CN104651510A - Probe, kit and method for detecting bacterial contamination in water body - Google Patents
Probe, kit and method for detecting bacterial contamination in water body Download PDFInfo
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- CN104651510A CN104651510A CN201510077109.XA CN201510077109A CN104651510A CN 104651510 A CN104651510 A CN 104651510A CN 201510077109 A CN201510077109 A CN 201510077109A CN 104651510 A CN104651510 A CN 104651510A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6841—In situ hybridisation
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Abstract
The invention provides a probe for detecting bacterial contamination in a water body. The probe is a molecular beacon probe, shows a hairpin structure without hybridizing with a target site, can not generate fluorescence and can generate fluorescence only when being specifically combined with the target site. The invention also provides a method for detecting the bacterial contamination in the water body. The detection method is high in signal strength, strong in specificity, simple in operation and low in detection cost and is capable of efficiently and rapidly detecting the bacterial contamination in the water body. The invention also provides a kit for detecting the bacterial contamination in the water body.
Description
Technical field
The present invention relates to a kind of probe detected, being specifically related to a kind of probe, test kit and method for detecting bacterial contamination in water body.
Background technology
By the major hidden danger that water-borne microbial diseases is harm humans health and ecological safety, the whole world about has the disease of 1/4 directly or indirectly to be caused by disease caused by infectious water pathogenic microorganism.In the U.S., every year by pathogenic bacterium in water diseases induced cause productivity to decline and the financial loss that causes up to 2,000,000,000 dollars.Estimate according to WHO, the whole world has 2,600,000,000 people not drink sanitary water, and along with the growing tension of world water resources, the utilization of reuse water can constantly increase, and the microbiological pollution of water body can be more serious.
Salmonella typhi, Shigellae, vibrio cholerae and Escherichia coli O 157 etc. are had by water-borne common pathogen, because the height of these bacteriums is infectious and highly pathogenic, in engine hygiene safety control index, need to carry out comprehensive monitoring and control to multiple pathogenic microorganisms.Therefore, setting up easy, quick, sensitive detection technique, propagating the infection risk of pathogenic bacterium for evaluating water, the hygienic safety ensureing water surrounding all tool is of great significance.
In water body, the traditional method of Bacteria Detection is culture method, and this method is long for detection time, and specificity is not high, and needs special laboratory and technician, to experience and technical requirements high.And nature has part bacterium, cannot on existing artificial medium incubation growth, therefore the bacterial species that detects of culture method and quantity all can lower than the actual values in environment.Use PCR or quantitative fluorescent PCR equimolecular biology techniques, can pathogenic bacteria in Direct Identification water body, without the need to culture of isolated bacterium on artificial medium, make can shorten to 4 ~ 5 hours from one week detection time.But the false positive issue on the one hand in amplification process never well solves in the evolution of round pcr, natural water body on the other hand, often containing the composition in a large number PCR reaction being had to inhibition in the water body particularly polluted, remaining of these materials of DNA extraction process, be then easy to cause false-negative result.Therefore, PCR detection method reality accuracy and non-specifically is desirable.
Summary of the invention
The object of the invention is to overcome weak point that prior art exists and provide a kind of probe, described probe can be used for detecting the bacterium polluted in water body, by the means of fluorescence in situ hybridization, present invention also offers a kind of quick, sensitive, test kit and method of detecting common bacteria in water body specifically.
For achieving the above object, the technical scheme taked: a kind of probe for detecting bacterial contamination in water body, 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 by the principle of nucleic acid hybridization the pathogenic bacterium brought because of contaminated in water body.
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 invention provides a kind of test kit based on bacterial contamination in fluorescence in situ hybridization rapid detection water body, described test kit comprises:
(1) probe described above;
(2) hybridization solution;
(3) washings;
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.
Present invention also offers the method for bacterial contamination in a kind of rapid detection water body, described method adopts test kit described above to detect, and said method comprising the steps of:
(1) water sample 1 ~ 5mL, 6000g to be checked is got centrifugal 2 minutes;
(2) stay the resuspended throw out of 20uL supernatant fluid, discard all the other supernatant fluid;
(3) getting the re-suspension liquid obtained in step (2) drops on slide glass, then dries;
(4) soak in the slide glass obtained in step (3) immersion methyl alcohol, then dry;
(5) suspected location on slide glass after steeping adds hybridization solution and probe, is placed in the hybridization of hybrid heater lucifuge;
(6) slide glass obtained in step (5) is immersed in the washings of preheating and wash, then dry;
(7) 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.
