CN102108375A - Method for detecting bacteria - Google Patents
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- CN102108375A CN102108375A CN 201010593081 CN201010593081A CN102108375A CN 102108375 A CN102108375 A CN 102108375A CN 201010593081 CN201010593081 CN 201010593081 CN 201010593081 A CN201010593081 A CN 201010593081A CN 102108375 A CN102108375 A CN 102108375A
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Abstract
The invention relates to the field of biotechnology, and discloses a method for detecting bacteria, which comprises the following steps of: providing a control bacterium sample; fully reacting a solid support fixed with more than one lectin with a sample to be detected and the control bacterium sample respectively, and washing to remove the uncombined sample from the solid support; fully reacting the washed solid support with lectin-modified gold nanoparticles, and washing to remove uncombined gold nanoparticles from the solid support; and detecting resonance light scattering signals of the gold nanoparticles combined to the solid support, comparing with resonance light scattering signals of the control bacterium sample, and analyzing the type of the bacterium in the sample to be detected. The method for detecting the bacteria has the advantages of universality, high sensitivity, low consumption of samples, and high stability, and can realize qualitative and semi-quantitative analysis of the bacteria. The invention also provides lectin-modified gold nanoparticles with high stability and good biocompatibility, and a kit for detecting bacteria, which is simple in preparation method and convenient to use.
Description
Technical field
The present invention relates to biological technical field, relate to detection method and a kind of Phytoagglutinin modified golden nanometer particle and a kind of test kit that is used for Bacteria Detection that is used for Bacteria Detection of a kind of bacterium specifically.
Background technology
Current society, the viral infection disease becomes worse, and threatens greatly but infectation of bacteria remains one of human health.For example the death that causes of tuberculosis still occupies the No. 1 in the world, and the sign that stages a comeback is arranged; Salmonellas, listeria spp and the intestinal bacteria etc. in food source are the important factors that causes diarrhoea; Cause that several cause of diseases of pneumonia such as Klebsiella pneumonia, pneumococcus, Chlamydia pneumoniae, mycoplasma pneumoniae etc. often constitute very big threat to lower for the resistance, old or young crowd; Infection of staphylococcus aureus is plain stubborn, and resistance is very general; The encephalitis coccus threatens bigger to the still very imperfect children of resistibility.Along with the continuous enhancing of bacterial drug resistance, dimly visible and food contamination of bioterrorism etc. influences daily life, and the threat of transmissible disease more and more is subjected to people's attention, so the development of effective method of detecting bacterium is significant to public health.
The detection method of bacterium is varied, the ordinary method that division bacteria is at present identified mainly depends on microbial culture, dyeing and microscopic examination technology, but bacterium need separate, cultivation and a series of biological chemistry detect and make this method (Manual of Clinical Microbiology very consuming time, 9th ed., Am.Soc.Microbiol.:Washington, D.C., 2007).In addition, also have nucleic acid (dna marker probe, PCR etc.) technology and antibody test (ELISA enzyme linked immunosorbent detection, immunofluorescence etc.) technology etc., but aforesaid method belongs to man-to-man detecting pattern, promptly a kind of label probe can only detect a kind of bacterium.Therefore, demand developing the detection technique of bacterium identification efficiently urgently.
Biochip is the important tool of present genomics, proteomics research.Biochip is with its high-throughput, sensitivity, characteristic of accurate, be applied to more and more the dynamics research that bacterium rapid detection and bacterium sugar group learns (Nat.Chem.Biol., 2006,2,153-157).But at present, biochip is a detection means with fluorescence mainly, remains in certain shortcoming at aspects such as detection sensitivity, selectivity, thus the detection novel method of development biochip to its great significance (Nature, 2007,4,437-444).
Summary of the invention
In view of this, the present invention seeks to provide the detection novel method of the higher bacterium of a kind of sensitivity and resolution at conventional detection sensitivity and the not good enough defective of selectivity.
