CN113039283B - Method and reagent for separating and/or enriching host source nucleic acid and pathogenic nucleic acid and preparation method thereof - Google Patents
Method and reagent for separating and/or enriching host source nucleic acid and pathogenic nucleic acid and preparation method thereof Download PDFInfo
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C—CHEMISTRY; METALLURGY
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- 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
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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Abstract
A method and reagent for isolating and/or enriching host-derived nucleic acids and pathogenic nucleic acids and a method for preparing the same. The method comprises a step selected from the following (a) or (b): (a) Mixing and incubating the host source probe immobilized on the solid phase carrier with a liquid sample containing host source nucleic acid and pathogenic nucleic acid, so that the host source probe hybridizes with the host source nucleic acid; performing solid-liquid separation to obtain host source nucleic acid bound to the solid carrier and pathogenic nucleic acid remained in the liquid sample; (b) Mixing and incubating a host source probe and a pathogen probe immobilized on a solid carrier with a liquid sample containing host source nucleic acid and pathogen nucleic acid, so that the host source probe hybridizes with the host source nucleic acid and the pathogen probe hybridizes with the pathogen nucleic acid; performing solid-liquid separation to obtain pathogenic nucleic acids bound to the solid support and host-derived nucleic acids remaining in the liquid sample. The method uses host source probes to specifically capture host source nucleic acids, and enrichment of pathogenic nucleic acids is achieved by isolating host source nucleic acids.
Description
Technical Field
The invention relates to the technical field of nucleic acid separation and enrichment, in particular to a method and a reagent for separating and/or enriching host source nucleic acid and pathogenic nucleic acid and a preparation method thereof.
Background
In recent years, DNA sequencing technology has made a major breakthrough in sequencing speed, throughput, and cost. Therefore, the gene sequencing technology has been widely applied to the field of medical health detection, including embryo implantation screening, prenatal examination, cancer screening, detection and prevention and control of infectious diseases, and the like. Among them, the presence and continued evolution of infectious agents further make the use of second generation sequencing to detect infectious diseases a very valuable and growing area of interest to more and more researchers.
At present, detection of species, abundance and functional characteristics of each component in a pathogenic microorganism population can be realized by utilizing gene sequencing and analysis technology, so that the correlation between people and pathogenic microorganisms is researched. However, these studies have been limited to complex components of the sample, including bacteria, fungi, viruses, protozoa, and excessive amounts of host human nucleic acids. Amplicon sequencing (e.g., 16S ribosomal RNA sequencing) in combination with PCR and sequencing techniques can allow for rapid, high-resolution detection of pathogenic nucleic acids in complex samples. Such amplicon sequencing can provide great advantages in terms of sequencing coverage as well as depth, but amplicon sequencing does not allow for research on whole epidemiology and identification of pathogenicity.
With respect to the limitations of amplicon sequencing, meta sequencing has been widely used in the detection of pathogenic viruses and pathogenic microorganisms to discover new pathogens and to genotype pathogens. Meta sequencing, in combination with corresponding rapid detection techniques, can also better reveal the transmission pathways of the causative agent, help in disease control, prevent infection outbreaks, etc. The use of blood as a sample for Meta sequencing to identify pathogenic nucleic acids in blood has been the mainstay of research in Meta sequencing. However, since there is an excessive amount of human nucleic acid in blood, of which only about 1% of pathogenic nucleic acids are present, it is difficult to detect pathogenic nucleic acids present in the excessive amount of human nucleic acid using conventional sequencing methods, and it is more difficult to give diagnostic guidelines such as drug resistance and infectivity for the corresponding conditions. Meanwhile, detection of pathogenic nucleic acids at low frequencies using conventional methods requires a high sequencing depth, which clearly increases the sequencing cost. Therefore, the choice of sample processing methods prior to blood Meta sequencing has been one of the key points of Meta sequencing technology.
Traditional methods for enriching pathogenic nucleic acids fall into two main categories. The first is to perform nucleic acid separation according to different methylation sites of human and pathogenic nucleic acids to enrich pathogenic nucleic acids, but separation principle based on methylation state limits the kinds of pathogenic nucleic acids that can be enriched. The second type of enrichment method is a ribonuclease-based human nucleic acid depletion method. The method can be suitable for enriching pathogenic nucleic acid such as virus, fungus, parasite and the like. However, enrichment of nucleic acids in bacteria by the human nucleic acid consumption method cannot be used to study the interaction mechanism of bacteria and their host cells due to the inconsistency of the composition and content of transcripts of bacteria and their host cells.
Methods for enriching pathogenic nucleic acids have been commercialized and mainly classified into the following categories. First was the NEB next generation microbial DNA concentration kit (NEBNext Microbiome DNA Enrichment kit) by Neon England Biotechnology (New England Biolabs) in the United states. The kit is designed according to the fact that the human nucleic acid contains more methylation sites, and the difference between the methylation sites of the human nucleic acid and the pathogenic nucleic acid is utilized. Fusion proteins prepared using the methylation site binding protein MBD2 and human immunoglobulin specifically remove human nucleic acids from a sample. Another type of kit is based on the principle that enrichment of pathogenic nucleic acids is achieved by means of the removal of human nucleic acids. Such commercial kits include MolYIs from Molzym, QIAamp DNA microbiome Kit (QIAamp DNA Microbiome Kit) from Qiagen, and microbiome enrichment Kit (MICROBEnrich Kit) from Simer Feichi technologies (Thermo Fisher Scientific). MolYIs and QIAamp DNA microbiome kit (QIAamp DNA Microbiome Kit) is mainly used for removing human DNA, the main principle of which is to selectively lyse human cells and remove human DNA by DNase. Microbiome enrichment Kit (MICROBEnrich Kit) is mainly used for removing human RNA, and its main principle is divided into two categories. The first type is to selectively lyse human cells and separate microorganisms such as bacteria and human RNA by centrifugation; the second is to lyse human and pathogenic cells at the same time, and then to adsorb 18S, 28S rRNA and mRNA in the human RNA by using magnetic beads with oligonucleotide chains to remove the human RNA. In recent years, other non-commercial methods have been developed during the course of research. In 2016, MR Hasan et al utilized low concentrations of non-ionic surfactant saponin to selectively lyse human cells and utilized Turbo DNase (Turbo DNase) to remove human DNA.
Drawbacks of the prior art include:
(1) The separation principle of NEB next generation microorganism DNA concentration kit (NEBNext Microbiome DNA Enrichment kit) is based on the difference between human source and pathogen methylation sites, so the method can not realize the enrichment of unknown pathogen.
(2) Protocols that utilize prior lysis of human cells to remove human nucleic acids such as MolYsis, QIAamp DNA microbiome Kit (QIAamp DNA Microbiome Kit) and microbiome enrichment Kit (microbench Kit) lose free pathogenic DNA or RNA present in the sample during removal of human nucleic acids with DNase or RNase. In addition, the sample may be lost during centrifugation or the like. Meanwhile, molYsis show a relatively large difference for different sample types. For the treatment of cerebrospinal fluid samples, the pathogenic nucleic acid is greatly reduced while the human nucleic acid is greatly reduced. For sample treatment of nasopharyngeal respiratory tract substances, the effect of removing the human DNA is not obvious, and a large amount of pathogenic RNA is lost while the human RNA is removed.
(3) In a scheme for removing human RNA by simultaneously lysing both human and pathogenic cells and adsorbing 18S, 28S rRNA and mRNA in human RNA using magnetic beads with oligonucleotide chains, this scheme is only applied to enrich for pathogenic RNA, since the isolation is mainly based on the specificity of human ribosomal RNA and mRNA.
