CN112063729B - Kit and method for detecting listeria monocytogenes - Google Patents
Kit and method for detecting listeria monocytogenes Download PDFInfo
- Publication number
- CN112063729B CN112063729B CN202010855361.XA CN202010855361A CN112063729B CN 112063729 B CN112063729 B CN 112063729B CN 202010855361 A CN202010855361 A CN 202010855361A CN 112063729 B CN112063729 B CN 112063729B
- Authority
- CN
- China
- Prior art keywords
- magnetic
- listeria monocytogenes
- biotin
- probe
- kit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/682—Signal amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
- G01N24/08—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using nuclear magnetic resonance
- G01N24/088—Assessment or manipulation of a chemical or biochemical reaction, e.g. verification whether a chemical reaction occurred or whether a ligand binds to a receptor in drug screening or assessing reaction kinetics
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- High Energy & Nuclear Physics (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kit and a method for detecting listeria monocytogenes. The kit comprises a capture probe modified by MNPs, a biotin-labeled signal probe, ALP-SA, AAP, potassium permanganate and the like. The principle of the method of the invention is as follows: the genome DNA of the listeria monocytogenes is hybridized with a capture probe modified by MNPs and a biotin-marked signal probe to form a double-chain sandwich complex, ALP-SA is connected to the double-chain sandwich complex through a biotin-avidin system, ALP can remove phosphate groups in AAP to convert the AAP into AA, and the AA can convert MnO 4 ‑ Reduction to Mn 2+ Conversion of Mn (VI) to Mn (II) causes a transverse relaxation time (T) 2 ) Significant variation of T 2 Is read as a signal indicative of listeria monocytogenes. The invention has high sensitivity, high accuracy, quick response and simple operation, and avoids the complex process of PCR and the multi-step operation of in situ hybridization.
Description
Technical Field
The invention relates to detection of microorganisms, in particular to a kit and a method for detecting listeria monocytogenes.
Background
In recent years, with the improvement of the living standard of people, food safety is increasingly concerned, and the food safety becomes an important issue of public policy. Especially pathogen-induced food-borne diseases have severely threatened public health safety and human health. Listeria monocytogenes (Listeria monocytogenes), abbreviated as Listeria monocytogenes, is one of the major food-borne pathogens, and can cause influenza-like diseases and serious complications to humans, including meningitis, septicemia, and even spontaneous abortion in fragile people. Therefore, to ensure public food safety, it is very important and necessary to establish a rapid and reliable detection method for food-borne pathogens such as listeria monocytogenes.
Various analytical methods have been widely used for detection of food-borne pathogens, including microbial culture, chromatography-mass spectrometry, enzyme-linked immunosorbent assay (ELISA) and assays based on Polymerase Chain Reaction (PCR). Although these conventional methods have high sensitivity and specificity, there are still some limitations. For example, bacterial culture requires time-consuming and laborious procedures and laborious tasks; chromatography-mass spectrometry requires expensive equipment and complex pretreatment procedures; PCR methods can encounter problems associated with sample matrices, as the sample matrices can contain PCR inhibitors commonly found in biological samples. Compared with the method, the ELISA method is simple to operate and suitable for on-site detection, but lacks sensitivity and anti-interference performance. Therefore, a new or improved method is needed to achieve a rapid, sensitive, accurate detection of pathogenic bacteria and with a high resistance to transformation.
In order to develop detection techniques to overcome the current limitations, various studies have been made, wherein biosensors have become a promising and important tool in food analysis and biological detection. Magnetic relaxation switch time (MRS) biosensors have two distinct advantages over electrochemical, electrochemiluminescent, colorimetric, and fluorescent related biosensors (1) signal tags are easy to separate or do not require a separation step; (2) Because the magnetic signal background of the food sample is low, the magnetic signal can avoid the interference of complex sample matrix to the signal. Among them, paramagnetic ion-mediated MRS biosensors have better stability and lower cost because of paramagnetic ions such as Fe 3+ /Fe 2+ 、Cu 2+ /Cu + And Mn of 7+ /Mn 2+ The valence change of (2) results in a significant change in the T1 or T2 signal. However, consider Fe-based 3+ /Fe 2+ Or Cu 2+ /Cu + Magnetic signal variation of the mediated magnetic biosensor is limited, mn 7+ /Mn 2+ Mediated magnetic biosensors have been demonstrated to detectA highly sensitive and reliable method for pathogens, even small molecules. However, the past MRS biosensors have relied primarily on expensive antibodies, which limit their further applications.
