CN102816850B - Method for simultaneously detecting salmonella typhimurium, escherichia coli O157:H7 and listeria monocytogenesis - Google Patents
Method for simultaneously detecting salmonella typhimurium, escherichia coli O157:H7 and listeria monocytogenesis Download PDFInfo
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- CN102816850B CN102816850B CN201210308227.3A CN201210308227A CN102816850B CN 102816850 B CN102816850 B CN 102816850B CN 201210308227 A CN201210308227 A CN 201210308227A CN 102816850 B CN102816850 B CN 102816850B
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Abstract
The invention discloses a method for simultaneously and quickly detecting salmonella typhimurium, escherichia coli O157:H7 and listeria monocytogenesis in food. The method comprises the following steps of: preparing nano immunomagnetic particles of the targeted typhimurium, escherichia coli O157:H7 and listeria monocytogenesis so as to capture target bacteria from a detection sample; obtaining the target bacteria by magnetic field separation; treating by adopting propylmercuric bromide azide (PMA) to eliminate the interference of killed bacteria; and finally, extracting DNA (Deoxyribose Nucleic Acid) to carry out multiple PCR (Polymerase Chain Reaction) detection. Compared with a classical bacteria separation and identifying and serological typing method, the method disclosed by the invention is quick and accurate, can be used for only detecting viable bacteria and can be used for simultaneously detecting and identifying the three pathogens.
Description
Technical field
The invention belongs to microorganism detection field, relate to a kind of detection method, particularly relate to a kind of method of rapid detection Salmonella typhimurium (Salmonella typhimurium), intestinal bacteria (EnteroheamorrhagicE.coli) O157:H7, listeria monocytogenes (Listeria monocytogenes) simultaneously.
Background technology
Salmonella typhimurium, Escherichia coli O 157: H7, listeria monocytogenes are all the pathogenic mushrooms be often found in food and drinking-water, are also the large sources of infection of the mankind modal three, cause serious harm to human health.
Salmonellas is that the whole world causes the topmost a kind of pathogenic bacterium of gastro-enteritis, and can cause 1,400,000 people's morbidities every year, salmonellosis is also one of most important zoonosis simultaneously, and it almost can adapt to the host of almost any type.Salmonella-polluted food and drinking-water are the infected main sources of the mankind.Wherein, Salmonella typhimurium is a kind of important pathogenic bacterium causing food poisoning and salmonellosis, and its Major Epidemic is in developing country, and for immunodefiiciency is individual, Salmonella Typhimurium Infection is usually fatal.Salmonella typhimurium mainly in enteron aisle internal breeding, adheres to Intestinal epithelial cells, and then invades lamina propria, release toxin, causes it to occur the acute inflammatory reactions such as hyperemia, oedema, petechial hemorrhage.Its diverse clinical manifestations, the normal person of immunologic function shows as gastro-enteritis type more.And in infant and weakling, then can occur septicemia after generation gastrointestinal symptom, and cause visceral lesion further.
Escherichia coli O 157: H7 is the principal causative bacterial strain of enterohemorrhagic Escherichia coli, also a lot of food poisoning can be caused every year, nineteen eighty-two finds breaking out of the hemorrhagic enteritis that O157:H7 intestinal bacteria cause in the U.S. first, within 1996, causes a maximum in the world outbreak of epidemic in Japan.Escherichia coli O 157: H7 is main after entering human body invades small intestine distal end and colon, kidney, lung, spleen and brain.Cause the pathology of intestinal mucosa oedema, hemorrhage, fluid accumulation, intestinal cells oedema, necrosis and kidney, spleen and brain.At Escherichia coli O 157: in the case that H7 infects, the infected patient of 5 ~ 10% can develop into Hemolytic Uremic Syndrome, and this disease can be destroyed hemocyte and finally cause renal failure.Compared with general coli strain, Escherichia coli O157 not containing general enterotoxin genes password, but can produce the stronger shiga-like toxin of virulence, causes the toxicity symptoms such as high fever.
Listeria monocytogenes is also a kind of pathogenic bacteria of zoonosis.It can cause the listeriosis of people, animal, after infecting, main manifestations is septicemia, in addition, also the diseases such as meningitis, encephalitis, endocarditis, miscarriage, abscess and purulence damage locally can be caused, the individuality of listeria monocytogenes main infection immunocompromised, comprise pregnant woman, newborn infant, hypoimmunity crowd and the elderly, and its mortality ratio can up to 30%, its pathogenic substance is bacillin (monocins) and bacterial strain surface composition mainly.Listeria monocytogenes still can growth and breeding under the environment of 4 DEG C, is refrigerated food food safety important hidden danger.
