Detection method based on super-multiplex PCR amplification technology and high-throughput sequencing technology

Abstract

The invention discloses a detection method based on a super-multiplex PCR amplification technology and a high-throughput sequencing technology. The detection method comprises the following steps: step 1, collection and pretreatment of a detection sample; step 2, carrying out ultra-multiple PCR (Polymerase Chain Reaction) amplification treatment; step 3, performing high-throughput sequencing treatment; step 4, analyzing the detection data; and step 5, interpreting a detection result, carrying out targeted amplification and enrichment on a target sequence by adopting a super-multiplex PCR library building system, and carrying out targeted high-throughput sequencing by adopting a next-generation sequencing technology to realize pathogen broad-spectrum accurate detection. Therefore, various genital tract infection pathogens and various HPV subtypes can be comprehensively and accurately evaluated through a multi-targeted amplification-high-throughput sequencing method, the genital tract infection diagnosis level is improved, and particularly, the female genital tract infection diagnosis level is improved, and the female genital tract infection diagnosis level is effectively improved for repeated genital tract infection caused by unknown reasons. The recurrence rate and the secondary infection rate of reproductive tract infectious diseases are reduced, and the medical burden of the masses is reduced, so that the reproductive tract infection is really and effectively treated.

Description

Technical Field

[0001] The present invention relates to the field of targeted sequencing technology, and specifically to a detection method based on super-multiplex PCR amplification technology and high-throughput sequencing technology. Background Art

[0002] Female reproductive tract infection is a common gynecological disease that troubles women around the world. More than 1 million people are infected with sexually transmitted diseases every day in the world, most of whom have no symptoms. About 311,000 people die from cervical cancer caused by human papillomavirus infection every year. At present, the hazards of common reproductive tract pathogens infection mainly include repeated reproductive tract infection affecting the quality of life, infertility affecting family happiness, adverse perinatal pregnancy outcomes, and cervical lesions co-factors accelerating HPV infection;

[0003] Current technology can achieve very complete testing, but on the one hand, the patient's privacy information is incomplete and the previous onset of the disease is unclear. On the other hand, it is uncertain whether the patient itself is a non-target target. Now we can obtain the amplicons of target targets and non-target targets, and through amplicon library construction and high-throughput sequencing, we can obtain the genomic cutting efficiency of multiple sgRNA target and off-target sites. However, the database is incomplete, and the relevant data of women and the subsequent testing cycle are long, and the efficiency and accuracy of the test cannot be guaranteed. Summary of the invention

[0004] The present invention provides a detection method based on super-multiplex PCR amplification technology and high-throughput sequencing technology, which can effectively solve the problem proposed in the above background technology that the current technology can achieve very complete detection, but on the one hand, the patient's privacy information is incomplete and the previous onset pattern is unclear; on the other hand, it is uncertain whether the patient itself is a non-target target. Now, amplicons of target targets and non-target targets can be obtained, and the genome cutting efficiency of multiple sgRNA target points and off-target sites can be obtained by building a library for the amplicons and high-throughput sequencing, but the database is incomplete, the female-related data and the subsequent detection cycle are long, and the efficiency and accuracy of the detection cannot be guaranteed.

[0005] To achieve the above-mentioned purpose, the present invention provides the following technical solutions: a detection method based on super-multiplex PCR amplification technology and high-throughput sequencing technology, using a super-multiplex PCR library construction system to perform targeted amplification and enrichment of target sequences, using second-generation sequencing technology, and performing targeted high-throughput sequencing to achieve broad-spectrum and accurate detection of pathogens, comprising the following steps:

[0006] Step 1: Collection and pretreatment of test samples;

[0007] Step 2: Super multiplex PCR amplification process;

[0008] Step 3: high-throughput sequencing processing;

[0009] Step 4: Analyze the test data;

[0010] Step 5: Interpret the test results.

