Preparation method of high-throughput sequencing library with low background nucleic acid ratio and method for reducing background nucleic acid ratio during high-throughput sequencing
By adding relevant fragmented background nucleic acid interference materials to the amplification or hybrid capture system of the high-throughput sequencing library, the problem of excessive proportion of background nucleic acids in high-throughput sequencing is solved, and more efficient target sequence detection is achieved, which significantly improves data quality.
Patent Information
- Application Number
- CN202411732051.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-28
- Publication Date
- 2025-03-07
AI Technical Summary
In high-throughput sequencing, the proportion of background nucleic acids is too high, especially in pathogenic microbial detection and low-frequency gene variant analysis. It is difficult for the prior art to effectively reduce the proportion of background nucleic acids.
By adding fragmented background nucleic acid interference with the nucleic acid to be built in the library amplification system or hybrid capture system, the background nucleic acid is inhibited from entering the sequencing library, thereby reducing the proportion of background nucleic acid sequences in the sequencing data.
It effectively reduces the proportion of background nucleic acids in high-throughput sequencing, improves the detection efficiency of target sequences, and significantly improves data quality in pathogenic microbial detection and low-frequency gene variant analysis.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of high-throughput sequencing library construction and high-throughput sequencing, and specifically relates to a method for preparing a high-throughput sequencing library with a low background nucleic acid ratio and a method for reducing the background nucleic acid ratio during high-throughput sequencing. Background Art
[0002] High-throughput sequencing technology greatly increases the scale of biological information acquisition by sequencing a large number of nucleic acids in parallel at the same time. With the development of technology, the cost of sequencing has gradually decreased, and information with a lower proportion in the sample is easier to analyze through a larger sequencing volume. However, for the analysis needs of low-frequency gene variants, pathogenic microorganisms, etc., the cost is still high, and it is necessary to combine targeted enrichment technology to increase the proportion of target sequences in order to achieve the required target sequencing volume within the cost range. However, although targeted enrichment technology is also constantly developing and improving, there are still many scenarios where even with targeted enrichment, the proportion of target sequences is still too low and the proportion of background nucleic acid sequences is still too high. For example, in pathogen detection, if the proportion of pathogen nucleic acid is originally one in a million, even if the targeted enrichment multiple is ten thousand times, 99% of the sequences will still be background sequences in the end.
[0003] Furthermore, when hybridization capture technology is used for human gene sequencing, most of the non-target sequences are repetitive sequences in the human genome. When performing hybridization capture sequencing, the proportion of target sequences can be further increased by adding Human Cot-1 DNA to the hybridization system. As a hybridization blocker, Human Cot-1 DNA is rich in repetitive DNA sequences, such as Alu and Kpn family members. It is extracted and prepared in large quantities from the placenta. When it is incorporated into the hybridization system in large quantities, it plays a good role in competing with the probe for binding to non-target library fragments. However, the hybridization capture of pathogenic microorganism sequences is different. Both the repetitive and non-repetitive regions of the human genome are background interferences, and the background microorganisms contained in healthy human samples are also parts that need to be excluded. Therefore, the current conventional hybridization blockers cannot meet the needs of non-human gene detection scenarios including pathogenic microorganisms or low-frequency gene mutations, and the problem of excessively high proportion of background nucleic acids in some high-throughput sequencing scenarios still cannot be overcome. Summary of the invention
[0004] The object of the present invention is to provide a method for preparing a high-throughput sequencing library with a low background nucleic acid ratio and a method for reducing the background nucleic acid ratio during high-throughput sequencing. The preparation method can inhibit the background nucleic acid from entering the sequencing library, thereby reducing its ratio in the sequencing results.
[0005] The present invention provides a method for preparing a high-throughput sequencing library with a low background nucleic acid ratio, comprising the following steps:
[0006] Connecting the nucleic acid to be constructed into a library with a sequencing adapter to obtain an adapter-connected product;
[0007] The adapter ligation product is subjected to one or both of the following operations to obtain the high-throughput sequencing library with a low background nucleic acid ratio:
[0008] 1) mixing the adapter ligation product with the interfering substance and then performing library amplification;
[0009] 2) performing library amplification on the adapter-ligated product to obtain a detection library; mixing the hybridization capture system containing the detection library with an interfering substance and then performing hybridization capture;
[0010] The interferor is a fragmented background nucleic acid that is related to the nucleic acid to be constructed.