Nucleic acid-based analysis techniques provide specific detection of pathogens due to the specificity and stability of deoxyribonucleic acid (DNA). Thus, high recognition efficiency of DNA can be used for DNA biosensors for pathogen detection. DNA biosensors based on optical and electrochemical methods have been successful in detecting a variety of pathogens. However, most DNA biosensors employ PCR amplification to increase sensitivity, resulting in long detection periods and high costs. Therefore, it is very necessary to develop a DNA biosensor that has high sensitivity, high accuracy, quick response, and simple operation. Therefore, the combination of MRS method and DNA recognition is a promising method for detecting food-borne pathogenic bacteria.
Disclosure of Invention
The invention aims to provide a kit for detecting listeria monocytogenes and a method for detecting listeria monocytogenes.
The aim of the invention is achieved by the following technical scheme:
a kit for detecting listeria monocytogenes comprising: magnetic Nanoparticle (MNPs) modified capture probes, biotin-labeled signaling probes, alkaline phosphatase-labeled streptavidin (ALP-SA), 2-phospho-L-ascorbate trisodium salt (AAP), potassium permanganate, and the like.
The capture probes and the signal probes can be specifically combined with the genome DNA of the listeria monocytogenes. Further, the capture probe and the signal probe can be specifically combined with the virulence gene hly of the listeria monocytogenes. Further, the sequences of the capture probe and the signal probe are as follows:
capture probe HLY1:5'-CACGAGAGCACCTGGAT-3';
signaling probe HLY2:5'-GACAGGAAGAACATCGGG-3'.
The capture probes modified by the Magnetic Nano Particles (MNPs) are as follows:
MNPs-CO-NH-(CH 2 ) 6 -TTTTTTTTTTTTTCACGAGAGCACCTGGAT。
the signal probe marked by the biotin is as follows:
GACAGGAAGAACATCGGGTTTTTTTTTTTT-(CH 2 ) 7 -Biotin。
the kit for detecting listeria monocytogenes further comprises: saline sodium citrate buffer.
A method of detecting listeria monocytogenes comprising the steps of:
(1) Extracting genome DNA of the sample to be detected.
(2) Performing thermal denaturation treatment on the capture probes modified by the magnetic nano particles, the signal probes marked by the biotin and the genome DNA extracted in the step (1), adding the thermal denaturation treatment into a physiological saline sodium citrate buffer solution for incubation to enable target DNA to hybridize with the probes, and collecting the magnetic adsorbate 1 through magnetic separation.
(3) Adding alkaline phosphatase-labeled streptavidin into the magnetic adsorbate 1 obtained in the step (2) to react so as to connect the streptavidin and the biotin, and collecting the magnetic adsorbate 2 through magnetic separation.
(4) Adding 2-phosphoric acid-L-ascorbic acid trisodium salt into the magnetic adsorbate 2 obtained in the step (3) to react to remove the phosphate group, and collecting the supernatant through magnetic separation.
(5) Adding potassium permanganate into the supernatant collected in the step (4) to react so as to obtain MnO 4 - Reduction to Mn 2+ After the reaction, T of the reaction mixture was measured by low-field nuclear magnetic resonance (LF-NMR) 2 Value according to T 2 The change in value is used to perform qualitative and quantitative detection on listeria monocytogenes.
Further, the method for detecting listeria monocytogenes comprises the following steps:
(1) Extracting genome DNA of the sample to be detected.
(2) 80-120 mu L of capture probe modified by magnetic nano particles with concentration of 1mg/mL and modification amount of 0.15-0.3nmol/g (capture probe/magnetic nano particles), 80-120 mu L of signal probe marked by 1.5nmol/L biotin, 20-100 mu L of genome DNA extracted in the step (1) are subjected to heat denaturation treatment at 90-100 ℃ for 5-10min, and then added into 100-180 mu L of physiological saline sodium citrate buffer solution to be incubated for 1-1.5h at 50-60 ℃ so as to enable target DNA to hybridize with the probe, and magnetic adsorbate 1 is collected through magnetic separation.
(3) Adding 50-150mL of 1 mug/mL alkaline phosphatase labeled streptavidin into the magnetic adsorbate 1 obtained in the step (2), shaking and reacting at 36.5-37.5 ℃ for 0.5-1h to connect the streptavidin and the biotin, and collecting the magnetic adsorbate 2 through magnetic separation.
(4) 50-150 mu L of 25mmol/L of 2-phosphoric acid-L-ascorbic acid trisodium salt is added to the magnetic adsorbate 2 obtained in the step (3), the phosphate group is removed by shaking reaction for 0.5-1h at 36.5-37.5 ℃, and the supernatant is collected by magnetic separation.
(5) Adding 25-75 mu L of 0.5mmol/L potassium permanganate into the supernatant collected in the step (4), reacting for 5-10min at normal temperature, adding MnO 4 - Reduction to Mn 2+ T of the reaction mixture was then measured by low field NMR 2 Value according to T 2 The change in value is used to perform qualitative and quantitative detection on listeria monocytogenes.