(4) The need to use modified DNase in the low concentration of non-ionic surfactant saponin solution may complicate the process or increase the cost. In addition, this scheme is applied only to analog samples, and processing of real samples has not been achieved yet.
Disclosure of Invention
The method for enriching the pathogenic nucleic acid provided by the invention uses the host source probe to specifically capture the host source nucleic acid in the sample, and realizes the enrichment of the pathogenic nucleic acid by a method for separating the host source nucleic acid. The method can be widely applied to enrichment of different types of pathogenic nucleic acid, and can also realize the purpose of simultaneous detection of a host source and the pathogenic nucleic acid, thereby enriching the knowledge of the action mechanism of pathogens and host cells and providing a new way for disease prevention, control and treatment.
Accordingly, the present invention provides a method and reagent for isolating and/or enriching host-derived and pathogenic nucleic acids and a method for preparing the same.
According to a first aspect, there is provided in one embodiment a method of isolating and/or enriching a host source nucleic acid and a pathogenic nucleic acid, the method comprising a step selected from the group consisting of (a) or (b) below:
(a) Incubating a host-derived probe immobilized on a solid support with a liquid sample comprising host-derived nucleic acids and pathogenic nucleic acids, and hybridizing the host-derived probe to the host-derived nucleic acids; performing solid-liquid separation to obtain host-derived nucleic acids bound to the solid support and pathogenic nucleic acids retained in the liquid sample;
(b) Mixing and incubating a host source probe, a pathogenic probe immobilized on a solid support, and a liquid sample containing a host source nucleic acid and a pathogenic nucleic acid, so that the host source probe hybridizes with the host source nucleic acid, and the pathogenic probe hybridizes with the pathogenic nucleic acid; performing solid-liquid separation to obtain pathogenic nucleic acids bound to the solid support and host-derived nucleic acids retained in the liquid sample.
In a preferred embodiment, the host-derived probe is a human-derived probe, and the host-derived nucleic acid is a human-derived nucleic acid.
In a preferred embodiment, the human probe in (a) above is a DNA nanosphere, a fragmented genomic sequence amplification product or a fragmented genomic sequence prepared from human genomic DNA as a raw material; wherein the fragmented genomic sequence amplification product and the fragmented genomic sequence are provided with a biotin label, and the solid support is provided with a streptavidin label.
In a preferred embodiment, the human probe in (b) above is a DNA nanosphere prepared from human genomic DNA as a raw material.
In a preferred embodiment, the solid support in (a) above is selected from a chip or a magnetic bead.
In a preferred embodiment, the solid support in (b) above is a digitally encoded magnetic bead.
In a preferred embodiment, the pathogenic probe of (b) above is a conserved region of the pathogenic genomic sequence.
In a preferred embodiment, the liquid sample is a nucleic acid extract from a blood, plasma, saliva, cerebrospinal fluid, or alveolar lavage sample.
In a preferred embodiment, the step (a) further includes: the host-derived nucleic acid bound to the solid phase carrier is denatured and eluted to obtain the free host-derived nucleic acid.
In a preferred embodiment, the step (b) further comprises: the pathogenic nucleic acid bound to the solid phase carrier is denatured and eluted to obtain free pathogenic nucleic acid.
According to a second aspect, there is provided in one embodiment an agent for isolating and/or enriching a host source nucleic acid and a pathogenic nucleic acid, the agent comprising a component selected from the group consisting of (a) or (b) below:
(a) A host-derived probe immobilized on a solid support, for mixed incubation with a liquid sample containing host-derived nucleic acids and pathogenic nucleic acids, hybridizing the host-derived probe with the host-derived nucleic acids, and obtaining host-derived nucleic acids bound to the solid support and pathogenic nucleic acids retained in the liquid sample by solid-liquid separation;
(b) A host-derived probe and a pathogenic probe immobilized on a solid support, which are used for mixed incubation with a liquid sample containing a host-derived nucleic acid and a pathogenic nucleic acid, hybridizing the host-derived probe with the host-derived nucleic acid, hybridizing the pathogenic probe with the pathogenic nucleic acid, and obtaining a pathogenic nucleic acid bound to the solid support and a host-derived nucleic acid remaining in the liquid sample by solid-liquid separation.
In a preferred embodiment, the host-derived probe is a human-derived probe, and the host-derived nucleic acid is a human-derived nucleic acid.
In a preferred embodiment, the human probe in (a) above is a DNA nanosphere, a fragmented genomic sequence amplification product or a fragmented genomic sequence prepared from human genomic DNA as a raw material; wherein the fragmented genomic sequence amplification product and the fragmented genomic sequence are provided with a biotin label, and the solid support is provided with a streptavidin label.
In a preferred embodiment, the human probe in (b) above is a DNA nanosphere prepared from human genomic DNA as a raw material.
In a preferred embodiment, the solid support in (a) above is selected from a chip or a magnetic bead.
In a preferred embodiment, the solid support in (b) above is a digitally encoded magnetic bead.
In a preferred embodiment, the pathogenic probe of (b) above is a conserved region of the pathogenic genomic sequence.
In a preferred embodiment, the liquid sample is a nucleic acid extract from a blood, plasma, saliva, cerebrospinal fluid, or alveolar lavage sample.
According to a third aspect, there is provided in one embodiment a method of preparing an agent of the second aspect, the method comprising a step selected from the group consisting of (a) or (b) below:
(a) Preparing a host source probe and fixing the host source probe on the surface of a solid carrier to obtain the host source probe fixed on the solid carrier;
(b) Preparing a host source probe, preparing a pathogen probe, and fixing the pathogen probe on a solid carrier to obtain the pathogen probe fixed on the solid carrier.
In a preferred embodiment, the host source probe in (a) is a DNA nanosphere prepared from human genomic DNA as a raw material, and the preparation method of the host source probe immobilized on a solid carrier comprises the steps of:
breaking the human genome DNA by using a transposase and connecting joints to obtain fragmented human genome DNA with joints at two ends;
performing notch translation and PCR amplification on the fragmented human genome DNA to obtain an amplified product;
denaturing the amplification product and adding an anchor oligonucleotide strand and a DNA ligase that match the ends of the adaptor to form a single-stranded loop;
hybridizing an extension primer in the joint region of the single-stranded ring, adding polymerase with a strand displacement function, and performing rolling ring amplification to obtain a linear amplification product of the single-stranded human genome fragment, and forming a DNA nanosphere through intramolecular hydrogen bonds;
and loading the DNA nanospheres on the surface of a chip to prepare the chip loaded with the DNA nanospheres.
In a preferred embodiment, the host source probe in (a) is a fragmented genomic sequence amplification product prepared from human genomic DNA, the fragmented genomic sequence amplification product has a biotin label thereon, the solid phase carrier has a streptavidin label thereon, and the method for preparing the host source probe immobilized on the solid phase carrier comprises the steps of:
breaking the human genome DNA by using a transposase and connecting joints to obtain fragmented human genome DNA with joints at two ends;
performing notch translation on the fragmented human genome DNA, and performing PCR (polymerase chain reaction) amplification by using a primer with a biotin label to obtain an amplification product with the biotin label;
and adding the magnetic beads with streptavidin marks into the amplification products with biotin marks, mixing and incubating to obtain the human probes with the amplification products with fragmented human genome sequences, which are fixed on the magnetic beads.
