CN114791491A - A method for rapid detection of Staphylococcus aureus using biofilm interference technology - Google Patents

Abstract

The invention discloses a method for rapidly detecting staphylococcus aureus by utilizing a biomembrane interference technology, which comprises the following steps: step one, sensor balancing; secondly, activating a sensor; step three, immobilizing the antibody; step four, sealing once; step five, secondary sealing; and step six, sample detection and result analysis. The invention has the beneficial effects that: the method has the advantages of few steps, no mark, real-time monitoring, simplicity, convenience and rapidness, and simple result judgment; the operation is simple and easy to learn, the prepared reagent and the prepared sample are only added into the sample plate to be detected on a computer, and the existence of staphylococcus aureus can be judged by reading the binding signal in real time; the antibody diluted by the acetic acid-sodium acetate buffer solution can be recycled, so that the detection cost is reduced; the high flux is detected, and the simultaneous detection of up to 95 samples can be realized by matching with a corresponding biomolecule interaction instrument and a matched sample plate; has better specificity, and can eliminate the interference of non-target bacteria in the sample.

Description

A method for rapid detection of Staphylococcus aureus using biofilm interference technology

technical field

The present invention relates to a rapid detection method of Staphylococcus aureus, in particular to a rapid detection method of Staphylococcus aureus by antibody combined with biofilm interference technology.

Background technique

Staphylococcus aureus is a facultative anaerobic gram-positive bacteria that widely exists in nature and human skin. Food is susceptible to its contamination, resulting in human food poisoning, the main symptoms of which are fever, diarrhea and nausea and vomiting. Staphylococcus aureus secretes more than 20 kinds of toxic proteins, of which there are 7 common ones. It has been reported that adults can cause food poisoning by consuming only about 100 ng of enterotoxin A. In recent years, food poisoning incidents caused by Staphylococcus aureus in my country have ranked fourth in the world. At present, the traditional detection method of Staphylococcus aureus has the advantages of high sensitivity and low cost, but the steps of selective pre-enrichment, selective plate culture, staining microscopy and plasma coagulase identification are complicated and time-consuming, which cannot meet the requirements of rapid detection. needs. The detection time of immunological detection methods (such as enzyme-linked immunosorbent technology) is significantly shortened, but the selectivity of reagents is high. Therefore, in order to reduce the occurrence of related foodborne diseases, a novel detection method of Staphylococcus aureus that is fast, convenient, sensitive, accurate and easy to operate needs to be developed.

Biolayer interferometry (BLI) technology realizes the interaction analysis or detection of biomolecules by monitoring the changes of optical interference signals in real time. The Octet used in this technology is a multi-channel detection device, the entire detection process is fully automated, the temperature of the detection module can be controlled, and the samples can be recovered after detection. The technology has the advantages of high sensitivity, no microfluidic system, label-free, rapid, simple, and real-time detection results, and is very suitable for rapid and sensitive detection of food-borne pathogens.

Antibodies are the most widely used recognition elements in biosensors. According to their preparation methods and principles, they can be divided into polyclonal antibodies, monoclonal antibodies and genetically engineered antibodies. Antigen recognition using antibodies is known as immunoassay, and it is used in various biosensor formats. However, there are few reports on the use of antibodies as the biological recognition element of BLI biosensors and the application of this technology to the rapid detection of food-borne pathogens.

In the present invention, the antibody is used as the biological recognition element of the second-generation amino-coupled sensor to recognize and detect Staphylococcus aureus. The second-generation amino-conjugated sensor increases the binding density and stabilizes the curing conditions while substantially reducing nonspecific binding. The sensor surface is suitable for a wide range of pH and salt conditions, providing robustness and flexibility for the development of regeneration conditions for higher throughput applications. The surface of the second-generation amino-coupled sensor is modified with carboxyl groups. When the antibody is immobilized, the carboxyl groups on the surface of the sensor are firstly replaced by 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC ) and N-hydroxysuccinimide (NHS) mixed activation reagent to form NHS ester after activation; NHS ester can react with the amino group on the antibody to form an extremely stable amide bond to fix the antibody on the second-generation amino-coupled connected to the sensor surface. If there is Staphylococcus aureus in the sample, the bacteria will be specifically recognized and bound by the antibody immobilized on the surface, so that the thickness of the optical layer of the sensor increases, the interference spectrum is shifted, and the generated response signal is detected and recorded by the spectrometer in real time to achieve golden yellow Real-time label-free rapid detection of staphylococci.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a rapid detection method of Staphylococcus aureus based on biofilm interference technology in order to solve the problems of many steps and complicated operations in the traditional detection method of Staphylococcus aureus.