The T was acquired at 35℃in step (5) above using the Carr-Purcell-Meiboom-Gill pulse sequence 2 The parameters are as follows: nuclear magnetic resonance frequency 19.894MHz;90 DEG pulse width, 13 mus; 180 DEG pulse width, 26 mus; tw=12000 ms; nech=12000 ms; sw=100 KHz; te=1.0 ms; ns=2. Three analyses were performed for each point (n=3), and for each interval T was calculated using the following formula 2 Value change (DeltaT) 2 )：ΔT 2 ＝|T 2 sample -T 2 blank |。
The principle of the invention is shown in fig. 1, and mainly comprises the following steps: the extracted genome DNA of the listeria monocytogenes is changed into a single-stranded structure from a double-helix structure through heat denaturation treatment; the MNPs-HLY1 conjugate and biotinylated HLY2 were fully extended by thermal denaturation treatment. HLY1 and HLY2 can bind to a target DNA sequence to form a double-stranded sandwich complex according to the base complementary pairing principle. ALP-SA is attached to the double-stranded sandwich complex by the biotin-streptavidin system. ALP can remove phosphate groups in AAP to convert it to Ascorbic Acid (AA). Finally, the generated AA willMnO is added to 4 - Reduction to Mn 2+ While the conversion of Mn (VI) to Mn (II) leads to a transverse relaxation time (T) 2 ) Significant variation of T 2 The change in (c) may be read as a signal indicative of listeria monocytogenes.
The invention has the following advantages and beneficial effects:
(1) The invention uses a magnetic relaxation time (MRS) sensing system, the signal label is easy to separate or does not need a separation step, the magnetic signal background of the food sample is low, and the interference of complex sample matrixes on the signal can be avoided;
(2) The invention selects paramagnetic ions Mn (VII)/Mn (II), the paramagnetic ions have better stability and lower cost; the magnetic signal change caused by Mn (VII)/Mn (II) is most remarkable, and the sensitivity is higher;
(3) The invention designs a specific probe and a primer for detection by utilizing the virulence gene-hly of the listeria monocytogenes and the high recognition efficiency of DNA, and replaces the expensive antibody commonly used in the prior DNA sensor;
(4) The magnetic sensor prepared by the invention is cascaded by using a biotin-streptavidin system, and utilizes ALP to catalyze AAP to generate AA, so that Mn (VII)/Mn (II) signal amplification is realized, and the complex process of PCR and multi-step operation of in-situ hybridization are avoided.
Drawings
FIG. 1 is a schematic diagram of a method of detecting Listeria monocytogenes according to the present invention.
FIG. 2 is a graph of Mn (VII)/Mn (II) transitions used as signal readouts to verify MnPS-based magnetic relaxation; a is ALP enzyme-mediated Mn (VI)/Mn (II) transformation induction (high ΔT) 2 Low delta T 2 ) Principle of (2); b is MnO 4 - With Mn 2+ Delta T in aqueous solution 2 A change in value; c is ALP vs KMnO with different concentrations 4 Influence of the solution; d is KMnO 4 Color change by reaction with aqueous AA; e is AA to KMnO with different concentrations 4 Influence of the solution.
FIG. 3 is a representation and analysis of MNPs-HLY1 conjugates using UV-Vis spectroscopy and DLS; a is MNPs and complexes of MNPs and DNA probe HLY1 characterized by differential scanning calorimeter (DLS) at 1000nm size; b is the Zeta potential characterizing MNPs and MNPs-HLY1 complexes by differential scanning calorimetry (DLS); c is the ultraviolet-visible absorption spectrum of MNPs, HLY1 and MNPs-HLY1 complexes.
FIG. 4 is a schematic representation of the sensitivity, specificity of the invention for detecting Listeria monocytogenes; a is a standard curve for detection of Listeria monocytogenes; b is a linear range for detection of Listeria monocytogenes; c is the principle of probe-DNA induced hybridization and enzyme-mediated Mn (VII)/Mn (II) transformation induction (high ΔT) 2 Low delta T 2 ) Principle of magnetic relaxation switch sensing; d is the specificity of the detection method of the invention. (the total number of colonies was 10 5 CFU/mL, error bars show standard deviation of three experiments).
Detailed Description
The technical scheme of the invention is further described below with reference to the embodiment and the attached drawings. It should be apparent to those skilled in the art that the examples are merely provided to aid in understanding the present invention and should not be construed as limiting the invention in any way.
