Targeted sequencing method for multiple pathogenic microorganisms

Abstract

The invention discloses a targeted sequencing method for various pathogenic microorganisms. The targeted sequencing method specifically comprises the following steps: collecting a biological detection material containing the pathogenic microorganisms; extracting nucleic acid in the pathogenic microorganisms to be detected; designing specific primers and probes according to the target pathogenic microorganisms; amplifying a nucleic acid sequence through a PCR (Polymerase Chain Reaction) technology; carrying out deep sequencing based on a high-throughput sequencer; according to the present invention, the specific primers and the probe are designed, such that the nucleic acid sequence of the target pathogenic microorganism can be efficiently enriched, and the high-throughput sequencing technology is adopted to perform the deep sequencing so as to significantly improve the sensitivity and the accuracy of the detection of the plurality of pathogenic microorganisms; the method can be used for simultaneously detecting various different types of pathogenic microorganisms such as bacteria, viruses, fungi and the like, is simple and convenient to operate, short in time consumption and low in cost, and can provide powerful support for quickly and accurately identifying the pathogenic microorganisms and timely taking effective prevention and control measures.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogenic microorganism detection, and specifically provides a targeted sequencing method for multiple pathogenic microorganisms. Background Art

[0002] Pathogenic microorganisms refer to a general term for a class of microorganisms that can invade and cause organisms to suffer from infections or even infectious diseases. They are diverse in types, including bacteria, fungi, viruses, etc. Bacteria and viruses are the most harmful. The etiology of diseases caused by pathogenic microorganisms is complex, and clinically it can lead to symptoms such as allergies, infections, tumors, and even death. It is currently the main disease threatening global human health. Therefore, accurate, efficient, and multi-index detection of pathogenic microorganisms is crucial for timely discovery, prevention, and blocking of the outbreak and spread of related diseases. With the development of molecular biology technology, detection techniques represented by real-time fluorescence quantitative polymerase chain reaction have solved the problems of traditional detection to a certain extent, but there are also problems such as inability to provide pathogen gene sequences and low detection throughput. The combination of ultra-multiplex PCR amplification and high-throughput sequencing technologies is used to obtain a targeted sequencing library by selectively amplifying target genes or sequences on the target genome through multiplex PCR, and then sequencing and downstream analysis are carried out to detect the microorganisms contained in the sample. This technology has an important position in multiple fields such as pathogenic microorganism detection, gene detection, and environmental microorganism detection, and is an emerging technology currently applied clinically;

[0003] However, the traditional methods for detecting pathogenic microorganisms mainly rely on serology and microbial culture at present, which have the phenomena of long cycle and inaccurate results. Moreover, the traditional diagnostic detection methods often require more time from sample collection to pathogen culture and then to result identification. Due to time delay, the best diagnosis and treatment stage is missed, which brings troubles to patients and medical staff. In addition, after obtaining the analysis result information, due to the lack of means to verify and confirm the analysis result, the accuracy and reliability of the analysis result cannot be guaranteed, affecting the actual clinical treatment effect. Summary of the Invention

[0004] The present invention provides a targeted sequencing method for multiple pathogenic microorganisms, which can effectively solve the problems put forward in the above background art that the traditional methods for detecting pathogenic microorganisms mainly rely on serology and microbial culture at present, with the phenomena of long cycle and inaccurate results. Moreover, the traditional diagnostic detection methods often require more time from sample collection to pathogen culture and then to result identification. Due to time delay, the best diagnosis and treatment stage is missed, which brings troubles to patients and medical staff. In addition, after obtaining the analysis result information, due to the lack of means to verify and confirm the analysis result, the accuracy and reliability of the analysis result cannot be guaranteed, affecting the actual clinical treatment effect.

[0005] To achieve the above object, the present invention provides the following technical solution: A targeted sequencing method for multiple pathogenic microorganisms, which enriches the nucleic acid sequences of specific pathogenic microorganisms by designing specific primers and probes, and then uses high-throughput sequencing technology for sequencing to simultaneously detect multiple microorganisms, so as to improve the sensitivity and accuracy of detection;

[0006] Specifically, it includes the following steps:

[0007] Step 1. Collect biological detection materials containing pathogenic microorganisms;

[0008] Step 2. Extract nucleic acids from the pathogenic microorganisms to be detected;

[0009] Step 3. Design specific primers and probes according to the target pathogenic microorganisms;

[0010] Step 4. Amplify the nucleic acid sequences by PCR technology;

[0011] Step 5. Perform deep sequencing based on a high-throughput sequencer;

[0012] Step 6. Perform bioinformatics analysis on the detection results.

