CN112255397B - Kit for detecting Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium and preparation method thereof - Google Patents
Kit for detecting Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium and preparation method thereof Download PDFInfo
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- CN112255397B CN112255397B CN202011107494.5A CN202011107494A CN112255397B CN 112255397 B CN112255397 B CN 112255397B CN 202011107494 A CN202011107494 A CN 202011107494A CN 112255397 B CN112255397 B CN 112255397B
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
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- B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
- B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
- B29C33/00—Moulds or cores; Details thereof or accessories therefor
- B29C33/38—Moulds or cores; Details thereof or accessories therefor characterised by the material or the manufacturing process
- B29C33/3842—Manufacturing moulds, e.g. shaping the mould surface by machining
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
- B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
- B29C39/00—Shaping by casting, i.e. introducing the moulding material into a mould or between confining surfaces without significant moulding pressure; Apparatus therefor
- B29C39/003—Shaping by casting, i.e. introducing the moulding material into a mould or between confining surfaces without significant moulding pressure; Apparatus therefor characterised by the choice of material
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
- B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
- B29C39/00—Shaping by casting, i.e. introducing the moulding material into a mould or between confining surfaces without significant moulding pressure; Apparatus therefor
- B29C39/02—Shaping by casting, i.e. introducing the moulding material into a mould or between confining surfaces without significant moulding pressure; Apparatus therefor for making articles of definite length, i.e. discrete articles
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/255—Salmonella (G)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/28—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Vibrionaceae (F)
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- 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
Abstract
The invention provides a kit for simultaneously detecting Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium and a preparation method thereof, belonging to the field of kits. The kit comprises a self-priming valve separation type chip for real-time detection; the real-time detection self-priming valve separation type chip comprises: the kit comprises a self-suction valve separation type chip, a FAM dye modification specific aptamer of Listeria monocytogenes, a FAM dye modification specific aptamer of Vibrio parahaemolyticus, a FAM dye modification specific aptamer of Salmonella typhimurium, a blank control FAM dye modification aptamer, HNB dye and graphene oxide. The minimum detection concentrations of the listeria monocytogenes, the vibrio parahemolyticus and the salmonella typhimurium are 46.8, 22.9 and 32.3CFU/mL respectively, the standard recovery rate reaches 97.11-10.40%, and the method is high in sensitivity and good in stability.
Description
Technical Field
The invention belongs to the field of kits, and particularly relates to a kit for detecting Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium and a preparation method thereof.
Background
With the development of economy and the improvement of the living standard of people, the population growth speed is continuously accelerated, the increase of floating population and the change of dietary habits are caused, the demand of consumers on fresh and low-processed food is improved, and the high adaptability of food-borne pathogenic bacteria to the environment is brought, so that the food-borne pathogenic bacteria are one of the main public health problems of various countries all the time, and huge economic loss is caused every year. An assessment of one survey in the united states shows that approximately 940 million people are infected with food-borne diseases each year, more than one third of which are caused by food-borne pathogenic bacterial infections. In the case of listeria monocytogenes, the pathogenic bacteria can cause gastroenteritis, fever and other symptoms, but because conventional fecal culture cannot detect the pathogenic bacteria, the clinical diagnosis sometimes does not take the pathogenic bacteria infection as a diagnosis result, thereby causing delay of disease, and early spontaneous abortion or abortion related to the pathogenic bacteria can be ignored. Therefore, it is important to establish a rapid and effective food-borne pathogenic bacterium detection method.
The traditional separation and identification method is simple and stable, but the detection time of one week is not enough to meet the requirement of rapid detection. Furthermore, the conventional immunological methods usually use antigen and antibody binding, and detect different target substances by different techniques, such as enzyme-linked immunosorbent assay, immunofluorescence assay, immunocolloidal gold assay, etc. However, the production of antibodies usually takes a long time, its titer needs to be characterized before use, and it is also a matter of consideration for its activity maintenance in a long-term storage state. In contrast, the advantages of aptamers are naturally embodied, not only are they easy to synthesize, but also they are excellent in performance in terms of low cost and high stability, and properties comparable to antibodies in terms of specificity make them widely used in practical applications in recent years.
