Respiratory pathogenic microorganism and drug resistance gene nucleic acid testing kit (gene chip method)

Abstract

The present invention relates to a respiratory pathogenic microorganism and drug resistance gene nucleic acid testing kit (gene chip method), comprising a kit body. The kit body comprises a PCR amplification reaction reagent, PCR amplification reaction consumables, gene chip hybridization reaction reagents, gene chip hybridization reaction consumables, and a specific gene probe. The gene chip hybridization reaction reagents include a hybridization solution and a 1000X cleaning solution; the gene chip hybridization reaction consumables include a gene chip and a coverslip; the PCR amplification reaction reagent is a PCR buffer solution; and the PCR amplification reaction consumables are PCR tubes. On the basis of gene chip technology in combination with novel multiplex PCR amplification technology, the present invention provides a novel respiratory pathogenic bacterium and drug resistance gene testing technology which has high testing throughput and sensitivity and good identification accuracy, is simple and easy to operate and low-cost, and is widely used as a conventional testing means; and testing results are used for guiding treatment plans and medications for patients suffering from respiratory diseases and even acute and severe patients.

Description

Respiratory Pathogens and Drug Resistance Gene Nucleic Acid Detection Kit (Gene Chip Method) Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection kits, in particular to a nucleic acid detection kit for respiratory pathogenic microorganisms and drug-resistant genes (gene chip method). Background Art

[0002] The existing technology for detecting respiratory pathogens is mainly based on culture methods. Different types of pathogens are cultured on culture media using different types of test kits. Most respiratory pathogens can also be detected for their drug resistance, and the cost is low. However, the detection time and detection throughput of the culture method are very serious shortcomings: conventional culture methods can only detect pathogens or drug resistance one by one, and it takes 3-7 days. For patients who are already seriously infected and admitted to the ICU due to acute and severe illness, it is impossible to wait for such a long detection time. At the same time, some patients with respiratory infections may carry more than one pathogen. Several different pathogens need to be fully tested with clear results, and drug resistance testing may also be required. As far as the culture method is concerned, it is not only inconvenient to operate, but the culture media are cultured one by one, and it takes considerable human resources to process.

[0003] In addition to culture methods, PCR amplification methods are also increasingly being used to detect respiratory pathogens and drug-resistant genes. Fluorescence quantitative PCR can shorten the detection time to 3 hours, meeting the needs of rapid detection of acute and severe cases. At the same time, the PCR method has a higher detection sensitivity than the culture method, and the cost of a single test is low, making it suitable for routine testing. However, the PCR method has a low detection throughput and usually requires the use of different primers to amplify different pathogen genes and drug-resistant genes. Therefore, in situations where the number of pathogens and test samples is large, it will be inconvenient to operate, just like the culture method. If a large number of different primers are mixed for amplification, the different sensitivities between the primers will cause some genes to be poorly amplified or unable to be amplified, resulting in low detection sensitivity and poor accuracy. When the number of samples tested is large, it will also cause a large amount of testing costs to be accumulated, making it difficult for users to bear, resulting in the inability of the technical means to be used in practice.

[0004] Compared with the above two methods, second-generation sequencing has extremely high detection throughput and sensitivity, and can detect a variety of respiratory pathogen genes with extremely high accuracy. However, the advantages of second-generation sequencing are mostly reflected in the sequencing of wild-type genes. For drug-resistant genes with more mutant genes, there will be problems such as low sensitivity and poor accuracy. The cost of second-generation sequencing is high and the operation is difficult. It requires professionals in the field who have received relevant training to operate. At the same time, it still takes a long time, 48 hours. It is still difficult to use as a routine detection method and a rapid detection method for acute and severe diseases. Therefore, its popularity is far less than the previous two.

[0005] However, traditional gene chip nucleic acid detection has the following disadvantages:

[0006] Traditional microarray gene chips have average detection sensitivity and throughput. Although the accuracy of the test results is acceptable, they may also produce high background signals, resulting in poor result accuracy. Traditional gene chips are difficult to operate, the operation process is relatively complicated, and may require harsh reaction conditions. They are not suitable for use as a technical means of routine and rapid testing in medical treatment, and are mostly used in scientific research experiments.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to provide a nucleic acid detection kit for respiratory pathogens and drug-resistant genes (gene chip method) to solve the problems mentioned in the above background technology, such as the general sensitivity and throughput of traditional microarray gene chips. Although the accuracy of the test results is acceptable, a high background signal may be generated, resulting in poor result accuracy. The operation of traditional gene chips is difficult, the operation process is relatively complicated, and harsh reaction conditions may be required. They are not suitable for use as a technical means for routine and rapid detection in medical treatment, and are mostly used in scientific research experiments.