[0013] According to the above technical solution, in step 1, when collecting biological detection materials, it is necessary to collect detection samples containing pathogenic microorganisms from the patient's body according to the actual detection requirements. The detection samples specifically include sputum, bronchoalveolar lavage fluid, nasal swabs, and throat swabs. When collecting detection samples, it is necessary to follow aseptic sampling operations to avoid sample contamination.

[0014] According to the above technical solution, in step 1, after collecting the detection samples, it is also necessary to perform preliminary processing on the detection samples, specifically including centrifuging, lysing, and filtering the detection samples to remove impurities in the DNA / RNA and ensure the purity of the subsequent detection samples. After the samples are processed, it is necessary to select a suitable detection sample storage method according to the type and quantity of microorganisms to ensure that the samples will not be contaminated during the storage process before detection.

[0015] According to the above technical solution, in step 2, use existing reagents and extraction equipment to extract nucleic acids from the pathogenic microorganisms to be detected in the detection samples, specifically to extract high-quality and high-purity DNA or RNA from complex biological detection samples;

[0016] During the extraction process, adopt means of efficient lysis, purification, and removal of residual inhibitors to remove proteins and polysaccharides in the biological detection samples, and use the extraction means of a broad-spectrum extraction kit to ensure that only the nucleic acids of the target microorganisms are in the subsequent extracted detection samples.

[0017] According to the above technical solution, in step three, targeted sequencing of specific pathogens is achieved by designing specific primers and probes;

[0018] When designing specific primers and probes, targeted design is carried out based on the genomic data of the target pathogenic microorganism. Specific primers are designed according to the target pathogenic microorganism, and at the same time, probes are designed for subsequent fluorescence labeling and detection;

[0019] The designed specific primers and probes need to be strictly verified to judge the specificity of the specific primers and probes and avoid the amplification of sequences of non-target pathogenic microorganisms.

[0020] According to the above technical solution, in step four, after obtaining specific primers and probes, the nucleic acid sequence of the target pathogenic microorganism is amplified by the designed specific primers in combination with the PCR amplification technology to achieve the amplification of the nucleic acid fragment of the target pathogenic microorganism, so that the nucleic acid fragment of the target pathogen can be distinguished among different species in the subsequent process;

[0021] After the nucleic acid fragment of the target pathogen is amplified, the amplified nucleic acid fragment needs to be pretreated. The specific pretreatment means include end repair, quality control, and adapter ligation. By pretreating the nucleic acid fragment, the nucleic acid fragment can be more conveniently applied to subsequent high-throughput sequencing;

[0022] Finally, the processed nucleic acid fragment is ligated to the adapter required by the sequencing platform. During the entire amplification process, processing process, and ligation process, operating procedures need to be strictly followed to prevent cross-contamination.

[0023] According to the above technical solution, in step five, high-throughput sequencing is carried out by using a high-throughput sequencer. Specifically, through high-throughput sequencing technology, in combination with the designed specific primers and probes, deep high-throughput sequencing is performed on the amplified nucleic acid fragment, and high-quality sequencing data is provided by using the high-throughput sequencer to obtain the sequence information of the nucleic acid fragment.

[0024] According to the above technical solution, in step five, during the sequencing process of the high-throughput sequencer, by labeling the sequencing samples, each sample to be sequenced is marked with a unique marker character, so that after sequencing, the sequencing data can be correctly associated with the sequencing samples to prevent data confusion and affect the detection results.

[0025] According to the above technical solution, in step six, after high-throughput sequencing is completed, special bioinformatics tools need to be used to perform bioinformatics sequence alignment analysis on the obtained sequencing data;

[0026] Before analyzing the sequencing data, quality control processing of the sequencing data is required. During the processing of the sequencing data, it includes filtering and cleaning the original sequencing data and removing low-quality sequencing data to ensure the quality of the sequencing data through processing;

[0027] After obtaining the sequencing data after quality control, the sequence information of the sequencing data is compared and analyzed with a reference database, which is a database of known pathogenic microorganisms. Through the comparison and analysis, the sequence information in the target region is identified, and all types of pathogenic microorganisms contained in the test sample are judged based on the aligned sequence information. With the help of bioinformatics tools, the quantity and abundance of the target pathogenic microorganisms are calculated, and all detected pathogens are listed in the form of a list.

