CN109593837A - A method of the nanogold colorimetric method based on magnetic capture quickly detects the rugged Cronobacter sakazakii of slope - Google Patents
A method of the nanogold colorimetric method based on magnetic capture quickly detects the rugged Cronobacter sakazakii of slope Download PDFInfo
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Abstract
The invention discloses a kind of methods that the nanogold colorimetric method based on magnetic capture quickly detects the rugged Cronobacter sakazakii of slope, belong to technical field of food safety.This method is that the somatic cells in substance to be checked are carried out to break process to release DNA, obtains after abstraction impurity removal and slightly mentions DNA solution, amino magnetic bead is added in DNA solution to slightly mentioning, is incubated for capture dna；The rugged Cronobacter sakazakii specific PCR reaction of slope is established, carries out PCR amplification by template of the DNA of capture；The DNA probe for designing the rugged Cronobacter sakazakii of slope, colloidal gold and DNA probe are incubated for, and are then carried out aging and are obtained colloidal gold probe, and by PCR product and colloidal gold probe bulk crossing, MgSO is added4Solution observes color change.The method of the present invention step is simple, and specificity is good, and the used time is shorter, may be implemented quickly to detect, at low cost without expensive reagents such as antibody.Slope rugged Cronobacter sakazakii of the present invention suitable for quickly detection food.
Description
Technical field
The present invention relates to a kind of methods that the nanogold colorimetric method based on magnetic capture quickly detects the rugged Cronobacter sakazakii of slope, belong to
In technical field of food safety.
Background technique
The rugged Cronobacter sakazakii of slope (Cronobacter sakazakii), former Enterobacter sakazakii (Enterobacter
Sakazakii), it is a kind of flagellum containing the whole body, can moves and the nonspore-bearing tubbiness bacillus of Gram-negative of amphimicrobian.The bacterium
It can cause Infant and child deaths, the threat of baby especially light to premature, birth weight or immunocompromised host baby are maximum, seriously
Person can lead to septicemia, meningitis or necrotizing enterocolitis, and for the case fatality rate of reported cases up to 20%~50%, survivor is normal
There is serious nervous system sequelae.The rugged Cronobacter sakazakii of slope is the main microbial safety index of infant formula, and slope is rugged
The Detection & Controling of Cronobacter sakazakii have become the key factor for influencing baby food safety.Therefore it establishes a kind of efficient, fast
The detection method of speed is particularly important.
Compared with traditional detection method based on microculture, polymerase chain reaction (PCR) is the one of Bacteria Detection
A milestone, PCR reaction is a kind of for amplifying the Protocols in Molecular Biology for expanding specific DNA fragmentation, its maximum feature is just
It is that micro DNA can be significantly increased.Therefore in theory, seldom bacterium can be detected in a short period of time.
However when PCR is used to detect the primary pollution source milk powder of the rugged Cronobacter sakazakii of slope, since the matrix in milk powder is more complicated, make
The DNA purity that must be obtained is not high, and containing substances such as more fat proteins, and these substances can generate strong suppression to PCR
Production is used, so will appear during PCR it is dense even if thallus DNA, remain on expansion not shaping band the phenomenon that, therefore
The separation and concentration that sample is carried out before PCR is necessary.Relatively common to the method for the separation and concentration of sample is to exempt from
Epidemic disease magnetic separation technique, but immune magnetic separation technique needs the antibody of specificity, Antibody preparation difficulty is very big, at high cost etc. asks
Topic all limits its development.Simultaneously round pcr there are also a big urgent problems, i.e., by the testing result of PCR need through
Agarose gel electrophoresis is crossed to observe as a result, the method needs the large-scale instrument of complex and expensive, while needing using easy system poison
Coloring agent EB, in addition to this, time-consuming for the method, complex steps, including the processes such as glue, point sample, race electrophoresis, and whole process is extremely
The time of a hour is needed less.
Paramagnetic particle method nucleic acid purification techniques use nanoscale magnetic bead microballon, a kind of official of the surface markers of this magnetic bead microballon
Such as amino can be rolled into a ball, adsorption reaction can occur with nucleic acid.Nanogold colorimetric method is to assemble front and back by nanogold, and absorption peak occurs red
Colloidal gold color change observes the result of PCR amplification caused by shifting.
CN105950471 A discloses a kind of method based on immunomagnetic bead technique fast Acquisition Cronobacter sakazakii and answers
With this method is direct using monoclonal antibody and carboxyl magnetic bead the progress covalent coupling adaptive immune magnetic bead of the rugged Cronobacter sakazakii of slope
The rugged Cronobacter sakazakii thallus of slope is captured, it is at high cost that there are antibody in this method, and sensitivity is low, detection the disadvantages of time-consuming.
Summary of the invention
Method to solve to capture Cronobacter sakazakii based on immunomagnetic bead technique in the prior art needs the antibody of specificity,
And Antibody preparation difficulty is very big, at high cost, the problem of not being suitable for batch on-site test, the present invention provides one kind to be based on magnetic
The method that the nanogold colorimetric method of capture quickly detects the rugged Cronobacter sakazakii of slope, the technical solution adopted is as follows:
The purpose of the present invention is to provide a kind of nanogold colorimetric methods based on magnetic capture quickly to detect the rugged Cronobacter bar of slope
The method of bacterium, this method are to be crushed the bacterium in matrix, carry out PCR amplification after the DNA released with amino magnetic bead capture,
Amplified production is hybridized with the colloidal gold that DNA probe is modified, eventually by the direct naked eyes of variation for adding colloidal gold color after salt
Observe testing result.