[0011] According to the above technical scheme, in the step 1, when collecting test samples, the subjects of the collection include: people with high-risk HPV positive, people with cervical / vaginal intraepithelial neoplasia, people with recurrent unexplained reproductive tract infections, and people undergoing cervical cancer screening. When collecting, aseptic operation should be followed to reduce contamination by foreign bacteria and avoid contamination of the specimen. Chlamydia trachomatis reproduces in host cells, and as many epithelial cells as possible are collected during collection;

[0012] During the specific sample collection process, the vagina is expanded with a vaginal dilator, and after collecting cervical exfoliated cells with a sterile sampling brush, secretions from the 4 cm inner wall or posterior fornix of the vaginal opening are collected at the same time. Be careful not to touch the skin when the sterile sampling brush is withdrawn to avoid contamination;

[0013] In addition, when collecting samples, body fluids from the infected area should be selected for testing to increase the sensitivity of detection, and specimens should be collected before the use of antimicrobial drugs as much as possible.

[0014] According to the above technical solution, in the step 1, when pre-treating the collected sample, the collected sample tissue needs to be crushed first, generally by mechanical crushing or chemical crushing method, and the mechanical crushing method uses a homogenizer for crushing, and the chemical crushing method uses an enzymatic reagent for crushing;

[0015] The tissue cells are lysed through crushing to release nucleic acids, and then a nucleic acid extraction kit is used to extract the nucleic acids. The extracted nucleic acids are subject to quality and concentration testing. During the quality and concentration testing, a nucleic acid detector is mainly used to measure the concentration and purity of the nucleic acids.

[0016] According to the above technical solution, in step one, after the collected samples are pre-processed, the sample liquid is stored in a test container, and the sample type and collection site are clearly marked on the test container, and the patient's identification information is also marked. The identification information usually includes the test code, the patient's name and the department where the sample was sent for inspection.

[0017] According to the above technical solution, in the step 2, in the specific processing process, specific primers are designed according to the detection target. When detecting multiple pathogens, it is necessary to design multiple pairs of primers for each pathogen according to the detection target. The design of the primers should take into account their length, GC content and annealing temperature. The length is 18-25bp and the GC content is 40%-60% to ensure efficient and specific amplification;

[0018] The extracted test sample solution is mixed with primers, dNTPs, DNA polymerase and buffer, the buffer provides appropriate pH and ionic strength to ensure activity, and PCR amplification is performed under appropriate thermal cycle conditions, and the condition parameters of the thermal cycle include denaturation temperature, annealing temperature, extension time and extension temperature;

[0019] Among them, the denaturation temperature is 94-98°C, the annealing temperature is 50-70°C, the extension time is 15-30 seconds, and the extension temperature is 70-75°C.

[0020] According to the above technical solution, in step 3, the product after super-multiplex PCR amplification needs to be purified to remove excess primers, including dNTPs and enzymes. The purification method uses column purification. Column purification uses a special adsorption column to adsorb the amplified product on the column, while impurities are eluted, and the amplified product is subsequently eluted;

[0021] Constructing a sequencing library, usually connecting sequencing adapters at both ends of the purified amplified product. The sequencing adapters contain sequence information compatible with the sequencing platform. The adapter ligation reaction requires the use of a ligase and is performed under appropriate buffer and temperature conditions;

[0022] The constructed sequencing library is quantitatively processed and sequenced on a high-throughput sequencing platform. By comparing with the standard, the concentration of the sequencing library can be accurately determined, and the sequencing data is subjected to bioinformatics analysis, including sequence alignment and variation detection.

[0023] According to the above technical solution, in step three, during the high-throughput sequencing process, it is also necessary to select a suitable high-throughput sequencing platform according to the detection requirements. After selecting the high-throughput sequencing platform, the sequencing library that has passed the quantitative and quality tests is sequenced on the machine to obtain sequencing data.

[0024] According to the above technical solution, in step 4, when analyzing the obtained sequencing data, it is first necessary to use software tools to perform quality control on the sequencing data, remove low-quality sequencing sequences, and use comparison software to compare the quality-controlled sequencing sequences with a known gene sequence database, and use the comparison to determine the position of the sequencing sequences in the genome, as well as their identity and similarity;

[0025] In the case of detecting pathogens, the type and genotype of the pathogen can be determined based on the comparison results. When detecting gene mutations, the type, location and frequency of the mutations can be analyzed to provide a basis for disease diagnosis.

