CN105372186B - The method for detecting microorganism in blood - Google Patents
The method for detecting microorganism in blood Download PDFInfo
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- CN105372186B CN105372186B CN201410404534.0A CN201410404534A CN105372186B CN 105372186 B CN105372186 B CN 105372186B CN 201410404534 A CN201410404534 A CN 201410404534A CN 105372186 B CN105372186 B CN 105372186B
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Abstract
The present invention provides a kind of method for detecting microorganism in blood, and this method includes the following steps: that 2,3, 5-Triphenyltertrazoliumchloride (TTC) is directly added into Blood culture bottle by (1)；Or 2,3, 5-Triphenyltertrazoliumchloride is mixed into silicon rubber oxygen-enriched film, is prepared into solid indicator, and is fixed on Blood culture bottle bottom；The TTC concentration is 0.01-0.03g/mL；(2) blood meida is added into Blood culture bottle, and in 121 DEG C, sterilize 20min；(3) blood sample is added into Blood culture bottle, after cultivating 0-5 days under the conditions of 35 DEG C；(4) the light reflected intensity for indicating band in Blood culture bottle is detected, light reflected intensity curve is drawn, microorganism growth result is determined according to light reflected intensity curve.The present invention has many advantages, such as that at low cost, operating process is simple, detection time is short, positive rate is high in clinical application, there is biggish application value.
Description
Technical field
The present invention relates to biotechnology, the side of microorganism in specific micro- raw detection method more particularly to a kind of detection blood
Method.
Background technique
Blood culture is important one of the detection project of clinical labororatory, since bacteremia, the death rate of septicemia are up to 30-
50%, therefore, cultivation results accurately and timely to clinical detection and are treated significant.Bacterium in blood culture in Chinese at present
Positive rate be only 10%, possible cause are as follows: 1, the bacteria containing amount in blood it is low；2, the antibacterial material in blood；3, resist
The application of raw element；4, the limitation of condition of culture.Blood culture testing product currently on the market is mainly with CO2It is main based on detection method
The determination techniques wanted have radioactive labeling techniques, detection technique of fluorescence and spectrophotometry.Because bacterium is in its growth metabolism mistake
Cheng Zhonghui generates CO2, the principle of these technologies be all by detection blood sample during the cultivation process in whether have CO2Generation
To determine whether there is microorganism in blood.But it is this that CO is generated based on bacterium2Detection method, can because it is some cannot using sugar
The microorganism (such as non-zymocyte) of metabolism, metabolic process does not generate CO2Or the CO generated2Measure it is extremely low, so as to cause measurement result
It is negative.
2,3, 5-Triphenyltertrazoliumchloride (TTC) can capture cell as a kind of special oxidation-reduction indicator
Or the electronics generated during bacterial respiratory, become empurpled reduced form from colourless oxidized form, whereby it can be detected that blood
In do not generate CO2Microorganism.There are positive rate height, the short advantage of detection time, for improving blood training as indicator with TTC
Feeding technology has valuable help.
Summary of the invention
The technical problem to be solved in the present invention is that researching and designing uses 2,3,5 triphenyltetrazolium chlorid (TTC) conduct
A kind of special oxidation-reduction indicator detects in blood whether contain microorganism, achievees the effect that improve blood culture technology.
The present invention provides a kind of method for detecting microorganism in blood, and this method includes the following steps:
(1) Blood culture bottle is added in 2,3, 5-Triphenyltertrazoliumchloride (TTC) and blood meida, adds 35-40mL
Blood meida and 2-4g resin particle, 2,3, 5-Triphenyltertrazoliumchloride concentration in blood meida and resin particle mixture
For 0.01-0.03g/mL；Or
2,3, 5-Triphenyltertrazoliumchloride (TTC) is mixed into silicon rubber oxygen-enriched film, is prepared into solid indicator, and solid
It is scheduled on Blood culture bottle bottom, adds 35-40mL blood meida and 2-4g resin particle；The 2,3,5 triphenyltetrazolium chlorid
Concentration is 0.01-0.03g/mL in silicon rubber oxygen-enriched film；
(2) Blood culture bottle is in 121 DEG C, and sterilize 20min；
(3) blood sample is added into Blood culture bottle, is cultivated 0-5 days under the conditions of 34-36 DEG C；
(4) the light reflected intensity for indicating band in Blood culture bottle is detected, light reflected intensity curve is drawn, according to light reflected intensity
Curve determines microorganism growth result.