[0011] Preferably, the step of preparing the interferor comprises: performing gene amplification on the background nucleic acid, and fragmenting the obtained gene amplification product to obtain the interferor.
[0012] Preferably, after the fragmentation, the fragmented gene amplification products are connected to sequencing adapters for PCR amplification, and the obtained PCR amplification products are fragmented to obtain the interferors; the sequencing adapters are sequencing adapters that do not match the high-throughput sequencing platform used for sequencing and do not exist in the target sequence set to be detected.
[0013] Preferably, the length of the interferor is no greater than the length of the fragment of the high-throughput sequencing library with a low background nucleic acid ratio.
[0014] Preferably, the length of the interferor is 50 to 500 bp.
[0015] Preferably, the interferor is DNA.
[0016] The present invention also provides a method for reducing the proportion of background nucleic acids during high-throughput sequencing, and a high-throughput sequencing library with a low proportion of background nucleic acids is prepared by the preparation method described in the above technical solution;
[0017] The high-throughput sequencing library with low background nucleic acid ratio is subjected to high-throughput sequencing.
[0018] The present invention also provides the preparation method described in the above technical scheme, or the use of a high-throughput sequencing library with a low background nucleic acid ratio prepared by the preparation method described in the above technical scheme, or the method described in the above technical scheme in preparing products for low-frequency gene detection or pathogenic microorganism detection.
[0019] Preferably, the product comprises a kit or a reagent.
[0020] Beneficial effects:
[0021] The present invention provides a method for preparing a high-throughput sequencing library with a low background nucleic acid ratio, wherein the preparation method provides an interfering substance of background nucleic acid in a library amplification system or a hybridization capture system in library preparation, thereby inhibiting the background nucleic acid from entering the high-throughput sequencing library, thereby reducing the ratio of background nucleic acid sequences in sequencing data. The preparation method of the interfering substance is simple and fast, making the preparation method of the high-throughput sequencing library with a low background nucleic acid ratio of the present invention easy to promote and use. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.
[0023] Figure 1 This is a diagram showing the reduction in the proportion of human genome after adding interfering substances to the PCR system for Chinese library preparation in Example 1;
[0024] Figure 2 This is a diagram showing the reduction in the proportion of human genome after adding interfering substances to the PCR system for Chinese library preparation in Example 2;
[0025] Figure 3 This is a graph showing the effect of adding interference substances on the proportion of bacterial data in the PCR stage and the targeted hybridization capture stage of Chinese library preparation in Example 3;
[0026] Figure 4 This is a graph showing the effect of adding interfering substances to RNA targeted capture sequencing in Example 4 on the improvement of the proportion of new coronavirus data. DETAILED DESCRIPTION
[0027] The present invention provides a method for preparing a high-throughput sequencing library with a low background nucleic acid ratio, comprising the following steps:
[0028] Connecting the nucleic acid to be constructed into a library with a sequencing adapter to obtain an adapter-connected product;
[0029] The adapter ligation product is subjected to one or both of the following operations to obtain the high-throughput sequencing library with a low background nucleic acid ratio:
[0030] 1) mixing the adapter ligation product with the interfering substance and then performing library amplification;
[0031] 2) performing library amplification on the adapter-ligated product to obtain a detection library; mixing the hybridization capture system containing the detection library with an interfering substance and then performing hybridization capture;
[0032] The interferor is a fragmented background nucleic acid that is related to the nucleic acid to be constructed.
[0033] The present invention connects the nucleic acid to be constructed with a sequencing adapter to obtain an adapter connection product. As an embodiment, the nucleic acid to be constructed in the present invention can be DNA or RNA. As an embodiment, the nucleic acid to be constructed in the present invention can be a fragmented nucleic acid.
[0034] After obtaining the adapter ligation product, the present invention performs one or both of the following operations on the adapter ligation product to obtain the high-throughput sequencing library with a low background nucleic acid ratio:
[0035] 1) mixing the adapter ligation product with the interfering substance and then performing library amplification;
[0036] 2) amplifying the adapter-ligated product to obtain a detection library; mixing the hybridization capture system containing the detection library with an interfering substance and then performing hybridization capture; the interfering substance is a non-target measurement nucleic acid that is correlated with the nucleic acid to be constructed.
