CN115010184A - Staphylococcus aureus recombinase polymerase amplification detection method based on immunomagnetic beads - Google Patents
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Abstract
The invention provides a staphylococcus aureus recombinase polymerase amplification detection method based on immunomagnetic beads, which comprises the steps of firstly preparing superparamagnetic carboxylated ferroferric oxide magnetic beads by a solvothermal method, then obtaining rabbit anti-staphylococcus aureus polyclonal antibodies by taking heat-inactivated staphylococcus aureus as immunogen, connecting the rabbit anti-staphylococcus aureus polyclonal antibodies by means of a carbodiimide method, and optimizing coupling conditions and the mass ratio of magnetic bead antibodies to obtain high-performance immunomagnetic beads; secondly, screening the best primer of the recombinase polymerase amplification technology, detecting the sensitivity and specificity of the primer, and finally evaluating the application effect of the immunomagnetic bead-recombinase polymerase amplification detection method on artificially contaminated samples and clinical samples. Fully verified, the detection method has the characteristics of high sensitivity, high specificity, simplicity, convenience and rapidness, and is an effective real-time monitoring technology for staphylococcus aureus in animal products.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a staphylococcus aureus recombinase polymerase amplification detection method based on immunomagnetic beads.
Background
Staphylococcus aureus (s. aureus) is a common pathogenic bacterium that is a pathogenic condition that harms the health of humans and animal husbandry. It is widely distributed in nature, such as in soil, in the air, on skin surfaces, in the anterior nasal cavity and even in the gastrointestinal tract, and it has been found that almost 30% of the population are permanent colonizers of Staphylococcus aureus [1] . The staphylococcus aureus is one of main pathogenic bacteria causing food poisoning and mastitis of cattle and sheep. In recent years, Methicillin-resistant Staphylococcus aureus (MRSA) has been detected in milk, dairy products and meat [2-5] And started to emerge the food chain-related and animal husbandry-related cross-host epidemic [6] . The staphylococcus aureus has more serious harm to human and animals, and the prevention, detection, control and the like of the staphylococcus aureusIs of importance. The currently common staphylococcus aureus detection methods comprise the traditional separation culture method, the liquid chromatography, the ELISA and the PCR method, but have the defects of long time consumption, complex operation, high equipment requirement, large influence of a sample matrix and the like, so that the application of the field rapid detection of the staphylococcus aureus is limited.
The basic principle of the immunomagnetic separation technology is to recognize and capture target antigens by virtue of specific antibodies coupled on the surfaces of magnetic beads, and separate and enrich the target antigens from various complex biological fluids by utilizing superparamagnetism of magnetic nanoparticles. The sensitivity, specificity and timeliness of a subsequent detection method are greatly influenced by a processing mode before biological sample detection, in a typical magnetic separation process, magnetic beads marked with antibodies and aptamers can be specifically combined to target objects (cells, proteins, DNA, RNA, bacteria and viruses), and then specific target objects are separated from a complex matrix through separation of an external magnetic field, so that an enrichment and concentration effect is achieved. The existing microorganism separation technology in food samples comprises a traditional separation culture method, a low-custom centrifugal precipitation method and a filter membrane filtration method. The traditional separation culture is still the gold standard for microbial detection, but the method has the biggest defects of complicated process and long time consumption; the popular centrifugal precipitation method has the defects of equipment dependence and low sensitivity; the filtration method is laborious when pushing food samples with a syringe and extracting the filter membrane from the filter. The fast and efficient enrichment and separation of Immunomagnetic beads (IMBs) has great advantages in pretreatment of a sample to be detected containing a small amount of target substances, namely simplicity, rapidness, high sensitivity and strong specificity.