[0009] To achieve the above-mentioned purpose, the present invention provides the following technical solution: a respiratory pathogenic microorganism and drug-resistant gene nucleic acid detection kit (gene chip method), comprising a kit body, the kit body comprising PCR amplification reaction reagents, PCR amplification reaction consumables, gene chip hybridization reaction reagents, gene chip hybridization reaction consumables and specific gene probes, the gene chip hybridization reaction reagents comprising hybridization solution and 1000X cleaning solution, the gene chip hybridization reaction consumables comprising a gene chip and a cover sheet, the PCR amplification reaction reagent is a PCR buffer, and the PCR amplification reaction consumables are PCR tubes.

[0010] As a preferred technical solution of the present invention, the hybridization solution includes formamide, sodium citrate buffer, sodium lauryl sulfate, PEG35K, TritonX-100, internal reference and purified water.

[0011] As a preferred technical solution of the present invention, the 1000X cleaning solution includes cysteamine hydrochloride and purified water.

[0012] As a preferred technical solution of the present invention, the PCR buffer includes Tris-HCl, KCl, (NH4)SO4, MgSO4, Triton X-100, D-trehalose (dihydrate), dNTP, Taq HS enzyme, PCR amplification primers, positive quality control and sterile water.

[0013] As a preferred technical solution of the present invention, the specific gene probes include gene probes that can capture pathogenic bacteria and gene probes that can capture drug resistance.

[0014] The method for using the nucleic acid detection kit for respiratory pathogenic microorganisms and drug-resistant genes of the present invention comprises the following steps:

[0015] Step 1: Determine reagents and consumables: All reagents and consumables are classified according to their functions, and the reagent components are integrated and optimized to obtain PCR amplification reaction reagents, PCR amplification reaction consumables, gene chip hybridization reaction reagents, and gene chip hybridization reaction consumables;

[0016] Step 2: Performing a PCR amplification reaction: performing a PCR amplification reaction using PCR amplification reaction reagents and PCR amplification reaction consumables;

[0017] Step 3: Perform hybridization reaction: Perform hybridization reaction on the amplified product using gene chip hybridization reaction reagents and gene chip hybridization reaction consumables; during hybridization, the probes on the gene chip hybridize and bind to 47 respiratory tract infection pathogens and 11 drug resistance genes in a sample;

[0018] Step 4: PCR reaction monitoring: monitoring the PCR reaction (results) by simultaneously hybridizing the amplified product of the positive quality control DNA template;

[0019] Step 5: Hybridization reaction monitoring: After hybridization, the gene chip is cleaned with a cleaning solution and dried, and the hybridization signals of the probes on the chip are scanned and the data is processed to obtain the test results.

[0020] As a preferred technical solution of the present invention, when the PCR amplification reaction in step 2 is specifically primer amplification, the primers are specially designed to accurately capture the target DNA in the sample and bind to and amplify it. The primers are grouped in advance according to sensitivity, prepared into a buffer solution, and then matched with the PCR tubes contained in the kit to allow multiple primers to perform PCR amplification reactions simultaneously.

[0021] As a preferred technical solution of the present invention, the hybridization reaction in step three is specifically that during hybridization, an internal reference modified with cy5 fluorescence is added to bind to the probe on the gene chip, and the probes that capture the target gene and the probes that do not capture the target gene are distinguished based on the strength of different fluorescence signals to obtain hybridization result data.

[0022] As a preferred technical solution of the present invention, the 47 respiratory tract infection pathogens and 11 respiratory pathogenic microorganisms with drug-resistant genes in step three include Gram-positive bacteria, Gram-negative bacteria and fungi.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. This invention designs amplification primers and gene probes for specific microbial genes and drug-resistant genes. The primers are used to amplify DNA in the sample, and the amplified DNA is hybridized using a gene chip. This allows for the simultaneous detection of 47 respiratory pathogens and 11 drug-resistant genes, covering Gram-positive bacteria, Gram-negative bacteria, and fungi, which is beyond the capabilities of any existing technology.

[0025] 2. The present invention overcomes the shortcomings of traditional microarray gene chips / gene chips, such as low detection sensitivity, insufficient accuracy, high background signal, high operational difficulty, and complicated procedures, and is applicable to routine and rapid medical testing, whereas traditional microarray gene chips / gene chips are basically only used in scientific research experiments;

[0026] 3. The detection process of the present invention takes 2-3 hours, which is shorter than the 48 hours required for second-generation sequencing, and its sensitivity and accuracy are even better than that of second-generation sequencing;

[0027] 4. The present invention has high detection throughput and is easy to operate. It only requires training on how to use the kit, and does not require professional personnel in the field to handle large-scale sample testing with relatively small manpower. At the same time, the cost of use of the present invention is much lower than that of second-generation sequencing, and it is not easy to cause excessive cost increases in the use scenario of large-scale sample testing. It is suitable for large-scale use in routine testing and rapid detection in emergency and critical care settings.