The method of these 3 kinds of pathogenic bacterium of existing detection is mainly by traditional cultural method, and have the time cycle long, the shortcomings such as complicated operation, and major part detects respectively, efficiency is lower.Also detect while having occurred adopting multiple PCR technique to carry out at present, but detect while being often two kinds of pathogenic bacterium, and when to when more multi-cultur es detects simultaneously, then easily occur that specificity is not strong, the problems such as accuracy is lower, in addition the influence factor due to multiplex PCR is quite complicated, template, primer, reaction system and loop parameter etc. all can play very large impact to final detected result, and the selection simultaneously limited goal gene, therefore multiplex PCR is when detecting multiple bacterial classification simultaneously, a lot of difficulty is all there is in the selection and design of primers etc. of goal gene.
As the large pathogenic infection source of the mankind modal three, have for Salmonella typhimurium, Escherichia coli O 157: H7 and listeria monocytogenes more efficiently, more fast, more accurately and the needs of the detection method that can simultaneously carry out.
Summary of the invention
The object of the present invention is to provide a kind of simple, fast, high specificity, highly sensitive can detect Salmonella typhimurium alive, Escherichia coli O 157 simultaneously: the method for H7, listeria monocytogenes.The method can be used for living in food samples Salmonella typhimurium, Escherichia coli O 157: H7, listeria monocytogenes primary dcreening operation detect, it is easy and simple to handle, result is objective and accurate, avoids existing method complex operation, wastes time and energy, shortcoming with high costs.
The present invention is achieved by the following technical solutions:
In first aspect, the invention provides a kind of method adopting the magnetic bead connecting polyclonal antibody to collect detection Salmonella typhimurium while object bacteria, nitrine bromination third ingot (PMA) process and multiplex PCR combine, Escherichia coli O 157: H7 and listeria monocytogenes.
Method of the present invention can comprise the following step:
(1) Salmonella typhimurium, Escherichia coli O 157: H7, the polyclonal antibody of listeria monocytogenes purifying optionally, is prepared;
(2) by the nanometer magnetic bead coupling of the polyclonal antibody of the Salmonella typhimurium of purifying, Escherichia coli O 157: H7, listeria monocytogenes and surface carboxyl groups, by the immunomagnetic beads of described coupling and detection sample contact, collect object bacteria, and by Beads enrichment, wash-out, collects elutriant;
(3) with the described elutriant of PMA process, centrifugal, collect bacterial sediment and extract DNA; With
(4) multiplex PCR detection is carried out to gained DNA, to determine whether object bacteria exists.
In the method for the invention, the primer used in described multiplex PCR is as follows: object bacteria is the primer pair fliC-F:CGCTGTTGACCAGAATAACCT(SEQ ID NO:1 of Salmonella typhimurium) and fliC-R:TCCTTACCACCCTTAATGGCAC(SEQ ID NO:2), object bacteria is the primer pair rfbE-F:TTTATACGGACATCCATGTGA(SEQ ID NO:3 of Escherichia coli O 157: H7) and rfbE-R:TTAATTCCA CGCCAACCA(SEQ ID NO:4) and/or object bacteria be the primer pair hly-F:TGAATGCAATTTCGAGCCTA(SEQ ID NO:5 of listeria monocytogenes) and hly-R:CGCCGAAGTTTACATTCAAGC(SEQ ID NO:6).