Example 1
AAP can be converted to AA using a widely used marker enzyme, alkaline phosphatase (ALP), providing a platform for enzymatic Mn (VII)/Mn (II) conversion (FIG. 2A). Thus, reacting the double-stranded sandwich complex formed with ALP-SA converts AAP to AA by cleavage of the phosphate group, and the resulting AA will convert MnO 4 - Reduction to Mn 2+ Whereas the conversion of Mn (VII) to Mn (II) leads to a transverse relaxation time (. DELTA.T) 2 ) Is a significant variation of (a). By measuring MnO of different concentration gradients 4 - And Mn of 2+ Delta T in aqueous solution 2 The value of DeltaT is clearly seen 2 Depending on the concentration of Mn (II), mn (VII) vs. DeltaT 2 The effect of (a) is negligible (fig. 2B). Thus, mnO is selected 4 - As a reaction substrate. In addition, by altering different solubility gradients, different concentrations of AA and ALP versus KMnO were validated 4 The effect of the solution (FIGS. 2C and 2E). In the presence of AA, mn (VII) reduced to Mn (II), the solution color changed significantly, and the reddish Mn (VII) turned to colorless Mn (II) (fig. 2D). By this method, the target DNA sequence can be efficiently detected.
Example 2
The equipment and materials used in this example to detect listeria monocytogenes are as follows:
the device comprises: oscillating by using an MS-3 oscillator; performing magnetic separation and washing steps by using a SuperMag magnetic separator; 0.5T low field nuclear magnetic resonance spectrometer (LF-NMR) measurement T 2 A signal; UV-1800 obtained ultraviolet-visible (UV-Vis) absorption spectra; dynamic Light Scattering (DLS) was performed by a Markov Zeta particle size analyzer (Nano-ZS), measuring hydrodynamic size and Zeta potential; the concentration and purity of DNA are obtained by a nanometer photometer-N60 Touch; performing hybridization reaction by using an HL-2000 hybridization linker (UVP); t100 TM Thermal cycler and GelDoc xr+ systems were used for amplification product and gel imaging, respectively.
Materials: magnetic nanoparticles (MNPs, diameter: 1000nm; solid phase content: 10mg/mL, with carboxyl modification) were purchased from Ocean nanoTech (USA); ascorbic Acid (AA), 2-phospho-L-ascorbic acid trisodium salt (AAP), N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2- (N-morpholin) ethanesulfonic acid, and Bovine Serum Albumin (BSA) were all purchased from Sigma-Aldrich (usa); alkaline phosphatase labeled streptavidin was purchased from Beyotime Biotechnology (1 mg/mL, ALP-SA, shanghai, china); saline sodium citrate buffer (20 XSSC, pH 7.0) and bacterial genomic DNA kit were purchased from ZOMANBIO (China, beijing); trypsin Soybean Broth (TSB) and Tryptone Soybean Agar (TSA) media were purchased from Qingdao Hope Bio-Technology co., ltd (china, peninsula); tris-EDTA buffer (TE, pH 8.0) for DNA solubilization was purchased from Shanghai Double-Heix Biotech Co., ltd. (China, shanghai); all other chemicals were analytical grade, purchased from Sinopharm Chemical co., ltd. (china, shanghai); milli-Q water was used throughout the experiment.
The oligonucleotide sequences purified by HPLC were purchased from TsingKe Biological Technology (marchantia in china) and were as follows:
TABLE 1
(1) Preparation of MNPs-HLY1 conjugates
The capture probe HLY1 is coupled to the carboxyl modified magnetic nanoparticle through EDC/NHS activation, and the specific process is as follows: mu.L MNPs (1000 nm,10mg/mL, modified with carboxyl groups) were taken into 1.5mL centrifuge tubes and washed 2 times with 500. Mu.L MEST (10 mmol/L MES, pH=6.0, 0.05% Tween-20) followed by first magnetic separation. After removal of the supernatant, 200. Mu.L of 5mg/mL EDC and 200. Mu.L of 5mg/mL NHS were added to the centrifuge tube and reacted with MNPs, and the mixture was gently shaken in a vortex shaker at 37℃for 30 minutes, subjected to a second magnetic separation and washed 2 times with MEST. Next, 150. Mu.L of 1.5nmol/L of oligonucleotide probe HLY1 (PBST as solvent) was added to the above centrifuge tube, the volume was adjusted to 500. Mu.L with PBST (0.01 mol/L PBS, pH=7.4, 0.05% Tween-20), the mixture was gently shaken in a vortex shaker at 37℃for 3 hours, the supernatant was removed by performing a third magnetic separation, then 1mL of 1% BSA-containing PBST was added to the MNPs-HLY1 conjugate, the residual site was blocked by reaction at 37℃for 30 minutes, and the fourth magnetic separation was performed and washed 4 times with 500. Mu.L of PBST. Finally, MNPs-HLY1 was obtained and dispersed in 1mL of PBST containing 0.5% BSA, and stored at 4℃for use.