In a preferred embodiment, the host source probe in (a) is a fragmented genomic sequence prepared from human genomic DNA, the fragmented genomic sequence is provided with a biotin label, the solid phase carrier is provided with a streptavidin label, and the preparation method of the host source probe immobilized on the solid phase carrier comprises the following steps:
Breaking the human genome DNA by using a transposase and adding biotin-labeled linkers to obtain fragmented human genome DNA with the linkers at two ends;
performing notch translation and extension on the fragmented human genome DNA to obtain a fragmented human genome DNA product with biotin marks;
and adding the magnetic beads with streptavidin marks into the fragmented human genome DNA products with the biotin marks, and mixing and incubating to obtain the human probes with the fragmented human genome sequence products fixed on the magnetic beads.
In a preferred embodiment, the host source probe in (b) above is a DNA nanosphere prepared from human genomic DNA as a raw material; the preparation method of the host source probe comprises the following steps:
breaking the human genome DNA by using a transposase and connecting joints to obtain fragmented human genome DNA with joints at two ends;
performing notch translation and PCR amplification on the fragmented human genome DNA to obtain an amplified product;
denaturing the amplification product and adding an anchor oligonucleotide strand and a DNA ligase that match the ends of the adaptor to form a single-stranded loop;
hybridizing an extension primer in the joint region of the single-stranded loop, adding polymerase with a strand displacement function, and performing rolling loop amplification to obtain a linear amplification product of the single-stranded human genome fragment, and forming the DNA nanospheres through intramolecular hydrogen bonds.
In a preferred embodiment, the pathogenic probe of (b) above is a conserved region of the pathogenic genomic sequence; the preparation method of the pathogen probe immobilized on the solid carrier comprises the following steps:
the sequence of the conserved region of the pathogenic nucleic acid with biotin marks is mixed with digital magnetic beads with streptavidin marks for incubation, so that the pathogenic probe for capturing the pathogenic nucleic acid in the sample is obtained.
The method for separating and/or enriching host source nucleic acid and pathogenic nucleic acid has the following beneficial effects:
(1) The scheme does not need to know the methylation site of pathogenic nucleic acid in advance, and can detect known and unknown pathogens simultaneously.
(2) The method does not require the step of digesting the host source nucleic acid with DNase and RNase, and thus does not largely lose free pathogenic nucleic acids present in the sample, and can enrich for all pathogenic nucleic acids in the sample.
(3) The scheme is applied to the capture probe for separating host source nucleic acid, preferably host source genome DNA, and can simultaneously hybridize host source DNA and RNA fragments in a sample, thereby realizing the simultaneous enrichment of pathogenic DNA and RNA.
(4) The chip and the magnetic bead preparation method for enrichment are simple and quick, the reagent used in the process is a mature commercial reagent, the complex processes such as transformation on wild enzyme are not needed, the operability is high, and the cost is low.
(5) The method for enriching the pathogenic nucleic acid can obviously increase the relative content of the pathogenic nucleic acid in the sample, and can reduce the number of read lengths (reads) and detection time and cost when applied to a downstream metagenome detection scheme.
Drawings
FIG. 1 is a schematic diagram of a transfer seat assembly according to an embodiment of the present invention;
FIG. 2 is a flowchart of a method for preparing a human capture probe chip for removing human nucleic acid from a Meta sample according to an embodiment of the present invention;
FIG. 3 is a flowchart of a method for preparing a humanized capture probe microsphere for removing a humanized nucleic acid from a Meta sample according to an embodiment of the present invention;
FIG. 4 is a flowchart of a method for preparing a humanized capture probe microsphere for removing a humanized nucleic acid from a Meta sample according to an embodiment of the present invention;
FIG. 5 is a flowchart (1) of a method for preparing a DNA nanosphere with a human capture probe for removing human nucleic acid from a Meta sample according to an embodiment of the invention, and a schematic diagram (2) of a pathogen capture digital magnetic bead capable of combining different types of pathogen nucleic acid.
Detailed Description
The invention will be described in further detail below with reference to the drawings by means of specific embodiments. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present invention. However, one skilled in the art will readily recognize that some of the features may be omitted in various situations, or replaced by other materials, methods.
Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.
Aiming at the defects of the pathogenic nucleic acid enrichment schemes in the current market, the invention designs a plurality of different schemes, and the separation of the host source and the pathogenic nucleic acid is realized by utilizing a chip or a magnetic bead for modifying the host source genome sequence or the pathogenic nucleic acid conservation region sequence so as to achieve the purpose of enriching the pathogenic nucleic acid. The technical effects of the enrichment of pathogenic nucleic acid by utilizing the scheme of the invention are as follows:
(1) The scheme does not need to know the methylation site of the pathogenic nucleic acid in advance, and can realize the detection of unknown pathogens.
(2) This protocol does not require digestion of the host source nucleic acid with DNase (DNase) and RNase (RNase) and therefore does not lose free pathogenic nucleic acids present in the sample.
(3) The capture probe of the scheme applied to enriching host source nucleic acid can be host source genome DNA, and can simultaneously hybridize host source DNA and RNA fragments in a sample, so that the simultaneous enrichment of pathogenic DNA and RNA is realized.
(4) The chip and the magnetic bead preparation method for enrichment are simple and quick, the reagent used in the process is a mature commercial reagent, the complex processes such as transformation on wild enzyme are not needed, the operability is high, and the cost is low.
Embodiments of the present invention provide a method for isolating and/or enriching host-derived nucleic acids and pathogenic nucleic acids. In the present invention, "isolating" means that the host nucleic acid and the pathogenic nucleic acid are separated to some extent, and may be separated entirely (for example, 100%) or partially (for example, in unequal proportions of 10% to 90%). By "enriching" is meant increasing the absolute or relative amount, e.g. absolute or relative concentration, etc., of at least one of the host source nucleic acid and the pathogenic nucleic acid.
In the present invention, the term "host-derived nucleic acid" refers to any nucleic acid of various sources such as host cells, host tissues or host individuals, which are parasitized or infected by a pathogen, including but not limited to animal-derived nucleic acids and plant-derived nucleic acids, etc., especially animal-derived nucleic acids, especially mammalian-derived nucleic acids, most typically human-derived nucleic acids. Such nucleic acids may be DNA or RNA, etc.
In the present invention, by "pathogenic nucleic acid" is meant a pathogenic microorganism that infects or infects any host cell, host tissue, or host individual, etc., including, but not limited to, bacteria, fungi, etc.
In one embodiment the invention provides a method for isolating and/or enriching host-derived nucleic acids and pathogenic nucleic acids, comprising the steps of: mixing and incubating the host source probe immobilized on the solid phase carrier with a liquid sample containing host source nucleic acid and pathogenic nucleic acid, so that the host source probe hybridizes with the host source nucleic acid; performing solid-liquid separation to obtain host-derived nucleic acid bound to the solid support and pathogenic nucleic acid retained in the liquid sample.
In embodiments of the invention, the host-derived probe is a probe that hybridizes to a host-derived nucleic acid, typically a human-derived probe that hybridizes to a human-derived nucleic acid. In a preferred embodiment, the human probe in the embodiment of the present invention is a DNA nanosphere, a fragmented genomic sequence amplification product or a fragmented genomic sequence prepared from human genomic DNA as a raw material. The amplified products of the fragmented genome sequence and the fragmented genome sequence are provided with biotin marks, and the solid phase carrier is provided with streptavidin marks.
In embodiments of the present invention, the host-derived probes have a degree of general property in that, for nucleic acids of the same host origin, host-derived nucleic acids (e.g., human-derived nucleic acids) of different host individuals (e.g., different humans) can be hybridized using the same host-derived probe (e.g., human-derived probe) prepared in advance. The prepared host source probe may be stored under appropriate conditions for a certain period of time, for example, may be stored at 4℃for 1 month, and used during that period.
