WO2020118543A1 - 分离和/或富集宿主源核酸和病原核酸的方法和试剂及其制备方法 - Google Patents

分离和/或富集宿主源核酸和病原核酸的方法和试剂及其制备方法 Download PDF

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WO2020118543A1
WO2020118543A1 PCT/CN2018/120468 CN2018120468W WO2020118543A1 WO 2020118543 A1 WO2020118543 A1 WO 2020118543A1 CN 2018120468 W CN2018120468 W CN 2018120468W WO 2020118543 A1 WO2020118543 A1 WO 2020118543A1
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nucleic acid
host
probe
pathogenic
human
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PCT/CN2018/120468
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French (fr)
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崔淼
汪为茂
章文蔚
陈奥
王欧
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深圳华大生命科学研究院
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Priority to CN201880099543.XA priority Critical patent/CN113039283B/zh
Priority to PCT/CN2018/120468 priority patent/WO2020118543A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material

Definitions

  • the invention relates to the technical field of nucleic acid separation and enrichment, in particular to a method and reagent for separating and/or enriching host-origin nucleic acid and pathogenic nucleic acid, and a preparation method thereof.
  • DNA sequencing technology has made major breakthroughs in sequencing speed, throughput, and cost. Therefore, gene sequencing technology has been widely used in the field of medical and health testing, including embryo implantation screening, prenatal screening, cancer screening, infection disease testing and prevention and control. Among them, the existence and continuous evolution of infectious pathogens further make the use of next-generation sequencing to detect infectious diseases a very valuable field that is gradually concerned by more and more researchers.
  • Meta sequencing has been widely used in the detection of pathogenic viruses and pathogenic microorganisms to discover new pathogens and to conduct germline genotyping of pathogens. Combined with the corresponding rapid detection technology, Meta sequencing can also better reveal the path of transmission of pathogens, help disease control, and prevent outbreaks of infectious diseases.
  • Using blood as a sample for Meta sequencing to identify pathogenic nucleic acids in blood has been the mainstream research direction in Meta sequencing.
  • the first category is to separate nucleic acids according to different methylation sites of human origin and pathogenic nucleic acids to achieve the purpose of enriching pathogenic nucleic acids, but the separation principle based on the methylation state limits the types of pathogenic nucleic acids that can be enriched.
  • the second type of enrichment method is a ribonuclease-based human nucleic acid depletion method. This method can be applied to the enrichment of pathogenic nucleic acids such as viruses, fungi and parasites.
  • enrichment of nucleic acid in bacteria using human-derived nucleic acid depletion method cannot be used to study the interaction mechanism of bacteria and their host cells.
  • NEBNext Microbiome DNA Enrichment kit launched by New England Biolabs.
  • This kit is designed based on the fact that human-derived nucleic acids contain more methylation sites, using the difference between the methylation sites of human-derived and pathogenic nucleic acids.
  • the fusion protein prepared by using methylation site binding protein MBD2 and human immunoglobulin can specifically remove human nucleic acid from the sample.
  • the principle of another type of kit is to achieve the enrichment of pathogenic nucleic acid by the method of removing human-derived nucleic acid.
  • Such commercial kits include MolYsis by Molzym, QIAamp DNA Microbiome Kit (QIAamp DNA Kit) by Qiagen, and Microbiome Enrichment by Thermo Fisher Scientific. Kit (MICROBEnrich Kit).
  • MolYsis and QIAamp DNA Microbiome Kit are mainly used to remove human DNA.
  • the main principle is to selectively lyse human cells and use DNase to remove human DNA.
  • the microbiome enrichment kit (MICROBEnrichKit) is mainly used to remove human-derived RNA, and its main principles are divided into two categories.
  • the first type is to selectively lyse human-derived cells, to separate microorganisms such as bacteria and human-derived RNA by centrifugation; the second type is to lyse human-derived and pathogenic cells at the same time, and then use magnetic beads with oligonucleotide chains to adsorb humans
  • the 18S, 28S rRNAs and mRNA in the source RNA achieve the purpose of removing human source RNA.
  • other uncommercialized methods have been gradually developed in the research process.
  • MR Hasan et al. used low concentration nonionic surfactant saponin to selectively lyse human cells and used Turbo DNase to remove human DNA.
  • the method for enriching pathogenic nucleic acid uses a host-derived probe to specifically capture the host-derived nucleic acid in the sample, and achieves the enrichment of the pathogenic nucleic acid by a method of isolating the host-derived nucleic acid.
  • This method can be widely used for enrichment of different types of pathogenic nucleic acids, and can also achieve the purpose of simultaneous detection of host sources and pathogenic nucleic acids, thereby enriching the understanding of the mechanism of pathogens and host cells, and providing new ways for disease prevention and treatment .
  • the present invention provides a method and reagent for separating and/or enriching host-derived nucleic acid and pathogenic nucleic acid and a preparation method thereof.
  • an embodiment provides a method for isolating and/or enriching a host-derived nucleic acid and a pathogenic nucleic acid, the method comprising the steps selected from (a) or (b) as follows:
  • the aforementioned host-derived probe is a human-derived probe
  • the aforementioned host-derived nucleic acid is a human-derived nucleic acid
  • the human probe in (a) above is a DNA nanosphere made of human genomic DNA as a raw material, a fragmented genomic sequence amplification product or a fragmented genomic sequence; wherein the fragmented genomic sequence is expanded
  • the bioproduct markers are carried on the augmented product and fragmented genomic sequences, and the streptavidin markers are carried on the solid phase carrier.
  • the human probe in (b) above is a DNA nanosphere made of human genomic DNA as a raw material.
  • the solid phase carrier in (a) above is selected from chips or magnetic beads.
  • the solid phase carrier in (b) above is a digitally encoded magnetic bead.
  • the pathogenic probe in (b) above is a conserved region of the pathogenic genome sequence.
  • the aforementioned liquid sample is a nucleic acid extract in a sample of blood, plasma, saliva, cerebrospinal fluid, or alveolar lavage fluid.
  • the above (a) further includes: denaturing and eluting the host-derived nucleic acid bound to the solid-phase carrier to obtain free host-derived nucleic acid.
  • the above (b) further includes: denaturing and eluting the pathogenic nucleic acid bound to the solid-phase carrier to obtain free pathogenic nucleic acid.
  • an embodiment provides an agent for isolating and/or enriching host-derived nucleic acid and pathogenic nucleic acid, the agent comprising a component selected from (a) or (b) as follows:
  • a host-derived probe immobilized on a solid-phase carrier which is used to mix and incubate with a liquid sample containing host-originated nucleic acid and pathogenic nucleic acid to hybridize the host-originated probe to the host-originated nucleic acid and separate them by solid-liquid separation Obtain the host-derived nucleic acid bound to the solid phase carrier and the pathogenic nucleic acid retained in the liquid sample;
  • a host-derived probe and a pathogenic probe immobilized on a solid support which are used to mix and incubate with a liquid sample containing the host-derived nucleic acid and the pathogenic nucleic acid to hybridize the host-derived probe with the host-derived nucleic acid;
  • the pathogenic probe is hybridized with the above-mentioned pathogenic nucleic acid, and the pathogenic nucleic acid bound to the above-mentioned solid-phase carrier and the host-derived nucleic acid retained in the above-mentioned liquid sample are obtained by solid-liquid separation.
  • the aforementioned host-derived probe is a human-derived probe
  • the aforementioned host-derived nucleic acid is a human-derived nucleic acid
  • the human probe in (a) above is a DNA nanosphere made of human genomic DNA as a raw material, a fragmented genomic sequence amplification product or a fragmented genomic sequence; wherein the fragmented genomic sequence is expanded
  • the bioproduct markers are carried on the augmented product and fragmented genomic sequences, and the streptavidin markers are carried on the solid phase carrier.
  • the human probe in (b) above is a DNA nanosphere made of human genomic DNA as a raw material.
  • the solid phase carrier in (a) above is selected from chips or magnetic beads.
  • the solid phase carrier in (b) above is a digitally encoded magnetic bead.
  • the pathogenic probe in (b) above is a conserved region of the pathogenic genome sequence.
  • the aforementioned liquid sample is a nucleic acid extract in a sample of blood, plasma, saliva, cerebrospinal fluid, or alveolar lavage fluid.
  • an embodiment provides a method of preparing the reagent of the second aspect, the method comprising the steps selected from (a) or (b) as follows:
  • the host-derived probe in (a) above is a DNA nanosphere made of human genomic DNA as a raw material
  • the preparation method of the host-originated probe fixed on a solid-phase carrier includes:
  • transposase to interrupt the above human genomic DNA and connect the linker to obtain the fragmented human genomic DNA with linkers at both ends;
  • the DNA nanosphere is loaded on the surface of the chip to make a chip carrying the DNA nanosphere.
  • the host-derived probe in (a) above is a fragmented genomic sequence amplification product made of human genomic DNA as a raw material, and the fragmented genomic sequence amplification product carries a biotin label on it.
  • the solid-phase carrier is labeled with streptavidin.
  • the preparation method of the host-based probe fixed on the solid-phase carrier includes:
  • transposase to interrupt the above human genomic DNA and connect the linker to obtain the fragmented human genomic DNA with linkers at both ends;
  • the host-derived probe in (a) above is a fragmented genomic sequence made of human genomic DNA as a raw material, the fragmented genomic sequence has a biotin tag on it, and the solid phase carrier has The streptavidin label, and the preparation method of the host source probe fixed on the solid phase carrier include:
  • transposase to interrupt the above human genomic DNA and add a biotin-labeled linker to obtain fragmented human genomic DNA with linkers at both ends;
  • the magnetic beads with streptavidin labeling were added to the above-mentioned biotin-labeled fragmented human genomic DNA product and mixed and incubated to obtain a human source probe with the fragmented human genomic sequence product fixed on the above-mentioned magnetic beads needle.
  • the host probe in (b) above is a DNA nanosphere made of human genomic DNA as a raw material; the preparation method of the host probe includes:
  • transposase to interrupt the above human genomic DNA and connect the linker to obtain the fragmented human genomic DNA with linkers at both ends;
  • the extension primer is hybridized at the junction region of the single-stranded loop, and a polymerase with a strand displacement function is added to perform rolling circle amplification to obtain a linear amplification product of the single-stranded human genome fragment, and DNA nanospheres are formed by intramolecular hydrogen bonding.
  • the conserved region sequence of the pathogenic nucleic acid labeled with biotin is mixed and incubated with the digital magnetic beads labeled with streptavidin to obtain the pathogenic probe for capturing the pathogenic nucleic acid in the sample.
  • the method for separating and/or enriching host-origin nucleic acid and pathogenic nucleic acid of the present invention has the following beneficial effects:
  • This scheme does not require prior knowledge of the methylation site of the pathogenic nucleic acid, and can simultaneously detect known and unknown pathogens.
