CN102816850A - 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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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 the microorganism detection field; Relate to a kind of detection method, relate in particular to the method for a kind of while rapid detection Salmonella typhimurium (Salmonella typhimurium), intestinal bacteria (EnteroheamorrhagicE.coli) O157:H7, listeria monocytogenes (Listeria monocytogenes).
Background technology
Salmonella typhimurium, Escherichia coli O 157: H7, listeria monocytogenes all are frequent found pathogenic mushrooms in food and drinking-water, also are the human modal three big sources of infection, and human health has been caused serious harm.
Salmonellas is that the whole world causes the topmost a kind of pathogenic bacterium of gastro-enteritis, can cause 1,400,000 people morbidity every year, and salmonellosis also is one of most important zoonosis simultaneously, and it almost can adapt to the almost host of any kind.Salmonella-polluted food and drinking-water are human infected main sources.Wherein, Salmonella typhimurium is a kind of important pathogenic bacterium that cause food poisoning and salmonellosis, and it is main popular in developing country, and the Salmonella typhimurium infection usually is fatal for immunodefiiciency is individual.Salmonella typhimurium adheres to the intestinal mucosa epithelial cell mainly in the enteron aisle internal breeding, and then invades lamina propria, discharges toxin, causes it acute inflammatory reactions such as hyperemia, oedema, petechial hemorrhage to occur.Its clinical manifestation is various, and the normal person of immunologic function shows as the gastro-enteritis type more.And in infant and weakling, then can after gastrointestinal symptom takes place, septicemia occur, and further cause visceral lesion.
Escherichia coli O 157: H7 is the main pathogenic strains of EHEC; Also can cause a lot of food poisonings every year; Nineteen eighty-two is found breaking out of hemorrhagic enteritis that the O157:H7 intestinal bacteria cause in the U.S. first, causes a maximum in the world outbreak of epidemic in Japan in 1996.Escherichia coli O 157: H7 mainly invades small intestine distal end and colon, kidney, lung, spleen and brain after getting into human body.Cause the pathology of intestines mucosa oedema, hemorrhage, fluid accumulation, intestinal cells oedema, necrosis and kidney, spleen and brain.In the case that Escherichia coli O 157: H7 infects, 5 ~ 10% infected patient can develop into the hemolytic uremic syndrome syndromes, and this disease can be destroyed hemocyte and finally cause renal failure.Compare Escherichia coli O 157 with general coli strain: H7 does not contain general enterotoxin genes password, but can produce the stronger shiga-like toxin of virulence, causes toxicity symptoms such as high fever.
Listeria monocytogenes also is a kind of pathogenic bacteria of zoonosis.It can cause the listeriosis of people, animal; Mainly show as septicemia after the infection, in addition, also can cause diseases such as meningitis, encephalitis, endocarditis, miscarriage, abscess and partial purulence damage; The individuality that the main infection immunity of listeria monocytogenes is low; Comprise pregnant woman, newborn infant, hypoimmunity crowd and the elderly, and its mortality ratio can be up to 30%, its pathogenic material mainly is bacteriocin (monocins) and bacterial strain surface composition.But listeria monocytogenes growth and breeding still under 4 ℃ environment is important hidden danger of refrigerated food food safety.
The method of these 3 kinds of pathogenic bacterium of existing detection mainly is through traditional cultural method, and it is long to have a time cycle, shortcomings such as complicated operation, and also major part is to detect respectively, efficient is lower.Detect when also having occurred adopting multiple PCR technique to carry out at present; But detect when often being two kinds of pathogenic bacterium, and when more bacterial classifications were detected simultaneously, it was not strong then to occur specificity easily; Problems such as accuracy is lower; Because the influence factor of multiplex PCR is quite complicated, template, primer, reaction system and loop parameter or the like all can play very big influence to final detected result, and have limited the selection to goal gene simultaneously in addition; Therefore multiplex PCR is when detecting a plurality of bacterial classifications simultaneously, all has a lot of difficulties at aspects such as the selection of goal gene and design of primers.
As the mankind modal three big pathogenic infection sources, 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 carry out simultaneously.