In embodiments of the invention, host-derived probes, e.g., human-derived probes, may be prepared by any suitable method using human-derived nucleic acids, e.g., human genomic DNA. For example, the human genome DNA is physically or enzymatically broken to obtain fragmented genome DNA, and then the fragmented genome DNA is specifically marked (e.g. biotin marked) to be fixed on the surface of a solid carrier, or the fragmented genome DNA is cyclized and rolling circle amplified to obtain DNA Nanospheres (DNB) to be loaded on the surface of the solid carrier (e.g. chip) to realize the probe fixation.
As shown in FIG. 1, in the case of disrupting genomic DNA using a transposase, the transposase forms a transposable complex with transposons (A transposon and B transposon), and a double transposon transposable complex containing both the A transposon and the B transposon is shown in FIG. 1. In other embodiments, the transposable complex may be a Shan Zhuai transposon transposable complex containing only one transposon (either the a transposon or the B transposon).
In the case where it is desired to label the fragmented genomic DNA with a label such as biotin label, this can be achieved in various ways. One way is to interrupt the genomic DNA using the biotin-labeled transposon complex of FIG. 1 (2), in which the biotin-labeled A transposon and the biotin-labeled B transposon form a transposase complex together, and to add a transposon sequence with a biotin label at both ends of the fragmented genomic DNA while interrupting the genomic DNA, and to effect probe immobilization by binding to streptavidin on a solid support.
In the embodiment of the invention, the human source nucleic acid is used as host source nucleic acid, and the human source probe is used as host source probe, so that the following three methods for separating and/or enriching the host source nucleic acid and the pathogenic nucleic acid are provided.
The method comprises the following steps: the method for enriching pathogenic nucleic acid based on human probe (DNA nanospheres) has the following principle and characteristics:
the DNA nanospheres fixed on the silicon-based chip are used as human probes to hybridized and adsorb human nucleic acid in the sample to be detected, and enrichment of pathogenic nucleic acid in the sample is realized through solid-liquid separation. The preparation method of the DNA nanosphere humanized probe fixed on the silicon-based chip is shown in figure 2:
Firstly, taking human genome DNA as a raw material, and obtaining a product of fragmented human genome DNA with specific linkers at two ends by breaking with transposase and adding the linkers. A large number of amplified products can be obtained by nick translation and PCR amplification. The amplified human genome fragment can then be prepared into a single-stranded loop by denaturing the double-stranded product and adding an anchor oligonucleotide strand and a DNA ligase that match the ends of the adaptor. Hybridizing an extension primer in the joint region of the single-stranded loop, and adding phi29 polymerase to obtain a single-stranded human genome fragment linear amplification product by a rolling loop amplification mode. The obtained long single chains can form DNA nanospheres through intramolecular hydrogen bonds. Adding a DNA nanosphere loading buffer solution into the DNA nanospheres, uniformly mixing, putting into a loading system, and incubating at room temperature to load the prepared DNA nanospheres onto a chip of a silicon substrate with the tail end modified by amino groups.
And loading the sample to be treated on a probe chip, hybridizing a human probe on the chip with human nucleic acid in the sample, and separating the solid-phase chip from the liquid-phase sample to be treated to enrich pathogenic nucleic acid in the sample.
The first method has the following advantages:
(1) Human genome DNA is taken as a raw material, and after fragmentation, a probe is prepared by amplification to capture human nucleic acid in a sample, so that hybridization separation is realized without utilizing specific methylation sites or conserved regions.
(2) The proposal is applied to the capture probe for separating the human nucleic acid from the human genome DNA, can simultaneously hybridize the human DNA and RNA fragments in the sample, and realizes the simultaneous enrichment of pathogenic DNA and RNA.
(3) The probes are present in the form of DNA nanospheres and are present in a copy number of about several hundred copies (e.g., 300 copies or more, preferably 500 copies) of the fragmented human genomic DNA product, increasing the efficiency of hybridization capture of the human nucleic acid in the sample.
The second method is as follows: the principle and characteristics of the method for enriching pathogenic nucleic acid based on human probes (fragmented genome sequence amplification products) are as follows:
the amplification product of the fragmented human genome sequence fixed on the magnetic beads is used as a human probe to hybridized adsorb human nucleic acid in a sample to be detected, and enrichment of pathogenic nucleic acid in the sample is realized through solid-liquid separation. The preparation method of the human probe of the amplification product of the fragmented human genome sequence immobilized on the magnetic beads is shown in fig. 3:
firstly, taking human genome DNA as a raw material, and obtaining a product of fragmented human genome DNA with specific linkers at two ends by breaking with transposase and adding the linkers. A large number of amplified products of fragmented human genome sequences with biotin labels can be obtained by nick translation and PCR amplification using biotin-labeled primers. Adding the magnetic beads with streptavidin marks into an amplification product solution with biotin marks and fragmenting human genome sequences, stirring and incubating at room temperature to obtain the human probe with the amplification products of the fragmenting human genome sequences, wherein the human probe is fixed on the magnetic beads.
Adding the magnetic beads fixed with the human probes into a sample to be treated, hybridizing the human probes on the magnetic beads with human nucleic acids in the sample, and separating the solid-phase magnetic beads from the liquid-phase sample to be treated to enrich pathogenic nucleic acids in the sample.
The second method has the following advantages:
(1) Human genome DNA is taken as a raw material, and after fragmentation, a probe is prepared by amplification to capture human nucleic acid in a sample, so that hybridization separation is realized without utilizing specific methylation sites or conserved regions.
(2) The proposal is applied to the capture probe for separating the human nucleic acid from the human genome DNA, can simultaneously hybridize the human DNA and RNA fragments in the sample, and realizes the simultaneous enrichment of pathogenic DNA and RNA.
(3) The PCR method is utilized to realize the marking of functional groups (biotin) and the amplification of the human probes in one step, thereby realizing the fixation of the human probes and further increasing the capture efficiency of human nucleic acids in the sample.
And a third method: the principle and characteristics of the method for enriching pathogenic nucleic acid based on human probes (fragmented genome sequences) are as follows:
the product of the fragmented human genome sequence fixed on the magnetic beads is used as a human probe to hybridized adsorb human nucleic acid in a sample to be detected, and enrichment of pathogenic nucleic acid in the sample is realized through solid-liquid separation. The preparation method of the humanized probe of the product of fragmenting human genome sequence immobilized on magnetic beads is shown in FIG. 4:
Firstly, taking human genome DNA as a raw material, and obtaining a product of fragmented human genome DNA with specific linkers at two ends by breaking with transposase and adding biotin-labeled linkers. The product of the fragmented human genome DNA with the biotin label can be obtained through incision translation and extension. And adding the magnetic beads with streptavidin marks into a product solution with the biotin marks of the fragmented human genome sequences, stirring and incubating at room temperature to obtain the human probe with the fragmented human genome sequences products fixed on the magnetic beads.
Adding the magnetic beads fixed with the human probes into a sample to be treated, hybridizing the human probes on the magnetic beads with human nucleic acids in the sample, and separating the solid-phase magnetic beads from the liquid-phase sample to be treated to enrich pathogenic nucleic acids in the sample.
The third advantage of the method comprises:
(1) The human genome DNA is taken as a raw material, the fragmented product is prepared into a probe for capturing human nucleic acid in a sample, and hybridization separation is realized without utilizing specific methylation sites or conserved regions.