  • This solution does not require the use of DNase and RNase to digest the host-derived nucleic acid, so it will not lose a large amount of free pathogenic nucleic acid present in the sample, and can target all pathogenic nucleic acids in the sample Enrichment.
  • the capture probe applied to isolate host-derived nucleic acid is preferably host-derived genomic DNA, which can simultaneously hybridize host-derived DNA and RNA fragments in the sample to achieve the simultaneous enrichment of pathogenic DNA and RNA.
  • the preparation method of the chips and magnetic beads used in the enrichment involved in this scheme is simple and fast.
  • the reagents used in the process are mature commercial reagents, which do not require complex processes such as modification of wild enzymes, and have strong operability and cost. Lower.
  • the pathogenic nucleic acid enrichment method of this scheme can significantly increase the relative content of pathogenic nucleic acids in the sample, which can reduce the number of reads and reduce the detection time and cost when applied to the downstream metagenomics detection scheme.
  • FIG. 1 is a schematic diagram of the structure and marking of a transposable complex in an embodiment of the present invention
  • FIG. 2 is a flow chart of a method for preparing a human capture probe chip for removing human nucleic acid in a Meta sample in an embodiment of the present invention
  • FIG. 3 is a flow chart of a method for preparing a human capture probe microsphere for removing human nucleic acid in a Meta sample according to an embodiment of the present invention
  • FIG. 4 is a flow chart of a method for preparing a human capture probe microsphere for removing human nucleic acid in a Meta sample in an embodiment of the present invention
  • FIG. 5 is a flow chart (1) of a method for preparing a human capture probe DNA nanosphere for removing human nucleic acid in a Meta sample according to an embodiment of the present invention and a schematic diagram of pathogen capture digital magnetic beads that can be combined with different types of pathogen nucleic acids (2).
  • the present invention designs a variety of different schemes, using chips or magnetic beads that modify the host-derived genome sequence or the pathogenic nucleic acid conserved region sequence to achieve the separation of the host source and the pathogenic nucleic acid In order to achieve the purpose of enriching pathogenic nucleic acid.
  • the enrichment of pathogenic nucleic acids by the scheme of the present invention can achieve the following technical effects:
  • This scheme does not require prior knowledge of the methylation site of the pathogenic nucleic acid, and can realize the detection of unknown pathogens.
  • the capture probe applied to enrich the host-derived nucleic acid can be host-derived genomic DNA, which can simultaneously hybridize the host-derived DNA and RNA fragments in the sample to achieve the simultaneous enrichment of pathogenic DNA and RNA.
  • the preparation method of the chips and magnetic beads used in the enrichment involved in this scheme is simple and fast.
  • the reagents used in the process are mature commercial reagents, which do not require complex processes such as modification of wild enzymes, and have strong operability and cost. Lower.
  • Embodiments of the present invention provide a method for isolating and/or enriching host-origin nucleic acid and pathogenic nucleic acid.
  • isolation refers to the separation of the host-originated nucleic acid and the pathogenic nucleic acid to a certain extent, which may be separated in whole (for example, 100%) or in part (for example, 10% to 90%, etc.).
  • enrichment refers to increasing the absolute or relative amount of at least one of the host-derived nucleic acid and the pathogenic nucleic acid, such as absolute concentration or relative concentration.
  • host-derived nucleic acid refers to any source of nucleic acid from various sources such as host cells, host tissues or host individuals parasitized or infected by pathogens, including but not limited to animal-derived nucleic acids and plant-derived nucleic acids, especially animals Source nucleic acids, especially those derived from mammals, are most typically human.
  • Such nucleic acid may be DNA or RNA.
  • the "pathogenic nucleic acid” refers to a pathogenic microorganism that parasitizes or infects any host cell, host tissue, or host individual, including but not limited to bacteria, fungi, and the like.
  • An embodiment of the present invention provides a method for isolating and/or enriching host-derived nucleic acid and pathogenic nucleic acid.
  • the method includes the steps of: fixing the host-derived probe immobilized on a solid-phase carrier and containing the host-derived nucleic acid and pathogen.
  • the liquid sample of the nucleic acid is mixed and incubated to hybridize the host-derived probe with the host-derived nucleic acid; solid-liquid separation is performed to obtain the host-derived nucleic acid bound to the solid-phase carrier and the pathogenic nucleic acid retained in the liquid sample.
  • the host-derived probe is a probe to which the host-derived nucleic acid hybridizes, typically, a human-derived probe that hybridizes to the human-derived nucleic acid.
  • the human probe in the embodiment of the present invention is a DNA nanosphere made of human genomic DNA as a raw material, an amplified product of a fragmented genome sequence, or a fragmented genome sequence. Fragmented genome sequence amplification products and fragmented genome sequences are marked with biotin, and the solid phase carrier is marked with streptavidin.
  • the host-derived probe has a certain degree of general characteristics, that is, for the same host-derived nucleic acid, host-derived nucleic acid (eg, human-derived nucleic acid) of different host individuals (eg, different humans) can be prepared in advance It is good to hybridize with the same host-derived probe (for example, a human-derived probe).
  • the pre-prepared host-derived probe can be stored under appropriate conditions for a certain period of time. For example, it can be stored at 4°C for 1 month and used within this period.
  • the host-derived probe for example, the human-derived probe can be prepared by a suitable method using human-derived nucleic acid, such as human genomic DNA.
  • human genomic DNA can be fragmented genomic DNA by physical or enzymatic interruption, and then the fragmentation can be specifically labeled (such as biotin labeling) to fix it on the surface of the solid phase carrier, or the fragmented genomic DNA DNA nanospheres (DNB) obtained by circularization and rolling circle amplification are loaded onto the surface of a solid phase carrier (such as a chip) to achieve probe immobilization.
  • a solid phase carrier such as a chip
  • the transposase and the transposon form a transposition complex, as shown in Figure 1(1) A double-transposon transposon complex containing two types of transposons, A transposon and B transposon, was produced.
  • the transposition complex may be a single transposon transposition complex containing only one transposon (A transposon or B transposon).
  • the fragmented genomic DNA needs to be labeled with a biotin label or the like, it can be implemented in various ways.
  • One way is to use the biotin-labeled transposon complex in Figure 1(2) to interrupt the genomic DNA.
  • the biotin-labeled A transposon and the biotin-labeled B transposon are formed together with the transposase
  • the transposition complex adds biotin-labeled transposon sequences to both ends of the fragmented genomic DNA while interrupting the genomic DNA, and the probe is fixed by binding to streptavidin on the solid phase carrier.
  • the human source nucleic acid is used as the host source nucleic acid and the human source probe is used as the host source probe to provide the following three methods for separating and/or enriching the host source nucleic acid and the pathogenic nucleic acid.
  • Method 1 The method of enriching pathogenic nucleic acid based on human probe (DNA nanosphere), the principle and characteristics are as follows:
  • the DNA nanospheres fixed on the silicon-based chip are used as human probes to hybridize and adsorb the human nucleic acid in the sample to be tested, and to achieve the enrichment of the pathogenic nucleic acid in the sample by solid-liquid separation.
  • the preparation method of the DNA nanosphere human probe fixed on the silicon-based chip is shown in Figure 2:
  • human genomic DNA is used as a raw material, and a product of fragmented human genomic DNA with specific linkers at both ends is obtained by using a transposase to interrupt and add a linker.
  • a large number of amplification products can be obtained through incision translation and PCR amplification.
  • the amplified human genome fragments can be prepared into single-stranded loops.
  • Linearly amplified products of single-stranded human genomic fragments can be obtained by hybridizing extension primers in the single-stranded loop linker region and adding phi29 polymerase by rolling circle amplification.
  • DNA nanosphere loading buffer to the DNA nanospheres, mix them and put them into the loading system. After incubation at room temperature, the prepared DNA nanospheres can be loaded onto the silicon-based chip modified with amino groups at the end.
  • the sample to be processed is loaded on the probe chip, and the human probe on the chip is hybridized with the human nucleic acid in the sample, and the enrichment of the pathogenic nucleic acid in the sample can be achieved by separating the solid phase chip and the liquid sample to be processed.
  • the capture probe used to isolate human nucleic acid is derived from human genomic DNA, which can simultaneously hybridize the human DNA and RNA fragments in the sample to achieve the simultaneous enrichment of pathogenic DNA and RNA.
  • the probe exists in the form of DNA nanospheres, and there are about hundreds of copies (for example, more than 300 copies, preferably 500 copies) of the fragmented human genomic DNA product copy number, increasing the efficiency of hybridization to capture human nucleic acid in the sample .
  • Method 2 The method of enriching pathogenic nucleic acid based on human probe (fragmented genomic sequence amplification product), the principle and characteristics are as follows:
  • the amplified product of the fragmented human genomic sequence fixed on the magnetic beads is used as a human probe to hybridize and adsorb the human nucleic acid in the sample to be tested and solid-liquid separation to enrich the pathogenic nucleic acid in the sample.
  • the preparation method of the humanized probe of the amplified product of the fragmented human genome sequence fixed on the magnetic beads is shown in FIG. 3:
  • human genomic DNA is used as a raw material, and a product of fragmented human genomic DNA with specific linkers at both ends is obtained by using a transposase to interrupt and add a linker.
  • a transposase to interrupt and add a linker.
  • biotin-labeled primers Through incision translation and PCR amplification using biotin-labeled primers, a large number of biotin-labeled fragmented human genome sequence amplification products can be obtained.
  • Add the streptavidin-labeled magnetic beads to the biotin-labeled fragmented human genome sequence amplification product solution and stir and incubate at room temperature to obtain the fragmented human immobilized on the magnetic beads Human-derived probes for amplification products of genomic sequences.
  • the capture probe used to isolate human nucleic acid is derived from human genomic DNA, which can simultaneously hybridize the human DNA and RNA fragments in the sample to achieve the simultaneous enrichment of pathogenic DNA and RNA.
  • the PCR method is used to realize the labeling of functional groups (biotin) and the amplification of human probes in one step, which can achieve the fixation of human probes and further increase the capture efficiency of human nucleic acids in samples.
  • Method 3 Based on human probe (fragmented genomic sequence) to enrich pathogenic nucleic acid, its principle and characteristics are as follows:
  • the product of the fragmented human genomic sequence immobilized on the magnetic beads is used as a human probe to hybridize and adsorb the human nucleic acid in the sample to be tested and to enrich the pathogenic nucleic acid in the sample by solid-liquid separation.
  • the preparation method of the humanized probe of the product of the fragmented human genome sequence fixed on the magnetic beads is shown in FIG. 4:
  • a transgenic enzyme is used to interrupt and add a biotin-labeled linker to obtain a fragmented human genomic DNA product with specific linkers at both ends.