In one embodiment, method of the present invention can comprise:
A (), under aseptic technique, obtain and detect sample, mill with damping fluid and make homogenate, get supernatant and obtain sample liquid, wherein said damping fluid is preferably phosphate buffered saline buffer;
B () gets the nanometer magnetic bead of surface carboxyl groups, after the MEST washing of ethanol and aseptic pH5.0-6.5, add diimine/N-hydroxy-succinamide (EDC/NHSS), vibration activation under room temperature, supernatant is removed after Magneto separate, with aseptic pH5.0-6.0, magnetic bead is collected after preferred pH5.7MEST washing, get the Salmonella typhimurium of purifying, Escherichia coli O 157: H7, listeria monocytogenes polyclonal antibody, pH is adjusted to 8.3 ~ 9.2, preferably 8.5 ~ 9.0, more preferably 8.7, join rapidly in the rear magnetic bead of activation, shaken at room temperature reaction 2 ~ 4h, preferably 2.5 ~ 3.5h, more preferably 3h, Magneto separate, obtain the coupling immunomagnetic beads of antibody, wash with MEST, for subsequent use,
C described immunomagnetic beads adds in described sample liquid and collects object bacteria by (), be separated magnetic bead and wash-out, collect elutriant, add PMA solution afterwards, the whole mass concentration of PMA is made to be 2.5 ~ 3.5 μ g/mL, preferably 2.8 ~ 3.2 μ g/mL, more preferably 3 μ g/mL, mixing, lucifuge under mixed sample liquid chamber temperature is cultivated 280 ~ 320 seconds, preferably 290 ~ 310 seconds, more preferably 300 seconds, the halogen lamp of 500W is utilized to expose 280 ~ 320 seconds, preferably 290 ~ 310 seconds, more preferably 300 seconds, when illumination is cross-linked, described mixed sample liquid is placed on ice, and at distance light source 20cm place, suspension after crosslinked is centrifugal, the precipitation of acquisition is carried out DNA extraction, wherein, preferably carry out DNA extraction with Boiling bath method, with
D () gained DNA carries out multiplex PCR detection, to determine whether have object bacteria in described detection sample.
Wherein, room temperature can be 20 DEG C ~ 30 DEG C, preferably 22 DEG C ~ 28 DEG C, more preferably 23 DEG C ~ 27 DEG C, most preferably 25 DEG C.
Wherein, the condition of described multiplex PCR can be: template DNA solution prepared by 4 μ L, 2 μ L5 × buffer, and the amount of every bar primer is 15pmol, 1U Taq archaeal dna polymerase; Reaction conditions is: 95 DEG C of 10min, then 94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 40 circulations, and last 72 DEG C extend 10min.
In the method for the invention, pcr amplification result is detected with agarose gel electrophoresis after described multiplex PCR, if wherein there is 784bp band, then illustrating has Salmonella typhimurium to exist, if there is 627bp band, then illustrate there is Escherichia coli O 157: H7 exists, if there is 360bp band, then illustrates and have listeria monocytogenes to exist.
In the method for the invention, described antibody can be that business is bought.
In second aspect, the invention provides a kind of for detecting Salmonella typhimurium, Escherichia coli O 157 simultaneously: H7 and listeria monocytogenes test kit, it is the primer pair of object bacteria with Salmonella typhimurium, Escherichia coli O 157: H7, listeria monocytogenes respectively that described test kit comprises..
Test kit of the present invention also can comprise Salmonella typhimurium, the Escherichia coli O 157 of purifying: the polyclonal antibody of H7, listeria monocytogenes.
In test kit of the present invention, described primer pair is as follows: the primer pair rfbE-F:SEQ ID NO:3 of object bacteria to be the primer pair fliC-F:SEQ ID NO:1 of Salmonella typhimurium and fliC-R:SEQ ID NO:2, object bacteria be Escherichia coli O 157: H7 and rfbE-R:SEQ ID NO:4 and/or object bacteria are primer pair hly-F:SEQ ID NO:5 and the hly-R:SEQ ID NO:6 of listeria monocytogenes.
Test kit of the present invention also can also comprise PMA solution.
In 3rd of the present invention, provide the detection method described in first aspect or the application of the test kit described in second aspect in tap water, food, healthcare products or feed detect.
Compared with prior art, the present invention has following beneficial effect:
1, can detect Salmonella typhimurium, Escherichia coli O 157: H7, listeria monocytogenes, efficiency is higher simultaneously, can obtain result in 4 hours;
2, the present invention is directed to Salmonella typhimurium, Escherichia coli O 157: the specific gene design Auele Specific Primer of H7, listeria monocytogenes, and by condition optimizing, can simultaneously, detect object bacterium in this sample efficiently, easy and simple to handle, result judges simple, reduces testing cost;
3, the magnetic bead connecting antibody collects thalline, avoid in ordinary method and collect thalline, qualification by cultivating, highly sensitive, greatly can improve the sensitivity of PCR reaction, compared with the antiserum(antisera) that must use in traditional detection method, can also be used for detecting the bacterial strain that some antiserum(antisera) can not detect, as the bacterial strain that antigen can not be expressed, make up the defect of immunodetection, more there is practicality;
4, the present invention simultaneously has also used PMA treatment process, to remove the interference of dead bacterium, overcomes the defect that general molecular biology for detection cannot distinguish dead bacterium and viable bacteria, only detects and has pathogenic viable bacteria.