For realizing purpose of the present invention, the present invention adopts following technical scheme:
The detection method of a kind of bacterium may further comprise the steps:
Step 1: the contrast bacteria samples is provided;
Step 2: the solid support that is fixed with more than one lectins fully acts on testing sample and contrast bacteria samples respectively, and unconjugated sample on the solid support is removed in washing;
Step 3: solid support after the washing and Phytoagglutinin modified golden nanometer particle fully act on, and unconjugated golden nanometer particle on the solid support is removed in washing;
Step 4: detect the resonant light scattering signal of bonded golden nanometer particle on the solid support, compare, analyze the testing sample bacterial species with bacterium standard substance resonant light scattering signal.
Bacterium surface is covered with multiple different sugar conjugate, as glycoprotein, glycolipid, glycosaminoglycan and protein-polysaccharide etc., and the kind of different bacterium surface coverage glycosyl is incomplete same, as the intestinal bacteria surface coverage sialic acid is arranged, Fucose, terminal acetylamino galactosamine, α-semi-lactosi, glycosyls such as terminal acetyl glucosamine and α-seminose, and it is different with intestinal bacteria, the subtilis surface is removed and is coated with sialic acid, Fucose, terminal acetylamino galactosamine, α-semi-lactosi, outside the glycosyls such as terminal acetyl glucosamine and α-seminose, also there is glucose-β-1,4 glucose oligopolymer.
Lectin (lectin) is the glycoprotein or the sugared albumen of combination of purifying from each kind of plant, invertebrates and higher animal, can discern glycosyl sequence specific in monose or the oligosaccharides, and combination with it, be usually used in the separation and purification of oligosaccharides and saccharide complex and the structural analysis of sugar chain.The aggegation of finding at present have kind more than 100, and commonly used is that (Phytoagglutin, PNA), with its plant that is extracted name, as concanavalin A, wheat germ element, peanut agglutinin and soybean agglutinin etc., lectin is their general name to phytohemagglutinin usually.A kind of lectin can be discerned one or more specific glycosyl sequences, and combination with it.Can discern semi-lactosi-β (1 as ricinus agglutinin I, 4) glucose (Gal β (1,4) GlcNAc β 1) glycosyl, can specific recognition terminal acetylamino galactosamine (Terminal GalNAc) glycosyl of soybean agglutinin, wheat germ agglutinin can specific recognition β-acetylglucosamine, sialic acid and acetylamino galactosamine glycosyl (β-GlcNAc, sialic acid, GalNAc).
The detection method of bacterium of the present invention is fixed with more than one different types of lectins on solid support, utilize lectin-glucide to interact and discern bacterium with different glycosyls, to be fixed on the solid support with lectin bonded bacterium, with Phytoagglutinin modified golden nanometer particle as the bacterium marker, utilize the resonant light scattering signal of resonant light scattering technology for detection bacterium surface golden nanometer particle, compare with the resonant light scattering signal that contrasts bacteria samples by resonant light scattering signal then testing sample, analyze the testing sample bacterial species, realize the qualitative analysis of bacterium.Wherein, Phytoagglutinin modified golden nanometer particle is that golden nanometer particle directly or indirectly combines the golden nanometer particle that has lectin that forms with lectin.
Lectin of the present invention can be in the lectin of finding at present any one.Wherein, as preferably, described lectin is ricinus agglutinin I (Ricinus Communis Agglutinin), Albizzia kalkora amurensin lectin I and II (Maackia Amurensis Agglutinin), Ulex europaeus lectin I (Ulex Europaeus Agglutinin), soybean agglutinin (Soybean Agglutinin), cockscomb sweet viburnum lectin (Erythrina Cristagalli Lectin), Williams Elder Twig lectin (Sambucus Nigra Lectin), BSLi I and II (Griffonia simplicifolia), wheat germ agglutinin (Wheat Germ Agglutinin), concanavalin A (Conconvalina), Aleuria lectin (Aleuria Aurantia Lectin), daffadowndilly's lectin (Narcissus Pseudonarcissus Lectin), stramonium lectin (Datura stramonium Lectin), lens culinaris agglutinin (Lens Culinaris Agglutinin) or peanut agglutinin (Phaseolus Vulgaris Agglutinin).