  • the biotin-tagged fragmented human genomic DNA product can be obtained by nick translation and extension.
  • the capture probe used to isolate human nucleic acid is derived from human genomic DNA, which can simultaneously hybridize the human DNA and RNA fragments in the sample to achieve the simultaneous enrichment of pathogenic DNA and RNA.
  • This scheme uses transposase to interrupt human genomic DNA in one step and add a linker to obtain a human probe.
  • the preparation scheme is simple and the steps are few, so the efficiency of preparing human probe is high and the cost is low.
  • An embodiment of the present invention provides a method for isolating and/or enriching host-origin nucleic acid and pathogenic nucleic acid.
  • the method includes the following steps:
  • the host-derived probe, the pathogenic probe fixed on the solid phase carrier and the liquid sample containing the host-derived nucleic acid and the pathogenic nucleic acid are mixed and incubated to hybridize the host-derived probe with the host-originated nucleic acid and the pathogenic probe with the pathogenic nucleic acid;
  • the solid-liquid separation obtains the pathogenic nucleic acid bound to the solid phase carrier and the host-derived nucleic acid retained in the liquid sample.
  • the host-derived probe for example, the human-derived probe can be prepared by a suitable method using human-derived nucleic acid, such as human genomic DNA.
  • the host source probe is used as a liquid phase probe to bind to the host source nucleic acid in the sample; the pathogenic probe is fixed on the solid phase carrier, combined with the pathogenic nucleic acid in the sample, and then the host source is realized by solid-liquid separation Separation of nucleic acids and pathogenic nucleic acids.
  • the human source nucleic acid is used as the host source nucleic acid and the human source probe is used as the host source probe to provide the following fourth method for separating and/or enriching the host source nucleic acid and the pathogenic nucleic acid.
  • Method 4 A method for enriching pathogenic nucleic acids based on human-derived probes (DNA nanospheres) and pathogenic probes (digitally encoded magnetic beads). The principles and characteristics are as follows:
  • human genomic DNA is used as a raw material, and a product of fragmented human genomic DNA with specific linkers at both ends is obtained by using a transposase to interrupt and add a linker.
  • a large number of amplification products can be obtained through incision translation and PCR amplification.
  • the amplified human genome fragments can be prepared into single-stranded loops.
  • Linearly amplified products of single-stranded human genomic fragments can be obtained by hybridizing extension primers in the single-stranded loop linker region and adding phi29 polymerase by rolling circle amplification. These long single strands can form DNA nanospheres through intramolecular hydrogen bonding, that is, human probes used for hybridization to remove human nucleic acids in samples.
  • Pathogen probes with conserved regions of pathogenic genomic sequences are prepared by mixing biotin-labeled conserved region sequences of pathogenic nucleic acids with streptavidin-labeled digital magnetic beads and incubating at room temperature to be used. A pathogenic probe for capturing pathogenic nucleic acids in a sample to be tested.
  • the prepared DNA nanosphere human probe and the digital magnetic beads immobilized with the pathogen probe are added to the sample to be processed, so that the human nucleic acid in the sample to be tested is hybridized and captured into the liquid phase DNA nanosphere human probe On the needle, the pathogenic nucleic acid in the sample to be tested is hybridized and captured on the digital magnetic beads to which the pathogenic probe is fixed, and the enrichment of the pathogenic nucleic acids in the sample can be achieved by separating the solid-phase digital magnetic beads and the liquid phase sample to be processed.
  • a human probe is prepared to capture human nucleic acid in the sample, and no specific methylation site or conserved region is needed to achieve hybridization separation.
  • the capture probe used to isolate human nucleic acid is derived from human genomic DNA, which can simultaneously hybridize the human DNA and RNA fragments in the sample to achieve the simultaneous enrichment of pathogenic DNA and RNA.
  • Human probes exist in the form of DNA nanospheres, and there are about hundreds of copies (for example, more than 300 copies, preferably 500 copies) of fragmented human genomic DNA product copy number, increasing the amount of human nucleic acid in the hybrid capture sample s efficiency.
  • This scheme utilizes a two-way selection method, that is, a scheme in which a human probe and a pathogen probe are simultaneously hybridized and captured for enrichment, which can further increase the specificity and efficiency of pathogen enrichment.
  • the enzyme for DNA fragmentation of the present invention is not limited to the Tn5 transposase used in this embodiment, and may be other enzymes of the Tn transposase family, such as Tn7; or other transposase families, such as Mu family; not even limited to transposases or enzyme preparations, as long as it can fragment DNA while linking a sequence to DNA.
  • the solid-phase carrier used for adsorption according to the present invention is not limited to the silicon-based chips and ferroferric oxide beads used in the examples, and may be polymer or glass microspheres, and may be solid, hollow or porous. It can even be the solid surface and interior of any other materials and morphology, as long as it can modify the probe oligonucleotide chain.
  • the surface modification is not limited to streptavidin in the examples, and the modification may be a hydroxyl group, a carboxyl group, an amino group and other molecules that can be covalently linked to a DNA oligonucleotide (oligo).
  • the lattice shape and number of solid-phase chips used for adsorption in the present invention are not limited to circular and 200M, and may be any shape and number.
  • the size and number of added magnetic beads are not limited to 20 ⁇ m and 200M, and can be any size and number.
  • the length and sequence information of the transposon sequence used in the present invention are not limited.
  • the transposon adaptor used in the present invention that interrupts genomic DNA and is added at both ends may be a single sequence adaptor or two different sequence adaptors.
  • the size of the fragment of the human probe obtained by interrupting the transposition complex added in the present invention is not limited, as long as the probe can effectively remove the human genome fragment in the sample.
  • the ligase used in the present invention is not limited to a specific ligase, but can be T4 DNA ligase, Taq DNA ligase, T7 DNA ligase, T3 DNA ligase, E. Coli DNA ligase and HiFi Taq DNA Ligase, as long as it can connect a nick to form a single chain loop.
  • the exonuclease used in the present invention is not limited to a specific exonuclease, and may be exonuclease I, exonuclease III, lambda exonuclease and T7 exonuclease, as long as it can digest the cyclization product The remaining linear DNA strands are sufficient.
  • the polymerase for preparing DNA nanospheres used in the present invention is not limited to phi29 polymerase, and may be other polymerases having strand displacement activity and capable of rolling circle amplification, and may be large fragments of Bst and Bsu, E.Coli DNA polymerase I, etc., as long as it can produce DNA nanospheres.
  • the material, size, shape and surface pattern of the digital magnetic beads used in the present invention are not limited to one, the material may be a polymer material, such as inorganic ferric oxide, etc.; the size may be 1 ⁇ m to 1 mm; the shape and surface
  • the pattern and surface modification method are not limited, as long as it can modify the conserved sequence of the pathogenic nucleic acid to achieve the purpose of enriching the pathogenic nucleic acid and can realize multiple detection.
  • the four methods of enriching pathogenic nucleic acids in the present invention are not limited to the enrichment of pathogenic nucleic acids in blood samples, but can also be applied to the enrichment of pathogenic nucleic acids in samples such as saliva, cerebrospinal fluid, and alveolar lavage fluid.
  • the enrichment method of four pathogenic nucleic acids in the present invention is not limited to the enrichment of pathogenic nucleic acids in sequencing sample processing, but can also be applied to the detection of pathogens based on methods such as qPCR, ddPCR, aptamer hybridization, binding protein, etc.
  • the detection signal method is not limited, and it can be fluorescence method, electrochemical method, chemiluminescence method, colorimetric method, etc.
  • Example 1 A method for enriching pathogenic nucleic acids based on human-derived probes (DNA nanospheres)
  • nucleic acid extracted in step (6) to the chip prepared in step (5) for hybridization at 25°C for 30 minutes. Collect the clear solution and rinse the surface of the chip with TE buffer to remove non-specifically adsorbed nucleic acids. All solutions and washing solutions can be combined. Get enriched pathogenic nucleic acid.
  • Humanized nucleic acid can be obtained by denaturing the nucleic acid hybridized with the chip and collecting it with TE buffer.
  • Example 2 A method for enriching pathogenic nucleic acids based on human-derived probes (fragmented genomic sequence amplification products)
  • 0.5 ⁇ 5pmol transposition complex was used to interrupt the genomic sequence and the linker sequence was added, and the reaction was carried out at 55°C for 5 ⁇ 20 minutes. After the reaction, the reaction was terminated by using NT buffer at room temperature for 3 ⁇ 10min to terminate the reaction. The products are subjected to terminal extension at 72°C for 5 to 15 minutes to obtain human genome fragments with linker sequences.
  • step (1) Add 1-2 ⁇ M biotin-labeled primers, DNA polymerase and dNTPs to the human genomic fragment with linker sequence prepared in step (1), extend at 72°C for 5 min, and then perform 8-15 polymerase chain reactions After circulation, 0.8-2.5X magnetic beads can be purified to obtain the amplified product of the human genome fragment with the biotin-labeled linker sequence.
  • step (3) Add the magnetic beads prepared in step (3) to the free nucleic acids extracted in step (4) and hybridize at 25°C for 30 minutes. Collect the supernatant and wash the non-specifically adsorbed nucleic acid on the surface of the magnetic beads with TE buffer. Combined to obtain enriched pathogenic nucleic acid.
  • Human nucleic acid can be obtained by denaturing the nucleic acid hybridized with the magnetic beads and collecting it with TE buffer.
  • Example 3 A method for enriching pathogenic nucleic acid based on human probe (fragmented genomic sequence)
  • step (1) Add the human genomic fragment with the biotin-labeled linker sequence prepared in step (1) to the streptavidin-labeled magnetic beads and mix well. Incubate for 1 hour at room temperature on a vertical mixer to obtain a human Magnetic beads of source probe (fragmented genomic sequence).
  • step (2) Add the magnetic beads prepared in step (2) to the free nucleic acids extracted in step (3) and hybridize at 25°C for 30 minutes. Collect the clear solution and wash the non-specifically adsorbed nucleic acid on the surface of the magnetic beads with TE buffer solution. Combined to obtain enriched pathogenic nucleic acid.
  • Human nucleic acid can be obtained by denaturing the nucleic acid hybridized with the magnetic beads and collecting it with TE buffer.
  • Example 4 A method for enriching pathogenic nucleic acids based on human-derived probes (DNA nanospheres) and pathogenic probes (digitally encoded magnetic beads)
  • the synthetic nucleic acid chain with conserved region of biotin-labeled pathogenic nucleic acid was added to the streptavidin-modified digital magnetic beads and mixed, and incubated for 1 hour at room temperature on a vertical mixer.
