CN112063729A

CN112063729A - Kit and method for detecting listeria monocytogenes

Info

Publication number: CN112063729A
Application number: CN202010855361.XA
Authority: CN
Inventors: 吴龙; 周书弘; 陈翊平
Original assignee: Hubei University of Technology
Current assignee: Hubei University of Technology
Priority date: 2020-08-24
Filing date: 2020-08-24
Publication date: 2020-12-11
Anticipated expiration: 2040-08-24
Also published as: CN112063729B

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 Listeria monocytogenes genome DNA, the capture probe modified by MNPs and the biotin-labeled signal probe form a double-stranded sandwich complex through hybridization, ALP-SA is connected to the double-stranded sandwich complex through a biotin-avidin system, and the ALP can be removedRemoval of the phosphate group in AAP to convert it to AA, which will convert the MnO to MnO₄ ^‑Reduction to Mn²⁺The conversion of Mn (VI) to Mn (II) causes a transverse relaxation time (T)₂) Of significant change, T₂As a signal indicative of listeria monocytogenes. The method has the advantages of high sensitivity, high accuracy, quick response and simple operation, and avoids the complex process of PCR and multi-step operation of in-situ hybridization.

Description

Kit and method for detecting listeria monocytogenes

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 receiving more and more attention, and the food safety becomes an important issue of public policy. Especially food-borne diseases caused by pathogens have seriously threatened public health safety and human health. Listeria monocytogenes (Listeria monocytogenes), abbreviated as Listeria monocytogenes, is one of the main food-borne pathogens, and can cause symptoms such as influenza-like diseases and serious complications to human beings, including meningitis and septicemia, and even spontaneous abortion occurs in vulnerable people. Therefore, in order to ensure the safety of public food, it is very important and necessary to establish a fast and reliable method for detecting food-borne pathogens such as Listeria monocytogenes.

Various analytical methods have been widely used for the detection of food-borne pathogens, including microbial culture, chromatography-mass spectrometry, enzyme-linked immunosorbent assays (ELISA) and Polymerase Chain Reaction (PCR) -based assays. Despite the high sensitivity and specificity of these conventional methods, several limitations still exist. For example, bacterial culture requires time-consuming and laborious procedures and laborious tasks; chromatography-mass spectrometry requires expensive equipment and complex pre-treatment procedures; PCR methods may suffer from problems associated with sample matrices, as the sample matrices may contain PCR inhibitors commonly found in biological samples. Compared with the method, the ELISA method is simple to operate and suitable for field detection, but lacks sensitivity and anti-interference performance. Therefore, there is a need for a new or improved method for rapid, sensitive, accurate detection of pathogenic bacteria with high conversion resistance.

In order to develop detection techniques to overcome the current limitations, various studies have been conducted, in which biosensors have become food analysis and biologyOne promising important tool in detection. Magnetic relaxation switching time (MRS) biosensors have two distinct advantages over biosensors related to electrochemistry, electrochemiluminescence, colorimetry, and fluorescence, (1) signal labels are easily separated or no separation step is required; (2) because the background of the magnetic signal of the food sample is low, the magnetic signal can avoid the interference of complex sample matrix to the signal. Among them, the paramagnetic ion-mediated MRS biosensor has better stability and lower cost because of paramagnetic ions such as Fe³⁺/Fe²⁺、Cu²⁺/Cu⁺And Mn⁷⁺/Mn²⁺The change in valence state of (a) results in a significant change in the T1 or T2 signal. However, Fe-based is considered³⁺/Fe²⁺Or Cu²⁺/Cu⁺Magnetic Signal Change Limited in mediated magnetic biosensors Mn⁷⁺/Mn²⁺Mediated magnetic biosensors have proven to be a highly sensitive and reliable method for detecting pathogens and even small molecules. However, previous MRS biosensors rely mainly on expensive antibodies, which limits their further applications.