(2) The proposal is applied to the capture probe for separating the human nucleic acid from the human genome DNA, can simultaneously hybridize the human DNA and RNA fragments in the sample, and realizes the simultaneous enrichment of pathogenic DNA and RNA.
(3) According to the scheme, the human probe is obtained by breaking human genome DNA in one step and adding the linker, the preparation scheme is simple, the steps are fewer, so that the efficiency of preparing the human probe is higher, and the cost is lower.
In one embodiment the invention provides a method for isolating and/or enriching host-derived nucleic acids and pathogenic nucleic acids, comprising the steps of:
mixing and incubating a host source probe and a pathogen probe immobilized on a solid carrier with a liquid sample containing host source nucleic acid and pathogen nucleic acid, so that the host source probe hybridizes with the host source nucleic acid and the pathogen probe hybridizes with the pathogen nucleic acid; performing solid-liquid separation to obtain pathogenic nucleic acids bound to the solid support and host-derived nucleic acids remaining in the liquid sample.
In embodiments of the invention, host-derived probes, e.g., human-derived probes, may be prepared by any suitable method using human-derived nucleic acids, e.g., human genomic DNA. In this embodiment, 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 a solid phase carrier and combined with pathogenic nucleic acid in a sample, and then the separation of host source nucleic acid and pathogenic nucleic acid is realized through solid-liquid separation.
In the embodiment of the invention, the human source nucleic acid is used as the host source nucleic acid, the human source probe is used as the host source probe, and the following fourth method for separating and/or enriching the host source nucleic acid and the pathogenic nucleic acid is provided.
The method four: the principle and the characteristics of the method for enriching pathogenic nucleic acid based on human probes (DNA nanospheres) and pathogenic probes (digitally encoded magnetic beads) are as follows:
the DNA nanospheres prepared based on human genome existing in liquid phase are used as human probes to hybridize and adsorb human nucleic acid in a sample to be detected, meanwhile, a conserved region of a pathogenic genome sequence fixed on digital magnetic beads is used as a pathogenic probe to hybridized and capture pathogenic nucleic acid in the sample to be detected, and enrichment of the pathogenic nucleic acid in the sample is realized through solid-liquid separation. Wherein, the preparation methods of the human probe of the DNA nanosphere prepared based on human genome and the pathogenic probe with the pathogenic genome sequence conservation area fixed on the digital magnetic bead are shown in figure 5:
firstly, taking human genome DNA as a raw material, and obtaining a product of fragmented human genome DNA with specific linkers at two ends by breaking with transposase and adding the linkers. A large number of amplified products can be obtained by nick translation and PCR amplification. The amplified human genome fragment can then be prepared into a single-stranded loop by denaturing the double-stranded product and adding an anchor oligonucleotide strand and a DNA ligase that match the ends of the adaptor. Hybridizing an extension primer in the joint region of the single-stranded loop, and adding phi29 polymerase to obtain a single-stranded human genome fragment linear amplification product by a rolling loop amplification mode. The obtained long single chains can form DNA nanospheres through intramolecular hydrogen bonds, namely human probes for removing human nucleic acids in samples by hybridization.
The preparation method of the pathogenic probe with the pathogenic genome sequence conservation region comprises the steps of mixing the pathogenic nucleic acid conservation region sequence with biotin marks with digital magnetic beads with streptavidin marks and incubating at room temperature to obtain the pathogenic probe for capturing the pathogenic nucleic acid in the sample to be detected.
Adding the prepared DNA nanosphere humanized probe and the digital magnetic beads fixed with the pathogenic probes into a sample to be treated, capturing the humanized nucleic acid hybridization in the sample to be detected onto the DNA nanosphere humanized probe in a liquid phase, capturing the pathogenic nucleic acid hybridization in the sample to be detected onto the digital magnetic beads fixed with the pathogenic probes, and separating the solid-phase digital magnetic beads from the liquid-phase sample to be treated to realize the enrichment of the pathogenic nucleic acid in the sample.
The third advantage of the method comprises:
(1) Human genome DNA is taken as a raw material, and after fragmentation, a human probe is amplified to capture human nucleic acid in a sample, and hybridization separation is realized without utilizing specific methylation sites or conserved regions.
(2) The proposal is applied to the capture probe for separating the human nucleic acid from the human genome DNA, can simultaneously hybridize the human DNA and RNA fragments in the sample, and realizes the simultaneous enrichment of pathogenic DNA and RNA.
(3) The presence of the human probes in the form of DNA nanospheres, with the presence of about several hundred copies (e.g., 300 copies or more, preferably 500 copies) of the fragmented human genomic DNA product, increases the efficiency of hybridization capture of the human nucleic acids in the sample.
(4) Because the two-dimensional code marks exist on the digital magnetic beads, different pathogenic nucleic acid conservation areas can be modified on the different marked magnetic beads to realize enrichment of different pathogenic nucleic acids on the different marked digital magnetic beads, so that enrichment of multiple types is realized respectively.
(5) The proposal utilizes a bidirectional selection method, namely a proposal of simultaneous hybridization and capture of a human probe and a pathogen probe to carry out enrichment, which can further increase the specificity and efficiency of pathogen enrichment.
The following detailed description of the present invention is provided by way of example only, and should not be construed as limiting the scope of the invention.
It should be noted that the methods of embodiments of the present invention are exemplary, and that various elements and materials in the above methods have a variety of other alternatives. For example, (1) the enzyme for DNA fragmentation of the present invention is not limited to Tn5 transposase used in the present example, and may be other enzymes of the Tn transposase family, such as Tn7; or other families of transposases, such as the Mu family; and is not even limited to transposases or enzyme preparations, as long as it is capable of fragmenting DNA while ligating a stretch of sequence to the DNA. (2) The solid phase carrier for adsorption according to the present invention is not limited to the silicon-based chip and the ferroferric oxide beads used in the examples, and may be polymer or glass microsphere, solid, hollow or porous form, and even solid surface and interior of any other materials and forms, so long as it can modify the probe oligonucleotide chains. The surface modification is not limited to streptavidin in the examples, and the modification may be a molecule to which a DNA oligonucleotide (oligo) can be covalently linked, such as a hydroxyl group, a carboxyl group, an amino group, or the like. (3) The shape and number of the dot matrix of the solid phase chip for adsorption of the present invention are not limited to a circle and 200M, and may be any shape and number. The size and number of the magnetic beads to be added are not limited to 20 μm and 200M, and may be any size and number. (4) The length and sequence information of the transposon sequences used in the present invention are not limited. (5) The transposon linkers used in the present invention to break genomic DNA and added at both ends may be single sequence linkers or two different sequence linkers. (6) The fragment size of the human-derived probe obtained by breaking the added transposition complex is not limited, so long as the probe can effectively remove the human-derived genome fragment in the sample. (7) The ligase used in the present invention is not limited to a specific ligase, and may 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 join nicks (nicks) to form a single-stranded loop. (8) The exonuclease used in the present invention is not limited to a specific one, and may be exonuclease I, exonuclease III, lambda exonuclease and T7 exonuclease as long as it can digest the linear DNA strand remaining in the circularized product. (9) The polymerase used in the present invention for preparing the DNA nanospheres is not limited to phi29 polymerase, and may be other polymerases having strand displacement activity and capable of rolling circle amplification, and may be Bst, large fragments of Bst, e.coli DNA polymerase I, etc., as long as it can prepare the DNA nanospheres. (10) The material, size, shape and surface pattern of the digital magnetic beads used in the invention are not limited to one, and the material can be a high polymer material such as inorganic ferroferric oxide and the like; the size may be 1 μm to 1mm; the shape and surface pattern and the surface modification method are not limited as long as they can modify the conserved sequence of the pathogenic nucleic acid to achieve the purpose of enriching the pathogenic nucleic acid and can achieve multiple detection. (11) The enrichment method of the four pathogenic nucleic acids is not only limited to enrichment of the pathogenic nucleic acids in blood samples, but also can be applied to enrichment of the pathogenic nucleic acids in saliva, cerebrospinal fluid, alveolar lavage fluid and other samples. (12) The enrichment method of the four pathogenic nucleic acids is not limited to enrichment of the pathogenic nucleic acids in sequencing sample treatment, can be applied to sample pretreatment for detecting the pathogenic nucleic acids based on qPCR, ddPCR, aptamer hybridization, binding protein and other methods, and can be a fluorescence method, an electrochemical method, a chemiluminescence method, a colorimetric method and the like.
