阅读说明：本技术 一种鉴别临床多种多重耐药细菌的荧光检测法 (Fluorescence detection method for identifying multiple clinical drug-resistant bacteria ) 是由 徐兆超 龙双双 苗露 于 2018-07-16 设计创作，主要内容包括：本发明提供一种鉴别临床多种多重耐药细菌的荧光检测法,属于生物分析检测领域。该方法通过单一小分子荧光探针实现多种临床分离的耐药菌的鉴别,具体按照如下步骤进行：(1)细菌培养；(2)细菌收集：在10000-14000rpm条件下离心5-10分钟,弃上清,用20mM、pH＝7.4的HEPES缓冲液清洗细菌2-3次,最后用HEPES重悬细菌；(3)在待检测细菌中加入荧光探针,用荧光光谱仪检测,以荧光增强(△S/S<Sub>0</Sub>)和荧光比率变化(I<Sub>482</Sub>/I<Sub>375</Sub>)作为探针响应的二维信号,用于鉴别不同种类细菌。本发明利用单一小分子荧光探针能够灵敏识别细菌表面的微小变化的性能,实现对不同耐药细菌表面的识别,具有操作简单,检测灵敏快速,普适性强的特点,性能优于已有的耐药细菌检测法。(The invention provides a fluorescence detection method for identifying multiple clinical multiple drug-resistant bacteria, which belongs to the field of biological analysis and detection and comprises the following steps of (1) bacteria culture, (2) bacteria collection, namely centrifugation for 5-10 minutes under the conditions of 10000 and 14000rpm, supernatant abandoning, washing the bacteria for 2-3 times by using HEPES buffer solution with the concentration of 20mM and the pH value of 7.4, and finally resuspending the bacteria by using HEPES, (3) adding a fluorescence probe into the bacteria to be detected, detecting by using a fluorescence spectrometer, and enhancing the fluorescence (△ S/S) 0 ) And change in fluorescence ratio (I) 482 /I 375 ) Two-dimensional signals as probe responses for the identification of different species of bacteria. The invention can sensitively identify the property of micro-change on the surface of bacteria by using the single small-molecule fluorescent probe, realizes the identification on the surfaces of different drug-resistant bacteria, and has the characteristics of simple operation, sensitive and quick detection and strong universalityThe performance is better than the existing drug-resistant bacteria detection method.)

1. A fluorescence detection method for identifying multiple clinical drug-resistant bacteria is characterized in that the method realizes identification of multiple clinical separated drug-resistant bacteria through a single small-molecule fluorescent probe, and specifically comprises the following steps:

(1) culturing bacteria;

(2) and (3) bacteria collection: centrifuging at 10000-;

(3) adding fluorescent probe into bacteria to be detected, detecting with fluorescence spectrometer, detecting fluorescence at 345nm excitation wavelength to obtain fluorescence spectrogram, and enhancing with fluorescence △ S/S₀And change in fluorescence ratio I₄₈₂/I₃₇₅Two-dimensional signals as probe responses for identifying different species of bacteria;

the molecular structural formula of the fluorescent probe is as follows:

2. the fluorescence detection method for identifying multiple clinically resistant bacteria according to claim 1, wherein: the concentration range of bacteria to be detected is more than 10⁵CFU/mL, the concentration of the probe is more than or equal to 5 mu M.

3. The fluorescence detection method for identifying multiple clinically resistant bacteria according to claim 1, wherein: the bacteria are gram-positive pathogenic bacteria or gram-negative pathogenic bacteria;

the gram-positive pathogenic bacteria are staphylococcus aureus, staphylococcus epidermidis, enterococcus faecalis, bacillus cereus, viridans streptococcus, enterococcus raffinose, hemolytic staphylococcus, bacillus subtilis, enterococcus faecium and streptococcus pneumoniae;

the gram-negative pathogenic bacteria are Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter baumannii, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter lofei, Haemophilus influenzae and Salmonella enteritidis.

4. The fluorescence detection method for identifying multiple clinically resistant bacteria according to claim 1, wherein: culturing the bacteria to OD in the step (1)₆₀₀＝0.1-2.0。

5. The fluorescence detection method for identifying multiple clinically resistant bacteria according to claim 1, wherein: resuspending to OD of bacteria with HEPES in step (2)₆₀₀＝0.05-2.0。

6. The fluorescence detection method for identifying multiple clinical drug-resistant bacteria according to any claim of claims 1 to 5, which is used for identifying wild-type bacteria and artificial drug-resistant bacteria thereof.