[0028] According to the above technical solution, in step six, after obtaining the analysis result through bioinformatics analysis of the test result, the analysis result needs to be verified and confirmed;

[0029] When verifying the analysis result, other molecular biology techniques can be used to detect the presence of specific pathogenic microorganisms in the analysis result to achieve side verification of the analysis result;

[0030] When confirming the analysis result, the accuracy and reliability of the analysis result can be confirmed by repeatedly performing the targeted sequencing process multiple times and comparing the analysis results obtained multiple times.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows: The structure of the present invention is scientific and reasonable, and it is safe and convenient to use:

[0032] 1. By designing specific primers and probes, the nucleic acid sequences of target pathogenic microorganisms can be efficiently enriched, and deep sequencing is carried out using high-throughput sequencing technology, significantly improving the sensitivity and accuracy of detecting multiple pathogenic microorganisms. It can simultaneously detect various types of pathogenic microorganisms such as bacteria, viruses, and fungi, and is particularly suitable for the early, rapid, and accurate diagnosis of infectious diseases. This method is simple to operate, time-consuming, and low-cost. Compared with traditional microbial culture and serological detection, it has the advantages of fast speed and wide coverage, and can provide strong support for quickly and accurately identifying pathogenic microorganisms and taking effective prevention and control measures in a timely manner.

[0033] 2. By preliminarily processing the detection samples to remove impurities in DNA / RNA, the purity of the subsequent detection samples is ensured. By preprocessing the amplified nucleic acid fragments, the high-quality nucleic acid fragments after processing can be more conveniently and effectively applied to subsequent high-throughput sequencing. By labeling the sequencing samples, each sample to be sequenced is marked with a unique marker character, so that the sequencing data can be correctly associated with the sequencing samples after sequencing, preventing data confusion and affecting the detection results.

[0034] By performing quality control processing on the sequencing data to ensure the quality of the sequencing data, and by verifying and validating the analysis results, not only is the side verification of the analysis results achieved, but also the accuracy and reliability of the analysis results can be ensured. Through the above-mentioned processing, the mutual interference between samples can be reduced, and the sensitivity and accuracy of the detection can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation to the present invention.

[0036] In the drawings:

[0037] Figure 1 is a step flow chart of the targeted sequencing method of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0038] The following is a description of the preferred embodiments of the present invention with reference to the drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0039] Embodiment: As Figure 1 shown, the present invention provides a technical solution, a targeted sequencing method for multiple pathogenic microorganisms. By designing specific primers and probes to enrich the nucleic acid sequences of specific pathogenic microorganisms, and then using high-throughput sequencing technology for sequencing, multiple microorganisms can be detected simultaneously to improve the sensitivity and accuracy of the detection;

[0040] Specifically, it includes the following steps:

[0041] Step 1. Collect biological detection materials containing pathogenic microorganisms;

[0042] Step 2. Extract nucleic acids from the pathogenic microorganisms to be detected;

[0043] Step 3. Design specific primers and probes according to the target pathogenic microorganisms;

[0044] Step 4. Amplify the nucleic acid sequences by PCR technology;

[0045] Step 5: Perform deep sequencing based on a high-throughput sequencer;

[0046] Step 6: Perform bioinformatics analysis on the test results.

[0047] Based on the above technical solution, in Step 1, when collecting biological test materials, it is necessary to collect test samples containing pathogenic microorganisms from the patient's body according to the actual test requirements. The test samples specifically include sputum, bronchoalveolar lavage fluid, nasal swabs, and throat swabs. When collecting test samples, it is necessary to follow aseptic sampling operations to avoid sample contamination.

[0048] Based on the above technical solution, in Step 1, after collecting the test samples, it is also necessary to perform preliminary processing on the test samples, specifically including centrifuging, lysing, and filtering the test samples to remove impurities in the DNA / RNA and ensure the purity of the subsequent test samples. After the samples are processed, it is necessary to select a suitable test sample storage method according to the type and quantity of microorganisms to ensure that the samples are not contaminated during the storage process before testing.

[0049] Based on the above technical solution, in Step 2, use existing reagents and extraction equipment to extract the nucleic acids of the pathogenic microorganisms to be detected from the test samples, specifically extract high-quality and high-purity DNA or RNA from complex biological test samples;

[0050] During the extraction process, adopt efficient means of lysis, purification, and removal of residual inhibitors to remove proteins and polysaccharides in the biological test samples, and use the extraction means of a broad-spectrum extraction kit to ensure that only the nucleic acids of the target microorganisms are in the subsequent extracted test samples.