Meanwhile, the PDMS material is widely recognized and applied by virtue of its advantages of good light transmittance, low cost, optical transparency, excellent plasticity and elasticity, and high fidelity obtained from a mold, for example, the combination of the microfluidic chip and the mature PCR technology shows many excellent performances in the aspects of amplification speed, detection limit, sample requirement and detection precision. Therefore, how to realize the detection of target pathogenic bacteria by means of the existing high-advantage materials following the research trend can simplify the operation, and the accurate detection result obtained in a short time becomes the target of the next detection technology, and is also very important for determining the infection source and the patient.
Disclosure of Invention
The invention aims to provide a kit for detecting Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium and a preparation method thereof.
The invention firstly provides a kit for detecting Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium, which comprises a self-priming valve separation type chip for real-time detection;
the real-time detection self-priming valve separation type chip comprises: the kit comprises a self-suction valve separation type chip, a specific adapter for FAM dye modification of Listeria monocytogenes, a specific adapter for FAM dye modification of Vibrio parahaemolyticus, a specific adapter for FAM dye modification of Salmonella typhimurium, a blank control FAM dye modification adapter, HNB dye and graphene oxide.
The invention also provides a preparation method of the kit for detecting Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium, which comprises the following steps:
the method comprises the following steps: preparation of self-priming valve separation type chip
1) Preparation of the mold
Designing a channel and cavity pattern of a self-sucking valve separation type chip by using CorelDRAW X8, printing the pattern on a transparent film as a mask, coating a negative photoresist on a silicon wafer by using a spin coater, coating two layers in total, then placing the silicon wafer on a single-sided photoetching machine below the mask, adjusting the position of the mask to enable the pattern to be positioned in the middle of the wafer, baking the silicon wafer after exposure, finally, washing the treated silicon wafer by using a developer until a mould pattern completely appears, and baking the mould again to obtain the mould;
2) manufacture of self-priming valve separation type chip
Treating a mould by using trimethylchlorosilane, mixing a polydimethylsiloxane component pre-curing agent A and a cross-linking agent B to remove bubbles, pouring the mixture into the mould, screwing a bolt and a nut to one end of the mixture to be flush, then placing the flush end downwards at the position of a valve of the mould, removing polydimethylsiloxane from the surface of the mould after baking and curing by using a heating plate, punching a sample adding hole by using a puncher, and finally, simultaneously treating a channel surface of the polydimethylsiloxane and the surface of a glass sheet by using plasma, and baking and curing the treated planes after mutually jointing to form a self-suction valve partition type chip;
step two: preparation of graphene oxide
Adding graphite powder into H2SO4And sodium nitrate, stirring in an ice bath, slowly adding potassium permanganate into the mixture under vigorous stirring, continuously stirring at 30-40 ℃, then heating to 140-150 ℃, stirring, adding deionized water to terminate the reaction system, and neutralizing H with sodium hydroxide in the ice bath2SO4Adjusting the pH value to 5.5-6, filtering the obtained light yellow solution through an ion filtering membrane to remove large particles, and dialyzing in a dialysis bag for 2-3 days to remove salt, so as to obtain graphene oxide;
step three: preparation of real-time detection self-suction valve partition type chip
Closing the valve, covering the top of the chip by using a transparent adhesive tape, placing the chip in a vacuum pump for degassing treatment, mixing the graphene oxide obtained in the second step with the FAM dye modification specific aptamer of Listeria monocytogenes, the FAM dye modification specific aptamer of Vibrio parahaemolyticus, the FAM dye modification specific aptamer of Salmonella typhimurium or blank control FAM dye modification aptamer and HNB respectively, uniformly distributing the mixture into the sample holes through four substrate sample inlet holes under a negative pressure condition, removing the adhesive tape on the top of the chip, placing the chip on a 65 ℃ heating plate for drying, taking the chip down after the substrates are completely dried, opening the valve, re-attaching the chip with the adhesive tape on the top of the chip, placing the chip in the vacuum pump for vacuumizing, and obtaining the self-priming valve partition type chip for real-time detection.