The method of the invention includes the following steps:
1) the bacteria-containing somatic cells of institute in substance to be checked are subjected to break process and release DNA, obtained after abstraction impurity removal thick
DNA solution is mentioned,
2) activated amino magnetic bead is added in DNA solution to slightly mentioning for step 1), is incubated for capture and slightly mentions in DNA solution
DNA；
3) the specific PCR amplified reaction for establishing the rugged Cronobacter sakazakii of slope is carried out using the DNA that step 2) captures as template
PCR amplification, obtains PCR product；
4) DNA probe for designing the rugged Cronobacter sakazakii of slope, colloidal gold and DNA probe are incubated for, phosphoric acid is then added
Buffer carries out aging, obtains colloidal gold probe；
5) PCR product and step 4) obtained step 3) obtains colloidal gold probe and carries out bulk crossing, is then added
MgSO4Color change is observed after solution in 5min, if solution is still presented red and shows to contain the rugged Crow of slope in the substance to be detected
Promise bacillus shows in the substance to be detected if solution becomes bluish violet by red without containing the rugged Cronobacter sakazakii of slope.
Further, step 1) is will to be resuspended in the TE buffer solution that pH is 4-11 after substance to be checked centrifugation, addition
0 μ L-50 μ L Proteinase K is incubated for, and then carries out ice bath after high-temperature process at 90 DEG C -100 DEG C, then with volume ratio is 25:24:1
Phenol chloroform-isoamyl alcohol extracted, obtain and slightly mention DNA solution.Wherein centrifugal condition can for 4 DEG C, 6000g,
20min, the incubation conditions of Proteinase K can be 56 DEG C, 30min, and the high-temperature process time can be 10min, and the ice bath time can be with
For 8min.
Further, the pH of the TE buffer solution is 9.Effect is best under this condition.
Further, the additive amount of the Proteinase K is 30 μ L.Effect is best under this condition.
Further, the additive amount of step 2) the activated amino magnetic bead are as follows: every 10mL, which is slightly mentioned in DNA solution, to be added
The activated amino magnetic bead of 0.1mg.0.1mg, the magnetic bead of the activation of 5ml/mg are such as added into the DNA solution slightly mentioned.
Further, step 2) the incubation capture is that capture 15min-1h is incubated at 40 DEG C -80 DEG C.Wherein at 60 DEG C
Lower incubation 30min, effect are best.
Further, in the specific PCR amplified reaction of the rugged Cronobacter sakazakii of the step 3) slope primer pair nucleotide
Column are learned as shown in SEQ ID NO.1 and SEQ ID NO.2；The nucleotide of the DNA probe of the rugged Cronobacter sakazakii of the step 4) slope
Sequence is as shown in SEQ ID NO.3.
Further, the step 4) colloidal gold corresponds to the rugged Cronobacter sakazakii of 0.5nmol slope according to every 900 μ L colloidal gold
DNA probe is incubated for, and wherein the diameter of colloidal gold is 15nm.
Further, described be incubated for of step 4) is to be incubated for 22h under conditions of 50 DEG C, 150rpm.
Further, step 4) the addition phosphate buffer, which carries out aging, is: 100 μ L 0.1M phosphoric acid buffers are first added
Then liquid is slowly added to the phosphate buffer of 0.01M several times, reach the concentration of NaCl in final colloidal gold probe solution
0.7M, the wherein ingredient of 0.1M phosphate buffer are as follows: 0.1M Na2HPO4, 0.1M NaH2PO4, 0.1% (m/v) SDS, pH 8；
The ingredient of 0.01M phosphate buffer are as follows: 0.01M Na2HPO4, 0.01M NaH2PO4, 2M NaCl, 0.01% (m/v) SDS, pH
It is 8.
Further, the volume ratio of the step 5) colloidal gold probe and PCR product is 3:7-7:3.
Further, the volume ratio of the step 5) colloidal gold probe and PCR product is 5:5.
Further, the step 5) MgSO4The concentration of solution is 150mM-350mM, and additive amount is 5 μ L.
Further, MgSO described in step 5)4The concentration of solution is 300mM, and additive amount is 5 μ L.
Further, hybridization time described in step 5) is 5min.
Test substance of the present invention can be the rugged Cronobacter sakazakii pure culture of slope, cream or dairy products, every time required inspection
It is measured as 10mL.
The diameter of colloidal gold can only use 15nm in the present invention, if changing colloidal gold and the DNA spy of other other partial sizes into
Needle binding capacity will be greatly reduced.
The testing principle of the method for the present invention:
The principle of amino magnetic bead capture dna is that the amino group based on magnetic bead surfaces modification passes through electrostatic interaction and hydrogen
Key effect combines DNA；Colloidal gold colorimetric method is that nano gold sol that partial size is 5-20nm is utilized since surface plasma is total
Effect of shaking has apparent absorption peak, the aobvious red of solution colour under 520nm wavelength.Under high level salt solution, nanogold occurs
Red shift can occur for aggregation, absorption peak, and colloidal sol color can become purple even blue.It is visited when in nanogold surface markers target gene
Needle, probe will hybridize with DNA of pathogenic, to make under state with high salt, solution still takes on a red color.
