WO2018192452A1 - 基于外泌体核酸进行微生物检测的方法及其应用 - Google Patents

基于外泌体核酸进行微生物检测的方法及其应用 Download PDF

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WO2018192452A1
WO2018192452A1 PCT/CN2018/083230 CN2018083230W WO2018192452A1 WO 2018192452 A1 WO2018192452 A1 WO 2018192452A1 CN 2018083230 W CN2018083230 W CN 2018083230W WO 2018192452 A1 WO2018192452 A1 WO 2018192452A1
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nucleic acid
species
sample
data
sequence
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French (fr)
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卢森
高雅
姬敬开
麻锦敏
赵佳
黄国栋
陈芳
蒋慧
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深圳华大生命科学研究院
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Publication of WO2018192452A1 publication Critical patent/WO2018192452A1/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

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  • the present invention relates to the field of microbial detection technology, and in particular to a method for microbial detection based on exosomal nucleic acid.
  • pathogenic microorganisms invade human tissues directly or indirectly cause harm to the human body; these pathogenic microorganisms include bacteria, fungi, viruses, parasites and the like.
  • these pathogenic microorganisms include bacteria, fungi, viruses, parasites and the like.
  • the rapid and accurate detection of unknown pathogenic microorganisms is the accurate identification of pathogenic microorganisms in clinical practice. It is essential for clinical correct diagnosis and effective prevention and treatment.
  • Pregnancy infection (such as TORCH screening widely carried out in pre-pregnancy stage in China) leads to premature delivery, abortion and pregnancy. An important factor in common diseases such as high blood pressure.
  • Metagenomics is a research technique and method for the collection of all microbial genomes in the environment. According to different research methods, metagenomics can be divided into the classification and identification of ribosomal rDNA, as well as the analysis and identification of all metagenomic DNA sequences.
  • nasopharyngeal swabs sputum, urine, blood (whole blood, serum, plasma), etc.
  • relative nasopharyngeal swabs, sputum, urine, blood is disturbed by the external environment. Smaller, less likely to be contaminated, more suitable as a sample for pathogen identification.
  • the method of pathogen identification using blood is usually to extract free DNA from plasma, undergo PCR amplification, and then sequence these free DNA by high-throughput sequencing.
  • the inventors of the present invention found in the course of research that by means of high-throughput sequencing technology, blood samples are used for pathogen identification, since the proportion of microbial sequences in blood is much lower than that of detecting individuals (such as humans), It is often the case that the proportion of people in the sequencing results is higher and the proportion of microorganisms is lower, and the sequencing saturation is far from enough. Based on this, it is an object of the present invention to provide a method for detecting a pathogenic microorganism based on high accuracy and high sensitivity of exosome DNA. The method can enrich the sequence of the microorganism to a certain extent, thereby significantly improving the accuracy of the microbial sequencing information in the sequencing process, so that the accuracy of the microbial detection result is greatly improved. Moreover, this enrichment can be achieved by means of exosomes, which is simple and convenient.
  • a method of detecting microorganisms in a sample to be tested comprising the steps of:
  • the microbial database described in the present invention is derived from an existing microbial database, and these databases are not static and can be continuously supplemented and improved as technology advances.
  • the microbial database can use Refseq data from NCBI, including bacterial/paleobacterial data, viral data, fungal data, protozoal data, plasmid data, and the like, as desired.
  • the method of detecting microorganisms in a sample to be tested by the present invention can be used to determine microorganisms in an individual or a certain species. Moreover, the method can be used not only to detect pathogenic microorganisms, but also to detect beneficial microorganisms in individuals or species, and to provide reference and reference for individuals to improve or adjust physical condition or constitution.
  • the exosomal nucleic acid is selected from the group consisting of exosomal DNA, exosomal RNA, or a combination thereof; preferably exosomal DNA.
  • the first species is selected from the group consisting of a mammal, a bird, or a reptile, and more preferably the mammal is a human.
  • the nucleic acid sequence information of the human can be removed with reference to hg19, the refMrna database in the UCSC, and/or the Yanhuang database.
  • the sample is a sample from a human (including males and females).
  • the sample to be tested is a sample from a normal individual.
  • the sample to be tested is a sample from a fever subject.
  • the sample to be tested is a sample from a pregnant individual.
  • the exosomal nucleic acid comprises exosomal nucleic acid of a pregnant woman's own origin, fetal exogenous nucleic acid of the pregnant woman, or a combination thereof.