Preferably, in the step one, the silicon wafer is baked at 60-70 ℃ for 1-3 min, and then baked at 85-95 ℃ for 10 min.
Preferably, the treatment time of the trimethylchlorosilane in the step one is 10-20 min.
Preferably, the mass ratio of the polydimethylsiloxane component pre-curing agent A to the cross-linking agent B in the first step is 10: 1.
preferably, the condition for removing bubbles by mixing the polydimethylsiloxane component in the first step is 1500-2000 rpm for 1-2 min.
Preferably, the temperature of the baking and curing in the step one is 70-90 ℃, and the time is 30-40 min.
Preferably, the size of the graphene oxide prepared in the second step is 550-600 nm.
Preferably, the degassing treatment time in the third step is 40-50 min.
Preferably, the concentration of the graphene oxide in the third step is 0.4-0.5 mg/mL-1The concentration of the four FAM-modified specific aptamers is 1-1.2 mu M, and the concentration of HNB is 0.16-0.25 mu M.
The invention has the advantages of
The invention provides a kit for detecting Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium and a preparation method thereof, the kit is characterized in that a self-suction valve separation type chip is firstly prepared, graphene oxide, specific aptamer modified by FAM dye and HNB dye are mixed, the mixture is injected into a sample chamber of the chip under the condition of negative pressure, liquid to be detected is injected into the chip under the condition of negative pressure after drying, if target bacteria exist, the liquid to be detected can be combined with the specific aptamer of a corresponding detection area and is far away from the surface of the graphene oxide, FAM fluorescence is recovered and appears green fluorescence, if no target bacteria exist, the FAM fluorescence is quenched, and the system appears red fluorescence, so that positive and negative differentiation is realized, and the three target bacteria can be quantified according to the intensity of the fluorescence intensity. The invention provides a mode of specifically combining pathogenic bacteria with FAM modified specific aptamers and introducing HNB dye, realizes dual fluorescence signal differentiation of negative and positive, can quantify target bacteria according to fluorescence intensity, shortens detection time, has small time variation coefficient of quantitative detection, has minimum detection concentrations of 46.8, 22.9 and 32.3CFU/mL for Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium respectively, has a standard recovery rate of 97.11-10.40%, and has high sensitivity and good stability.
Drawings
FIG. 1 is a graph showing the standard test for Listeria monocytogenes in example 5.
FIG. 2 is a graph showing the standard curve for detecting Vibrio parahaemolyticus in example 5.
FIG. 3 is a graph showing the standard curve of the Salmonella typhimurium detection in example 5.
FIG. 4 is a fluorescence image of specific detection of Listeria monocytogenes in example 5.
FIG. 5 is a fluorescent image of the specific detection of Vibrio parahaemolyticus in example 5.
FIG. 6 is a fluorescent image of the specific detection of Salmonella typhimurium in example 5.
FIG. 7 is a graph showing the standard curve of the detection of Listeria monocytogenes in example 6.
FIG. 8 is a graph showing the standard curve for detecting Vibrio parahaemolyticus in example 6.
FIG. 9 is a graph showing the standard test for Salmonella typhimurium in example 6.
FIG. 10 is a flow chart of the present invention for real-time detection of Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium using self-priming valve separation chip.
Detailed Description
The invention firstly provides a kit for detecting Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium, which comprises a self-priming valve separation type chip for real-time detection;
the real-time detection self-priming valve separation type chip comprises: the kit comprises a FAM dye modification specific aptamer of Listeria monocytogenes, a FAM dye modification specific aptamer of Vibrio parahaemolyticus, a FAM dye modification specific aptamer of Salmonella typhimurium, a blank control FAM dye modification aptamer, HNB dye and graphene oxide.