Step described in the method for the present invention (1) silicon rubber oxygen-enriched film are as follows: the dimethyl silicone polymer of 25mL, the 750 positive cinnamic acids of μ L
Ethyl ester, 100 μ L dibutyl tin dilaurates and 1.25mL cyclohexane mixtures.
Step (1) TTC first with etc. the single of quality steam water dissolution and reapply.
Step (1) the blood meida 35-40mL mixing 2-4g resin particle；The blood meida be selected from broth bouillon,
Soybean casein culture medium or trypticase soy broth, preferably 35mL broth bouillon；The resin particle is 0.2-
The cation exchange resin grain or non-ionic adsorption resin grain of 0.8mm partial size, the cation exchange resin grain-by-grain seed selection from 732 sun from
Sub-exchange resin grain, strongly acidic styrene type cation exchange resin, superpower property styrene type cation exchange resin, macropore are strong
Basic quaternary ammonium type cation exchange resin；Non-ionic adsorption resin grain-by-grain seed selection self-polystyrene type adsorbs resin or Amberlite
The non-ionic macroreticular resin of XAD-4.
The light wave of step (4) the 590nm/750nm dual wavelength is for detecting 2,3,5 triphenyltetrazolium chlorid
(TTC) culture sample in Blood culture bottle is added with blood meida and resin particle；570-610nm optical wavelength for detecting 2,
3,5- benzyltriphenylphosphonium chloride tetrazoles (TTC) are mixed into silicon rubber oxygen-enriched film and are prepared into solid indicator, and are fixed on Blood culture bottle
Bottom adds the culture sample of blood meida and resin particle.
Instruction band described in step (4) are as follows: 2,3,5 triphenyltetrazolium chlorid (TTC) is directly added into the blood of culture medium
Culture bottle method refers to the culture medium at the 2-3cm of culture medium surface layer；2,3,5 triphenyltetrazolium chlorid (TTC) is mixed
Refer to the silicon rubber oxygen-enriched film of culture bottle bottom to the Blood culture bottle of silicon rubber oxygen-enriched film.
105 clinical samples are carried out with the present invention blood culture the experimental results showed that, positive rate of the invention is
20%.The prior art is that recall rate is 5%.
Beneficial effects of the present invention:
(1) what the present invention innovated has used TTC as the indicator of detection microorganism, and feature can capture micro- for TTC
The electronics of the Microbiological release of amount is reduced shape, to show the strong purple of color, experimental result is made to be easy observation.
(2) present invention is to have the characteristics of strong absworption peak at 590nm in wavelength using reduced form TTC, selects 570-610nm wave
Long light wave can accurately judge whether contain microorganism in blood as Detection wavelength.
(3) present invention is in clinical application at low cost, operating process is simple, detection time is short, positive rate is high
The advantages that, there is biggish application value.
Detailed description of the invention
Fig. 1 is the blood culture testing result of embodiment 1, electric signal of the ordinate for light emission intensity transformation in figure, unit:
mv.Abscissa is time, unit: min.Straight line is threshold value at ordinate value 1.1 in figure.Meet: (1) detected signal value is greater than threshold
Value；(2) there are two conditions of serpentine curve, as positive signal in detection curve.Fig. 1 illustrates testing result for the positive, blood culture
There is bacterial growth in bottle, positive detection time is 13 hours or so.
Fig. 2 is the blood culture testing result of embodiment 2, electric signal of the ordinate for light emission intensity transformation in figure, unit:
mv.Abscissa is time, unit: min.Straight line is threshold value at ordinate value 1.2 in figure.Meet: (1) detected signal value is greater than threshold
Value；(2) there are two conditions of serpentine curve, as positive signal in detection curve.Fig. 2 illustrates testing result for the positive, blood culture
There is bacterial growth in bottle, positive detection time is 13 hours or so.
Fig. 3 is the blood culture testing result of embodiment 2, electric signal of the ordinate for the transformation of light reflected intensity in figure, unit:
mv.Abscissa is time, unit: min.Straight line is threshold value at ordinate value 1.2 in figure.Meet: (1) detected signal value is greater than threshold
Value；(2) there are two conditions of serpentine curve, as positive signal in detection curve.Fig. 3 illustrates testing result for the positive, blood culture
There is bacterial growth in bottle, positive detection time is 62 hours or so.