[0026] According to the above technical solution, the step five specifically refers to interpreting the results of data analysis. In pathogen detection, when information on the type and load of pathogens is detected in the report sample, it can be specifically estimated through sequencing depth data, and the test results are organized into a standardized test report and provided to relevant medical personnel;

[0027] In the detection of genetic diseases, the discovery of known pathogenic gene mutations can indicate the patient's risk of developing the corresponding genetic disease.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention adopts a super-multiplex PCR library construction system, performs targeted amplification and enrichment of target sequences, and adopts second-generation sequencing technology to perform targeted high-throughput sequencing to achieve broad-spectrum and accurate detection of pathogens. In this way, multiple reproductive tract infection pathogens and multiple HPV subtypes can be used to more comprehensively and accurately evaluate female reproductive tract infectious diseases through multiple targeted amplification-high-throughput sequencing, improve the diagnosis level of reproductive tract infections, especially for recurrent unexplained reproductive tract infections, reduce the recurrence rate and secondary infection rate of reproductive tract infectious diseases, and reduce the medical burden on the masses, thereby truly and effectively treating reproductive tract infections.

[0030] In addition, by merging the overall data and then coordinating it with automated big data, analysis can be performed based on patient information during testing to improve efficiency and accuracy. Targeted technology can be used to enrich target pathogen sequences from nucleic acids to achieve accurate, rapid, and sensitive pathogen sequencing. Multiple primers track pathogens, and low detection limits make detection more accurate. Only target sequences are detected, shortening experimental time and making detection faster. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0032] In the attached picture:

[0033] Figure 1 is a flow chart of the steps of the detection method of the present invention;

[0034] Figure 2 is a schematic diagram of the targeted sequencing technology of the present invention;

[0035] Figure 3 is a schematic diagram of high-throughput sequencing of the present invention;

[0036] Figure 4 It is a detection flow chart of the targeted sequencing technology of the present invention. DETAILED DESCRIPTION

[0037] The preferred embodiments of the present invention are described below in conjunction with the accompanying 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.

[0038] Example: Figure 1-4 As shown, the present invention provides a technical solution, a detection method based on super-multiplex PCR amplification technology and high-throughput sequencing technology, using a super-multiplex PCR library construction system to perform targeted amplification and enrichment of target sequences, using second-generation sequencing technology, and performing targeted high-throughput sequencing to achieve broad-spectrum and accurate detection of pathogens, including the following steps:

[0039] Step 1: Collection and pretreatment of test samples;

[0040] Step 2: Super multiplex PCR amplification process;

[0041] Step 3: high-throughput sequencing processing;

[0042] Step 4: Analyze the test data;

[0043] Step 5: Interpret the test results.

[0044] Based on the above technical solution, in step 1, when collecting test samples, the subjects of collection include: people with high-risk HPV positive, people with cervical / vaginal intraepithelial neoplasia, people with recurrent unexplained reproductive tract infections, and people undergoing cervical cancer screening. When collecting, aseptic operation should be followed to reduce contamination by foreign bacteria and avoid contamination of specimens. Chlamydia trachomatis reproduces in host cells, and as many epithelial cells as possible should be collected during collection;

[0045] During the specific sample collection process, the vagina is expanded with a vaginal dilator, and after collecting cervical exfoliated cells with a sterile sampling brush, secretions from the 4 cm inner wall or posterior fornix of the vaginal opening are collected at the same time. Be careful not to touch the skin when the sterile sampling brush is withdrawn to avoid contamination;

[0046] In addition, when collecting samples, body fluids from the infected area should be selected for testing to increase the sensitivity of detection, and specimens should be collected before the use of antimicrobial drugs as much as possible.

[0047] Based on the above technical solution, in step 1, when pre-treating the collected sample, the collected sample tissue needs to be crushed first, generally by mechanical crushing or chemical crushing method, and the mechanical crushing method uses a homogenizer for crushing, and the chemical crushing method uses an enzymatic reagent for crushing;

[0048] The tissue cells are lysed through crushing to release nucleic acids, and then a nucleic acid extraction kit is used to extract the nucleic acids. The extracted nucleic acids are subject to quality and concentration testing. During the quality and concentration testing, a nucleic acid detector is mainly used to measure the concentration and purity of the nucleic acids.

[0049] Based on the above technical solution, in step one, after the collected samples are pre-processed, the sample liquid is stored in a test container, and the sample type and collection site are clearly marked on the test container, and the patient's identification information is also marked. The identification information usually includes the test code, the patient's name and the department where the sample was sent for inspection.

