阅读说明：本技术 一种血流感染病原阴性菌微量肉汤荧光药敏方法 (Blood stream infection pathogen negative bacteria trace broth fluorescence drug sensitization method ) 是由 赵志军 周云花 杨宁爱 康宇婷 于 2021-01-25 设计创作，主要内容包括：本申请提供一种血流感染病原阴性菌微量肉汤荧光药敏方法,包括：挑取单个菌落于培养基中过夜培养并进行12个梯度设计,测试菌浓度的OD-(625)值；用无菌去离子水将缓冲液稀释,得到稀释后的缓冲液；将20μL ApoH磁珠、缓冲液以及培养后的菌落在37℃细菌培养摇床300rpm孵育15min,得到细菌-ApoH磁珠混合物；将细菌-ApoH磁珠混合物于4℃、12000rpm离心、弃上清以及重悬得到菌悬液；将菌悬液置于MS分离柱上过柱,得到过柱后的反应液；使用反应液进行96孔板荧光药敏试验。本申请提供所述方法检测血流感染病原菌时,由于利用的是磁珠富集原理来进行检测,而且磁珠富集细菌能够提高检测灵敏度,从而提高检测的阳性率和检出率。(The application provides a blood stream infection pathogen negative bacteria trace broth fluorescence drug sensitivity method, which comprises the following steps: single colonies were picked and grown overnight in medium and subjected to 12 gradient design to test OD of bacterial concentration 625 A value; diluting the buffer solution by using sterile deionized water to obtain a diluted buffer solution; incubating 20 mu L of ApoH magnetic beads, buffer solution and cultured colonies for 15min at the temperature of 37 ℃ by a bacteria culture shaker at 300rpm to obtain a bacteria-ApoH magnetic bead mixture; magnetic treatment of bacterium-ApoHCentrifuging the bead mixture at 4 ℃ and 12000rpm, discarding the supernatant and resuspending to obtain a bacterial suspension; putting the bacterial suspension on an MS separation column to pass through the column to obtain reaction liquid after passing through the column; the reaction solution was used to perform a 96-well plate fluorescence susceptibility test. When the method is used for detecting the pathogenic bacteria infected by the blood stream, the detection is carried out by utilizing the magnetic bead enrichment principle, and the detection sensitivity can be improved by enriching bacteria by the magnetic beads, so that the positive rate and the detectable rate of the detection are improved.)

1. A blood stream infection pathogen negative bacteria trace broth fluorescence drug sensitization method is characterized by comprising the following steps:

single colonies were picked and grown overnight in medium and subjected to 12 gradient design to test OD of bacterial concentration₆₂₅A value;

diluting the buffer solution by using sterile deionized water to obtain a diluted buffer solution;

incubating 20 mu L of ApoH magnetic beads, the buffer solution and the cultured colony for 15min at the temperature of 37 ℃ by a bacteria culture shaker at 300rpm to obtain a bacteria-ApoH magnetic bead mixture;

centrifuging the bacterium-ApoH magnetic bead mixture at 12000rpm at 4 ℃, discarding the supernatant and resuspending to obtain a bacterium suspension;

putting the bacterial suspension on an MS separation column to pass through the column to obtain reaction liquid after passing through the column;

the reaction solution was used to perform a 96-well plate fluorescence susceptibility test.

2. The method of claim 1, wherein the single colony is cultured overnight in a culture medium and subjected to 12 gradient design to test OD of bacteria concentration₆₂₅The values include:

picking a single colony of Escherichia coli (ATCC-25922) in an Erlenmeyer flask containing a proper amount of LB broth for shaking overnight, wherein the temperature is 37 ℃ and the rotating speed is 180 rpm/min; then, 5mL of physiological saline with the mass fraction of 0.9 percent is used for carrying out gradient design of bacterial colony amount; the colonies added thereto (CFU/mL) were: 1X 10⁹、1.5×10⁸、1×10⁸、1×10⁷、1×10⁶、1×10⁵、1×10⁴、1×10³、1×10²、1×10¹、1×10⁰And a 0.9% normal saline control group;

OD for measuring concentration of corresponding bacteria by using multifunctional microplate monitor₆₂₅The value is obtained.

