CN109554437B - Dual-wavelength microorganism biochemical reaction dynamic interpretation detection method - Google Patents
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Abstract
The invention discloses a dual-wavelength microorganism biochemical reaction dynamic interpretation detection method. The method is characterized in that the absorbance of a microbial biochemical identification test hole is dynamically detected in real time by adopting double wavelengths, after the ratio of the absorbance of the double wavelengths is piecewise fitted, the positive and negative of the reaction hole can be pre-judged by solving the speed and the acceleration of a curve, and then the bacterial species can be rapidly identified, wherein the method for pre-judging the positive and negative of the reaction hole comprises the following steps: and (3) solving the ratio of the absorbance of the two wavelengths, carrying out piecewise rolling fitting on a curve, solving the velocity or the acceleration of the curve, and solving the problems that the positive is monotonously increased (decreased), and the negative is basically unchanged or monotonously decreased (increased). The method is particularly suitable for the microbial biochemical reaction with simultaneously changing color and turbidity, the comprehensive change of the turbidity and the color in the reaction hole is dynamically monitored, the ratio of dual-wavelength absorbance is fitted in a segmented mode, the speed or the acceleration of a curve is calculated, the positive and the negative of the test hole can be accurately judged in advance, and the quick identification of bacteria can be realized.
Description
Technical Field
The invention relates to the field of microorganism identification, in particular to a dual-wavelength microorganism biochemical reaction dynamic interpretation detection method.
Background
The microorganism identification can provide an important diagnosis and treatment basis for infectious diseases, so that patients can be treated timely and effectively. The bacteria identification refers to the process of putting unknown bacteria into a proper position in a system according to biological characteristics, comparing the similarity with known strains, and determining the classification status of the bacteria by a comparative analysis method. The bacteria identification method comprises biochemical identification, nucleic acid detection, serological identification, automated instrument identification, mass spectrometry technology and the like. The biochemical identification is the most important one in bacteria identification, and is mainly used for identifying bacteria according to the difference of bacteria in nutrient decomposition capacity and metabolites thereof, and comprises a protein decomposition product test, a sugar decomposition product test, a catalase test, an oxidase test, a coagulase test and the like.
Biochemical identification is also the most important identification method in the current commercial identification cards, which can change the color, turbidity or substrate color in the culture medium through the above-mentioned various tests, and the identification instrument mainly identifies the bacterial species by monitoring the color or turbidity change in the reaction well through the transmission method.
However, the current transmission method has the problem that the negative and positive of the reaction hole which changes color and turbidity simultaneously are difficult to be judged. Biochemical identification of microorganisms assay a, colourless to yellowish assay, such as the beta galactosidase assay; b. examples of the color change include a change from colorless to brown, such as a p-nitroanilide decarboxylase test, a change from green to blue or yellow, such as a maltose fermentation test using bromothymol blue as an indicator, and a change from yellow to red, such as an arginine decarboxylase test using phenol red as an indicator. The turbidity is greatly changed when the color is changed, so that the problem of false negative or false positive is easy to occur by adopting a single-wavelength transmission detection method.
In addition, most of the existing microorganism identification adopts an end-point interpretation method, namely, microorganisms are cultured in a constant temperature environment of about 35 ℃ for a long time, and after the tests in biochemical identification reaction holes fully react, an instrument can analyze and identify the species of the microorganisms and report the results, and the identification speed can not meet the current market demand.
Disclosure of Invention
The invention aims to overcome the defects of the background technology and provides a dual-wavelength microorganism biochemical reaction dynamic interpretation detection method.
In order to realize the purpose of the invention, the dual-wavelength microbial biochemical reaction interpretation and detection method dynamically detects the absorbance of the microbial biochemical identification test hole in real time by adopting dual wavelengths, and after the ratio of the dual-wavelength absorbance is piecewise fitted, the negative and positive of the reaction hole can be prejudged by solving the speed and the acceleration of a curve, so as to rapidly identify the bacterial species.
Furthermore, in the dynamic real-time detection, the data acquisition period is 15-30 minutes.
Further, the method for pre-judging whether the reaction hole is positive or negative comprises the following steps: and (3) calculating the ratio of the absorbance of the double wavelengths, fitting a curve according to 2-10 points, such as 3-7 points, and for example, 5 points by means of piecewise rolling, and calculating the speed or the acceleration, wherein the positive value is monotonically increased (decreased), and the negative value is basically unchanged or monotonically decreased (increased).