The Recombinase Polymerase Amplification (RPA) is a technology capable of rapidly amplifying a target fragment under isothermal condition, and has the greatest advantages that the target fragment can be rapidly amplified at 37-42 ℃, the result can be observed in only 10-20 minutes, and the Amplification product can be observed in various ways: agarose gel electrophoresis, real-time quantitative fluorometer, and lateral flow test strip. The RPA gets rid of the complex processes of traditional PCR amplification denaturation and annealing, has the advantages of convenience, high sensitivity and specificity, and is a promising method for rapidly detecting pathogenic bacteria. Compared with LAMP which is an isothermal amplification technology, the design of the primers required by RPA is simpler. Through the rapid development in recent years, RPA has been widely used in various fields of detection, such as pathogen detection, food identification, and the like.
[1]Kluytmans J,Van B A,Verbrugh H.Nasal carriage of Staphylococcus aureus:epidemiology,underlying mechanisms,and associated risks[J].Clinical Microbiology Reviews,1997,10(3):505-520.
[2]Cui M,Li J,Ali T,et al.Emergence of livestock-associated MRSA ST398 from bulk tank milk,China[J].Journal of Antimicrobial Chemotherapy,2020,75(12):3471-3474.
[3]Soltan Dallal M M,Salehipour Z,Sharifi Yazdi M K,et al.Phenotypic and Genotypic Characteristics of Methicillin-Resistant Staphylococcus aureus Isolated from Dairy and Meat Products in Iran[J].Journal of Food Quality and Hazards Control,2020,7:108-114.
[4]Tanomsridachchai W,Changkaew K,Changkwanyeun R,et al.Antimicrobial Resistance and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus Isolated from Slaughtered Pigs and Pork in the Central Region of Thailand[J].Antibiotics(Basel),2021,10(2),206.
[5]Lin Q,Sun H,Yao K,et al.The Prevalence,Antibiotic Resistance and Biofilm Formation of Staphylococcus aureus in Bulk Ready-To-Eat Foods[J].Biomolecules,2019,9(10),524.
[6]Nunez J,Reynolds S J,Bisha B,et al.JA:2021-20.Source Attribution of MRSA Exposure and Carriage among Dairy Workers[J].Journal of Agromedicine,2020,25(3):246-247.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a staphylococcus aureus recombinase polymerase amplification detection method based on immunomagnetic beads. Connecting a rabbit anti-staphylococcus aureus polyclonal antibody to the surface of a self-made carboxylated ferroferric oxide magnetic bead to obtain an immunomagnetic bead for staphylococcus aureus, aiming at capturing staphylococcus aureus in a sample to be detected and realizing enrichment and purification of the staphylococcus aureus; the separated staphylococcus aureus is detected by combining recombinase polymerase amplification technology, a rapid detection method of staphylococcus aureus IMBs-RPA in animal products is established, and technical support is provided for on-site rapid detection of pathogenic bacteria in food.
In order to achieve the above purpose, the solution of the invention is as follows:
a preparation method of carboxylated ferroferric oxide magnetic beads comprises the following steps:
(1) respectively dissolving ferric trichloride hexahydrate and sodium acetate in ethylene glycol, stirring until the ferric trichloride and the sodium acetate are dissolved, mixing, pouring the mixed solution into a reaction kettle, heating for reaction, cooling to room temperature after the reaction is finished, cleaning, and drying in vacuum to obtain the carboxylated ferroferric oxide magnetic beads.
Preferably, the molar concentration of ferric trichloride hexahydrate is 0.1-0.15mol/L, and the molar concentration of sodium acetate is 0.5-1 mol/L.
Preferably, the rotation speed of the stirring is 250-500rpm, preferably 300 rpm; the time is 3-6 h.
Preferably, the temperature of the temperature-rising reaction is 160-220 ℃, and the reaction time is 4-8 h.
Preferably, the temperature of vacuum drying is 50-70 ℃ and the time is 6-9 h.
Carboxylated ferroferric oxide magnetic beads are obtained by the preparation method.