  • Enriched pathogenic nucleic acid can be obtained by denaturing the nucleic acid hybridized with the digital magnetic beads and collecting it with TE buffer.
  • Comparative Example The samples without pathogen nucleic acid enrichment were constructed and sequenced under the same library construction conditions as in the example.
  • Example 1 Example 2 Example 3 Example 4 Number of sequencing reads (M) 80 40 40 40 40 40 Number of pathogens read (Reads) 19 19 twenty two 20 17

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Abstract

一种分离和/或富集宿主源核酸和病原核酸的方法和试剂及其制备方法。所述方法包括选自如下(a)或(b)的步骤：(a)将固定在固相载体上的宿主源探针与含有宿主源核酸和病原核酸的液体样品混合孵育，使宿主源探针与宿主源核酸杂交；进行固液分离得到结合在固相载体上的宿主源核酸和保留在液体样品中的病原核酸；(b)将宿主源探针、固定在固相载体上的病原探针与含有宿主源核酸和病原核酸的液体样品混合孵育，使宿主源探针与宿主源核酸杂交、病原探针与病原核酸杂交;进行固液分离得到结合在固相载体上的病原核酸和保留在液体样品中的宿主源核酸。该方法使用宿主源探针特异性捕获宿主源核酸，通过分离宿主源核酸来实现病原核酸的富集。

Description

分离和/或富集宿主源核酸和病原核酸的方法和试剂及其制备方法 技术领域
本发明涉及核酸分离和富集技术领域，具体涉及一种分离和/或富集宿主源核酸和病原核酸的方法和试剂及其制备方法。
背景技术
近几年，DNA测序技术在测序速度、通量以及成本上取得了重大突破。因此，基因测序技术已经广泛应用于医疗健康检测领域，包括胚胎植入筛查、产前检查、癌症筛查、感染病的检验与防控等。其中，感染病原的存在以及不断的演变进一步使利用二代测序来检测感染疾病成为一个非常有价值并逐渐被越来越多的研究者关注的领域。
目前，利用基因测序及分析技术已经可以实现针对病原微生物种群中各组分的种属，丰度以及功能特性的检测，进而研究人与病原微生物之间的关联。但是，这些研究一直受限于样本的复杂组分，包括细菌、真菌、病毒、原生生物以及过量的宿主人源核酸。结合PCR和测序技术的扩增子测序(例如16S核糖体RNA测序)可以实现在复杂样本中对病原核酸进行快速、高分辨的检测。这种扩增子测序可以在测序覆盖度以及深度方面提供极大优势，但是扩增子测序无法实现对整个流行病学的研究以及致病性的鉴定。
针对扩增子测序的局限性，Meta测序已经被广泛应用到病原病毒以及病原微生物的检测中，用以发现新的致病体以及对病原体进行种系基因分型。结合相应的快速检测技术，Meta测序也可以更好地揭示致病体的传播途径，帮助疾病控制，预防传染病爆发等。利用血液为样本进行Meta测序来鉴定血液中的病原核酸一直是Meta测序中的主流研究方向。但是，由于血液中存在过量的人源核酸，其中只有约1％的病原核酸，利用传统的测序方法很难检测到存在于过量人源核酸中的病原核酸，更加难以针对相应的情况给予例如耐药性和感染性等诊断指导。同时，利用传统方法检测低频的病原核酸需要很高的测序深度，这无疑也会增加测序成本。因此，针对血液Meta测序前的样本处理方法的选择一直是Meta测序技术的关键点之一。
传统的富集病原核酸的方法主要分为两类。第一类是根据人源和病原核酸的不同甲基化位点来进行核酸分离从而达到富集病原核酸的目的，但是基于甲基化状态的分离原理限制了可富集的病原核酸的种类。第二类富集方法是基于核糖核酸酶的人源核酸消耗法。这种方法可以适用于病毒、真菌以及寄生虫等病原核酸的富集。但是由于细菌和其宿主细胞的转录本的组成及含量的不一致性，利用人源核酸消耗法富集细菌内核酸无法用以研究细菌及其宿主细胞的相互作用机理。
已经商品化的富集病原核酸的方法主要分为以下几类。首先是美国纽英伦生物技术公司(New England Biolabs)推出的NEB下一代微生物DNA浓缩试剂盒(NEBNext Microbiome DNA Enrichment kit)。这种试剂盒是根据人源核酸含有更多的甲基化位点，利用人源和病原核酸甲基化位点的不同来设计的。利用甲基化位点结合蛋白MBD2与人免疫球蛋白制备的融合蛋白可特异性去除样本中的人源核酸。另一类试剂盒的原理是利用去除人源核酸的方法实 现病原核酸的富集。这类商业化的试剂盒包括Molzym公司推出的MolYsis，Qiagen公司推出的QIAamp DNA微生物组试剂盒(QIAamp DNA Microbiome Kit)，以及赛默飞世尔科技公司(Thermo Fisher Scientific)推出的微生物组富集试剂盒(MICROBEnrich Kit)。MolYsis和QIAamp DNA微生物组试剂盒(QIAamp DNA Microbiome Kit)主要用于去除人源DNA，其主要原理是选择性裂解人源细胞并利用DNA酶(DNase)去除人源DNA。微生物组富集试剂盒(MICROBEnrich Kit)主要用于去除人源RNA，其主要原理分为两类。第一类是选择性裂解人源细胞，利用离心法分离细菌等微生物和人源RNA；第二类是对人源和病原细胞同时裂解，再利用带有寡核苷酸链的磁珠吸附人源RNA中的18S、28S rRNAs和mRNA达到去除人源RNA的目的。近几年，另一些未商品化的方法也在研究过程中逐渐被开发出来。2016年，MR Hasan等人利用低浓度的非离子型表面活性剂皂素选择性地裂解人源细胞并利用Turbo DNA酶(Turbo DNase)除去人源DNA。
现有技术的缺点包括：
(1)NEB下一代微生物DNA浓缩试剂盒(NEBNext Microbiome DNA Enrichment kit)的分离原理，是基于人源和病原甲基化位点的差异，所以此方法无法实现未知病原的富集。
(2)利用先裂解人源细胞来去除人源核酸的方案例如MolYsis、QIAamp DNA微生物组试剂盒(QIAamp DNA Microbiome Kit)和微生物组富集试剂盒(MICROBEnrich Kit)在用DNase或者RNase去除人源核酸的过程会丢失存在于样本中的游离病原DNA或者RNA。此外，样本在离心处理等过程中会出现丢失的问题。同时，MolYsis对于不同样本类型表现差异比较大。对于脑脊液样本的处理，在大幅降低人源核酸的同时也会大幅降低病原核酸。对于鼻咽呼吸道物质的样本处理，去除人源DNA效果不明显，去除人源RNA的同时也损失了大量的病原RNA。
(3)利用同时裂解人源和病原细胞并利用带有寡核苷酸链的磁珠吸附人源RNA中的18S、28S rRNAs和mRNA达到去除人源RNA的方案中，由于分离主要是基于人源核糖体RNA和mRNA的特异性，所以此方案只应用于富集病原RNA。
(4)利用低浓度的非离子型表面活性剂皂素方案中需要利用改造后的DNase，可能会使工艺更复杂或者成本升高。另外，此方案只应用到了模拟样本上，还未实现在真实样本的处理。
发明内容
本发明提供的富集病原核酸的方法，使用宿主源探针来特异性捕获样本中宿主源核酸，通过分离宿主源核酸的方法来实现病原核酸的富集。该方法可以广泛应用于不同种类的病原核酸的富集，也可实现宿主源及病原核酸同时检测的目的，从而丰富了病原与宿主细胞作用机理的认识，为疾病防控及治疗提供新的途径。
因此，本发明提供一种分离和/或富集宿主源核酸和病原核酸的方法和试剂及其制备方法。
根据第一方面，一种实施例中提供一种分离和/或富集宿主源核酸和病原核酸的方法，该方法包括选自如下(a)或(b)的步骤：
(a)将固定在固相载体上的宿主源探针与含有宿主源核酸和病原核酸的液体样品混合孵育，使上述宿主源探针与上述宿主源核酸杂交；进行固液分离得到结合在上述固相载体上的宿主源核酸和保留在上述液体样品中的病原核酸；
(b)将宿主源探针、固定在固相载体上的病原探针与含有宿主源核酸和病原核酸的液体样品混合孵育，使上述宿主源探针与上述宿主源核酸杂交、上述病原探针与上述病原核酸杂交；进行固液分离得到结合在上述固相载体上的病原核酸和保留在上述液体样品中的宿主源核酸。
在优选实施例中，上述宿主源探针是人源探针，相应地，上述宿主源核酸是人源核酸。
在优选实施例中，上述(a)中的人源探针是以人基因组DNA为原料制成的DNA纳米球、片段化基因组序列扩增产物或片段化基因组序列；其中上述片段化基因组序列扩增产物和片段化基因组序列上带有生物素标记，上述固相载体上带有链霉亲和素标记。
在优选实施例中，上述(b)中的人源探针是以人基因组DNA为原料制成的DNA纳米球。
在优选实施例中，上述(a)中的固相载体选自芯片或磁珠。
在优选实施例中，上述(b)中的固相载体是数位编码磁珠。
在优选实施例中，上述(b)中病原探针是病原基因组序列的保守区。
在优选实施例中，上述液体样品是血液、血浆、唾液、脑脊液、或肺泡灌洗液样本中的核酸提取物。
在优选实施例中，上述(a)中还包括：将结合在上述固相载体上的宿主源核酸变性并洗脱下来得到游离的宿主源核酸。
在优选实施例中，上述(b)中还包括：将结合在上述固相载体上的病原核酸变性并洗脱下来得到游离的病原核酸。
根据第二方面，一种实施例中提供一种分离和/或富集宿主源核酸和病原核酸的试剂，该试剂包括选自如下(a)或(b)的组分：