[0051] Based on the above technical solution, in Step 3, achieve targeted sequencing of specific pathogens by designing specific primers and probes;

[0052] When designing specific primers and probes, perform targeted design based on the genomic data of the target pathogenic microorganisms. Design specific primers according to the target pathogenic microorganisms, and at the same time design probes for subsequent fluorescence labeling and detection;

[0053] The designed specific primers and probes need to be strictly verified to judge the specificity of the specific primers and probes and avoid the amplification of sequences of non-target pathogenic microorganisms.

[0054] Based on the above technical solution, in Step 4, after obtaining specific primers and probes, use the designed specific primers in combination with PCR amplification technology to amplify the nucleic acid sequences of the target pathogenic microorganisms, achieve the amplification of nucleic acid fragments of the target pathogenic microorganisms, and distinguish the nucleic acid fragments of the target pathogen from different species in the subsequent steps;

[0055] After the nucleic acid fragment of the target pathogen is amplified, it is necessary to preprocess the amplified nucleic acid fragment. The specific preprocessing means include end repair, quality control, and adapter ligation. By preprocessing the nucleic acid fragment, it can be more conveniently applied to subsequent high-throughput sequencing;

[0056] Finally, the processed nucleic acid fragment is ligated to the adapter required by the sequencing platform. During the entire amplification process, processing process, and ligation process, it is necessary to strictly follow the operating procedures to prevent cross-contamination.

[0057] Based on the above technical solution, in step five, high-throughput sequencing is performed by using a high-throughput sequencer. Specifically, through high-throughput sequencing technology, combined with the designed specific primers and probes, deep high-throughput sequencing is performed on the amplified nucleic acid fragment, and high-quality sequencing data is provided by sequencing with a high-throughput sequencer, that is, the sequence information of the nucleic acid fragment is obtained.

[0058] Based on the above technical solution, in step five, during the sequencing process of the high-throughput sequencer, by labeling the sequencing samples, each sample to be sequenced is marked with a unique marker character, so that after sequencing, the sequencing data can be correctly associated with the sequencing samples to prevent data confusion and affect the detection results.

[0059] Based on the above technical solution, in step six, after high-throughput sequencing is completed, it is necessary to use special bioinformatics tools to perform bioinformatics sequence alignment analysis on the obtained sequencing data;

[0060] Before analyzing the sequencing data, it is necessary to perform quality control processing on the sequencing data. During the processing of the sequencing data, it includes filtering and cleaning the original sequencing data and removing the low-quality sequencing data to ensure the quality of the sequencing data through processing;

[0061] After obtaining the sequencing data after quality control, the sequence information of the sequencing data is compared and analyzed with a reference database, which is a database of known pathogenic microorganisms. By comparing and analyzing, the sequence information in the target region is identified, and all the pathogenic microorganism species contained in the test sample are judged according to the aligned sequence information. With the help of bioinformatics tools, the quantity and abundance of the target pathogenic microorganism are calculated, and all the detected pathogens are listed in the form of a list.

[0062] Based on the above technical solution, in step six, after obtaining the analysis result by performing bioinformatics analysis on the detection result, it is also necessary to verify and confirm the analysis result;

[0063] When verifying the analysis results, other molecular biology techniques can be used to detect the presence of specific pathogenic microorganisms in the analysis results, so as to verify the analysis results from the side;

[0064] When confirming the analysis results, the accuracy and reliability of the analysis results can be confirmed by repeating the targeted sequencing process multiple times and comparing the analysis results obtained multiple times.

[0065] Finally, it should be noted that the above are only preferred examples of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A targeted sequencing method for multiple pathogenic microorganisms, characterized in that: Enrich the nucleic acid sequences of specific pathogenic microorganisms by designing specific primers and probes, and then use high-throughput sequencing technology for sequencing to detect multiple microorganisms simultaneously; Specifically, it includes the following steps: Step 1: Collect biological detection materials containing pathogenic microorganisms; Step 2: Extract the nucleic acids from the pathogenic microorganisms to be detected; Step 3: Design specific primers and probes based on the target pathogenic microorganisms; Step 4: Amplify the nucleic acid sequences by PCR technology; Step 5: Perform deep sequencing based on a high-throughput sequencer; Step 6: Conduct bioinformatics analysis on the detection results.

2. The targeted sequencing method for multiple pathogenic microorganisms according to claim 1, characterized in that: In Step 1, when collecting biological detection materials, it is necessary to collect detection samples containing pathogenic microorganisms from the patient's body according to the actual detection requirements. The detection samples specifically include sputum, bronchoalveolar lavage fluid, nasal swabs, and throat swabs. When collecting detection samples, aseptic sampling operations need to be followed.