The somatic cells of whole bacterium contained in substance to be detected are crushed first, are released DNA, so
It is obtained afterwards by impurity such as the preliminary removing protein of extraction, fat and slightly mentions DNA solution；Pass through hydrogen using the amino magnetic bead after activation
Key effect and electrostatic interaction capture slightly mention DNA present in DNA solution, and the DNA after capture is then carried out the rugged Crow of slope
The specific PCR amplified reaction of promise bacillus, the DNA will if containing the rugged Cronobacter sakazakii DNA of slope in the DNA of capture at this time
By specific amplification, specific amplification segment will not be obtained if not containing the rugged Cronobacter sakazakii DNA of slope in the DNA of capture；It is logical
It crosses and the DNA probe label of the rugged Cronobacter sakazakii of slope is obtained into colloidal gold probe on colloidal gold；Then by the production after PCR amplification
Object is hybridized with colloidal gold probe, if not containing the rugged Cronobacter sakazakii of slope in matrix to be checked, in the DNA of amino magnetic bead capture
The rugged Cronobacter sakazakii DNA of slope is not contained then, will not be expanded during the specific PCR amplified reaction of the rugged Cronobacter sakazakii of slope
Increase, i.e., the rugged Cronobacter sakazakii DNA of slope is not contained in pcr amplification product, in pcr amplification product and colloidal gold probe hybrid process
In, it can not be hybridized with colloidal gold probe since the rugged Cronobacter sakazakii DNA of slope is not present in system, Adlerika is added
Afterwards, colloidal gold can be assembled under high level salt solution, and red shift can occur for absorption peak, and colloidal sol color can become bluish violet (including indigo plant
Color or purple)；If containing the rugged Cronobacter sakazakii of slope in matrix to be checked, then contain the rugged Crow of slope in the DNA of amino magnetic bead capture
Promise bacillus DNA can be also amplified, i.e., in pcr amplification product during the specific PCR amplified reaction of the rugged Cronobacter sakazakii of slope
Containing the rugged Cronobacter sakazakii DNA of slope, in pcr amplification product and colloidal gold probe hybrid process, the rugged Cronobacter sakazakii DNA of slope with
It is complementary that colloidal gold probe carries out hybridization, so that colloidal gold probe is protected in situation with high salt, no after Adlerika is added
Colloidal gold precipitating can be generated, so that final testing result is red.During atual detection, it is possible that because of bacterium
Concentration is relatively low, and part colloidal gold precipitating occurs in aubergine phenomenon, or appear in PCR product and Adlerika
After addition, red can because of the slight thin out phenomenon of diluting effect, but these above-mentioned colors still can have with feminine gender it is more bright
Aobvious difference.
Since magnetic bead can capture out DNA in complicated matrix, and it is set to be separated by the magnetism of magnetic bead, because
And higher sensitivity can be realized in complicated newborn matrix；The PCR amplification primer of specificity determines that the method is good
Specificity；After the thallus DNA of amino magnetic bead capture carries out the PCR amplification of specificity, it may be implemented fastly by colloidal gold colorimetric method
The naked-eye observation of speed, colorimetric process is in 10min it is achieved that also avoiding the use of large-scale instrument simultaneously；This reality simultaneously
Test and directly DNA captured by magnetic bead, avoid the use of antibody in immunomagnetic beads method, greatly reduce experiment at
This, also avoids the difficulty for preparing antibody.The above speciality feature determines that the method can be widely applied to testing agency, enterprise
Industry.This detection technique has exploitation at the prospect of kit simultaneously, at the same it is also potential may be implemented Multiple detection, whole process consumption
When it is short, it is at low cost, may be implemented to visually observe, therefore also can satisfy the needs of grass-roots unit.Therefore, the method has good
Application prospect, and have the ability to bring considerable economic benefit.In terms of science, magnetic capture nucleic acid method and colloidal gold colorimetric method
Combination technology quickly detects the difficult disadvantage at high cost of Antibody preparation that Cronobacter sakazakii compensates for immune magnetic catching method, breaches
The problem of time-consuming for agarose gel electrophoresis method for detecting, instrument is complicated and reagent malicious using easily system, substantially reduces detection time,
It reduces costs.
The invention has the advantages that:
It exists in the prior art and is obtained using monoclonal antibody and carboxyl magnetic bead the progress covalent coupling of the rugged Cronobacter sakazakii of slope
The rugged Cronobacter sakazakii of immunomagnetic beads Direct Acquisition slope method, monoclonal antibody must be used just to be able to achieve thallus in this method
Capture, it is big that monoclonal antibody prepares difficulty, at high cost, and the method for the present invention is by using rugged gram of amino magnetic bead Direct Acquisition slope
The DNA of Luo Nuo bacillus is made by the hydrogen bond action and electrostatic interaction between amino magnetic bead and the rugged Cronobacter sakazakii DNA of slope
Target dna can be in complicated matrix by amino magnetic bead Direct Acquisition, and compared to existing method, the method for the present invention directly uses ammonia
Base magnetic capture bacterium DNA is not necessarily to any antibody, antibody is omitted, and overcomes Antibody preparation hardly possible, antibody in immune magnetic catching method
Problem at high cost, meanwhile, pretreatment process of the present invention only needs 1-2 hour, and increases bacterium before tradition and need 3-5 days, overcomes
Preceding increasing bacterium time of conventional method long drawback, substantially reduces the pre-treatment time.
