Detection method oriented to microbial drug sensitive test and based on threshold time and total number of viable bacterial colonies combined calibration curve

Abstract

The invention provides a detection method based on a combined calibration curve of threshold time (TT) and total number of viable bacterial colonies (TVC) for a microbial drug sensitive test. A microbial growth curve is obtained by detecting carbon dioxide (CO2) released by microbial respiration, and a TT-TVC calibration curve is further constructed to determine the longest culture time (TTmax) of a drug sensitive test (AST). The method is based on a tunable semiconductor laser absorption spectrum (TDLAS) technology, has the characteristics of high sensitivity, strong anti-interference capability and high response speed, and can avoid pollution and remarkably shorten the detection period. Staphylococcus sciuri is taken as a test object, wide applicability and accuracy of the method are verified, and the minimum inhibitory concentration (MIC) of the method is measured by combining with an ampicillin solution.

Description

Technical Field

[0001] The invention relates to the field of clinical microbiology and drug sensitivity detection, and in particular to a detection method for a microbial drug sensitivity test based on a threshold time and a combined calibration curve of the total number of viable bacterial colonies. Background Art

[0002] Antimicrobial resistance (AMR) has been identified as one of the three major factors threatening human health. AMR not only leads to prolonged disease course, increased complications, increased risk of death, etc., but also has many adverse effects on the healthcare system and social economy, including prolonged hospital stay and increased medical expenses.

[0003] Studies have shown that the misuse and abuse of antimicrobial drugs is the primary cause of AMR. Antibiotics are used comprehensively, continuously and for a long time in clinical medicine, animal husbandry and animal disease prevention and control. In particular, the drug sensitivity test (AST), which is used to determine the ability of drugs to inhibit or kill microorganisms under in vitro conditions and is usually used to guide the clinical selection of antibiotic types and determine dosages, is usually time-consuming. Clinicians often prescribe based on empirical diagnosis without waiting for the test results. This has led to an increase in the types and numbers of drug-resistant microorganisms, especially highly resistant bacteria, and the degree of drug resistance has continued to worsen, seriously threatening human life and health safety, and has attracted the attention of various fields around the world.

[0004] Currently, common AST detection methods include paper diffusion method, dilution method (including agar and broth dilution method), antibiotic concentration gradient method (E-test method), polymerase chain reaction (PCR), etc. However, there are problems such as long measurement cycle, unstable data and complicated detection procedures.

[0005] Therefore, there is an urgent need to provide a new detection method to solve the problem of time-consuming microbial drug sensitivity experiments. Summary of the invention

[0006] The purpose of the present invention is to provide a microbial drug sensitivity test method that can be simultaneously applicable to specific microbial drug sensitivity tests and viable bacteria count tests, does not require the addition of a third-party medium, has a short response time, is simple to operate, has strong anti-interference ability, and does not require frequent calibration to systematically detect the target microorganisms, and can solve the problems of traditional drug sensitivity tests that are time-consuming.

[0007] To achieve the above object, the present invention provides the following solutions: (1) First, we start with the metabolites of microbial growth and use the changes in carbon dioxide (CO2), a product of microbial metabolism, to represent the growth status of microorganisms. By continuously monitoring the CO2 concentration produced by microorganisms based on tunable semiconductor laser absorption spectroscopy (TDLAS) technology, we can obtain the growth curves of microorganisms under different concentration gradients.

[0008] (2) Subsequently, in order to determine a good linear relationship between the total viable colony count (TVC) and the threshold time (TT) and to improve the detection time and efficiency, it is necessary to determine an optimal signal value as the threshold and measure the time when the CO2 concentration reaches this threshold, thereby establishing a calibration relationship between TVC and TT.

[0009] (3) The possibility of a calibration relationship between TVC and TT in different samples was investigated using a general form of linear model:

[0010] in [h] represents the threshold time, [log10(CFU / mL)] represents the total number of colonies in the sample (i.e. the total microbial content in the sample). represents the overall mean of the overall threshold time, is the measurement error.