A method for preparing staphylococcus aureus immunomagnetic beads comprises the following steps:
respectively dissolving 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in precooled 2- (N-morpholine) ethanesulfonic acid (MES) to prepare EDC solution and NHS solution; centrifuging the carboxylated ferroferric oxide magnetic beads, carrying out magnetic separation, discarding the supernatant, and washing for several times; then adding EDC solution and mixing uniformly, adding NHS solution and mixing uniformly, and activating;
(2) washing the magnetic beads treated in the step (1) by using an MES solution, adding the MES solution and a rabbit anti-staphylococcus aureus polyclonal antibody, placing the mixture in an incubator for incubation, carrying out magnetic separation, and discarding the supernatant;
(3) adding Phosphate Buffer Solution (PBS) containing Bovine Serum Albumin (BSA), placing on a mixing instrument, and sealing;
(4) by using sodium azide (NaN) 3 ) And bovine serum albumin solution, and taking a phosphate buffer solution as an immunomagnetic bead preservation solution to resuspend the immunomagnetic beads, and preserving for later use.
Preferably, in step (1), the activation temperature is 25-37 ℃ and the activation time is 0.5-1 h.
Preferably, in the step (2), the temperature of the incubator is 37 ℃ and the time is 0.5-1.5 h.
Preferably, in step (3), the blocking temperature is 37 ℃ and the time is 0.5-1.5 h.
Staphylococcus aureus immunomagnetic beads obtained by the method.
A method for detecting staphylococcus aureus immunomagnetic bead-recombinase polymerase amplification, which comprises the following steps: combining with recombinase polymerase amplification technology, and amplifying and detecting staphylococcus aureus separated by staphylococcus aureus immunomagnetic beads by using a designed primer;
wherein the staphylococcus aureus immunomagnetic bead is the staphylococcus aureus immunomagnetic bead.
Preferably, the sequence of the primer is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO. 16.
Due to the adoption of the scheme, the invention has the beneficial effects that:
the immunomagnetic bead for separating and enriching staphylococcus aureus is obtained based on self-made ferroferric oxide magnetic particles, and has the advantages of high capture rate of 92.75 percent, high sensitivity of 2.5CFU/mL and strong specificity on staphylococcus aureus. The immunomagnetic bead can efficiently, quickly, conveniently and accurately separate and concentrate staphylococcus aureus from milk and pork homogenate so as to be further used for rapid detection of RPA, and has the advantages of concentrating the concentration of staphylococcus aureus in a sample to be detected, improving the detection sensitivity, eliminating the influence of complex matrixes, improving the detection accuracy, reducing the sample processing time and being non-dependent on high equipment. The combination of the IMBs and the RPA further shortens the detection time of the staphylococcus aureus in the animal product samples, and the whole process of immune magnetic separation, DNA extraction and RPA detection can be controlled within 2.5 h. In addition, the IMBs-RPA has higher sensitivity and specificity, and the sensitivity is about 1000 times of that of the PCR method which is only used.
Drawings
FIG. 1 is a schematic diagram of the rapid detection process of Staphylococcus aureus RPA based on IMBs according to the present invention.
FIG. 2 shows carboxylated Fe in example 1 of the present invention 3 O 4 Morphological structure of magnetic beads (A: Fe) 3 O 4 Scanning Electron microscopy of magnetic beads B Fe 3 O 4 Transmission electron microscopy of magnetic beads).
FIG. 3 shows carboxylated Fe in example 1 of the present invention 3 O 4 Fourier infrared spectrum of magnetic beads.
FIG. 4 shows carboxylated Fe in example 1 of the present invention 3 O 4 Hysteresis curves of the magnetic beads.
FIG. 5 is a graph showing the sensitivity of Staphylococcus aureus immunomagnetic beads in example 2 of the present invention.
Fig. 6 is a transmission electron microscope (sem) morphology of staphylococcus aureus (a), immunomagnetic beads, and s.
FIG. 7 is a specificity diagram of Staphylococcus aureus immunomagnetic beads in example 2 of the present invention.
FIG. 8 is a schematic diagram of the screening of the RPA primer in example 3 of the present invention.
FIG. 9 is a graph showing the sensitivity of PCR detection in example 3 of the present invention.
FIG. 10 is a graph showing the sensitivity (A) and specificity (B) of RPA detection in example 3 of the present invention.