According to the invention, the FAM dye modification specific aptamer of Listeria monocytogenes, the FAM dye modification specific aptamer of Vibrio parahaemolyticus, the FAM dye modification specific aptamer of Salmonella typhimurium and the blank control FAM dye modification aptamer are synthesized by Shanghai bio-corporation;
the sequence of the Listeria monocytogenes resistant aptamer is as follows: 5 '-6-FAM-TTTTTTTTTTATCCATGGGGCGGAGATGAGGGGGAGGAGGGCGGGTACCCGGTTGAT-3';
the sequence of the vibrio parahaemolyticus-resistant aptamer is as follows: 5 '-6-FAM-ATAGG AGTCA CGACG ACCAG AATCT AAAAA TGGGC AAAGA AACAG TGACT CGTTG AGATA CTTAT GTGCG TCTAC CTCTT GACTA AT-3';
the sequence of the anti-salmonella typhimurium aptamer is as follows: 5 '-6-FAM-AAAAAAGGTTGATTTCCTGATCG-3';
the FAM dye modified aptamer sequence of the blank control is as follows: 5 '-6-FAM-GGG CTG GCC AGA TCA GAC CCC GGA TGA TCA TCC TTG TGA GAA CCA-3'.
The invention also provides a preparation method of the kit for detecting Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium, which comprises the following steps:
the method comprises the following steps: preparation of self-priming valve separation type chip
1) Preparation of the mold
Designing a channel and chamber pattern of a self-suction valve separated chip by using CorelDRAW X8, printing the pattern on a transparent film as a mask, preferably coating two layers of negative photoresist on a silicon wafer considering the height of the chamber, coating the negative photoresist on a clean and dry silicon wafer by using a Spincoat G3P-8 spin coater under the conditions of 2000-3000 rpm for 30s-1min, repeating the step again to form a second layer, placing the silicon wafer on a single-sided lithography machine of 'h 49-25c 4' type under the mask, adjusting the position of the mask before exposure to locate the pattern in the middle of the wafer for subsequent chip manufacture, preferably 25 s-40 s, baking the silicon wafer at 60-70 ℃ for 1-3 min, and then baking at 85-95 ℃ for 10min, finally, the treated silicon wafer is rinsed with developer until the mold pattern is completely developed; the developer is preferably SU-8, and is preferably baked at 300 ℃ for 20-40min to complete the preparation of the die in order to enable the die to have better mechanical properties;
2) manufacture of self-sucking valve separated chip
Treating the mold with trimethylchlorosilane (MACKLIN, china) for a preferred treatment time of 10-20min to prevent adhesion of the polydimethylsiloxane, and then mixing the polydimethylsiloxane component pre-cure agent a and the cross-linking agent B, preferably in a mixing mass ratio of 10: 1, rotating at 1500-2000 rpm for 1-2 min preferably to remove bubbles generated during mixing, pouring the bubbles into a mold, removing the bubbles in polydimethylsiloxane after a chip is static for about 1min, screwing a bolt and a nut to one end to be flush, then placing the flush end downwards at the position of a valve of the mold, paying attention to avoid introducing the bubbles into the polydimethylsiloxane so as not to influence the performance of the chip, baking the chip on a heating plate at 70-90 ℃ preferably for 30-40 min preferably, carefully peeling the polydimethylsiloxane layer from the mold after the polydimethylsiloxane layer is completely cured, and punching sample adding holes by a 1.0/1.2mm puncher preferably; finally, simultaneously carrying out plasma pretreatment on the channel surface of the polydimethylsiloxane and the surface of the glass sheet, wherein the plasma pretreatment condition is preferably 200V for 1min, and after the treated planes are mutually attached, preferably baking at 90 ℃ for 30min to form a firm and stable chip; the chip top was then covered with scotch tape to obtain a self-priming valve compartmentalized chip.