Accompanying drawing explanation
Fig. 1 is multiplexed PCR amplification result.
Wherein, M:DL1000DNA Marker; 1: multiplex PCR detects Salmonella typhimurium, Escherichia coli O 157 simultaneously: H7, listeria monocytogenes; 2: multiplex PCR detects Salmonella typhimurium, Escherichia coli O 157 simultaneously: H7; 3: multiplex PCR detects Salmonella typhimurium, listeria monocytogenes simultaneously; 4: multiplex PCR detects listeria monocytogenes, Escherichia coli O 157 simultaneously: H7; 5: multiplex PCR single detection Salmonella typhimurium; 6: the single detection Escherichia coli O 157 of multiplex PCR: H7; 7: the single detection listeria monocytogenes of multiplex PCR.
Fig. 2 shows the interference that PMA removes dead bacterium.(A) PMA treatment combination mode; (B) multiplex PCR detected result.In each group, the concentration of Salmonella typhimurium, Escherichia coli O 157: H7, listeria monocytogenes is 10
6cFU/mL.
Fig. 3 is that multiplex PCR detects Salmonella typhimurium, Escherichia coli O 157 simultaneously: the lowest detection line of H7.(A) Salmonella typhimurium; (B) Escherichia coli O 157: H7; (C) listeria monocytogenes.
Specific embodiments
Below in conjunction with specific embodiment, explain the present invention further, should be understood that these embodiments are only not used in for illustration of the present invention and limit the scope of the invention.In the following example, unreceipted actual conditions is experimental technique, condition in usual conveniently condition, or according to the suggestion condition of manufacturer, the bacterial strain related in embodiment all belongs to prior art, and those skilled in the art can obtain from disclosed commercial channel easily.
Embodiment
Embodiment 1, the preparation of polyclonal antibody and purifying
The preparation of 1.1 immunity thalline
LB solid plate line activation Salmonella typhimurium and Escherichia coli O 157: H7, picking list bacterium colony carries out LB liquid 37 DEG C of shaking culture; BHI solid plate line activated mononuclear hyperplasia Listeria, picking list bacterium colony carries out BHI liquid 37 DEG C of shaking culture.4000g, 5min collected by centrifugation thalline; , washing thalline 2 time resuspended with PBS, final PBS is resuspended and carry out ultrasonication; 13000g, 10min centrifugal enrichment bacterial chip, PBS is resuspended, after washing 2 times, resuspended; Add formalin solution (37% formaldehyde) by 0.7: 100, after mixing, room temperature is placed, thalline deactivation 24h; 13000g, 10min are centrifugal, get precipitation, and PBS washs 2 times (washing most formaldehyde); Centrifugal collecting precipitation ,-80 DEG C save backup (after can first weighing, packing is for subsequent use).
The preparation of 1.2 immunizing antigens
Weigh bacterial sediment, be respectively 2.5mg by each immunity amount of every rabbit for the every strain bacterial chip of 5mg() carry out mixing packing, resuspended with 250 μ L PBS ,-70 DEG C store for future use; Before first immunisation, add the mixing of isopyknic (250 μ L) complete Freund's adjuvant, injecting immune is for subsequent use.Before follow-up immunization, add the incomplete Freund's adjuvant mixing of isopyknic (250 μ L), injecting immune is for subsequent use.(each immunity amount of every rabbit should≤500 μ L) 1.3 injecting immune rabbits
Buy and be heavily about 2kg, female, Japanese screech owl rabbit 4, first raises 4 ~ 5 days before immunity, is convenient to it and adapts to new environment; Before first immunisation, get blood from auricular vein, every rabbit gets 100 ~ 200 μ L, as negative serum control; Serum process: after getting blood, places 3h for 37 DEG C; Then be placed in 4 DEG C and place 2h; Sucking-off serum, adds isopyknic 100% glycerine, and after mixing, it is for subsequent use to be placed in-20 DEG C of storages; During first immunisation, the immunizing antigen selecting complete Freund's adjuvant to mix carries out subcutaneous multi-point injection; After 2 weeks, carry out second time immunity.Equally, before immunity, get blood, treatment process and step same as above.During second time immunity, the immunizing antigen selecting incomplete Freund's adjuvant to mix carries out subcutaneous multi-point injection; After 2 weeks, carry out third time immunity.Concrete steps are with second time immunity; After 2 weeks, carry out the 4th immunity.Concrete steps are with second time immunity; The mensuration that the antiserum(antisera) got before selecting the 4th immunity carries out tiring, as needs, then carries out the 5th immunity after 2 weeks.Otherwise the 4th immunity, after 1 week, carries out arterial blood extracting.