Further, the dual wavelength is two of 550nm plus or minus 20nm, 450nm plus or minus 20nm and 630nm plus or minus 20 nm; preferably, the dual wavelength is two of 550nm, 450nm and 630 nm.
Preferably, the biochemical identification of the microorganism is a test that does not change color to yellow, such as a beta galactosidase test;
preferably, the biochemical identification of the microorganism is carried out without a color-changing brown test, such as a p-nitroanilide decarboxylase test,
preferably, the microorganism is biochemically identified as a blue or yellow color, such as a maltose fermentation test using bromothymol blue as an indicator;
preferably, the biochemical identification of the microorganism is a test for reddening of yellow, such as an arginine decarboxylase test using phenol red as an indicator.
Further preferably, in the microorganism identification phenylalanine decarboxylase test, the interpretation that no color change brown is positive and no color change is negative is carried out by adopting 630nm and 450nm for detection.
Further preferably, in the microorganism identification arginine decarboxylase test, the interpretation that the yellow color changes to red is positive, does not change to the red or changes to orange-red is carried out by adopting 630nm and 550nm for detection.
Further preferably, in the fermentation test for identifying maltose by microorganisms, the green turns yellow to positive and blue to negative, and the detection is carried out by adopting 630nm and 450 nm.
Preferably, the detection light path adopted by the detection method provided by the invention comprises a light-emitting source (1), a Fresnel lens (2), a stepping wheel (3), an optical filter (4), an optical fiber (5), a diaphragm (7), a 96-hole test card (8), a silicon photocell (9) and a post-processing circuit.
Preferably, the light source (1) is a halogen lamp or an LED point light source, emitted light is focused by the lens (2), is coupled into the optical fiber (5) in one division way after being filtered by the optical filter, light coming out of the optical fiber is focused by the lens (2) and is refracted to enter a reaction hole in the microorganism identification drug sensitive test card, and transmitted light is received by the silicon photocell (9) after passing through the diaphragm (7), wherein the optical fiber is an optical fiber with 8-96 paths, and 1 path represents a detection channel and corresponds to a detection hole on the test card.
Further, the wavelength of the optical filter adopts three wavelengths of 550nm +/-20 nm, 450nm +/-20 nm and 630nm +/-20 nm; the optical filter (4) is fixed on the stepping wheel (3) and is driven to switch by a stepping motor.
The method is particularly suitable for the microbial biochemical reaction with simultaneously changing color and turbidity, the comprehensive change of the turbidity and the color in the reaction hole is dynamically monitored, the ratio of dual-wavelength absorbance is fitted in a segmented mode, the speed or the acceleration of a curve is calculated, the positive and the negative of the test hole can be accurately judged in advance, and the quick identification of bacteria can be realized.
Drawings
FIG. 1 is a diagrammatic optical path of a measurement and detection system;
FIG. 2 is a device for rapid identification and detection of microorganisms.
The reference numerals are explained below:
the method comprises the following steps of 1-a light source, 2-Fresnel lenses 1 and 3-a stepping wheel, 4-a light filter, 5-an optical fiber, 6-Fresnel lenses 2 and 7-a diaphragm, 8-a test card, 9-a silicon photocell, 10-an incubation module, 11-a measurement and detection module, 12-a transmission module and 13-a test card.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention. It is to be understood that the following description is only illustrative of the present invention and is not to be taken in a limiting sense.
The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.
The conjunction "consisting of …" excludes any unspecified elements, steps or components. If used in a claim, the phrase is intended to claim as closed, meaning that it does not contain materials other than those described, except for the conventional impurities associated therewith. When the phrase "consisting of …" appears in a clause of the claim body and not immediately after the subject matter, it defines only the elements described in the clause; other elements are not excluded from the claims as a whole.
In the examples of the present invention, the definitions are as follows:
1. detection device and optical path
The detection device has the constant temperature function of 35 +/-2 ℃ and the function of automatically sending and reading cards.
The detection light path is as shown in FIG. 1, incident light IOThe light enters from the bottom of the reaction well, is refracted by the bottom of the reaction well and the solution, and the transmitted light intensity is I, and the absorbance is A ═ log (I)0I). Incident light IOTwo monochromatic wavelengths.