[0037] As an embodiment, in step 1), the mass ratio of the interfering substance to the linker ligation product is greater than 10;
[0038] As an embodiment, in step 2), the mass ratio of the interferor to the detection library is not less than 10; in a specific embodiment, the mass ratio of the interferor to the detection library can be 10:1.
[0039] In the present invention, the interferor is a fragmented background nucleic acid that is correlated with the nucleic acid to be built. The correlation with the nucleic acid to be built means that based on the sample to be tested, the skilled person in the art can distinguish and know the nature of the background nucleic acid through common technical common sense. For example, in a specific embodiment, the target nucleic acid is the nucleic acid sequence of a pathogenic microorganism, and the nucleic acid to be built is a human nasal swab nucleic acid sample. Then the background microorganisms contained in the human genome repetitive region and non-repetitive region and the healthy human nasal swab nucleic acid sample are all background nucleic acids, and the fragmented background nucleic acid that is correlated with the nucleic acid to be built can be used as an interferor, which can be distinguished by the skilled person in the art. For example, in another specific embodiment, the high-throughput sequencing platform used for sequencing is the Illumina Novaseq 6000 sequencing platform, and the library connected with the connector matching the Illumina Novaseq 6000 sequencing platform is the range of the target detection nucleic acid, and the library connected with the sequencing connector matching the MGI sequencing platform belongs to the background nucleic acid, and the fragmented background nucleic acid that is correlated with the nucleic acid to be built can be used as an interferor, which can also be distinguished by the skilled person in the art.
[0040] It is precisely by providing background interferents in a targeted manner in a high-throughput sequencing library that the background nucleic acid can be effectively inhibited from entering the high-throughput sequencing library, thereby achieving the purpose of a high-throughput sequencing library with a low background nucleic acid ratio.
[0041] As an embodiment, the preparation step of the interferor of the present invention includes: performing gene amplification on the background nucleic acid, and fragmenting the obtained gene amplification product to obtain the interferor.
[0042] As an embodiment, when preparing the interferor, after the fragmentation, the fragmented gene amplification product can also be connected to a sequencing adapter for PCR amplification, and the obtained PCR amplification product is fragmented to obtain the interferor. As an embodiment, the sequencing adapter is a sequencing adapter that does not match the high-throughput sequencing platform used for sequencing and does not exist in the target sequence set to be detected; in a specific embodiment, the high-throughput sequencing platform used for sequencing is the Illumina Novasek 6000 sequencing platform, and when preparing the interferor, the connected sequencing adapter is a sequencing adapter that matches the MGI sequencing platform.
[0043] As an embodiment, the length of the interferor of the present invention is not greater than the fragment length of the high-throughput sequencing library with low background nucleic acid proportion, which has the advantage of increasing the molar amount of the interferor, such as: a 200bp interferor fragment binds to a 200bp library fragment, inhibiting its amplification. If the interferor is broken into two 100bp fragments, it can bind to the two library fragments, and the library fragments will have an amplification inhibition effect as long as there is an interferor in the middle. As another embodiment, the length of the interferor of the present invention can be 50 to 500bp.
[0044] As an embodiment, the interferor is DNA; that is, when the non-target measurement nucleic acid is RNA, the non-target measurement nucleic acid is reverse transcribed into cDNA and then used to prepare the interferor.
[0045] Unless otherwise specified, the present invention has no particular limitations on the reagents, systems and procedures used in the linker connection, library amplification, hybridization capture and PCR amplification processes, and conventional reagents, systems and procedures in the art may be used.
[0046] The preparation method of the present invention provides background nucleic acid interferors in a targeted manner in a library amplification system or a hybridization capture system for library preparation, thereby inhibiting background nucleic acids from entering a high-throughput sequencing library, thereby reducing the proportion of background nucleic acid sequences in sequencing data.