[0028] 5. The present invention classifies reagents and consumables according to their functions, and integrates all the reagent components required for PCR reactions into one tube of solution except for the template DNA, making the kit extremely convenient to use. When using the kit, there is no need to prepare any additional reagents and consumables, nor is there any need to prepare the reagents one by one according to the ingredients. All reactions can be completed in one box, greatly improving the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 is a schematic diagram of the structure of the kit of the present invention;

[0030] FIG2 is a schematic diagram of the structure of the gene chip hybridization reaction reagent of the present invention;

[0031] FIG3 is a schematic diagram of the structure of the hybridization solution of the present invention;

[0032] FIG4 is a schematic diagram of the structure of the 1000X cleaning solution of the present invention;

[0033] FIG5 is a schematic diagram of the structure of the PCR buffer of the present invention;

[0034] FIG6 is a schematic diagram of the structure of a specific gene probe of the present invention;

[0035] FIG7 is a flowchart of the use of the present invention;

[0036] FIG8 is a diagram showing the distribution of primers in eight tubes of the present invention;

[0037] FIG9 is a diagram showing the distribution of positive quality control DNA in eight tubes of the present invention;

[0038] FIG10 is a fluorescence signal image of a Haemophilus influenzae sample of the present invention;

[0039] FIG11 is a fluorescence signal image of a Serratia marcescens sample of the present invention;

[0040] FIG12 is a fluorescence signal image of the mixed bacteria sample of the present invention. DETAILED DESCRIPTION

[0041] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described 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 making creative efforts are within the scope of protection of the present invention.

[0042] Please refer to Figures 1-12. The present invention provides a respiratory pathogenic microorganism and drug-resistant gene nucleic acid detection kit (gene chip method), including a kit body, which includes PCR amplification reaction reagents, PCR amplification reaction consumables, gene chip hybridization reaction reagents, gene chip hybridization reaction consumables and specific gene probes. The gene chip hybridization reaction reagents include hybridization solution and 1000X cleaning solution. The gene chip hybridization reaction consumables include a gene chip and a cover sheet. The PCR amplification reaction reagent is a PCR buffer, and the PCR amplification reaction consumables are PCR tubes.

[0043] The hybridization solution includes formamide, sodium citrate buffer, sodium dodecyl sulfate, PEG35K, TritonX-100, internal control and purified water.

[0044] 1000X cleaning solution consists of cysteamine hydrochloride and purified water.

[0045] PCR buffer includes Tris-HCl, KCl, (NH4)SO4, MgSO4, Triton X-100, D-trehalose (dihydrate), dNTPs, Taq HS enzyme, PCR amplification primers, positive quality control and sterile water.

[0046] Specific gene probes include probes that can capture pathogenic bacteria genes and probes that can capture drug-resistance genes.

[0047] The method for using the nucleic acid detection kit for respiratory pathogenic microorganisms and drug-resistant genes of the present invention comprises the following steps:

[0048] Step 1: Determine reagents and consumables: All reagents and consumables are classified according to their functions, and the reagent components are integrated and optimized to obtain PCR amplification reaction reagents, PCR amplification reaction consumables, gene chip hybridization reaction reagents, and gene chip hybridization reaction consumables;

[0049] Step 2: Performing a PCR amplification reaction: performing a PCR amplification reaction using PCR amplification reaction reagents and PCR amplification reaction consumables;

[0050] Step 3: Perform hybridization reaction: Perform hybridization reaction on the amplified product using gene chip hybridization reaction reagents and gene chip hybridization reaction consumables; during hybridization, the probes on the gene chip hybridize and bind to 47 respiratory tract infection pathogens and 11 drug resistance genes in a sample;

[0051] Step 4: PCR reaction monitoring: monitoring the PCR reaction (results) by simultaneously hybridizing the amplified product of the positive quality control DNA template;

[0052] Step 5: Hybridization reaction monitoring: After hybridization, the gene chip is cleaned with a cleaning solution and dried, and the hybridization signals of the probes on the chip are scanned and the data is processed to obtain the test results.