Summary of the invention
The object of the present invention is to provide a kind of simple, fast, high specificity, the highly sensitive method that can detect Salmonella typhimurium alive, Escherichia coli O 157: H7, listeria monocytogenes simultaneously.This method can be used in the food samples living primary dcreening operation of Salmonella typhimurium, Escherichia coli O 157: H7, listeria monocytogenes detects, and it is easy and simple to handle, the result is objective and accurate, has avoided existing method complex operation, wastes time and energy, shortcoming with high costs.
The present invention realizes through following technical scheme:
In first aspect, the invention provides a kind of method that detects Salmonella typhimurium, Escherichia coli O 157: H7 and listeria monocytogenes when the magnetic bead that connects polyclonal antibody is collected object bacteria, nitrine bromination third ingot (PMA) is handled and multiplex PCR combines that adopts.
Method of the present invention can comprise the following step:
(1) randomly, the polyclonal antibody and the purifying of preparation Salmonella typhimurium, Escherichia coli O 157: H7, listeria monocytogenes;
(2) with Salmonella typhimurium, the Escherichia coli O 157 of purifying: the nanometer magnetic bead coupling that the polyclonal antibody of H7, listeria monocytogenes is carboxylated with the surface; Said link coupled immunomagnetic beads is contacted with detecting sample, collect object bacteria, and magnetic bead is separated; Wash-out is collected elutriant;
(3) handle said elutriant with PMA, centrifugal, collect bacterial sediment and extract DNA; With
(4) gained DNA is carried out multiplex PCR and detect, to confirm whether object bacteria exists.
In the method for the invention, employed primer is following in the said multiplex PCR: object bacteria is that the primer of Salmonella typhimurium is that the primer of Escherichia coli O 157: H7 is that the primer of listeria monocytogenes is to hly-F:TGAATGCAATTTCGAGCCTA (SEQ ID NO:5) and hly-R:CGCCGAAGTTTACATTCAAGC (SEQ ID NO:6) to rfbE-F:TTTATACGGACATCCATGTGA (SEQ ID NO:3) and rfbE-R:TTAATTCCA CGCCAACCA (SEQ ID NO:4) and/or object bacteria to fliC-F:CGCTGTTGACCAGAATAACCT (SEQ ID NO:1) and fliC-R:TCCTTACCACCCTTAATGGCAC (SEQ ID NO:2), object bacteria.
In one embodiment, method of the present invention can comprise:
(a) under aseptic technique, obtain the detection sample, mill with damping fluid and process homogenate, to get supernatant and get sample liquid, wherein said damping fluid is preferably phosphate buffered saline buffer;
(b) get the carboxylated nanometer magnetic bead in surface, after the MEST washing of ethanol and aseptic pH5.0-6.5, add diimine/N-hydroxy-succinamide (EDC/NHSS), the activation of vibrating under the room temperature; Magnetic removes supernatant after separating, and with aseptic pH5.0-6.0, magnetic bead is collected in preferred pH5.7MEST washing back; Get Salmonella typhimurium, the Escherichia coli O 157 of purifying: H7, listeria monocytogenes polyclonal antibody, pH is adjusted to 8.3 ~ 9.2, preferred 8.5 ~ 9.0; More preferably 8.7, join after the activation in the magnetic bead room temperature oscillatory reaction 2 ~ 4h rapidly; 2.5 ~ 3.5h preferably, 3h more preferably, magnetic separates; The immunomagnetic beads of antibody that obtained coupling, with the MEST washing, subsequent use;
(c) said immunomagnetic beads is added in the said sample liquid collect object bacteria, separate magnetic bead and wash-out, collect elutriant, add PMA solution afterwards; The whole mass concentration that makes PMA is 2.5 ~ 3.5 μ g/mL, preferred 2.8 ~ 3.2 μ g/mL, more preferably 3 μ g/mL, mixing; Lucifuge under the mixed sample liquid chamber temperature was cultivated 280 ~ 320 seconds, preferred 290 ~ 310 seconds, more preferably 300 seconds; Utilize the halogen lamp of 500W to make public 280 ~ 320 seconds, preferred 290 ~ 310 seconds, more preferably 300 seconds; Said mixed sample liquid placed on ice when illumination was crosslinked, and apart from light source 20cm place, and the suspension-s after crosslinked is centrifugal; The deposition that obtains is carried out DNA extraction, wherein, preferably carry out DNA extraction with the boiling water bath method; With
(d) gained DNA carries out the multiplex PCR detection, to confirm in the said detection sample whether object bacteria being arranged.