Distinguishing features such as resonant light scattering technology (RLS) is the new instrument analysis technology that in recent years develops rapidly, and is highly sensitive because of it, simple to operate and widespread use.Along with the environmental change around the golden nanometer particle, the intensity difference of its resonant light scattering signal thus can bacterial detection surface golden nanometer particle bonding strength.Because of golden nanometer particle is rapid to the reacting condition of surrounding environment, thus the sensitivity and the accuracy height that detect, so this method also can realize the semi-quantitative analysis of bacterium, and can be applicable to detect the bacterium under the varying environment.
As preferably, described golden nanometer particle radius is 10nm～13nm.
Preferably, also comprise the enhancing signal step before the described detection resonant light scattering signal, with further raising detection sensitivity.
As preferably, described enhancing signal step is specially and silver enhancement solution reaction 5～10min, 18.2M Ω water washing, wherein, described silver enhancement solution is that the silver enhancement solution A that U.S. Sigma (Sigma) company produces mixed gained in 1: 1 by volume with silver enhancement solution B, and promptly system is promptly used.
18.2M Ω water of the present invention is meant absolute pure water.M Ω is meant resistivity of water, and the parameter of expression high purity water water quality refers to that cross-sectional area is 1cm under a certain temperature
2Length is the resistance of the water of 1cm, and its unit is * centimetre (Ω * CM) of ohm, and the high more salt that shows of resistivity is few more.Absolute pure water is 18.2M Ω * CM 25 ℃ theoretical value, and measured value is relevant with temperature, and temperature is high more, and resistivity is low more, otherwise high more.
The present invention also provides a kind of Phytoagglutinin modified golden nanometer particle that is used for Bacteria Detection, and golden nanometer particle directly or indirectly combines with lectin.
Golden nanometer particle of the present invention and lectin can combine in several ways, comprise that golden nanometer particle combines the Phytoagglutinin modified golden nanometer particle of formation with lectin by directly or indirectly forming ionic linkage, covalent linkage or coordinate bond.
The present invention also provides a kind of preparation method of Phytoagglutinin modified golden nanometer particle, comprising:
Step 1: the reaction of golden nanometer particle and polypeptide, obtain peptide modified golden nanometer particle, described polypeptide be Cys-Ala-Leu-Asn-Asn and
Mol ratio is 9: 1 a mixture;
Step 2: peptide modified golden nanometer particle and avidin reaction obtain the avidin gold nano-particles modified;
Step 3: vitamin H and lectin react, and obtain the lectin of biotin modification;
Step 4: the lectin reaction of avidin gold nano-particles modified and biotin modification obtains Phytoagglutinin modified golden nanometer particle.
Polypeptide mixture of the present invention be Cys-Ala-Leu-Asn-Asn (cystyl alanyl leucyl asparaginyl l-asparagine) and
(cystyl alanyl leucyl asparaginyl asparaginyl glycyl lysyl (vitamin H) glycine) mol ratio is 9: 1 a mixture.
Cys-Ala-Leu-Asn-Asn and gold surface have very strong affinity, can form fine and close protective layer, make golden nanometer particle water-soluble, and can maintaining a long-term stability property.
Avidin is a kind of glycoprotein, can be by extracting in the egg white, each molecule is made up of 4 subunits, can with 4 intimate combinations of biotin molecule, the key that both form in conjunction with the back is stable to be difficult to fracture.Avidin-vitamin H is the most stable title complex of finding at present.