(a)固定在固相载体上的宿主源探针，其用于与含有宿主源核酸和病原核酸的液体样品混合孵育，使上述宿主源探针与上述宿主源核酸杂交，并通过固液分离得到结合在上述固相载体上的宿主源核酸和保留在上述液体样品中的病原核酸；
(b)宿主源探针和固定在固相载体上的病原探针，其用于与含有宿主源核酸和病原核酸的液体样品混合孵育，使上述宿主源探针与上述宿主源核酸杂交、上述病原探针与上述病原核酸杂交，并通过固液分离得到结合在上述固相载体上的病原核酸和保留在上述液体样品中的宿主源核酸。
在优选实施例中，上述宿主源探针是人源探针，相应地，上述宿主源核酸是人源核酸。
在优选实施例中，上述(a)中的人源探针是以人基因组DNA为原料制成的DNA纳米球、片段化基因组序列扩增产物或片段化基因组序列；其中上述片段化基因组序列扩增产物和片段化基因组序列上带有生物素标记，上述固相载体上带有链霉亲和素标记。
在优选实施例中，上述(b)中的人源探针是以人基因组DNA为原料制成的DNA纳米球。
在优选实施例中，上述(a)中的固相载体选自芯片或磁珠。
在优选实施例中，上述(b)中的固相载体是数位编码磁珠。
在优选实施例中，上述(b)中病原探针是病原基因组序列的保守区。
在优选实施例中，上述液体样品是血液、血浆、唾液、脑脊液、或肺泡灌洗液样本中的核酸提取物。
根据第三方面，一种实施例中提供一种制备第二方面的试剂的方法，该方法包括选自如下(a)或(b)的步骤：
(a)制备宿主源探针并将上述宿主源探针固定在固相载体表面，得到固定在固相载体上的宿主源探针；
(b)制备宿主源探针，以及制备病原探针并将上述病原探针固定在固相载体上，得到固定在固相载体上的病原探针。
在优选实施例中，上述(a)中的宿主源探针是以人基因组DNA为原料制成的DNA纳米球，上述固定在固相载体上的宿主源探针的制备方法包括：
利用转座酶打断上述人基因组DNA并连接接头得到两端带有接头的片段化人基因组DNA；
对上述片段化人基因组DNA进行缺口平移以及PCR扩增获得扩增产物；
变性上述扩增产物并加入与上述接头两端匹配的锚定寡核苷酸链和DNA连接酶将上述扩增产物制成单链环；
在上述单链环的接头区域杂交延伸引物并加入具有链置换功能的聚合酶进行滚环扩增得到单链人基因组片段线性扩增产物，通过分子内氢键形成DNA纳米球；
将上述DNA纳米球加载到芯片表面上制成载有上述DNA纳米球的芯片。
在优选实施例中，上述(a)中的宿主源探针是以人基因组DNA为原料制成的片段化基因组序列扩增产物，该片段化基因组序列扩增产物上带有生物素标记，上述固相载体上带有链霉亲和素标记，上述固定在固相载体上的宿主源探针的制备方法包括：
利用转座酶打断上述人基因组DNA并连接接头得到两端带有接头的片段化人基因组DNA；
对上述片段化人基因组DNA进行缺口平移，并且利用带有生物素标记的引物进行PCR扩增获得带有生物素标记的扩增产物；
将带有链霉亲和素标记的磁珠加入上述带有生物素标记的扩增产物中混合孵育，得到固定在上述磁珠上的带有片段化人基因组序列的扩增产物的人源探针。
在优选实施例中，上述(a)中的宿主源探针是以人基因组DNA为原料制成的片段化基因组序列，该片段化基因组序列上带有生物素标记，上述固相载体上带有链霉亲和素标记，上述固定在固相载体上的宿主源探针的制备方法包括：
利用转座酶打断上述人基因组DNA并加入生物素标记的接头的方式得到两端带有接头的片段化人基因组DNA；
对上述片段化人基因组DNA进行缺口平移及延伸得到带有生物素标记的片段化人基因组DNA产物；
将带有链霉亲和素标记的磁珠加入上述带有生物素标记的片段化人基因组DNA产物中混合孵育，得到固定在上述磁珠上的带有片段化人基因组序列产物的人源探针。
在优选实施例中，上述(b)中的宿主源探针是以人基因组DNA为原料制成的DNA纳米球；上述宿主源探针的制备方法包括：
利用转座酶打断上述人基因组DNA并连接接头得到两端带有接头的片段化人基因组DNA；
对上述片段化人基因组DNA进行缺口平移以及PCR扩增获得扩增产物；
变性上述扩增产物并加入与上述接头两端匹配的锚定寡核苷酸链和DNA连接酶将上述扩增产物制成单链环；
在上述单链环的接头区域杂交延伸引物并加入具有链置换功能的聚合酶进行滚环扩增得到单链人基因组片段线性扩增产物，通过分子内氢键形成DNA纳米球。
在优选实施例中，上述(b)中的病原探针是病原基因组序列的保守区；上述固定在固相载体上的病原探针的制备方法包括：
利用带有生物素标记的病原核酸保守区序列与带有链霉亲和素标记的数位磁珠混合孵育，得到用于捕获样本中病原核酸的病原探针。
本发明的分离和/或富集宿主源核酸和病原核酸的方法具有如下有益效果：
(1)该方案不需要预先知晓病原核酸的甲基化位点，可以同时进行已知及未知病原的检测。
(2)该方案不需要应用DNA酶(DNase)和RNA酶(RNase)对宿主源核酸进行消化的步骤，因此不会大量丢失掉存在于样本中的游离病原核酸，可针对样本中所有病原核酸进行富集。
(3)该方案应用于分离宿主源核酸的捕获探针优选宿主源基因组DNA，可同时杂交样本中的宿主源DNA及RNA片段，实现病原DNA和RNA的同时富集。
(4)该方案涉及的富集所用的芯片及磁珠制备方法简单快速，过程中所使用的试剂有成熟的商业化试剂，不需要针对野生酶进行改造等复杂过程，可操作性强，成本较低。
(5)该方案的病原核酸富集方法可使样本中的病原核酸的相对含量显著增加，应用到下游的宏基因组检测方案时可以降低读长(reads)数，降低检测时间和成本。
附图说明
图1为本发明实施例中转座复合体的结构及标记示意图；
图2为本发明实施例中去除Meta样本中人源核酸的人源捕获探针芯片的制备方法流程图；
图3为本发明实施例中去除Meta样本中人源核酸的人源捕获探针微球的制备方法流程图；
图4为本发明实施例中去除Meta样本中人源核酸的人源捕获探针微球的制备方法流程图；
图5为本发明实施例中去除Meta样本中人源核酸的人源捕获探针DNA纳米球的制备方 法流程图(1)及可以结合不同种类病原核酸的病原捕获数位磁珠示意图(2)。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。在以下的实施方式中，很多细节描述是为了使得本发明能被更好的理解。然而，本领域技术人员可以毫不费力的认识到，其中部分特征在不同情况下是可以省略的，或者可以由其他材料、方法所替代。
另外，说明书中所描述的特点、操作或者特征可以以任意适当的方式结合形成各种实施方式。同时，方法描述中的各步骤或者动作也可以按照本领域技术人员所能显而易见的方式进行顺序调换或调整。因此，说明书和附图中的各种顺序只是为了清楚描述某一个实施例，并不意味着是必须的顺序，除非另有说明其中某个顺序是必须遵循的。
针对目前市场上出现的病原核酸富集方案的种种不足，本发明设计了多种不同方案，利用修饰宿主源基因组序列或者病原核酸保守区序列的芯片或磁珠来实现宿主源和病原核酸的分离以达到富集病原核酸的目的。利用本发明的方案进行病原核酸的富集均可实现如下技术效果：
(1)此方案不需要预先知晓病原核酸的甲基化位点，可以实现未知病原的检测。
(2)此方案不需要应用DNA酶(DNase)和RNA酶(RNase)对宿主源核酸进行消化，因此不会丢失掉存在于样本中的游离病原核酸。
(3)此方案应用于富集宿主源核酸的捕获探针可以是宿主源基因组DNA，可同时杂交样本中的宿主源DNA及RNA片段，实现病原DNA和RNA的同时富集。
(4)该方案涉及的富集所用的芯片及磁珠制备方法简单快速，过程中所使用的试剂有成熟的商业化试剂，不需要针对野生酶进行改造等复杂过程，可操作性强，成本较低。
本发明实施例提供一种分离和/或富集宿主源核酸和病原核酸的方法。本发明中，所谓“分离”是指将宿主源核酸和病原核酸在一定程度上分开，可以是全部(例如100％)分开，也可以是部分(例如10％至90％等不等比例)分开。所谓“富集”是指提高宿主源核酸和病原核酸中的至少一种的绝对量或相对量，例如绝对浓度或相对浓度等。
本发明中，所谓“宿主源核酸”是指任何被病原寄生或感染的宿主细胞、宿主组织或宿主个体等各种来源的核酸，包括但不限于动物源核酸和植物源核酸等，尤其是动物源核酸，特别是哺乳动物来源的核酸，最典型的是人源核酸。这种核酸可以是DNA或RNA等。
本发明中，所谓“病原核酸”是指寄生或感染任何宿主细胞、宿主组织或宿主个体等的病原微生物，包括但不限于细菌、真菌等。
本发明一种实施例中提供一种分离和/或富集宿主源核酸和病原核酸的方法，该方法包括如下步骤：将固定在固相载体上的宿主源探针与含有宿主源核酸和病原核酸的液体样品混合孵育，使宿主源探针与宿主源核酸杂交；进行固液分离得到结合在固相载体上的宿主源核酸和保留在液体样品中的病原核酸。
本发明实施例中，宿主源探针是针对宿主源核酸与其杂交的探针，典型的是，与人源核酸杂交的人源探针。在优选实施例中，本发明实施例中的人源探针是以人基因组DNA为原料制成的DNA纳米球、片段化基因组序列扩增产物或片段化基因组序列。片段化基因组序 列扩增产物和片段化基因组序列上带有生物素标记，固相载体上带有链霉亲和素标记。
本发明实施例中，宿主源探针具有一定程度的通用特性，即针对同一种宿主来源的核酸，不同宿主个体(例如，不同人)的宿主源核酸(例如，人源核酸)可以使用预先制备好的同一种宿主源探针(例如，人源探针)杂交。预先制备好的宿主源探针可以在合适的条件下保存一定时间，例如，可以在4℃下保存1个月，并在该期限内使用。
本发明实施例中，宿主源探针，例如人源探针可以使用来源于人的核酸，如人基因组DNA，通过适当的方法制备得到。例如，将人基因组DNA通过物理打断或酶打断得到片段化基因组DNA，再对片段化进行特定标记(例如生物素标记)以将其固定到固相载体表面，或者对片段化基因组DNA进行环化和滚环扩增得到DNA纳米球(DNB)加载到固相载体(例如芯片)表面实现探针固定。