Wherein, room temperature can be 20 ℃ ~ 30 ℃, preferred 22 ℃ ~ 28 ℃, and more preferably 23 ℃ ~ 27 ℃, most preferably 25 ℃.
Wherein, the condition of said multiplex PCR can be the template DNA solution of: 4 μ L preparation, 2 μ L5 * buffer, and the amount of every primer is 15pmol, 1U Taq archaeal dna polymerase; Reaction conditions is: 95 ℃ of 10min, follow 94 ℃ of 30s, and 50 ℃ of 30s, 40 circulations of 72 ℃ of 1min, last 72 ℃ are extended 10min.
In the method for the invention; Detect the pcr amplification result with agarose gel electrophoresis after the said multiplex PCR, wherein if the 784bp band is arranged, then explanation has Salmonella typhimurium to exist; If the 627bp band is arranged; Then explanation has Escherichia coli O 157: H7 to exist, if the 360bp band is arranged, then explanation has listeria monocytogenes to exist.
In the method for the invention, said antibody can be commercial the purchase.
In second aspect; The invention provides a kind of be used for detecting simultaneously Salmonella typhimurium, Escherichia coli O 157: H7 and listeria monocytogenes test kit, it is that the primer of object bacteria is right that said test kit comprises respectively with Salmonella typhimurium, Escherichia coli O 157: H7, listeria monocytogenes..
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, said primer is to as follows: object bacteria is that the primer of Salmonella typhimurium is that the primer of Escherichia coli O 157: H7 is that the primer of listeria monocytogenes is to hly-F:SEQ ID NO:5 and hly-R:SEQ ID NO:6 to rfbE-F:SEQ ID NO:3 and rfbE-R:SEQ ID NO:4 and/or object bacteria to fliC-F:SEQ ID NO:1 and fliC-R:SEQ ID NO:2, object bacteria.
Test kit of the present invention also can also comprise PMA solution.
Aspect the 3rd of the present invention, the application in tap water, food, healthcare products or feed detect of described detection method of first aspect or the described test kit of second aspect is provided.
Compared with prior art, the present invention has following beneficial effect:
1, can detect Salmonella typhimurium, Escherichia coli O 157: H7, listeria monocytogenes simultaneously, efficient is higher, can in 4 hours, obtain the result;
2, the present invention is directed to the specific gene design specific primers of Salmonella typhimurium, Escherichia coli O 157: H7, listeria monocytogenes; And through condition optimizing, can be simultaneously, detect purpose bacterium in this sample efficiently, easy and simple to handle; The result judges simply, has reduced the detection cost;
3, the magnetic bead that connects antibody is collected thalline, has avoided collecting thalline, evaluation through cultivating in the ordinary method, and is highly sensitive; Can improve the sensitivity of PCR reaction greatly; Than the antiserum(antisera) that must use in the traditional detection method, can also be used to detect some antiserum(antisera) can not detected bacterial strain, the bacterial strain that can not express like antigen; Remedy the defective of immunodetection, have practicality more;
4, the present invention has simultaneously also used the PMA treatment process, to remove the interference of dead bacterium, has overcome the defective that general molecular Biological Detection method can't be distinguished dead bacterium and viable bacteria, only detects to have pathogenic viable bacteria.
Description of drawings
Fig. 1 is the multiplex PCR amplification.
Wherein, M:DL1000DNA Marker; 1: multiplex PCR detects Salmonella typhimurium, Escherichia coli O 157: H7, listeria monocytogenes simultaneously; 2: multiplex PCR detects Salmonella typhimurium, Escherichia coli O 157: H7 simultaneously; 3: multiplex PCR detects Salmonella typhimurium, listeria monocytogenes simultaneously; 4: multiplex PCR detects listeria monocytogenes, Escherichia coli O 157: H7 simultaneously; 5: the single detection of multiplex PCR 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 has shown that PMA removes the interference of dead bacterium.(A) PMA treatment combination mode; (B) multiplex PCR detected result.The concentration of Salmonella typhimurium, Escherichia coli O 157: H7, listeria monocytogenes is 10 in each group
6CFU/mL.