MNPs-HLY1 conjugates were characterized using Dynamic Light Scattering (DLS) and ultraviolet-visible spectroscopy (UV-Vis). After modification of HLY1, the average hydrodynamic diameter (1411 nm) of MNPs-HLY1 conjugates was significantly increased compared to unmodified MNPs (1036 nm) (fig. 3A). For surface potential, the surface modification was followed by a slight increase from-27 mV to-25 mV (FIG. 3B). This slight potential change may be due to the fact that the oligonucleotide probe itself has a negative potential. In addition, FIG. 3C shows that the absorbance spectra of MNPs-HLY1 conjugates have a maximum absorbance peak at 278nm, whereas the original MNPs have no absorbance peak at any wavelength. Furthermore, the absorbance spectrum of DNA has a maximum absorbance peak at 260nm due to the electron interaction between bases in the DNA molecule, and therefore, the maximum absorbance peak of DNA oligonucleotide (HLY 1) is located around 260nm (FIG. 3C). Clearly, the adsorption peak of the MNPs-HLY1 conjugate was red shifted 18nm relative to that of the pure DNA oligonucleotide (HLY 1), indicating that HLY1 adsorbed on the MNPs surface and that an amino linkage was formed between MNPs and HLY 1. These results indicate that HLY1 was successfully modified to the surface of MNPs.
(2) Detection of listeria monocytogenes
From different concentrations (20-2X 10) 7 CFU/mL) of Listeria monocytogenes culture, and dissolving the DNA with Tris-EDTA buffer (TE, pH 8.0). 4 pathogenic bacteria of streptococcus enteritis, escherichia coli, staphylococcus aureus and vibrio parahaemolyticus are selected, and the concentration of each pathogenic bacteria is 10 5 1mL of the bacterial liquid of CFU/mL, extract genome DNA, as negative control.
100. Mu.L of the MNPs-HLY1 solution obtained in step (1), 100. Mu.L of 1.5nmol/L of biotinylated oligonucleotide probe HLY2 (1 XSSC, pH7.0 as solvent), 40. Mu.L of genomic DNA of different concentrations and 160. Mu.L of physiological saline sodium citrate buffer (1 XSSC, pH 7.0) were added to 1.5mL centrifuge tubes and mixed, and the complex in each centrifuge tube was heat-treated at 95℃for 10 minutes before hybridization, immediately cooled in ice for 5 minutes, cooled and placed in an oven at 53℃for continuous shaking incubation for 1 hour to hybridize the target DNA with the probe. After the first magnetic separation and washing of the complex in the centrifuge tube, 100. Mu.L of 1. Mu.g/mL ALP-SA (1 XSSC as solvent to dilute the stock solution) was added, and the reaction was gently shaken at 37℃for 30 minutes, followed by the second magnetic separation and washing. The supernatant was removed in the first magnetic separation and each tube complex was washed 3 times with 0.5 XSSC buffer, respectively; the second magnetic separation was performed by washing with TBST buffer 4 times and pure water 1 time to remove the excess ALP-SA. Subsequently, 100. Mu.L of a 25mmol/L aqueous AAP solution was added to the centrifuge tube, and the reaction was gently shaken at 37℃for 30 minutes, and the supernatant of each tube was collected after the third magnetic separation. Finally, the supernatant was reacted with 0.5mmol/L potassium permanganate aqueous solution at a volume ratio of 2:1 for 5min, and the T of the reaction mixture was measured by LF-NMR of 0.5T 2 Values.
T was acquired at 35℃using the Carr-Purcell-Meiboom-Gill pulse sequence 2 The parameters are as follows: nuclear magnetic resonance frequency 19.894MHz;90 DEG pulse width, 13 mus; 180 DEG pulse width, 26 mus; tw=12000 ms; nech=12000 ms; sw=100 KHz; te=1.0 ms; ns=2. Three analyses were performed for each point (n=3), and for each interval T was calculated using the following formula 2 Variation of (DeltaT) 2 )：ΔT 2 ＝|T 2 sample -T 2 blank |。
The results are shown in FIG. 4: FIG. 4A shows a range from 20 to 2X 10 7 As a result of the concentration of Listeria monocytogenes of CFU/mL, the more target bacteria, the obtained ΔT 2 The higher the value, the result shows that the target DNA sequence can be enriched by capturing MNPs-HLY1 and hybridization reaction, and the signal can be amplified by Mn (VII)/Mn (II) interconversion mediated by enzyme catalytic reaction. Fig. 4B also shows Δt 2 The value and the concentration of the target bacteria are 2 multiplied by 10 2 ～2×10 7 The linear relationship is found in the CFU/mL range, and the linear equation is Y=62.45X+35.67 (X=log ([ Listeria monocytogenes (CFU/mL))]，R 2 0.9976) with a detection limit (S/n=3) of 10 2 CFU/mL. Delta T for Listeria monocytogenes detection 2 The values were far higher than the negative control (FIG. 4D), also indicating that the oligonucleotide probe designed based on the base complementary pairing principle had better specificity for the target sequence, i.e.the genomic DNA of Listeria monocytogenes could be specifically recognized and captured by MNPs-HLY1 conjugates and HLY2 (FIG. 4C).