Example 1: method for enriching pathogenic nucleic acid based on human probe (DNA nanosphere)
(1) The preparation method of the human genome segment with the linker sequence comprises the following steps:
50ng of human genome DNA is used as a raw material and homogenized to 10 mu L, 10 mu L of 0.8 mu M transposition complex is added to break the genome sequence in 1 XTAG buffer solution, meanwhile, a joint sequence is added, the reaction is carried out for 7 minutes at 55 ℃, and after the reaction is finished, 5 mu L of NT solution is added for reaction for 5 minutes at room temperature, so that the reaction is stopped.
(2) Amplification method of human genomic fragment with linker sequence:
adding 5 mu L of 20 mu M primer into the human genome segment with the joint sequence prepared in the step (1) to enable the final concentration to be 1 mu M, carrying out extension on DNA polymerase and dNTPs at 72 ℃ for 5min, then carrying out polymerase chain reaction for 8 cycles, and purifying by using 0.8X magnetic beads to obtain the amplified human genome segment. 350ng of amplification product was homogenized to 60. Mu.L.
(3) The preparation method of the single-chain ring based on the human genome segment comprises the following steps:
adding 10 mu L of 10 mu M anchored oligonucleotide chain into the amplified fragment prepared in the step (2), carrying out high-temperature denaturation at 95 ℃ for 3min, rapidly cooling and renaturation, adding 1.2 mu L of 100mM ATP and 0.4 mu L of 600U/. Mu.L of T4 ligase, and reacting in 1 XTA buffer for 0.5 h at 37 ℃. To the above product, 3.9. Mu.L of 20U/. Mu.L of exonuclease I and 1.3. Mu.L of 100U/. Mu.L of exonuclease III were added, and the remaining uncycled single and double strands were digested at 37℃for 30 minutes. After completion of the reaction, the digestion reaction was terminated by adding 6. Mu.L of 0.5M EDTA. The single-chain ring based on the human genome segment can be obtained after purification by using 2.5X magnetic beads. 6ng of single-stranded loop product was homogenized to 20. Mu.L.
(4) The preparation method of the human probe (DNA nanospheres) comprises the following steps:
adding 20 mu L of 1 mu M primer into the single-stranded loop prepared in the step (3), annealing at 95 ℃ 1min,65 ℃ 1min and 40 ℃ 1min, and carrying out isothermal amplification for 20 minutes by using 4 mu L of 10U/. Mu.L phi 29 polymerase and 40 mu L of 2 XSSB at 30 ℃ to obtain the human probe (DNA nanosphere).
(5) The preparation method of the chip with the human probe (DNA nanospheres) comprises the following steps:
adding a DNA nanosphere loading buffer solution into the DNA nanospheres prepared in the step (4), uniformly mixing, putting into a loading system, loading the prepared DNA nanospheres onto a chip of a silicon substrate with the tail end amino group modified, and incubating for 1 hour at room temperature.
(6) Method for extracting nucleic acid from whole blood:
5ml of whole blood was used as a starting sample, and after anticoagulation by adding 0.04M EDTA, the upper plasma and the leukocyte layer were sucked by centrifugation. The collected product was centrifuged at 2000rpm for 5min and the upper plasma was collected in centrifuge tube No. 1 and the white blood cell layer in centrifuge tube No. 2. 10% formalin solution was added to the plasma, and after incubation with proteinase K for 30 minutes, the free nucleic acids in the plasma were adsorbed by an adsorption column. Washing with 70% ethanol solution twice, eluting the adsorbed nucleic acid with eluent to obtain free nucleic acid.
(7) Enrichment method of pathogenic nucleic acid:
Adding the nucleic acid extracted in the step (6) to the chip prepared in the step (5), hybridizing for 30 minutes at 25 ℃, collecting clear liquid, flushing the surface of the chip with TE buffer solution to remove nonspecifically adsorbed nucleic acid, and combining all the solutions with the washing solution to obtain the enriched pathogenic nucleic acid.
(8) The collection method of the human nucleic acid comprises the following steps:
the nucleic acid hybridized with the chip is denatured and collected by TE buffer solution to obtain the human nucleic acid.
Example 2: method for enriching pathogenic nucleic acid based on human probe (fragmented genome sequence amplification product)
(1) The preparation method of the human genome segment with the linker sequence comprises the following steps:
50ng of human genome DNA is taken as a raw material, 0.5-5 pmol of transposition complex is utilized to break the genome sequence, simultaneously a joint sequence is added, the reaction is carried out for 5-20 minutes at 55 ℃, and after the reaction is finished, the NT buffer solution is utilized to react for 3-10 minutes at room temperature to terminate the reaction. The product is subjected to terminal extension at 72 ℃ for 5-15 minutes, and the human genome segment with the linker sequence can be obtained.
(2) Amplification method of human genomic fragment with linker sequence:
adding 1-2 mu M biotin-marked primer, DNA polymerase and dNTPs into the human genome segment with the joint sequence prepared in the step (1), performing 72 ℃ extension for 5min, performing polymerase chain reaction for 8-15 cycles, and purifying by using 0.8-2.5X magnetic beads to obtain an amplification product of the human genome segment with the joint sequence marked by biotin.
(3) The preparation method of the magnetic beads with the human probes (fragmented genome sequence amplification products) comprises the following steps:
adding the amplified product of the human genome fragment with the biotin-marked joint sequence prepared in the step (2) into streptavidin-marked magnetic beads, uniformly mixing, and incubating for 1 hour at room temperature on a vertical mixer to obtain the magnetic beads with the human probes (fragmented genome sequence amplified product).
(4) Method for extracting free nucleic acid from whole blood:
5ml of whole blood was used as a starting sample, and after anticoagulation by adding 0.04M EDTA, the upper plasma and the leukocyte layer were sucked by centrifugation. The collected product was centrifuged at 2000rpm for 5min and the upper plasma was collected in centrifuge tube No. 1 and the white blood cell layer in centrifuge tube No. 2. 10% formalin solution was added to the plasma, and after incubation with proteinase K for 30 minutes, the free nucleic acids in the plasma were adsorbed by an adsorption column. Washing with 70% ethanol solution twice, eluting the adsorbed nucleic acid with eluent to obtain free nucleic acid.
(5) Enrichment method of pathogenic nucleic acid:
adding the magnetic beads prepared in the step (3) into the free nucleic acid extracted in the step (4), hybridizing for 30 minutes at 25 ℃, collecting clear liquid, washing nonspecifically adsorbed nucleic acid on the surfaces of the magnetic beads by using TE buffer solution, and combining all clear liquid and washing solution to obtain the enriched pathogenic nucleic acid.