[0047] Based on the above advantages, the present invention also provides a method for reducing the proportion of background nucleic acids during high-throughput sequencing, comprising: preparing a high-throughput sequencing library with a low background nucleic acid proportion using the preparation method described in the above technical scheme; and performing high-throughput sequencing on the high-throughput sequencing library with a low background nucleic acid proportion.
[0048] The present invention has no special limitation on the platform and strategy of the high-throughput sequencing, and the sequencing adapters in the high-throughput sequencing library with the low background nucleic acid ratio can be matched.
[0049] As an embodiment, after the high-throughput sequencing, the obtained sequencing data is further subjected to bioinformatics analysis. The present invention has no particular limitation on the type and method of the bioinformatics analysis, and the type and method of conventional bioinformatics analysis in the art can be used.
[0050] The present invention also provides the preparation method described in the above technical solution or the high-throughput sequencing library with low background nucleic acid ratio prepared by the preparation method described in the above technical solution or the use of the method described in the above technical solution in preparing products for low-frequency gene detection or pathogenic microorganism detection. As an embodiment, the product described in the present invention can be a kit or a reagent.
[0051] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0052] Example 1
[0053] 10 ng of human genomic DNA standard (Promega, G1471) was taken and multiple displacement amplification was performed using phi29-XT WG A Kit (NEB#E1604S, the kit includes random primers). 5 μg of amplified product was taken and sheared for 500 s using microTUBE-130 on an M220 Focused-ultrasonicator (Covaris). The sheared product was 50 to 150 bp long as an interference, which was used to reduce the proportion of human genome in the sequencing library prepared from mixed samples. The principle is that the sheared product can interfere with the amplification of human genomic DNA template and the binding with the probe, thereby reducing the proportion of human genome in the sequencing library prepared from mixed samples.
[0054] The human genomic DNA standard (Promega, G1471) and the reference containing 20 bacterial DNAs (ATCC, MSA-1003) were mixed at a mass ratio of 9:1 to obtain a DNA template mixture. The Rapid DNA Digestion Library Construction Kit v2 for Illumina (Nanodata, #1002601) was used to perform the steps of fragmentation, end repair & A addition, adapter ligation and adapter ligation product purification according to the standard operation in the instruction manual to obtain the purified adapter ligation product.
[0055] The purified adapter ligation product was divided into two parts, one part with interference added, denoted as Lib2, and the other part without interference added, denoted as Lib1. Then Lib1 and Lib2 were PCR amplified separately. The PCR amplification system is shown in Table 1, and the PCR amplification procedure was carried out according to the instructions in the kit.
[0056] Table 1 PCR amplification system of the adapter ligation product in Example 1
[0057]
[0058] The two libraries were sequenced on Illumina Novasek 6000 in PE150 mode. After the sequencing data were aligned with the combined reference genome containing the human genome and 20 bacterial genomes, the proportion of bacteria and humans in the data was calculated. The results are as follows: Figure 1 Compared with the library sequencing data without the addition of interfering substances, the addition of interfering substances significantly inhibited the proportion of human genome, from 86% to 45%, which is about 8 times higher than the ratio of bacteria to humans.
[0059] Example 2
[0060] In this example, PCR was used to amplify the background nucleic acid. 10 ng of human genomic DNA standard (Promega, G1471) was taken. The Rapid DNA Digestion Library Construction Kit v2 for MGI (Naonda, #1002601) was used to perform fragmentation, end repair & A addition, adapter ligation and adapter ligation product purification steps according to the standard operations described in the instruction manual to obtain the purified adapter ligation product.
[0061] The purified adapter ligation product was subjected to PCR amplification. In the PCR amplification step, the matching index primer was not used, but the forward primer with the sequence of 5'-CTCACAGAACGACATGGCTACGATC-3' (SEQ ID NO: 1) and the reverse primer with the sequence of 5'-TCCTAAGACCGCTTGGCCTC-3' (SEQ ID NO: 2) were used for PCR amplification (the amplification system and procedure were carried out according to the kit instructions), and the PCR amplification product was obtained. The two ends of this PCR amplification product fragment have partial sequences of the MGI library, and since it is subsequently sequenced on the Illumina sequencing platform, it meets the requirements of being an interference.
