CN204690016U - A kind of probe for detecting bacterium in food and test kit - Google Patents
A kind of probe for detecting bacterium in food and test kit Download PDFInfo
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- CN204690016U CN204690016U CN201520108675.8U CN201520108675U CN204690016U CN 204690016 U CN204690016 U CN 204690016U CN 201520108675 U CN201520108675 U CN 201520108675U CN 204690016 U CN204690016 U CN 204690016U
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- 238000012360 testing method Methods 0.000 title claims abstract description 19
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- 238000001514 detection method Methods 0.000 abstract description 11
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- 238000000034 method Methods 0.000 description 15
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- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 2
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The utility model provides a kind of probe for detecting bacterium in food, and this probe is molecular beacon probe, is hairpin structure, can not sends fluorescence when not hybridizing with target site, only have with target site specificity combine after, just have fluorescence to produce; Detection method strength of signal of the present utility model is high, high specificity, and simple to operate, testing cost is low, can detect the bacterium in food efficiently, rapidly; The utility model additionally provides a kind of test kit for detecting bacterium in food.
Description
Technical field
The utility model relates to a kind of probe detected, and is specifically related to a kind of probe for detecting bacterium in food and test kit.
Background technology
Namely food bacterium refers to be everlasting the bacterium existed in food, and natural bacterial species is various, but due to food physico-chemical property, the restriction of the factors such as residing envrionment conditions and processing treatment, the bacterium existed in food is the sub-fraction of nature bacterium.Food bacterium comprises pathogenic bacterium and non-pathogenic bacteria: pathogenic bacterium can cause food origin disease after referring to and entering human body with food, and common person is as Salmonellas, Shigellae etc., directly relevant with disease, and general provision does not allow to detect in food.Non-pathogenic bacteria does not generally cause human diseases, but wherein a part is spoilage organism, as pseudomonas, has substantial connection with food spoilage, is the important indicator evaluating food hygiene quality.
In the food hygienic standard of China, measure the method for bacterial number in food, carry out under the cultural method and culture condition of strict regulation, make each living bacterial cells adapting to these conditions can generate a macroscopic bacterium colony, namely the total number of bacterial colony generated is the total plate count in this food.In food, the kind of bacterium is a lot, and their physiological property and required culture condition are not quite similar.If adopt the method for cultivation to count all bacterial species and quantity in food, must adopt different substratum and culture condition, its workload is very large, usually can adopt the method for Testing index bacterium to substitute.
Such as, pathogen enterobacteria such as salmonella and Shigella are the important pathogenic bacterias causing food poisoning, but these pathogenic bacterium often comparatively small amt and be difficult to cultivate, carry out by batch to check piece by piece and impossible to food, in view of coliform is originated identical with pathogen enterobacteria, and general in external environment survival time also consistent with primary bowel pathogenic bacteria, so coliform is usual just as the indicator of enteric pathogenic bacteria contaminated food products.The pathogenic bacterium existence in food is roughly judged by Testing index bacterium, to can yet be regarded as a kind of short-cut method, but obviously, Bacteria Indicators not completes parallel relation with the existence of pathogenic bacteria---detect coliform in food, the possibility that enteric pathogenic bacteria exists can only be described, do not detect coliform in food, can not get rid of completely in food and may have enteropathogenic body pollution.
As can be seen here, current food method of detecting bacterium can not meet well easy, detect demand fast and accurately, difficulty to be cultivated, the food source pathogenic bacterium of difficult qualification, needs more efficient detection means.
Utility model content
The purpose of this utility model is to overcome weak point that prior art exists and provides a kind of probe, described probe can be used for detecting bacterium in food, by the means of fluorescence in situ hybridization, the utility model additionally provides a kind of quick, sensitive, test kit and method of detecting common bacteria in food specifically.