3. The method of claim 1, wherein diluting the buffer with sterile deionized water to obtain a diluted buffer comprises diluting 10 XTTGB-10 buffer with sterile deionized water to 2 XTTGB-10 buffer.

4. The method of claim 1, wherein the step of incubating 20 μ L ApoH magnetic beads, the buffer solution and the cultured colony for 15min at 37 ℃ on a bacterial shaker at 300rpm to obtain a bacterial-ApoH magnetic bead mixture comprises:

first, the concentration of the bacteria contained in the culture medium (CFU/mL) was 1X 10⁹、1.5×10⁸、1×10⁸、1×10⁷、1×10⁶、1×10⁵、1×10⁴、1×10³、1×10²、1×10¹、1×10⁰And 0.9% saline control 5mL of 0.9% saline added 5mL of 2 XTGB-10 buffer, then 20. mu.L of ApoH magnetic beads to each respective tube;

the tube was incubated at 37 ℃ for 15min with a bacterial shaker at 300rpm to obtain a bacterial-ApoH magnetic bead mixture.

5. The method of claim 1, wherein the step of centrifuging the bacterium-ApoH magnetic bead mixture at 12000rpm at 4 ℃, discarding the supernatant, and resuspending the bacterium suspension to obtain the fluorescence sensitivity of the blood stream infection pathogen negative bacteria comprises:

centrifuging the bacterium-ApoH magnetic bead mixture at 4 ℃ and 12000rpm for 10min, and removing the supernatant;

resuspending the mixture with the supernatant in 1mL PBS solution with pH 7.4, centrifuging at 12000rpm for 5min under 4 deg.C, and discarding the supernatant;

the suspension after discarding the supernatant was resuspended in 1mL of PBS to obtain a bacterial suspension.

6. The method for fluorescence susceptibility of blood stream infection pathogen negative bacteria broth as claimed in claim 1, wherein the step of placing the bacterial suspension on an MS separation column and passing the bacterial suspension through the column to obtain the reaction solution after passing through the column comprises:

placing the bacterial suspension on an MS separation column, washing the column by using 1mL of washing buffer solution, wherein the reaction solution contains unlabeled bacteria;

taking out the MS separation column from the separation device, placing the MS separation column into a new centrifuge tube, and sucking 1mL of washing buffer solution;

the reaction mixture was centrifuged at 12000rpm at 4 ℃ for 5min, and the supernatant was discarded and then resuspended in 100. mu.L of PBS to obtain a resuspended reaction mixture.

7. The method for performing fluorescence susceptibility testing on blood stream infection pathogen negative bacteria broth as claimed in claim 1, wherein the 96-well plate fluorescence susceptibility testing step using the reaction solution comprises:

taking out antibiotic powder, dissolving and diluting, adding 200 mu L of antibacterial drug with the concentration of 64 mu g/mL into the first hole of the pore plate, respectively adding 100 mu L of MH broth culture medium into the second hole to the eleventh hole, sucking 100 mu L from the first hole, adding the mixture into the second hole, uniformly mixing, sucking 100 mu L to the third hole, and repeating the steps, sucking 100 mu L from the tenth hole and discarding; the drug concentration in each well is as follows: 128. 64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25. mu.g/mL, 200. mu. LMH broth medium was added to column 11 as a negative control, and 200. mu.L of the reaction solution was added to column 12 as a positive control; wherein the concentration of the reaction solution is 1X 10⁵CFU/mL；

The reaction solution was aspirated and its concentration was adjusted to 1X 10 with MH broth⁸CFU/mL, diluted 100-fold with MH broth to reach a reaction concentration of 1X 10⁶CFU/mL, adding 100 mu L of CFU/mL into each concave hole in the drug sensitive plate according to 1-10 rows per hole, so that the final bacterial suspension concentration in each hole is 1 multiplied by 10⁵CFU/mL；

Labeling the antibody with a fluorescent kit;

placing the inoculated drug sensitive plate on37 ℃ and 5% CO₂Culturing in an incubator;

the drug sensitive plate is at 37 ℃ and 5% CO₂After incubation in an incubator for 10h, 5. mu.L of fluorescent antibody was added to each well and the incubation was continued at 37 ℃ and 5% CO₂Incubating in incubator for 30 min;

and (4) detecting the fluorescence intensity by using a multifunctional microplate monitor, and determining the MIC.