The present invention is with avidin and peptide modified golden nanometer particle covalent attachment, utilize the stable coordinate bond that forms between avidin and vitamin H again, combine with the lectin of biotin modification, lectin is connected on the peptide modified golden nanometer particle, obtain Phytoagglutinin modified golden nanometer particle, can be used as marker specific recognition glucide, be applied to the identification and the detection of glucide.The lectin of biotin modification of the present invention is the GS-II lectin of commercially available biotin modification in a specific embodiments.
Preferably, described golden nanometer particle radius is 10nm～13nm.
The present invention also provides the Phytoagglutinin modified golden nanometer particle that utilizes above-mentioned preparation method's preparation.
The present invention also provides a kind of test kit that is used for Bacteria Detection, comprise Phytoagglutinin modified golden nanometer particle and lectin biochip, described lectin biochip is fixed on the solid support by the lectin with more than one and obtains, and described Phytoagglutinin modified golden nanometer particle is above-mentioned direct or indirect and lectin bonded golden nanometer particle.
The lectin biochip belongs to glucide biochip, is a kind of very promising Measurement for Biotechnique that grows up after gene chip, protein chip, organization chip etc., has that the test sample consumption is few, high-throughput and a specificity advantages of higher.
In one embodiment, described lectin biochip is 4 * 4 16 subarrays of arranging, and wherein each subarray comprises 4 binding sites.
As preferably, described solid support is a slide.According to the present invention, slide is preferably silica glass sheet well known in the art or organosilicon semi-conductor slide, and the present invention does not do qualification, and the finishing of described slide has epoxide group, can with lectin generation covalent reaction.
The preparation method who is used for the lectin biochip of Bacteria Detection of the present invention, for getting different types of lectin point sample on solid support, 25 ℃～37 ℃ leave standstill 4h～16h, obtain the lectin biochip.
Preferably, the described preparation method who is used for the lectin biochip of Bacteria Detection also comprises washing and sealing step.Described sealing is to react 1h～2h at 25 ℃～37 ℃ with lock solution, to seal the not combining site of lectin, to reduce the influence of non-specific binding background.Contain skim-milk, calf serum etc. in the confining liquid commonly used.
As preferably, lock solution of the present invention is the phosphate buffered saline buffer that comprises 40mmol/L～60mol/L bovine serum albumin, 0.1mol/L～0.2mol/L thanomin and 0.15mol/L～0.2mol/L sodium-chlor.
In one embodiment, the described lectin biochip preparation process that is used for Bacteria Detection is: with 16 kinds of different lectin speckings in 4 * 4 epoxy slides of arranging 16 subarrays, each subarray comprises 4 binding sites, respectively the identical lectin of specking.Afterwards specking lectin slide 25 ℃～37 ℃ the reaction 4h～16h, phosphate buffer soln washing slide, be soaked in then in the lock solution of the phosphate buffer soln that contains 40mM～60mM bovine serum albumin (BSA), 0.1M～0.2M thanomin and 0.15M～0.2M sodium-chlor, 25 ℃～37 ℃ are reacted 1h～2h down, make the lectin biochip.
In a specific embodiments, the present invention serves as the contrast bacteria samples with intestinal bacteria, subtilis, enterobacter cloacae and staphylococcus aureus, utilize the detection method of bacterium of the present invention to detect resonant light scattering finger printing and the strength of signal that contrasts bacteria samples and testing sample respectively, by comparing testing sample and the resonant light scattering finger printing and the strength of signal that contrast bacteria samples, the bacterial species of qualitative analysis testing sample.
In a specific embodiments, the present invention also detects intestinal bacteria, subtilis, enterobacter cloacae and the staphylococcus aureus of different concns respectively with described detection method, and the result shows that intestinal bacteria, staphylococcus aureus and subtilis detection are limited to 10
3, enterobacter cloacae is surveyed and is limited to 10
4, and its resonant light scattering finger printing of the contrast bacteria samples of different concns is similar, and resonant light scattering strength of signal difference shows that the detection method test sample consumption of bacterium of the present invention is few, and the detection sensitivity height can be realized the bacterium semi-quantitative analysis.