[0053] In step 2, the PCR amplification reaction is specifically primer amplification. The primers are specially designed to accurately capture the target DNA in the sample, bind to it, and amplify it. The primers are grouped in advance according to sensitivity, prepared into a buffer solution, and then paired with the PCR tubes contained in the kit to allow multiple primers to perform PCR amplification reactions simultaneously.

[0054] In the hybridization reaction in step 3, during hybridization, an internal reference modified with cy5 fluorescence is added to bind to the probes on the gene chip, and the probes that capture the target gene and those that do not are distinguished based on the strength of different fluorescence signals to obtain hybridization result data.

[0055] The respiratory pathogens with 47 types of respiratory infection pathogens and 11 types of drug-resistant genes in step three cover Gram-positive bacteria, Gram-negative bacteria and fungi.

[0056] Example 1:

[0057] In the present invention, DNA is first amplified with special primers, and then hybridized with specific gene probes and gene chips, and finally multiple respiratory pathogens and drug-resistant genes in a sample are detected simultaneously; during primer amplification, the primers with specially designed sequences that accurately capture the target DNA in the sample and bind to and amplify it are grouped in advance according to sensitivity and prepared into a buffer solution, which is then matched with the PCR tubes contained in the kit to allow multiple primers to perform PCR amplification reactions simultaneously; the amplification buffer contains a positive quality control DNA template, and the PCR reaction is controlled by hybridizing the amplified products of the positive quality control DNA template. Monitoring; specific gene probes include probes that capture pathogenic bacteria genes / drug-resistance genes; during hybridization, by adding an internal reference modified with cy5 fluorescence, it is combined with the probes on the gene chip, and the probes that capture the target gene and those that do not are distinguished based on the strength of different fluorescent signals to obtain hybridization result data; after hybridization, the gene chip is cleaned and dried with a cleaning solution, the hybridization signal is scanned and the data is processed to obtain the test results; the above technology has been applied to the detection and identification of 47 respiratory tract infection pathogens and 11 drug-resistant genes, covering Gram-positive bacteria, Gram-negative bacteria and fungi (as shown in Table 1).

[0058] Table 1. 47 respiratory tract infection pathogens and 11 drug resistance genes involved in the detection range of the present invention

[0059] Example 2:

[0060] In the present invention, the kit contains all reagents and consumables required for PCR amplification reaction and gene chip hybridization reaction. The specific contents are shown in Table 2 and Table 3. The ingredients, proportions and concentrations of each component are obtained through experimental optimization for use in the present invention.

[0061] Table 2. Kit structure and main components

[0062] Table 3. Components, proportions, and final concentrations of each reagent in the kit

[0063] Next, this example further expands on the PCR buffer, gene chip, cover glass and hybridization solution.

[0064] The PCR buffer contains PCR primers specially designed for amplifying target bacterial species and drug-resistant gene DNA. In addition to the components required for the standard PCR reaction system, the present invention adds 1% Triton X-100, D-trehalose (dihydrate) with a final system concentration of 0.625 mol / L, and positive quality control DNA to the PCR buffer in addition to the amplification primers, compared to the standard reaction. The composition of the buffer and the proportions and concentrations of its components are all optimized to achieve the ideal performance of the present invention. Its design has the specific purpose of ensuring the effectiveness of the present invention.

[0065] Amplification primers: PCR amplification primers were designed and divided into 8 groups, named groups AH (as shown in Table 4). Each primer sequence was designed based on a specific region on the target pathogen gene and drug resistance gene (DNA) to accurately capture the corresponding template DNA. At the same time, the reason for the special grouping of primers is that different primers have different sensitivities in binding to template DNA. If primers with similar sensitivities are not grouped, some primers with relatively low sensitivity will not easily bind to template DNA under the influence of high-sensitivity primers, resulting in a small number of PCR amplification products, low amplification efficiency, and subsequent missed detections, low detection sensitivity, and poor accuracy. Therefore, primers with similar sensitivities are selected as a group, and PCR reactions are carried out in an isolated independent environment with other groups of primers to effectively solve this problem. At the same time, PCR amplification primers are specially designed to carry cy3 fluorescent modification on the product during amplification, which cooperates with the internal reference in subsequent hybridization to produce an effect.

[0066] Table 4. Eight primer combinations

[0067] In actual use, the eight primer sets are grouped in the kit packaging. Based on the above-mentioned PCR buffer components, each set is prepared into an independent solution and placed in one of the eight PCR tubes. The eight tubes correspond to eight PCR buffers containing different primer sets. In conjunction with Figure 8 of the specification, when performing a PCR reaction, a certain amount of template DNA from the same sample is added to each of the eight tubes. Then, the eight tubes are placed in the PCR instrument to achieve simultaneous multi-primer amplification of a single sample. At the same time, the problem of poor PCR amplification due to excessive primers in the reaction system or large differences in primer sensitivity is avoided.