7. The fluorescence detection method for identifying multiple clinically-resistant bacteria according to any of claims 1 to 5, characterized in that the method is used for identifying multiple clinically-isolated drug-resistant bacteria.

Technical Field

The invention belongs to the field of biological analysis and detection, and particularly relates to a fluorescence detection method for identifying multiple clinical multiple drug-resistant bacteria.

Background

Nosocomial infections refer to infections that occur during hospital admission to a patient, including infections that occur during hospital admission to a patient or infections that occur after hospital discharge. Nosocomial infections have become the greatest threat to hospitalized patients, and bacterial infections are considered to be one of the leading causes of nosocomial infections. Most of bacterial strains causing bacterial infection are strains resistant to antibacterial drugs, even strains resistant to multiple drugs, for example, methicillin-resistant staphylococcus aureus (MRSA) is a common drug-resistant gram-positive coccus in clinical separation, and the separation rate is as high as more than 50%; among gram-negative bacteria, drug-resistant bacteria expressing extended-spectrum beta-lactamase (CTX-M-ESBLS) are the most common, such as Escherichia coli and Klebsiella bacteria. The long-term overuse of antibacterial drugs in China causes the generation of various drug-resistant bacteria, and particularly the number of infected people in various hospitals caused by multiple drug-resistant bacteria accounts for about 30 percent of the total number of infected people in pathogenic bacteria of hospital patients. After infection with multiple resistant bacteria, the clinical treatment is difficult, and even higher death rate is caused. An effective way to control the occurrence of bacterial infections in a patient in a hospital is to rapidly and effectively identify drug-resistant bacteria followed by purposeful medication.

At present, the commonly used drug-resistant bacteria detection method is that bacteria culture medium culture or polymerase chain reaction method is used for separating and determining the species of bacteria, then the bacteria are respectively incubated and cultured with a plurality of drugs, and then the Minimum Inhibitory Concentration (MIC) of the bacteria is detected and the drug resistance of the bacteria is determined. Therefore, a simple and rapid detection method with broad spectrum is urgently needed for detecting drug-resistant bacteria. The fluorescent probe has the advantages of rapidness, simplicity, sensitivity, real-time monitoring and the like, and is widely applied to the detection of bacteria. However, the fluorescence detection method commonly used at present is to load a probe molecule to a recognition unit, such as an antibody, an aptamer, a polypeptide, etc., and recognize bacteria by recognizing a specific biological target of the bacteria. However, this method can only detect a single bacterium with one probe, and cannot detect multiple bacteria simultaneously without universality.

Since the bacterial surface is composed of a plurality of components, and any change in the bacterial gene may cause a slight change in the cell surface, a method for distinguishing different bacteria by monitoring the slight change in the bacterial surface has been developed. Its response signal is generated by the nonspecific interaction of multiple fluorescent probes with different properties and the surfaces of various bacteria. The plurality of response signals form a two-dimensional map by an array method, which is called a two-dimensional sensor array method. 5 aggregation-induced emission probes with specific properties have been reported as an array sensor to be used for distinguishing 8 kinds of bacteria. However, this method requires the simultaneous use of multiple compounds to be able to achieve the differentiation of small differences between different species, and no probe has been reported for identifying multiple drug-resistant bacteria. Meanwhile, the two-dimensional sensor array method has the problems of complex operation, poor repeatability and small application range. Therefore, it is a current challenge to develop a simple and rapid method for identifying multiple drug-resistant bacteria.

Disclosure of Invention

The invention aims to provide a fluorescence detection method for identifying multiple clinical multi-drug-resistant bacteria, which utilizes the difference of the surface action modes of a single small-molecule fluorescent probe and different bacteria, forms a two-dimensional map to identify the clinical drug-resistant bacteria by taking the fluorescence enhancement and the fluorescence ratio change of the probe as signals, has the characteristics of simple operation, sensitive and rapid detection and strong universality, and has better performance than the existing bacteria detection probe.