FIG. 11 is a diagram showing the detection of IMBs-RPA and IMBs-PCR in the spiked samples (detection of artificially contaminated samples of pork (D) by IMBs-RPA on milk (A), IMBs-PCR on milk (B), IMBs-RPA on pork (C) and IMBs-PCR on pork (D)) in example 3 of the present invention.
FIG. 12 is a graph showing the results of detection of IMBs-RPA in clinical samples (milk (A) and pork (B) clinical samples by IMBs-RPA) in example 3 of the present invention.
FIG. 13 is a graph showing the results of detection of IMBs-PCR in clinical samples (detection of milk (A) and pork (B) clinical samples by IMBs-PCR) in example 3 of the present invention.
Detailed Description
The invention provides a rapid detection method of staphylococcus aureus RPA based on IMBs.
As shown in FIG. 1, the present invention first utilizes a solvothermal method to prepare carboxylated Fe 3 O 4 Magnetic beads, coupling the magnetic beads with rabbit anti-staphylococcus aureus polyclonal antibody by means of a carbodiimide method to obtain staphylococcus aureus specific immunomagnetic beads; then adding staphylococcus aureus immunomagnetic beads into milk and pork homogeneous liquid to be detected to capture staphylococcus aureus, applying an external magnetic field to realize separation and enrichment of staphylococcus aureus, and combining an RPA amplification technology to realize rapid, high-sensitivity and specific detection of staphylococcus aureus in an actual sample.
Example 1:
1. solvothermal preparation of carboxylated Fe 3 O 4 Magnetic bead
Method for preparing Fe by solvothermal one-pot method 3 O 4 Nano particles, the process is as follows: 3.51g of FeCl 3 ·6H 2 O and 7.46g NaAc are respectively dissolved in 40mL of glycol and stirred at a high speed for 30min until the NaAc is fully dissolved; mixing the two solutions, supplementing 100mL of ethylene glycol, continuously stirring at high speed for 3h, and respectively obtaining Fecl with final concentration of 0.13mol/L 3 ·6H 2 O, 0.91mol/L NaAc; finally pouring the mixture into a sealed high-temperature reaction kettle, wherein the temperature is 200 ℃, and the reaction time is 5 hours; and after the reaction temperature is reduced to room temperature, pouring out the black target product from the reaction kettle, alternately cleaning the product with absolute ethyl alcohol and water for three times respectively, placing the product in a vacuum drying oven, and drying for 8 hours at the temperature of 60 ℃.
2. Detection of physicochemical Properties of magnetic beads
(1) Surface morphology detection
Fixing a thin layer of dried magnetic powder on a double-sided adhesive tape of a silicon wafer, carefully blowing off particles which are not adhered to the surface, directly fixing the particles on a microscope support of a field emission scanning electron microscope, and sending the particles into a cabin to start scanning and observing the surface morphology and the particle size distribution of the magnetic particles.
(2) Structural component detection
Dissolving magnetic powder in absolute ethyl alcohol to enable the concentration of the magnetic powder to be 1mg/mL, performing ultrasonic dispersion for 3 times, wherein each time is 20min, using a suction tube to drip the uniformly mixed alcoholic solution of the magnetic iron oxide on a carbon film copper net, and observing the structure and aggregation state of the magnetic nanoparticles by using a high-resolution field transmission electron microscope after the magnetic nanoparticles are naturally dried.
(3) Surface functional group detection
Weighing 1mg of the dried magnetic powder sample and KBr, uniformly grinding the sample and the KBr in a mortar, preparing a tabletting sample by using a tabletting machine, and performing 400-4000cm by using a Fourier transform infrared spectrometer -1 The elemental composition of the magnetic powder is detected in the wavenumber range.
(4) Magnetic detection
30mg of dried magnetic powder sample is sent into a vibration sample magnetometer, and a magnetic hysteresis loop of the magnetic powder sample under the action of an external magnetic field of (-10000) -10000 Oe is detected at 25 ℃ and is used for determining the superparamagnetism and the saturation magnetic strength of the magnetic particles.