For achieving the above object, the technical scheme taked: a kind of probe for detecting bacterium in food, described probe is molecular beacon structure, namely probe sequence is made up of stem, ring two portions, total length 25 ~ 60 bases, ring Sequence wherein and bacterium 16s rRNA or 23s rRNA sequence match, and two ends stem portion is 5 ~ 8 complementary DNA or PNA sequences, and probe can be annealed formation hairpin structure at normal temperatures automatically; 5 ' end fluorophor of probe marks, 3 ' end fluorescent quenching group mark, probe free under normality is owing to being hairpin structure, fluorophor is because of adjacent fluorescent quenching group, and do not have fluorescence and send, only have after on probe and target sequence successful cross, fluorophor and fluorescent quenching group are separated, and just can send fluorescence;
Described probe detects bacterium in food by the principle of nucleic acid hybridization, and described bacterium comprises the bacterium and the contaminated rear pathogenic bacterium brought of food of normally carrying in food.
Preferably, the fluorophor that described probe 5 ' is held is the one in FITC, FAM and Cy3, and the fluorescent quenching group that described probe 3 ' is held is the one in DABCYL, BDH and TANRA.
The utility model provide a kind of for detect bacterium in food test kit, described test kit comprises:
(1) probe described above;
(2) resuspended stationary liquid;
(3) hybridization solution;
(4) washings;
Described resuspended stationary liquid comprises:
Preferably, described hybridization solution comprises 5% (w/v) PEG, 50mM NaCl, 40% (v/v) methane amide, 0.5% (w/v) ammonium persulphate, 1% (w/v) DEPC, 1% (w/v) ficoll, the Tris-HCl of 50mM EDTA, 0.1% (v/v) TritonX-100,50mM pH 8.0;
Described washings comprises 50mM Tris-HCl (pH 10.0), 0.5M NaCl, 0.1% (v/v) NP-40,0.5% (v/v) Triton X-100.
The utility model additionally provides the method for bacterium in a kind of rapid detection food, and described method adopts test kit described above to detect, and said method comprising the steps of:
(1a) get food samples to be checked, join in resuspended stationary liquid, concussion mixing;
(2a) get the fluid drips after mixing in step (1a) on slide glass, then dry;
(3a) soak in the slide glass obtained in step (2a) immersion methyl alcohol, then dry;
(4a) suspected location on slide glass after steeping adds hybridization solution and probe, is placed in the hybridization of hybrid heater lucifuge;
(5a) slide glass obtained in step (4a) is immersed in the washings of preheating and wash, then dry;
(6a) after dripping mountant, with fluorescence microscopy, with the pan of 10 × object lens and counting, ne ar is observed with 40 or 100 × object lens;
Or said method comprising the steps of:
(1b) get food samples to be checked, join in resuspended stationary liquid, concussion mixing;
(2b) filtration obtains filtrate;
(3b) getting the filtrate obtained in step (2b) drops on slide glass, then dries;
(4b) soak in the slide glass obtained in step (3b) immersion methyl alcohol, then dry;
(5b) suspected location on slide glass after steeping adds hybridization solution and probe, is placed in the hybridization of hybrid heater lucifuge;
(6b) slide glass obtained in step (5b) is immersed in the washings of preheating and wash, then dry;
(7b) after dripping mountant, with fluorescence microscopy, with the pan of 10 × object lens and counting, ne ar is observed with 40 or 100 × object lens.
Step described above (1a), (1b) make the bacterium in food samples separate with food ingredient by resuspended stationary liquid, and make bacterial suspension separately in resuspended stationary liquid, step described above (2a), (3b) are to make bacteria adhension in food samples to be checked on slide glass.
Preferably, described step (1a), (1b) volume ratio of food samples to be checked described in and described resuspended stationary liquid is 1:1 ~ 1:5, the volume that liquid described in described step (2a) drips is 10 μ L, the volume of filtrate added drop-wise described in described step (3b) is 10 μ L, described step (2a), (3a), (5a), (3b), (4b), (6b) bake out temperature in is 52 ~ 58 DEG C, described step (3a), (4b) soak time described in is 5 minutes, described step (5a), (6b) in, the preheating temperature of washings is 52 ~ 58 DEG C, described step (5a), (6b) washing time described in is 5 ~ 20 minutes.
Preferably, the volume that adds of hybridization solution described in described step (3a), (4b) is 20 μ L, and the concentration after described probe adds is 20ng/L.
Preferably, in described step (3a), (4b), hybridization temperature is 52 DEG C, and hybridization time is 30 minutes.