[0062] A portion of the obtained PCR amplification product was further amplified using the above primers to produce a larger amount of interference. 5 μg of the amplification product was taken and interrupted for 300 s using a microTUBE-130 on an M220 Focused-ultrasonicator (Covaris). The length of the interrupted product as an interference was 50 to 150 bp.
[0063] In accordance with the operation in Example 1, human genomic DNA standard (Promega, G1471) and reference material containing 20 bacterial DNAs (ATCC, MSA-1003) were mixed at a ratio of 9:1 to obtain a DNA template mixture. 20 ng of the DNA template mixture was taken and The Rapid DNA Digestion Library Construction Kit v2 for Illumina (Nanodata, #1002601) was used to perform the steps of fragmentation, end repair & A addition, adapter ligation and adapter ligation product purification according to the standard operation in the instruction manual to obtain the purified adapter ligation product.
[0064] The purified adapter ligation product was divided into two parts, one part with interference added, denoted as Lib4, and the other part without interference added, denoted as Lib3. Then Lib3 and Lib4 were PCR amplified separately. The PCR amplification system is shown in Table 2, and the PCR amplification procedure was carried out according to the instructions in the kit.
[0065] Table 2 PCR amplification system of the adapter ligation product in Example 2
[0066]
[0067] Both libraries were sequenced on Illumina Novasek 6000 in PE150 mode.
[0068] After comparing the sequencing data with the reference genome containing the human genome and 20 bacterial genomes, the proportion of bacteria and humans in the data was counted. The results are as follows: Figure 2 As shown, it can be seen that the interference prepared by PCR amplification of background nucleic acid also increased the proportion of bacterial data. Compared with the library sequencing data without the addition of interference, the addition of interference significantly inhibited the proportion of human genome, from 88% to 52%.
[0069] Example 3
[0070] A reference substance containing 20 kinds of bacterial DNA (ATCC, MSA-1003) was spiked into a human genomic DNA standard (Promega, G1471) at a mass ratio of 0.001% to obtain a DNA template mixture.
[0071] Treatment 1: Take 10 ng of DNA template mixture and The library was prepared according to the standard operation of the instruction manual using the Rapid DNA Enzyme Digestion Library Construction Kit v2 for Illumina (Nanoda, #1002601), and the NEX-tPanel v1.0 (Nanoda) probes targeting multiple pathogens were used. ES Hybrid Capture Reagent (Naonda) was used for hybridization capture to enrich bacterial fragments.
[0072] Treatment 2: Prepare the library according to the steps in treatment 1. During the PCR amplification stage of library preparation, add 1 μg of interferor (the interferor is the same as in Example 2) according to the method in Example 2. Other steps are the same as the first treatment.
[0073] Treatment 3: Prepare the library according to the steps in Treatment 1, add 5 μg of interferor to the hybridization capture system (interferor is the same as Example 2), and the rest is the same as the first treatment.
[0074] Treatment 4: Prepare the library according to the steps in Treatment 1. In the PCR amplification stage of library preparation, add 1 μg of interferor (the interferor is the same as in Example 2) in the manner of Example 2, and add 5 μg of interferor (the interferor is the same as in Example 2) to the hybridization capture system.
[0075] The specific method of adding interference to the hybrid capture system is as follows: ES Hybrid Capture Reagent was used for hybridization capture. In the vacuum concentration step, the interference was combined with the library and other components according to the operation in Table 3 and then vacuum concentrated. The subsequent steps were carried out in full accordance with the instructions.
[0076] Table 3 Hybrid capture system with interference
[0077]
[0078] The post-capture libraries obtained from the four treatment methods were sequenced on Illumina Novasek 6000 in PE150 mode.
[0079] After comparing the sequencing data with the reference genome containing the human genome and 20 bacterial genomes, the proportion of bacteria and humans in the data was counted. The results are as follows: Figure 3As shown, although bacterial DNA was incorporated in a trace amount of one hundred thousandth, hybridization capture enrichment made the bacterial data account for about 1%; after the introduction of interfering substances in library preparation, the bacterial data accounted for about 7%; after the introduction of interfering substances in the hybridization step, the bacterial data accounted for about 4%; after the introduction of interfering substances in both the library preparation and hybridization steps, the bacterial data accounted for about 28%.