[0050] Based on the above technical solution, in step 2, in the specific processing process, specific primers are designed according to the detection target. When detecting multiple pathogens, it is necessary to design multiple pairs of primers for each pathogen according to the detection target, such as specific gene sequences and pathogen genes. The design of the primers should take into account their length, GC content and annealing temperature. The length is 21bp and the GC content is 50% to ensure efficient and specific amplification;

[0051] The extracted test sample solution is mixed with primers, dNTPs, DNA polymerase and buffer, the buffer provides appropriate pH and ionic strength to ensure activity, and PCR amplification is performed under appropriate thermal cycle conditions, and the condition parameters of the thermal cycle include denaturation temperature, annealing temperature, extension time and extension temperature;

[0052] Among them, the denaturation temperature is 96°C, the annealing temperature is determined according to the Tm value of the primer, which is 60°C, the extension time is determined according to the length of the amplified fragment, which is 20 seconds, and the extension temperature is 72°C.

[0053] Based on the above technical solution, in step 3, the product after super-multiplex PCR amplification needs to be purified to remove excess primers, including dNTPs and enzymes. The purification method uses column purification. Column purification uses a special adsorption column to adsorb the amplified product on the column, while the impurities are eluted, and the amplified product is eluted later;

[0054] Constructing a sequencing library, usually connecting sequencing adapters at both ends of the purified amplified product. The sequencing adapters contain sequence information compatible with the sequencing platform. The adapter ligation reaction requires the use of a ligase and is performed under appropriate buffer and temperature conditions;

[0055] The constructed sequencing library is quantitatively processed and sequenced on a high-throughput sequencing platform. By comparing with the standard, the concentration of the sequencing library can be accurately determined, and the sequencing data is subjected to bioinformatics analysis, including sequence alignment and variation detection.

[0056] Based on the above technical solution, in step three, during the high-throughput sequencing process, it is also necessary to select a suitable high-throughput sequencing platform according to the detection requirements. After selecting the high-throughput sequencing platform, the sequencing library that has passed the quantitative and quality tests is sequenced on the machine to obtain sequencing data.

[0057] Based on the above technical solution, in step 4, when analyzing the obtained sequencing data, it is necessary to first use software tools to perform quality control on the sequencing data, remove low-quality sequencing sequences, and use comparison software to compare the quality-controlled sequencing sequences with the known gene sequence database, and use the comparison to determine the position of the sequencing sequence in the genome, as well as its identity and similarity;

[0058] In the case of detecting pathogens, the type and genotype of the pathogen can be determined based on the comparison results. When detecting gene mutations, the type, location and frequency of the mutations can be analyzed to provide a basis for disease diagnosis.

[0059] Based on the above technical solution, step five specifically refers to interpreting the results of data analysis. In pathogen detection, when information on pathogen type and load is detected in the report sample, it can be estimated through sequencing depth data, and the test results are organized into a standardized test report and provided to relevant medical personnel;

[0060] In the detection of genetic diseases, the discovery of known pathogenic gene mutations can indicate the patient's risk of developing the corresponding genetic disease.

[0061] Finally, it should be noted that the above description is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A detection method based on super multiplex PCR amplification technology and high-throughput sequencing technology, characterized in that: The ultra-multiplex PCR library construction system is used to perform targeted amplification and enrichment of the target sequence, and the second-generation sequencing technology is used for targeted high-throughput sequencing to achieve broad-spectrum and accurate detection of pathogens, including the following steps: Step 1: Collection and pretreatment of test samples; Step 2: Super multiplex PCR amplification process; Step 3: high-throughput sequencing processing; Step 4: Analyze the test data; Step 5: Interpret the test results.

2. The detection method based on super multiplex PCR amplification technology and high-throughput sequencing technology according to claim 1, characterized in that: In the step 1, when collecting test samples, the subjects of the samples include: people with high-risk HPV positive, people with cervical / vaginal intraepithelial neoplasia, people with recurrent unexplained reproductive tract infections, and people undergoing cervical cancer screening. Aseptic operation should be followed during collection. Chlamydia trachomatis reproduces in host cells, and epithelial cells are collected during collection; During the specific sample collection process, the vagina is expanded with a vaginal dilator, and after collecting cervical exfoliated cells with a sterile sampling brush, secretions from the inner wall or posterior fornix 4 cm from the vaginal opening are collected at the same time; In addition, when collecting samples, body fluids from the infected area should be selected for testing, and specimens should be collected before antimicrobial drugs are used.