Technical Field

The application relates to the technical field of pathogenic microorganisms, in particular to a blood stream infection pathogenic negative bacteria trace broth fluorescence drug sensitization method.

Background

Blood Stream Infection (BSI) refers to a serious systemic infectious disease in which various pathogenic microorganisms (including bacteria, viruses or fungi) invade the blood, multiply in the blood and produce and release metabolites such as toxins, and induce cytokine release, causing systemic infection, poisoning and inflammatory reactions, and possibly even causing changes in the coagulation and fibrinolysis systems, resulting in systemic multiple organ dysfunction syndrome. Therefore, control of bloodstream infections is receiving increasing attention.

Generally, the blood stream infection is more dangerous, if the anti-infection treatment cannot be carried out in time, the prognosis is poorer, the morbidity and the mortality of clinical patients can be increased, and researches show that the early diagnosis can reduce the disease mortality caused by the blood stream infection. Bloodstream infections are a time critical medical emergency and should be as urgent as wound care or myocardial infarction with a definite response time. Antibiotic Susceptibility Test (AST) is a currently accepted method for determining bacterial susceptibility to antibiotics by determining the efficacy of antibacterial agents in inhibiting the growth of pathogenic microorganisms in vitro. Therefore, after bacteria are captured, dead bacteria floating in the supernatant are removed by using a density gradient centrifugation method, and in order to achieve the purpose of rapid detection and treatment of blood stream infection, only the fact that a patient has blood stream infection is proved, an antibacterial drug sensitivity test result is rapidly given, then the rapid fluorescence culture of trace broth is directly carried out, and the purpose of purifying the bacteria by picking a single colony for culture is not needed.

At present, the methods for carrying out drug sensitivity tests in clinical microbiological laboratories mainly comprise a paper diffusion method, a dilution method, an antibiotic concentration method, an automatic instrument and the like. Many clinical laboratories use the paper diffusion method as a routine drug susceptibility test, which has the disadvantage of not being suitable for slow growing strains and some fastidious bacteria; the main disadvantage of the dilution method is the cumbersome operation; the cost of the antibiotic concentration method is too high to be suitable for routine test application; automated instruments require instrumentation to perform. These methods have low blood culture positive rate and detection rate for pathogenic bacteria of clinical bloodstream infection, the positive rate is about 9%, the detection rate is about 85%, and blood culture is mainly used for diagnosing bloodstream infection, but the total turnover time of traditional antibacterial drug sensitivity detection of blood samples in positive bloody culture bottles of patients with bloodstream infection is more than three days, because the method needs three overnight culture steps: blood culture, subculture, and antibiotic susceptibility test culture, so that treatment is delayed and contamination by microorganisms is easy. Approximately 30% -50% of blood culture results show negatives in those patients who appear to have an infection. These negative blood cultures may be due to low pathogen counts or the presence of non-culturable infectious agents in the blood samples. And in the positive blood culture bottle, the bacteria are mixed with a large amount of blood cells, and the concentration of the bacteria is within 10⁷To 10⁹CFU/mL, unpredictable. Since conventional AST systems detect changes in the bacterial population by measuring the Optical Density (OD) of antibiotic-administered pathogen cultures, they are very sensitively dependent on the amount of the initial inoculum strain, and the initial concentration of bacteria should be strictly controlled. Therefore, blood culture positive bottles cannot be used directly for antibiotic susceptibility testing using conventional methods, and subculture on agar plates is required to obtain pure strains at accurate concentrations in order to perform an overnight antibiotic susceptibility testing process using bacterial stock. Due to the low detection sensitivity, a certain contamination rate exists, so that the diagnosis and treatment of early blood stream infection are influenced.