The technical essential of test kit described in the utility model and detection method or principle: the bacterium in food can separate with food ingredient by resuspended stationary liquid described in the utility model fast, and make it to be easy to be attached on slide glass.Fluorescence in situ hybridization (Flourescence in situ Hybridization, FISH) is the probe that a kind of application is marked with fluorescent substance, is detected the method for cell or tissue internal specific DNA or RNA by the method for hybridization; Molecular beacon probe is the probe that one has uniqueness " hair clip " space structure, not with target sequence in conjunction with time, molecular beacon is in " hair clip " structure, there are a ring sequence (loop) and a stem sequence (stem), wherein ring sequence is and target site, the i.e. base sequence of bacterium 16sRNA complementation, and stem sequence is the complementary sequence irrelevant with target site; Fluorophor and quenching of fluorescence group is marked with respectively at the two ends of probe, when probe is in hairpin structure, fluorophor is adjacent with quencher, and generate energy resonance transfer effect, makes fluorophor by quencher, fluorescent signal can not be produced, and when probe and target site in conjunction with time, hairpin structure is opened, and fluorophor and quencher are separately, produce fluorescent signal, this fluorescent signal can be detected by fluorescent microscope.
The utility model is groped by great many of experiments, determines that the optimum temps of fluorescence in situ hybridization in detection method described in the utility model is 52 DEG C, and the optimum concn of methane amide is 20% (v/v), and the optimal final concentration of probe is 20ng/L.
The sample range that probe of the present utility model, test kit and detection method detect, includes but not limited to the varieties of food items such as veterinary antibiotics, meat, flour, candy, cake, condiment, beverage.
The bacterium that probe of the present utility model, test kit and detection method detect, includes but not limited to intestinal bacteria common in food, Salmonellas, Shigellae, Clostridium botulinum, pseudomonas and other bacteriums that may pollute food.
The beneficial effects of the utility model are: the utility model, based on fluorescence in situ hybridization technique, provides a kind of probe for detecting bacterium in food, this probe specially for molecular beacon probe, its high specificity; The utility model additionally provides a kind of test kit for detecting bacterium in food, and this test kit comprises the resuspended stationary liquid that the bacterium in food and food ingredient can be made to separate fast; The utility model additionally provides the method for bacterium in a kind of rapid detection food, and detection method strength of signal of the present utility model is high, high specificity, and simple to operate, testing cost is low, can detect the bacterium in food efficiently, rapidly.
Accompanying drawing explanation
Fig. 1 is the structural representation of molecular beacon probe unbound state and hybridized state.
Embodiment
For the purpose of this utility model, technical scheme and advantage are better described, below in conjunction with specific embodiment, the utility model is described in further detail.Unreceipted actual conditions person in embodiment, conveniently conditioned disjunction manufacturers suggestion condition is carried out.Instrument or the unreceipted production firm person of reagent, be the conventional products that can buy on the market.The hybridization solution used in the utility model embodiment comprises 5% (w/v) PEG, 50mM NaCl, 40% (v/v) methane amide, 0.5% (w/v) ammonium persulphate, 1% (w/v) DEPC, 1% (w/v) ficoll, 50mM EDTA, the Tris-HCl of 0.1% (v/v) TritonX-100,50mM pH 8.0; Washings comprises 50mM Tris-HCl (pH 10.0), 0.5M NaCl, 0.1% (v/v) NP-40,0.5% (v/v) Triton X-100; Resuspended stationary liquid comprises:
Embodiment 1: for detecting the Design and synthesis of colibacillary specific molecular beacon probe
The structural representation of molecular beacon probe unbound state and hybridized state is as Fig. 1, by the comparison to bacterium 16s rRNA, filter out and can detect colibacillary probe sequence specifically, 5 '-CAAAGAGCAAGCTTCTTCC-3 ', this section of sequence is the loop section of molecular beacon probe, on this basis, adds 5 complementary bases at these sequence two ends respectively, form the stem portion of probe, just constitute complete molecular beacon probe:
Beacon E.coli
(5’-FAM-TGAGACAAAGAGCAAGCTTCTTCCTCTCA-DABCAL-3’)
This molecular beacon is by the distinguished sequence with loop-stem structure of based composition, wherein 5 ' end FAM mark, 3 ' end DABCTL mark, and fluorophor requires excitation wavelength 495nm, determined wavelength 520nm; Engineer's synthetic molecules beacon probe and with the oligonucleotide of its loop section complete complementary (5 '-GGAAGAAGCTTGCTCTTTG-3 ').By doing thermal denaturation curve experiment to molecular beacon probe and oligonucleotide, determine that the optimal reaction temperature of fluorescence in situ hybridization is 52 DEG C, the optimum concn of deionized formamide is 20%.