Preferably, the volume that re-suspension liquid described in described step (3) drips is 10 ~ 20 μ L, bake out temperature in described step (3), (4), (6) is 50 ~ 60 DEG C, described in described step (4), soak time is 5 minutes, in described step (6), the preheating temperature of washings is 55 DEG C, and described in described step (6), washing time is 5 ~ 20 minutes.The volume that adds of hybridization solution described in described step (5) is 20 μ L, and the final concentration after described probe adds is 20ng/L.
Preferably, in described step (5), hybridization temperature is 52 DEG C, and hybridization time is 30 ~ 60 minutes.
The technical essential of test kit of the present invention and detection method or principle: fluorescence in situ hybridization (Flourescence in situ Hybridization, FISH) be the probe that a kind of application is marked with fluorescent substance, 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 present invention is groped by great many of experiments, determines that the optimum temps of fluorescence in situ hybridization in detection method of the present invention is 52 DEG C, and the optimum concn of methane amide is 30% (v/v), and the optimal final concentration of probe is 20ng/L.
The sample range that probe of the present invention, test kit and detection method detect, includes but not limited to the detection of bacterial contamination situation in the water sample such as the natural water bodies such as river, He Hu, sea, underground water and tap water, sanitary sewage, industrial sewage.
The bacterium that probe of the present invention, test kit and detection method detect, include but not limited to intestinal bacteria common in water, salmonella typhi, Shigellae and other may cause the bacterium of water pollutions.
Beneficial effect of the present invention is: the present invention is based on fluorescence in situ hybridization technique, provides a kind of probe for detecting bacterial contamination in water body, this probe specially for molecular beacon probe, its high specificity; Present invention also offers the method for bacterial contamination in a kind of rapid detection water body, detection method strength of signal of the present invention is high, high specificity, and simple to operate, testing cost is low, can detect the bacterium polluted in water body efficiently, rapidly; Present invention also offers a kind of test kit for detecting bacterial contamination in water body.
Accompanying drawing explanation
Fig. 1 is the structural representation of molecular beacon probe unbound state and hybridized state;
Fig. 2 is the schematic diagram of a kind of embodiment of slide glass in test kit of the present invention;
Fig. 3 is the fluorescence microscopy result figure being detected colibacillary a kind of embodiment in water body by detection method of the present invention.
Embodiment
For better the object, technical solutions and advantages of the present invention being described, below in conjunction with specific embodiment, the invention will be further described.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.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, 50mM EDTA, the Tris-HCl of 0.1% (v/v) TritonX-100,50mM pH 8.0; Described washings comprises 50mM Tris-HCl (pH10.0), 0.5M NaCl, 0.1% (v/v) NP-40,0.5% (v/v) Triton X-100.
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, as Fig. 1, by the comparison to bacterium 16srRNA, filters 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, forms 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 sanitary sewage of design and synthesis in embodiment 1
Detection method:
(1) get 2mL water sample to be measured, centrifugal 2 minutes of 6000g, with the resuspended throw out of 200 μ L supernatant fluid, discard all the other supernatant fluid;
(2) drawing resuspension fluids in 10 μ L steps (1) 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, hybridize 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, as Fig. 3.
Embodiment 3: use molecular beacon probe of the present invention to detect in the water quality of sewage draining exit downstream have which bacterial contamination.
(1) get water sample 20mL to be checked, with filter paper filtering, remove macroscopic impurity;
(2) centrifugal 2 minutes of 6000g, with the resuspended throw out of 200 μ L supernatant fluid, discards all the other supernatant fluid;
(3) draw re-suspension liquid be applied on 8 hole slide glasss, shown 8 hole slide glasss as shown in Figure 2, every hole 10 μ L, 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 water body.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 technical scheme of the present invention but not limiting the scope of the invention; although be explained in detail the present invention with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can modify to technical scheme of the present invention or equivalent replacement, and not depart from essence and the scope of technical solution of the present invention.
Claims (8)
1. one kind for detecting the probe of bacterial contamination in water body, 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, ring Sequence wherein and bacterium 16s rRNA or 23s rRNA sequence match, 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 by the principle of nucleic acid hybridization the pathogenic bacterium brought because of contaminated in water body.
2. the probe for detecting bacterial contamination in water body according to claim 1, it is characterized in that, 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.