Fig. 3 is the lowest detection line that multiplex PCR detects Salmonella typhimurium, Escherichia coli O 157: H7 simultaneously.(A) Salmonella typhimurium; (B) Escherichia coli O 157: H7; (C) listeria monocytogenes.
Specific embodiments
Below in conjunction with specific embodiment, further explain the present invention, should be understood that these embodiment only be used to the present invention is described and be not used in the restriction scope of the present invention.Unreceipted actual conditions is an experimental technique in the following example; Usually according to the condition in the normal condition; Or according to the suggestion condition of manufacturer, the bacterial strain that relates among the embodiment all belongs to prior art, and those skilled in the art can obtain from disclosed commercial channel at an easy rate.
Embodiment
1.1 immunity is with the preparation of thalline
LB solid plate line activation Salmonella typhimurium and Escherichia coli O 157: H7, picking list bacterium colony carries out 37 ℃ of shaking culture of LB liquid; BHI solid plate line activation listeria monocytogenes, picking list bacterium colony carries out 37 ℃ of shaking culture of BHI liquid.4000g, the centrifugal collection thalline of 5min; , washing thalline 2 time resuspended with PBS, finally resuspended and carry out ultrasonication with PBS; 13000g, 10min centrifugal enrichment bacterial chip, PBS is resuspended, after the washing 2 times, and is resuspended; Added formalin solution (37% formaldehyde) by 0.7: 100, behind the mixing, room temperature is placed, thalline deactivation 24h; 13000g, 10min is centrifugal, gets deposition, and PBS washs 2 times (washing most formaldehyde); Centrifugal collecting precipitation ,-80 ℃ of preservations subsequent use (after can carrying out weighing earlier, packing is subsequent use).
1.2 the preparation of immunizing antigen
The weighing bacterial sediment mixes packing by the each immunity amount of every rabbit for 5mg (every strain bacterial chip is respectively 2.5mg), and resuspended with 250 μ L PBS ,-70 ℃ store for future use; Before the first immunisation, add isopyknic (250 μ L) complete Freund's adjuvant mixing, injecting immune is subsequent use.Before the follow-up immunization, add the incomplete Freund's adjuvant mixing of isopyknic (250 μ L), injecting immune is subsequent use.(every each immunity amount of rabbit is answered≤500 μ L) 1.3 injecting immune rabbits
Purchase heavily is about 2kg, and is female, and 4 of Japanese screech owl rabbits were raised 4 ~ 5 days earlier before the immunity, is convenient to the new environment of its adaptation; Before the first immunisation, get blood from auricular vein, every rabbit is got 100 ~ 200 μ L, contrasts as negative serum; Serum is handled: after getting blood, place 3h for 37 ℃; Place 4 ℃ to place 2h then; Sucking-off serum adds isopyknic 100% glycerine, behind the mixing, places-20 ℃ of storages subsequent use; During first immunisation, select for use the immunizing antigen of complete Freund's adjuvant mixing to carry out subcutaneous multi-point injection; After 2 weeks, carry out the immunity second time.Equally, get blood before the immunity, treatment process and step are with the above.During immunity for the second time, select for use the immunizing antigen of incomplete Freund's adjuvant mixing to carry out subcutaneous multi-point injection; After 2 weeks, carry out immunity for the third time.Concrete steps are with immunity for the second time; After 2 weeks, carry out the 4th immunity.Concrete steps are with immunity for the second time; The mensuration of selecting for use the preceding antiserum(antisera) of being got of the 4th immunity to tire like needs, is then carried out the 5th immunity after 2 weeks.Otherwise, after the 4th 1 week of immunity, carry out arterial blood extracting.
2.1 accurately prepare 1mg/mL BSA solution with the 0.005M borate buffer solution.
2.2 get the 5mg magnetic bead with washing with alcohol once (flush away tensio-active agent).
2.3 with aseptic MEST (pH5.5 ~ 6.0) washing twice, magnetic removes supernatant after separating.
2.4 add EDC/NHSS (each 5mg), vibration activation 30min under the room temperature.
2.5 magnetic removes supernatant after separating, and moves into new centrifuge tube with the resuspended back of aseptic MEST (pH5.5 ~ 6.0), washs three times.