Disclosure of Invention

The application provides a blood stream infection pathogenic negative bacteria trace broth fluorescence drug sensitivity method, which aims to solve the problems that the diagnosis and treatment of early blood stream infection are influenced due to low detection sensitivity and certain pollution rate.

The application provides a blood stream infection pathogen negative bacteria trace broth fluorescence drug sensitivity method, which comprises the following steps:

single colonies were picked and grown overnight in medium and subjected to 12 gradient design to test OD of bacterial concentration₆₂₅A value;

diluting the buffer solution by using sterile deionized water to obtain a diluted buffer solution;

incubating 20 mu L of ApoH magnetic beads, the buffer solution and the cultured colony for 15min at the temperature of 37 ℃ by a bacteria culture shaker at 300rpm to obtain a bacteria-ApoH magnetic bead mixture;

centrifuging the bacterium-ApoH magnetic bead mixture at 12000rpm at 4 ℃, discarding the supernatant and resuspending to obtain a bacterium suspension;

putting the bacterial suspension on an MS separation column to pass through the column to obtain reaction liquid after passing through the column;

the reaction solution was used to perform a 96-well plate fluorescence susceptibility test.

Alternatively, single colonies were picked and grown overnight in medium and subjected to 12 gradient design to test the OD of the bacterial concentration₆₂₅The values include:

experiment picking single colony of Escherichia coli (ATCC-25922) in an erlenmeyer flask filled with a proper amount of LB broth culture medium, shaking for overnight, wherein the temperature is 37 ℃ and the rotating speed is 180 rpm/min; then, 5mL of physiological saline with the mass fraction of 0.9 percent is used for carrying out gradient design of bacterial colony amount; the colonies added thereto (CFU/mL) were: 1X 10⁹、1.5×10⁸、1×10⁸、1×10⁷、1×10⁶、1×10⁵、1×10⁴、1×10³、1×10²、1×10¹、1×10⁰And a 0.9% normal saline control group;

OD for measuring concentration of corresponding bacteria by using multifunctional microplate monitor₆₂₅The value is obtained.

Optionally, the buffer solution is diluted with sterile deionized water to obtain a diluted buffer solution, which includes diluting the 10 × TTGB-10 buffer solution with sterile deionized water to 2 × TTGB-10 buffer solution.

Optionally, the step of incubating 20 μ L of ApoH magnetic beads, the buffer and the cultured colony for 15min at 37 ℃ in a bacterial shaker at 300rpm to obtain a bacterial-ApoH magnetic bead mixture comprises:

first, the concentration of the bacteria contained in the culture medium (CFU/mL) was 1X 10⁹、1.5×10⁸、1×10⁸、1×10⁷、1×10⁶、1×10⁵、1×10⁴、1×10³、1×10²、1×10¹、1×10⁰And 0.9% saline control 5mL of 0.9% saline added 5mL of 2 XTGB-10 buffer, then 20. mu.L of ApoH magnetic beads to each respective tube;

the tube was incubated at 37 ℃ for 15min with a bacterial shaker at 300rpm to obtain a bacterial-ApoH magnetic bead mixture.

Optionally, the step of centrifuging the bacterium-ApoH magnetic bead mixture at 12000rpm at 4 ℃, discarding the supernatant, and resuspending to obtain a bacterial suspension comprises:

centrifuging the bacterium-ApoH magnetic bead mixture at 4 ℃ and 12000rpm for 10min, and removing the supernatant;

resuspending the mixture with the supernatant in 1mL PBS solution with pH 7.4, centrifuging at 12000rpm for 5min under 4 deg.C, and discarding the supernatant;

the suspension after discarding the supernatant was resuspended in 1mL of PBS to obtain a bacterial suspension.