3. The targeted sequencing method for multiple pathogenic microorganisms according to claim 2, characterized in that: In Step 1, after the detection samples are collected, preliminary processing of the detection samples is also required, specifically including centrifugation, lysis, and filtration of the detection samples to remove impurities in the DNA / RNA. After the samples are processed, it is necessary to select a suitable detection sample preservation method according to the types and quantities of microorganisms.

4. A targeted sequencing method for multiple pathogenic microorganisms according to claim 1, characterized in that: In Step 2, use existing reagents and extraction equipment to extract the nucleic acids from the pathogenic microorganisms to be detected from the detection samples, specifically to extract high-quality and high-purity DNA or RNA from complex biological detection samples; During the extraction process, adopt means of efficient lysis, purification, and removal of residual inhibitors to remove proteins and polysaccharides in the biological detection samples, and rely on the extraction means of a broad-spectrum extraction kit to ensure that only the nucleic acids of the target microorganisms are in the subsequent extracted detection samples.

5. A targeted sequencing method for multiple pathogenic microorganisms according to claim 1, characterized in that: In Step 3, achieve targeted sequencing of specific pathogens by designing specific primers and probes; When designing specific primers and probes, conduct targeted design based on the genomic data of the target pathogenic microorganisms. Design specific primers according to the target pathogenic microorganisms, and at the same time design probes for subsequent fluorescence labeling and detection; The designed specific primers and probes need to be strictly verified to judge the specificity of the specific primers and probes and avoid the amplification of sequences of non-target pathogenic microorganisms.

6. The targeted sequencing method for multiple pathogenic microorganisms according to claim 1, wherein: In Step 4, after obtaining specific primers and probes, use the designed specific primers and combine with PCR amplification technology to amplify the nucleic acid sequences of the target pathogenic microorganisms, achieve the amplification of the nucleic acid fragments of the target pathogenic microorganisms, and distinguish the nucleic acid fragments of the target pathogen from different species in the subsequent process; After the nucleic acid fragments of the target pathogen are amplified, it is necessary to perform pretreatment on the amplified nucleic acid fragments. The specific pretreatment means include end repair, quality control, and adapter ligation. By pretreating the nucleic acid fragments, the nucleic acid fragments can be more conveniently applied to subsequent high-throughput sequencing; Finally, connect the processed nucleic acid fragments to the adapters required by the sequencing platform. During the entire amplification process, processing process, and connection process, it is necessary to strictly abide by the operating procedures to prevent cross-contamination.

7. A targeted sequencing method for multiple pathogenic microorganisms according to claim 1, characterized in that: In step five, high-throughput sequencing is performed using a high-throughput sequencer. Specifically, through high-throughput sequencing technology, combined with designed specific primers and probes, deep high-throughput sequencing is carried out on the amplified nucleic acid fragments, and thus the sequence information of the nucleic acid fragments is obtained.

8. A targeted sequencing method for multiple pathogenic microorganisms according to claim 7, characterized in that: In step five, during the process of sequencing by the high-throughput sequencer, by performing a labeling process on the sequencing samples, each sample to be sequenced is provided with a unique labeling character, so that after sequencing, the sequencing data can be correctly associated with the sequencing samples.

9. A targeted sequencing method for multiple pathogenic microorganisms according to claim 7, characterized in that: In step six, after the high-throughput sequencing is completed, specialized bioinformatics tools need to be used to perform bioinformatics sequence alignment analysis on the obtained sequencing data; Before analyzing the sequencing data, quality control processing of the sequencing data is required. During the processing of the sequencing data, it includes filtering and cleaning the original sequencing data and removing low-quality sequencing data, and the quality of the sequencing data is ensured through the processing. After obtaining the sequencing data after quality control, the sequence information of the sequencing data is compared and analyzed with a reference database, which is a database of known pathogenic microorganisms. Through the comparison and analysis, the sequence information in the target region is identified, and all the types of pathogenic microorganisms contained in the test sample are judged according to the aligned sequence information. With the help of bioinformatics tools, the quantity and abundance of the target pathogenic microorganisms are calculated, and all the detected pathogens are listed in the form of a list.

10. A targeted sequencing method for multiple pathogenic microorganisms according to claim 9, characterized in that: In step six, after obtaining the analysis result through bioinformatics analysis of the test result, the analysis result also needs to be verified and confirmed; When verifying the analysis result, other molecular biology techniques can be used to detect the presence of specific pathogenic microorganisms in the analysis result to achieve a side verification of the analysis result; When confirming the analysis result, the accuracy and reliability of the analysis result can be confirmed by comparing the analysis results obtained multiple times through repeating the targeted sequencing process multiple times.

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