如图1所示，在采用转座酶打断基因组DNA的情况下，转座酶与转座子(A转座子和B转座子)形成转座复合体，图1(1)中示出了含有A转座子和B转座子两种转座子的双转座子转座复合体。在其他实施方案中，转座复合体可以是仅含有一个转座子(A转座子或B转座子)的单转座子转座复合体。
在需要将片段化基因组DNA标记上生物素标记等标记的情况下，可以有多种方式实现。其中一种方式是使用图1(2)中生物素标记的转座复合体打断基因组DNA，图中生物素标记的A转座子和生物素标记的B转座子与转座酶一起形成转座复合体，在打断基因组DNA的同时在片段化的基因组DNA两端添加带有生物素标记的转座子序列，通过与固相载体上的链霉亲和素结合实现探针固定。
本发明实施例中，以人源核酸作为宿主源核酸、人源探针作为宿主源探针，提供如下三种分离和/或富集宿主源核酸和病原核酸的方法。
方法一：基于人源探针(DNA纳米球)来富集病原核酸的方法，其原理及特点如下：
利用固定在硅基芯片上的DNA纳米球为人源探针来杂交吸附待测样本中的人源核酸并通过固液分离来实现样本中病原核酸的富集。其中，固定在硅基芯片上的DNA纳米球人源探针的制备方法如图2所示：
首先，以人基因组DNA为原料，利用转座酶打断并加入接头的方式得到两端带有特定接头的片段化的人基因组DNA的产物。通过切口平移以及PCR扩增可获得大量扩增产物。之后，通过变性双链产物并加入与接头两端匹配的锚定寡核苷酸链和DNA连接酶可将扩增后的人基因组片段制备成单链环。在单链环接头区域杂交延伸引物并加入phi29聚合酶通过滚环扩增的方式即可得到单链人基因组片段线性扩增的产物。这些得到的长单链通过分子内氢键即可形成DNA纳米球。在DNA纳米球中加入DNA纳米球加载缓冲液，混匀后放入加载系统中，室温孵育后即可将制备的DNA纳米球加载到末端氨基修饰的硅基底的芯片上。
将待处理样本加载到探针芯片上，使芯片上的人源探针与样本中的人源核酸杂交，通过分离固相芯片和液相待处理样本即可实现样本中病原核酸的富集。
上述方法一的优势包括：
(1)以人基因组DNA为原料，片段化后扩增制备探针用来捕获样本中的人源核酸，不需要利用特异性的甲基化位点或者保守区来实现杂交分离。
(2)此方案应用于分离人源核酸的捕获探针来源于人源基因组DNA，可同时杂交样本中的人源DNA及RNA片段，实现病原DNA和RNA的同时富集。
(3)探针存在的形式为DNA纳米球，存在大约数百拷贝(例如，300拷贝以上，优选500拷贝)的片段化人基因组DNA产物的拷贝数，增加杂交捕获样本中人源核酸的效率。
方法二：基于人源探针(片段化基因组序列扩增产物)来富集病原核酸的方法，其原理及特点如下：
利用固定在磁珠上的片段化人基因组序列的扩增产物作为人源探针来杂交吸附待测样本中的人源核酸并通过固液分离来实现样本中病原核酸的富集。其中，固定在磁珠上的片段化人基因组序列的扩增产物的人源探针的制备方法如图3所示：
首先，以人基因组DNA为原料，利用转座酶打断并加入接头的方式得到两端带有特定接头的片段化的人基因组DNA的产物。通过切口平移，并且利用带有生物素标记的引物进行PCR扩增即可获得大量带有生物素标记的片段化人基因组序列的扩增产物。将带有链霉亲和素标记的磁珠加入到带有生物素标记的片段化人基因组序列的扩增产物溶液中搅拌并室温孵育，即可得到固定在磁珠上的带有片段化人基因组序列的扩增产物的人源探针。
将固定了人源探针的磁珠加入到待处理的样本中，使磁珠上的人源探针与样本中的人源核酸杂交，通过分离固相磁珠和液相待处理样本即可实现样本中病原核酸的富集。
上述方法二的优势包括：
(1)以人基因组DNA为原料，片段化后扩增制备探针用来捕获样本中的人源核酸，不需要利用特异性的甲基化位点或者保守区来实现杂交分离。
(2)此方案应用于分离人源核酸的捕获探针来源于人源基因组DNA，可同时杂交样本中的人源DNA及RNA片段，实现病原DNA和RNA的同时富集。
(3)利用PCR方法一步实现官能基团(生物素)的标记和人源探针的扩增，可实现人源探针的固定以及进一步增加样本中人源核酸的捕获效率。
方法三：基于人源探针(片段化基因组序列)来富集病原核酸的方法，其原理及特点如下：
利用固定在磁珠上的片段化人基因组序列的产物作为人源探针来杂交吸附待测样本中的人源核酸并通过固液分离来实现样本中病原核酸的富集。其中，固定在磁珠上的片段化人基因组序列的产物的人源探针的制备方法如图4所示：
首先，以人基因组DNA为原料，利用转座酶打断并加入生物素标记的接头的方式得到两端带有特定接头的片段化的人基因组DNA的产物。通过切口平移及延伸即可得到带有生物素标记的片段化的人基因组DNA的产物。将带有链霉亲和素标记的磁珠加入到带有生物素标记的片段化人基因组序列的产物溶液中搅拌并室温孵育，即可得到固定在磁珠上的带有片段化人基因组序列产物的人源探针。
将固定了人源探针的磁珠加入到待处理的样本中，使磁珠上的人源探针与样本中的人源核酸杂交，通过分离固相磁珠和液相待处理样本即可实现样本中病原核酸的富集。
上述方法三的优势包括：
(1)以人基因组DNA为原料，片段化产物制备探针用来捕获样本中的人源核酸，不需 要利用特异性的甲基化位点或者保守区来实现杂交分离。
(2)此方案应用于分离人源核酸的捕获探针来源于人源基因组DNA，可同时杂交样本中的人源DNA及RNA片段，实现病原DNA和RNA的同时富集。
(3)此方案利用转座酶一步打断人基因组DNA并加接头的方式得到人源探针，制备方案简单，步骤较少，所以制备人源探针的效率较高，成本较低。
本发明一种实施例中提供一种分离和/或富集宿主源核酸和病原核酸的方法，该方法包括如下步骤：
将宿主源探针、固定在固相载体上的病原探针与含有宿主源核酸和病原核酸的液体样品混合孵育，使宿主源探针与宿主源核酸杂交、病原探针与病原核酸杂交；进行固液分离得到结合在固相载体上的病原核酸和保留在液体样品中的宿主源核酸。
本发明实施例中，宿主源探针，例如人源探针可以使用来源于人的核酸，如人基因组DNA，通过适当的方法制备得到。本实施例中，宿主源探针作为液相探针与样本中的宿主源核酸结合；而病原探针固定在固相载体上，与样本中的病原核酸结合，再通过固液分离实现宿主源核酸和病原核酸的分离。
本发明实施例中，以人源核酸作为宿主源核酸、人源探针作为宿主源探针，提供如下第四种分离和/或富集宿主源核酸和病原核酸的方法。
方法四：基于人源探针(DNA纳米球)以及病原探针(数位编码磁珠)来富集病原核酸的方法，其原理及特点如下：
利用存在于液相的基于人基因组制备的DNA纳米球为人源探针杂交吸附待测样本中的人源核酸，同时利用固定在数位磁珠上的病原基因组序列的保守区作为病原探针来杂交捕获待测样本中的病原核酸并通过固液分离来实现样本中病原核酸的富集。其中，基于人基因组制备的DNA纳米球的人源探针和固定在数位磁珠上的带有病原基因组序列保守区的病原探针的制备方法如图5所示：
首先，以人基因组DNA为原料，利用转座酶打断并加入接头的方式得到两端带有特定接头的片段化的人基因组DNA的产物。通过切口平移以及PCR扩增可获得大量扩增产物。之后，通过变性双链产物并加入与接头两端匹配的锚定寡核苷酸链和DNA连接酶可将扩增后的人基因组片段制备成单链环。在单链环接头区域杂交延伸引物并加入phi29聚合酶通过滚环扩增的方式即可得到单链人基因组片段线性扩增的产物。这些得到的长单链通过分子内氢键即可形成DNA纳米球，即用于杂交去除样本中人源核酸的人源探针。
带有病原基因组序列保守区的病原探针的制备方法为，利用带有生物素标记的病原核酸保守区序列与带有链霉亲和素标记的数位磁珠混合并室温孵育，即可得到用于捕获待测样本中病原核酸的病原探针。
将制备好的DNA纳米球人源探针和固定了病原探针的数位磁珠加入到待处理的样本中，使待测样本中的人源核酸杂交捕获到液相的DNA纳米球人源探针上，待测样本中的病原核酸杂交捕获到固定了病原探针的数位磁珠上，通过分离固相数位磁珠和液相待处理样本即可实现样本中病原核酸的富集。
上述方法三的优势包括：
(1)以人基因组DNA为原料，片段化后扩增制备人源探针用来捕获样本中的人源核酸，不需要利用特异性的甲基化位点或者保守区来实现杂交分离。
(2)此方案应用于分离人源核酸的捕获探针来源于人源基因组DNA，可同时杂交样本中的人源DNA及RNA片段，实现病原DNA和RNA的同时富集。
(3)人源探针存在的形式为DNA纳米球，存在大约数百拷贝(例如，300拷贝以上，优选500拷贝)的片段化人基因组DNA产物的拷贝数，增加杂交捕获样本中人源核酸的效率。
(4)由于数位磁珠上存在二维码标记，可通过在不同标记的磁珠上修饰不同的病原核酸保守区实现不同种类的病原核酸富集在不同标记种类的数位磁珠上，从而实现多种类分别富集。
(5)此方案利用了双向选择法，即人源探针和病原探针同时杂交捕获的方案进行富集，可以进一步增加病原富集的特异性和效率。
以下通过实施例详细说明本发明的技术方案，应当理解，实施例仅是示例性的，不能理解为对本发明保护范围的限制。