3. The detection method based on super multiplex PCR amplification technology and high-throughput sequencing technology according to claim 2, characterized in that: In the step 1, when pre-treating the collected sample, the collected sample tissue needs to be crushed first, using a mechanical crushing method or a chemical crushing method, wherein the mechanical crushing method uses a homogenizer for crushing, and the chemical crushing method uses an enzymatic reagent for crushing; The tissue cells are lysed through crushing to release nucleic acids, and then a nucleic acid extraction kit is used to extract the nucleic acids. The extracted nucleic acids are subject to quality and concentration testing. During the quality and concentration testing, a nucleic acid detector is mainly used to measure the concentration and purity of the nucleic acids.

4. The detection method based on super multiplex PCR amplification technology and high-throughput sequencing technology according to claim 2, characterized in that: In step one, after the collected samples are pre-processed, the sample liquid is stored in a test container, and the sample type and collection site are clearly marked on the test container, and the patient's identification information is also marked. The identification information usually includes the test code, the patient's name and the department where the sample was sent for testing.

5. The detection method based on super multiplex PCR amplification technology and high-throughput sequencing technology according to claim 1, characterized in that: In the step 2, in the specific processing process, specific primers are designed according to the detection target. When detecting multiple pathogens, it is necessary to design multiple pairs of primers for each pathogen according to the detection target. The design of the primers should take into account their length, GC content and annealing temperature. The length is 18-25bp and the GC content is 40%-60%; The extracted test sample solution is mixed with primers, dNTPs, DNA polymerase and buffer, the buffer provides appropriate pH and ionic strength, and PCR amplification is performed under thermal cycle conditions, and the condition parameters of the thermal cycle include denaturation temperature, annealing temperature, extension time and extension temperature; Among them, the denaturation temperature is 94-98°C, the annealing temperature is 50-70°C, the extension time is 15-30 seconds, and the extension temperature is 70-75°C.

6. The detection method based on super multiplex PCR amplification technology and high-throughput sequencing technology according to claim 1, characterized in that: In the step 3, the product after the super-multiplex PCR amplification needs to be purified to remove excess primers, including dNTPs and enzymes. The purification method uses column purification. The column purification uses a special adsorption column to adsorb the amplification product on the column, while the impurities are eluted, and the amplification product is subsequently eluted; Construct a sequencing library, connect sequencing adapters to both ends of the purified amplified product. The sequencing adapters contain sequence information compatible with the sequencing platform. The adapter ligation reaction requires the use of a ligase and is performed under appropriate buffer and temperature conditions; The constructed sequencing library is quantitatively processed and sequenced on a high-throughput sequencing platform. The concentration of the sequencing library is determined by comparison with the standard, and the sequencing data is subjected to bioinformatics analysis, including sequence alignment and variation detection.

7. The detection method based on super multiplex PCR amplification technology and high-throughput sequencing technology according to claim 6, characterized in that: In step three, during the high-throughput sequencing process, it is also necessary to select a suitable high-throughput sequencing platform according to the detection requirements. After the high-throughput sequencing platform is selected, the sequencing library that has passed the quantitative and quality tests is sequenced on the machine to obtain sequencing data.

8. The detection method based on super multiplex PCR amplification technology and high-throughput sequencing technology according to claim 1, characterized in that: In step 4, when analyzing the obtained sequencing data, it is first necessary to perform quality control on the sequencing data through software tools, remove low-quality sequencing sequences, and use comparison software to compare the quality-controlled sequencing sequences with a known gene sequence database, and through comparison, determine the position of the sequencing sequences in the genome, and their identity and similarity; In the case of detecting pathogens, the type and genotype of the pathogen can be determined based on the comparison results. When detecting gene mutations, the type, location and frequency of the mutations can be analyzed to provide a basis for disease diagnosis.

9. The detection method based on super multiplex PCR amplification technology and high-throughput sequencing technology according to claim 1, characterized in that: The step five specifically refers to interpreting the results of data analysis. In pathogen detection, when information on the type and load of pathogens is detected in the report sample, it can be estimated specifically through sequencing depth data, and the test results are organized into a standardized test report and provided to relevant medical personnel; In the detection of genetic diseases, the discovery of known pathogenic gene mutations indicates that the patient is at risk of developing the corresponding genetic disease.

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