CN105349664A - Gene chip and kit for detecting pathogenic bacteria in cerebrospinal fluid of central nervous system bacterial infected person - Google Patents

Abstract

This application discloses a gene chip and a kit for detecting pathogenic bacteria in the cerebrospinal fluid of patients with central nervous system bacterial infection, wherein the gene chip includes: a solid phase carrier and an oligonucleotide probe immobilized on the solid phase carrier. The oligonucleotide probes include the set bacteria 16SrRNA universal probe, Gram-positive bacteria universal probe, Gram-negative bacteria universal probe and species- and genus-specific probes for identifying bacteria. Use the designed primers to perform PCR amplification on the genomic DNA of the sample to be detected, and use the above-mentioned gene chip for hybridization, and then use the interpretation software Baio? Array? Doctor? V? 2.0 Analysis and identification of hybridization results. The invention has the advantages that it can quickly, accurately and reliably detect one or more pathogenic bacteria in the cerebrospinal fluid of patients with central nervous system bacterial infection.

Description

Gene chip and kit for detecting pathogenic bacteria in cerebrospinal fluid of patients with central nervous system bacterial infection

technical field

The invention relates to the technical field of biomedicine, in particular to a multi-site gene detection chip, especially a rapid diagnostic gene chip integrated detection method suitable for bacterial central nervous system infection pathogens, in which various bacterial probes are set on the chip, These include some common pathogenic bacteria in the cerebrospinal fluid of bacterial CNS infection. PCR amplification products are used for hybridization test detection and identification of test samples.

Background technique

Central nervous system (central nervous system, CNS) infection is one of the main disease spectrum of the nervous system, the clinical manifestations are meningitis (encephalitis) symptoms, such as fever, headache, vomiting, disturbance of consciousness and meningeal irritation, etc., the fatality rate and disability rate High, early and clear etiological diagnosis is the key to correct clinical anti-infection treatment. There are many types of pathogenic microorganisms that cause CNS infection, and bacterial pathogens are one of the more common ones. For a long time, the methods used in clinical microbiology laboratories to diagnose bacterial CNS infection pathogens mainly include phenotypic methods such as cerebrospinal fluid smear microscopy and culture identification, cerebrospinal fluid immunological detection methods, biochemical marker detection, and polymerase chain reaction (PCR). ) technology, etc. There are also reports at home and abroad that use the structural characteristics of the bacterial 16SrRNA gene to construct a gene chip to identify pathogens.

Cerebrospinal fluid smear microscopy and culture and other phenotypic methods are still the most commonly used detection methods for the diagnosis of central nervous system infection in clinical laboratories. The detection of pathogenic bacteria is the "gold standard" for judging infection, but these conventional methods have the following shortcomings : (1) Due to laboratory detection methodology, the infectious pathogen cannot be detected or the detection cycle is too long, which lags behind the clinical needs, making the diagnosis difficult and missing the opportunity for early or critical treatment: ① Microscopic examination of cerebrospinal fluid smear, low detection rate , the sensitivity is poor; ②It takes a long time to culture bacteria in cerebrospinal fluid (2-5 days), and the positive rate of culture is low (about 10-15%); ③Limited by the use of antibiotics and the conditions of pathogens themselves, it is impossible to screen for broad-spectrum pathogens investigation and monitoring. (2) Due to the inability or rapid and accurate isolation and identification of pathogens, it leads to blindness in treatment, increases the number of drug-resistant bacteria and increases the burden on patients: ①The current automatic bacterial identification instrument may make mistakes in the identification of certain bacteria, and the identification is accurate ②Mixed infections are easy to be misdiagnosed or missed; ③Mass spectrometer identification still needs to obtain positive colonies first, and direct detection and identification of pathogens still needs a large number of samples to verify; ④The database of commercial phenotypic identification system is limited, it is difficult to distinguish between Types of similar pathogenic bacteria.