Monoclonal antibody and carboxyl magnetic bead existing in the prior art using the rugged Cronobacter sakazakii of slope carries out covalent coupling
The method of the rugged Cronobacter sakazakii of adaptive immune magnetic bead Direct Acquisition slope, this method need to count or combine further combined with rubbing method
Final testing result can just be known by agarose gel electrophoresis after molecules amplification method, and the present invention uses colloidal gold ratio
Color method, before and after being assembled by nanogold caused by absorption peak generation red shift the characteristics of colloidal gold color change, it is only necessary to very
Clock can observe by the naked eye final color and determine, without not needing the testing staff of profession further combined with other operations
Final result can be obtained with equipment, and then realize the purpose quickly detected, solve time-consuming in traditional plate count method
The problem of long complex steps, also solves the large-scale instrument that complex and expensive is needed in common agarose gel electrophoresis method for detecting, together
When need using easily system poison coloring agent EB the problem of.
The method of the present invention does not have directly to use traditional preparation process during colloidal gold probe production plus salt aging
In common 0.8M or 1M final concentration, but the ultimate density of salt will be added to control in 0.7M, the colloidal gold probe property of acquisition
More stable, colour generation is more preferable.
In the prior art during preparing colloidal gold probe, it will usually 4-5h is incubated for after adding salt aging, to obtain
Stability is high, the better colloidal gold probe of colour generation, and the present invention has been surprisingly found that during the experiment, omits the incubation process, system
The colouring effect of standby colloidal gold probe is more preferable, overcomes in the prior art during preparing colloidal gold probe, it is necessary to
Add incubation 4-5h after salt aging that could obtain stability height, the prejudice of the good colloidal gold probe of colour generation.
The method of the present invention operating procedure is simple, is not necessarily to expensive reagent, low in cost, is suitable for batch detection.Side of the present invention
Method is suitable for the sample to be tested with complex matrices, such as milk powder, cow's milk etc., can effectively avoid in complex matrices it is complicated at
Fast enriching thallus DNA is realized in the influence divided.
The method of the present invention combines magnetic capture nucleic acid method and colloidal gold colorimetric method, establishes complete detection architecture, takes
It obtained unexpected technical effect: reaching 10 for the detection sensitivity of the rugged Cronobacter sakazakii of slope in pure culture1Cfu/mL,
The sensitivity of the rugged Cronobacter sakazakii of slope reaches 10 in cream2Cfu/mL, compared to the existing monoclonal with the rugged Cronobacter sakazakii of slope
The method sensitivity that antibody and carboxyl magnetic bead carry out the rugged Cronobacter sakazakii of covalent coupling adaptive immune magnetic bead Direct Acquisition slope improves
10 times；The method of the present invention step is simple simultaneously, and specificity is good, and the used time is shorter, and entire detection architecture can within short 5 hours
To be fully completed, detection time is substantially reduced, may be implemented quickly to detect；The present invention provides one for the detection of pathogenic bacteria
New way becomes the new technology that can substitute traditional detection method.
Detailed description of the invention
The pH value that Fig. 1 is TE buffer determines；
(M, DNA Marker D2000；0, blank control；1, pH 4；2, pH 5；3, pH 6；4, pH 7；5, pH 8；6,
pH 9；7, pH 10；8, pH 11).
Fig. 2 is the determination of Proteinase K additive amount；
(M, DNA Marker D2000；0, blank control；1,0 μ L；2,10 μ L；3,20 μ L；4,30 μ L；5,40 μ L；6,
50μL)。
Fig. 3 is the determination for capturing incubation time；
(M, DNA Marker D2000；1,15min；2,30min；3,1h；4,2h；5,3h；6,4h；7,5h).Fig. 4 is to catch
Obtain the optimization of incubation temperature；
(M, DNA Marker D2000；0 DEG C of Isosorbide-5-Nitrae；2,50 DEG C；3,60 DEG C；4,70 DEG C；5,80 DEG C；6,90 DEG C).Fig. 5 is
MgSO4The determination of concentration.
Fig. 6 is that colloidal gold probe and PCR product volume ratio determine.
Fig. 7 is the pure bacterium solution testing result of various concentration；
(M, DNA Marker；1-8, bacterial concentration are respectively 3.5 × 107CFU/mL, 3.5 × 106CFU/mL, 3.5 ×
105CFU/mL, 3.5 × 104CFU/mL, 3.5 × 103CFU/mL, 3.5 × 102CFU/mL, 3.5 × 101CFU/mL, 3.5 ×
100CFU/mL)。
Fig. 8 is the bacterium solution testing result of various concentration in newborn system；
(M, DNA Marker；1-8, bacterial concentration are respectively 3.5 × 108CFU/mL, 3.5 × 107CFU/mL, 3.5 ×
106CFU/mL, 3.5 × 105CFU/mL, 3.5 × 104CFU/mL, 3.5 × 103CFU/mL, 3.5 × 102CFU/mL, 3.5 ×
101CFU/mL)。
Fig. 9 is specific test testing result；
(M, DNA Marker；1-3,3 plants of rugged Cronobacter sakazakiis of slope；4, clostridium perfringen ATCC 13048；5, cloaca intestines bar
Bacterium ATCC 13047；6, Escherichia coli CMCC B4413；7, Listeria monocytogenes CMCC 54006).
Specific embodiment
The present invention will be further described combined with specific embodiments below, but the present invention should not be limited by the examples.
