CN102121042A - Method for identifying pathogenic bacteria of drinking water by using surface enhanced Raman spectroscopy - Google Patents
Method for identifying pathogenic bacteria of drinking water by using surface enhanced Raman spectroscopy Download PDFInfo
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- CN102121042A CN102121042A CN 201010579950 CN201010579950A CN102121042A CN 102121042 A CN102121042 A CN 102121042A CN 201010579950 CN201010579950 CN 201010579950 CN 201010579950 A CN201010579950 A CN 201010579950A CN 102121042 A CN102121042 A CN 102121042A
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Abstract
The invention relates to a surface enhanced Raman spectroscopy identification method for pathogenic bacteria of drinking water. The method comprises the following steps of: detecting three specified pathogenic bacteria, namely enterococcus faecalis, pseudomonas aeruginosa and clostridium perfringens by adopting gold or silver nano sol as an enhancement reagent and using surface enhanced Raman spectroscopy, and performing cluster analysis on the detection results to realize identification of the pathogenic bacteria of the drinking water. The method is high in sensitivity, good in selectivity, easiness in operation, high in detection result and low in cost, can realize large-scale online detection of the pathogenic bacteria of the drinking water, and is widely applicable to the technical fields of food safety, environment monitoring and the like.
Description
Technical field
The present invention relates to the detection method of a kind of tap water pathogenic bacterium, relate in particular to a kind of method of utilizing surface enhanced Raman spectroscopy and cluster analysis that the tap water pathogenic bacterium are differentiated.
Background technology
In the water quality monitoring (WQM)s such as source water, Urban water supply and tap water, the detected result of polluting indicator is normally estimated the important indicator of water body quality.Coliform generally is stored in the enteron aisle of people and other warm-blooded animal, propagates by the enteron aisle movement, can cause disease, therefore, generally assesses the situation that water quality is subjected to fecal pollution by the quantity that detects coliform in the national standard.About the quantity that studies show that coliform and the actual state of fecal pollution do not have good dependency, in various fresh water and ocean water, the disease that streptococcus faecium causes has surpassed coliform but in recent years.For this reason, replaced the GB8537-2008 " natural mineral water " of GB 8537-1995 in microbiological indicator, to increase streptococcus faecium (Enterococcus faecalis), Pseudomonas aeruginosa Pseudomonas aeruginosa), the detection of clostridium perfringens (Clostridium perfringens), in GB/T 8538-2008 " the natural mineral water method of inspection ", all adopt filter membrane method to measure above three kinds of pathogenic bacterium simultaneously.This testing process comprises culture experiment and confirmatory test, reliable results, but complicated operation, and sense cycle is longer.
The existing conventional detection techniques of food microorganism comprises that increasing bacterium, bacterium colony repeatedly separates and multiple biochemistry and serology identification experiment, or realize by polymerase chain reaction (PCR).Though these detection methods are very professional and classical, but its step complexity, consuming time longer, the cost height, and its topmost weak point is to increase bacterium in advance, and determine that from being sampled to qualitative still is the quantitative identification microorganism, the time of subsistence level more than one day, therefore be difficult to adapt to the modern food production of develop rapidly and the needs of circulation.
Summary of the invention
The objective of the invention is to propose the surface enhanced Raman spectroscopy discrimination method of a kind of tap water pathogenic bacterium, it is highly sensitive, easy handling, and detection speed is fast, and is with low cost, can realize large-scale online detection, thereby overcome deficiency of the prior art.
For achieving the above object, the present invention has adopted following technical scheme:
The surface enhanced Raman spectroscopy discrimination method of a kind of tap water pathogenic bacterium, it is characterized in that, this method is: adopt gold or silver-colored Nano sol as strengthening reagent, with surface enhanced Raman spectroscopy the tap water pathogenic bacterium are detected, through detected result is carried out cluster analysis, realize discriminating to the tap water pathogenic bacterium.
Say that further this method is specially:
Drink at least a activation bacterial strain standard model of making in the water pathogenic bacterium, and detect with Raman spectrum after will activating bacterial strain standard model and gold or silver-colored Nano sol thorough mixing, detected result carried out cluster analysis, and differentiate the kind of tap water pathogenic bacterium thus thereafter;
Described tap water pathogenic bacterium comprise streptococcus faecium, Pseudomonas aeruginosa and clostridium perfringens;
The usage quantity of described gold or silver-colored Nano sol and the consumption of pathogenic bacterium are at 1 mmole: 100 viable counts～1 mmole: between 30000 viable counts.