The present invention provides a rapid detection method of Staphylococcus aureus by antibody combined with biofilm interference technology. The method includes sensor balance, sensor activation, antibody immobilization, primary blocking, secondary blocking, sample detection and result analysis, and the specific steps are as follows:

Step 1. Sensor balance: first immerse the end of the second-generation amino-coupled sensor in pure water to pre-wet for at least 10 minutes to balance the sensor;

Step 2. Sensor activation: Immerse the equilibrated second-generation amino-coupled sensor in 20 mM 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 10 mM N-hydroxysuccinimide Activated in the mixed reagent for 300-450 seconds;

Step 3. Antibody immobilization: Immerse the activated second-generation amino-coupled sensor in an antibody solution with a concentration of 12.5-100 μg/mL diluted with pH 5-7 acetic acid-sodium acetate buffer solution for 400-900 seconds for immobilization ;

Step 4. One-time blocking with ethanolamine hydrochloride solution: Immerse the second-generation amino-coupled sensor after immobilizing the antibody in ethanolamine hydrochloride solution (pH 8.5, 1mol/L) for 300-450 seconds;

Step 5. Secondary blocking of bovine serum albumin solution: immerse the second-generation amino-coupled sensor after blocking with ethanolamine hydrochloride in 1% bovine serum albumin solution for 300-450 seconds;

Step 6. Sample detection and result analysis: After blocking in bovine serum albumin solution, immerse the second-generation amino-coupled sensor in PBS phosphate buffer solution containing 0.02% Tween-20 for 150-450 seconds, and then equilibrate for 150-450 seconds. It is immersed in the sample to detect Staphylococcus aureus, reads the binding signal in real time, and analyzes the detection result according to the binding signal.

Beneficial effects of the present invention:

The rapid detection method for Staphylococcus aureus based on the biofilm interference technology provided by the invention has few steps, no label, real-time monitoring, simple and quick, and simple result judgment; the operation is simple and easy to learn, and only needs to add the configured reagents to the sample plate and prepare Good samples can be tested on the machine, and the presence of Staphylococcus aureus can be determined by reading the binding signal in real time; the antibody diluted with acetic acid-sodium acetate buffer solution can be recovered and reused to reduce the detection cost; The corresponding biomolecular interaction instrument and the supporting sample plate can realize the simultaneous detection of up to 95 samples; it has good specificity and can eliminate the interference of non-target bacteria in the samples.

Description of drawings

FIG. 1 is a schematic diagram of the specificity detection result of the detection method of the present invention.

Figure 2 is a schematic diagram of the detection of different concentrations of Staphylococcus aureus in the detection method of the present invention.

Detailed ways

The specific examples are written below, and more detailed implementation steps are given. The examples are all implemented according to this technical solution. The experimental methods that do not specify specific conditions in the examples are usually in accordance with conventional conditions or as suggested by the reagent manufacturer. conditions of.

The main materials and reagents used in the examples are shown in Table 1, and the main instruments and equipment are shown in Table 2.

Table 1 Information table of main materials and reagents

Table 2 Main instrument and equipment information table

Embodiment 1: the specificity investigation of this technical scheme

Taking Staphylococcus aureus as the target bacteria, and taking Salmonella, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Listeria monocytogenes as the non-target bacteria for detection and analysis, the specific steps are as follows:

(1) Inoculate Staphylococcus aureus, Salmonella, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Listeria monocytogenes into sterilized tryptone soybean broth medium with an inoculation loop, respectively, at 37°C , 200rpm shaking culture for 15-18 hours. The bacterial suspension was centrifuged, the supernatant was discarded, washed twice with PBS phosphate buffered solution, and then the concentration of the bacterial suspension of the six bacteria was adjusted to 1× with PBS phosphate buffered solution containing 0.02% Tween-20. 10 ⁶ CFU/mL, to be tested.