Step two: preparation of graphene oxide
Adding graphite powder into H2SO4And sodium nitrate, stirring for 25-30 min in an ice bath, slowly adding 3.0-4.0 g of potassium permanganate into the mixture (keeping the temperature at 0 ℃ but completing within 10 min) under vigorous stirring, stirring for 1.5-2H, continuously stirring for 1.5-2H at 30-40 ℃, then heating to 140-150 ℃, stirring for 2-3H, adding deionized water to terminate the reaction system, and then neutralizing H with sodium hydroxide in the ice bath2SO4Adjusting the pH value to 5.5-6, filtering the obtained light yellow solution through an ion filtering membrane of 0.22 mu m to remove large particles, and dialyzing in a dialysis bag (MWCO 1000da) for 2-3 days to remove salt, so as to obtain graphene oxide; the mass ratio of the graphite powder to the sodium nitrate is preferably 1.0-1.5: 40 to 46, H2SO4Volume mL: the mass g of the sodium nitrate is preferably 80-100: 40-46; the size of the prepared graphene oxide is 550-600 nm.
Step three: preparation of real-time detection self-suction valve partition type chip
Closing the valve, covering the top of the chip by using a transparent adhesive tape, placing the chip in a vacuum pump for degassing treatment, wherein the degassing time is preferably 40-50 min, and then mixing the graphene oxide obtained in the second step with the four FAM-modified aptamers and HNB respectively, wherein the preferred concentration of the graphene oxide is preferably 0.4-0.5 mg/mL-1The concentration of four FAM modified specific aptamers is 1-1.2 mu M, the concentration of HNB is 0.16-0.25 mu M, the volume of the four mixtures is preferably 6.5-7 mu L, the mixtures are uniformly distributed in a sample detection chamber through four substrate sample feeding holes under the condition of negative pressure, the adhesive tape on the top of the chip is removed, and the chip is placed on a 65 ℃ heating plate for drying, preferably for 2-2.5 hours. And (3) taking down the chip after the substrate is completely dried, opening a valve, reattaching the top of the chip by using an adhesive tape, placing the chip in a vacuum pump, and vacuumizing for 40min preferably to obtain the real-time detection self-suction valve partition type chip.
The kit is used for detecting Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium, and comprises the following specific steps:
a needle is taken to pierce the adhesive tape at the sample inlet of the real-time detection self-suction valve partition type chip in a negative pressure state manually, then 26 mu L of sample to be detected is rapidly injected, the sample is rapidly distributed into each detection cell of the real-time detection self-suction valve partition type chip in the negative pressure condition, the sample is placed in a small animal imager after 10min, and a fluorescence image is acquired under the excitation condition of 455nm blue light.
The present invention is described in further detail below with reference to specific examples, wherein the starting materials are all commercially available
EXAMPLE 1 preparation of self-priming valve divided chip
The mold was treated with trimethylchlorosilane (MACKLIN, China) for 10min to prevent adhesion of the polydimethylsiloxane. Polydimethylsiloxane component precure a and crosslinker B (# RTV 615, Momentive, usa) were then mixed at a ratio of 10: 1, removing air bubbles and pouring into a mould. After the chip was allowed to stand for about 1min, the bubbles generated in the PDMS during pouring were removed. The bolts and nuts are screwed to one end flush and then the flush end is placed down at the valve position of the mold, taking care to avoid introducing air bubbles into the polydimethylsiloxane so as not to affect the chip performance. Then, the substrate was baked on a hot plate at 90 ℃ for 30min, and after the polydimethylsiloxane layer was completely cured, the substrate was carefully peeled off from the mold, and the sample application hole was punched using a 1.2mm punch. And finally, simultaneously carrying out plasma pretreatment on the channel surface of the polydimethylsiloxane and the surface of the glass sheet by using a name constant plasma cleaner (PDC-MG), attaching the treated planes to each other, and then baking and curing at 90 ℃ for 30min to obtain the self-priming valve separation type chip.