The detection method of bacterium of the present invention has versatility, and highly sensitive, few, the good stability of sample consumption, can realize the qualitative and semi-quantitative analysis of bacterium.High, the good biocompatibility of Phytoagglutinin modified golden nanometer particle stability that is used for Bacteria Detection of the present invention, the preparation method is simple, quick.The test kit preparation method who is used for Bacteria Detection of the present invention is simple, easy to use, is fit to extensively promote and use.
Description of drawings
Fig. 1 shows lectin biochip microarray Pareto diagram, and the lectin of corresponding numbering representative sees Table 1;
Fig. 2 shows the colibacillary resonant light scattering finger printing of embodiment 6 contrast bacteria samples;
Fig. 3 shows the colibacillary resonant light scattering intensity map of embodiment 6 contrast bacteria samples;
Fig. 4 shows the resonant light scattering finger printing of embodiment 6 contrast bacteria samples subtilises;
Fig. 5 shows the resonant light scattering intensity map of embodiment 6 contrast bacteria samples subtilises;
Fig. 6 shows the resonant light scattering finger printing of embodiment 6 contrast bacteria samples enterobacter cloacaes;
Fig. 7 shows the resonant light scattering intensity map of embodiment 6 contrast bacteria samples enterobacter cloacaes;
Fig. 8 shows the resonant light scattering finger printing of embodiment 6 contrast bacteria samples staphylococcus aureus;
Fig. 9 shows the resonant light scattering intensity map of embodiment 6 contrast bacteria samples staphylococcus aureus;
Figure 10 shows the resonant light scattering finger printing of embodiment 6 testing samples;
Figure 11 shows the resonant light scattering intensity map of embodiment 6 testing samples;
Figure 12 shows the colibacillary resonant light scattering finger printing of embodiment 7 different concns, and e. coli concentration is respectively: a, 10
6, b, 10
5, c, 10
4, d, 10
3, e, 10
2
Embodiment
The embodiment of the invention discloses a kind of bacterium detection method, be used for the Phytoagglutinin modified golden nanometer particle and preparation method thereof of Bacteria Detection and be used for the test kit of Bacteria Detection.Those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as being included in the present invention.Product of the present invention and method are described by preferred embodiment, the related personnel obviously can be in not breaking away from content of the present invention, spirit and scope to product as herein described with method is changed or suitably change and combination, realize and use the technology of the present invention.
In order further to understand the present invention, the present invention is described in detail below in conjunction with embodiment.
Embodiment 1: the preparation of lectin biochip
Make and handle the lectin biochip in the SmartArrayer48 chip manufacturing system that provides by Beijing Bo Ao Bioisystech Co., Ltd.The lectin specking that to get 16 kinds of concentration be 1mg/mL reacts 4h under 25 ℃～37 ℃ vacuum conditions on epoxy group modified slide, the glycosyl of lectin kind and specific recognition thereof sees Table 1.Lectin biochip microarray Pareto diagram is seen Fig. 1.Be that 0.05% tween 20,0.15mol/L NaCl and pH value are 8.0 50mol/L phosphate buffer soln washing slide with containing volumetric concentration then, again with the phosphate buffer soln that contains 60mol/L bovine serum albumin (BSA), 0.1mol/L thanomin and 0.2mol/L sodium-chlor, 25 ℃～37 ℃ are reacted 1h down, and the sealing chip obtains the lectin biochip.Be array point that obtains and the activity that keeps biomolecules, it is 15% glycerine that sampling liquid contains volumetric concentration, 10mol/L monose, and 0.15mol NaCl and pH value are 8.0 50mol/L phosphate buffer soln.