Example 2:
1. preparation of staphylococcus aureus immunomagnetic beads:
carboxylation of Fe using EDC-NHS 3 O 4 And activating carboxyl groups on the surfaces of the magnetic beads, wherein the activated intermediate ester structure can react with amino groups on the rabbit anti-staphylococcus aureus antibody, and then connecting the activated intermediate ester structure and the rabbit anti-staphylococcus aureus antibody together to prepare the immunomagnetic beads capable of specifically separating and enriching staphylococcus aureus.
The method comprises the following steps:
washing: by taking carboxylated Fe 3 O 4 Magnetic beads are put in a centrifugal tube for magnetic separation, and supernatant is discarded; washed once with absolute ethanol, 0.1 mol/L2- (N-morpholine) ethanesulfonic acid (MES) solution containing 0.05% Tween-20 and pH 5.02 times.
And (3) activation: respectively preparing EDC solution and NHS solution with the concentration of 100mg/mL by using precooled 0.1mol/L MES; taking 100 mu L of carboxylated Fe 3 O 4 Magnetic beads are put in a 1.5mL centrifuge tube for magnetic separation, and the supernatant is discarded; washing with 300 μ L absolute ethanol once and 300 μ L MEST Buffer 2 times; then 200 mul EDC solution is added into the tube and mixed evenly and waits for 2min, then 200 mul NHS solution is added into the solution and mixed evenly, and the mixture is put on a rotary mixer to be activated for 30min at 37 ℃.
Coupling: resuspending the activated magnetic beads in PBS solution with pH 7.2, 0.01mol/L, MES solution with pH 5.0, 0.1mol/L, PBS solution with pH 7.2 at 0.1mol/L, pH, respectively; then adding a certain amount of antibody, fixing on a rotary blending machine, and incubating for 1h at 37 ℃; and (3) magnetically separating, measuring the concentration of free immunoglobulin in the supernatant, calculating the coupling rate of the antibody, and determining the optimal buffer solution for antibody coupling.
And (3) sealing: performing orthogonal test on magnetic beads with the mass of 5mg, 6mg, 7mg and 8mg and antibodies with the final concentration of 1mg/mL, 1.5mg/mL and 2mg/mL to determine the optimal dosage of the magnetic bead antibody; incubating for 1h at 37 ℃ on a rotary mixer; washing with PBS for three times by means of a magnetic frame; then adding 500 mu L of immunomagnetic bead blocking solution (PBS containing 5% BSA), and blocking for 1h at 37 ℃ on a rotary mixer; repeating the washing step; get 10 3 Adding the bacterial liquid of the CFU into immunomagnetic beads, supplementing 1mL with PBS, placing on a mixing machine, and incubating for 1h in a 37 ℃ incubator; and (3) carrying out magnetic separation to obtain a bacterial magnetic bead immune complex, washing the bacterial magnetic bead immune complex with PBS for three times, carrying out plate counting on the magnetic bead bacterial liquid immune complex to calculate the bacterial capture rate, and determining the optimal magnetic bead antibody mass ratio.
And (3) storage: with a solution containing 0.02% NaN 3 0.1% BSA at pH 7.2, 0.01mol/L PBS solution as preservation solution of immunomagnetic beads resuspension concentration of immunomagnetic beads is 10mg/mL, 4 ℃ preservation for use. Before blocking with 1% BSA, the immunomagnetic beads were spotted on SDS-PAGE to detect whether the antibody was successfully coupled to the beads.
2. And (3) detecting the performance of the immunomagnetic beads:
(1) sensitive detection of immunomagnetic beads
6mg of prepared immunomagnetic beads were respectively added to 2.5X 10 0 、2.5×10 1 、2.5×10 2 、2.5×10 3 、2.5×10 4 、2.5×10 5 And (3) capturing the CFU/mL staphylococcus aureus, determining the capture rate of the immunomagnetic beads by using a plate counting method, and determining the sensitivity of the immunomagnetic beads.