Embodiment 1:
1 materials and methods
1.1 main bacterial strains
This experiment bacterial strain uses therefor is as shown in table 1 below:
1 strain information of table
Slope rugged Cronobacter sakazakii reference culture ATCC 29544, ATCC 29004, ATCC 12868, clostridium perfringen ATCC
13048, enterobacter cloacae ATCC 3503, Escherichia coli CMCC B4413, Listeria monocytogenes CMCC 54006 are purchased from respectively
American Type Culture collection warehousing ATCC and Chinese medicine Culture Collection CMCC.
1.2 experimental method
1.2.1 the culture of bacterial strain
Take the rugged Cronobacter sakazakii ATCC29544 of slope as reference culture, the rugged Cronobacter sakazakii reference strain of 2 plants of slopes and other
4 plants of non-rugged Cronobacter sakazakii difference activation cultures of slope are in NB fluid nutrient medium, and 37 DEG C of overnight incubations are to get in logarithm life
The rugged Cronobacter sakazakii bacterium solution of long-term slope, to be used for subsequent experimental.
1.2.2 DNA in amino magnetic bead Acquisition Detection system
It takes 10mL measuring samples to be centrifuged 20min in 4 DEG C of revolving speeds with 6000g, precipitating is resuspended in containing 5% (w/v)
In the 500 μ L TE buffer (10mM contains 1.0mM EDTA, pH 9) of Triton X-100,30 μ L albumen are added thereto
Enzyme K is incubated for 30min under the conditions of 56 DEG C, boils rapid ice bath 8min after 10min, then pass through phenol chloroform-isoamyl alcohol
(volume ratio 25:24:1) is extracted, the DNA solution slightly mentioned.
Amino magnetic bead used in this experiment is purchased from Luoyang Hui Er nanosecond science and technology Co., Ltd, and activation process is to specifications
Described in: please bead suspension is mixed well before use, is taken 100 μ l bead suspensions (containing magnetic bead 1mg), in Magneto separate abandoning
Clearly；2. 1ml PBS (0.01M, pH 7.4), which is added, washs magnetic bead, repeated washing is primary；In above-mentioned 200 μ l bead suspension,
800 μ l 25% (g/g) glutaraldehydes are added, under room temperature, the concussion activation 1h on sample mixed instrument；Magneto separate abandons supernatant；
It is cleaned magnetic bead 3 times of activation with 1ml PBS；The magnetic bead of activation is dispersed in 200 μ l PBS, the magnetic bead activated suspends
Liquid.
0.1mg is added into the DNA solution slightly mentioned, the magnetic bead of the activation of 5ml/mg is incubated at a temperature of 60 DEG C and shakes
30min after being cleaned three times with TE buffer, is resuspended 100 μ L and is used for subsequent detection.
1.2.3 the foundation of PCR reaction system
PCR reaction system, primers F 3 and B3 sequence are established for the rugged Cronobacter sakazakii ESA_04347-ESA_04349 gene of slope
Column are shown in Table 2.PCR takes 5 μ L amplified productions after reaction, carries out 1% agarose gel electrophoresis.PCR reaction system such as table 3:
2 PCR primer of table
3 PCR reaction system of table
Reaction cycle parameter is as follows: 94 DEG C initial denaturation 3 minutes,
1.2.4 the preparation of colloidal gold probe
For test DNA probe nucleotide sequence used as shown in SEQ ID NO.3, particular sequence is (5 ' -3 ')
AAAAAAAAAATCGTGCTGCGAGTTTGAGAGACTCTGACACACCGCG.By 900 μ L colloidal golds and 0.5nmolDNA probe
At 50 DEG C, 0.1M phosphate buffer (0.1M Na is transferred to after being incubated for 22h under the conditions of 150rpm2HPO4, 0.1M NaH2PO4,
0.1% (m/v) SDS, pH 8), it is slowly added to 0.01M phosphate buffer (the 0.01M Na that pH is 8 several times2HPO4, 0.01M
NaH2PO4, 2M NaCl, mass concentration is the SDS of 0.01% (m/v)) and aging is carried out, until in final colloidal gold probe solution
NaCl concentration reaches 0.7M, is centrifuged 30min after aging with the revolving speed of 20000g, supernatant is removed, twice by washing of precipitate, to remove
The DNA probe being not bound with is removed, precipitating is finally resuspended in the 0.01M phosphate buffer (0.01M that the pH of 100 μ L is 8
Na2HPO4, the NaH of 0.01M2PO4, the NaCl of 0.7M, the SDS of 0.01% (m/v)) in.
1.2.5 colloidal gold colorimetric method observes testing result
The colloidal gold probe for taking 5 μ L pcr amplification products to prepare with 5 μ L mixes, and stands 5min at room temperature, then to system
The middle MgSO that 5 μ L, 300mM are added4Solution stands observing colloid gold color change after 5min at room temperature.
1.2.6 magnetic catches the sensitivity of the rugged Cronobacter sakazakii of DNA association colloid gold colorimetric determination slope
It takes 1mL to be in the rugged Cronobacter sakazakii pure culture liquid of slope of logarithmic growth phase, is carried out with 0.85% (m/v) physiological saline
10 times of gradient dilutions 100-108Times, and dilution-plate method is used to calculate initial bacterial concentration as 3.5 × 108CFU/mL.With
It states method the rugged Cronobacter sakazakii of the slope of various concentration is captured and expanded respectively, 5 μ L amplified productions is taken to pass through colloidal gold ratio
Color method observation experiment result.