The specificity combination of table 1. lectin and corresponding glycosyl
Embodiment 2: the preparation of Phytoagglutinin modified golden nanometer particle
Cys-Ala-Leu-Asn-Asn and
The mixing solutions of 9: 1 in molar ratio preparation polypeptide mixture, the Trisodium Citrate gold nano-particles modified is mixed with mol ratio with the polypeptide mixing solutions at 1: 50000, behind room temperature reaction 1h～2h,, obtain peptide modified golden nanometer particle by centrifugal purified reaction product.Peptide modified golden nanometer particle and avidin react 1h～2h at ambient temperature, and centrifugal afterwards purified reaction product obtains the avidin gold nano-particles modified.(available from Vector Laboratory Ltd., USA) reaction 1h～2h behind centrifugal purified reaction product, obtains Phytoagglutinin modified golden nanometer particle to the GS II lectin of avidin gold nano-particles modified and biotin modification.Phytoagglutinin modified golden nanometer particle is dispersed in 2～8 ℃ of preservations down in the phosphate buffer soln.
Embodiment 3: the preparation of Phytoagglutinin modified golden nanometer particle
Cys-Ala-Leu-Asn-Asn and
The mixing solutions of 9: 1 in molar ratio preparation polypeptide mixture, the Trisodium Citrate gold nano-particles modified is mixed with mol ratio with the polypeptide mixing solutions at 1: 50000, behind room temperature reaction 1h～2h,, obtain peptide modified golden nanometer particle by centrifugal purified reaction product.Peptide modified golden nanometer particle and avidin react 1h～2h at ambient temperature, and centrifugal afterwards purified reaction product obtains the avidin gold nano-particles modified.Vitamin H and RCA120 react, and get the lectin of biotin modification.The lectin reaction 1h～2h of avidin gold nano-particles modified and biotin modification behind centrifugal purified reaction product, obtains Phytoagglutinin modified golden nanometer particle.Phytoagglutinin modified golden nanometer particle is dispersed in 2～8 ℃ of preservations down in the phosphate buffer soln.
Embodiment 4: the test kit that is used for Bacteria Detection
A kind of test kit that is used for Bacteria Detection comprises according to the lectin biochip of embodiment 1 described preparation method's preparation and the Phytoagglutinin modified golden nanometer particle for preparing according to embodiment 2 described preparation methods.
Embodiment 5: the test kit that is used for Bacteria Detection
A kind of test kit that is used for Bacteria Detection comprises according to the lectin biochip of embodiment 1 described preparation method's preparation and the Phytoagglutinin modified golden nanometer particle for preparing according to embodiment 3 described preparation methods.
Embodiment 6: the detection of testing sample
1, the cultivation of contrast bacteria samples and testing sample:
Contrast bacteria samples intestinal bacteria (DH5 α, available from Beijing ancient cooking vessel state biotechnology limited liability company), subtilis (Bacillus subtilis), enterobacter cloacae (Enterobacter cloacae, from Chinese common micro-organisms culture presevation administrative center), staphylococcus aureus (Staphyloccocus aureus Rosenbach, from Sino-Japanese Party Hospital, Jilin Univ.) and after testing sample cultivates 12h～16h respectively, centrifugal under 4000g～5000g, clean with phosphoric acid salt, repeat 2～4 times, each 5～8min, being dispersed in afterwards and making concentration in the phosphate buffer soln is 10
6Bacteria suspension.
2, the detection of contrast bacteria samples and testing sample:
The lectin biochip that embodiment 1 prepares be 10 with concentration respectively
6The contrast bacteria samples and testing sample at 30 ℃ of reaction 2h, phosphate buffer soln cleans not in conjunction with sample, centrifuge dripping is 2 * 10 with the concentration that embodiment 2 prepares then
-9The Phytoagglutinin modified solution of gold nanoparticles of mol/L is reacted 1h at 30 ℃, phosphate buffer soln cleans not joining gold nanoparticle, centrifuge dripping, drip on the solid support promptly make after promptly the silver enhancement solution of usefulness makes it cover the golden nanometer particle mark fully, reaction 10min is with the flushing of 18.2M Ω water.Utilize chrominance arrays scanner (U.S. TeleChem.International Inc. company) to detect the respective resonant light scattering signal, utilize Image J computed in software resonant light scattering strength of signal then.Corresponding resonant light scattering signal identification finger printing and resonant light scattering intensity are shown in Fig. 2～11.When the resonant light scattering strength of signal less than 3000 or strength of signal during less than three times of signal standards deviations, the resonant light scattering signal is covered by background, may detect less than fingerprint signal on the resonant light scattering finger printing.