Embodiment 2: use the intestinal bacteria in the probe in detecting milk of design and synthesis in embodiment 1
Detection method:
(1) get 0.1mL milk, add the resuspended stationary liquid of 0.3mL, mixing;
(2) liquid drawn in 10L step (1) after mixing is applied on slide glass, and hybrid heater is heated to 52 DEG C of oven dry;
(3) slide glass is immersed in methyl alcohol, soak 5 minutes, put after taking-up on hybrid heater, 52 DEG C of oven dry;
(4) add the hybridization solution of 20 μ L containing embodiment 1 Middle molecule beacon probe, the concentration after probe adds is 20ng/L, hybridizes 30 minutes for 52 DEG C;
(5) be dipped in washings by slide glass, 55 DEG C are washed 5 minutes, put on hybrid heater, 52 DEG C of oven dry after taking-up.
(6) after dripping mountant, with fluorescence microscopy, with the pan of 10 × object lens and counting, ne ar is observed with 40 or 100 × object lens.In dark background, intestinal bacteria send green fluorescence.
Embodiment 3: use the bacterium in molecular beacon probe described in the utility model detection beef
(1) get beef 1g to be checked, chopping, is soaked into 2mL and fixes in re-suspension liquid, fully shake;
(2) with filter paper filtering, beef residue is removed;
(3) drawing filtrate is applied on 8 hole slide glasss, 8 hole slide glass every hole 10 μ L, and hybrid heater is heated to 52 DEG C of oven dry;
(4) slide glass is immersed in methyl alcohol, soak 5 minutes, put after taking-up on hybrid heater, 52 DEG C of oven dry;
(5) add 20 μ L hybridization solutions in each hole and detect the corresponding probe of all kinds of bacterium, hybridizing 30 minutes for 52 DEG C;
(6) be dipped in washings by slide glass, 55 DEG C are washed 5 minutes, and put after taking-up on hybrid heater, 52 DEG C air-dry.
(7), after dripping mountant, with fluorescence microscopy, according to the numbering in hole and the difference of fluorescence color, come there is which bacterial contamination in interpretation beef.With the pan of 10 × object lens and counting, observe ne ar with 40 or 100 × object lens.
Finally to should be noted that; above embodiment is only in order to illustrate the technical solution of the utility model but not restriction to the utility model protection domain; although be explained in detail the utility model with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can modify to the technical solution of the utility model or equivalent replacement, and not depart from essence and the scope of technical solutions of the utility model.
Claims (2)
1. for detecting a probe for bacterium in food, it is characterized in that: described probe is molecular beacon structure, namely probe sequence is made up of stem, ring two portions, total length 25 ~ 60 bases, and probe can be annealed formation hairpin structure at normal temperatures automatically.
2. for detecting a test kit for bacterium in food, it is characterized in that, described test kit comprises:
(1) probe described in claim 1.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201520108675.8U CN204690016U (en) | 2015-02-13 | 2015-02-13 | A kind of probe for detecting bacterium in food and test kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201520108675.8U CN204690016U (en) | 2015-02-13 | 2015-02-13 | A kind of probe for detecting bacterium in food and test kit |
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| Publication Number | Publication Date |
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| CN204690016U true CN204690016U (en) | 2015-10-07 |
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| CN201520108675.8U Expired - Fee Related CN204690016U (en) | 2015-02-13 | 2015-02-13 | A kind of probe for detecting bacterium in food and test kit |
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| Country | Link |
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2015
- 2015-02-13 CN CN201520108675.8U patent/CN204690016U/en not_active Expired - Fee Related
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| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20151007 |