[0068] Triton X-100: As an active agent in the PCR reaction, its specific function is to promote more primers to bind to the target template DNA, thereby improving the efficiency of the PCR reaction by increasing the amount of PCR product;

[0069] D-trehalose (dihydrate): acts as a protective agent for Taq HS enzyme, protecting the activity of the enzyme contained in the present invention, so that the enzyme can be used for the desired PCR reaction after a certain period of frozen transportation or storage;

[0070] Positive quality control: 8 specially designed single-stranded DNA templates with different sequences, whose specific function is to monitor whether the PCR reaction is successful. By adding positive quality control to the PCR buffer in the kit of the present invention, during the PCR reaction, the primers will also bind to these positive quality control DNAs and produce subsequent amplification reactions. Therefore, if the PCR reaction is successful, in addition to the amplified products of the target DNA, the product should also contain amplified products of the positive quality control. By placing probes for detecting positive quality control DNA on the gene chip, the positive quality control products in the PCR products are detected during the gene chip hybridization reaction to observe whether the PCR reaction is successful. The traditional PCR method requires the addition of a gel electrophoresis detection step after amplification, and the amplification quality is monitored by the bands generated by electrophoresis. The present invention combines the advantages of PCR amplification and gene chip methods, and monitors the amplification quality during the gene chip hybridization reaction. There is no need for additional gel electrophoresis, which saves equipment and labor costs and improves the overall operating efficiency. The 8 positive quality controls are named Interncontrltemp 1-8 (as shown in Table 5), according to the sequence number 1-8 corresponding to the primer set AH (A-1, B-2 ~ H-8), respectively, add each primer set to the PCR buffer, put it in eight tubes, and refer to Figure 9 of the instruction manual.

[0071] Table 5. Eight PCR-positive quality control DNAs

[0072] 2. Gene Chips and Cover Slips

[0073] Based on the above detection range, the present invention includes at least one gene probe that can detect each pathogenic bacteria or drug-resistant gene for each pathogenic bacteria and drug-resistant gene. The probes capture pathogenic bacteria genes / drug-resistant genes and are applied to the detection and identification of 47 respiratory pathogens and 11 drug-resistant genes, covering Gram-positive bacteria, Gram-negative bacteria, and fungi. All probes, or probe library sequences, are designed based on specific regions on target pathogenic bacteria genes and drug-resistant genes, accurately pairing with corresponding genes. The probes are colonized on a microscope slide with a low fluorescence background to form a gene chip. When in use, the cover glass included in the present invention is used to cover the gene chip, and a hybridization solution mixed with amplification products is added between the two layers to facilitate hybridization reactions in subsequent steps.

[0074] 3. Hybridization Solution

[0075] The hybridization solution is mainly composed of hybridization buffer and internal reference. When used, the product DNA obtained by PCR amplification is mixed with the hybridization solution, and then the hybridization solution is added to the gene chip and coverslip, which can be used for subsequent hybridization reactions.

[0076] Hybridization buffer: The components and proportions are shown in Table 2. Formamide lowers the temperature required for the binding of the amplified product and the probe to 37°C; sodium citrate buffer prevents significant changes in the solution pH when the other components are added to the solution; sodium dodecyl sulfate, PEG35K, and Triton X-100 act as active agents, improving the binding efficiency of the product and the probe. The high viscosity of PEG35K also ensures that the surface tension of the solution on the gene chip during hybridization is not excessive, resulting in good fluidity. Based on the above components, the gene chip hybridization reaction of the present invention does not require stringent environmental requirements; hybridization can be completed by simply rotating the solution at a constant temperature of 37°C for 45 minutes, yielding clear and accurate results.

[0077] Internal reference: The internal reference is a specially designed short DNA sequence modified with a cy5 fluorescent dye at its 5' end. When the amplified product DNA is amplified, the primer with the cy3 fluorescent modification hybridizes with the gene chip probe DNA. The internal reference also hybridizes with the probe, so that the probes that successfully capture the target gene and those that fail to capture the target gene show different fluorescence signal intensities. The former will have a strong cy3 fluorescent signal, while the latter will only have a cy5 fluorescent signal, resulting in a clear contrast result to facilitate data analysis. Specific internal reference information is shown in Table 6.