Specific embodiment
Embodiment 1 (the instruction agent method present invention uses TTC as detection microorganism)
(1) take the dimethyl silicone polymer of 25mL, the positive ethyl cinnamate of 750 μ L, 100 μ L dibutyl tin dilaurates,
The TTC (TTC is first dissolved with the mono- steaming water of 0.5g) of 1.25mL hexamethylene and 0.5g are uniformly mixed in container, and are added to blood culture
Bottom of bottle portion solidifies at 35 DEG C, is prepared into silicon rubber oxygen-enriched film；
(2) 40mL broth bouillon and (the Chinese medicines group life of 732 cation exchange resin grain of 4g are added into Blood culture bottle
Produce), and in 121 DEG C, sterilize 20min；
(3) in Shanghai City Tung Wah hospital inpatient department, acquisition has fever (>=38 DEG C) or low temperature (≤36 DEG C), shivers with cold, leucocyte
Increase the blood preparation of the patient of sign for testing.10mL blood sample is added into Blood culture bottle；
(4) Blood culture bottle that blood sample is added is placed in 35 in U.S. company BD BACTEC9050 Full-automatic blood culture instrument
DEG C culture, and with 590nm/750nm dual wavelength light wave detect Blood culture bottle bottom silicon rubber oxygen-enriched film light reflected intensity.
(5) it cultivates 13 hours, detection signal shows that light reflected intensity is more than threshold value, and light reflectivity curve has apparent " S "
Shape curve (such as Fig. 1), thus illustrates there is microorganism growth in Blood culture bottle.
Embodiment 2 (the instruction agent method present invention uses TTC as detection microorganism)
(1) 35mL broth bouillon, the non-ionic macroreticular resin grain of 2g Amberlite XAD-4 (Rhom and Hass are taken
Production) and 1.05g TTC (TTC first uses the single of 1.05g to steam water dissolution) it is uniformly mixed in container, be added in Blood culture bottle simultaneously
In 121 DEG C, sterilize 20min；
(2) in Shanghai City Tung Wah hospital inpatient department, acquisition has fever (>=38 DEG C) or low temperature (≤36 DEG C), shivers with cold, leucocyte
Increase (cell density be greater than 10.0 × 109A/L) sign patient blood preparation for testing.It is added into Blood culture bottle
5mL blood sample；
(3) Blood culture bottle that blood sample is added is placed in 37 in U.S. company BD BACTEC9050 Full-automatic blood culture instrument
DEG C culture, and the culture medium at 590nm/750nm wavelength light wave monitoring distance culture medium surface layer 2-3cm.
(4) 13 or so are cultivated, detection signal shows that light reflected intensity is more than threshold value, and light reflectivity curve has apparent " S "
Shape curve (such as Fig. 2), this two signals show that cultivation results for the positive, thus illustrate there is microorganism growth in Blood culture bottle.
Embodiment 3 (colorimetric method)
(1) " the aerobic Blood culture bottle of Zhuhai Deere (colorimetric method) " is taken to be tested.
(2) in Shanghai City Tung Wah hospital inpatient department, acquisition has fever (>=38 DEG C) or low temperature (≤36 DEG C), shivers with cold, leucocyte
Increase and (counts and be greater than 10.0 × 109/ L) sign patient blood preparation for testing.Blood preparation is by Clinical microorganism reality
It tests room technician and acquires clinical patient blood sample.5mL blood sample is added into Blood culture bottle；
(3) Blood culture bottle that blood sample is added is placed in 35 in U.S. company BD BACTEC9050 Full-automatic blood culture instrument
DEG C culture, and with the mating detection device detection of BACTEC9050 Full-automatic blood culture instrument；
(4) cultivate 62 hours, detection signal shows that light reflected intensity is more than threshold value (such as Fig. 3), and light reflectivity curve have it is bright
Aobvious serpentine curve, this two signals show that cultivation results for the positive, thus illustrate there is microorganism growth in Blood culture bottle.
Above-mentioned comparative experiments choose altogether 105 from from each section of Shanghai City Tung Wah hospital be hospitalized suspected case patient acquisition
Blood culture sample, blood culture as the result is shown the utility model positive detection time be 13 hours, Zhuhai Deere blood culture
The positive detection time of bottle is 62 hours；The utility model detects 21 positives, positive rate 20%, the training of Zhuhai Deere blood
It is positive to support bottle detection 6, positive rate 5%.
