatoxin B1 aptamer is 5'-AAAAAGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3'.
The ssDNA of the single-stranded signal probe is 5'-CCCCCCACACCCGATCCCCCCTGTGGGCCTAGCG-3'.
The DNA amplification system comprises a buffer solution, dNTP and Phi29 DNA polymerase, wherein the buffer solution is prepared from Tris-HCl and MgCl2、 (NH4)2SO4And (4) forming.
The exonuclease is Exo III exonuclease.
The reducing agent in the silver ion reduction detection system is borohydride.
The borohydride is sodium borohydride.
The invention also provides an aflatoxin B1 nucleic acid aptamer for detecting aflatoxin B1, wherein the base sequence of the aptamer is 5'-AAAAAGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3'.
THE ADVANTAGES OF THE PRESENT INVENTION
According to the detection method and the kit, the interference of background fluorescence is eliminated, and the detection sensitivity and the detection precision of the aflatoxin B1 are improved.
Drawings
FIG. 1 shows the relationship between the ssDNA-Ag nanocluster fluorescence intensity and aflatoxin B1 concentration.
Detailed Description
Example 1
A kit for detecting aflatoxin B1 at least comprises: the kit comprises an aflatoxin B1 aptamer, a single-stranded signal probe ssDNA, a DNA amplification system, exonuclease and a silver ion reduction detection system. The aflatoxin B1 aptamer is 5' -AAAAAGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3'. The ssDNA of the single-stranded signal probe is 5'-CCCCCCACACCCGATCCCCCCTGTGGGCCTAGCG-3'. The DNA amplification system comprises buffer solution (Tris-HCl, MgCl)2、 (NH4)2SO4) dNTP and Phi29 DNA polymerase. The exonuclease is Exo III exonuclease. The reducing agent in the silver ion reduction detection system is borohydride, such as sodium borohydride.
Example 2
A method for detecting aflatoxin B1 comprises the following specific operation processes:
each DNA stock was heat-treated at 95 ℃ for 5 minutes and allowed to stand at room temperature for 30 minutes before use. Then, 40. mu.L of a hybridization buffer solution containing 3.0. mu. mol of aflatoxin B1 aptamer Apt and 40. mu.L of a hybridization buffer solution containing 3.0. mu. mol of signal probe ssDNA were placed in a 2ml centrifuge tube, and hybridization was carried out at 37 ℃ for 1 hour to produce an aflatoxin B1 aptamer-signal probe hybrid (Apt-ssDNA).
And at 37 ℃, sequentially adding aflatoxin B1 with the concentration of 0-2 ng/mL into the Apt-ssDNA solution, reacting aflatoxin B1 with an aflatoxin B1 aptamer to generate the aptamer-aflatoxin B1, and releasing ssDNA. At this time, ssDNA, the remaining (unreacted) APT-ssDNA, and the aptamer-aflatoxin B1 were present in the system.
10 μ L of buffer (buffer composition 50 mM Tris-HCl, 10 mM MgCl) was added2，10 mM(NH4)2SO4pH 7.5), followed by addition of dNTP (10 mM) 18 μ L. Then 2 muL Phi29 DNA polymerase (10 u/muL) is added into the system and reacted for 15 minutes at 37 ℃, so that aflatoxin B1 aptamer-signal probe hybridization sequence (Ap-ssDNA) is used as a template to be amplified into double-stranded DNA. The Phi29 DNA was inactivated by holding at 65 ℃ for 10 minutes.
mu.L of Exo III exonuclease (20 u/. mu.L) was added to the reaction system and reacted at 37 ℃ for 30 minutes to remove the double-stranded DNA selectively by hydrolysis into mononucleotides, and the single-stranded probe ssDNA was retained without hydrolysis due to the extremely slow reaction rate.
To the reaction solution were added 25. mu.L of 1 mmol silver nitrate and 180. mu.L of sodium citrate buffer (10 mM, pH 7.0). Then, after the mixture was left at room temperature in the dark or dark for 10 minutes, 100. mu.L of a newly prepared sodium borohydride solution with a concentration of 200. mu.M was added under rapid stirring. And then reacting for 5-10 min at 45 ℃. The solution was transferred to a microcuvette, the fluorescence intensity of the system was measured, and aflatoxin B1 was quantitatively measured, and the results are shown in fig. 1.
The linear range is 0.008-0.20 ng/mL, the detection limit is 0.9 pg/mL, and the recovery rate is 95.8-106.6%. The detection of other biological micromolecules such as mycotoxin, vitamin C, glucose and the like, namely aflatoxin B1, is free from interference.
<110> Hunan university of science and technology
<120> detection method and detection kit for aflatoxin B1
<160>2
<210>1
<211>55
<212>DNA
<213> aflatoxin B1 nucleic acid aptamer
<400>1
5’-AAAAAGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3’
<210>2
<211>35
<212>DNA
<213> Single-stranded Signal Probe
<400>2
5’-CCCCCCACACCCGATCCCCCCCTGTGGGCCTAGCG-3’