Example 2 preparation of graphene oxide
1.0g of graphite powder was added to 100mL of H2SO4And 46g of sodium nitrate, in an ice bath for 30 min. 3.0g of potassium permanganate was slowly added to the mixture (temperature was kept at 0 ℃ C., but completed within 10 min) with vigorous stirring for 2 h. Stirring was continued for 2h at 40 ℃ and then heating to 150 ℃ and stirring for 3 h. The reaction was terminated by adding 200mL of deionized water. H was then neutralized with sodium hydroxide in an ice bath2SO4The pH was adjusted to 6. The resulting pale yellow solution was filtered through an ion filtration membrane of 0.22 μm to remove large particles, and then dialyzed in a dialysis bag (MWCO 1000da) for 3 days to remove salts, to obtain graphene oxide.
Example 3 preparation of a self-priming valve compartmentalized chip for real-time detection
The valve was closed, the top of the chip was covered with scotch tape, and placed in a vacuum pump for degassing for 40 min. Then 0.4mg/mL of graphene oxide was mixed with 1. mu.M of the above four FAM-modified aptamers and 0.25. mu.M of HNB, respectively, and the volume of the four mixtures was 7. mu.L, and the mixtures were uniformly distributed in the sample detection cell under negative pressure conditions through the four substrate sample injection holes, respectively. And removing the adhesive tape on the top of the chip, and drying on a 65 ℃ heating plate for 2 h. And (3) taking down the chip after the substrate is completely dried, opening the valve, reattaching the top of the chip by using an adhesive tape, placing the chip in a vacuum pump, and vacuumizing for 40min to obtain the real-time detection self-suction valve separation type chip.
Example 4 preparation of bacterial liquid Standard
Taking a strain stored at minus 80 ℃, activating the strain, streaking and purifying the activated strain on a 3% sodium chloride trypsin vein agar plate, culturing at 37 ℃ for 18-24h, selecting a single colony, inoculating a 3% sodium chloride tryptone liquid culture medium, and performing shake culture at 37 ℃ for 12-18 h. And taking 1mL of bacterial liquid by a 10-time dilution plate pouring method for viable bacteria counting. Inactivating the rest bacteria solution with 1% formaldehyde at room temperature for 10min, centrifuging the inactivated bacteria solution at 3000rpm for 3min, collecting thallus, mixing with PBS solution to obtain bacteria suspension, and regulating the bacteria suspension concentration to 10 with PBS solution9CFU/mL, stored in a 4 ℃ refrigerator for use.
Example 5 method for detecting Listeria monocytogenes, Vibrio parahaemolyticus, and Salmonella typhimurium based on real-time detection of self-priming valve compartmentalized chips
The detection process is as shown in fig. 10, a needle is taken to pierce the adhesive tape at the sample inlet of the real-time detection self-suction valve partition type chip in a negative pressure state manually, then 26 mu L of sample to be detected is rapidly injected, the sample is rapidly distributed into each detection cell of the real-time detection self-suction valve partition type chip under the negative pressure condition, the sample is placed in a small animal imager after 10min, and a fluorescence image and a fluorescence signal are collected under the excitation condition of 455nm blue light. Referring to the standard curve diagrams 1-3, the numbers of Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium in the samples were determined, respectively. The concentration range of the bacteria is quantitatively detected to be 10-107CFU/mL。
FIG. 1 is a standard curve diagram of the detection of Listeria monocytogenes in example 6, wherein the abscissa is the logarithmic value of the concentration of Listeria monocytogenes, and the ordinate is the gray scale value. Detection concentration range: 10-107CFU/mL。
FIG. 2 is a graph showing the standard curve for detecting Vibrio parahaemolyticus in example 6, wherein the abscissa is the logarithm of the concentration of Vibrio parahaemolyticus and the ordinate is the gray scale value. Detection concentration range: 10-107CFU/mL。
FIG. 3 is a graph of the standard curve for detecting Salmonella typhimurium in example 6, wherein the abscissa is the logarithmic value of the Salmonella typhimurium concentration and the ordinate is the gray scale value. Detection concentration range: 10-107CFU/mL。
The method has the advantages of stable detection, 46.8, 22.9 and 32.3CFU/mL detection limits on Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium, short detection time, simple operation and good detection effect. When a sample to be detected is detected, the specific aptamer presents green fluorescence positive reaction when corresponding to the detected target bacteria, and the rest is negative, (note: green represents that pathogenic bacteria in the corresponding area are positive, and red represents that the pathogenic bacteria in the corresponding area are negative.) the method has strong specificity, and false positive and false negative results are not seen, and the results are shown in fig. 4-6.