(2) Specific detection of immunomagnetic beads
Taking immunomagnetic beads, and respectively adding the immunomagnetic beads into the mixture with the concentration of 1mL being 10 4 And (3) capturing fresh bacterium liquid of CFU/mL salmonella typhimurium, listeria monocytogenes, escherichia coli and staphylococcus aureus, and determining the capturing performance of the immunomagnetic beads on different bacteria according to plate counting so as to judge the specificity of the immunomagnetic beads.
Example 3:
establishment based on IMBs-RPA method
(1) Design and screening of RPA primers
The core of the technology lies in the design and screening of primers. The Nuc gene sequence for designing the RPA primer of the staphylococcus aureus is downloaded from GenBank, and the gene expresses the heat-resistant nuclease of the staphylococcus aureus and is a highly conserved and specific sequence. 8 pairs of RPA primers (shown in Table 1) were designed according to the recommendations of the TwinDx RPA design guidelines, and the designed primers were evaluated for amplification. The specific operation steps of the RPA reaction are carried out according to the instructions of Twist @ basic kit, and the amplification product is extracted and purified by phenol-chloroform and detected by DNA gel electrophoresis. Finally, the optimal primer nuc-2.1 is screened out.
(2) RPA sensitivity and specificity detection
Optimal primer nuc-2.1, pair 10 was used 4 、10 3 、10 2 And 10 1 The DNA of CFU s cells was amplified for detection of RPA sensitivity. At the same time, pair 10 5 And (3) amplifying genomes of the CFU salmonella typhimurium, the Escherichia coli and the Listeria monocytogenes, and researching the specificity of the RPA detection method. The amplification product was purified and observed by gel electrophoresis.
(3) Detection performance of IMBs-RPA in artificially contaminated milk and pork
Pasteurized milk to be purchased from supermarketsAnd performing the test of the staphylococcus aureus on the fresh pork sample by referring to national standard GB 4789.10-2016, and selecting the sample with the test result of the staphylococcus aureus negativity for preparing the artificial pollution sample. Diluting milk and pork homogenate with PBS at a ratio of 1:10 in a sterile operating station, taking 10mL of diluted sample, and adding into the diluted sample with final concentration of 10 4 、10 3 、10 2 And 10 1 CFU/mL Staphylococcus aureus cultures. The IMBs were used to capture Staphylococcus aureus in artificially contaminated samples and to extract genomic DNA from Staphylococcus aureus cultures. The extracted DNA was used as a template and the detection was carried out by the established RPA method. And verifying the results of the IMBs-RPA by using PCR, and observing the amplified products in gel electrophoresis after purifying the amplified products.
(4) Effect of application of IMBs-RPA in clinical specimens
20 parts of raw milk from different dairy farms and 20 parts of chilled pork from different vegetable markets were diluted or homogenized with PBS in a ratio of 1:9, respectively. And detecting the IMBs-RPA by using the established IMBs-RPA method, and simultaneously verifying the result of the IMBs-RPA by using PCR. The amplification results were analyzed using DNA gel electrophoresis.
TABLE 1
As a result:
(1) morphological and structural analysis
The carboxylated Fe was observed under SEM (A in FIG. 2) and TEM (B in FIG. 2) 3 O 4 The size of the magnetic bead particles is 200nm, and the black nano particles are uniform in appearance and are monodisperse.
(2) Distribution of surface functional groups
As shown in FIG. 3, the FT-IR spectrum showed the magnetic beads to be 584cm -1 The characteristic absorption peak of Fe-O bond appears at 1536cm -1 And 1411cm -1 Peaks corresponding to v (COO-) asymmetric vibration and v (COO-) symmetric vibration are observed; at 3436cm -1 、2961cm -1 And 1042cm -1 Characteristic absorption bands were observed separatelyThis can be attributed to stretching vibrations of the O-H bond, C-H bond and C-O bond, respectively. Finally, 1627cm -1 The absorption band at (a) corresponds to the out-of-plane bending vibration of the-OH group (γ OH · O out-of-plane). Therefore, the method successfully prepares the magnetic particles with various functional groups on the surface.