2.6 get a 1.5mL centrifuge tube, add 100 μ L 1mg/mL purifying polyclonal antibodies respectively, complement to 1mL with the 0.005M borate buffer solution, transfer pH to 8.5 ~ 9.0 with NaOH, join room temperature oscillatory reaction 3h in the firm activatory magnetic bead rapidly.
2.7 magnetic separates back sucking-off supernatant, the magnetic bead of coupling antibody is preserved subsequent use with MEST washing three times.
We are example with the food sample, and the process of sample process in the method for the present invention is described.
Take by weighing the food sample of 1g, add the PBS of 9mL, mill and process homogenate, the centrifugal 5min of 900g removes the swill that strengthens, and gets supernatant (the necessary aseptic technique of this process).
Get 1mL food sample liquid, add immunomagnetic beads 2.0mg, 37 ℃ are softly rocked and hatch 45min altogether, separate with the magnetic force frame, and PBST washs twice, and isolating magnetic bead is resuspended with 50 μ L PBS.
PMA is dissolved in the methyl-sulphoxide (DMSO), is mixed with the PMA solution of 0.5mg/mL, and-20 ℃ " keep in Dark Place.Get the bacteria suspension that 500 μ L prepare and place the 1.5mL Eppendorf tube, add the PMA solution of 3 μ L 0.5mg/mL, the whole mass concentration that makes PMA is 3 μ g/mL; After PMA and bacteria suspension mix at ambient temperature lucifuge cultivate 5min; Utilize the halogen lamp exposure 5min of 500W, sample placed on ice (avoiding overheated) when illumination was crosslinked, and apart from light source 20cm place; Suspension-s after crosslinked is in the centrifugal 5min of 10000g, and the gained deposition is used for the extraction of DNA.
The sample that obtains through step 3 is at 10000g, and centrifugal 5min under 4 ℃ of conditions abandons supernatant; And carefully siphon away residual liquid; Add the aseptic deionized water of 30 μ L and boil 10min for 100 ℃, 12000g after cooling, centrifugal 5min under 4 ℃ of conditions; Get supernatant as the mPCR reaction template, the template of preparation should be used for detecting immediately.
Multiplex PCR why do by difficulty, and key of problem is that a plurality of target spot amplification conditions are incompatible.Each target spot all needs the primer on both sides to cooperate simultaneously.Therefore; The present invention chooses Salmonella typhimurium, Escherichia coli O 157: H7, listeria monocytogenes specific gene respectively; Adopt Oligo7 software design multiple PCR primer, adjust each the annealing temperature between the primer, and regulate primer between the amplification cooperate degree; Make each consistent as far as possible to the annealing temperature of primer, amplification rate equates as far as possible.
And through verifying the expanding effect of multiplex PCR, the optimum primer of the condition of choosing to as following detection with primer (table 2).And the bacterial strain that provides of employing table 1 carries out the primer specificity checking, and table 1 is seen in the numbering and the source of bacterial strain.
Table 2 strains tested and detected result
aJX-CDC, Jiangxi Province's disease prevention and control center, China;
bNCTC, national typical DSMZ, Britain
cATCC, U.S. typical case DSMZ, 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, wherein, the template DNA solution of 4 μ L preparation, 2 μ L5 * buffer, the amount of every primer is 15pmol, 1U Taq archaeal dna polymerase.Reaction conditions is: 95 ℃ of 10min, and then 40 circulations (94 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 1min), last 72 ℃ are extended 10min.
Table 2 detects uses primer
aThe GenBank number of including: fliC, JN587177; RfbE, S83460.1; Hly, FJ030609. step 7, the mPCR amplification detects
After the mPCR reaction finishes, draw 5 μ L reaction solutions with the rifle head, be added in the upward appearance hole of 2% sepharose, 85V electrophoresis 30min takes out gel piece, in the ultraviolet imagery system, observes electrophoretic band, and Taking Pictures recording.Confirm whether to have in the sample the concrete standard of object bacteria to be: to detect the pcr amplification result with agarose gel electrophoresis; If the 784bp band is arranged; Then explanation has Salmonella typhimurium to exist, if the 627bp band is arranged, then explanation has Escherichia coli O 157: H7 to exist; If the 360bp band is arranged, then explanation has listeria monocytogenes to exist.