Example 3
This embodiment is as in basic embodiment 2, except that: in step (1), the concentration of HLY1 was changed to 1nmol/L, 2nmol/L and 3nmol/L, respectively, and as a result, it was found that all conjugates had an adsorption peak at 278 nm. Meanwhile, when the concentration of HLY1 is more than 1nmol/L, the absorption spectrum of the conjugate MNPs-HLY1 is almost unchanged. Therefore, HLY1 of 1.5nmol/L is adopted for coupling with MNPs to improve the coupling efficiency.
Example 4
This embodiment is as in basic embodiment 2, except that: in the step (2), the concentration of AAP added was changed to 20mmol/L and 30mmol/L, respectively. At 20 to 2 multiplied by 10 7 In the seven control data of CFU/mL, deltaT was compared to the initial 25mmol/L concentration 2 The value is obviously reduced, the difference value fluctuates in 0-150ms, and the value is extremely unstable. Therefore, the optimum concentration of AAP is 25mmol/L.
Example 5
This embodiment is as in basic embodiment 2, except that: in step (2), the mixture was admixed with 0.5mmol/L KMnO 4 Reaction of aqueous solutionsThe volume ratio is changed to 1:1. In the seven control data, Δt was compared to the initial 2:1 volume ratio 2 The values decrease significantly, the difference is large, and the fluctuation occurs in 150-300 ms. Thus, the mixture was admixed with 0.5mmol/L KMnO 4 The optimal reaction volume ratio of (2) to (1).
Example 6
This example compares the invention with conventional PCR amplification methods for detection of Listeria monocytogenes. First, pure culture of Listeria monocytogenes was subjected to 10-fold gradient dilution with sterile phosphate buffer to a final concentration of 10 8 About 10CFU/mL, and then 1mL of bacterial solutions with different concentrations are added into a 1.5mL centrifuge tube to extract genome DNA. The total reaction volume was 25. Mu.L, including 2.5. Mu.L of 10 XPCR buffer, 0.25. Mu.L of Taq DNA polymerase (5U/mL), 2.0. Mu.L of 2.5mmol/L dNTP mix, 1. Mu.L of genomic DNA, 18.25. Mu.L of double distilled water, 0.5. Mu.L of forward primer (hly AF, 10. Mu. Mol/L) and 0.5. Mu.L of reverse primer (hly AR, 10. Mu. Mol/L), the primer sequences are shown in Table 1. The PCR reaction conditions were: pre-denaturation at 95℃for 5min, then denaturation at 95℃for 1min, annealing at 62℃for 1min, extension at 72℃for 1min, total of 35 cycles, and extension at 72℃for 8min. The final PCR products were detected by 1% agarose gel electrophoresis.
As a result, it was found that the sensitivity of the PCR method for detecting Listeria monocytogenes was about 10 3 CFU/mL. In gel imaging of extracted DNA without PCR amplification, the DNA is only from 10 8 CFU/mL and 10 7 Only the genomic DNA extracted from the CFU/mL bacterial liquid shows a weak band. Therefore, the magnetic DNA biosensor developed by the invention can not only avoid complex operation steps such as PCR operation and the like, but also provide a promising method with less time consumption and high sensitivity for detecting food-borne pathogenic bacteria.
Example 7
In the embodiment, the ham sample marking recovery test is carried out, and the ham homogenate samples added with the listeria monocytogenes with different concentrations are detected. Genomic DNA was extracted and tested as described above. The recovery rate is 87.2% -101.3%, and the variation coefficient is 6.46% -9.54% (Table 2), which shows the potential and feasibility of the constructed biosensor for detecting listeria monocytogenes in complex samples.
TABLE 2 recovery of Listeria monocytogenes in ham at different concentration levels
The applicant states that the process of the invention is illustrated by the above examples, but the invention is not limited to, i.e. does not mean that the invention must be carried out in dependence on the above process steps. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.
Sequence listing
<110> university of Hubei industries
<120> A kit and method for detecting Listeria monocytogenes
<141> 2020-08-24
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
cacgagagca cctggat 17
<210> 2
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
gacaggaaga acatcggg 18
<210> 3
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
gcctgcaagt cctaagacgc caatc 25
<210> 4
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
cttgcaactg ctctttagta acagc 25
Claims (8)
1. A kit for detecting listeria monocytogenes, comprising: comprising: magnetic nanoparticle modified capture probes, biotin-labeled signaling probes, alkaline phosphatase-labeled streptavidin, 2-phospho-L-ascorbate trisodium salt, and potassium permanganate;
the capture probes and the signal probes can be specifically combined with the genome DNA of the listeria monocytogenes; the capture probes and signaling probes are capable of binding to the target DNA sequence to form a double-stranded sandwich complex.