Cerebrospinal fluid immunological detection methods (such as streptococcal latex agglutination detection) can only detect specific pathogens, but cannot detect unknown bacteria in cerebrospinal fluid and classify pathogenic bacteria, and have the disadvantages of high false positive rate. PCR detection methods mainly use fluorescent quantitative PCR technology and deoxyribose nucleic acid sequencing technology. The defect of the former is that it is limited by the number of fluorescence detection channels. At present, only one or a few bacterial nucleic acids can be detected at the same time, and the throughput is low; the latter is complicated to operate. , It is not convenient to carry out routinely in laboratory laboratories, and there is a certain risk of error in the determination of sequencing results, which requires multi-copy bidirectional sequencing. At present, many gene chips are mostly prepared with different carriers such as nitrocellulose membrane and nylon membrane, and methods such as digoxin and colloidal gold color development, or are based on bacterial 16SrRNA micro-oligonucleotide chips to detect pathogenic bacteria in cerebrospinal fluid, but there are insufficient coverage of pathogenic bacteria. Insufficiencies such as comprehensiveness and lack of large-scale clinical validation data limit its clinical application.

At present, the traditional bacterial culture identification used in clinical microbiology laboratories has many objective shortcomings, such as relying on phenotypic identification, low sensitivity, long detection cycle, and easy to be limited by the application of antibiotics. requirements for rapid diagnosis. The current methods for strain typing mainly include polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), sequencing, sequence-specific primer PCR, fluorescent quantitative PCR and other methods, which are not only cumbersome to operate, but also take a long time to detect , The throughput is small, and the detection process has many influencing factors and is difficult to control. It is impossible to type multiple pathogens at the same time, and it is difficult to meet the requirements of clinical testing. It has made it impossible to carry out broad-spectrum and integrated detection of bacterial pathogens in central nervous system infection.

The gene chip is one of the major scientific and technological advances with the most epochal characteristics in the high-tech field in recent years. Its main principle is to combine gene probes (oligonucleotide probes, cDNA clones) , PCR products, etc.) in a certain order and density fixed on a carrier (such as glass slide or silicon wafer, nylon membrane, nitrocellulose membrane, plastic sheet, etc.) to form an array, and hybridize with the actual sample (or nucleic acid amplification product) The reaction, through hybridization signal analysis, can obtain high-throughput information of all genes to be detected. With its advantages of high throughput, rapidity, and high accuracy, this technology provides a new solution for the diagnosis of infectious etiology, and plays an important role in the clinical treatment and prognosis evaluation of infectious diseases in patients.

Bacterial ribosomal RNA (rRNA) is a sign of bacterial life, and its 16S rRNA gene exists in multiple copies in all bacterial chromosome genes. In the 1990s abroad, it was reported that gene chips were constructed using the structural characteristics of bacterial 16SrRNA genes and applied to bacterial identification. In recent years, some people in China have devoted themselves to the clinical detection of cerebrospinal fluid bacteria using oligonucleotide chip technology prepared by different carriers such as nitrocellulose membrane and nylon membrane, digoxin, colloidal gold and other methods, but there are common clinical cerebrospinal fluid Incomplete coverage of pathogenic bacteria, long hybridization time and other deficiencies, especially the lack of large-sample clinical verification data limit its clinical application value. At present, the most important gene chip products on the market are medium and low-density gene chips for gene expression detection prepared by spotting and other methods. An important development trend of gene chips is the standardization of chip preparation, sample processing, hybridization, detection, and data analysis to improve the accuracy and reliability of gene chips. Therefore, how to ensure both high-throughput detection and superior chip performance is one of the bottleneck issues. The use of multi-site microarrays based on the bacterial 16SrRNA gene to detect a variety of bacterial pathogens of central nervous system infection has important practical significance for the clinical analysis of bacterial pathogens and types of central nervous system infection by taking advantage of its characteristics of simple operation, large throughput, and accurate results.

Contents of the invention

The purpose of the present invention is to overcome the above-mentioned deficiencies, and provide a gene chip and kit for detecting pathogenic bacteria in the cerebrospinal fluid of patients with bacterial infection of the central nervous system, which solves the problem of using a multi-site chip based on the bacterial 16SrRNA gene to detect a variety of central nervous system infection bacteria The problem of pathogens.

An object of the present invention is to provide a gene chip for detecting pathogenic bacteria in the cerebrospinal fluid of central nervous system bacterial infection, comprising a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, characterized in that: Oligonucleotide probes include bacterial 16SrRNA universal probes, gram-positive bacterial universal probes, gram-negative bacterial universal probes, and species- and genus-specific probes for identifying bacteria set on the substrate. Species and genus-specific probes are DNA fragments of the base sequence shown in SEQIDNO: 1-NO: 14, and the bacterial 16SrRNA universal probe is a DNA fragment of the base sequence shown in SEQ ID NO: 15. The universal probe for Gram-positive bacteria is a DNA fragment of the base sequence shown in SEQ ID NO:16, and the universal probe for Gram-negative bacteria is a DNA fragment of the base sequence shown in SEQ ID NO:17.

