CN104152531A - Method for establishing pathogenic bacterium fingerprint by using Terahertz waves - Google Patents
Method for establishing pathogenic bacterium fingerprint by using Terahertz waves Download PDFInfo
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Abstract
The invention discloses a method for establishing pathogenic bacterium fingerprint by using Terahertz waves. The method comprises the following concrete steps: subjecting a to-be-detected bacterial strain to enrichment culture, carrying out cleaning with normal saline and collection and adjusting turbidity of a bacterial liquid to 0.5 McFarland (MCF) by using a bacterial nephelometer or turbidity meter; and then carrying out detection under the conditions that Terahertz frequency is 0.2 to 3 THz, wavelength is 100 to 1500 mu m, average wavelength is 300 mu m, nitrogen is used for flushing, ambient humidity is less than 5%, and ambient temperature is 22 DEG C. The method is simple and fast, can detect liquid samples and is capable of identifying the variety of the to-be-detected bacterial strain according to the established fingerprint.
Description
Technical field
The invention belongs to detection field, be specifically related to utilize THz wave to set up the method for pathogenic bacterium finger printing.
Background technology
In the researching and analysing of the biomolecules such as carbohydrate, protein, nucleic acid, spectroscopy is being brought into play very important effect.Terahertz (Terahertz Waves, THz) ripple refers to the hertzian wave of frequency between 0.1～10THz (1THz=1012Hz) (wavelength is at 30～3000 μ m), and its wave band is between microwave and infrared light.The generation developing rapidly as THz pulse of ultrafast laser technique and semiconductor material science and technology provides stable, reliable excitation light source in recent ten years, has promoted the application of THz radiation aspect spectroscopy and imaging technique.
Than ultraviolet-visible absorption spectroscopy, the conventional arts such as x-ray imaging analysis, THz spectrum has its unique advantage: the characteristic absorbance of (1) spectrum: the interaction (hydrogen bond a little less than between many molecules, Van der Waals force etc.), the skeletal vibration of biomacromolecule, in the rotation of dipole and vibrational transition and crystal, the low-frequency vibration of lattice absorbs just in time in THz frequency band range, and the fine difference that THz spectroscopic techniques exists detecting material structure and variation are very sensitive, the fingerprint characteristic with reflection compound structure, therefore THz spectroscopic techniques is having wide prospect aspect analysis and research biomacromolecule, (2) security is good: the photon energy of THz electromagnetic radiation is low, only has milli electron-volt, can not destroy because of ionization biomolecules, because of but a kind of lossless detection method safely and effectively, (3) highly sensitive: the typical pulse-widths of THz pulse is at picosecond magnitude, not only can carry out temporal resolution research to biological sample, and can effectively suppress the interference of far infrared background radiation noise, the signal to noise ratio of radiant intensity measurement and detectivity are far above Fourier transform infrared spectroscopy, (4) broadband property: THz impulse source only comprises the electromagnetic oscillation in several cycles conventionally, and the frequency band of individual pulse can cover the scope of GHz to tens THz, is convenient to the spectral quality of amalyzing substances in large scope, (5) coherency: the coherent measurement technology of THz time-domain spectroscopic technology (THz-TDS) can directly be measured amplitude and the phase of THz electric field, can extract specific refractory power, the uptake factor of sample easily.But in prior art, THz wave can only have no the report for detection of liquid sample for detection of solid sample, more has no the report for detection of pathogenic bacterium finger printing in liquid sample.
Summary of the invention
In view of this, the object of the present invention is to provide the method for utilizing THz wave to set up pathogenic bacterium finger printing, following technical scheme be provided:
Utilize THz wave to set up the method for pathogenic bacterium finger printing, bacterial strain to be checked is increased after bacterium cultivation, with physiological saline, clean and collect, with bacterial turbidity meter or turbidimeter, reconcile the bacterium liquid turbidity Wei0.5 (McFarland of Maxwell unit, MCF), in Terahertz frequency, be then that 0.2～3THz, wavelength are that 100-1500 μ m, mean wavelength are that 300 μ m, nitrogen wash, ambient moisture <5%, temperature are to detect under 22 ℃ of conditions.