(6) The collection method of the human nucleic acid comprises the following steps:
the nucleic acid hybridized with the magnetic beads is denatured and collected by TE buffer solution to obtain the human nucleic acid.
Example 3: method for enriching pathogenic nucleic acid based on human probe (fragmented genome sequence)
(1) A method for preparing a human genome fragment with a biotin-labeled linker sequence, comprising the steps of:
2. Mu.g of human genomic DNA was used as a raw material and homogenized to 400. Mu.L, 400. Mu.L of 0.8. Mu.M transposition complex was added to break the genomic sequence in 1 XTAG buffer while the biotin-labeled linker sequence was added thereto, and the reaction was stopped after completion of the reaction by adding 200. Mu.L of NT solution and reacting at room temperature for 5 minutes.
(2) The preparation method of the magnetic beads with the human probes (fragmented genome sequences) comprises the following steps:
and (3) adding the human genome fragment with the biotin-marked joint sequence prepared in the step (1) into the streptavidin-marked magnetic beads, uniformly mixing, and incubating for 1 hour at room temperature on a vertical mixer to obtain the magnetic beads with the human probes (fragmented genome sequences).
Adding the amplified product of the human genome fragment with the biotin-marked joint sequence prepared in the step (2) into streptavidin-marked magnetic beads, uniformly mixing, and incubating for 1 hour at room temperature on a vertical mixer to obtain the magnetic beads with the human probes (fragmented genome sequence amplified product).
(3) Method for extracting free nucleic acid from whole blood:
5ml of whole blood was used as a starting sample, and after anticoagulation by adding 0.04M EDTA, the upper plasma and the leukocyte layer were sucked by centrifugation. The collected product was centrifuged at 2000rpm for 5min and the upper plasma was collected in centrifuge tube No. 1 and the white blood cell layer in centrifuge tube No. 2. 10% formalin solution was added to the plasma, and after incubation with proteinase K for 30 minutes, the free nucleic acids in the plasma were adsorbed by an adsorption column. Washing with 70% ethanol solution twice, eluting the adsorbed nucleic acid with eluent to obtain free nucleic acid.
(4) Enrichment method of pathogenic nucleic acid:
adding the magnetic beads prepared in the step (2) into the free nucleic acid extracted in the step (3), hybridizing for 30 minutes at 25 ℃, collecting clear liquid, washing nonspecifically adsorbed nucleic acid on the surfaces of the magnetic beads by using TE buffer solution, and combining all clear liquid and washing solution to obtain the enriched pathogenic nucleic acid.
(5) The collection method of the human nucleic acid comprises the following steps:
the nucleic acid hybridized with the magnetic beads is denatured and collected by TE buffer solution to obtain the human nucleic acid.
Example 4: method for enriching pathogenic nucleic acid based on human-derived probes (DNA nanospheres) and pathogenic probes (digitally encoded magnetic beads)
(1) The preparation method of the human genome segment with the linker sequence comprises the following steps:
50ng of human genome DNA is used as a raw material and homogenized to 10 mu L, 10 mu L of 0.8 mu M transposition complex is added to break the genome sequence in 1 XTAG buffer solution, meanwhile, a joint sequence is added, the reaction is carried out for 7 minutes at 55 ℃, and after the reaction is finished, 5 mu L of NT solution is added for reaction for 5 minutes at room temperature, so that the reaction is stopped.
(2) Amplification method of human genomic fragment with linker sequence:
adding 5 mu L of 20 mu M primer into the human genome segment with the joint sequence prepared in the step (1) to enable the final concentration to be 1 mu M, carrying out extension on DNA polymerase and dNTPs at 72 ℃ for 5min, then carrying out polymerase chain reaction for 8 cycles, and purifying by using 0.8X magnetic beads to obtain the amplified human genome segment. 350ng of amplification product was homogenized to 60. Mu.L.
(3) The preparation method of the single-chain ring based on the human genome segment comprises the following steps:
adding 10 mu L of 10 mu M anchored oligonucleotide chain into the amplified fragment prepared in the step (2), carrying out high-temperature denaturation at 95 ℃ for 3min, rapidly cooling and renaturation, adding 1.2 mu L of 100mM ATP and 0.4 mu L of 600U/. Mu.L of T4 ligase, and reacting in 1 XTA buffer for 0.5 h at 37 ℃. To the above product, 3.9. Mu.L of 20U/. Mu.L of exonuclease I and 1.3. Mu.L of 100U/. Mu.L of exonuclease III were added, and the remaining uncycled single and double strands were digested at 37℃for 30 minutes. After completion of the reaction, the digestion reaction was terminated by adding 6. Mu.L of 0.5M EDTA. The single-chain ring based on the human genome segment can be obtained after purification by using 2.5X magnetic beads. 6ng of single-stranded loop product was homogenized to 20. Mu.L.
(4) The preparation method of the human probe (DNA nanospheres) comprises the following steps:
adding 20 mu L of 1 mu M primer into the single-stranded loop prepared in the step (3), annealing at 95 ℃ 1min,65 ℃ 1min and 40 ℃ 1min, and carrying out isothermal amplification for 20 minutes by using 4 mu L of 10U/. Mu.L phi 29 polymerase and 40 mu L of 2 XSSB at 30 ℃ to obtain the human probe (DNA nanosphere).
(5) The preparation method of the digital magnetic beads with pathogenic probes (conserved regions of pathogenic nucleic acids) comprises the following steps:
the synthesized nucleic acid chain with the biotin-marked pathogenic nucleic acid conservation region is added into the streptavidin-modified digital magnetic beads to be uniformly mixed, and the mixture is incubated for 1 hour at room temperature on a vertical mixer.
(6) Method for extracting nucleic acid from whole blood:
5ml of whole blood was used as a starting sample, and after anticoagulation by adding 0.04M EDTA, the upper plasma and the leukocyte layer were sucked by centrifugation. The collected product was centrifuged at 2000rpm for 5min and the upper plasma was collected in centrifuge tube No. 1 and the white blood cell layer in centrifuge tube No. 2. 10% formalin solution was added to the plasma, and after incubation with proteinase K for 30 minutes, the free nucleic acids in the plasma were adsorbed by an adsorption column. Washing with 70% ethanol solution twice, eluting the adsorbed nucleic acid with eluent to obtain free nucleic acid.
(7) The collection method of the human nucleic acid comprises the following steps:
Adding the magnetic beads prepared in the step (5) and the human probes (DNA nanospheres) prepared in the step (4) into the free nucleic acid extracted in the step (6) for hybridization at 25 ℃ for 30 minutes, collecting clear liquid, washing the nucleic acid nonspecifically adsorbed on the surfaces of the magnetic beads by using TE buffer solution, and combining all clear liquid and washing solution to obtain the human nucleic acid.
(8) Enrichment method of pathogenic nucleic acid:
the nucleic acid hybridized with the digital magnetic beads is denatured and collected by TE buffer solution, thus obtaining the enriched pathogenic nucleic acid.
Comparative example: samples not subjected to pathogen nucleic acid enrichment were library constructed and sequenced under the same banking conditions as in the examples
5ml of whole blood was used as a starting sample, and after anticoagulation by adding 0.04M EDTA, the upper plasma and the leukocyte layer were sucked by centrifugation. The collected product was centrifuged at 2000rpm for 5min and the upper plasma was collected in centrifuge tube No. 1 and the white blood cell layer in centrifuge tube No. 2. 10% formalin solution was added to the plasma, and after incubation with proteinase K for 30 minutes, the free nucleic acids in the plasma were adsorbed by an adsorption column. Washing with 70% ethanol solution twice, eluting the adsorbed nucleic acid with eluent to obtain free nucleic acid. Downstream pooling is continued directly with this free nucleic acid.