需要说明的是，本发明实施例的方法均是示例性的，上述方法中各种元素和材料有多种其他替代性选择。例如，(1)本发明用来进行DNA片段化的酶不限于本实施例所使用的Tn5转座酶，可以为Tn转座酶家族的其他酶，如Tn7；或者其他转座酶家族，如Mu家族；甚至不限于转座酶或者酶制剂，只要其能够使DNA被片段化同时将一段序列连接至DNA上。(2)本发明用来进行吸附的固相载体不限于实施例所使用的硅基底的芯片和四氧化三铁磁珠，可以为聚合物或者玻璃微球，可以为实心、中空或者多孔形态，甚至可以为任何其他材料和形态的固相表面及内部，只要其能够修饰上探针寡核苷酸链。其表面修饰不限于实施例中的链霉亲和素，其修饰可以是羟基、羧基、氨基等可以共价键连接DNA寡核苷酸(oligo)的分子。(3)本发明用来进行吸附的固相芯片的点阵形状及个数不限于圆形和200M，可以为任何形状及个数。加入的磁珠尺寸及个数不限于20μm和200M，可以为任意尺寸及个数。(4)本发明使用的转座子序列的长度和序列信息不限。(5)本发明使用的打断基因组DNA并在两端加入的转座子接头可以是单一序列的接头或者两种不同序列的接头。(6)本发明中加入的转座复合体打断得到的人源探针的片段大小没有限制，只要其探针可以有效去除样本中的人源基因组片段即可。(7)本发明使用的连接酶不限于某种特定的连接酶，可以为T4 DNA连接酶、Taq DNA连接酶、T7 DNA连接酶、T3 DNA连接酶、E.Coli DNA连接酶和HiFi Taq DNA连接酶，只要其可以连接切口(nick)形成单链环即可。(8)本发明使用的外切酶不限于某种特定的外切酶，可以为外切酶I、外切酶III、λ外切酶和T7外切酶，只要其可以消化环化产物中剩余的线性DNA链即可。(9)本发明使用的制备DNA纳米球的聚合酶不限于phi29聚合酶，可以为其他具有链置换活性并能进行滚环扩增的聚合酶，可以为Bst、Bsu的大片段、E.Coli DNA聚合酶I等，只要其可以制备出DNA纳米球即可。(10)本发明中使用的数位磁珠的材料、尺寸、形状以及表面图案不限于一种，材料可以为高分子材料，如无机四氧化三铁等；尺寸可以为1μm到1mm；形状和表面图案以及表面修饰方法不限，只要其可以修饰上病原核酸的保守序列从而实现富集病原核酸的目的及可实现多重检测即可。(11)本发明中四 种病原核酸的富集方法不仅限于血液样本中的病原核酸的富集，同样可应用于唾液、脑脊液、肺泡灌洗液等样本中病原核酸的富集。(12)本发明中四种病原核酸的富集方法不仅限于应用于测序样本处理中病原核酸的富集，同样可应用于基于qPCR、ddPCR、适体(aptamer)杂交、结合蛋白等方法检测病原核酸的样本前处理，检测信号方法不限，可以是荧光法、电化学法、化学发光法、比色法等。
实施例1：一种基于人源探针(DNA纳米球)来富集病原核酸的方法
(1)带有接头序列的人源基因组片段的制备方法：
以50ng人源基因组DNA为原料均一化到10μL，加入10μL 0.8μM转座复合体在1×TAG缓冲液中打断基因组序列同时加入接头序列，55℃反应7分钟，反应完后加入5μL NT溶液室温反应5min终止反应。
(2)带有接头序列的人源基因组片段的扩增方法：
将步骤(1)制备的带有接头序列的人源基因组片段中加入5μL 20μM引物使终浓度为1μM，DNA聚合酶，dNTPs先进行72℃延伸5min，随后进行聚合酶链式反应8个循环，0.8X磁珠纯化后即可得扩增的人源基因组片段。取350ng扩增产物均一化至60μL。
(3)基于人源基因组片段的单链环的制备方法：
将步骤(2)制备的扩增片段中加入10μL 10μM的锚定寡核苷酸链后95℃高温变性3min，快速冷却复性后加入1.2μL 100mM ATP和0.4μL 600U/μL的T4连接酶，在1X TA缓冲液中37℃反应0.5小时。在上述产物中加入3.9μL 20U/μL外切酶I和1.3μL 100U/μL外切酶III在37℃消化剩余未环化的单链及双链30分钟。反应完成后加入6μL 0.5M EDTA终止消化反应。利用2.5X磁珠纯化后即可得基于人源基因组片段的单链环。取6ng单链环产物均一化至20μL。
(4)人源探针(DNA纳米球)的制备方法：
将步骤(3)制备的单链环中加入20μL 1μM引物，95℃ 1min，65℃ 1min，40℃ 1min退火，利用4μL 10U/μL的phi 29聚合酶和40μL 2X SSB 30℃等温扩增20分钟即可得人源探针(DNA纳米球)。
(5)带有人源探针(DNA纳米球)芯片的制备方法：
将步骤(4)制备的DNA纳米球中加入DNA纳米球加载缓冲液，混匀后放入加载系统中，将制备的DNA纳米球加载到末端氨基修饰的硅基底的芯片上，室温孵育1小时。
(6)全血中提取核酸的方法：
以5ml全血为起始样品，加入0.04M EDTA抗凝后离心吸取上层血浆和白细胞层。将收集到的产物利用2000rpm离心5min后收集上层血浆于1号离心管中，白细胞层于2号离心管中。在血浆中加入10％福尔马林溶液，经裂解液以及蛋白酶K孵育30分钟后用吸附柱吸附血浆中的游离核酸。70％乙醇溶液洗涤两次后利用洗脱液将吸附的核酸洗脱，可得游离核酸。
(7)病原核酸的富集方法：
将步骤(6)提取的核酸加到步骤(5)制备的芯片上25℃杂交30分钟，收集清液并用TE缓冲液冲洗芯片表面去除非特异性吸附的核酸，将所有溶液和洗涤液合并即可得到富集的 病原核酸。
(8)人源核酸的收集方法：
将与芯片杂交的核酸变性处理并用TE缓冲液收集即可得到人源核酸。
实施例2：一种基于人源探针(片段化基因组序列扩增产物)来富集病原核酸的方法
(1)带有接头序列的人源基因组片段的制备方法：
以50ng人源基因组DNA为原料，利用0.5～5pmol转座复合体打断基因组序列同时加入接头序列，55℃反应5～20分钟，反应完后利用NT缓冲液室温反应3～10min终止反应。产物进行末端延伸72℃反应5～15分钟，即可得到带有接头序列的人源基因组片段。
(2)带有接头序列的人源基因组片段的扩增方法：
将步骤(1)制备的带有接头序列的人源基因组片段中加入1～2μM生物素标记的引物，DNA聚合酶，dNTPs先进行72℃延伸5min，随后进行聚合酶链式反应8～15个循环，0.8～2.5X磁珠纯化后即可得带有生物素标记的接头序列的人源基因组片段的扩增产物。
(3)带有人源探针(片段化基因组序列扩增产物)的磁珠的制备方法：
将步骤(2)制备的带有生物素标记的接头序列的人源基因组片段的扩增产物加入到链霉亲和素标记的磁珠中混匀，在垂直混匀仪上室温孵育1小时即可得带有人源探针(片段化基因组序列扩增产物)的磁珠。
(4)全血中提取游离核酸的方法：
以5ml全血为起始样品，加入0.04M EDTA抗凝后离心吸取上层血浆和白细胞层。将收集到的产物利用2000rpm离心5min后收集上层血浆于1号离心管中，白细胞层于2号离心管中。在血浆中加入10％福尔马林溶液，经裂解液以及蛋白酶K孵育30分钟后用吸附柱吸附血浆中的游离核酸。70％乙醇溶液洗涤两次后利用洗脱液将吸附的核酸洗脱，可得游离核酸。
(5)病原核酸的富集方法：
将步骤(3)制备的磁珠加到步骤(4)提取的游离核酸中25℃杂交30分钟，收集清液并用TE缓冲液清洗磁珠表面非特异性吸附的核酸，将所有清液和洗涤液合并即可得到富集的病原核酸。
(6)人源核酸的收集方法：
将与磁珠杂交的核酸变性处理并用TE缓冲液收集即可得到人源核酸。
实施例3：一种基于人源探针(片段化基因组序列)来富集病原核酸的方法
(1)带有标记生物素的接头序列的人源基因组片段的制备方法：
以2μg人源基因组DNA为原料均一化到400μL，加入400μL 0.8μM转座复合体在1×TAG缓冲液中打断基因组序列同时加入标记了生物素的接头序列，55℃反应7分钟，反应完后加入200μL NT溶液室温反应5min终止反应。
(2)带有人源探针(片段化基因组序列)的磁珠的制备方法：
将步骤(1)制备的带有生物素标记的接头序列的人源基因组片段加入到链霉亲和素标记的磁珠中混匀，在垂直混匀仪上室温孵育1小时即可得带有人源探针(片段化基因组序列)的磁珠。
将步骤(2)制备的带有生物素标记的接头序列的人源基因组片段的扩增产物加入到链霉亲和素标记的磁珠中混匀，在垂直混匀仪上室温孵育1小时即可得带有人源探针(片段化基因组序列扩增产物)的磁珠。
(3)全血中提取游离核酸的方法：
以5ml全血为起始样品，加入0.04M EDTA抗凝后离心吸取上层血浆和白细胞层。将收集到的产物利用2000rpm离心5min后收集上层血浆于1号离心管中，白细胞层于2号离心管中。在血浆中加入10％福尔马林溶液，经裂解液以及蛋白酶K孵育30分钟后用吸附柱吸附血浆中的游离核酸。70％乙醇溶液洗涤两次后利用洗脱液将吸附的核酸洗脱，可得游离核酸。
(4)病原核酸的富集方法：
将步骤(2)制备的磁珠加到步骤(3)提取的游离核酸中25℃杂交30分钟，收集清液并用TE缓冲液清洗磁珠表面非特异性吸附的核酸，将所有清液和洗涤液合并即可得到富集的病原核酸。
(5)人源核酸的收集方法：
将与磁珠杂交的核酸变性处理并用TE缓冲液收集即可得到人源核酸。
实施例4：一种基于人源探针(DNA纳米球)以及病原探针(数位编码磁珠)来富集病原核酸的方法
(1)带有接头序列的人源基因组片段的制备方法：
以50ng人源基因组DNA为原料均一化到10μL，加入10μL 0.8μM转座复合体在1×TAG缓冲液中打断基因组序列同时加入接头序列，55℃反应7分钟，反应完后加入5μL NT溶液室温反应5min终止反应。
(2)带有接头序列的人源基因组片段的扩增方法：
将步骤(1)制备的带有接头序列的人源基因组片段中加入5μL 20μM引物使终浓度为1μM，DNA聚合酶，dNTPs先进行72℃延伸5min，随后进行聚合酶链式反应8个循环，0.8X磁珠纯化后即可得扩增的人源基因组片段。取350ng扩增产物均一化至60μL。
(3)基于人源基因组片段的单链环的制备方法：
将步骤(2)制备的扩增片段中加入10μL 10μM的锚定寡核苷酸链后95℃高温变性3min，快速冷却复性后加入1.2μL 100mM ATP和0.4μL 600U/μL的T4连接酶，在1X TA缓冲液中37℃反应0.5小时。在上述产物中加入3.9μL 20U/μL外切酶I和1.3μL 100U/μL外切酶III在37℃消化剩余未环化的单链及双链30分钟。反应完成后加入6μL 0.5M EDTA终止消化反应。利用2.5X磁珠纯化后即可得基于人源基因组片段的单链环。取6ng单链环产物均一化至20μL。
(4)人源探针(DNA纳米球)的制备方法：
将步骤(3)制备的单链环中加入20μL 1μM引物，95℃ 1min，65℃ 1min，40℃ 1min退火，利用4μL 10U/μL的phi 29聚合酶和40μL 2X SSB 30℃等温扩增20分钟即可得人源探针(DNA纳米球)。