Nucleic acid-based analytical techniques provide specific detection of pathogens due to the specificity and stability of deoxyribonucleic acid (DNA). Therefore, high recognition efficiency of DNA can be used for a DNA biosensor for pathogen detection. DNA biosensors based on optical and electrochemical methods have successfully detected various pathogens. However, most DNA biosensors use PCR amplification to improve sensitivity, resulting in long detection period and high cost. Therefore, it is necessary to develop a DNA biosensor with high sensitivity, high accuracy, fast response and simple operation. Therefore, the MRS method is combined with DNA recognition, and the method is a promising method for detecting the 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 purpose of the invention is realized by the following technical scheme:

a kit for detecting listeria monocytogenes, comprising: capture probes modified by Magnetic Nanoparticles (MNPs), biotin-labeled signal probes, alkaline phosphatase-labeled streptavidin (ALP-SA), 2-phospho-L-ascorbic acid trisodium salt (AAP), potassium permanganate, and the like.

The capture probe and the signal probe can be specifically combined with the genome DNA of the listeria monocytogenes. Furthermore, the capture probe and the signal probe can be specifically combined with a virulence gene hly of the listeria monocytogenes. Furthermore, the sequences of the capture probe and the signal probe are as follows:

capture probe HLY 1: 5'-CACGAGAGCACCTGGAT-3', respectively;

signaling probe HLY 2: 5'-GACAGGAAGAACATCGGG-3' are provided.

The capture probe modified by the Magnetic Nanoparticles (MNPs) is as follows:

MNPs-CO-NH-(CH₂)₆-TTTTTTTTTTTTTCACGAGAGCACCTGGAT。

the biotin-labeled signal probe is as follows:

GACAGGAAGAACATCGGGTTTTTTTTTTTT-(CH₂)₇-Biotin。

the kit for detecting listeria monocytogenes further comprises: physiological saline sodium citrate buffer.

A method for detecting Listeria monocytogenes, comprising the steps of:

(1) and extracting the genome DNA of the sample to be detected.

(2) And (2) performing thermal denaturation on the capture probe modified by the magnetic nanoparticles, the biotin-labeled signal probe and the genomic DNA extracted in the step (1), adding the thermally denatured product into a normal saline sodium citrate buffer solution for incubation so as to enable the target DNA to be hybridized with the probe, and collecting the magnetic adsorbate 1 through magnetic separation.

(3) Adding alkaline phosphatase-labeled streptavidin to the magnetic adsorbate 1 obtained in step (2), reacting to link the streptavidin to the biotin, and collecting the magnetic adsorbate 2 by magnetic separation.

(4) And (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 supernatant through magnetic separation.

(5) Adding potassium permanganate into the supernatant collected in the step (4) to react so as to obtain MnO₄ ^-Reduction to Mn²⁺After the reaction, the T of the reaction mixture was measured by low-field nuclear magnetic resonance (LF-NMR)₂Value according to T₂And qualitatively and quantitatively detecting the listeria monocytogenes through the change of the value.

Further, the method for detecting listeria monocytogenes comprises the following steps:

(1) and extracting the genome DNA of the sample to be detected.

(2) 80-120 mu L of capture probe modified by magnetic nanoparticles with the concentration of 1mg/mL and the modification amount of 0.15-0.3nmol/g (capture probe/magnetic nanoparticles), 80-120 mu L of 1.5nmol/L biotin-labeled signal probe and 20-100 mu L of genomic DNA extracted in the step (1) are subjected to heat denaturation treatment at 90-100 ℃ for 5-10min, and then added into 180 mu L of 100-saline sodium citrate buffer solution to be incubated at 50-60 ℃ for 1-1.5h after the heat denaturation treatment, so that the target DNA is hybridized with the probe, and the magnetic adsorbate 1 is collected through magnetic separation.

(3) And (3) adding 50-150mL of alkaline phosphatase-labeled streptavidin in a concentration of 1. mu.g/mL to the magnetic adsorbate 1 obtained in the step (2), reacting the mixture at 36.5-37.5 ℃ for 0.5-1h with shaking to link the streptavidin and the biotin, and collecting the magnetic adsorbate 2 by magnetic separation.

(4) Adding 50-150 μ L of 25mmol/L trisodium 2-phosphate-L-ascorbate to the magnetic adsorbate 2 obtained in the step (3), shaking the mixture at 36.5-37.5 ℃ for 0.5-1h to remove the phosphate group, and collecting the supernatant through 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, and adding MnO₄ ^-Reduction to Mn²⁺T of the reaction mixture was then measured by low-field nuclear magnetic resonance₂Value according to T₂And qualitatively and quantitatively detecting the listeria monocytogenes through the change of the value.