Experimental results:
The sample treatment protocol of examples 1 to 4 was used to enrich for pathogenic nucleic acids in samples, and libraries were created and sequenced under the same banking conditions as samples not subjected to pathogenic nucleic acid enrichment of comparative examples, and the sequencing results were compared as shown in table 1 below:
TABLE 1
| Parameters (parameters) | Comparative example | Example 1 | Example 2 | Example 3 | Example 4 |
| Sequencing Total read Length (Reads) number (M) | 80 | 40 | 40 | 40 | 40 |
| Detecting the number of pathogenic Reads (bars) | 19 | 19 | 22 | 20 | 17 |
The foregoing description of the invention has been presented for purposes of illustration and description, and is not intended to be limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art to which the invention pertains, based on the idea of the invention.
Claims (14)
1. A method of isolating and/or enriching a host source nucleic acid and a pathogenic nucleic acid, the method comprising a step selected from the group consisting of (a) or (b):
(a) Incubating a host-derived probe immobilized on a solid support in combination with a liquid sample containing host-derived nucleic acids and pathogenic nucleic acids, such that the host-derived probe hybridizes to the host-derived nucleic acids; performing solid-liquid separation to obtain host-derived nucleic acids bound to the solid support and pathogenic nucleic acids retained in the liquid sample;
(b) Mixing and incubating a host source probe, a pathogen probe immobilized on a solid support, and a liquid sample containing a host source nucleic acid and a pathogen nucleic acid, so that the host source probe hybridizes with the host source nucleic acid and the pathogen probe hybridizes with the pathogen nucleic acid; performing solid-liquid separation to obtain pathogenic nucleic acids bound to the solid support and host-derived nucleic acids retained in the liquid sample;
the host source probe is a human source probe, and correspondingly, the host source nucleic acid is a human source nucleic acid; the human probe is a DNA nanosphere prepared by taking human genome DNA as a raw material, and a fragmented genome sequence amplification product or a fragmented genome sequence;
wherein, the preparation method of the DNA nanospheres comprises the following steps: breaking the human genome DNA by using a transposase and connecting joints to obtain fragmented human genome DNA with joints at two ends; performing notch translation and PCR amplification on the fragmented human genome DNA to obtain an amplification product; denaturing the amplified product and adding an anchor oligonucleotide strand and a DNA ligase that match the ends of the adaptor to form a single-stranded loop; hybridizing an extension primer in the joint region of the single-stranded ring, adding polymerase with a strand displacement function, and performing rolling ring amplification to obtain a linear amplification product of the single-stranded human genome fragment, and forming a DNA nanosphere through intramolecular hydrogen bonds;
The preparation method of the fragmented genome sequence amplification product comprises the following steps: breaking the human genome DNA by using a transposase and connecting joints to obtain fragmented human genome DNA with joints at two ends; performing notch translation on the fragmented human genome DNA, and performing PCR (polymerase chain reaction) amplification by using a primer with a biotin label to obtain an amplification product with the biotin label;
the preparation method of the fragmented genome sequence comprises the following steps: breaking the human genome DNA by using a transposase and adding biotin-labeled linkers to obtain fragmented human genome DNA with the linkers at two ends; and carrying out notch translation and extension on the fragmented human genome DNA to obtain a fragmented human genome DNA product with biotin marks.
2. The method of claim 1, wherein the fragmented genomic sequence amplification product and fragmented genomic sequence bear a biotin tag and the solid support bears a streptavidin tag.
3. The method of claim 1, wherein the solid support in (a) is selected from a chip or a magnetic bead.
4. The method of claim 1, wherein the solid support in (b) is a digitally encoded magnetic bead.
5. The method of claim 1, wherein the pathogenic probe in (b) is a conserved region of a pathogenic genomic sequence.
6. The method of claim 1, wherein the liquid sample is blood, plasma, saliva, cerebrospinal fluid, or a nucleic acid extract in an alveolar lavage sample.
7. The method of claim 1, wherein (a) further comprises: the host-derived nucleic acid bound to the solid support is denatured and eluted to yield the free host-derived nucleic acid.
8. The method of claim 1, wherein (b) further comprises: the pathogenic nucleic acid bound to the solid support is denatured and eluted to yield free pathogenic nucleic acid.
9. An agent for isolating and/or enriching a host source nucleic acid and a pathogenic nucleic acid, wherein the agent comprises a component selected from the group consisting of (a) or (b):
(a) A host-derived probe immobilized on a solid support for mixed incubation with a liquid sample containing host-derived nucleic acids and pathogenic nucleic acids, hybridizing the host-derived probe with the host-derived nucleic acids, and obtaining host-derived nucleic acids bound to the solid support and pathogenic nucleic acids retained in the liquid sample by solid-liquid separation;
(b) A host source probe and a pathogenic probe immobilized on a solid support, for mixed incubation with a liquid sample containing host source nucleic acid and pathogenic nucleic acid, hybridizing the host source probe with the host source nucleic acid, hybridizing the pathogenic probe with the pathogenic nucleic acid, and obtaining a pathogenic nucleic acid bound to the solid support and a host source nucleic acid retained in the liquid sample by solid-liquid separation;
the host source probe is a human source probe, and correspondingly, the host source nucleic acid is a human source nucleic acid; the human probe is a DNA nanosphere prepared by taking human genome DNA as a raw material, and a fragmented genome sequence amplification product or a fragmented genome sequence;
wherein, the preparation method of the DNA nanospheres comprises the following steps: breaking the human genome DNA by using a transposase and connecting joints to obtain fragmented human genome DNA with joints at two ends; performing notch translation and PCR amplification on the fragmented human genome DNA to obtain an amplification product; denaturing the amplified product and adding an anchor oligonucleotide strand and a DNA ligase that match the ends of the adaptor to form a single-stranded loop; hybridizing an extension primer in the joint region of the single-stranded ring, adding polymerase with a strand displacement function, and performing rolling ring amplification to obtain a linear amplification product of the single-stranded human genome fragment, and forming a DNA nanosphere through intramolecular hydrogen bonds;
The preparation method of the fragmented genome sequence amplification product comprises the following steps: breaking the human genome DNA by using a transposase and connecting joints to obtain fragmented human genome DNA with joints at two ends; performing notch translation on the fragmented human genome DNA, and performing PCR (polymerase chain reaction) amplification by using a primer with a biotin label to obtain an amplification product with the biotin label;
the preparation method of the fragmented genome sequence comprises the following steps: breaking the human genome DNA by using a transposase and adding biotin-labeled linkers to obtain fragmented human genome DNA with the linkers at two ends; and carrying out notch translation and extension on the fragmented human genome DNA to obtain a fragmented human genome DNA product with biotin marks.
10. The reagent of claim 9, wherein the fragmented and genomic sequence amplification products bear a biotin label and the solid support bears a streptavidin label.
11. The reagent of claim 9, wherein the solid support in (a) is selected from a chip or a magnetic bead.
12. The reagent of claim 9, wherein the solid support in (b) is a digitally encoded magnetic bead.
13. The agent of claim 9, wherein the pathogenic probe of (b) is a conserved region of a pathogenic genomic sequence.
14. The reagent of claim 9, wherein the liquid sample is blood, plasma, saliva, cerebrospinal fluid, or nucleic acid extract in an alveolar lavage sample.
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