[0080] Example 4
[0081] Take 20ng RNA sample from nasal swab of healthy person Total RNA-To-DNA Module (Naonda, #1002401) was reverse transcribed into cDNA and used as background nucleic acid to prepare interferors according to the steps in Example 2. The interferors mainly contained human RNA sequences and RNA sequences of healthy human nasal flora.
[0082] Take 20 ng of RNA sample from a healthy person's nasal swab and add 10 copies of SARS-CoV-2 RNA quality control product (Jingliang, GW-CRPM002) as a template for RNA library preparation; DNA Universal Library Construction Kit (for ) (Naonda, #1002101) was used with NadPrep Total RNA-To-DNA Module (Naonda, #1002401) to prepare the total RNA library; the total RNA library was hybridized and captured using NanoRVPanel-TechAccess (Naonda, #1001121) probe to obtain a control library.
[0083] The treated library was prepared according to the preparation steps of the control library, except that 1 μg of interferor was added in the PCR amplification step of the library preparation, and 5 μg of interferor was added in the hybridization capture step in the manner of Example 3 to inhibit the nonspecific binding of background nucleic acid to the probe to obtain the treated library.
[0084] The control library and the treated library were sequenced on the Illumina Novasek 6000 in PE150 mode. The read counts of the novel coronavirus, human RNA, and bacterial 16S ribosomal RNA in the sequencing data of the control library and the treated library were counted. Since the background bacteria lacked a complete reference sequence, the bacterial 16S ribosomal RNA was compared and counted using the SILVA SSU database to reflect the changes in the proportion of background bacterial RNA. The results are shown in Figure 2. Figure 4 As shown, it can be seen that after being processed by the method in Example 4, the proportion of human RNA and background bacterial RNA data has decreased significantly, and the proportion of new coronavirus data has increased from less than 1% to more than 10%.
[0085] It can be concluded from the above examples that the preparation method can inhibit background nucleic acids from entering the sequencing library, thereby reducing their proportion in the sequencing results.
[0086] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A method for preparing a high-throughput sequencing library with a low background nucleic acid ratio, characterized in that: The steps include: Connecting the nucleic acid to be constructed into a library with a sequencing adapter to obtain an adapter-connected product; The adapter ligation product is subjected to one or both of the following operations to obtain the high-throughput sequencing library with a low background nucleic acid ratio: 1) mixing the adapter ligation product with the interfering substance and then performing library amplification; 2) performing library amplification on the adapter-ligated product to obtain a detection library; mixing the hybridization capture system containing the detection library with an interfering substance and then performing hybridization capture; The interferor is a fragmented background nucleic acid that is related to the nucleic acid to be constructed.
2. The preparation method according to claim 1, characterized in that: The preparation step of the interferor includes: performing gene amplification on the background nucleic acid, and fragmenting the obtained gene amplification product to obtain the interferor.
3. The preparation method according to claim 2, characterized in that: After the fragmentation, the fragmented gene amplification products are connected to sequencing adapters for PCR amplification, and the obtained PCR amplification products are fragmented to obtain the interferors; the sequencing adapters are sequencing adapters that do not match the high-throughput sequencing platform used for sequencing and do not exist in the target sequence set to be detected.
4. The preparation method according to claim 1, characterized in that: The length of the interferent is no greater than the length of the fragment of the high-throughput sequencing library with a low background nucleic acid ratio.
5. The preparation method according to claim 4, characterized in that: The length of the interferor is 50 to 500 bp.
6. The preparation method according to any one of claims 1 to 5, characterized in that: The interferor is DNA.
7. A method for reducing the proportion of background nucleic acids in high-throughput sequencing, characterized in that: A high-throughput sequencing library with a low background nucleic acid ratio is prepared by the preparation method according to any one of claims 1 to 6; The high-throughput sequencing library with low background nucleic acid ratio is subjected to high-throughput sequencing.
8. Use of the preparation method according to any one of claims 1 to 6, or a high-throughput sequencing library with a low background nucleic acid ratio prepared by the preparation method according to any one of claims 1 to 6, or the method according to claim 7 in preparing products for pathogenic microorganism detection.
9. The use according to claim 8, characterized in that: The products include kits or reagents.