As shown in fig. 1, the detection light path specifically comprises a light source (1), a fresnel lens (2), a stepping wheel (3), a filter (4), an optical fiber (5), a diaphragm (7), a 96-hole test card (8), a silicon photocell (9) and a post-processing circuit, as shown in fig. 1.
The light source (1) is a halogen lamp or an LED point light source, emitted light is focused by the lens (2), is coupled into a multi-path optical fiber (5) after being filtered by the optical filter, light coming out of the optical fiber is focused by the lens (2) and is refracted into a reaction hole in the microorganism identification drug sensitive test card, and the transmitted light is received by the silicon photocell (9) after passing through the diaphragm (7).
The optical fiber is divided into 8-96 paths, and 1 path represents a detection channel corresponding to a detection hole on the test card.
The wavelength of the optical filter adopts three types of 550nm +/-20 nm, 450nm +/-20 nm and 630nm +/-20 nm, and the optical filter (4) is fixed on the stepping wheel (3) and is driven to switch by a stepping motor.
2. Yin-yang interpretation method
(1) Wavelength selection
The dual wavelength adopted in the microbial biochemical identification test refers to two of 550nm +/-20 nm, 450nm +/-20 nm and 630nm +/-20 nm;
for example: identification of microorganism in enzyme reaction test, wherein the absence of color change and brown is positive, and the absence of color change and negative are judged, such as phenylalanine decarboxylase test, etc., and 630nm and 450nm are adopted for detection.
For example: and identifying the interpretation that the yellow color turns red to be positive, does not turn red or turns to orange red in an enzymatic reaction test by microorganisms, such as an arginine decarboxylase test and the like, and adopting 630nm and 550nm for detection.
For example: the sugar decomposition product test shows that the green turns yellow to be positive and the blue to be negative, such as maltose fermentation test and the like, and 630nm and 450nm are adopted for detection.
(2) Interpretation method
The fast negative and positive judging method for test hole includes fitting curve sectionally based on the ratio of double wavelength absorbance, calculating its speed or acceleration, and pre-judging the negative and positive results of the reaction hole based on certain rule. The specific method comprises the following steps:
rate method:
and collecting data once every 15-30 minutes to obtain the ratio of the absorbance of the double wavelengths. And (3) piecewise fitting a straight line according to 2-10 points, for example 3-7 points, and for example 5 points, and obtaining a first differentiation to obtain the slope of the curve. If the following conditions are met, the system prompts to report the sun; otherwise, continuing to measure and performing rolling analysis.
The slope of each piecewise fit is continuously greater or less than a particular threshold;
the slopes of the three continuous unit bands are calculated according to points, and the ratio of the absorbance of the double wavelengths can be increased or decreased to a specific threshold value;
an acceleration method:
data are collected once every 15-30 minutes to obtain the ratio of the absorbance of the double wavelengths, and a quadratic curve is piecewise fitted according to 2-10 points, such as 3-7 points, and 5 points, so as to obtain the quadratic differential. If the following conditions are met, the system prompts to report the sun; otherwise, continuing to measure and performing rolling analysis.
The acceleration value of the middle point of each piecewise fitting is continuously greater than or less than a specific threshold value;
the accelerations of three continuous unit bands are calculated according to points, and the ratio of the absorbance of the double wavelengths can be increased or decreased to a specific threshold value;
(3) strain identification method
And (4) rapidly identifying the species of the strains by adopting a bifidus matrix method according to positive and negative results pre-judged in advance by each test hole and the established strain database.
3. Microorganism identification device
The microorganism rapid identification and detection device adopting the method comprises an incubation component, a transmission component, a measurement and detection module, hardware and software. The front incubation and the back interpretation layout are adopted, and the incubation assembly adopts a rotating structure. The incubation assembly can store 30-90 microorganism test cards and has the function of keeping the constant temperature of 35 ℃; mainly detecting the turbidity or the color combination change in the dynamic growth process of the microorganism; the transmission module is used for realizing the periodic transmission of the test card at the incubation and detection positions; the hardware and the software realize the ordered operation of the whole system and achieve the expected purpose.
And (3) rapidly identifying the species of the strains by adopting a bifidus matrix method according to positive and negative results pre-judged in advance by each test hole and the established strain database, thereby realizing rapid identification of the bacteria.