In the step (5), T is collected at 35 ℃ by using a Carr-Purcell-Meiboom-Gill pulse sequence₂The parameters are as follows: nuclear magnetic resonance frequency 19.894MHz; pulse width of 90 °, 13 μ s; pulse width 180 °, 26 μ s; TW 12000 ms; NECH 12000 ms; SW is 100 KHz; TE is 1.0 ms; NS is 2. Three analyses were performed for each point (n-3) and for each interval, T was calculated using the following formula₂Variation of value (. DELTA.T)₂)：ΔT₂＝|T_{2 samples}-T_{2 blank}|。

The principle of the invention is shown in figure 1, which mainly comprises the following steps: the extracted genome DNA of the listeria monocytogenes is changed from a double-helix structure into a single-strand structure through thermal denaturation; the MNPs-HLY1 conjugate and biotinylated HLY2 were fully extended by heat denaturation treatment. According to the base complementary pairing principle, HLY1 and HLY2 can be combined with a target DNA sequence to form a double-stranded sandwich complex. ALP-SA was attached to the double stranded sandwich complex by a biotin-streptavidin system. ALP removes the phosphate group in the AAP to convert it to Ascorbic Acid (AA). Finally, the AA formed will be MnO₄ ^-Reduction to Mn²⁺While the conversion of Mn (VI) to Mn (II) causes a transverse relaxation time (T)₂) Of significant change, T₂Can 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 labels are easy to separate or do not need separation steps, the magnetic signal background of the food sample is low, and the interference of a complex sample matrix to signals 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 obvious, and the sensitivity is higher;

(3) the invention utilizes the high recognition efficiency of the virulence gene-hly and DNA of the Listeria monocytogenes to design a specific probe and a primer for detection, and replaces the expensive antibody commonly used by the prior DNA sensor;

(4) the magnetic sensor prepared by the invention uses a biotin-streptavidin system for cascade connection, and utilizes ALP to catalyze AAP to generate AA, thereby realizing Mn (VII)/Mn (II) signal amplification and avoiding a complex process of PCR and multi-step operation of in-situ hybridization.

Drawings

FIG. 1 is a schematic diagram of the method for detecting Listeria monocytogenes of the present invention.

FIG. 2 is a graph demonstrating MnPS based magnetic relaxation using the Mn (VII)/Mn (II) transition as a signal readout; a is ALP enzyme mediated Mn (VI)/Mn (II) conversion induction (high Δ T)₂Low Δ T₂) The principle of (1); b is MnO₄ ^-With Mn²⁺Delta T in aqueous solution₂A change in value; c is ALP vs KMnO at different concentrations₄The influence of the solution; d is KMnO₄Color change caused by reaction with aqueous AA; e is AA to KMnO at various concentrations₄Influence 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, which are characterized by a differential scanning calorimeter (DLS) in a size of 1000 nm; 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 complex.

FIG. 4 shows the sensitivity and specificity of detecting Listeria monocytogenes according to the present invention; a is a standard curve for detecting Listeria monocytogenes; b is the linear range for detecting Listeria monocytogenes; c is the principle of probe-DNA induced hybridization and enzyme-mediated Mn (VII)/Mn (II) conversion induction (high Δ T)₂Low Δ T₂) The principle of magnetic relaxation switching sensing of (1); d is the specificity of the detection method of the invention. (the total number of colonies was 10)⁵CFU/mL, error bars show standard deviation of three experiments).

Detailed Description

The technical solution of the present invention is further described below with reference to the following embodiments and the accompanying drawings. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