This method comprises the steps:
(1) preparation of activation bacterial strain standard model
At least a bacterial classification of drinking in the water pathogenic bacterium is streak culture on the LB solid medium, cultivate 24-48h down for 30-37 ℃, with the single bacterium colony of aseptic inoculation ring picking dispersive, place distilled water to vibrate, then at 5000-10000rmp centrifugal treating 5-30min, centrifugal sediment is made activation bacterial strain standard model after washing, described tap water pathogenic bacterium comprise streptococcus faecium, Pseudomonas aeruginosa and clostridium perfringens;
(2) Raman spectrum detects
Raman spectrum surface is strengthened reagent and above-mentioned activation bacterial strain standard model with 1 mmole: 100 viable counts～1 mmole: the usage ratio of 30000 viable counts is mixed, is vibrated, and makes both abundant combinations, then detects with Raman spectrum;
(3) data processing
The Raman spectrum detected result of standard model is carried out cluster analysis, differentiate the kind of tap water pathogenic bacterium thus.
The condition determination of Raman spectrometer is made as in the step (2): laser power scope: 20-300mw, sweep time: 2-20s.
Activation bacterial strain standard model is 2-60s with the time that strengthens reagent mix, vibration in the step (2).
Be to differentiate tap water pathogenic bacterium kind in the step (3) by each peak-to-peak value in the Raman spectrogram of comparison tap water pathogenic bacterium.
The preparation method of described gold nano colloidal sol is:
It is to boil under 105-135 ℃ the condition that 0.30-1.50mg/mL potassium chloraurate solution is inserted temperature, and the sodium citrate solution concentration of adding is 0.1wt%-10wt%, boils 5-95min, makes gold nano colloidal sol.
Say that further this method comprises following concrete steps:
(1) preparation of activation bacterial strain standard model
The bacterial classification of getting streptococcus faecium, Pseudomonas aeruginosa and clostridium perfringens is streak culture on the LB solid medium respectively, cultivate 24-48h down for 30-37 ℃, with the single bacterium colony of aseptic inoculation ring picking dispersive, place distilled water to vibrate, then at 5000-10000rmp centrifugal treating 5-30min, centrifugal sediment is made three kinds of activation bacterial strain standard models after washing;
(2) Raman spectrum detects
The Raman spectrum surface is strengthened reagent mix, vibrate, make both abundant combinations, then detect with Raman spectrum with above-mentioned activation bacterial strain standard model;
(3) data processing
The Raman spectrum detected result of each standard model is carried out cluster analysis, set up the cluster analysis figure that can be used for differentiating and distinguishing the kind of tap water pathogenic bacterium thus;
(4) with reference to the operation of step (2), detect more than one unknown bacterial classification samples with Raman spectrum, the Raman spectrum detected result and step (3) the gained cluster analysis figure of gained Raman spectrum detected result and the above-mentioned standard model of step (2) gained are compared, thereby identify the kind of unknown bacterial classification.
Description of drawings
Fig. 1 is the surface enhanced Raman spectroscopy comparison diagram of three kinds of tap water pathogenic bacterium;
Fig. 2 is the cluster analysis figure of the 24 strain bacteria samples Raman spectrum data of the unknown;
I shown in the above figure is that streptococcus faecium (Enterococcus faecalis), II are that Pseudomonas aeruginosa (Pseudomonas aeruginosa), III are clostridium perfringens (Clostridium perfringens).
Embodiment
This case contriver is through studying for a long period of time and putting into practice discovery, because different bacterial species is different aspect their cellular biochemistry composition, each molecule all has a characteristic spectrum, can detect the feature hertzian wave of molecule by Raman spectrometer.Utilize Raman spectrum can obtain in the complete viable cell direct evidence of 4 kinds of main biomacromolecule structures and variation thereof simultaneously, comprise the phosphoric acid skeleton of nucleic acid, ribodesose (or ribose) and base: proteinic master, side chain; Trans and the guache conformation of the fatty acid carbon chain hydrogen chain of adipose membrane and the carbohydrate of various not isomorphic maps etc.Thereby, can just can identify the kind of bacterium according to their Raman spectrum.
According to above-mentioned discovery, this case contriver has proposed the tap water pathogenic bacterium discrimination method based on surface enhanced Raman spectroscopy, and its technical scheme is roughly as follows:
(1) preparation of microbiological specimens
Activated drinking water pathogenic bacterium bacterial strain washs thalline after the cultivation in distilled water, make activated drinking water pathogenic bacterium bacterial strain standard model, treats sample introduction after finishing.