3. for detecting a test kit for bacterial contamination in water body, it is characterized in that, described test kit comprises:
(1) probe described in claim 1 or 2;
(2) hybridization solution;
(3) washings.
4. the test kit for detecting bacterial contamination in water body according to claim 3, it is characterized in that, 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, 50mM EDTA, the Tris-HCl of 0.1% (v/v) TritonX-100,50mM pH 8.0;
Described washings comprises the Tris-HCl of 50mM pH 10.0,0.5M NaCl, 0.1% (v/v) NP-40,0.5% (v/v) Triton X-100.
5. for detecting a method for bacterial contamination in water body, it is characterized in that, said method comprising the steps of:
(1) water sample 1 ~ 5mL, 6000g to be checked is got centrifugal 2 minutes;
(2) stay the resuspended throw out of 20uL supernatant fluid, discard all the other supernatant fluid;
(3) getting the re-suspension liquid obtained in step (2) drops on slide glass, then dries;
(4) soak in the slide glass obtained in step (3) immersion methyl alcohol, then dry;
(5) suspected location on slide glass after steeping adds hybridization solution and probe, is placed in the hybridization of hybrid heater lucifuge;
(6) slide glass obtained in step (5) is immersed in the washings of preheating and wash, then dry;
(7) 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.
6. the method for detecting bacterial contamination in water body according to claim 5, it is characterized in that, the volume that re-suspension liquid described in described step (3) drips is 10 ~ 20 μ L, bake out temperature in described step (3), (4), (6) is 50 ~ 60 DEG C, described in described step (4), soak time is 5 minutes, in described step (6), the preheating temperature of washings is 55 DEG C, and described in described step (6), washing time is 5 ~ 20 minutes.
7. the method for detecting bacterial contamination in water body according to claim 5, is characterized in that, the volume that adds of hybridization solution described in described step (5) is 20 μ L, and the final concentration after described probe adds is 20ng/L.
8. the method for detecting bacterial contamination in water body according to claim 7, is characterized in that, in described step (5), hybridization temperature is 52 DEG C, and hybridization time is 30 ~ 60 minutes.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105116078A (en) * | 2015-08-10 | 2015-12-02 | 中国热带农业科学院热带生物技术研究所 | Method for treating gram bacterium protein for mass spectrum identification and buffer solution of method |
| WO2018196691A1 (en) * | 2017-04-24 | 2018-11-01 | 倪燕翔 | Precise recognition method for nucleic acid |
| CN110964841A (en) * | 2019-12-20 | 2020-04-07 | 宁波美康盛德医学检验所有限公司 | Molecular beacon probe and kit for detecting septicemia and detection method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104017889A (en) * | 2014-06-19 | 2014-09-03 | 方华成 | Molecular beacon probe for rapid detection of Mycobacterium tuberculosis and method for detecting Mycobacterium tuberculosis |
| CN104032023A (en) * | 2014-06-19 | 2014-09-10 | 洪冉 | Molecular beacon probe for quickly detecting non-Mycobacterium tuberculosis and detection method using same |
| CN104313174A (en) * | 2014-11-12 | 2015-01-28 | 方华成 | Molecular beacon probe for rapidly detecting streptococcus pneumoniae and detection method |
-
2015
- 2015-02-13 CN CN201510077109.XA patent/CN104651510A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017889A (en) * | 2014-06-19 | 2014-09-03 | 方华成 | Molecular beacon probe for rapid detection of Mycobacterium tuberculosis and method for detecting Mycobacterium tuberculosis |
| CN104032023A (en) * | 2014-06-19 | 2014-09-10 | 洪冉 | Molecular beacon probe for quickly detecting non-Mycobacterium tuberculosis and detection method using same |
| CN104313174A (en) * | 2014-11-12 | 2015-01-28 | 方华成 | Molecular beacon probe for rapidly detecting streptococcus pneumoniae and detection method |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105116078A (en) * | 2015-08-10 | 2015-12-02 | 中国热带农业科学院热带生物技术研究所 | Method for treating gram bacterium protein for mass spectrum identification and buffer solution of method |
| WO2018196691A1 (en) * | 2017-04-24 | 2018-11-01 | 倪燕翔 | Precise recognition method for nucleic acid |
| CN110964841A (en) * | 2019-12-20 | 2020-04-07 | 宁波美康盛德医学检验所有限公司 | Molecular beacon probe and kit for detecting septicemia and detection method thereof |
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