Optionally, the step of placing the bacterial suspension on an MS separation column and passing through the column to obtain a reaction solution after passing through the column comprises:

placing the bacterial suspension on an MS separation column, washing the column by using 1mL of washing buffer solution, wherein the reaction solution contains unlabeled bacteria;

taking out the MS separation column from the separation device, placing the MS separation column into a new centrifuge tube, sucking 1mL of washing buffer solution, and reacting to obtain the required components;

the reaction mixture was centrifuged at 12000rpm at 4 ℃ for 5min, and the supernatant was discarded and then resuspended in 100. mu.L of PBS to obtain a resuspended reaction mixture.

Optionally, the step of performing a 96-well plate fluorescence drug sensitivity test by using the reaction solution includes:

taking out antibiotic powder, dissolving and diluting, adding 200 mu L of antibacterial drug with the concentration of 64 mu g/mL into the first hole of the pore plate, respectively adding 100 mu L of MH broth culture medium into the second hole to the eleventh hole, sucking 100 mu L from the first hole, adding the mixture into the second hole, uniformly mixing, sucking 100 mu L to the third hole, and repeating the steps, sucking 100 mu L from the tenth hole and discarding; the drug concentration in each well is as follows: 128. 64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25. mu.g/mL, 200. mu. LMH broth medium was added to column 11 as a negative control, and 200. mu.L of the reaction solution was added to column 12 as a positive control; wherein the concentration of the reaction solution is 1X 10⁵CFU/mL；

The reaction solution was aspirated and its concentration was adjusted to 1X 10 with MH broth⁸CFU/mL, diluted 100-fold with MH broth to reach a reaction concentration of 1X 10⁶CFU/mL, adding 100 mu L of CFU/mL into each concave hole in the drug sensitive plate according to 1-10 rows per hole, so that the final bacterial suspension concentration in each hole is 1 multiplied by 10⁵CFU/mL；

Labeling the antibody with a fluorescent kit;

placing the inoculated drug sensitive plate at 37 ℃ and 5% CO₂Culturing in an incubator;

the drug sensitive plate is at 37 ℃ and 5% CO₂After incubation in an incubator for 10h, 5. mu.L of fluorescent antibody was added to each well and the incubation was continued at 37 ℃ and 5% CO₂Incubating in incubator for 30 min;

and (4) detecting the fluorescence intensity by using a multifunctional microplate monitor, and determining the MIC.

According to the technical scheme, the application provides a blood stream infection pathogen negative bacteria trace broth fluorescence drug sensitization method, which comprises the following steps: single colonies were picked and grown overnight in medium and subjected to 12 gradient design to test OD of bacterial concentration₆₂₅A value; diluting the buffer solution by using sterile deionized water to obtain a diluted buffer solution; incubating 20 mu L of ApoH magnetic beads, the buffer solution and the cultured colony for 15min at the temperature of 37 ℃ by a bacteria culture shaker at 300rpm to obtain a bacteria-ApoH magnetic bead mixture; centrifuging the bacterium-ApoH magnetic bead mixture at 4 ℃ and 12000rpm, discarding the supernatant and resuspending to obtain a bacterium suspension; placing the bacterial suspension on an MS separation columnPassing through a column to obtain a reaction solution after passing through the column; the reaction solution was used to perform a 96-well plate fluorescence susceptibility test.

According to the method, when pathogenic bacteria infected by blood flow are detected, overnight culture and flat plate purification culture of the blood specimen are not needed after blood collection, and bacteria are enriched from the blood specimen by applying ApoH magnetic beads directly by using the magnetic bead enrichment principle.

When the method is used for detecting the pathogenic bacteria infected by the blood stream, the magnetic bead enrichment principle is utilized for detection, and the magnetic bead enrichment bacteria can improve the detection sensitivity, so that the detection positive rate and the detection rate are improved, and the detection method is improved compared with the traditional antibacterial drug sensitivity detection system.

The method for detecting the pathogenic bacteria of the bloodstream infection is based on the direct magnetic bead enrichment principle, and by applying the combination of the ApoH magnetic beads and the bacteria in the blood sample, only the bacterial infection in the blood sample is detected and the drug sensitive result can be determined, so that the clinical treatment can be carried out, and the antibacterial drug sensitive test can be directly carried out without the need of bacterial purification.