Experimental result such as Fig. 1, Fig. 2, shown in Figure 3, Fig. 1 are the multiplex PCR amplification.M:DL1000DNA Marker; 1: multiplex PCR detects Salmonella typhimurium, Escherichia coli O 157: H7, listeria monocytogenes simultaneously; 2: multiplex PCR detects Salmonella typhimurium, Escherichia coli O 157: H7 simultaneously; 3: multiplex PCR detects Salmonella typhimurium, listeria monocytogenes simultaneously; 4: multiplex PCR detects listeria monocytogenes, Escherichia coli O 157: H7 simultaneously; 5: the single detection of multiplex PCR 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 has shown that PMA removes the interference of dead bacterium, (A) PMA treatment combination mode; (B) multiplex PCR detected result.The concentration of Salmonella typhimurium, moscow' paratyphi B, salmonella typhi is 106CFU/mL in each group.Fig. 3 is the lowest detection line that multiplex PCR detects Salmonella typhimurium, Escherichia coli O 157: H7 simultaneously.(A) Salmonella typhimurium; (B) Escherichia coli O 157: H7; (C) salmonella typhi.
The test of the strains tested through table 2 proves that the detection method of embodiment of the present invention detects has excellent specificity, and good detection effect is arranged.
Applicant's statement; The present invention explains detailed content of the present invention through the foregoing description; But the present invention is not limited to concrete component and parameter in the above-mentioned specific embodiment etc., does not mean that promptly the present invention must rely on above-mentioned concrete component and parameter could be implemented.The person of ordinary skill in the field should understand; To any improvement of the present invention; To the interpolation of the equivalence replacement of employed concrete component and parameter in method of the present invention and the product and ancillary component, the selection of concrete mode etc., all drop within protection scope of the present invention and the open scope.
Claims (10)
1. a magnetic bead that adopts to connect polyclonal antibody is collected the method that detects Salmonella typhimurium (Salmonella typhimurium), intestinal bacteria (Enteroheamorrhagic E.coli) O157:H7 and listeria monocytogenes (Listeria monocytogenes) when object bacteria, nitrine bromination third ingot (PMA) processing and multiplex PCR combine.
2. the method for claim 1, said method comprises the following step:
(1) randomly, the polyclonal antibody and the purifying of preparation Salmonella typhimurium, Escherichia coli O 157: H7, listeria monocytogenes;
(2) with Salmonella typhimurium, the Escherichia coli O 157 of purifying: the nanometer magnetic bead coupling that the polyclonal antibody of H7, listeria monocytogenes is carboxylated with the surface; Said link coupled immunomagnetic beads is contacted with detecting sample, collect object bacteria, and magnetic bead is separated; Wash-out is collected elutriant;
(3) handle said elutriant with PMA, centrifugal, collect bacterial sediment and extract DNA; With
(4) gained DNA is carried out multiplex PCR and detect, to confirm whether object bacteria exists.
3. according to claim 1 or claim 2 method, employed primer is following in the wherein said multiplex PCR: object bacteria is that the primer of Salmonella typhimurium is that the primer of Escherichia coli O 157: H7 is that the primer of listeria monocytogenes is to hly-F:TGAATGCAATTTCGAGCCTA and hly-R:CGCCGAAGTTTACATTCAAGC to rfbE-F:TTTATACGGACATCCATGTGA and rfbE-R:TTAATTCCACGCCAACCA and/or object bacteria to fliC-F:CGCTGTTGACCAGAATAACCT and fliC-R:TCCTTACCACCCTTAATGGCAC, object bacteria.