Further, the 5' end of the species- and genus-specific probes for identifying bacteria above contains amino-modified poly-dT strings, and the amino-modified poly-dT strings are 16-merized polydeoxythymidylic acid.

Further, the gene chip also includes a DNA sequence marked with biotin dots as shown in SEQ ID NO:18.

Further, the solid phase carrier is a modified glass or silicon wafer.

Another object of the present invention is to provide a kit for detecting pathogenic bacteria in the cerebrospinal fluid of patients with central nervous system bacterial infection, which is characterized in that it includes the gene chip as claimed in claim 1 .

Further, the kit also includes primer sequences shown in SEQ ID NO: 19-NO: 20.

Further, the 5' end of the primer has a labeling group, and the labeling group includes: digoxigenin molecule, biotin molecule, fluorescein and its derivative molecules, Cy3, Cy5, alkaline phosphatase, pepper root peroxidase.

Another object of the present invention is to provide a method for detecting pathogenic bacteria infection in the cerebrospinal fluid of patients with central nervous system bacterial infection. It is characterized in that: comprising the following steps:

(1) Prepare template DNA by conventional methods (extract DNA samples containing possibly infected bacteria from the patient's cerebrospinal fluid);

(2) the template DNA prepared in the step (1) is amplified by PCR reaction with the primer sequence described in claim 6, and the 5' end has a marker group DNA amplification product;

(3) mixing the DNA amplification product in step (2) with the hybridization buffer;

(4) hybridization reaction is carried out with the DNA probe and Bio distributed above the gene chip described in claim 3 with the mixed solution gained in step (3);

(5) After the hybridization reaction in step (4), the hybridization signal of the gene chip is detected, and the information to be tested is obtained according to the position and intensity of the marker signal on the gene chip.

Further, before the step (4), pre-hybridize the gene chip with the pre-hybridization buffer.

Further, the step of the PCR reaction in the step (2) also includes:

1) Pre-denaturation at 94°C for 5 minutes; 2) Denaturation at 94°C for 45s; 3) Annealing at 58°C for 45s; 4) Extension at 72°C for 60s, repeating 2)-4) 30 times; 5) Last reaction step of 72°C extension for 7min.

The invention is suitable for directly extracting pathogen DNA from clinical specimens such as cerebrospinal fluid for PCR amplification, and using target genes for hybridization detection, and can be used for pathogen screening of suspected bacterial infections. The present invention uses the solid material carrier as the substrate, which is convenient for production and operation; the bacterial target gene is amplified by the bacterial universal primer PCR method, which avoids the time-consuming cultivation stage and greatly shortens the detection time (about 4 hours); the target sequence of the marker is used The identification method of hybridization with the oligonucleotide probe on the substrate is more accurate than the identification method based on physiology and biochemistry, which increases the detection accuracy and is not affected by the culture conditions and the physiological state of the bacteria; no matter whether the bacteria are "dead or alive", The results of gene chip detection all provide important clinical value. This technical method can not only confirm the presence or absence of bacteria in a short period of time, but also interpret the pathogenic bacteria of common clinical infectious diseases, which provides a basis for early diagnosis of diseases with bacterial infections but not obvious early clinical manifestations, which is beneficial to antibiotics. The use of antibiotics to avoid the abuse. With the increase of the number of specific probes, its ability to detect more samples or mixed infection samples will be higher and higher, and the detection time will be shorter and shorter, which will provide a new method for the diagnosis of clinical bacteria. The new, fast and sensitive method replaces the traditional bacterial detection and identification method to some extent.

In order to make the above and other objects, features and advantages of the present invention more comprehensible, preferred embodiments will be described in detail below together with the accompanying drawings.

Description of drawings

The accompanying drawings described here are used to provide a further understanding of the present invention, and constitute a part of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention, and do not constitute improper limitations to the present invention. In the attached picture:

Figure 1 is a schematic diagram of the arrangement of 18 probes of the gene chip of the present invention described in Example 2 of the present invention, wherein Represents DNA sequences labeled with biotin dots.

Fig. 2 is a graph showing the results of analysis of Enterococcus faecalis.

Fig. 3 is a diagram showing the results of the analysis of P. faecium.

Fig. 4 is a graph showing the results of analysis of Staphylococcus aureus.

Fig. 5 is a diagram showing the analysis results of Listeria monocytogenes.

Fig. 6 is a graph showing the results of analysis of Streptococcus pneumoniae.

Fig. 7 is a graph showing the analysis results of Escherichia coli.

Fig. 8 is a graph showing the analysis results of Klebsiella pneumoniae.

Fig. 9 is a graph showing the results of analysis of Pseudomonas aeruginosa.

Fig. 10 is a graph showing the analysis results of Acinetobacter baumannii.

Figure 11 is a graph showing the results for Stenotrophomonas maltophilia.

Figure 12 is a graph showing the results for Haemophilus influenzae.

Fig. 13 is a graph showing the results for bacteria belonging to the genus Enterobacter.

Figure 14 is a graph showing the results for the presence of Grammar bacteria.

Figure 15 is a graph showing the results of the presence of G+ bacteria.

Figure 16 Schematic diagram of the working principle of the gene chip.

detailed description

In order to further illustrate the gene chip and its detection method for detecting pathogenic bacteria in the cerebrospinal fluid of patients with central nervous system bacterial infection of the present invention, the following preferred examples are given for illustration. These examples are for illustration and not to limit the present invention in any form.

Design and preparation of embodiment one probe (taking Escherichia coli as an example)

1. Sequence acquisition: Log in to Genbank to retrieve the DNA sequence of the Escherichia coli standard sample, obtain the base sequence, and select the 16SrRNA gene of the bacterium as the target sequence;

2. probe design: considering factors such as probe length, GC% and Tm value, the present invention designs probe by PrimerPriemer5.0 software, designs the DNA fragment of base sequence as shown in SEQIDNO:4,

Further, in order to reduce the steric hindrance during hybridization, the present invention adds an amino-modified poly-dT string at the 5' end of the above-mentioned base sequence;

Specifically, the above-mentioned amino-modified poly dT string uses 16-mer polydeoxythymidylic acid;

3. Synthesis of gene probes: artificially synthesize (entrust Sangon Bioengineering (Shanghai) Co., Ltd.) the probes designed above.

The synthesis of embodiment two gene chips

1. Design the spotting mode of the bacterial chip: Repeat 3 spots for each probe to make a 7*12 DNA microarray as shown in Figure 1. The corresponding numbers of each probe in the illustration are shown in Table 1.

Table 1 Corresponding numbers of bacterial probes

The specific numbers in Figure 1 in Table 1 correspond to the strains

Spotting of gene chips:

1) On the iArrayer gene chip spotting instrument produced by Shanghai Bio AO Technology Co., Ltd., set the spotting program according to the pattern diagram of the chip;

2) Mix the diluted 100uM probe solution with Shanghai Bio-Tech Co., Ltd. The spotting buffer is mixed in a certain proportion and added to the designated position of the 384-well plate according to the spotting procedure;

3) Place the aldehyde-based substrates sequentially on the spotting platform of the spotting instrument;

4) Place the sample spotting plate in the spotting instrument, start the sample spotting program, and complete chip spotting;

5) The gene chip was left to stand for 12 hours under constant temperature and humidity conditions.

6) Quality inspection: Place the gene chips under a microscope in turn for observation. The array should be neat and complete without any leaks.

If the oligonucleotide probe does not contain amino modifications, its preparation method can also refer to: "Gene Diagnosis Technology-Non-radioactive Operation Manual" edited by Wang Shenwu; Derisi, JL, etc. in "Science" 278 (5338) in 1997 : 680-686 published "exploring the metabolic and genetic control of gene expression in the genome" (Dersi, JL, IyerVR, Brown PO. Exploring the metabolic and genetic control of gene expression on genetic scale. Science. 1997; 278 (5338): 680-686) and the biochip edited by Ma Liren et al. Chemical Industry Press.

Example three primer synthesis

1. Sequence acquisition: log into Genbank for Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Enterobacter, Haemophilus influenzae, Stenotrophomonas maltophilia, meningitis Neisseria, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pneumoniae, coagulase-negative staphylococcus DNA sequence retrieval and analysis.