(2) optimization of enhancing reagent preparation condition
The present invention can choose gold nano colloidal sol, silver-colored Nano sol or other similar reagents as strengthening reagent.Wherein, gold nano colloidal sol and silver-colored Nano sol can adopt the known method preparation of those skilled in the art.For example, gold nano colloidal sol can adopt the method preparation of Trisodium Citrate reduction hydrochloro-auric acid or its esters, and silver-colored Nano sol can adopt with NaBH
4The method preparation of reduction soluble silver salt.
(3) setting of Raman spectrometer sampling condition
(4) collection of tested bacterial classification Raman spectrum
Enhancing reagent in the activation bacterial strain standard model of preparation in the step (1) and the step (2) is mixed in proper ratio (this ratio is advisable to obtain preferably the surperficial enhancing rate of Raman spectrum), vibration is in conjunction with certain hour (bacterium and nanoparticle to sample fully are combined into suitable), measure according to step (3) among placing Raman spectrometer, the spectrogram that scanning obtains is converted to after treatment and can treats the follow-up data processing for the data sheet of data processing.
(5) data processing
The data of Raman spectrogram are carried out cluster analysis, differentiate the tap water pathogenic bacterium of different genera by each peak-to-peak value of Raman spectrogram of each tap water pathogenic bacterium standard model.
Aforesaid step can be preferably respectively:
The preparation of microbiological specimens: bacterial classification is streak culture on the LB solid medium, cultivates 24-48h down for 30-37 ℃, with the single bacterium colony of aseptic inoculation ring picking dispersive, place distilled water to vibrate, the centrifugal 5-30min of 5000-10000rmp, triplicate operation, then, finish with water washing.
Strengthen the optimization of reagent preparation condition: adopt nanometer gold as strengthening reagent, oil bath is warming up to about 105-135 ℃ and adds 0.30-1.50mg/mL potassium chloraurate solution and wait to seethe with excitement, the sodium citrate solution concentration that adds is 0.1%-10% (mass percent), and boiling the reaction times is 5-95min.
Sampling condition: the Raman spectrometer condition determination is set at: laser power scope: 20-300mw, sweep time: 2-20 second.Each bacteria samples is 1 with gold sol mixed volume ratio: 1-1: 5, and the two mixing time is consistent, and is 2-60s.
Data processing method: adopt SPSS (SPSS Inc.), SAS software (SAS Institute Inc.) or other similar softwares to the Raman spectrum diagram data by cluster analysis, thereby carry out the discriminating of tap water pathogenic bacterium.Below in conjunction with accompanying drawing and some preferred embodiments technical scheme of the present invention is elaborated, but the present invention is not limited only to following embodiment.
Embodiment 1: utilize surface enhanced Raman spectroscopy to differentiate three kinds of tap water pathogenic bacterium: streptococcus faecium (Enterococcus faecalis), Pseudomonas aeruginosa (Pseudomonas aeruginosa), clostridium perfringens (Clostridium perfringens), and its process is:
The frozen bacterial classification inoculation of getting above-mentioned three kinds of pathogenic bacterium activates to the TSB centrifuge tube, activate 24h at least at 30-37 ℃, treat the TSB muddiness, be seeded in the LB solid medium streak culturely, cultivate 24h～48h at 37 ℃, with the single bacterium colony of aseptic inoculation ring picking dispersive, place the distilled water of centrifuge tube to vibrate, in the centrifugal 5min of refrigerated centrifuge 8500rmp, repeat more than 3 times, get precipitation and finish with water washing and treat sample introduction.Get according to the gold nano colloidal sol 500 μ L of aforementioned optimal conditions preparation and the aforementioned thalline standard model of 300 μ L respectively at mixing 10s in the sample introduction bottle, Raman is measured 250mw, 10s obtains spectrogram, according to the every strain bacteria samples of aforesaid operations, collect collection of illustrative plates, carry out data processing and obtain Fig. 1, among this figure, 500cm
-1About the peak be the S-S key stretching vibration of protein peptide class; 1330cm
-1About the peak should be the characteristic peak of VITAMIN B4---be similar to the spectrogram of DNA; 1125cm
-1The appearance of characteristic peak shows that nano particle may enter the sex change that causes DNA behind the bacteria cell wall; All the other most peaks may be protein in the cell walls, peptide class and amino acid; The strongest 730cm
-1About the peak belong to NAG composition in the peptidoglycan structure, also might be the frequency multiplication peak of FAD; Streptococcus faecium and clostridium perfringens are gram-positive microorganisms, and its cell wall structure is similar, and be thicker more coarse than Pseudomonas aeruginosa cell walls, by 1330cm
-1The intensity at peak matches; Pseudomonas aeruginosa is a Gram-negative bacteria, and its cell walls is thinner than positive bacteria, and the vibration peak intensity of its lipid bilayer is less.