1.2.7 magnetic catches the rugged Cronobacter sakazakii of slope in DNA association colloid gold colorimetric determination artificial contamination's milk powder
Milk powder is purchased from local supermarket, confirms before artificial contaminated bacteria samples through National Standard Method and does not contain the rugged Cronobacter sakazakii of slope.According to GB/
478940 2010, it takes 25g powdered milk sample to be put into the sterile homogenizing bag for filling 225mL aqua sterilisa, 1:10 dilution is made.Add
Add 108CFU/mL-101The pure bacterium solution 1mL of the rugged Cronobacter sakazakii of slope of CFU/mL difference dilution respectively in 9 groups of 9mL dilutions,
The rugged Cronobacter sakazakii concentration of slope is 10 after homogeneous7CFU/mL-100CFU/mL, in aforementioned manners respectively in artificial contamination's milk powder
The bacterium solution of various concentration is captured and is expanded, and 5 μ L amplified productions is taken to pass through colloidal gold colorimetric method observation experiment result.
2 results and analysis
The determination of 2.1 TE buffer pH value
This test has carried out gradient optimizing, 8 groups of examinations based on method described in 1.2.2, for the pH value of wherein TE buffer
The pH value tested is respectively 4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0, then expands respectively to the DNA after capture
And agarose gel electrophoresis.
As a result as shown in Figure 1, electrostatic interaction may be influence that amino magnetic bead and genomic DNA combine it is main because
Element, when pH is equal to or more than 10, electrophoretic band is very dark, shows that only a small amount of DNA is integrated on amino magnetic bead.Occur this
As a result the reason of may be due to when the pH in solution is higher than the isoelectric point of amino magnetic bead (9.6) amino magnetic bead surface with negative
Charge cannot attract DNA in connection, therefore faint electrophoretic band can only occur when pH is equal to or more than 10.When solution
When pH is lower than the isoelectric point of amino magnetic bead, amino magnetic bead is positively charged to be tightly combined with negatively charged DNA.It can from figure
Out, when pH is closer to isoelectric point, band is brighter, thus pH be 9 be optimal TE buffer pH value.
The determination of 2.2 Proteinase K additive amounts
This test has carried out gradient optimizing, 6 groups of examinations based on method described in 1.2.2, for the additive amount of wherein Proteinase K
The Proteinase K additive amount tested is respectively 0 μ L, 10 μ L, 20 μ L, 30 μ L, 40 μ L, 50 μ L, then is expanded respectively the DNA after capture
Increasing and agarose gel electrophoresis.
As a result as shown in Fig. 2, electrophoretic band is most bright when the additive amount of Proteinase K is 30 μ L.When additive amount is less than
When 30 μ L, protein removal in matrix it is insufficient, therefore can suppression PCR to a certain extent.When Proteinase K additive amount is greater than
When 30 μ L, proteinase K digestion is be easy to cause to be not thorough, since Proteinase K itself is also a kind of protein, equally understands suppression PCR
Effect, therefore 30 μ L are selected as the additive amount of optimum protein enzyme K.
2.3 capturing the determination of incubation time
This test has carried out gradient optimizing, 7 groups of tests based on method described in 1.2.2, for wherein capture incubation time
Capture incubation time be respectively 15min, 30min, 1h, 2h, 3h, 4h, 5h, then respectively to the DNA after capture carry out amplification and
Agarose gel electrophoresis.
As a result as shown in figure 3, in 15min, in the capture time of 30min and 1h, electrophoretic band clearly becomes clear, and is being greater than 1h
Capture time in band present as the time increases gradually dimmed variation, it is too long for generating the possible cause of this phenomenon
Time causes amino magnetic bead that oxidation reaction has occurred.To reduce detection time, while considering the complexity of milk powder matrix, it is final to select
Selecting 30min is the best capture time.
The determination of 2.4 capture incubation temperatures
This test has carried out gradient optimizing, 5 groups of tests based on method described in 1.2.2, for wherein capture incubation temperature
Capture incubation temperature be respectively 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, then respectively to the DNA after capture carry out PCR amplification and
Agarose gel electrophoresis.
As a result as shown in figure 4, electrophoretic band is most bright under 60 DEG C of incubation temperature.60 DEG C are changed to from 40 DEG C in temperature
When, it may be since the more double-stranded DNAs of higher temperature trigger are opened that electrophoretic band, which obviously gradually brightens, another may
The reason of be the movement that amino magnetic bead and DNA molecular are promoted due to high temperature, so that magnetic bead and DNA molecular is easier to collide and is combined.
And when temperature is greater than 60 DEG C, electrophoresis strip had it is apparent dimmed, this is because amino magnetic bead is aoxidized in hot conditions,
Cause the loss of part magnetism.Therefore 60 DEG C are had finally chosen as best incubation temperature.