[0011] Due to the variability of the sample, the error is usually negligible, so the calculation relationship between TVC and TT in the sample is as follows:

[0012] in and It can be obtained from the calibration curve in Eq.

[0013] (4) From the formula, it can be obtained that when TVC, i.e., the microbial content in the culture bottle, approaches 0, , the threshold time at this time can be defined as TT max , that is, when the TT corresponding to the intercept is exceeded max When the CO2 concentration still does not reach the threshold, it can be determined that the microbial content in the culture bottle is close to 0. At this time, TT max It is the longest culture time of AST for this type of microorganism.

[0014] That is, the present invention is an AST detection method for shortening the detection time by constructing a TT-TVC function curve, which is characterized by comprising: obtaining a microbial growth curve in a culture bottle by detecting CO2 released by microbial respiration, and then obtaining the longest AST culture time TT by constructing a microbial TT-TVC function curve. max .

[0015] The AST detection method of the present invention does not need to add a third-party medium during the test process, thus avoiding contamination during the production process to a large extent, thereby ensuring the accuracy of the results. In addition, since a specific TT-TVC function curve for each strain is constructed, the step of ensuring complete cultivation of microorganisms is eliminated, so that the entire AST cycle can be greatly shortened and can be applied to multiple strains and multiple environments, with a wide range of applicable objects and high versatility. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a workflow diagram of the AST detection method based on the TT and TVC combined calibration curve of the present invention.

[0017] Figure 2 In the embodiment, the growth of Staphylococcus sciuri is measured at different concentrations using a Staphylococcus sciuri solution as a test sample.

[0018] Figure 3 In the embodiment, different spectrum signal values ​​are selected as thresholds, ranging from 0.25 V to 1.50 V, with a step length of 0.25 V. Relationship between TT and TVC at different thresholds.

[0019] Figure 4 In the embodiment, the squared residual and the coefficient of determination between the measured data and the fitted data are obtained at different thresholds.

[0020] Figure 5 TT-TVC function curve of Staphylococcus australis at the optimal threshold (0.75 V) in the embodiment.

[0021] Figure 6 In the example, it is a typical graph of the growth of Staphylococcus sciuri with an initial concentration of 0.5 MGH in a culture medium with different concentrations of ampicillin added. DETAILED DESCRIPTION

[0022] The method of the present invention is an AST detection method for shortening the detection time by constructing a TT-TVC function curve, characterized in that the method comprises obtaining a microbial growth curve in a culture bottle by detecting CO2 released by microbial respiration, and then obtaining the longest AST culture time TT by constructing a microbial TT-TVC curve. max .

[0023] Hereinafter, the present invention will be described in more detail based on examples etc. It should be noted that the following examples are used in the present invention, but are not intended to limit the scope of the present invention.

[0024] Step 1: Test strains Staphylococcus squirrelii ( Staphylococcus sciuri): IFO 12022 Step 2: Preparation of bacterial suspension Staphylococcus australis was cultured in nutrient broth (NB) medium. Under sterile conditions, the isolated strain of Staphylococcus australis was placed on mannitol sodium chloride medium and incubated at 37°C for 24 h. A typical colony of Staphylococcus australis was selected and placed in nutrient broth medium, and cultured in a constant temperature shaker at 37°C for 24 hours to obtain the initial bacterial solution for use.

[0025] Step 3: Preparation of inoculum Take 1 ml of the initial bacterial solution and count it by dilution plate method to get the initial bacterial solution of 1.62×10 8 CFU / ml. Then, 1 ml of the initial bacterial solution was placed in a test tube with 9 ml of nutrient broth medium and shaken well. Repeat the above operation 5 times to obtain 1.62×10 7 CFU / ml, 1.62×10 6 CFU / ml, 1.62×10 5 CFU / ml, 1.62×10 4 CFU / ml, 1.62×10 3 CFU / ml of five different concentrations of S. squirrelis bacterial solutions. Before the experiment, the bacterial suspension was kept in a refrigerator at a constant temperature of 4°C. In the experiment, 1 mL of S. squirrelis solution with different initial concentrations was added to a culture bottle containing 9 mL of nutrient broth medium and incubated at 37°C for growth detection. The WM-TDLAS technology was used to continuously monitor the changes in carbon dioxide concentration in the culture bottle to obtain the growth curves of S. squirrelis with different concentration gradients.