Embodiment 2, the preparation of immunomagnetic beads
2.1 accurately prepare 1mg/mL BSA solution with 0.005M borate buffer solution.
2.2 get 5mg magnetic bead washing with alcohol once (washes away tensio-active agent).
2.3 with aseptic MEST(pH5.5 ~ 6.0) washing twice, remove supernatant after Magneto separate.
2.4 add each 5mg of EDC/NHSS(), vibration activation 30min under room temperature.
Supernatant is removed, with aseptic MEST(pH5.5 ~ 6.0 after 2.5 Magneto separate) the new centrifuge tube of resuspended rear immigration, washs three times.
2.6 get a 1.5mL centrifuge tube, add 100 μ L 1mg/mL purified polyclonal antibodies respectively, complement to 1mL with 0.005M borate buffer solution, adjust pH to 8.5 ~ 9.0 with NaOH, join rapidly shaken at room temperature reaction 3h in the firm magnetic bead activated.
Sucking-off supernatant after 2.7 Magneto separate, the magnetic bead MEST of coupled antibody washs three times, saves backup.
Embodiment 3, sample preprocessing
We are for food sample, and the process of sample process in method of the present invention is described.
Take the food sample of 1g, add the PBS of 9mL, mill and make homogenate, the centrifugal 5min of 900g, remove the swill strengthened, get supernatant (the necessary aseptic technique of this process).
Step 4, the object bacteria in immunomagnetic ca pture food sample
Get 1mL food sample liquid, add immunomagnetic beads 2.0mg, 37 DEG C are softly rocked and hatch 45min altogether, are separated, PBST washing twice, by resuspended with 50 μ L PBS for the magnetic bead be separated with magnetic frame.
Embodiment 4, nitrine bromination third ingot (PMA) processes
PMA is dissolved in methyl-sulphoxide (DMSO), and be mixed with the PMA solution of 0.5mg/mL ,-20 DEG C " keep in Dark Place.Get the bacteria suspension that 500 μ L prepare and be placed in 1.5mL Eppendorf tube, add the PMA solution of 3 μ L 0.5mg/mL, make the whole mass concentration of PMA be 3 μ g/mL; After PMA mixes with bacteria suspension, lucifuge cultivates 5min at ambient temperature, utilize the halogen lamp exposure 5min of 500W, when illumination is cross-linked, sample is placed on ice (it is overheated to avoid), and at distance light source 20cm place, suspension after crosslinked is in the centrifugal 5min of 10000g, and gained precipitation is used for the extraction of DNA.
Embodiment 5, the extraction of genomic dna
Through step 3 obtain sample at 10000g, centrifugal 5min under 4 DEG C of conditions, abandon supernatant, and carefully siphon away residual liquid, add the aseptic deionized water of 30 μ L 100 DEG C and boil 10min, 12000g after cooling, centrifugal 5min under 4 DEG C of conditions, get supernatant as mPCR reaction template, the template of preparation should immediately for detecting.
Embodiment 6, chooses target spot and design primer
Multiplex PCR why do by difficulty, and the key of problem is that multiple target spot amplification condition is incompatible.Each target spot needs the primer on both sides to coordinate simultaneously.Therefore, the present invention chooses Salmonella typhimurium, Escherichia coli O 157 respectively: H7, listeria monocytogenes specific gene, adopt Oligo7 software design multiple PCR primer, adjust the annealing temperature between each pair of primer, and regulate the amplification cooperate degree between primer pair, make the annealing temperature of each pair of primer as far as possible consistent, amplification rate is as far as possible equal.
And verify the expanding effect of multiplex PCR by experiment, the primer pair choosing condition optimum is as detection primer (table 2) below.And the bacterial strain adopting table 1 to provide carries out primer specificity checking, the numbering of bacterial strain and source are in table 1.