A fluorescence detection method for identifying multiple clinical drug-resistant bacteria is characterized in that a fluorescence probe of multiple drug-resistant bacteria is adopted, and the method comprises the following steps:

(1) culturing bacteria;

(2) and (3) bacteria collection: centrifuging at 10000-;

(3) adding fluorescent probe into bacteria to be detected, detecting with fluorescence spectrometer, detecting fluorescence at 345nm excitation wavelength to obtain fluorescence spectrogram, and enhancing with fluorescence (△ S/S)₀) And change in fluorescence ratio (I)₄₈₂/I₃₇₅) As probe response signals for distinguishing different species of bacteria.

The molecular structural formula of the fluorescent probe for identifying multiple drug-resistant bacteria is as follows:

the probe is characterized in that two ends of bigeminy imidazolium salt are connected with two pyrene fluorophores which can emit pyrene monomer or pyrene exciplex fluorescence at 375nm and 482nm respectively; the compound has 2 positive charges, and fluorescence quenching is caused by the formation of nano aggregates due to hydrophobic interaction in aqueous solution.

The concentration range of the detected bacteria is more than 10⁵CFU/mL, the concentration of the probe is more than or equal to 5 mu M.

The bacteria are gram-positive pathogenic bacteria or gram-negative pathogenic bacteria:

the gram-positive pathogenic bacteria are staphylococcus aureus, staphylococcus epidermidis, enterococcus faecalis, bacillus cereus, viridans streptococcus, enterococcus raffinose, hemolytic staphylococcus, bacillus subtilis, enterococcus faecium and streptococcus pneumoniae;

the gram-negative pathogenic bacteria are Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter baumannii, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter lofei, Haemophilus influenzae and Salmonella enteritidis.

A fluorescence detection method for identifying multiple clinical drug-resistant bacteria, which is preferably as follows:

(1) bacteria were cultured to OD₆₀₀＝0.1-2.0。

(2) And (3) bacteria collection: centrifuging at 10000-₆₀₀＝0.05-2.0；

(3) Adding probe (concentration greater than 5 μ M) into bacteria, detecting with fluorescence spectrometer, detecting fluorescence at 345nm excitation wavelength to obtain fluorescence spectrogram, and changing with fluorescence enhancement and fluorescence ratio (I)₄₈₂/I₃₇₅) As probe response signals for distinguishing different species of bacteria.

The application of the fluorescence detection method for identifying multiple clinical drug-resistant bacteria can be used for identifying wild bacteria and artificial drug-resistant bacteria thereof.

The application of a fluorescence detection method for identifying multiple multi-drug-resistant bacteria in clinic can be used for identifying multiple multi-drug-resistant bacteria in clinic.

The invention has the advantages and beneficial effects that:

the small difference of the surface structures of different drug-resistant bacteria causes the action modes of the drug-resistant bacteria and probe molecules to be different, so that the probe forms monomers and dimers with different degrees, thereby generating the ratio change (I) of pyrene monomer and exciplex fluorescence₄₈₂/I₃₇₅). The fluorescence enhancement generated after the probe polymer is combined with bacteria and the fluorescence ratio change of the monomer and the exciplex are used as fluorescence signals to make a two-dimensional map, so that various clinically separated drug-resistant bacteria can be identified.

Drawings

FIG. 1 is a graph showing the growth of fluorescent probes in HEPES buffer (20mM, pH 7.4) for quantitative detection of different bacteria.

FIG. 2 shows the interaction of fluorescent probe with E.coli BL21 and E.coli BL21 artificial drug-resistant bacteria (OD 21) in HEPES buffer (20mM, pH 7.4)₆₀₀0.1) fluorescence spectra before and after the action and two-dimensional spectra.

FIG. 3 shows the interaction of fluorescent probe with E.coli DH 5a and its artificial drug-resistant bacterium (OD) in HEPES buffer (20mM, pH 7.4)₆₀₀0.1) fluorescence spectra before and after the action and two-dimensional spectra.

FIG. 4 shows fluorescent probes in HEPES buffer (20mM, pH 7.4) and clinically isolated 8 strains of multidrug/non-resistant Escherichia coli (OD)₆₀₀0.1) fluorescence spectra before and after the action and two-dimensional spectra.

FIG. 5 shows fluorescent probes against clinically isolated 6 strains of multidrug/non-resistant Staphylococcus aureus (OD) in HEPES buffer (20mM, pH 7.4)₆₀₀0.1) fluorescence spectra before and after the action and two-dimensional spectra.

Detailed Description

The following examples further illustrate the invention but are not intended to limit the invention thereto.