(3) Magnetic assay
As shown in FIG. 4, carboxylated Fe 3 O 4 A magnetic hysteresis loop of the magnetic bead passes through the origin, which shows that the magnetic bead has good paramagnetism; the saturation magnetic strength is 74.37emu/g, the coercive force is 80Oe, so that the magnetic material can be used for preparing immunomagnetic beads subsequently.
(4) Sensitivity of immunomagnetic beads
As shown in FIG. 5, the capture rate of the immunomagnetic beads can reach 92.75% at most, and the sensitivity is 2.5CFU/mL, which indicates that the immunomagnetic beads have high sensitivity. FIG. 6 is a morphological picture of Staphylococcus aureus captured by immunomagnetic beads under a transmission electron microscope, and it can be seen that the immunomagnetic beads are adsorbed on the surface of Staphylococcus aureus.
(5) Specificity of immunomagnetic beads
As shown in FIG. 7, the immunomagnetic beads prepared by the invention have extremely high capture rate on Staphylococcus aureus, and the non-specific capture rate on Salmonella typhimurium, Listeria monocytogenes and Escherichia coli is below 1%, which indicates that the specificity of the immunomagnetic beads is relatively good.
(6) RPA optimal primer
As shown in FIG. 8, the electrophoretic band in lane 3 is brightest and no dimer is present, so that the primer nuc-2.1 has the best amplification performance, and the size of the amplification product using this primer is 130 bp.
(7) RPA sensitivity and specificity
As shown in FIG. 9, the sensitivity of PCR to Staphylococcus aureus was 2.5X 10 3 CFU/mL; as shown in FIG. 10, the RPA was 2.5 CFU/mL. In addition, no band is found in the specificity detection of the RPA method on the typhimurium, the listeria monocytogenes and the escherichia coli. Therefore, the RPA method has higher sensitivity and specificity to the detection of staphylococcus aureus.
(8) Detection performance of IMBs-RPA in standard milk and pork
As shown in FIG. 11, the detection sensitivity of both IMBs-RPA and IMBs-PCR method for Staphylococcus aureus in both milk and pork samples was 2.5 CFU/mL. These results indicate that the sensitivity of the IMBs-RPA and IMBs-PCR methods is about 1000 times that of the PCR method alone. Meanwhile, the results were examined using IMBs-PCR and were consistent with RPA.
(9) Detection of IMBs-RPA in clinical samples
The presence of staphylococcus aureus was detected in 20 samples of raw milk and chilled pork from dairy and supermarket by the IMBs-RPA method. The result is shown in fig. 12, 1 milk sample staphylococcus aureus test is positive; the positive rate is 5%; the staphylococcus aureus of 2 cold fresh pork samples is detected to be positive, and the corresponding positive rate is 10%. These results were in good agreement with the results obtained by PCR (FIG. 13). It is noted that the detection time of the IMBs-RPA method is about 1h shorter than that of the IMB-PCR method.
As can be seen from the above, the present invention successfully produces carboxylated Fe having a particle size of 200nm 3 O 4 And the magnetic beads have higher capture rate compared with the purchased carboxyl magnetic beads. 2.5-2.5X 10 pairs with 5mg of IMBs in 1h 4 The staphylococcus aureus diluted by the (CFU)/mL gradient is captured, the average Capture Efficiency (CE) is between 62.74 and 92.75 percent, and the sensitivity and the specificity are higher. The detection sensitivity of the IMBs-RPA method to the artificially contaminated sample reaches 2.5CFU/mL, and the method can complete the whole processes of bacterial capture, DNA extraction, amplification, electrophoresis and the like within 2.5 h; clinical samples of 20 parts of raw milk and cold fresh pork were tested by IMBs-RPA method, and the detection rates were 5% (1/20) and 10% (2/20), respectively, which is consistent with the PCR results. In conclusion, the 200nm carboxylated Fe prepared by the invention 3 O 4 The magnetic beads can be used for separating and enriching bacteria in clinical samples, the established staphylococcus aureus IMBs-RPA method has the characteristics of high sensitivity, high specificity, simplicity and rapidness, and through full verification, the detection time is shortened, and the method has higher sensitivity and specificity, and is a very effective staphylococcus aureus monitoring technology in animal products.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Sequence listing
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Claims (8)
1. A preparation method of carboxylated ferroferric oxide magnetic beads is characterized by comprising the following steps: it includes:
respectively dissolving ferric trichloride hexahydrate and sodium acetate in ethylene glycol, stirring until the ferric trichloride and the sodium acetate are dissolved, mixing, pouring the mixed solution into a reaction kettle for heating reaction, cooling to room temperature after the reaction is finished, cleaning, and drying in vacuum to obtain the carboxylated ferroferric oxide magnetic beads.