(2) The mixed reagent of pure water, 20mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10mM N-hydroxysuccinimide was added in sequence to the corresponding position of the 96-well plate, Antibody solution at a concentration of 25 μg/mL diluted in acetic acid-sodium acetate buffer solution at pH 6, ethanolamine hydrochloride solution (pH 8.5, 1 mol/L), 1% bovine serum albumin solution, containing 0.02% Tween-20 PBS phosphate buffered solution and samples to be tested.

(3) Hydrate the second-generation amino-coupled sensor in advance (the end of the sensor is immersed in pure water) for 9 minutes, then put the loaded 96-well plate into the Octet Red 96 molecular interaction instrument, and run the system. The end of the second-generation amino-coupled sensor was immersed sequentially in pure water (60 seconds), 20 mM 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, and 10 mM N-hydroxysuccinimide. Mix reagents (300 sec), antibody solution at a concentration of 25 μg/mL diluted in acetic acid-sodium acetate buffer pH 6 (400 sec), ethanolamine hydrochloride solution (300 sec), 1% bovine serum albumin solution ( 300 seconds), PBS containing 0.02% Tween-20 phosphate buffer solution (180 seconds) and the sample to be tested (900 seconds).

(4) When the instrument is running, the detection signal value can be observed in real time. As shown in Figure 1, the signal value of the target bacteria Staphylococcus aureus sample is far above the effective signal value (0.0075nm), and the non-target bacteria Salmonella, Escherichia coli, subtilis The signal values of Bacillus, Pseudomonas aeruginosa, and Listeria monocytogenes were all negative, indicating that the method had high specificity.

Example 2: Detection of Staphylococcus aureus at different concentrations

(1) Inoculate Staphylococcus aureus into sterilized tryptone soybean broth medium with an inoculation loop, and culture with shaking at 37° C. and 200 rpm for 15-18 hours. The bacterial suspension was centrifuged, the supernatant was discarded, washed twice with PBS phosphate buffered solution, and then the concentration of bacterial suspension was adjusted to 10 ⁷ CFU/mL, 10 ⁶ CFU/mL, 10 ⁵ CFU/mL, to be tested.

Steps (2) (3) are the same as (2) (3) in Example 1.

(4) The detection signal value can be observed in real time when the instrument is running. As shown in Figure 2, the detection signal is significantly positively correlated with the concentration of Staphylococcus aureus, indicating that this method can not only be used for the qualitative detection of Staphylococcus aureus, but also for its quantitative detection.

Claims (1)

1. a method that utilizes biofilm interference technology to rapidly detect Staphylococcus aureus, it is characterized in that: comprise sensor balance, sensor activation, antibody immobilization, primary sealing, secondary sealing, sample detection and result analysis, and its concrete steps are as follows : Step 1. Sensor balance: Immerse the end of the second-generation amino-coupled sensor in pure water for at least 10 minutes to balance the sensor; Step 2. Sensor activation: Immerse the equilibrated second-generation amino-coupled sensor in 20 mM 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 10 mM N-hydroxysuccinimide Activated in mixed reagents for 300-450 seconds; Step 3. Antibody immobilization: Immerse the activated second-generation amino-coupled sensor in an antibody solution with a concentration of 12.5-100 μg/mL diluted with pH 5-7 acetic acid-sodium acetate buffer solution for 400-900 seconds for immobilization ; Step 4. One-time blocking with ethanolamine hydrochloride solution: Immerse the second-generation amino-coupled sensor after immobilizing the antibody in ethanolamine hydrochloride solution (pH 8.5, 1mol/L) for 300-450 seconds; Step 5. Secondary blocking of bovine serum albumin solution: immerse the second-generation amino-coupled sensor after blocking with ethanolamine hydrochloride in 1% bovine serum albumin solution for 300-450 seconds; Step 6. Sample detection and result analysis: After blocking in bovine serum albumin solution, immerse the second-generation amino-coupled sensor in PBS phosphate buffer solution containing 0.02% Tween-20 for 150-450 seconds, and then equilibrate for 150-450 seconds. It is immersed in the sample to detect Staphylococcus aureus, reads the binding signal in real time, and analyzes the detection result according to the binding signal.

Images