2. The kit of claim 1, wherein: the capture probe and the signal probe can be used for combining virulence genes of listeria monocytogeneshlySpecific binding.
3. The kit of claim 1, wherein: the sequences of the capture probe and the signal probe are as follows:
capture probe HLY1: -CACGAGAGCACCTGGAT;
signaling probe HLY2: GACAGGAAGAACATCGGG.
4. The kit of claim 1, wherein: the capture probe modified by the magnetic nano-particles is as follows: MNPs-CO-NH- (CH) 2 ) 6 -TTTTTTTTTTTTTCACGAGAGCACCTGGAT; the signal probe marked by the biotin is as follows: GACAGGAAGAACATCGGGTTTTTTTTTTTT- (CH) 2 ) 7 -Biotin。
5. The kit of claim 1, wherein: comprises physiological saline sodium citrate buffer.
6. A method of detecting listeria monocytogenes using the kit of any of claims 1-5, wherein: the method comprises the following steps:
(1) Extracting genome DNA of a sample to be detected;
(2) Performing thermal denaturation on the capture probes modified by the magnetic nano particles, the signal probes marked by the biotin and the genome DNA extracted in the step (1), adding the thermal denaturation into a physiological saline sodium citrate buffer solution for incubation to enable target DNA to hybridize with the probes, and collecting the magnetic adsorbate 1 through magnetic separation;
(3) Adding alkaline phosphatase marked streptavidin into the magnetic adsorbate 1 obtained in the step (2) to react so as to connect the streptavidin and the biotin, and collecting the magnetic adsorbate 2 through magnetic separation;
(4) Adding 2-phosphoric acid-L-ascorbic acid trisodium salt into the magnetic adsorbate 2 obtained in the step (3) to react to remove phosphate groups, and collecting supernatant through magnetic separation;
(5) Adding potassium permanganate into the supernatant collected in the step (4) to react so as to obtain MnO 4 - Reduction to Mn 2+ Measurement of T of the reaction mixture by Low field Nuclear magnetic resonance after the reaction 2 Value according to T 2 The change in value is used to perform qualitative and quantitative detection on listeria monocytogenes.
7. The method of detecting listeria monocytogenes of claim 6, wherein: the method comprises the following steps:
(1) Extracting genome DNA of a sample to be detected;
(2) 80-120 mu L of magnetic nanoparticle modified capture probe with the concentration of 1mg/mL and the modification amount of 0.15-0.3nmol/g, 80-120 mu L of 1.5nmol/L biotin-labeled signaling probe, 20-100 mu L of genome DNA extracted in the step (1) are subjected to heat denaturation at 90 for 5-10min, and then added into 100-180 mu L of physiological saline sodium citrate buffer solution to be incubated for 1-1.5h at 50, so that target DNA and the probe are hybridized, and magnetic adsorbate 1 is collected through magnetic separation;
(3) Adding 50-150mL of 1 mug/mL alkaline phosphatase labeled streptavidin into the magnetic adsorbate 1 obtained in the step (2), shaking at 36.5 for reaction for 0.5-1h to connect the streptavidin and the biotin, and collecting the magnetic adsorbate 2 through magnetic separation;
(4) Adding 50-150 mu L of 25 mmol/L2-phosphoric acid-L-ascorbic acid trisodium salt into the magnetic adsorbate 2 obtained in the step (3), shaking at 36.5 for reaction for 0.5-1h to remove phosphate groups, and collecting supernatant by magnetic separation;
(5) Adding 25-75 mu L of 0.5mmol/L potassium permanganate into the supernatant collected in the step (4), reacting for 5-10min at normal temperature, adding MnO 4 - Reduction to Mn 2+ T of the reaction mixture was then measured by low field NMR 2 Value according to T 2 The change in value is used to perform qualitative and quantitative detection on listeria monocytogenes.