Claims (4)

1. A method for detecting aflatoxin B1 is characterized by comprising the following steps:
(A) the aflatoxin B1 aptamer Apt is hybridized with a single-stranded signal probe ssDNA through complementary bases of underlined parts to form a hybrid chain;
(B) reacting the hybrid chain with a sample to be detected, and when aflatoxin B1 exists in the sample to be detected, specifically reacting an aflatoxin B1 aptamer in the hybrid chain with aflatoxin B1 to release a single-chain signal probe ssDNA;
(C) in order to eliminate the interference of the hybrid strand, DNA amplification is carried out by taking the hybrid strand as a template, so that the hybrid strand becomes double-stranded DNA, and then the double-stranded DNA is hydrolyzed into mononucleotide by using exonuclease to remove the double-stranded DNA, so that a single-stranded signal probe ssDNA is remained;
(D) the aflatoxin B1 aptamer-aflatoxin B1 combination cannot induce silver ions to reduce to generate near-infrared fluorescent silver nanoclusters, a single-stranded signal probe ssDNA in a system to be detected induces the silver ions to reduce to the near-infrared fluorescent silver nanoclusters, and the fluorescence intensity of the system is detected, so that the aflatoxin B1 content in a sample to be detected is determined;
the DNA amplification system comprises a buffer solution, dNTP and Phi29 DNA polymerase, wherein the buffer solution is prepared from Tris-HCl and MgCl2And (NH)4)2SO4Composition is carried out; the exonuclease is Exo III exonuclease;
the aflatoxin B1 aptamer is 5' -AAAAAGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTC GCTAGGCCCACA-3’；
The ssDNA of the single-stranded signal probe is 5' -CCCCCCACACCCGATCCCCCCTGTGGGCCTAGCG-3’。
2. A kit for detecting aflatoxin B1 is characterized by comprising: the kit comprises an aflatoxin B1 aptamer, a single-stranded signal probe ssDNA, a DNA amplification system, exonuclease and a silver ion reduction detection system; the aflatoxin B1 aptamer is 5'-AAAAAGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3';
the single-stranded signal probe DNA is 5'-CCCCCCACACCCGATCCCCCCTGTGGGCCTAGCG-3';
the DNA amplification system comprises a buffer solution, dNTP and Phi29 DNA polymerase, wherein the buffer solution is prepared from Tris-HCl and MgCl2And (NH)4)2SO4Composition is carried out;
the exonuclease is Exo III exonuclease.
3. The kit for detecting aflatoxin B1 of claim 2, wherein the reducing agent in the silver ion reduction detection system is borohydride.
4. The kit for detecting aflatoxin B1 of claim 3, wherein the borohydride is sodium borohydride.
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CN108444992B (en) * 2018-02-09 2020-11-13 河南工业大学 Aflatoxin quantitative detection kit and detection method thereof
CN109402128B (en) * 2018-12-21 2020-11-06 青岛农业大学 Aflatoxin B1The aptamer of (a), and aflatoxin B containing the aptamer1Detection kit and detection method

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CN104267192A (en) * 2014-03-06 2015-01-07 上海大学 Bio-electrochemical sensor for detecting thrombin as well as preparation method and application of bio-electrochemical sensor

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CN104267192A (en) * 2014-03-06 2015-01-07 上海大学 Bio-electrochemical sensor for detecting thrombin as well as preparation method and application of bio-electrochemical sensor

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