[0078] Table 6. Hybridization internal reference information

[0079] The above-mentioned respiratory pathogenic microorganism and drug-resistant gene nucleic acid detection kit (gene chip method) is used as follows: first, DNA is amplified using special primers, then hybridized with a specific gene probe and gene chip, and finally, multiple respiratory pathogenic microorganisms and drug-resistant genes in a sample are detected simultaneously. The structure and preparation method of the gene chip and gene probe involved have been separately patented by the applicant of the present invention in the past, with application numbers CN201110129845, CN201810939564 and CN201810941697 respectively. When the primers are amplified, Primers with specially designed sequences that accurately capture, bind, and amplify target DNA in the sample are grouped in advance by sensitivity and prepared into a buffer. This buffer is then combined with the eight-tube PCR strip included in the kit, allowing multiple different primers to perform PCR amplification reactions simultaneously under independent conditions. During hybridization, an internal reference modified with cy5 fluorescence is added, and the amplified DNA products are labeled with cy3 fluorescence during PCR. The internal reference DNA products simultaneously bind to the probes, causing them to bind to the probes on the gene chip. The strength of the different fluorescence signals is used to distinguish probes that capture the target gene from those that do not, and hybridization result data for analysis is obtained. After hybridization, the gene chip is cleaned with a cleaning solution and dried. The hybridization signal is scanned and the data is processed to obtain the test results. In actual use, all probes will hybridize with the internal reference, resulting in a certain green fluorescent signal (cy5). At the same time, the amplified products of the target DNA contained in the sample will hybridize with specific probes, causing these probes to also show a clear red signal (cy3) during the scan. The analysis software calculates the background value based on the signal value of the internal reference through a specific calculation program, and then combines the background value with the combined signal value. By comparing the positive signal value (i.e., cy3 fluorescence signal) of the probe that has bound the target DNA with the background value, it is possible to determine which probes have bound to the target DNA, thereby obtaining an obvious and clear detection result. In this embodiment, the present invention is used to detect and identify DNA samples of pathogenic bacteria within the detection range. The selected samples are DNA extracted from Haemophilus influenzae and DNA extracted from a standard strain of Serratia marcescens (bacterial concentration = 10^5 bacteria). The DNA of Haemophilus influenzae and Serratia marcescens are detected respectively. The detection process is PCR amplification, gene chip hybridization, scanning and analysis of hybridization signals.

[0080] Among them, the specific PCR primers and gene probes for 47 respiratory tract infection pathogens and 11 drug-resistant genes, the forward primers, the nucleic acid sequences of which are shown in SEQ ID NOs: 1 to 22; the reverse primers, the nucleic acid sequences of which are shown in SEQ ID NOs: 23 to 46; the gene probes, the nucleic acid sequences of which are shown in SEQ ID NOs: 48 to 154, are screened according to Genbank data and domestic and foreign literature to obtain the genome sequences of the 47 respiratory tract infection pathogens and 11 drug-resistant genes, and the genome sequences are aligned and analyzed using DNAMAN and Blast tools to obtain the specific sequences of the pathogen microorganisms and drug-resistant genes, and specific PCR primers (as shown in Table 7) and gene probes (as shown in Table 8) are designed based on these target sequences.

[0081] Table 7. Specific primers and sequences of the present invention

[0082] Table 8. Gene probes and sequences of the present invention

[0083] At the same time, a common primer for use in a PCR reaction was also designed based on the target sequence. The nucleic acid sequence of the common primer is shown in SEQ ID NO: 47. The 5' end of the common primer is modified with a cy3 fluorescent group. In the PCR reaction, the template DNA extracted from the sample to be tested is first amplified by the specific primers and then further amplified by the common primer, ultimately making the amplified PCR product DNA labeled with the cy3 fluorescent group. At the same time, the PCR reaction amplifies the template DNA to a level suitable for hybridization detection by chip probes. The common primer information is shown in Table 9.

[0084] Table 9. Common PCR primers and sequences of the present invention

[0085] 8 positive quality control nucleic acids and their corresponding gene probes were designed after analyzing and comparing the genomic sequences of 47 respiratory tract infection pathogens and 11 drug-resistant genes using DNAMAN and Blast tools. The nucleic acid sequences of the positive quality control nucleic acids are shown in SEQ ID NOs: 155 to 162; the nucleic acid sequences of the positive quality control corresponding gene probes are shown in SEQ ID NOs: 163 to 170; in the PCR reaction, the DNA of the positive quality control nucleic acid is linearly amplified by the above-mentioned common primers, which will not interfere with the amplification of other template DNAs; the amplified positive quality control PCR products hybridize with the above-mentioned corresponding probes in the hybridization reaction (the probes are fixed on the chip), and will not interfere with the hybridization of other probes, and therefore will not interfere with the detection. The positive quality control information is shown in Table 10; the positive quality control corresponding gene probes are shown in Table 11.