2.5 MgSO4The determination of concentration
This test is based on method described in 1.2.4, for wherein MgSO4Concentration has carried out gradient optimizing, 8 groups of tests
MgSO4Concentration is respectively 50mM, 100mM, 150mM, 250mM, 300mM, 350mM, 400mM, 500mM, then passes through negative and sun
The color contrast of property control group judges optium concentration.As a result as shown in figure 5, MgSO4When concentration is 50mM-150mM, positive pipe
Red is presented with negative tube, produces false positive results；In MgSO4Concentration is 150mM-250mM and 350mM-400mM
When, blue is presented in negative tube, and red is presented in positive pipe, but the color contrast degree of two pipes is not enough obviously clear to；In MgSO4Concentration
When for 500mM, bluish violet is presented in negative tube and positive pipe, produces false negative result；And in MgSO4Concentration is 300mM
When, positive pipe presents distinct red and negative tube presents the apparent hyacinthine of comparison, therefore finally chooses 300mM and be
MgSO4Optium concentration.
The determination of 2.6 colloidal gold probe solution and PCR product volume ratio
This test is based on method described in 1.2.4, for wherein colloidal gold probe solution and PCR product volume ratio (totally 10 μ
L gradient optimizing) has been carried out, the volume ratio of 9 groups of tests is respectively 9:1,8:2,7:3,6:4,5:5,4:6,3:7,2:8,1:9, then
Optium concentration is judged by negative and positive controls color contrast.As a result as shown in fig. 6, volume ratio is positive in 6:4-9:1
Property the red of pipe the trend gradually become shallower as is presented, and the blue of negative tube is also more and more shallow, and moves closer to red, two Guan Yan
Colour contrast is not obvious enough；Volume ratio in 4:6-1:9, gradually deepen by the red of positive pipe, the final false negative shape that purple is presented
State；And when colloidal gold probe solution and PCR product volume ratio are 5:5, the red of positive pipe and the hyacinthine pair of negative tube
It is more obvious than, therefore finally choosing 5:5 is best colloidal gold probe solution and PCR product volume ratio.
2.7 magnetic catch the sensitivity of the rugged Cronobacter sakazakii of DNA association colloid gold colorimetric determination slope
As seen from Figure 6, the rugged Cronobacter sakazakii pure culture concentration of slope is 107CFU/mL-101When CFU/mL, final detection
As a result positive pink is presented, and reaches 10 in pure culture concentration0When CFU/mL.Final testing result starts to show indigo plant
Purple, the magnetic that this experiment is established are caught DNA association colloid gold colorimetric method and are limited to the detection of the rugged Cronobacter sakazakii pure culture of slope
3.5×101CFU/mL。
2.8 magnetic catch the rugged Cronobacter sakazakii of slope in DNA association colloid gold colorimetric determination artificial contamination's milk powder
From fig.7, it can be seen that the concentration of the rugged Cronobacter sakazakii of slope is 10 in artificial contamination's milk powder7CFU/mL-102When CFU/mL most
Red is presented in whole solution, and when bacterial concentration reaches 3.5 × 101Solution starts that bluish violet is presented when CFU/mL, this experiment is established
Magnetic catch DNA association colloid gold colorimetric method 3.5 × 10 be limited to the detection of the rugged Cronobacter sakazakii of slope in artificial contamination's milk powder2CFU/
mL.Although the result shows that the method can effectively reduce influence of the newborn mesostroma to PCR, but fat cannot be released completely
Protein etc. is to the inhibiting effect of PCR, but the method greatly reduces detection time, before too long in conventional method
Increase the bacterium time, the quick detection development of pathogenic bacteria in dairy products is made great sense.
2.9 magnetic catch the specificity of the rugged Cronobacter sakazakii of DNA association colloid gold colorimetric determination slope
The present embodiment mainly has chosen the closer bacterium of Cronobacter sakazakii affiliation, i.e., on the Evaluation on specificity of antibody
Clostridium perfringen ATCC 13048, enterobacter cloacae ATCC 13047, Escherichia coli CMCC B4413, Listeria monocytogenes CMCC
54006, while having chosen the Cronobacter sakazakii of three plants of ATCC preservations.As seen from Figure 8, the centrifuge tube of three plants of rugged Cronobacter sakazakiis of slope
Pink is presented.Other the 4 plants non-rugged Cronobacter sakazakiis of slope are negative as a result, bluish violet, table is presented in centrifuge tube color
Bright the method specificity is good.
3 conclusions
The rugged Cronobacter sakazakii of slope can cause serious neonatal meningitis, enterocolitis and bacteremia, and the death rate is up to
50 or more percent, therefore the prevention and detection of Cronobacter sakazakii rugged for slope are very important.Traditional detection method
The longer preceding increasing bacterium time is needed, it is cumbersome.Commonly although immune magnetic separation technique shortens detection time, but antibody
The problems such as preparation is difficult, at high cost equally limits the development of fast inspection technology.And molecular biology inspection relatively popular at present
Most of survey technology needs to observe testing result with agarose gel electrophoresis, and this method needs large-scale instrument, and time-consuming, needs simultaneously
It to use and easily make malicious reagent etc..This experiment is special to the capture characteristic of nucleic acid and the colour developing of colloidal gold colorimetric method using amino magnetic bead
Property, detection time can be greatly shortened, and can complete directly to observe the naked eyes of testing result in 10 minutes, realize for
Simple, the quick detection of the rugged Cronobacter sakazakii of slope.