The technical features of the embodiments of the present invention may be combined with each other as long as they do not conflict with each other.
Example 1
In the protein decomposition product test, if the colorless change of brown is positive and the colorless change of brown is negative, such as phenylalanine decarboxylase test (PD test) and the like, the wavelength of 630nm and 450nm are adopted for detection, and the absorbance ratio is determined for interpretation.
TABLE 1 PD TEST SINGLE/DOUBLE WAVE YIN-YANG READING
As can be seen from Table 1, the 450nm single wavelength detection of the positive and negative absorbance data shows a simultaneous monotone increasing trend; the detection is carried out by adopting a single wavelength of 550nm, and the positive absorbance data shows the phenomena of big sequence and small middle; the detection is carried out by adopting a single wavelength of 630nm, the positive presentation trend is increased, and the negative presentation trend is decreased. The main reason for the above change is that the turbidity of the solution changes while the color changes gradually in the enzymatic reaction process, and the change and the mutual influence are caused. And adopting a 630nm and 450nm dual-wavelength ratio and a primary curve and a secondary curve which are fitted by positive data to respectively obtain the slope and the acceleration. The positive color shows obvious monotone increasing trend, the red component is increased (a figure which can not provide color in patent text and can not be seen from the figure of the invention), and the color is gradually changed into brown; the negative absorbance ratio is basically unchanged, namely the color is basically unchanged, so that the negative and positive of the reaction hole can be accurately distinguished.
Example 2
The determination that the yellow color turns red to be positive, does not turn red or turns to orange-red in the enzymatic reaction test, such as an arginine decarboxylase test (ADH test) and the like, adopts 630nm and 550nm for detection.
As can be seen from Table 2, the ADH test is judged to be positive when the color changes from yellow to red, does not change or changes to orange-red, and the color difference change is not obvious because the bottom color of the reaction hole is light yellow, and the color difference change is detected by adopting a single wavelength, for example, the positive and negative absorbance shows an increasing trend when the detection is carried out by adopting a single wavelength of 450 nm; the change trend of negative and positive can be preliminarily reflected by adopting 550nm single-wavelength detection, but the change quantity of OD value is about 2 times, and threshold value distinguishing is difficult to set; the detection is carried out by adopting a single wavelength of 630nm, the positive and negative absorbance is in a descending trend at the same time, and the change is not obvious. The main reason for the above change is that the turbidity of the solution changes while the color changes gradually, and the change and the mutual influence are caused. And adopting a 630nm and 550nm dual-wavelength ratio and a primary curve and a secondary curve which are fitted by positive data to respectively obtain the slope and the acceleration. The positive shows obvious monotonous decreasing trend, namely the red component is increased (a figure which can not provide color in patent text and can not be seen from the figure of the invention), and the yellow is gradually changed into red; the negative absorbance ratio is in a slowly decreasing trend, namely the red component is slightly increased, namely the yellow is gradually changed into orange red, so that the negative and positive of the reaction holes can be accurately distinguished.
Example 3
In the sugar decomposition product test, the interpretation that the green turns yellow to be positive and the blue to be negative, the maltose fermentation test (MAL test) and the like adopt the wavelength detection of 630nm and 450nm to determine the absorbance ratio for interpretation.