[0086] Table 10. The present invention relates to positive quality control

[0087] Table 11. Gene probes corresponding to positive quality control of the present invention

[0088] The nucleic acid sequence of the hybridization internal reference described in the original application of the present invention is shown in SEQ ID NO: 171:

[0089] Table 12. Hybridization internal references of the present invention

[0090] Example 3:

[0091] In the present invention, based on Example 1 and Example 2, this embodiment uses the present invention to detect and identify Haemophilus influenzae samples and Serratia marcescens samples, and the steps are as follows:

[0092] S1. Take out the eight-tube strips from the test kit. One row of eight tubes corresponds to one test sample. Each of the eight tubes contains eight sets of pre-made PCR buffer containing different primer sets.

[0093] S2. Add 5 μL of the extracted DNA of the sample to be tested to each PCR buffer reaction tube, and centrifuge briefly to collect the residual liquid on the tube wall to the bottom;

[0094] S3. Place the reaction tube in a PCR instrument and set the following reaction program for amplification: pre-denaturation at 95°C for 5 minutes; 5 cycles of 95°C for 15 seconds, 53°C for 1 minute and 30 seconds, and 72°C for 20 seconds; 30 cycles of 94°C for 15 seconds, 55°C for 35 seconds, and 72°C for 20 seconds;

[0095] S4. After amplification, 5 μL of each of the 8 PCR amplification products from the same sample DNA was mixed in an EB tube for gene chip hybridization reaction. 121 μL of the kit hybridization solution was added to the EB tube and mixed thoroughly.

[0096] S5. Place the EB tube at 100°C for 5 minutes, then remove it and place it in an ice water bath for 5 minutes;

[0097] S6. Cover the gene chip with the cover glass provided in the kit, then add the sample after the ice water bath along the gap between the cover glass and the edge of the gene chip, and then place it in the hybridization box;

[0098] S7. Place the hybridization box in the hybridizer for hybridization. After hybridization is completed, remove the gene chip and dilute the 1000X cleaning solution in the kit with purified water to 1X cleaning solution.

[0099] S8. One gene chip cleaning box requires 150 ml of 1X cleaning solution to clean the gene chip. Then put the hybridized gene chip into the cleaning box, close the box lid and rinse it up and down three times. Finally, pour in the cleaning solution to soak the gene chip.

[0100] S9. Scan the cleaned gene chip with a gene chip scanner to obtain an image with a fluorescent signal. Combined with Figures 10 and 11 of the specification, analyze the results.

[0101] Table 13. DNA test results of Haemophilus influenzae samples

[0102] Table 14. DNA test results of Serratia marcescens samples

[0103] According to Tables 13 and 14, the DNA extracted from Haemophilus influenzae and Serratia marcescens showed consistent results on the gene chip after being tested by the kit, and also showed drug-resistant gene results, proving that the kit detected the DNA of single pathogenic bacteria and the drug-resistant genes contained therein.

[0104] Example 4:

[0105] In the present invention, based on Example 1, the present invention is used to detect and identify DNA samples of pathogenic bacteria within the detection range. The selected samples are DNA extracted from mixed bacterial samples (bacteria concentration = 10^ 7The mixed bacterial sample was prepared by mixing standard strains of Legionella pneumophila, Streptococcus pyogenes, Klebsiella oxytoca and Candida albicans. The detection process was PCR amplification, gene chip hybridization, scanning and analysis of hybridization signals. The PCR amplification process and hybridization process were the same as those in Example 3. Finally, the cleaned gene chip was scanned with a gene chip scanner to obtain an image with a fluorescent signal. Combined with Figure 12 of the specification, and the results were analyzed, the experimental results are shown in Table 15.

[0106] Table 15. DNA test results of mixed bacterial samples

[0107] According to Table 15, the DNA extracted from Haemophilus influenzae and Serratia marcescens showed consistent results on the gene chip after being tested by the kit, and also showed drug-resistant gene results, proving that the kit can detect the DNA of the mixed pathogenic bacteria sample and the drug-resistant genes contained therein.