Fig. 2 is contrast bacteria samples intestinal bacteria resonant light scattering finger printing, wherein, other matrix all has fingerprint signal except that 11 and 12 work song matrixes, wherein 1,7 and 8 work song matrix fingerprint signals are the strongest, 6,10 and 14 work song matrix fingerprint signals are very faint, and other submatrix fingerprint signal intensity is placed in the middle.
Fig. 4 is contrast bacteria samples subtilis resonant light scattering finger printing, and wherein, 5,11,12,13 and 15 work song matrixes do not detect fingerprint signal, and other submatrix fingerprint signal is all stronger.
Fig. 6 is contrast bacteria samples enterobacter cloacae resonant light scattering finger printing, wherein, 5,11,13 and 15 work song matrixes do not detect fingerprint signal, and 4,6,8,9,10,12,14 and 16 work song matrix fingerprint signal intensity are very weak, and 1,2,3 and 7 work song matrix fingerprint signals are all stronger.
Fig. 8 is contrast bacteria samples staphylococcus aureus resonant light scattering finger printing, wherein, 4,5,8,13,14,15 and 16 work song matrixes do not detect fingerprint signal, and 9 work song matrix fingerprint signal intensity other submatrix head and shoulders above, though 10 work song matrixes detect fingerprint signal but be very faint, 1,2,3,6,7 and 12 work song matrix fingerprint signals are stronger than 10 work song matrix fingerprint signals.
Figure 10 is a testing sample resonant light scattering finger printing, and wherein, 11,12 and 14 work song matrixes do not detect fingerprint signal, and 6 work song matrix fingerprint signals are very weak, and other submatrix fingerprint signal intensity is placed in the middle.
Compare with the resonant light scattering finger printing of described contrast bacteria samples intestinal bacteria, subtilis, enterobacter cloacae and staphylococcus aureus, the result shows that the resonant light scattering finger printing of testing sample and described colibacillary resonant light scattering finger printing are comparatively approaching, only is that strength of signal is slightly different.As seen from Figure 11, testing sample resonant light scattering intensity map is consistent with intestinal bacteria intensity map trend, therefore judges that testing sample is intestinal bacteria.
Utilize conventional microbial culture, dyeing and microscopic examination that testing sample is detected, the result shows that testing sample is intestinal bacteria, and is consistent with detection method result of the present invention.
Embodiment 7: the detection of different concns bacterium
1, the cultivation of different concns bacterium:
Contrast bacteria samples intestinal bacteria (DH5 α, available from Beijing ancient cooking vessel state biotechnology limited liability company), subtilis (Bacillus subtilis), enterobacter cloacae (Enterobacter cloacae, from Chinese common micro-organisms culture presevation administrative center), staphylococcus aureus (Staphyloccocus aureus Rosenbach, from Sino-Japanese Party Hospital, Jilin Univ.) cultivate 12h～16h after, centrifugal under 4000g～5000g, clean with phosphoric acid salt, repeat 2～4 times, each 5～8min, being dispersed in afterwards and making concentration in the phosphate buffer soln is 10
6, 10
5, 10
4, 10
3With 10
2Bacteria suspension.
2, the detection of different concns bacterium:
The lectin biochip that embodiment 1 prepares and the contrast bacteria samples of different concns are at 30 ℃ of reaction 1h, and phosphate buffer soln cleans and centrifuge dripping, and the concentration with embodiment 3 preparations is 2 * 10 then
-9The Phytoagglutinin modified solution of gold nanoparticles of mol/L is reacted 2h at 30 ℃, and phosphate buffer soln cleans and centrifuge dripping.Drip and to make promptly promptly the silver enhancement solution of usefulness makes on its solid support that covers mark contrast bacteria samples fully, reaction 5min is with the flushing of 18.2M Ω water.Utilize the chrominance arrays scanner to detect the corresponding resonant light scattering identification of the contrast bacteria samples finger printing of different concns.