The conversion of AAP to AA is achieved using alkaline phosphatase (ALP), a widely used marker enzyme, providing a platform for enzymatic Mn (VII)/Mn (II) conversion (FIG. 2A). Thus, the pair to be formedReaction of the chain-sandwiched complexes with ALP-SA converts AAP to AA by cleavage of the phosphate group, and the resulting AA will convert MnO to MnO₄ ^-Reduction to Mn²⁺While the conversion of Mn (VII) to Mn (II) causes a transverse relaxation time (Δ T)₂) Significant variations in the temperature of the sample. By measuring MnO at different concentration gradients₄ ^-And Mn²⁺Delta T in aqueous solution₂Value of, then, Δ T is clearly seen₂Depending on the concentration of Mn (II), Mn (VII) vs. Δ T₂The effect of (c) is negligible (fig. 2B). Thus, MnO is selected₄ ^-As a reaction substrate. In addition, different concentrations of AA and ALP versus KMnO were verified by altering different solubility gradients₄Influence of the solution (fig. 2C and 2E). In the presence of AA, Mn (VII) is reduced to Mn (II), the solution undergoes a clear color change, and the pale red Mn (VII) is converted to colorless Mn (II) (FIG. 2D). By this method, the target DNA sequence can be efficiently detected.

Example 2

The equipment and materials used for detecting the listeria monocytogenes in the embodiment are as follows:

equipment: oscillating by using an MS-3 oscillator; carrying out magnetic separation and washing by a SuperMag magnetic separator; measuring T by 0.5T low-field nuclear magnetic resonance spectrometer (LF-NMR)₂A signal; UV-1800 obtains an ultraviolet-visible (UV-Vis) absorption spectrum; a Malvern Zeta particle size analyzer (Nano-ZS) for Dynamic Light Scattering (DLS), measuring hydrodynamic size and Zeta potential; obtaining the concentration and purity of DNA by a nanometer photometer-N60 Touch; performing hybridization reaction by using HL-2000 hybridization linker (UVP); t100^TMThermal cycler and GelDoc XR + systems were used for amplification products and gel imaging, respectively.

Materials: magnetic nanoparticles (MNPs, diameter: 1000 nm; solid content: 10mg/mL, with carboxyl modification) were purchased from Ocean NanoTech (USA); ascorbic Acid (AA), trisodium 2-phosphate-L-ascorbate (AAP), N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2- (N-morpholine) ethanesulfonic acid, and Bovine Serum Albumin (BSA) were purchased from Sigma-Aldrich (USA); alkaline phosphatase-labeled streptavidin was purchased from Beyotime Biotechnology (1mg/mL, ALP-SA, shanghai, china); physiological saline sodium citrate buffer (20 XSSC, pH7.0) and bacterial genomic DNA kit were purchased from ZOMANBIO (China, Beijing); tryptic Soy Broth (TSB) and Tryptone Soy Agar (TSA) media were purchased from Qingdao Hope Bio-Technology co., Ltd. (china, Qingdao); DNA lysis was purchased from Shanghai Double-Helix Biotech Co., Ltd. (China, Shanghai) using Tris-EDTA buffer (TE, pH 8.0); all other chemicals were analytical grade, purchased from Sinopharm Chemical co., Ltd. (china, shanghai); Milli-Q water was used throughout the experiment.

The sequences of the HPLC purified oligonucleotides were purchased from TsingKe Biological Technology (Wuhan, China) and were as follows:

TABLE 1

(1) Preparation of MNPs-HLY1 conjugates

Coupling the capture probe HLY1 to the carboxyl modified magnetic nanoparticle through EDC/NHS activation, and comprises the following specific processes: mu.L of MNPs (1000nm, 10mg/mL, modified with carboxyl groups) were taken out and washed 2 times in a 1.5mL centrifuge tube with 500. mu.L of MEST (10mmol/L MES, pH 6.0, 0.05% Tween-20) and then subjected to the first magnetic separation. After removing the supernatant, 200. mu.L of 5mg/mL EDC and 200. mu.L of 5mg/mL NHS were added to the 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 oligonucleotide probe HLY1(PBST as solvent) was added to the centrifuge tube, the volume was adjusted to 500. mu.L with PBST (0.01mol/L PBS, pH 7.4, 0.05% Tween-20), the mixture was gently shaken at 37 ℃ for 3h in a vortex shaker to remove the supernatant, then 1mL of PBST containing 1% BSA was added to the MNPs-HLY1 conjugate, reacted at 37 ℃ for 30min to block the residual sites, and the fourth magnetic separation was performed and washed 4 times with 500. mu.L PBST. MNPs-HLY1 were finally obtained and dispersed in 1mL PBST containing 0.5% BSA and stored at 4 ℃ until use.