TABLE 3 MAL TEST SINGLE/DOUBLE WAVE YIN-YANG INDICATION
As is clear from Table 3, the color change in the MAL test was clear when the color changed from yellow to positive and from blue to negative. The detection is carried out by adopting a single wavelength of 450nm, the positive absorbance presents an increasing trend, and the negative absorbance does not change obviously; the detection is carried out by adopting a single wavelength of 550nm, and the positive and negative absorbances are in a descending trend at the same time; the detection is carried out by adopting a single wavelength of 630nm, the positive and negative absorbance is in a descending trend at the same time, and the change is not obvious. The main reason for the above change is also the sugar fermentation reaction process, and the solution turbidity changes while the color changes gradually, and the mutual influence is generated. And adopting a 630nm and 450nm dual-wavelength ratio and a primary curve and a secondary curve which are fitted by positive data to respectively obtain the slope and the acceleration. The positive shows obvious monotone increasing trend, the red component is increased (the patent text can not provide colored drawings, so the colored drawings can not be seen in the drawings of the invention), namely, the green is gradually changed into yellow; the negative absorbance ratio shows a monotonous increasing trend, the blue component is increased, namely, the green is gradually changed into the blue, so that the negative and positive of the reaction hole can be accurately distinguished.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A dual-wavelength microorganism biochemical reaction dynamic interpretation detection method is characterized in that the method dynamically detects the absorbance of a microorganism biochemical identification test hole in real time by adopting dual wavelengths, and after the ratio of the dual-wavelength absorbance is piecewise fitted, the negative and positive of the reaction hole can be prejudged by solving the speed and the acceleration of a curve, so as to quickly identify the species of bacteria; the method for pre-judging the positivity and negativity of the reaction hole comprises the following steps: solving the ratio of the absorbance of the double wavelengths, fitting a curve of the absorbance in a segmented rolling manner according to 2-10 points, and solving the speed or the acceleration of the absorbance, wherein the positive value is increased or decreased monotonously, and the negative value is basically unchanged or decreased monotonously or increased; the dual wavelength is two of 550nm +/-20 nm, 450nm +/-20 nm and 630nm +/-20 nm.
2. The dual wavelength biochemical reaction dynamic interpretation detection method of claim 1, wherein the biochemical identification test of microorganism is a, no color change yellow test; or b, test for no color change to brown, or c, test for green to blue or yellow, or d, test for yellow to red.
3. The dual wavelength biochemical reaction dynamic interpretation detection method according to claim 1, wherein the biochemical identification test of the microorganism is a β -galactosidase test, or a p-nitroanilino decarboxylase test, or a maltose fermentation test using bromothymol blue as an indicator, or an arginine decarboxylase test using phenol red as an indicator.
4. The method for performing dynamic interpretation and detection of biochemical reactions of microorganisms with dual wavelengths according to claim 1, wherein the data acquisition period in the dynamic real-time detection is 15-30 minutes.
5. The method for detecting the dynamic interpretation of the biochemical reaction of the dual-wavelength microorganisms according to claim 1, wherein the method for pre-judging the positivity and negativity of the reaction wells comprises: and (3) calculating the ratio of the absorbance of the double wavelengths, fitting a curve of the double wavelengths by means of segmented rolling according to 3-7 points, and calculating the speed or the acceleration of the double wavelengths, wherein the positive values are monotonically increased or decreased, and the negative values are basically unchanged or monotonically decreased or increased.
6. The method for detecting the dynamic interpretation of the biochemical reaction of the dual-wavelength microorganisms according to claim 1, wherein the method for pre-judging the positivity and negativity of the reaction wells comprises: and (3) calculating the ratio of the absorbance of the double wavelengths, fitting a curve according to 5 point segmentation rolling, and calculating the speed or the acceleration of the curve, wherein the positive is monotonically increased or decreased, and the negative is basically unchanged or monotonically decreased or increased.
7. The method as claimed in claim 1, wherein said dual wavelength is two of 550nm, 450nm and 630 nm.
8. The dual-wavelength microorganism biochemical reaction dynamic interpretation detection method according to any one of claims 1 to 7, wherein a detection light path adopted by the detection method is composed of a light emitting source (1), a Fresnel lens (2), a stepping wheel (3), an optical filter (4), an optical fiber (5), a diaphragm (7), a 96-hole test card (8), a silicon photocell (9) and a post-processing circuit.
9. The method for the dual-wavelength dynamic interpretation and detection of the biochemical reaction of the microorganism according to claim 8, wherein the light source (1) is a halogen lamp or an LED point light source, the emitted light is focused by the lens (2), filtered by the optical filter, and coupled into the optical fiber (5) having a plurality of paths, the light from the optical fiber is focused by the lens (2) and refracted into the reaction hole of the microorganism identification drug sensitive test card, and the transmitted light is received by the silicon photocell (9) after passing through the diaphragm (7), wherein the optical fiber has 8 to 96 paths, and 1 path represents a detection channel corresponding to a detection hole of the test card.
10. The method for dual-wavelength dynamic interpretation and detection of microbial biochemical reactions according to claim 8, wherein the filter wavelength is 550nm ± 20nm, 450nm ± 20nm, or 630nm ± 20 nm; the optical filter (4) is fixed on the stepping wheel (3) and is driven to switch by a stepping motor.
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