PH value of the present invention for the TE buffer during magnetic capture nucleic acid, the additive amount of Proteinase K, capture are incubated
It educates the time, capture incubation temperature is optimized, and determines that the pH value of TE buffer is 9, the additive amount of Proteinase K is 30 μ L, is caught
Obtaining incubation time is 30min, and capture incubation temperature is 60 DEG C.This experiment simultaneously also optimizes during colloidal gold colorimetric method
MgSO4Concentration and colloidal gold probe and PCR product volume ratio, determine MgSO4Concentration be 300mM, colloidal gold probe and
The volume ratio 5:5 of PCR product.This experiment detection sensitivity is up to 101Cfu/mL, and with clostridium perfringen, enterobacter cloacae,
Escherichia coli, Listeria monocytogenes no cross reaction.Detection for the rugged Cronobacter sakazakii of slope in artificially contaminated milk, can reach
102Cfu/mL provides a kind of efficient, high specific method for the detection of the rugged Cronobacter sakazakii of slope in cream.
Although the present invention has been disclosed in the preferred embodiment as above, it is not intended to limit the invention, any to be familiar with this
The people of technology is not departing from spirit and scope of the invention, can do various change and modification, therefore, guarantor of the invention
Shield range should subject to the definition of the claims.
Sequence table
<110>Northeast Agricultural University
<120>a kind of method that the nanogold colorimetric method based on magnetic capture quickly detects the rugged Cronobacter sakazakii of slope
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<160> 3
<170> PatentIn version 3.5
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Claims (10)
1. a kind of method that the nanogold colorimetric method based on magnetic capture quickly detects the rugged Cronobacter sakazakii of slope, which is characterized in that packet
Include following steps:
1) the bacteria-containing somatic cells of institute in substance to be checked are subjected to break process and release DNA, obtained after abstraction impurity removal and slightly mention DNA
Solution,
2) activated amino magnetic bead is added in DNA solution to slightly mentioning for step 1), is incubated for the thick DNA mentioned in DNA solution of capture；
3) the specific PCR amplified reaction for establishing the rugged Cronobacter sakazakii of slope carries out PCR as template using the DNA that step 2) captures
Amplification obtains PCR product；
4) DNA probe for designing the rugged Cronobacter sakazakii of slope, colloidal gold and DNA probe are incubated for, phosphoric acid buffer is then added
Liquid carries out aging, obtains colloidal gold probe；
5) PCR product and step 4) obtained step 3) obtains colloidal gold probe and carries out bulk crossing, and MgSO is then added4It is molten
Color change is observed after liquid in 5min, if solution is still presented red and shows to contain the rugged Cronobacter sakazakii of slope in the substance to be detected,
Show in the substance to be detected if solution becomes bluish violet by red without containing the rugged Cronobacter sakazakii of slope.
2. the method according to claim 1, wherein it is 4- that step 1), which is by pH is resuspended in after substance to be checked centrifugation,
In 11 TE buffer solution, 0 μ L-50 μ L Proteinase K of addition is incubated for, and is then carried out after high-temperature process at 90 DEG C -100 DEG C
Ice bath, then extracted with phenol chloroform-isoamyl alcohol that volume ratio is 25:24:1, it obtains and slightly mentions DNA solution.
3. the method according to claim 1, wherein the additive amount of step 2) the activated amino magnetic bead
Are as follows: every 10mL slightly mentions the addition activated amino magnetic bead of 0.1mg in DNA solution.
4. the method according to claim 1, wherein step 2) the incubation capture is incubated at 40 DEG C -80 DEG C
Educate capture 15min-1h.
5. the method according to claim 1, wherein the step 4) colloidal gold is according to every 900 μ L colloidal gold pair
The rugged Cronobacter sakazakii DNA probe of 0.5nmol slope is answered to be incubated for, wherein the diameter of colloidal gold is 15nm.
6. the method according to claim 1, wherein step 4) the addition phosphate buffer progress aging is:
100 μ L0.1M phosphate buffers are first added, are then slowly added to the phosphate buffer of 0.01M several times, visit final colloidal gold
The concentration of NaCl reaches 0.7M in needle solution, wherein the ingredient of 0.1M phosphate buffer are as follows: 0.1M Na2HPO4, 0.1M
NaH2PO4, 0.1% (m/v) SDS, pH 8；The ingredient of 0.01M phosphate buffer are as follows: 0.01M Na2HPO4, 0.01M
NaH2PO4, 2M NaCl, 0.01% (m/v) SDS, pH 8.
7. the method according to claim 1, wherein the volume of step 5) colloidal gold probe and PCR product
Than for 3:7-7:3.
8. the method according to claim 1, wherein the step 5) MgSO4The concentration of solution is 150mM-
350mM, additive amount are 5 μ L.
9. the method according to claim 1, wherein hybridization time described in step 5) is 5min.
10. the method according to claim 1, wherein the specific PCR of the rugged Cronobacter sakazakii of the step 3) slope
The nucleotide of primer pair is arranged as shown in SEQ ID NO.1 and SEQ ID NO.2 in amplified reaction；The rugged Crow of the step 4) slope
The nucleotide sequence of the DNA probe of promise bacillus is as shown in SEQ ID NO.3.
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| CN117230069A (en) * | 2023-08-16 | 2023-12-15 | 江南大学 | Nucleic acid aptamer capable of specifically recognizing Cronobacter and application thereof |
| CN117434263A (en) * | 2023-08-16 | 2024-01-23 | 江南大学 | 3D-DNA walker aptamer sensor based on magnetic separation and Cronobacter sakazakii detection method |
| CN117230069B (en) * | 2023-08-16 | 2024-05-31 | 江南大学 | Nucleic acid aptamer capable of specifically recognizing Cronobacter and application thereof |
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Application publication date: 20190409 |