The result shows that intestinal bacteria, staphylococcus aureus and subtilis detect and be limited to 10
3, linearity range is 4 orders of magnitude, enterobacter cloacae is surveyed and is limited to 10
4, linearity range is 3 orders of magnitude.Figure 12 is the colibacillary resonant light scattering identification finger printing of different concns.
The explanation of above embodiment just is used for helping to understand method of the present invention and core concept thereof.Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention, can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of claim of the present invention.
Claims (9)
1. the detection method of a bacterium may further comprise the steps:
Step 1: the contrast bacteria samples is provided;
Step 2: the solid support that is fixed with more than one lectins fully acts on testing sample and contrast bacteria samples respectively, and unconjugated sample on the solid support is removed in washing;
Step 3: solid support after the washing and Phytoagglutinin modified golden nanometer particle fully act on, and unconjugated golden nanometer particle on the solid support is removed in washing;
Step 4: detect the resonant light scattering signal of bonded golden nanometer particle on the solid support, compare, analyze the testing sample bacterial species with contrast bacteria samples resonant light scattering signal.
2. according to the described method of claim 1, it is characterized in that described lectin is ricinus agglutinin I, Albizzia kalkora amurensin lectin, Ulex europaeus lectin, soybean agglutinin, cockscomb sweet viburnum lectin, Williams Elder Twig lectin, BSLi, wheat germ agglutinin, concanavalin A, Aleuria lectin, daffadowndilly's lectin, stramonium lectin, lens culinaris agglutinin or peanut agglutinin.
3. according to the described method of claim 1, it is characterized in that described golden nanometer particle radius is 10nm～13nm.
4. according to the described method of claim 1, it is characterized in that, also comprise the enhancing signal step before the described detection resonant light scattering signal.
5. according to the described method of claim 4, it is characterized in that described enhancing signal step is specially and silver enhancement solution reaction 5～10min, 18.2M Ω water washing.
6. a Phytoagglutinin modified golden nanometer particle that is used for Bacteria Detection is characterized in that, golden nanometer particle directly or indirectly combines with lectin.
7. the preparation method of a Phytoagglutinin modified golden nanometer particle comprises:
Step 1: the reaction of golden nanometer particle and polypeptide, obtain peptide modified golden nanometer particle, described polypeptide be Cys-Ala-Leu-Asn-Asn and
Mol ratio is 9: 1 a mixture;
Step 2: peptide modified golden nanometer particle and avidin reaction obtain the avidin gold nano-particles modified;
Step 3: vitamin H and lectin react, and obtain the lectin of biotin modification;
Step 4: the lectin reaction of avidin gold nano-particles modified and biotin modification obtains Phytoagglutinin modified golden nanometer particle.
8. the Phytoagglutinin modified golden nanometer particle of the described preparation method of claim 7 preparation.
9. a test kit that is used for Bacteria Detection comprises claim 6 or 8 described Phytoagglutinin modified golden nanometer particle and lectin biochips, and described lectin is fixed on the solid support by the lectin with more than one and obtains.
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| CN102944549A (en) * | 2012-11-23 | 2013-02-27 | 清华大学 | Electrogenerated chemiluminescence bacterium sensing method and multi-functional probe |
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| CN102944549A (en) * | 2012-11-23 | 2013-02-27 | 清华大学 | Electrogenerated chemiluminescence bacterium sensing method and multi-functional probe |
| CN102944549B (en) * | 2012-11-23 | 2015-04-15 | 清华大学 | Electrogenerated chemiluminescence bacterium sensing method and multi-functional probe |
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