Table 2 strains tested and detected result
ajX-CDC, disease prevention and control center, Jiangxi Province, China;
bnCTC, national Culture Collection, Britain
caTCC, American Type Culture preservation center, the U.S.;
dcMCC, Chinese medicine DSMZ, Chinese step 6, mPCR reaction system and reaction parameter
This multiplex PCR system comprises 10 μ L reaction solutions altogether, and wherein, template DNA solution prepared by 4 μ L, 2 μ L5 × buffer, the amount of every bar primer is 15pmol, 1U Taq archaeal dna polymerase.Reaction conditions is: 95 DEG C of 10min, then 40 circulations (94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min), and last 72 DEG C extend 10min.
Table 2 detection primer
athe GenBank number of including: fliC, JN587177; RfbE, S83460.1; Hly, FJ030609. step 7, mPCR amplification detects
After mPCR reaction terminates, draw 5 μ L reaction solutions with rifle head, be added in the loading hole of 2% sepharose, 85V electrophoresis 30min, take out gel piece, in ultraviolet imagery system, observe electrophoretic band, and Taking Pictures recording.Determine that the specific standards whether having object bacteria in sample is: detect pcr amplification result with agarose gel electrophoresis, if there is 784bp band, then illustrating has Salmonella typhimurium to exist, if there is 627bp band, then illustrate there is Escherichia coli O 157: H7 exists, if there is 360bp band, then illustrates and have listeria monocytogenes to exist.
As shown in Figure 1, Figure 2, Figure 3 shows, Fig. 1 is multiplexed PCR amplification result to experimental result.M:DL1000DNA Marker; 1: multiplex PCR detects Salmonella typhimurium, Escherichia coli O 157 simultaneously: H7, listeria monocytogenes; 2: multiplex PCR detects Salmonella typhimurium, Escherichia coli O 157 simultaneously: H7; 3: multiplex PCR detects Salmonella typhimurium, listeria monocytogenes simultaneously; 4: multiplex PCR detects listeria monocytogenes, Escherichia coli O 157 simultaneously: H7; 5: multiplex PCR single detection Salmonella typhimurium; 6: the single detection Escherichia coli O 157 of multiplex PCR: H7; 7: the single detection listeria monocytogenes of multiplex PCR.Fig. 2 shows the interference that PMA removes dead bacterium, (A) PMA treatment combination mode; (B) multiplex PCR detected result.In each group, the concentration of Salmonella typhimurium, moscow' paratyphi B, salmonella typhi is 106CFU/mL.Fig. 3 is that multiplex PCR detects Salmonella typhimurium, Escherichia coli O 157 simultaneously: the lowest detection line of H7.(A) Salmonella typhimurium; (B) Escherichia coli O 157: H7; (C) salmonella typhi.
Proved by the test of the strains tested of table 2, implement detection method of the present invention and carry out detection there is good specificity, have good Detection results.
Applicant states, the present invention illustrates detailed content of the present invention by above-described embodiment, but the present invention is not limited to concrete component and parameter etc. in above-mentioned specific embodiment, does not namely mean that the present invention must rely on above-mentioned concrete component and parameter could be implemented.Person of ordinary skill in the field should understand; any improvement in the present invention; to equivalence replacement and the interpolation of ancillary component, the concrete way choice etc. of the concrete component used in method of the present invention and product and parameter, all drop within protection scope of the present invention and open scope.