Embodiment described above only describe the preferred embodiments of the invention, not to model of the invention
It encloses and is defined, without departing from the spirit of the design of the present invention, those of ordinary skill in the art are to technical side of the invention
The various changes and improvements that case is made should all be fallen into the protection scope that claims of the present invention determines.
Claims (10)
1. the method for detecting microorganism in blood, which is characterized in that this method includes the following steps:
(1) Blood culture bottle is added in 2,3, 5-Triphenyltertrazoliumchloride and blood meida, add 35-40mL blood meida and
2-4g resin particle, 2,3, 5-Triphenyltertrazoliumchloride concentration in blood meida and resin particle mixture is 0.01-
0.03g/mL；Or
2,3, 5-Triphenyltertrazoliumchloride is mixed into silicon rubber oxygen-enriched film, is prepared into solid indicator, and is fixed on blood training
Bottom of bottle portion is supported, 35-40mL blood meida and 2-4g resin particle are added；The 2,3,5 triphenyltetrazolium chlorid is in silicon rubber
Concentration is 0.01-0.03g/mL in oxygen permeable membrane；
(2) Blood culture bottle is in 121 DEG C, and sterilize 20min；
(3) blood sample is added into Blood culture bottle, is cultivated 0-5 days under the conditions of 34-36 DEG C；
(4) the light reflected intensity for indicating band in Blood culture bottle is detected, light reflected intensity curve is drawn, according to light reflected intensity curve
Determine microorganism growth result.
2. detecting the method for microorganism in blood according to claim 1, which is characterized in that step (1) silicon rubber oxygen-enriched film
Are as follows: dimethyl silicone polymer, the positive ethyl cinnamate of 730-770 μ L, 90-110 μ L dibutyl tin dilaurate and the 1- of 23-27mL
The mixture of 1.5mL hexamethylene.
3. detecting the method for microorganism in blood according to claim 1, which is characterized in that the step (1) 2,3,5- tri-
First the single of the quality such as use steams water dissolution to tetraphenylphosphonium chloride tetrazole.
4. detecting the method for microorganism in blood according to claim 1, which is characterized in that step (2) blood meida
Selected from broth bouillon, soybean casein culture medium or trypticase soy broth.
5. detecting the method for microorganism in blood according to claim 1, which is characterized in that the resin particle of the step (2)
Cation exchange resin grain or non-ionic adsorption resin grain selected from 0.2-0.8mm.
6. the method for stating microorganism in detection blood according to claim 5, which is characterized in that the step (2) cation
Exchanger resin grain-by-grain seed selection is from 732 cation exchange resin grains, strongly acidic styrene type cation exchange resin, superpower property polystyrene
Cation exchange resin, macroporous strong basic quaternary ammonium cation exchanger resin；Non-ionic adsorption resin grain-by-grain seed selection self-polystyrene type
Adsorb resin, the non-ionic macroreticular resin of Amberlite XAD-4.
7. according to the method for microorganism in detection blood described in claim 1, which is characterized in that the step (4) is by 2,3,5- tri-
Light wave when tetraphenylphosphonium chloride tetrazole is directly added into the detection of the culture sample in Blood culture bottle is 590nm/750nm dual wavelength.
8. detecting the method for microorganism in blood according to claim 1, which is characterized in that the step (4) is by 2,3,5-
Light wave when benzyltriphenylphosphonium chloride tetrazole is mixed into the sample detection of silicon rubber oxygen-enriched film culture is 570-610nm wavelength.
9. detecting the method for microorganism in blood according to claim 1, which is characterized in that finger described in the step (4)
Show band are as follows: 2,3, 5-Triphenyltertrazoliumchloride is directly added into the Blood culture bottle of culture medium, is referred to apart from culture medium surface layer 2-
Culture medium at 3cm.
10. according to the method for microorganism in detection blood described in claim 1, which is characterized in that instruction described in the step (4)
Band are as follows: 2,3, 5-Triphenyltertrazoliumchloride is mixed into silicon rubber oxygen-enriched film, the Blood culture bottle for adding culture medium refers to training
Support the silicon rubber oxygen-enriched film in bottom of bottle portion.
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