In the process of detecting the blood stream infection pathogenic bacteria by the method provided by the application, the density gradient centrifugation method is utilized to deposit the live bacteria, so that the live bacteria and the dead bacteria are distinguished, and the dead bacteria are removed.

The method provided by the application is used for carrying out the drug sensitivity test of the pathogenic bacteria of the bloodstream infection, and the observation of the drug sensitivity result is easier by observing the change of the fluorescence intensity value.

The total turnover time from blood sampling to antibiotic susceptibility results for detecting bloodstream infection pathogens by the methods provided herein is within about 13 hours; the total turnaround time from positive blood culture to antibiotic susceptibility was approximately 26 h.

Drawings

In order to more clearly explain the technical solution of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious to those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a schematic flow chart of a blood stream infection pathogen negative bacteria broth fluorescence drug sensitivity method provided by the present application;

FIG. 2 is a graph comparing conventional bloodstream infection detection with the methods provided herein.

Detailed Description

Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, like numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following examples do not represent all embodiments consistent with the present application. But merely as exemplifications of systems and methods consistent with certain aspects of the application, as recited in the claims.

Referring to fig. 1, a schematic flow chart of a blood stream infection pathogen negative bacteria trace broth fluorescence drug sensitivity method is provided for the present application.

The application provides a blood stream infection pathogen negative bacteria trace broth fluorescence drug sensitivity method, which comprises the following steps:

1, weighing bacteria, putting the bacteria into a culture medium for culture, carrying out 12 gradient designs, and testing the OD of the concentration of the bacteria₆₂₅A value;

picking a single colony of Escherichia coli (ATCC-25922) in an Erlenmeyer flask containing a proper amount of LB broth for shaking overnight, wherein the temperature is 37 ℃ and the rotating speed is 180 rpm/min; then, 5mL of physiological saline with the mass fraction of 0.9 percent is used for carrying out gradient design of bacterial colony amount; the colonies added thereto (CFU/mL) were: 1X 10⁹、1.5×10⁸、1×10⁸、1×10⁷、1×10⁶、1×10⁵、1×10⁴、1×10³、1×10²、1×10¹、1×10⁰And a 0.9% normal saline control group;

OD for measuring concentration of corresponding bacteria by using multifunctional microplate monitor₆₂₅The value is obtained.

2, diluting the buffer solution by using sterile deionized water to obtain a diluted buffer solution;

diluting the buffer solution with sterile deionized water to obtain diluted buffer solution, wherein the diluting of the 10 XTGB-10 buffer solution to 2 XTGB-10 buffer solution with sterile deionized water is included.

3, incubating 20 mu L of ApoH magnetic beads, the buffer solution and the cultured colony for 15min at the temperature of 37 ℃ by a bacteria culture shaker at 300rpm to obtain a bacteria-ApoH magnetic bead mixture;

firstly, the concentration of the contained bacteria is 1 multiplied by 10⁹、1.5×10⁸、1×10⁸、1×10⁷、1×10⁶、1×10⁵、1×10⁴、1×10³、1×10²、1×10¹、1×10⁰And 0.9% saline control 5mL of 2 × TTGB-10 buffer in 0.9% saline, then 20 μ L of ApoH magnetic beads to each respective tube;

the tube was incubated at 37 ℃ for 15min with a bacterial shaker at 300rpm to obtain a bacterial-ApoH magnetic bead mixture.

4, centrifuging the bacterium-ApoH magnetic bead mixture at 4 ℃ and 12000rpm, discarding the supernatant and carrying out resuspension to obtain a bacterium suspension;

centrifuging the bacterium-ApoH magnetic bead mixture at 4 ℃ and 12000rpm for 10min, and removing the supernatant;

resuspending the mixture with the supernatant in 1mL PBS solution with pH 7.4, centrifuging at 12000rpm for 5min under 4 deg.C, and discarding the supernatant;

the suspension after discarding the supernatant was resuspended in 1mL of PBS to obtain a bacterial suspension.

5, placing the bacterial suspension on an MS separation column to pass through the column to obtain a reaction solution after passing through the column;

placing the bacterial suspension on an MS separation column, enabling the liquid to flow down naturally, enabling the reaction liquid to contain unlabeled bacteria, and washing the column by using 1mL of washing buffer liquid;

taking out the MS separation column from the separation device, placing the MS separation column into a new centrifuge tube, sucking 1mL of washing buffer solution, and reacting to obtain the required components;

the reaction mixture was centrifuged at 12000rpm at 4 ℃ for 5min, and the supernatant was discarded and then resuspended in 100. mu.L of PBS to obtain a reaction mixture.

6, a 96-well plate fluorescence drug sensitivity test was performed using the reaction solution.

Taking out antibiotic powder, dissolving and diluting, adding 200 μ L antibacterial agent with concentration of 64 μ g/mL into the first hole of the hole plate, and adding the second to the tenthAdding 100 μ L MH broth into one well, sucking 100 μ L from the first well, adding into the second well, mixing, sucking 100 μ L to the third well, repeating above steps, sucking 100 μ L from the tenth well, and discarding; the drug concentration in each well is as follows: 128. 64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25. mu.g/mL, 200. mu. LMH broth medium was added to column 11 as a negative control, and 200. mu.L of the reaction solution was added to column 12 as a positive control; wherein the concentration of the reaction solution is 1X 10⁵CFU/mL；

The reaction solution was aspirated and its concentration was adjusted to 1X 10 with MH broth⁸CFU/mL, diluted 100-fold with MH broth to reach a reaction concentration of 1X 10⁶CFU/mL, adding 100 mu L of CFU/mL into each concave hole in the drug sensitive plate according to 1-10 rows per hole, so that the final bacterial suspension concentration in each hole is 1 multiplied by 10⁵CFU/mL；

Labeling the antibody with a fluorescent kit;

placing the inoculated drug sensitive plate at 37 ℃ and 5% CO₂Culturing in an incubator;

the drug sensitive plate is at 37 ℃ and 5% CO₂After incubation in an incubator for 10h, 5. mu.L of fluorescent antibody was added to each well and the incubation was continued at 37 ℃ and 5% CO₂Incubating in incubator for 30 min;

and (3) detecting the fluorescence intensity by using a multifunctional microplate monitor, and determining the MIC (minimum inhibitory concentration).

Examples

Selecting standard strain Escherichia coli ATCC25922 to carry out rapid fluorescence drug sensitivity test of blood stream infection pathogenic negative bacteria trace broth, putting the trace broth drug sensitivity plate inoculated with gentamicin, tetracycline, ciprofloxacin and imipenem into a 35 ℃ incubator to be respectively cultured for 4h, 6h, 8h and 10h, then taking out, adding 5 mu L of Lipid A LPS Polyclonal Antibody (LPS Polyclonal Antibody) (1: 30) marked by Alexa Fluor Protein Labeling Kit (Alexa Fluor 488 Protein Labeling Kit) into each hole, continuously culturing for 30min at 35 ℃, and detecting the fluorescence intensity value by using a multifunctional microplate monitor to determine MIC. The changes of fluorescence intensity values of gentamicin, tetracycline, ciprofloxacin and imipenem drug sensitive holes at different culture time points are shown in table 1, it was found by examination that the fluorescence intensity values of MH broth inoculated with gentamicin, tetracycline, ciprofloxacin and imipenem were substantially gradually decreased from 4h to 10h of incubation until 10h of incubation in an incubator at 35 deg.C, after further incubation for 30min with 5. mu.l of fluorescent antibody, the drug sensitive wells with fluorescence intensity value of 0 mean that no bacteria survive in the drug sensitive wells containing antibiotics, and the fluorescence intensity value is basically gradually reduced along with the increase of the drug concentration, which indicates that the amount of bacteria is gradually reduced, according to CLSIM100 files, the results are sensitive, wherein the MIC of gentamicin is less than or equal to 4 mug/mL, the MIC of tetracycline is less than or equal to 4 mug/mL, the MIC of ciprofloxacin is less than or equal to 1 mug/mL, and the MIC of imipenem is less than or equal to 1 mug/mL.

TABLE 1 statistics of the results of the fluorescence susceptibility test on Escherichia coli ATCC-25922 susceptibility

According to the technical scheme, the application provides a blood stream infection pathogen negative bacteria trace broth fluorescence drug sensitization method, which comprises the following steps: single colonies were picked and grown overnight in medium and subjected to 12 gradient design to test OD of bacterial concentration₆₂₅A value; diluting the buffer solution by using sterile deionized water to obtain a diluted buffer solution; incubating 20 mu L of ApoH magnetic beads, the buffer solution and the cultured colony for 15min at the temperature of 37 ℃ by a bacteria culture shaker at 300rpm to obtain a bacteria-ApoH magnetic bead mixture; centrifuging the bacterium-ApoH magnetic bead mixture at 4 ℃ and 12000rpm, discarding the supernatant and resuspending to obtain a bacterium suspension; putting the bacterial suspension on an MS separation column to pass through the column to obtain reaction liquid after passing through the column; the reaction solution was used to perform a 96-well plate fluorescence susceptibility test.

In order to reduce the total turnaround time (TAT) of the current antibiotic drug resistance detection process, the method mainly aims to process the inoculation strain quantity with a wide dynamic range from a blood culture positive bottle under the condition of no separation and purification culture process to develop a rapid fluorescence drug sensitive system for pathogenic bacteria trace broth of blood stream infection, the total turnaround time from blood culture to antibacterial drug sensitive test is expected to be 27 hours under the condition of not influencing the accuracy of detection results, and the total turnaround time is shortened by 38 hours compared with the total turnaround time of the current clinical traditional antibacterial drug sensitive detection for pathogenic bacteria of blood stream infection; and the MIC is judged by detecting the fluorescence intensity value, so that the method is simpler and more convenient.

When the method is used for detecting the pathogenic bacteria infected by the blood stream, the magnetic bead enrichment principle is utilized for detection, and the magnetic bead enrichment bacteria can improve the detection sensitivity, so that the detection positive rate and the detection rate are improved, and the detection method is improved compared with the traditional antibacterial drug sensitivity detection system.

The method for detecting the pathogenic bacteria of the bloodstream infection is based on the direct magnetic bead enrichment principle, and by applying the combination of the ApoH magnetic beads and the bacteria in the blood sample, only the bacterial infection in the blood sample is detected and the drug sensitive result can be determined, so that the clinical treatment can be carried out, and the antibacterial drug sensitive test can be directly carried out without the need of bacterial purification.

The present application utilizes ApoH magnetic beads to enrich bacteria in a 5mL blood culture flask within 20 minutes (pathogen bound to ApoH magnetic beads for 15min, bead wash for 5min) and also has a detection limit of 1 CFU/mL. In the traditional trace broth dilution method, the pathogenic bacteria and the antibiotics are co-cultured for about 20h, the broth turbidity is observed, and the broth OD value is detected to judge the MIC value, while in the application, the pathogenic bacteria and the antibiotics are quickly co-cultured for 10h, then the fluorescent antibody is added for incubation for 0.5h, the fluorescent antibody is utilized to specifically bind the pathogenic bacteria, and the MIC value is determined by detecting the fluorescence intensity value, so that the time is saved, and the intuition is realized. FIG. 2 is a graph comparing conventional bloodstream infection detection with the methods provided herein. The total turnover time from blood sampling to antibiotic susceptibility results for detecting bloodstream infection pathogens by the methods provided herein is within about 13 hours; the total turnaround time from positive blood culture to antibiotic susceptibility was approximately 26 h.

The embodiments provided in the present application are only a few examples of the general concept of the present application, and do not limit the scope of the present application. Any other embodiments extended according to the scheme of the present application without inventive efforts will be within the scope of protection of the present application for a person skilled in the art.