[0026] Step 4: Construct the TT-TVC curve of Staphylococcus aureus (1) In order to determine the optimal threshold, the signal value of 0.25~1.5 V (interval of 0.25 V) was used as the threshold, and the residual sum of squares (SSR) and determination coefficient (R) between the measured data line and the linear fitting data at different thresholds were calculated. 2 ), thus selecting the optimal threshold as 0.75V.

[0027] (2) Under the optimal threshold conditions, the TT-TVC function curve of Staphylococcus aureus was constructed through a general linear model.

[0028]

[0029] in [h] represents the threshold time [ ] represents the total colony count of the sample (i.e. the total microbial content in the sample).

[0030] (3) According to the formula It can be seen that when TVC, i.e. the microbial content in the culture bottle, approaches 0, , the threshold time at this time can be defined as TT max , at this time TT max This is the longest culture time of AST.

[0031] Step 5: Determination of the minimum inhibitory concentration (MIC) value of Staphylococcus aureus against ampicillin 1 mL of a standard Staphylococcus aureus solution with an initial bacterial concentration of 0.5 MCF and 1 mL of ampicillin solution of different concentrations were added to a culture bottle containing 8 mL of nutrient broth (NB) medium and incubated at 37°C for growth monitoring. Continuous monitoring After 1 hour, if the carbon dioxide content in the culture bottle still does not reach the threshold value, it can be determined that there is no growth of Staphylococcus australis in the culture bottle. The minimum ampicillin concentration that meets this condition is the MIC value of Staphylococcus australis to ampicillin.

Claims

1. A detection method for microbial drug sensitivity test based on threshold time and total viable bacterial colony count combined calibration curve, characterized in that: The growth curve of the microorganisms in the culture bottle is obtained by detecting the carbon dioxide released by the respiration of the microorganisms. The sensitivity of carbon dioxide detection can reach the ppm level, ensuring real-time and accurate monitoring of the growth status of the microorganisms. A function curve of the threshold time of the microorganism and the total number of viable colonies is constructed to determine the maximum culture time of the microorganism's drug sensitivity test.

2. The detection method according to claim 1, characterized in that: Based on tunable semiconductor laser absorption spectroscopy technology, the changes in CO2 concentration in the culture bottle are continuously monitored to indicate the growth status of microorganisms.

3. The detection method according to claim 1 or 2, characterized in that: The TT-TVC calibration curve was constructed by a general form linear model and the signal value was used as the threshold to determine the optimal threshold.

4. The detection method according to claim 3, characterized in that: The residual sum of squares and determination coefficient methods were used to evaluate the calibration curve error under different threshold conditions by calculating the sum of square differences between the measured data and the fitted data and the proportion of the total variation explained by the fitted model, thereby determining the optimal threshold signal value and ensuring the accuracy and stability of the calibration.

5. The detection method according to any one of claims 1 to 4, characterized in that: When TTmax exceeds the intercept value of the calibration curve, it is determined that the content of microorganisms in the culture bottle is close to zero.

6. The detection method according to any one of claims 1 to 5, characterized in that: This method is applicable to a variety of microorganisms and a variety of culture conditions, and does not require the introduction of third-party media, thus avoiding the risk of contamination.

7. The detection method according to any one of claims 1 to 6, characterized in that: The wide applicability and accuracy of this method were verified by detecting the TT-TVC function curve of Staphylococcus aureus.

8. The detection method according to claim 7, characterized in that: The minimum inhibitory concentration of Staphylococcus aureus was determined by TT-TVC function curve using ampicillin solutions of different concentrations.

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