(5)带有病原探针(病原核酸的保守区)的数位磁珠的制备方法：
将合成的带有生物素标记的病原核酸保守区的核酸链加入到链霉亲和素修饰的数位磁珠中混匀，在垂直混匀仪上室温孵育1小时。
(6)全血中提取核酸的方法：
以5ml全血为起始样品，加入0.04M EDTA抗凝后离心吸取上层血浆和白细胞层。将收集到的产物利用2000rpm离心5min后收集上层血浆于1号离心管中，白细胞层于2号离心管中。在血浆中加入10％福尔马林溶液，经裂解液以及蛋白酶K孵育30分钟后用吸附柱吸附血浆中的游离核酸。70％乙醇溶液洗涤两次后利用洗脱液将吸附的核酸洗脱，可得游离核酸。
(7)人源核酸的收集方法：
将步骤(5)制备的磁珠和步骤(4)制备的人源探针(DNA纳米球)加到步骤(6)提取的游离核酸中25℃杂交30分钟，收集清液并用TE缓冲液清洗磁珠表面非特异性吸附的核酸，将所有清液和洗涤液合并即可得到人源核酸。
(8)病原核酸的富集方法：
将与数位磁珠杂交的核酸变性处理并用TE缓冲液收集即可得到富集的病原核酸。
对比例：未进行病原核酸富集的样本以与实施例相同的建库条件构建文库并测序
以5ml全血为起始样品，加入0.04M EDTA抗凝后离心吸取上层血浆和白细胞层。将收集到的产物利用2000rpm离心5min后收集上层血浆于1号离心管中，白细胞层于2号离心管中。在血浆中加入10％福尔马林溶液，经裂解液以及蛋白酶K孵育30分钟后用吸附柱吸附血浆中的游离核酸。70％乙醇溶液洗涤两次后利用洗脱液将吸附的核酸洗脱，可得游离核酸。利用此游离核酸直接继续进行下游建库。
实验结果：
利用上述实施例1至4中的样本处理方案对样本中的病原核酸进行富集，并与对比例未进行病原核酸富集的样本以相同的建库条件建立文库并测序，所得测序结果比较如下表1所示：
表1
参数 对比例 实施例1 实施例2 实施例3 实施例4
测序总读长(Reads)数(M) 80 40 40 40 40
检出病原读长(Reads)数(条) 19 19 22 20 17
以上应用了具体个例对本发明进行阐述，只是用于帮助理解本发明，并不用以限制本发明。对于本发明所属技术领域的技术人员，依据本发明的思想，还可以做出若干简单推演、变形或替换。

Claims (24)

  1. 一种分离和/或富集宿主源核酸和病原核酸的方法，其特征在于，所述方法包括选自如下(a)或(b)的步骤：
    (a)将固定在固相载体上的宿主源探针与含有宿主源核酸和病原核酸的液体样品混合孵育，使所述宿主源探针与所述宿主源核酸杂交；进行固液分离得到结合在所述固相载体上的宿主源核酸和保留在所述液体样品中的病原核酸；
    (b)将宿主源探针、固定在固相载体上的病原探针与含有宿主源核酸和病原核酸的液体样品混合孵育，使所述宿主源探针与所述宿主源核酸杂交、所述病原探针与所述病原核酸杂交；进行固液分离得到结合在所述固相载体上的病原核酸和保留在所述液体样品中的宿主源核酸。
  2. 根据权利要求1所述的方法，其特征在于，所述宿主源探针是人源探针，相应地，所述宿主源核酸是人源核酸。
  3. 根据权利要求2所述的方法，其特征在于，所述(a)中的人源探针是以人基因组DNA为原料制成的DNA纳米球、片段化基因组序列扩增产物或片段化基因组序列；其中所述片段化基因组序列扩增产物和片段化基因组序列上带有生物素标记，所述固相载体上带有链霉亲和素标记。
  4. 根据权利要求2所述的方法，其特征在于，所述(b)中的人源探针是以人基因组DNA为原料制成的DNA纳米球。
  5. 根据权利要求1所述的方法，其特征在于，所述(a)中的固相载体选自芯片或磁珠。
  6. 根据权利要求1所述的方法，其特征在于，所述(b)中的固相载体是数位编码磁珠。
  7. 根据权利要求1所述的方法，其特征在于，所述(b)中病原探针是病原基因组序列的保守区。
  8. 根据权利要求1所述的方法，其特征在于，所述液体样品是血液、血浆、唾液、脑脊液、或肺泡灌洗液样本中的核酸提取物。
  9. 根据权利要求1所述的方法，其特征在于，所述(a)中还包括：将结合在所述固相载体上的宿主源核酸变性并洗脱下来得到游离的宿主源核酸。
  10. 根据权利要求1所述的方法，其特征在于，所述(b)中还包括：将结合在所述固相载体上的病原核酸变性并洗脱下来得到游离的病原核酸。
  11. 一种分离和/或富集宿主源核酸和病原核酸的试剂，其特征在于，所述试剂包括选自如下(a)或(b)的组分：
    (a)固定在固相载体上的宿主源探针，其用于与含有宿主源核酸和病原核酸的液体样品混合孵育，使所述宿主源探针与所述宿主源核酸杂交，并通过固液分离得到结合在所述固相载体上的宿主源核酸和保留在所述液体样品中的病原核酸；
    (b)宿主源探针和固定在固相载体上的病原探针，其用于与含有宿主源核酸和病原核酸的液体样品混合孵育，使所述宿主源探针与所述宿主源核酸杂交、所述病原探针与所述病原核酸杂交，并通过固液分离得到结合在所述固相载体上的病原核酸和保留在所述液体样品中的宿主源核酸。
  12. 根据权利要求11所述的试剂，其特征在于，所述宿主源探针是人源探针，相应地， 所述宿主源核酸是人源核酸。
  13. 根据权利要求12所述的试剂，其特征在于，所述(a)中的人源探针是以人基因组DNA为原料制成的DNA纳米球、片段化基因组序列扩增产物或片段化基因组序列；其中所述片段化基因组序列扩增产物和片段化基因组序列上带有生物素标记，所述固相载体上带有链霉亲和素标记。
  14. 根据权利要求12所述的试剂，其特征在于，所述(b)中的人源探针是以人基因组DNA为原料制成的DNA纳米球。
  15. 根据权利要求11所述的试剂，其特征在于，所述(a)中的固相载体选自芯片或磁珠。
  16. 根据权利要求11所述的试剂，其特征在于，所述(b)中的固相载体是数位编码磁珠。
  17. 根据权利要求11所述的试剂，其特征在于，所述(b)中病原探针是病原基因组序列的保守区。
  18. 根据权利要求11所述的试剂，其特征在于，所述液体样品是血液、血浆、唾液、脑脊液、或肺泡灌洗液样本中的核酸提取物。
  19. 一种制备权利要求11所述的试剂的方法，其特征在于，所述方法包括选自如下(a)或(b)的步骤：
    (a)制备宿主源探针并将所述宿主源探针固定在固相载体表面，得到固定在固相载体上的宿主源探针；
    (b)制备宿主源探针，以及制备病原探针并将所述病原探针固定在固相载体上，得到固定在固相载体上的病原探针。
  20. 根据权利要求19所述的方法，其特征在于，所述(a)中的宿主源探针是以人基因组DNA为原料制成的DNA纳米球，所述固定在固相载体上的宿主源探针的制备方法包括：
    利用转座酶打断所述人基因组DNA并连接接头得到两端带有接头的片段化人基因组DNA；
    对所述片段化人基因组DNA进行缺口平移以及PCR扩增获得扩增产物；
    变性所述扩增产物并加入与上述接头两端匹配的锚定寡核苷酸链和DNA连接酶将所述扩增产物制成单链环；
    在所述单链环的接头区域杂交延伸引物并加入具有链置换功能的聚合酶进行滚环扩增得到单链人基因组片段线性扩增产物，通过分子内氢键形成DNA纳米球；
    将所述DNA纳米球加载到芯片表面上制成载有所述DNA纳米球的芯片。
  21. 根据权利要求19所述的方法，其特征在于，所述(a)中的宿主源探针是以人基因组DNA为原料制成的片段化基因组序列扩增产物，该片段化基因组序列扩增产物上带有生物素标记，所述固相载体上带有链霉亲和素标记，所述固定在固相载体上的宿主源探针的制备方法包括：
    利用转座酶打断所述人基因组DNA并连接接头得到两端带有接头的片段化人基因组DNA；
    对所述片段化人基因组DNA进行缺口平移，并且利用带有生物素标记的引物进行PCR扩增获得带有生物素标记的扩增产物；
    将带有链霉亲和素标记的磁珠加入所述带有生物素标记的扩增产物中混合孵育，得到固定在所述磁珠上的带有片段化人基因组序列的扩增产物的人源探针。
  22. 根据权利要求19所述的方法，其特征在于，所述(a)中的宿主源探针是以人基因组DNA为原料制成的片段化基因组序列，该片段化基因组序列上带有生物素标记，所述固相载体上带有链霉亲和素标记，所述固定在固相载体上的宿主源探针的制备方法包括：
    利用转座酶打断所述人基因组DNA并加入生物素标记的接头的方式得到两端带有接头的片段化人基因组DNA；
    对所述片段化人基因组DNA进行缺口平移及延伸得到带有生物素标记的片段化人基因组DNA产物；
    将带有链霉亲和素标记的磁珠加入所述带有生物素标记的片段化人基因组DNA产物中混合孵育，得到固定在所述磁珠上的带有片段化人基因组序列产物的人源探针。
  23. 根据权利要求19所述的方法，其特征在于，所述(b)中的宿主源探针是以人基因组DNA为原料制成的DNA纳米球；所述宿主源探针的制备方法包括：
    利用转座酶打断所述人基因组DNA并连接接头得到两端带有接头的片段化人基因组DNA；
    对所述片段化人基因组DNA进行缺口平移以及PCR扩增获得扩增产物；
    变性所述扩增产物并加入与上述接头两端匹配的锚定寡核苷酸链和DNA连接酶将所述扩增产物制成单链环；
    在所述单链环的接头区域杂交延伸引物并加入具有链置换功能的聚合酶进行滚环扩增得到单链人基因组片段线性扩增产物，通过分子内氢键形成DNA纳米球。
  24. 根据权利要求19所述的方法，其特征在于，所述(b)中的病原探针是病原基因组序列的保守区；所述固定在固相载体上的病原探针的制备方法包括：
    利用带有生物素标记的病原核酸保守区序列与带有链霉亲和素标记的数位磁珠混合孵育，得到用于捕获样本中病原核酸的病原探针。
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