8. The method for detecting listeria monocytogenes of claim 6 or 7, wherein: in step (5), the T is acquired by using a Carr-Purcell-Meiboom-Gill pulse sequence 2 The parameters are as follows: nuclear magnetic resonance frequency 19.894MHz;90 DEG pulse width, 13 mus; 180 DEG pulse width, 26 mus; tw=12000 ms; nech=12000 ms; sw=100 KHz; te=1.0 ms; ns=2; three analyses were performed for each point and for each interval T was calculated using the following formula 2 Value change T 2 ：∆T 2 =T 2 sample -T 2 blank 。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010855361.XA CN112063729B (en) | 2020-08-24 | 2020-08-24 | Kit and method for detecting listeria monocytogenes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010855361.XA CN112063729B (en) | 2020-08-24 | 2020-08-24 | Kit and method for detecting listeria monocytogenes |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112063729A CN112063729A (en) | 2020-12-11 |
| CN112063729B true CN112063729B (en) | 2023-05-09 |
Family
ID=73660389
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010855361.XA Active CN112063729B (en) | 2020-08-24 | 2020-08-24 | Kit and method for detecting listeria monocytogenes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112063729B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108333536A (en) * | 2017-01-20 | 2018-07-27 | 国家纳米科学中心 | The Magnetic Sensor and its construction method, purposes read based on longitudinal relaxation time signal |
| CN110726841A (en) * | 2019-10-21 | 2020-01-24 | 华中农业大学 | Method for detecting veterinary drug residues based on enzymatic click reaction signal amplification magnetic relaxation time immunosensor |
-
2020
- 2020-08-24 CN CN202010855361.XA patent/CN112063729B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108333536A (en) * | 2017-01-20 | 2018-07-27 | 国家纳米科学中心 | The Magnetic Sensor and its construction method, purposes read based on longitudinal relaxation time signal |
| CN110726841A (en) * | 2019-10-21 | 2020-01-24 | 华中农业大学 | Method for detecting veterinary drug residues based on enzymatic click reaction signal amplification magnetic relaxation time immunosensor |
Non-Patent Citations (1)
| Title |
|---|
| Zhilong Wang等.Background Signal-Free Magnetic Bioassay for Food-Borne Pathogen and Residue of Veterinary Drug via Mn(VII)/Mn(II) Interconversion.《ACS Sens》.2019,第4卷第2771−2777页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112063729A (en) | 2020-12-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jayan et al. | Recent development in rapid detection techniques for microorganism activities in food matrices using bio-recognition: A review | |
| Majdinasab et al. | Aptamer-based assays and aptasensors for detection of pathogenic bacteria in food samples | |
| Bu et al. | Ultrasensitive detection of pathogenic bacteria by CRISPR/Cas12a coupling with a primer exchange reaction | |
| Zhang et al. | Sensitive assay of Escherichia coli in food samples by microchip capillary electrophoresis based on specific aptamer binding strategy | |
| Hao et al. | An enhanced chemiluminescence resonance energy transfer aptasensor based on rolling circle amplification and WS2 nanosheet for Staphylococcus aureus detection | |
| Paniel et al. | Aptasensor and genosensor methods for detection of microbes in real world samples | |
| Hayat et al. | Recent advances in ochratoxin A-producing fungi detection based on PCR methods and ochratoxin A analysis in food matrices | |
| Mao et al. | A nanoparticle amplification based quartz crystal microbalance DNA sensor for detection of Escherichia coli O157: H7 | |
| Zhu et al. | A reusable DNA biosensor for the detection of genetically modified organism using magnetic bead-based electrochemiluminescence | |
| CN109207567B (en) | Method for determining staphylococcus aureus based on aptamer and strand displacement amplification reaction | |
| Hao et al. | A chemiluminescent aptasensor based on rolling circle amplification and Co2+/N-(aminobutyl)-N-(ethylisoluminol) functional flowerlike gold nanoparticles for Salmonella typhimurium detection | |
| Kim et al. | Development and characterization of a magnetic bead-quantum dot nanoparticles based assay capable of Escherichia coli O157: H7 quantification | |
| Ren et al. | An aptamer-exonuclease III (Exo III)–assisted amplification-based lateral flow assay for sensitive detection of Escherichia coli O157: H7 in milk | |
| Lu et al. | State of the art in CRISPR/Cas system-based signal conversion and amplification applied in the field of food analysis | |
| Sharif et al. | A novel impedimetric sensor for detecting LAMP amplicons of pathogenic DNA based on magnetic separation | |
| CN106990081A (en) | One kind is based on graphene oxide sensor and its to Hg2+The method of detection | |
| Petrucci et al. | On-site detection of food and waterborne bacteria–Current technologies, challenges, and future directions | |
| Pei et al. | A facile signal-on electrochemical DNA sensing platform for ultrasensitive detection of pathogenic bacteria based on Exo III-assisted autonomous multiple-cycle amplification | |
| Yuan et al. | A graphene oxide-based paper chip integrated with the hybridization chain reaction for peanut and soybean allergen gene detection | |
| Zhao et al. | Nanomaterials-assisted CRISPR/Cas detection for food safety: Advances, challenges and future prospects | |
| Hou et al. | Bio-barcode assay: A useful technology for ultrasensitive and logic-controlled specific detection in food safety: A review | |
| CN114480583A (en) | Colorimetric biosensor, preparation method thereof and method for detecting novel coronavirus | |
| CN112011597B (en) | Cadmium ion sensing method combining induced allosteric probe with rolling circle amplification | |
| CN112063729B (en) | Kit and method for detecting listeria monocytogenes | |
| Bai et al. | A label-free fluorescent sensor for Pb 2+ based on G-quadruplex and graphene oxide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |