Gene chip, kit and microbiological detection method

Abstract

The invention relates to the technical field of pathogenic microorganism detection, and discloses a gene chip, a kit and a microorganism detection method.According to the detection purpose, a microorganism sample is collected from an environment or an organism, microorganisms are extracted from the pretreated sample through an extraction method, and according to the detection purpose and microorganism characteristics, the gene chip and the kit are obtained. Results of microbial culture, immunological detection and molecular biological detection are obtained. The method comprises the following steps: collecting a microorganism sample, then extracting microorganisms, selecting a proper detection method according to the detection purpose and the characteristics of the microorganisms, comprehensively analyzing and determining the types of infection pathogens according to the results of microorganism culture, immunological detection and molecular biological detection, and further determining the types of the infection pathogens aiming at the determined pathogens. The method comprises the following steps of: selecting a specific gene sequence as a basis of probe design, designing a proper probe sequence according to the selected gene sequence, and scanning a hybridized chip by using a fluorescence scanner or other detection equipment, so as to complete pathogenic microorganism detection.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogenic microorganism detection, and in particular to a gene chip, a kit and a microorganism detection method. Background Art

[0002] Most patients with orthopedic infections are admitted to the hospital for surgical or interventional treatments, and the infections are mostly related to surgical incisions and wounds. Therefore, the types of orthopedic infections and their pathogens are relatively special. The results of many studies have shown that the common pathogens of orthopedic infections in my country are Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, etc., and these pathogens are often resistant to antibiotics. The detection of pathogens of orthopedic infections generally relies on culture methods, which rely on the clinician's prediction of the infectious pathogen. At the same time, the culture effect on specific pathogens such as tuberculosis and viruses is poor.

[0003] Gene chip technology is a molecular detection method developed in recent years. The essence of gene chip is high-density miniaturized molecular hybridization. On a solid phase carrier surface with an area of ​​only a few square centimeters, hundreds of DNA probe fragments can be fixed by covalent connection. The pre-designed probes are usually selected from specific genes or nucleic acid fragments of microorganisms. As the target gene for detection, it can complementarily bind with the same microbial nucleic acid in the sample, thereby obtaining information about the target pathogenic microorganism in the sample. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the deficiencies of the prior art, the present invention provides a gene chip, a kit and a microorganism detection method, which have the advantages of detecting pathogenic microorganisms and solve the problems mentioned in the above background technology.

[0006] (II) Technical solution

[0007] In order to achieve the purpose of pathogenic microorganism detection mentioned in the above background technology, the present invention provides the following technical solution: a microorganism detection method, comprising the following steps:

[0008] Step 1: Collect microbial samples from appropriate environments or organisms according to the purpose of the test;

[0009] Step 2: Extract microorganisms from the pretreated sample using an appropriate extraction method;

[0010] Step 3: Select appropriate detection methods based on the detection purpose and microbial characteristics;

[0011] Step 4: Based on the results of microbial culture, immunological testing and molecular biological testing, conduct a comprehensive analysis to determine the type of infectious pathogen.

[0012] Preferably, the purpose of the test should be clearly defined, and then the appropriate sample type should be selected, including blood samples, tissue samples, secretion samples, and puncture fluid samples. Multiple samples should be collected using sterile instruments and containers, and it should be ensured that each sample will not be contaminated with each other. The samples should then be sent to the laboratory for processing to avoid long-term storage that may lead to inactivation or contamination of microorganisms.

[0013] Preferably, the extracting of microorganisms from the pretreated sample comprises the following steps:

[0014] Step 1: Pretreatment: For solid samples, grinding and homogenization are required first. Joint fluid and bone marrow samples need to be centrifuged and filtered to remove impurities and enriched for culture.

[0015] Step 2: Purification of microorganisms: Spread the enriched culture sample evenly on a solid culture medium; after culture, select single colonies with obvious morphological and color characteristics for further purification, streak separation or subculture in liquid culture medium on the selected single colonies, and repeat the above steps until a pure culture is obtained.

[0016] Preferably, the microbial detection methods include: traditional culture methods, including bacterial culture, fungal culture and anaerobic culture;

[0017] Immunological methods: antigen-antibody reaction;

[0018] Molecular biology methods: polymerase chain reaction method and gene chip technology.

[0019] For RNA extracted from RNA viruses or nucleic acids co-extracted from bacteria and viruses, a reverse transcription step is required to convert RNA into cDNA. During the process, ensure that the nucleic acids and reaction reagents are placed on ice to prevent nucleic acid, especially RNA, degradation. The reverse transcription product can be stored at -20°C for a short term and at -80°C for a long term.

[0020] Traditional culture methods include: bacterial culture: inoculate the specimen on a suitable nutrient medium, culture under specific conditions, observe bacterial growth, and identify bacterial species through colony morphology, Gram staining, biochemical reactions, etc.;

[0021] Fungal culture: For patients suspected of fungal infection, fungal culture is performed using specific culture media to observe fungal growth characteristics and conduct identification;

[0022] Anaerobic culture: For patients suspected of anaerobic infection, anaerobic culture technology is used for testing.

[0023] Immunological method: antigen-antibody reaction, using the principle of specific antigen-antibody binding, to determine the type of infectious pathogen by detecting the presence of specific antibodies in the patient. Commonly used immunological methods include immunofluorescence technology and enzyme-linked immunosorbent assay;

[0024] Molecular biology methods: polymerase chain reaction: use specific primers to amplify pathogen DNA, and determine the type of pathogen by detecting the amplified product. PCR technology has the advantages of high sensitivity, high specificity, rapidity and accuracy;

[0025] Gene chip technology: A large number of gene fragments with known sequences are fixed on the chip, and hybridized with the DNA or RNA in the sample to be tested to achieve simultaneous detection of multiple pathogens.

[0026] Preferably, a gene chip comprises the following steps:

[0027] Step 1: Identify common pathogens in bone infection;

[0028] Step 2: For the identified pathogen, select its specific gene sequence as the basis for probe design and design a suitable probe sequence based on the selected gene sequence;

[0029] Step 3: Select appropriate carrier material and chemically treat the carrier;

[0030] Step 4: synthesize the probe in vitro using chemical synthesis technology, and fix the synthesized probe on the pre-treated carrier by spotting method or in situ synthesis method;

[0031] Step 5: extracting pathogenic nucleic acid from the sample of the bone infection patient, labeling the extracted pathogenic nucleic acid with fluorescence or other forms, and performing a hybridization reaction;

[0032] Step 6: Use a fluorescent scanner or other detection equipment to scan the hybridized chip, and analyze whether the target pathogen exists in the sample and the type and quantity of the pathogen based on the collected data.

[0033] Preferably, an appropriate nucleic acid extraction method is used to extract pathogenic nucleic acid from the sample. During the extraction process, the purity and integrity of the nucleic acid should be ensured to avoid degradation and loss. The extracted nucleic acid should be purified to remove proteins and polysaccharides. The labeled nucleic acid sample should be quality controlled, including concentration determination and purity inspection. According to the experimental requirements and the characteristics of the target pathogen, the conditions of the hybridization reaction, including temperature, time, salt concentration, and buffer composition, are optimized. The labeled nucleic acid sample is hybridized with the probe fixed on the chip. After the hybridization is completed, an appropriate eluent is used to remove unbound nucleic acid molecules and other impurities.

[0034] Preferably, scanning parameters such as laser power, gain, and exposure time need to be adjusted during the scanning process. After the scanning is completed, the fluorescence scanner converts the captured signal into digital image data, performs a preliminary quality control check on the acquired image data, performs background correction and signal enhancement preprocessing on the acquired image data, calculates the fluorescence intensity value of each probe point based on the preprocessed image data, performs cluster analysis on genes or samples with similar expression patterns, and performs functional enrichment analysis on differentially expressed genes.

[0035] Turn on and preheat the hybridization box at 45°C, and place the hybridization box and hybridization plate in the hybridization box for at least 30 minutes;

[0036] Incubate the reaction hybridization solution at 95℃ for 10 minutes on a PCR instrument, then cool it down to 70℃ for preservation (it can also be placed on ice for rapid cooling after the 95℃ incubation). Add the hybridization solution to the corresponding positions of the hybridization plate and chip;

[0037] Transfer the chip to the hybridization plate and check whether the liquid covers the entire chip; put the chip into the hybridization box, and then put the hybridization box into the 45℃ hybridization box for incubation for 2 hours. ;

[0038] Open and preheat the hybridization box at 30℃, place the plate washer and 30mL.Wash B (in a centrifuge tube) in the hybridization box for at least 30 minutes; then pour 30ml.Wash A into the plate washer at room temperature;

[0039] Take the chip out of the hybridization box and observe whether there are bubbles on the chip. Then transfer the chip to Wash A and wash it at room temperature for 5 minutes.

[0040] Pour Wash B into the washing plate, then transfer the chip plate to the Wash B washing plate and wash at 30°C for 5 minutes;

[0041] Dilute SAPE 1:100 with 4x SSC and add the diluted dye to the corresponding chip position on the well plate;

[0042] Take the chip out of Wash B, shake off the moisture on the chip surface, transfer the chip to the dye solution, and dye it at room temperature in the dark for 20 minutes;

[0043] Pour 4x SSC into the washing plate, and after staining, transfer the chip to the 4x SSC washing plate and wash for 2 minutes;

[0044] Select S2B RFID, select BF, green-AM1E-1s and green-AM1E-4s channels, and select the corresponding chip position for scanning.

[0045] Compared with the prior art, the present invention provides a gene chip, a kit and a microbial detection method, which have the following beneficial effects:

[0046] 1. The present invention collects microbial samples and then extracts microorganisms, selects appropriate detection methods according to the detection purpose and microbial characteristics, and comprehensively analyzes the results of microbial culture, immunological detection and molecular biological detection to determine the type of infectious pathogens. Then, for the determined pathogens, its specific gene sequence is selected as the basis for probe design and a suitable probe sequence is designed based on the selected gene sequence. Then, a fluorescent scanner or other detection equipment is used to scan the hybridized chip to complete the detection of pathogenic microorganisms.

[0047] 2. The present invention can detect dozens to hundreds of microorganisms according to the design of the number of probes, and the sequence-specific probes can basically eliminate the possibility of false positives.

[0048] 3. The present invention has flexible detection content, and the probe can be flexibly designed according to the usage scenario, such as the design of drug-resistant genes of different pathogens and the design of specific sequences for distinguishing strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a schematic diagram of the first process of the present invention;

[0050] Figure 2 This is a schematic diagram of the second process of the present invention.

[0051] Among them: 1. box body; 2. water outlet pipe; 3. cover plate; 4. installation plate; 5. limit block; 6. auxiliary plate; 7. connection box; 8. installation pipe; 9. plug rod; 10. auxiliary rod; 11. spring; 12. placement rack. DETAILED DESCRIPTION

[0052] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] A method for detecting microorganisms comprises the following steps:

[0054] Step 1: Collect microbial samples from appropriate environments or organisms according to the purpose of the test;

[0055] Step 2: Extract microorganisms from the pretreated sample using an appropriate extraction method;

[0056] Step 3: Select appropriate detection methods based on the detection purpose and microbial characteristics;

[0057] Step 4: Based on the results of microbial culture, immunological testing and molecular biological testing, conduct a comprehensive analysis to determine the type of infectious pathogen.

[0058] Specifically, Figure 1 As shown, clarify the purpose of the test, and then select the appropriate sample type, including blood samples, tissue samples, secretion samples, and puncture fluid samples, and then use sterile instruments and containers to collect multiple samples, and ensure that each sample will not be contaminated with each other. Then send the samples to the laboratory for processing to avoid long-term storage that may cause inactivation or contamination of microorganisms.

[0059] Specifically, Figure 1 As shown, the extraction of microorganisms from the pretreated sample comprises the following steps:

[0060] Step 1: Pretreatment: For solid samples, grinding and homogenization are required first. Joint fluid and bone marrow samples need to be centrifuged and filtered to remove impurities and enriched for culture.

[0061] Step 2: Purification of microorganisms: Spread the enriched culture sample evenly on a solid culture medium; after culture, select single colonies with obvious morphological and color characteristics for further purification, streak separation or subculture in liquid culture medium on the selected single colonies, and repeat the above steps until a pure culture is obtained.

[0062] Specifically, Figure 1 As shown, the microbial detection methods include: traditional culture methods, including bacterial culture, fungal culture and anaerobic bacteria culture;

[0063] Immunological methods: antigen-antibody reaction;

[0064] Molecular biology methods: polymerase chain reaction method and gene chip technology.

[0065] Through the above technical solution: For RNA extracted from RNA viruses or nucleic acids co-extracted from bacteria and viruses, a reverse transcription step is required to convert RNA into cDNA. During the process, ensure that the nucleic acid and reaction reagents are placed on ice to prevent nucleic acid, especially RNA, from degradation. The reverse transcription product can be stored at -20℃ for a short term and at -80℃ for a long term.

[0066] Traditional culture methods include: bacterial culture: inoculate the specimen on a suitable nutrient medium, culture under specific conditions, observe bacterial growth, and identify bacterial species through colony morphology, Gram staining, biochemical reactions, etc.;

[0067] Fungal culture: For patients suspected of fungal infection, fungal culture is performed using specific culture media to observe fungal growth characteristics and conduct identification;

[0068] Anaerobic culture: For patients suspected of anaerobic infection, anaerobic culture technology is used for testing.

[0069] Immunological method: antigen-antibody reaction, using the principle of specific antigen-antibody binding, to determine the type of infectious pathogen by detecting the presence of specific antibodies in the patient. Commonly used immunological methods include immunofluorescence technology and enzyme-linked immunosorbent assay;

[0070] Molecular biology methods: polymerase chain reaction: use specific primers to amplify pathogen DNA, and determine the type of pathogen by detecting the amplified product. PCR technology has the advantages of high sensitivity, high specificity, rapidity and accuracy;

[0071] Gene chip technology: A large number of gene fragments with known sequences are fixed on the chip, and hybridized with the DNA or RNA in the sample to be tested to achieve simultaneous detection of multiple pathogens.

[0072] Specifically, Figure 2 As shown, a gene chip comprises the following steps:

[0073] Step 1: Identify common pathogens in bone infection;

[0074] Step 2: For the identified pathogen, select its specific gene sequence as the basis for probe design and design a suitable probe sequence based on the selected gene sequence;

[0075] Step 3: Select appropriate carrier material and chemically treat the carrier;

[0076] Step 4: synthesize the probe in vitro using chemical synthesis technology, and fix the synthesized probe on the pre-treated carrier by spotting method or in situ synthesis method;

[0077] Step 5: extracting pathogenic nucleic acid from the sample of the bone infection patient, labeling the extracted pathogenic nucleic acid with fluorescence or other forms, and performing a hybridization reaction;

[0078] Step 6: Use a fluorescent scanner or other detection equipment to scan the hybridized chip, and analyze whether the target pathogen exists in the sample and the type and quantity of the pathogen based on the collected data.

[0079] Specifically, Figure 2As shown, use an appropriate nucleic acid extraction method to extract pathogens from samples. During the nucleic acid extraction process, the purity and integrity of the nucleic acid should be ensured to avoid degradation and loss. The extracted nucleic acid should be purified to remove proteins and polysaccharides. The labeled nucleic acid samples should be quality controlled, including concentration determination and purity inspection. According to experimental requirements and the characteristics of the target pathogen, the conditions of the hybridization reaction, including temperature, time, salt concentration, and buffer composition, should be optimized. The labeled nucleic acid samples should be hybridized with the probes fixed on the chip. After the hybridization is completed, an appropriate eluent should be used to remove unbound nucleic acid molecules and other impurities.

[0080] Specifically, Figure 2 As shown, scanning parameters such as laser power, gain, and exposure time need to be adjusted during the scanning process. After the scanning is completed, the fluorescence scanner converts the captured signal into digital image data, performs a preliminary quality control check on the acquired image data, performs background correction and signal enhancement preprocessing on the acquired image data, calculates the fluorescence intensity value of each probe point based on the preprocessed image data, performs cluster analysis on genes or samples with similar expression patterns, and performs functional enrichment analysis on differentially expressed genes.

[0081] By using the above technical solution, the hybridization box is turned on and preheated to 45°C, and the hybridization box and the hybridization plate are placed in the hybridization box for at least 30 minutes;

[0082] Incubate the reaction hybridization solution at 95℃ for 10 minutes on a PCR instrument, then cool it down to 70℃ for preservation (it can also be placed on ice for rapid cooling after the 95℃ incubation). Add the hybridization solution to the corresponding positions of the hybridization plate and chip;

[0083] Transfer the chip to the hybridization plate and check whether the liquid covers the entire chip; put the chip into the hybridization box, and then put the hybridization box into the 45℃ hybridization box for incubation for 2 hours. ;

[0084] Open and preheat the hybridization box at 30℃, place the plate washer and 30mL.Wash B (in a centrifuge tube) in the hybridization box for at least 30 minutes; then pour 30ml.Wash A into the plate washer at room temperature;

[0085] Take the chip out of the hybridization box and observe whether there are bubbles on the chip. Then transfer the chip to Wash A and wash it at room temperature for 5 minutes.

[0086] Pour Wash B into the washing plate, then transfer the chip plate to the Wash B washing plate and wash at 30°C for 5 minutes;

[0087] Dilute SAPE 1:100 with 4x SSC and add the diluted dye to the corresponding chip position on the well plate;

[0088] Take the chip out of Wash B, shake off the moisture on the chip surface, transfer the chip to the dye solution, and dye it at room temperature in the dark for 20 minutes;

[0089] Pour 4x SSC into the washing plate, and after staining, transfer the chip to the 4x SSC washing plate and wash for 2 minutes;

[0090] Select S2B RFID, select BF, green-AM1E-1s and green-AM1E-4s channels, and select the corresponding chip position for scanning.

[0091] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting microorganisms, characterized in that: The following steps are involved: Step 1: Collect microbial samples from appropriate environments or organisms according to the purpose of the test; Step 2: Extract microorganisms from the pretreated sample using an appropriate extraction method; Step 3: Select appropriate detection methods based on the detection purpose and microbial characteristics; Step 4: Based on the results of microbial culture, immunological testing and molecular biological testing, conduct a comprehensive analysis to determine the type of infectious pathogen.

2. A microbial detection method according to claim 1, characterized in that: Clarify the purpose of the test and then select the appropriate sample type, including blood samples, tissue samples, secretion samples, and puncture fluid samples. Use sterile instruments and containers to collect multiple samples and ensure that each sample will not be contaminated by each other. Then send the samples to the laboratory for processing to avoid long-term storage that may cause inactivation or contamination of microorganisms.

3. A microbial detection method according to claim 1, characterized in that: The method of extracting microorganisms from the pretreated sample comprises the following steps: Step 1: Pretreatment: For solid samples, grinding and homogenization are required first. Joint fluid and bone marrow samples need to be centrifuged and filtered to remove impurities and enriched for culture. Step 2: Purification of microorganisms: Spread the enriched culture sample evenly on a solid culture medium; after culture, select single colonies with obvious morphological and color characteristics for further purification, streak separation or subculture in liquid culture medium on the selected single colonies, and repeat the above steps until a pure culture is obtained.

4. A microbial detection method according to claim 1, characterized in that: The microbial detection methods include: traditional culture methods, including bacterial culture, fungal culture and anaerobic bacteria culture; Immunological methods: antigen-antibody reaction; Molecular biology methods: polymerase chain reaction method and gene chip technology.

5. A gene chip according to claim 1, characterized in that: The following steps are involved: Step 1: Identify common pathogens in bone infection; Step 2: For the identified pathogen, select its specific gene sequence as the basis for probe design and design a suitable probe sequence based on the selected gene sequence; Step 3: Select appropriate carrier material and chemically treat the carrier; Step 4: synthesize the probe in vitro using chemical synthesis technology, and fix the synthesized probe on the pre-treated carrier by spotting method or in situ synthesis method; Step 5: extracting pathogenic nucleic acid from the sample of the bone infection patient, labeling the extracted pathogenic nucleic acid with fluorescence or other forms, and performing a hybridization reaction; Step 6: Use a fluorescent scanner or other detection equipment to scan the hybridized chip, and analyze whether the target pathogen exists in the sample and the type and quantity of the pathogen based on the collected data.

6. A gene chip according to claim 5, characterized in that: Use appropriate nucleic acid extraction methods to extract pathogens from samples. During the nucleic acid extraction process, the purity and integrity of the nucleic acid should be ensured to avoid degradation and loss. The extracted nucleic acid should be purified to remove proteins and polysaccharides. The labeled nucleic acid samples should be quality controlled, including concentration determination and purity inspection. According to experimental requirements and the characteristics of the target pathogen, the conditions of the hybridization reaction, including temperature, time, salt concentration, and buffer composition, should be optimized. The labeled nucleic acid samples should be hybridized with the probes fixed on the chip. After the hybridization is completed, an appropriate eluent should be used to remove unbound nucleic acid molecules and other impurities.

7. A gene chip according to claim 1, characterized in that: Scanning parameters such as laser power, gain, and exposure time need to be adjusted during the scanning process. After the scan is completed, the fluorescence scanner converts the captured signal into digital image data, performs a preliminary quality control check on the acquired image data, performs background correction and signal enhancement preprocessing on the acquired image data, calculates the fluorescence intensity value of each probe point based on the preprocessed image data, performs cluster analysis on genes or samples with similar expression patterns, and performs functional enrichment analysis on differentially expressed genes.

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