Embodiment 2: utilize surface enhanced Raman spectroscopy to differentiate unknown tap water pathogenic bacterium sample
Present embodiment is got 24 unknown strain tap water pathogenic bacterium samples sample introduction respectively according to the method described above, collect gained Raman spectrum collection of illustrative plates, spectroscopic data is carried out the SPSS software processes obtain cluster analysis result, again the spectrogram and the cluster analysis result of spectroscopic data and cluster analysis result and aforesaid standards sample are compared, and determined that thus wherein label is 1,10,21,24 sample, 2,3,4,6,7,13,14,15,16,17,21,22 sample, 5,8,11,12,18,19,20,23 sample is respectively streptococcus faecium, clostridium perfringens and Pseudomonas aeruginosa, thus realized the evaluation of unknown tap water pathogenic bacterium sample kind.
Certainly, the process of present embodiment is only in order to illustrate technical scheme of the present invention, those skilled in the art are according to enlightenment of the present invention, also can be easy to expect other various bacteria except that above-mentioned three kinds of tap water pathogenic bacterium are distinguished with similar method and identified.But all those skilled in the art are because of technology involved in the present invention enlightenment, replace or technical scheme that the equivalent deformation mode forms all drops in protection scope of the present invention and adopt to be equal to.
Claims (8)
1. the surface enhanced Raman spectroscopy discrimination method of tap water pathogenic bacterium, it is characterized in that, this method is: adopt gold or silver-colored Nano sol as strengthening reagent, with surface enhanced Raman spectroscopy the tap water pathogenic bacterium are detected, through detected result is carried out cluster analysis, realize discriminating to the tap water pathogenic bacterium.
2. the surface enhanced Raman spectroscopy discrimination method of tap water pathogenic bacterium according to claim 1 is characterized in that, this method is specially:
Drink at least a activation bacterial strain standard model of making in the water pathogenic bacterium, and detect with Raman spectrum after will activating bacterial strain standard model and gold or silver-colored Nano sol thorough mixing, detected result carried out cluster analysis, and differentiate the kind of tap water pathogenic bacterium thus thereafter;
Described tap water pathogenic bacterium comprise three kinds of pathogenic bacterium of regulation among the GB 8537-2008 " natural mineral water ": streptococcus faecium (Enterococcus faecalis), Pseudomonas aeruginosa (pseudomonas aeruginosa), clostridium perfringens (Clostridium perfringens);
The usage quantity of described gold or silver-colored Nano sol and the consumption of pathogenic bacterium are at 1 mmole: 100 viable counts～1 mmole: between 30000 viable counts.
3. the surface enhanced Raman spectroscopy discrimination method of tap water pathogenic bacterium according to claim 1 and 2 is characterized in that this method comprises the steps:
(1) preparation of activation bacterial strain standard model
At least a bacterial classification of drinking in the water pathogenic bacterium is streak culture on the LB solid medium, cultivate 24-48h down for 30-37 ℃, with the single bacterium colony of aseptic inoculation ring picking dispersive, place distilled water to vibrate, then at 5000-10000rmp centrifugal treating 5-30min, centrifugal sediment is made activation bacterial strain standard model after washing, described tap water pathogenic bacterium comprise streptococcus faecium, Pseudomonas aeruginosa and clostridium perfringens;
(2) Raman spectrum detects
Raman spectrum surface is strengthened reagent and above-mentioned activation bacterial strain standard model with 1 mmole: 100 viable counts～1 mmole: the usage ratio of 30000 viable counts is mixed, is vibrated, and makes both abundant combinations, then detects with Raman spectrum;
(3) data processing
The Raman spectrum detected result of standard model is carried out cluster analysis, differentiate the kind of tap water pathogenic bacterium thus.
4. the surface enhanced Raman spectroscopy discrimination method of tap water pathogenic bacterium according to claim 3 is characterized in that, the condition determination of Raman spectrometer is made as in the step (2): laser power scope: 20-300mw, sweep time: 2-20s.
5. the surface enhanced Raman spectroscopy discrimination method of tap water pathogenic bacterium according to claim 3 is characterized in that, activation bacterial strain standard model is 2-60s with the time that strengthens reagent mix, vibration in the step (2).