Preferably, the method for described strain culturing be by inoculation to liquid nutrient medium, be then cultured to bacterial concentration 0.5 Maxwell unit.
Preferably, the method that described physiological saline cleans be by bacterium liquid centrifugal 5～10min under 1500～3000g condition, abandons supernatant liquor, precipitate and add physiological saline, and centrifugal 5～10min under 1500～3000g condition again after mixing, then abandon supernatant liquor.
Preferred, described bacterial strain to be checked is streptococcus aureus or escherichia coli.
Beneficial effect of the present invention is: the invention discloses the method for utilizing THz wave to set up pathogenic bacterium finger printing, utilize different bacterium the different characteristics absorption spectrum of THz frequency band to be set up to the finger printing of pathogenic bacterium, the method is simple, quick, can be applicable to liquid sample, and can utilize the finger printing of reference culture and the fingerprint chromatogram of bacterial strain to be measured to compare, thereby reach the object of identifying clinical common pathogenic bacteria.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearer, the invention provides following accompanying drawing:
Fig. 1 is finger printing (the X-coordinate representative detection frequency (unit: THz) in figure of streptococcus aureus and escherichia coli, ordinate zou represents absorption intensity (unit: a.u.), coccus represents streptococcus aureus, and bacillus represents escherichia coli).
Fig. 2 is poor (the X-coordinate representative detection frequency (unit: THz), ordinate zou represents absorption intensity (unit: a.u.)) in figure of streptococcus aureus and escherichia coli absorption intensity.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, conventionally according to normal condition or the condition of advising according to manufacturer.
Embodiment 1
Utilize THz wave to set up the method for pathogenic bacterium finger printing, concrete steps are as follows:
A. get and be accredited as escherichia coli and with transfering loop, be seeded in LB (Luria-Bertani) liquid nutrient medium after the lower freeze thawing of room temperature (18-25 ℃), then under 37 ℃ of conditions, incubated overnight to bacterial concentration is greater than 0.5 Maxwell unit and (is about 1.5 * 10
8cfu/mL);
B. the bacterium liquid centrifugal 5min under 1500g condition after step a being cultivated, abandons supernatant liquor, and precipitation adds physiological saline, centrifugal 5min under 1500g condition again after mixing; Abandon again supernatant liquor, add physiological saline vibration to mix the Maxwell unit to bacterium liquid turbidity Wei0.5;
C. by step c gained bacterium liquid, in Terahertz frequency, be that 0.2～3THz, wavelength are that 100-1500 μ m, mean wavelength are that 300 μ m, nitrogen wash, ambient moisture <5%, temperature are to detect under 22 ℃ of conditions, the finger printing of acquisition as shown in Figure 1.
Embodiment 2
Utilize THz wave to set up the method for pathogenic bacterium finger printing, concrete steps are as follows:
A. get and be accredited as streptococcus aureus and with transfering loop, be seeded in LB (Luria-Bertani) liquid nutrient medium after the lower freeze thawing of room temperature (18-25 ℃), then under 37 ℃ of conditions, incubated overnight to bacterial concentration is greater than 0.5 Maxwell unit and (is about 1.5 * 10
8cfu/mL);
B. the bacterium liquid centrifugal 10min under 3000g condition after step a being cultivated, abandons supernatant liquor, and precipitation adds physiological saline, centrifugal 10min under 3000g condition again after mixing; Abandon again supernatant liquor, add physiological saline vibration to mix the Maxwell unit to bacterium liquid turbidity Wei0.5;
C. by step c gained bacterium liquid, in Terahertz frequency, be that 0.2～3THz, wavelength are that 100-1500 μ m, mean wavelength are that 300 μ m, nitrogen wash, ambient moisture <5%, temperature are to detect under 22 ℃ of conditions, the finger printing of acquisition as shown in Figure 1.