The MNPs-HLY1 conjugates were characterized using Dynamic Light Scattering (DLS) and ultraviolet-visible spectroscopy (UV-Vis). After modification of HLY1, the mean hydrodynamic diameter (1411nm) of MNPs-HLY1 conjugates was significantly increased over unmodified MNPs (1036nm) (fig. 3A). For the surface potential, the surface modification was slightly increased from-27 mV to-25 mV (FIG. 3B). This slight change in potential may be due to the oligonucleotide probe itself having a negative potential. In addition, FIG. 3C shows that the absorption spectrum of the MNPs-HLY1 conjugate has a maximum absorption peak at 278nm, whereas the original MNPs have no absorption peak at any wavelength. Furthermore, the absorption spectrum of DNA has a maximum absorption peak at 260nm due to the electronic interaction between bases in the DNA molecule, and thus the maximum absorption peak of DNA oligonucleotide (HLY1) is located around 260nm (FIG. 3C). Apparently, the adsorption peak of the MNPs-HLY1 conjugate is red-shifted by 18nm relative to the adsorption peak of the pure DNA oligonucleotide (HLY1), which indicates that HLY1 is adsorbed on the surface of MNPs, and amino linkage is formed between MNPs and HLY 1. These results indicate that HLY1 successfully modified the surface of MNPs.

(2) Detection of Listeria monocytogenes

From different concentrations (20-2X 10)⁷CFU/mL) was extracted from the culture of Listeria monocytogenes, and Tris-EDTA buffer (TE, pH8.0) was used for DNA dissolution. Selecting 4 pathogenic bacteria of streptococcus enteritis, Escherichia coli, Staphylococcus aureus and Vibrio parahaemolyticus, and collecting the pathogenic bacteria with concentration of 10⁵1mL of CFU/mL of the bacterial solution was used as a negative control, and genomic DNA was extracted.

mu.L of the MNPs-HLY1 solution obtained in step (1), 100. mu.L of 1.5nmol/L biotinylated oligonucleotide probe HLY2(1 XSSC, pH7.0 as solvent), 40. mu.L of genomic DNA with different concentrations, and 160. mu.L of sodium citrate buffer (1 XSSC, pH7.0) in physiological saline were added to 1.5mL of centrifuge tubes and mixed, and the complex in each centrifuge tube before hybridization was heat-treated at 95 ℃ for 10min, immediately cooled in ice for 5min, and after cooling, placed in an oven for incubation at 53 ℃ for 1h with continuous shaking to allow the target DNA to hybridize 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 was used as a solvent to dilute the stock solution) was added and the reaction was gently shaken at 37 ℃ for 30min, followed by the second magnetic separation and washing. First, theRemoving supernatant during magnetic separation, and washing each tube of complex with 0.5 XSSC buffer solution for 3 times; the second magnetic separation was performed by washing 4 times with TBST buffer and 1 time with pure water to remove the excess ALP-SA. Then 100 μ L of 25mmol/L aqueous AAP solution is added into the centrifuge tube to react for 30min at 37 ℃ by gentle shaking, and after the third magnetic separation, the supernatant of each tube is collected. Finally, the supernatant was reacted with 0.5mmol/L aqueous potassium permanganate at a volume ratio of 2:1 for 5min, and the T of the reaction mixture was measured by LF-NMR of 0.5T₂The value is obtained.

T acquisition at 35 ℃ using Carr-Purcell-Meiboom-Gill pulse sequence₂The parameters are as follows: nuclear magnetic resonance frequency 19.894 MHz; pulse width of 90 °, 13 μ s; pulse width 180 °, 26 μ s; TW 12000 ms; NECH 12000 ms; SW is 100 KHz; TE is 1.0 ms; NS is 2. Three analyses were performed for each point (n-3) and for each interval, T was calculated using the following formula₂Change (Δ T)₂)：ΔT₂＝|T_{2 samples}-T_{2 blank}|。