6. the surface enhanced Raman spectroscopy discrimination method of tap water pathogenic bacterium according to claim 3 is characterized in that, in the step (3) is to differentiate tap water pathogenic bacterium kind by each peak-to-peak value in the Raman spectrogram of comparison tap water pathogenic bacterium.
7. the surface enhanced Raman spectroscopy discrimination method of tap water pathogenic bacterium according to claim 1 and 2 is characterized in that, the preparation method of described gold nano colloidal sol is:
It is to boil under 105-135 ℃ the condition that 0.30-1.50mg/mL potassium chloraurate solution is inserted temperature, and the sodium citrate solution concentration of adding is 0.1wt%-10wt%, boils 5-95min, makes gold nano colloidal sol.
8. the surface enhanced Raman spectroscopy discrimination method of tap water pathogenic bacterium according to claim 3 is characterized in that, this method comprises following concrete steps:
(1) preparation of activation bacterial strain standard model
The bacterial classification of getting streptococcus faecium, Pseudomonas aeruginosa and clostridium perfringens is streak culture on the LB solid medium respectively, cultivate 24-48h down for 30-37 ℃, with the single bacterium colony of aseptic inoculation ring picking dispersive, place distilled water to vibrate, then at 5000-10000rmp centrifugal treating 5-30min, centrifugal sediment is made three kinds of activation bacterial strain standard models after washing;
(2) Raman spectrum detects
The Raman spectrum surface is strengthened reagent mix, vibrate, make both abundant combinations, then detect with Raman spectrum with above-mentioned activation bacterial strain standard model;
(3) data processing
The Raman spectrum detected result of each standard model is carried out cluster analysis, set up the cluster analysis figure that can be used for differentiating and distinguishing the kind of tap water pathogenic bacterium thus;
(4) with reference to the operation of step (2), detect more than one unknown bacterial classification samples with Raman spectrum, the Raman spectrum detected result and step (3) the gained cluster analysis figure of gained Raman spectrum detected result and the above-mentioned standard model of step (2) gained are compared, thereby identify the kind of unknown bacterial classification.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103926235A (en) * | 2014-05-04 | 2014-07-16 | 广东工业大学 | Surface enhanced raman scattering method for authenticating Shewanella |
| CN104316509A (en) * | 2014-10-22 | 2015-01-28 | 上海大学 | Method for detecting 2-MIB (2-methylisoborneol) in water by utilizing graphene nano silver complex enhanced Raman scattering |
| CN104697977A (en) * | 2015-03-23 | 2015-06-10 | 苏州大学 | Silicon-based SERS multifunctional chip and preparation method thereof |
| CN112033949A (en) * | 2020-09-04 | 2020-12-04 | 中国农业大学 | Method for rapidly detecting aquatic product spoilage bacteria by SERS (surface enhanced Raman scattering) biosensor |
| CN114324296A (en) * | 2021-12-31 | 2022-04-12 | 海南大学 | Rapid identification method for powdery mildew conidia |
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2010
- 2010-12-09 CN CN 201010579950 patent/CN102121042A/en active Pending
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| 《Anal Bioanal Chem》 20061231 Atanu Sengupta et al Detection of bacteria by surface-enhanced Raman spectroscopy 摘要以及1379，1384页 1－8 第386卷, * |
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| 《科技信息》 20091231 赫丽娜等 纳米金溶胶的制备与应用研究 30－32页 7 , 第13期 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103926235A (en) * | 2014-05-04 | 2014-07-16 | 广东工业大学 | Surface enhanced raman scattering method for authenticating Shewanella |
| CN104316509A (en) * | 2014-10-22 | 2015-01-28 | 上海大学 | Method for detecting 2-MIB (2-methylisoborneol) in water by utilizing graphene nano silver complex enhanced Raman scattering |
| CN104697977A (en) * | 2015-03-23 | 2015-06-10 | 苏州大学 | Silicon-based SERS multifunctional chip and preparation method thereof |
| CN112033949A (en) * | 2020-09-04 | 2020-12-04 | 中国农业大学 | Method for rapidly detecting aquatic product spoilage bacteria by SERS (surface enhanced Raman scattering) biosensor |
| CN114324296A (en) * | 2021-12-31 | 2022-04-12 | 海南大学 | Rapid identification method for powdery mildew conidia |
| CN114324296B (en) * | 2021-12-31 | 2023-05-05 | 海南大学 | Rapid identification method for powdery mildew conidium |
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Application publication date: 20110713 |