[0108] Example 5:

[0109] In the present invention, based on Example 1, the present invention is used in this embodiment to detect and identify clinical DNA samples. The selected samples are pathogenic bacteria DNA extracted from sputum of patients with respiratory tract infections. The detection process is PCR amplification, gene chip hybridization, scanning and analysis of hybridization signals. The PCR amplification process and hybridization process are the same as those in Example 2. The experimental results are shown in Table 16:

[0110] Table 16. Pathogen DNA detection results of sputum samples from patients with respiratory tract infections

[0111] According to Table 16, the pathogenic bacteria DNA extracted from the sputum samples of patients with respiratory tract infection diseases showed corresponding results on the gene chip after the detection experiment by the kit, and also showed the drug-resistant gene results, proving that the kit detected the pathogenic bacteria DNA contained in the sputum samples of patients with respiratory tract infection diseases and the drug-resistant genes contained therein.

[0112] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A nucleic acid detection kit for respiratory pathogenic microorganisms and drug-resistant genes (gene chip method), comprising a kit body, characterized in that: The kit body includes PCR amplification reaction reagents, PCR amplification reaction consumables, gene chip hybridization reaction reagents, gene chip hybridization reaction consumables and specific gene probes. The gene chip hybridization reaction reagents include hybridization solution and 1000X cleaning solution. The gene chip hybridization reaction consumables include a gene chip and a cover sheet. The PCR amplification reaction reagent is a PCR buffer, and the PCR amplification reaction consumables are PCR tubes.

2. The respiratory pathogenic microorganism and drug resistance gene nucleic acid detection kit (gene chip method) according to claim 1, characterized in that: The hybridization solution includes formamide, sodium citrate buffer, sodium dodecyl sulfate, PEG35K, TritonX-100, internal reference and purified water.

3. The respiratory pathogenic microorganism and drug resistance gene nucleic acid detection kit (gene chip method) according to claim 1, characterized in that: The 1000X cleaning solution includes cysteamine hydrochloride and purified water.

4. The respiratory pathogenic microorganism and drug resistance gene nucleic acid detection kit (gene chip method) according to claim 1, characterized in that: The PCR buffer comprises Tris-HCl, KCl, (NH4)SO4, MgSO4, Triton X-100, D-trehalose (dihydrate), dNTP, Taq HS enzyme, PCR amplification primer, positive quality control and sterile water.

5. The respiratory pathogenic microorganism and drug resistance gene nucleic acid detection kit (gene chip method) according to claim 1, characterized in that: The specific gene probes include gene probes that can capture pathogenic bacteria and gene probes that can capture drug resistance.

6. The method for using the respiratory pathogenic microorganism and drug resistance gene nucleic acid detection kit (gene chip method) according to any one of claims 1 to 5, characterized in that: The following steps are involved: Step 1: Determine reagents and consumables: All reagents and consumables are classified according to their functions, and the reagent components are integrated and optimized to obtain PCR amplification reaction reagents, PCR amplification reaction consumables, gene chip hybridization reaction reagents and gene chip hybridization reaction consumables; Step 2: Perform PCR amplification reaction: PCR amplification reaction was performed on the material; Step 3, performing hybridization reaction: performing hybridization reaction on the amplified product through gene chip hybridization reaction reagent and gene chip hybridization reaction consumables; during hybridization, the probes on the gene chip hybridize and bind to 47 respiratory tract infection pathogens and 11 drug resistance genes in a sample; Step 4: PCR reaction monitoring: monitoring the PCR reaction (result) by simultaneously hybridizing the amplified product of the positive quality control DNA template; Step 5: Hybridization reaction monitoring: After hybridization, the gene chip is cleaned and dried with a cleaning solution, and the hybridization signals of the probes on the chip are scanned and the data is processed to obtain the test results.

7. The method for using the respiratory pathogenic microorganism and drug resistance gene nucleic acid detection kit (gene chip method) according to claim 6, characterized in that: When the PCR amplification reaction in step 2 is specifically primer amplification, the primers that accurately capture the target DNA in the sample and bind to and amplify it are grouped in advance according to sensitivity, prepared into a buffer solution, and then matched with the PCR tubes contained in the kit to allow multiple primers to perform PCR amplification reactions simultaneously.

8. The method for using the respiratory pathogenic microorganism and drug resistance gene nucleic acid detection kit (gene chip method) according to claim 6, characterized in that: The hybridization reaction in step three is specifically that during hybridization, an internal reference with cy5 fluorescence modification is added to make it bind to the probe on the gene chip, and the probes that capture the target gene and the probes that do not capture the target gene are distinguished based on the strength of different fluorescence signals to obtain hybridization result data.

9. The method for using the respiratory pathogenic microorganism and drug resistance gene nucleic acid detection kit (gene chip method) according to claim 6, characterized in that: The 47 respiratory tract infection pathogens and 11 respiratory pathogenic microorganisms with drug-resistant genes in step three cover gram-positive bacteria, gram-negative bacteria and fungi.

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