Claims (17)
1. one kind adopt while the magnetic bead connecting polyclonal antibody collects object bacteria, nitrine bromination third ingot process and multiplex PCR combine detect Salmonella typhimurium Salmonella typhimurium in tap water, food, healthcare products or feed sample), the method for intestinal bacteria (Enteroheamorrhagic E.coli) O157:H7 and listeria monocytogenes (Listeria monocytogenes), it is characterized in that, described method comprises the following step:
A (), under aseptic technique, obtains and detects sample, add damping fluid and mill and make homogenate, centrifugal, and get supernatant and obtain sample liquid, wherein said damping fluid is phosphate buffered saline buffer;
B () gets the nanometer magnetic bead of surface carboxyl groups, after the fatty acid methyl ester sulfonate-Tween buffer washing of ethanol and aseptic pH5.0 ~ 6.5, add diimine/N-hydroxy-succinamide, vibration activation at 20 DEG C ~ 30 DEG C, supernatant is removed after Magneto separate, magnetic bead is collected with after aseptic pH5.0 ~ 6.5 fatty acid methyl ester sulfonate-Tween buffer washing, get the Salmonella typhimurium of purifying, Escherichia coli O 157: H7, listeria monocytogenes polyclonal antibody, pH is adjusted to 8.3 ~ 9.2, join rapidly in the rear magnetic bead of activation, 20 DEG C ~ 30 DEG C oscillatory reaction 2 ~ 4h, Magneto separate, obtain the coupling immunomagnetic beads of antibody, with fatty acid methyl ester sulfonate-Tween buffer washing, for subsequent use,
C described immunomagnetic beads adds in described sample liquid and collects object bacteria by (), be separated magnetic bead and wash-out, collect elutriant, add nitrine bromination third ingot solution afterwards, the whole mass concentration of nitrine bromination third ingot is made to be 2.5 ~ 3.5 μ g/mL, mixing, by at mixed sample liquid 20 DEG C ~ 30 DEG C, lucifuge is cultivated 280 ~ 320 seconds, the halogen lamp of 500W is utilized to expose 280 ~ 320 seconds, when illumination is cross-linked, described mixed sample liquid is placed on ice, and at distance light source 20cm place, suspension after crosslinked is centrifugal, the precipitation of acquisition is carried out DNA extraction, wherein, DNA extraction is carried out with Boiling bath method, with
D () gained DNA carries out multiplex PCR, to determine whether have object bacteria in described detection sample; The primer used in wherein said multiplex PCR is as follows: the primer pair rfbE-F:TTTATACGGACATCCATGTGA that object bacteria is primer pair fliC-F:CGCTGTTGACCAGAATAACCT and fliC-R:TCCTTACCACCCTTAATGGCAC of Salmonella typhimurium, object bacteria is Escherichia coli O 157: H7 and rfbE-R:TTAATTCCA CGCCAACCA and object bacteria are primer pair hly-F:TGAATGCAATTTCGAGCCTA and hly-R:CGCCGAAGTTTACATTCAAGC of listeria monocytogenes.
2. the method for claim 1, is characterized in that, in step (b), the pH value of described fatty acid methyl ester sulfonate-Tween buffer is 5.5-6.0.
3. method as claimed in claim 2, it is characterized in that, in step (b), the pH value of described fatty acid methyl ester sulfonate-Tween buffer is 5.7.
4. the method for claim 1, is characterized in that, in step (b), the temperature of vibration activation is 22-28 DEG C.
5. method as claimed in claim 4, is characterized in that, in step (b), the temperature of vibration activation is 25 DEG C.
6. the method for claim 1, it is characterized in that, the Salmonella typhimurium of getting purifying described in step (b), Escherichia coli O 157: H7, listeria monocytogenes polyclonal antibody, adjust to 8.5 ~ 9.0 and join rapidly in the rear magnetic bead of activation by pH.
7. method as claimed in claim 6, it is characterized in that, described pH adjusts to 8.7.
8. the method for claim 1, is characterized in that, the oscillatory reaction time in step (b) is 2.5-3.5h.
9. method as claimed in claim 8, it is characterized in that, the described oscillatory reaction time is 3h.
10. the method for claim 1, is characterized in that, the whole mass concentration of nitrine bromination third ingot described in step (c) is 2.8 ~ 3.2 μ g/mL.
11. methods as claimed in claim 10, is characterized in that, the whole mass concentration of described nitrine bromination third ingot is 3 μ g/mL.
12. the method for claim 1, is characterized in that, the lucifuge culture temperature described in step (c) is 22-28 DEG C.
13. methods as claimed in claim 12, it is characterized in that, described lucifuge culture temperature is 25 DEG C.
14. the method for claim 1, is characterized in that, the described lucifuge incubation time of step (c) is 290-310 second.
15. methods as claimed in claim 14, is characterized in that, the described lucifuge incubation time of step (c) is 300 seconds.
16. the method for claim 1, is characterized in that, step (c) the described time shutter is 290-310 second.
17. the method for claim 1, pcr amplification result is detected with agarose gel electrophoresis after it is characterized in that described multiplex PCR, if wherein there is 784bp band, Salmonella typhimurium is then had to exist, if there is 627bp band, then there is Escherichia coli O 157: H7 exists, if there is 360bp band, then has listeria monocytogenes to exist.
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