2. Primer design: design primers with the base sequence shown in SEQ ID NO: 19-NO: 20 by PrimerPriemer 5.0 software;

Further, the 5' end of the primer can also be modified with a labeling group,

Specifically, digoxigenin molecule (DIG), biotin molecule (Bio), fluorescein and its derivative molecules (FITC, etc.), other fluorescent molecules (such as Cy3, Cy5, etc.), alkaline phosphatase (AP), pepper Root peroxidase (HRP) labeled primers;

3. Synthesis of primer sequences: artificially synthesize (entrust Sangon Bioengineering (Shanghai) Co., Ltd.) the primers designed above.

Example 4 Working principle of gene chip

The 3' end of the amplified fragment amplified by the designed primers is complementary to the specific probe immobilized on the chip, and the 5' end has a labeling group. If the probe is a probe complementary to the 3' end of the amplification product, one end of the amplification product hybridizes with the specific probe on the chip, and the other end has a labeling group, and the labeling group is subjected to color development , the signal can be detected, otherwise no signal can be detected. Fig. 16 is a schematic diagram of the working principle of the gene chip.

Embodiment 5 Detection kit and operation method for pathogenic bacteria in the cerebrospinal fluid of patients with central nervous system bacterial infection

In the method for detecting pathogenic bacteria in the cerebrospinal fluid of patients with bacterial infection of the central nervous system using the kit containing the prepared gene chip and primers, the present invention has made a series of experiments on the detection steps, detection conditions and other factors, such as bacteria The ratio of each component in the PCR reaction mixture, hybridization temperature, hybridization time, etc., and the formulation of main reagents, such as hybridization solution, washing solution, etc. (see the following examples for the formulation). The components in the PCR reaction mixture, especially the concentration of primers, the selection of Buffer, the concentration of Taq enzyme, the type and amount of luciferin, and the concentration of the template are the optimal combinations obtained after multiple comparative experiments. The annealing temperature, time, extension time and number of cycles used in PCR are also excellent conditions selected after gradient experiments. PCR amplification was subjected to 1) pre-denaturation at 94°C for 5 minutes; 2) denaturation at 94°C for 45s; 3) annealing at 58°C for 45s; 4) extension at 72°C for 60s, repeating 2)-4) 30 times; 5) final extension at 72°C for 7min the process of. The experimental process of the detection chip for detecting pathogenic bacteria in the cerebrospinal fluid of patients with bacterial infection of the central nervous system is also the result of optimization. The following is a preferred embodiment of using the above-mentioned kit to detect pathogenic bacteria in the cerebrospinal fluid of patients with central nervous system bacterial infection, and the specific instructions are as follows:

1. Preparation of positive control samples

(1) Apply TIANGEN kit to extract positive control bacterial genomic DNA

1) Take 5ml of Staphylococcus aureus (ATCC25923) and Escherichia coli (ATCC25922) bacterium solution prepared by sterile normal saline, make the bacterium solution into 0.5 McFarland unit, adopt 10-fold serial dilution method, make the concentration of bacteria suspension 10-10 ⁸ CFU/ml respectively;

2) Centrifuge the bacterial solution at 12,000 rpm/min for 5 minutes, discard the supernatant; add 200 μl of 20 mg/ml lysozyme, mix well, and bathe in 37°C water bath for 30 minutes;

3) Add 20 μl of proteinase K solution and mix well; add 220 μl of solution B, shake for 15 seconds and mix well, put in a 70°C metal bath for 10 minutes;

4) Add 220 μl of absolute ethanol, shake for 15 seconds to mix, transfer the resulting solution to a clean gel column, centrifuge at 12000 rpm/min for 30 seconds, and discard the waste liquid;

5) Add 500 μl solution D, centrifuge at 12000 rpm/min for 30 seconds, and discard the waste liquid;

6) Add 700 μl solution PW, centrifuge at 12,000 rpm/min for 30 seconds, and discard the waste liquid; repeat the above steps once and then centrifuge at 12,000 rpm/min for 2 minutes, discard the waste liquid, and place the remaining liquid at room temperature to dry;

7) Add 50 μl of eluent TE, place at room temperature for 2-5 minutes, centrifuge at 12000 r/min for 2 minutes, and the obtained supernatant is the bacterial genomic DNA.