The results are shown in FIG. 4: FIG. 4A shows a range from 20 to 2X 10⁷As a result of the CFU/mL Listeria monocytogenes concentration, the more target bacteria, the more Delta T is obtained₂The higher the value, the higher the result indicates that the target DNA sequence can be enriched by capturing MNPs-HLY1 and hybridization reactions, and the signal can be amplified by the enzyme-catalyzed reaction-mediated Mn (VII)/Mn (II) interconversion. FIG. 4B also shows Δ T₂The value is 2X 10 to the concentration of the target bacteria²～2×10⁷In a linear relationship in the CFU/mL range, the linear equation is Y-62.45X +35.67 (X-log ([ Listeria monocytogenes (CFU/mL))]，R²0.9976) detection limit (S/N3) is 10²CFU/mL. Delta T for detecting listeria monocytogenes₂The values are much higher than those of the negative control (FIG. 4D), which also indicates that the oligonucleotide probe designed based on the base complementary pairing principle has better specificity for the target sequence, i.e., the genomic DNA of Listeria monocytogenes can be specifically recognized and captured by the MNPs-HLY1 conjugate and HLY2 (FIG. 4C).

Example 3

This example is similar to example 2 except that: in step (1), the concentration of HLY1 added was changed to 1nmol/L, 2nmol/L and 3nmol/L, respectively, and all conjugates were found to have an adsorption peak at 278 nm. Meanwhile, when the concentration of HLY1 is greater than 1nmol/L, the absorption spectrum of the MNPs-HLY1 conjugate is almost unchanged. Thus, 1.5nmol/L of HLY1 was used for coupling to MNPs to increase coupling efficiency.

Example 4

This example is similar to example 2 except that: in the step (2), the concentration of the added AAP is changed to 20mmol/L and 30mmol/L respectively. At 20 to 2 x 10⁷Seven sets of CFU/mL control data, Δ T, vs. initial 25mmol/L concentration₂The value is obviously reduced, the difference value fluctuates in 0-150ms, and the value is extremely unstable. Therefore, the optimum concentration of AAP is 25 mmol/L.

Example 5

This example is similar to example 2 except that: in step (2), the mixture was mixed with 0.5mmol/L KMnO₄The reaction volume ratio of the aqueous solution was changed to 1: 1. In seven sets of control data, Δ T compared to the initial 2:1 volume ratio₂The values are significantly reduced, the difference is large, fluctuating in 150-300 ms. Thus, the mixture was mixed with 0.5mmol/L KMnO₄The optimal reaction volume ratio of (3) is 2: 1.

Example 6

This example compares the present invention with the conventional PCR amplification method for detecting Listeria monocytogenes. First, a pure culture of Listeria monocytogenes was diluted 10-fold in a gradient with sterile phosphate buffer to a final concentration of 10⁸10CFU/mL, then 1mL of different concentrations of the bacterial liquid into a 1.5mL centrifuge tube to extract genomic 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 genomic DNA, 18.25. mu.L double distilled water, 0.5. mu.L forward primer (hly AF, 10. mu. mol/L) and 0.5. mu.L reverse primer (hly AR, 10. mu. mol/L), with the primer sequences shown in Table 1. The PCR reaction conditions are as follows: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 1min, annealing at 62 deg.C for 1min, extension at 72 deg.C for 1min for 35 cycles, and extension at 72 deg.C for 8 min. The final PCR product was detected by electrophoresis on a 1% agarose gel.

Results found that PCR method for detecting Listeria monocytogenesSensitivity of the bacteria is about 10³CFU/mL. Gel imaging of extracted DNA without PCR amplification, only from 10⁸CFU/mL and 10⁷The genomic DNA extracted from the CFU/mL bacterial suspension showed a faint 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 the detection of food-borne pathogenic bacteria.

Example 7

In this example, a ham sample labeling recovery test of the present invention was performed to detect ham homogenate samples added with different concentrations of listeria monocytogenes. Genomic DNA was extracted and tested as described above. The recovery rate is 87.2% -101.3%, the coefficient of variation is 6.46% -9.54% (table 2), and the potential and feasibility of the constructed biosensor for detecting the listeria monocytogenes in the complex sample are shown.

TABLE 2 recovery of Listeria monocytogenes in ham at different concentration levels

The applicant states that the present invention is illustrated by the above examples of the process of the present invention, but the present invention is not limited to the above process steps, i.e. it is not meant that the present invention must rely on the above process steps to be carried out. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.

Sequence listing

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Claims

1. A kit for detecting Listeria monocytogenes, which is characterized in that: comprises the following steps: the kit comprises a capture probe modified by magnetic nanoparticles, a biotin-labeled signal probe, streptavidin labeled by alkaline phosphatase, 2-phosphoric acid-L-ascorbic acid trisodium salt and potassium permanganate.