Example 6 detection of a simulated sample
Preparing a pork simulation sample: 5g of fresh pork is cut into minced meat, and 15mL of sterile physiological saline is added to soak the minced meat at 4 ℃ overnight. The filtrate was filtered the next day through 0.22 μm filter membranes, 5 x 10 each2And 5 x 106CFU/mL of Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium are inoculated into the leachate and detected by the detection method.
A needle is taken to pierce through an adhesive tape at a sample inlet of the real-time detection self-suction valve partition type chip in a negative pressure state manually, then a pork sample to be detected is rapidly injected into 28 mu L, so that the pork sample to be detected is rapidly distributed into each detection cell of the real-time detection self-suction valve partition type chip in the negative pressure state, the pork sample is placed in a small animal imager after 10min, and a fluorescence image and a fluorescence signal are collected under the excitation condition of 455nm blue light.
Referring to the standard curve chart 7-FIG. 9, the number of Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium in the sample was determined, and the concentration of the bacteria was quantitatively determined in the range of 10-107CFU/mL. The method disclosed by the invention is stable in detection, and the recovery rate of the added standard reaches 97.11-10.40%.
FIG. 7 is a standard curve diagram for Listeria monocytogenes detection in example 7 of the present invention. The abscissa is the log value of the concentration of listeria monocytogenes in pork, and the ordinate is the gray value. Detection concentration range: 10-107CFU/mL。
FIG. 8 is a graph showing the standard curve for detecting Vibrio parahaemolyticus in example 7 of the present invention. The abscissa is the logarithmic value of the concentration of vibrio parahaemolyticus in the pork, and the ordinate is the gray value. Detection concentration range: 10-107CFU/mL。
FIG. 9 is a graph showing the standard curve of the detection of Salmonella typhimurium in example 7 of the present invention. The abscissa is the logarithmic value of the concentration of salmonella typhimurium in pork, and the ordinate is the gray value. Detection concentration range: 10-107CFU/mL。
Claims (10)
1. A kit for detecting Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium is characterized in that the kit comprises a self-priming valve separation type chip for real-time detection;
the real-time detection self-priming valve separation type chip comprises: the kit comprises a self-suction valve separation type chip, a specific adapter for FAM dye modification of Listeria monocytogenes, a specific adapter for FAM dye modification of Vibrio parahaemolyticus, a specific adapter for FAM dye modification of Salmonella typhimurium, a blank control FAM dye modification adapter, HNB dye and graphene oxide.