(2). PCR amplification template DNA

The amplification liquid in the kit of the present invention includes upstream and downstream primers (biotin-labeled) and various reagents required for PCR reaction (such as dNTP, H ₂ O, Buffer, etc.), and Taq enzyme is provided separately. The Taq enzyme is added to the amplification solution, and the extracted target DNA is rapidly amplified by PCR reaction for hybridization and color reaction. The composition of the 25 μl amplification reaction system is as shown in Table 2: amplification solution A (22 μl) + 2 μl extraction product (DNA) + 1 μl Taq enzyme.

Table 2 Bacterial PCR Amplification System Amplification Solution A Formula

Reagent name Dosage 10×Buffer 2.5μl dNTP (2.5mM each) 2.5μl Upstream primer (10uM) 0.3μl Downstream primer (10uM) 1μl DDW 15.7μl Total 22μl

Put the above mixture into the PCR machine and perform the following procedures:

94℃5min

94℃45s

58℃45s

72 ℃ 60s return to the second step for 30 cycles

72℃7min

2. Preparation of Negative Control Samples

Same as the preparation of the above-mentioned positive control sample, use 5ml sterile physiological saline instead of bacterial template DNA for operation.

3. Preparation of Clinical Samples

(1). The extraction of the genomic DNA of positively isolated bacteria in cerebrospinal fluid culture is the same as the preparation of the above-mentioned positive control group samples;

(2). DNA extraction from cerebrospinal fluid samples with clinically suspected infection was performed using the QIAampUCPPathogenMiniKit kit. Add 1.5ml cerebrospinal fluid into the PathogenLysisTube, centrifuge at 13400r/min for 5min, discard the supernatant, add the matching buffer ATL (including reagent DX) to suspend the particles, vortex for 10 minutes, centrifuge briefly, absorb 400μl supernatant in In sterile EP tubes, the subsequent experimental operations were performed according to the instructions of the QIAampUCPPathogenMiniKit. According to the PCR amplification conditions of the positive control sample, the nucleic acid DNA of the clinical sample with biotin label is amplified.

4. Hybridization

Using the Hybridization Chromogenic Kit (BST03021) produced by Shanghai Bio-Technology Co., Ltd., e-Hyb automatic hybridization instrument: in BSE03011, proceed as follows:

(1) Preparation of hybridization reaction solution: pipette 190 μl of hybridization buffer, 10 μl each of the amplification products prepared in the above steps 1-3, and mix well.

(2) According to Table 3, set the reaction program and dispense the reagents, run the program, and the hybridization color reaction will be carried out automatically.

Table 3. Hybridization system and reaction program

(3) Take out the gene chip and mark it

5. Detection of chip hybridization signal:

Put the hybridized and washed gene chip into BE-2.0 biochip reader detection, with Gene chip image analysis software BaioArrayDoctorV2.0 performs image scanning and data analysis, and outputs the results. As shown in Fig. 2-15, it is a scanning diagram of the detection result using the kit provided by the present invention.

The invention is suitable for directly extracting pathogen DNA from clinical specimens such as cerebrospinal fluid for PCR amplification, and using target genes for hybridization detection, and can be used for pathogen screening of suspected bacterial infections. The present invention uses the solid material carrier as the substrate, which is convenient for production and operation; the bacterial target gene is amplified by the bacterial universal primer PCR method, which avoids the time-consuming cultivation stage and greatly shortens the detection time (about 4 hours); utilizes the labeled target sequence The identification method of hybridization with the oligonucleotide probe on the substrate is more accurate than the identification method based on physiology and biochemistry, which increases the detection accuracy and is not affected by the culture conditions and the physiological state of the bacteria; regardless of whether the bacteria are "dead or alive", The results of gene chip detection all provide important clinical value. This technical method can not only confirm the presence or absence of bacteria in a short period of time, but also interpret the pathogenic bacteria of common clinical infectious diseases, which provides a basis for early diagnosis of diseases with bacterial infections but not obvious early clinical manifestations, which is beneficial to antibiotics. The use of antibiotics to avoid the abuse. With the increase of the number of specific probes, its ability to detect more samples or mixed infection samples will be higher and higher, and the detection time will be shorter and shorter, which will provide a new method for the diagnosis of clinical bacteria. The new, fast and sensitive method replaces the traditional bacterial detection and identification method to some extent.