2. The kit of claim 1, wherein: the capture probe and the signal probe can be specifically combined with the genome DNA of the listeria monocytogenes.

3. The kit of claim 1, wherein: the capture probe and the signal probe can be specifically combined with a virulence gene hly of the Listeria monocytogenes.

4. The kit of claim 1, wherein: the sequences of the capture probe and the signal probe are as follows:

capture probe HLY 1: 5'-CACGAGAGCACCTGGAT-3', respectively;

signaling probe HLY 2: 5'-GACAGGAAGAACATCGGG-3' are provided.

5. The kit of claim 1, wherein: the capture probe modified by the magnetic nanoparticles comprises: MNPs-CO-NH- (CH)₂)₆-TTTTTTTTTTTTTCACGAGAGCACCTGGAT; the biotin-labeled signal probe is as follows: GACAGGAAGAACATCGGGTTTTTTTTTTTT- (CH)₂)₇-Biotin。

6. The kit of claim 1, wherein: comprises normal saline sodium citrate buffer.

7. A method for detecting Listeria monocytogenes using the kit of any one of claims 1-6, wherein: the method comprises the following steps:

(1) extracting genome DNA of a sample to be detected;

(2) performing thermal denaturation treatment on the capture probe modified by the magnetic nanoparticles, the biotin-labeled signal probe and the genomic DNA extracted in the step (1), adding the obtained product into a normal saline sodium citrate buffer solution after the thermal denaturation treatment, incubating to enable the target DNA to be hybridized with the probe, 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 supernatant through magnetic separation;

(5) adding potassium permanganate into the supernatant collected in the step (4) to react so as to obtain MnO₄ ^-Reduction to Mn²⁺Measuring the T of the reaction mixture after the reaction by low-field nuclear magnetic resonance₂Value according to T₂And qualitatively and quantitatively detecting the listeria monocytogenes through the change of the value.

8. The method of detecting listeria monocytogenes of claim 7, wherein: the method comprises the following steps:

(1) extracting genome DNA of a sample to be detected;

(2) 80-120 mu L of capture probe modified by magnetic nanoparticles with the concentration of 1mg/mL and the modification amount of 0.15-0.3nmol/g, 80-120 mu L of signal probe labeled by 1.5nmol/L biotin and 20-100 mu L of genomic DNA extracted in the step (1) are subjected to thermal denaturation treatment at 90-100 ℃ for 5-10min, and then added into 100 plus 180 mu L of physiological saline sodium citrate buffer solution to be incubated at 50-60 ℃ for 1-1.5h after the thermal denaturation treatment, so that the target DNA is hybridized with the probe, and the magnetic adsorbate 1 is collected by magnetic separation;

(3) adding 50-150mL of streptavidin labeled with 1 microgram/mL alkaline phosphatase into the magnetic adsorbate 1 obtained in the step (2), shaking for reaction 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) adding 50-150 μ L of 25 mmol/L2-phosphoric acid-L-ascorbic acid trisodium salt into the magnetic adsorbate 2 obtained in the step (3), shaking for reaction at 36.5-37.5 ℃ for 0.5-1h to remove the phosphate group, and collecting the supernatant through 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, and adding MnO₄ ^-Reduction to Mn²⁺T of the reaction mixture was then measured by low-field nuclear magnetic resonance₂Value according to T₂Qualitative and quantitative analysis of Listeria monocytogenes by variation of valueAnd (6) detecting.

9. The method of claim 7 or 8, wherein the listeria monocytogenes is selected from the group consisting of: in the step (5), a Carr-Purcell-Meiboom-Gill pulse sequence is used for collecting T at 35 DEG C₂The parameters are as follows: nuclear magnetic resonance frequency 19.894 MHz; pulse width of 90 °, 13 μ s; pulse width 180 °, 26 μ s; TW 12000 ms; NECH 12000 ms; SW is 100 KHz; TE is 1.0 ms; NS is 2; three analyses were performed for each point and for each interval, T was calculated using the following formula₂Variation of value Δ T₂：ΔT₂＝|T_{2 samples}-T_{2 blank}|。