4. method as claimed in claim 3, said method comprises:
(a) under aseptic technique, obtain the detection sample, the adding damping fluid is milled and processed homogenate, and is centrifugal, gets supernatant and obtains sample liquid, and wherein said damping fluid is preferably phosphate buffered saline buffer;
(b) get the carboxylated nanometer magnetic bead in surface, ethanol and aseptic pH5.0 ~ 6.5, preferred pH5.5 ~ 6.0 are more preferably after the MES of pH5.7-tween damping fluid (MEST) washing; Add diimine/N-hydroxy-succinamide (EDC/NHSS), 20 ℃ ~ 30 ℃, preferred 22 ℃ ~ 28 ℃, more preferably 25 ℃ of vibration activation down; Magnetic removes supernatant after separating, with aseptic pH5.0 ~ 6.5, and preferred pH5.5 ~ 6.0, more preferably magnetic bead is collected in pH5.7MEST washing back; Get Salmonella typhimurium, the Escherichia coli O 157 of purifying: H7, listeria monocytogenes polyclonal antibody, pH is adjusted to 8.3 ~ 9.2, preferred 8.5 ~ 9.0, more preferably 8.7; Join rapidly after the activation in the magnetic bead, 20 ℃ ~ 30 ℃, preferred 22 ℃ ~ 28 ℃, more preferably 25 ℃ of oscillatory reaction 2 ~ 4h; Preferred 2.5 ~ 3.5h, more preferably 3h, magnetic separates; The immunomagnetic beads of antibody that obtained coupling, with the MEST washing, subsequent use;
(c) said immunomagnetic beads is added in the said sample liquid collect object bacteria, separate magnetic bead and wash-out, collect elutriant, add PMA solution afterwards; The whole mass concentration that makes PMA is 2.5 ~ 3.5 μ g/mL, and preferred 2.8 ~ 3.2 μ g/mL are 3 μ g/mL more preferably, and mixing is with 20 ℃ ~ 30 ℃ of mixed sample liquid; Preferred 22 ℃ ~ 28 ℃, more preferably 25 ℃ of following lucifuges were cultivated 280 ~ 320 seconds, and preferred 290 ~ 310 seconds, more preferably 300 seconds; Utilize the halogen lamp exposure 280 ~ 320 seconds of 500W, preferred 290-310 second, more preferably 300 seconds; Said mixed sample liquid placed on ice when illumination was crosslinked, and apart from light source 20cm place, and the suspension-s after crosslinked is centrifugal; The deposition that obtains is carried out DNA extraction, wherein, preferably carry out DNA extraction with the boiling water bath method; With
(d) gained DNA carries out multiplex PCR, to confirm in the said detection sample whether object bacteria being arranged.
5. like each described method among the claim 1-4; It is characterized in that detecting the pcr amplification result with agarose gel electrophoresis after the said multiplex PCR,, then have Salmonella typhimurium to exist wherein if the 784bp band is arranged; If the 627bp band is arranged; Then there is Escherichia coli O 157: H7 to exist,, then has listeria monocytogenes to exist if the 360bp band is arranged.
6. it is that the primer of object bacteria is right that test kit that is used for detecting simultaneously Salmonella typhimurium, Escherichia coli O 157: H7 and listeria monocytogenes, said test kit comprise respectively with Salmonella typhimurium, Escherichia coli O 157: H7, listeria monocytogenes.
7. test kit as claimed in claim 6, said test kit also comprise Salmonella typhimurium, the Escherichia coli O 157 of purifying: the polyclonal antibody of H7, listeria monocytogenes.
8. like claim 6 or 7 described test kits, said primer is to being that the primer of Salmonella typhimurium is that the primer of Escherichia coli O 157: H7 is that the primer of listeria monocytogenes is to hly-F:TGAATGCAATTTCGAGCCTA and hly-R:CGCCGAAGTTTACATTCAAGC to rfbE-F:TTTATACGGACATCCATGTGA and rfbE-R:TTAATTCCA CGCCAACCA and/or purpose thalline to fliC-F:CGCTGTTGACCAGAATAACCT and fliC-R:TCCTTACCACCCTTAATGGCAC, object bacteria for object bacteria.
9. like each described test kit among the claim 6-8, said test kit also comprises PMA solution.
10. like the application of each described test kit among each described detection method or the claim 6-9 among the claim 1-5 in tap water, food, healthcare products or feed detect.
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Denomination of invention: Method for simultaneous detection of Salmonella typhimurium, Escherichia coli O157: H7, and Listeria monocytogenes Effective date of registration: 20231107 Granted publication date: 20150121 Pledgee: Wuxi New District Chuang friends financing Company limited by guarantee Pledgor: WUXI ZODOLABS BIOTECH Co.,Ltd. Registration number: Y2023980064030 |
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