The above description shows and describes several preferred embodiments of the present application, but as mentioned above, it should be understood that the present application is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various Various other combinations, modifications and environments, and can be modified by the above teachings or the technology or knowledge in the related field within the scope of the application concept described herein. However, modifications and changes made by those skilled in the art do not depart from the spirit and scope of the present application, and should all be within the protection scope of the appended claims of the present application.

Claims (10)

1. A gene chip for detecting pathogenic bacteria in the cerebrospinal fluid of central nervous system bacterial infection, comprising a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, characterized in that: the oligonucleotide probe The needles include bacterial 16SrRNA universal probes, gram-positive bacterial universal probes, gram-negative bacterial universal probes, and species- and genus-specific probes for identifying bacteria set on the substrate. The species- and genus-specific probes for identifying bacteria The probe is a DNA fragment of the base sequence shown in SEQ ID NO: 1-NO: 14, and the bacterial 16SrRNA universal probe is a DNA fragment of the base sequence shown in SEQ ID NO: 15, and the Gram-positive bacteria are universal The probe is a DNA fragment with the base sequence shown in SEQ ID NO: 16, and the Gram-negative bacteria universal probe is a DNA fragment with the base sequence shown in SEQ ID NO: 17. 2. the gene chip of pathogenic bacteria in the cerebrospinal fluid of detection central nervous system bacterial infection according to claim 1, it is characterized in that: the kind of above-mentioned identification bacterium, genus-specific probe 5' end contains the polydT string of amino modification, The amino-modified poly-dT string is 16-merized polydeoxythymidylic acid. 3. The gene chip for detecting pathogenic bacteria in the cerebrospinal fluid of patients with central nervous system bacterial infection according to claim 1, characterized in that: the gene chip also includes a DNA sequence marked with biotin spots as shown in SEQ ID NO:18. 4. The gene chip for detecting pathogenic bacteria in the cerebrospinal fluid of patients with central nervous system bacterial infection according to claim 1, characterized in that: the solid phase carrier is a modified glass slide or a silicon wafer. 5. A kit for detecting pathogenic bacteria in the cerebrospinal fluid of patients with central nervous system bacterial infection, characterized in that: comprising the gene chip as claimed in claim 1. 6. The kit for detecting pathogenic bacteria in cerebrospinal fluid of patients with central nervous system bacterial infection according to claim 5, characterized in that: said kit also includes primer sequences shown in SEQ ID NO: 19-NO: 20. 7. The kit for detecting pathogenic bacteria in the cerebrospinal fluid of patients with central nervous system bacterial infection according to claim 6, characterized in that: the 5' end of the primer has a labeling group, and the labeling group includes: Digoxigenin molecule, biotin molecule, luciferin and its derivative molecule, Cy3, Cy5, alkaline phosphatase, horseradish peroxidase. 8. A method for detecting pathogenic bacteria in the cerebrospinal fluid of central nervous system bacterial infection, not used for diagnosis and treatment of disease, characterized in that: comprising the steps of: (1) Prepare template DNA by conventional methods (extract DNA samples containing possibly infected bacteria from the patient's cerebrospinal fluid); (2) the template DNA prepared in the step (1) is amplified by PCR reaction with the primer sequence described in claim 6, and the 5' end has a marker group DNA amplification product; (3) mixing the DNA amplification product in step (2) with the hybridization buffer; (4) hybridization reaction is carried out with the DNA probe and Bio distributed above the gene chip described in claim 3 with the mixed solution gained in step (3); (5) After the hybridization reaction in step (4), the hybridization signal of the gene chip is detected, and the information to be tested is obtained according to the position and intensity of the marker signal on the gene chip. 9. The method for detecting pathogenic bacteria in the cerebrospinal fluid of central nervous system bacterial infection according to claim 8, characterized in that: before the step (4), the gene chip and the pre-hybridization buffer are pre-hybridized. 10. the method for detecting pathogenic bacteria in the cerebrospinal fluid of central nervous system bacterial infection according to claim 8, is characterized in that: the step of PCR reaction in the described step (2), also comprises: 1) Pre-denaturation at 94°C for 5 minutes; 2) Denaturation at 94°C for 45s; 3) Annealing at 58°C for 45s; 4) Extension at 72°C for 60s, repeating 2)-4) 30 times; 5) Last reaction step of 72°C extension for 7min.