2. The method for preparing carboxylated ferroferric oxide magnetic beads according to claim 1, wherein the method comprises the following steps: the molar concentration of the ferric trichloride hexahydrate is 0.1-0.15mol/L, and the molar concentration of the sodium acetate is 0.5-1 mol/L; and/or the presence of a gas in the gas,
the stirring speed is 250-500rpm, and the time is 3-6 h; and/or the presence of a gas in the atmosphere,
the temperature of the temperature rise reaction is 160-220 ℃, and the reaction time is 4-8 h; and/or the presence of a gas in the gas,
the temperature of the vacuum drying is 50-70 ℃, and the time is 6-9 h.
3. A carboxylated ferroferric oxide magnetic bead is characterized in that: which is obtained by the production method according to claim 1 or 2.
4. A method for preparing staphylococcus aureus immunomagnetic beads by using the carboxylated ferroferric oxide magnetic beads as claimed in claim 3, which is characterized in that: the method comprises the following steps:
(1) respectively dissolving 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and N-hydroxysuccinimide in precooled 2- (N-morpholine) ethanesulfonic acid to prepare a 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide solution and an N-hydroxysuccinimide solution; centrifuging the carboxylated ferroferric oxide magnetic beads, carrying out magnetic separation, discarding the supernatant, and washing for several times; then adding 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide solution, mixing uniformly, adding N-hydroxysuccinimide solution into the solution, mixing uniformly, and activating;
(2) washing the magnetic beads treated in the step (1) by using a 2- (N-morpholine) ethanesulfonic acid solution, then adding the 2- (N-morpholine) ethanesulfonic acid solution and a rabbit anti-staphylococcus aureus polyclonal antibody, placing the mixture in an incubator for incubation, carrying out magnetic separation, and removing supernatant;
(3) adding a phosphate buffer solution containing bovine serum albumin, and placing the mixture on a mixing machine for sealing;
(4) and resuspending the immunomagnetic beads by using a solution containing sodium azide and bovine serum albumin and a phosphate buffer solution as an immunomagnetic bead preservation solution, and preserving for later use.
5. The method of claim 4, wherein the method comprises the steps of: in the step (1), the activation temperature is 25-37 ℃, and the activation time is 0.5-1 h; and/or the presence of a gas in the gas,
in the step (2), the temperature of the incubator is 37 ℃ and the time is 0.5-1.5 h; and/or the presence of a gas in the gas,
in the step (3), the sealing temperature is 37 ℃ and the sealing time is 0.5-1.5 h.
6. A staphylococcus aureus immunomagnetic bead is characterized in that: obtained by the process of claim 4.
7. A detection method for staphylococcus aureus immunomagnetic bead-recombinase polymerase amplification is characterized by comprising the following steps: it includes: combining with recombinase polymerase amplification technology, and amplifying and detecting staphylococcus aureus separated by staphylococcus aureus immunomagnetic beads by using a designed primer;
the staphylococcus aureus immunomagnetic bead is the staphylococcus aureus immunomagnetic bead of claim 6.
8. The detection method according to claim 7, characterized in that: the sequence of the primer is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO. 16.
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