As shown in Figure 1, escherichia coli is different with the finger printing of streptococcus aureus in 0.2～1THz wave band, and streptococcus aureus obtains absorption intensity and is greater than escherichia coli and obtains absorption intensity.From charateristic avsorption band, the absorption peak of escherichia coli and streptococcus aureus is roughly the same, but near 0.8THz, charateristic avsorption band appears in streptococcus aureus.The above results has shown that different bacterium liquid has different characteristics absorption spectrum, can utilize aforesaid method to obtain the finger printing of reference culture, then the collection of illustrative plates of bacterial strain to be measured is contrasted in standard diagram, thereby identifies the kind of bacterial strain to be measured.
The absorption value of the finger printing of streptococcus aureus in Fig. 1 and escherichia coli is subtracted each other, obtain streptococcus aureus and escherichia coli optical density difference value, result as shown in Figure 2.Result demonstration, the difference of the optical density of streptococcus aureus and escherichia coli all, more than zero coordinate, shows that the absorption intensity of streptococcus aureus is all greater than escherichia coli.
Finally explanation is, above preferred embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is described in detail by above preferred embodiment, but those skilled in the art are to be understood that, can to it, make various changes in the form and details, and not depart from the claims in the present invention book limited range.
Claims (4)
1. utilize THz wave to set up the method for pathogenic bacterium finger printing, it is characterized in that: bacterial strain to be checked is increased after bacterium cultivation, with physiological saline, clean and collect, with bacterial turbidity meter or turbidimeter, reconciling bacterium liquid turbidity Wei0.5 Maxwell unit, is then that 0.2～3THz, wavelength are that 100-1500 μ m, mean wavelength are that 300 μ m, nitrogen wash, ambient moisture <5%, temperature are to detect under 22 ℃ of conditions in Terahertz frequency.
2. method according to claim 1, is characterized in that: the method for described strain culturing be by inoculation to liquid nutrient medium, then increase bacterium and be cultured to bacterial concentration and be greater than 0.5 Maxwell unit.
3. method according to claim 1, it is characterized in that: the method that described physiological saline cleans, for by bacterium liquid centrifugal 5～10min under 1500～3000g condition, is abandoned supernatant liquor, precipitation adds physiological saline, centrifugal 5～10min under 1500～3000g condition again after mixing, then abandon supernatant liquor.
4. according to the method described in claim 1-3 any one, it is characterized in that: described bacterial strain to be checked is streptococcus aureus or escherichia coli.
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Cited By (3)
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| CN104483409A (en) * | 2014-12-12 | 2015-04-01 | 河北农业大学 | Fingerprint based staphylococcus aureus identification method |
| CN104502303A (en) * | 2015-01-20 | 2015-04-08 | 中国人民解放军第三军医大学第一附属医院 | Sub-THz nano-biosensor for quickly frame-detecting bacteria and detection method thereof |
| CN105588820A (en) * | 2015-12-15 | 2016-05-18 | 中国人民解放军第三军医大学第一附属医院 | Method for detecting trace live bacteria on basis of terahertz metamaterial |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104483409A (en) * | 2014-12-12 | 2015-04-01 | 河北农业大学 | Fingerprint based staphylococcus aureus identification method |
| CN104483409B (en) * | 2014-12-12 | 2017-01-11 | 河北农业大学 | Fingerprint based staphylococcus aureus identification method |
| CN104502303A (en) * | 2015-01-20 | 2015-04-08 | 中国人民解放军第三军医大学第一附属医院 | Sub-THz nano-biosensor for quickly frame-detecting bacteria and detection method thereof |
| CN104502303B (en) * | 2015-01-20 | 2017-05-31 | 中国人民解放军第三军医大学第一附属医院 | For the Asia-Pacific hertz nano biological sensor and its detection method of fast frame inspection bacterium |
| CN105588820A (en) * | 2015-12-15 | 2016-05-18 | 中国人民解放军第三军医大学第一附属医院 | Method for detecting trace live bacteria on basis of terahertz metamaterial |
| CN105588820B (en) * | 2015-12-15 | 2018-06-05 | 中国人民解放军第三军医大学第一附属医院 | The method that micro bacterium living is detected based on Terahertz Meta Materials |
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