2. The method for preparing a kit for detecting listeria monocytogenes, vibrio parahaemolyticus and salmonella typhimurium according to claim 1, comprising:
the method comprises the following steps: preparation of self-priming valve separation type chip
1) Preparation of the mold
Designing a channel and cavity pattern of a self-sucking valve separated chip by using CorelDRAW X8, printing the pattern on a transparent film as a mask, coating a negative photoresist on a silicon wafer by using a spin coater, coating two layers in total, then placing the silicon wafer on a single-sided photoetching machine below the mask, adjusting the position of the mask to enable the pattern to be positioned in the middle of the wafer, baking the silicon wafer after exposure, finally, washing the treated silicon wafer by using a developer until a mould pattern completely appears, and baking the mould again to obtain the mould;
2) manufacture of self-sucking valve separated chip
Treating a mould by using trimethylchlorosilane, mixing a polydimethylsiloxane component pre-curing agent A and a cross-linking agent B to remove bubbles, pouring the mixture into the mould, screwing a bolt and a nut to one end of the mixture to be flush, then placing the flush end downwards at the position of a valve of the mould, removing polydimethylsiloxane from the surface of the mould after baking and curing by using a heating plate, punching a sample adding hole by using a puncher, and finally, simultaneously treating a channel surface of the polydimethylsiloxane and the surface of a glass sheet by using plasma, and baking and curing the treated planes after mutually jointing to form a self-suction valve partition type chip;
step two: preparation of graphene oxide
Adding graphite powder into H2SO4And sodium nitrate, stirring in an ice bath, slowly adding potassium permanganate into the mixture under vigorous stirring, continuously stirring at 30-40 ℃, then heating to 140-150 ℃, stirring, adding deionized water to terminate the reaction system, and neutralizing H with sodium hydroxide in the ice bath2SO4Adjusting the pH value to 5.5-6, filtering the obtained light yellow solution through an ion filtering membrane to remove large particles, and dialyzing in a dialysis bag for 2-3 days to remove salt, so as to obtain graphene oxide;
step three: preparation of real-time detection self-suction valve partition type chip
Closing the valve, covering the top of the chip by using a transparent adhesive tape, placing the chip in a vacuum pump for degassing treatment, mixing the graphene oxide obtained in the second step with the FAM dye modification specific aptamer of Listeria monocytogenes, the FAM dye modification specific aptamer of Vibrio parahaemolyticus, the FAM dye modification specific aptamer of Salmonella typhimurium or blank control FAM dye modification aptamer and HNB respectively, uniformly distributing the mixture into the sample holes through four substrate sample inlet holes under a negative pressure condition, removing the adhesive tape on the top of the chip, placing the chip on a 65 ℃ heating plate for drying, taking the chip down after the substrates are completely dried, opening the valve, re-attaching the chip with the adhesive tape on the top of the chip, placing the chip in the vacuum pump for vacuumizing, and obtaining the self-priming valve partition type chip for real-time detection.
3. The method for preparing a kit for detecting Listeria monocytogenes, Vibrio parahaemolyticus, and Salmonella typhimurium according to claim 2, wherein the baking conditions for the silicon wafer in the first step are 60-70 ℃ for 1-3 min, followed by 85-95 ℃ for 10 min.
4. The method for preparing a kit for detecting Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium according to claim 2, wherein the time for the trimethylchlorosilane treatment in the first step is 10-20 min.
5. The method for preparing a kit for detecting listeria monocytogenes, vibrio parahaemolyticus and salmonella typhimurium according to claim 2, wherein the mass ratio of polydimethylsiloxane component precuring agent a to cross-linking agent B in the step one is 10: 1.
6. the method of claim 2, wherein the conditions for removing bubbles by mixing the polydimethylsiloxane component in the step one are 1500-2000 rpm for 1-2 min.
7. The method for preparing a kit for detecting Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium according to claim 2, wherein the temperature for curing is 70-90 ℃ for 30-40 min in the step one.
8. The method for preparing a kit for detecting Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium according to claim 2, wherein the graphene oxide prepared in the second step has a size of 550-600 nm.
9. The method for preparing a kit for detecting Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium according to claim 2, wherein the degassing treatment time in the third step is 40-50 min.
10. The method for preparing the kit for detecting Listeria monocytogenes, Vibrio parahaemolyticus and Salmonella typhimurium according to claim 2, wherein the graphene oxide concentration in the step three is 0.4-0.5 mg-mL-1The concentration of the four FAM-modified specific aptamers is 1-1.2 mu M, and the concentration of HNB is 0.16-0.25 mu M.
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