  • the sample to be tested is a sample from a pregnant individual
  • the obtained exosomal nucleic acid contains not only the exosome nucleic acid derived from the pregnant woman itself, but also the fetal-derived exosomal nucleic acid in the pregnant woman.
  • the analysis of exogenous nucleic acid in pregnant women can be used to judge the pathogen infection of pregnant women and fetuses.
  • the exosomal nucleic acid comprises fetal-derived exocytic DNA.
  • the exosomal nucleic acid is explant DNA derived from the placenta and the gestational tissue.
  • the microorganism is selected from the group consisting of a virus, a bacterium, a fungus, a parasite, a Chlamydia, a Mycoplasma, or a combination thereof.
  • the microbial detection result includes the kind of the microorganism, and the quantity or abundance of the microorganism.
  • the sample to be tested is selected from the group consisting of a blood sample, or a body fluid sample.
  • the blood sample is selected from the group consisting of plasma, serum, or a combination thereof.
  • the body fluid sample is selected from the group consisting of urine, saliva, pleural effusion, cerebrospinal fluid, sweat, amniotic fluid, cell culture fluid, or a combination thereof.
  • the blood sample is a pretreated blood sample.
  • the blood sample is a supernatant obtained by centrifuging a blood sample.
  • the supernatant is prepared in a two-step process:
  • step (a) exosomes are first isolated from the sample to be tested, and then exosomal nucleic acids are obtained or prepared from the exosomes.
  • step (a) the separating comprises the steps of:
  • the separation employs a magnetic bead separation method, an affinity separation method, or a combination thereof.
  • the magnetic bead separation method is performed using magnetic beads having a surface labeled with a CD63 antibody or a PLAPI antibody.
  • the isolating comprises sorting or capturing using a specific antibody against at least one of the following antigens: PLAP, CD9, CD63, or CD81.
  • the sequencing comprises high throughput sequencing, and more preferably, sequencing using the BGISEQ series or the MGISEQ series sequencing platform.
  • step (c) the following sub-steps are included:
  • the predetermined alignment parameter is 3 bases maximum fault tolerance, or a maximum of 3 base insertions or deletions.
  • the predetermined fault tolerance is 15% to 25%.
  • step (c) when the first species is human, the nucleic acid sequence information of the first species is the Hg19 genomic sequence.
  • step (d) the microbial species classification is performed using the Kraken method (e.g., Kraken-0.10.5-beta version).
  • step (d) the classification of the microbial species using the Kraken method comprises:
  • step (d2) interrupting each of the sequences into segments of a predetermined length K-mer based on the removed processed sequence data obtained in step (c);
  • the K-mer database includes sequence data for the following species: human genome, bacteria/archaea, virus, fungus, protozoa, plasmid.
  • the method further comprises, for the step (c), performing a genetic detection analysis on the excluded nucleic acid sequence corresponding to the first species, thereby obtaining a corresponding The genetic test results of a species.
  • the genetic test results corresponding to the first species include: non-invasive prenatal genetic test results.
  • the non-invasive prenatal genetic test result may be a condition in which the fetal chromosome genetic abnormality in the pregnant woman's abdomen is reflected, such as fetal chromosome aneuploidy, fetal chromosome microdeletion, fetal chromosome microduplication, and the like.
  • the invention provides a system for determining microorganisms in a sample to be tested.
  • the system includes: a nucleic acid separation device that separates the sample to be tested to obtain an exosomal nucleic acid, wherein the sample to be tested is from a first species; a sequencing device, the sequencing device and The exosomal nucleic acid separating device is connected, the sequencing device sequencing the exosomal nucleic acid to obtain a sequencing result composed of nucleic acid sequence data; and the data screening device, the data screening device is connected to the sequencing device, The data screening device excludes a nucleic acid sequence corresponding to the first species from the sequencing result based on nucleic acid sequence information of the first species, thereby obtaining removed sequence data; and a data analysis device, a data analysis device is coupled to the data screening device, the data analysis device comparing the removed processed sequence data with a nucleic acid sequence of a microbial database, and performing microbial species classification to obtain microbial detection in the sample
  • the exosome nucleic acid is selected from the group consisting of exosomal DNA, exosomal RNA, or a combination thereof; preferably exosomal DNA.
  • the first species is selected from the group consisting of mammals, birds or reptiles.
  • the mammal is preferably a human.
  • the sample to be tested is a sample from a normal individual, a sample of a fever individual, or a sample from a pregnant individual.
  • the exosomal nucleic acid comprises exosomal nucleic acid derived from a pregnant woman, exogenous nucleic acid derived from a fetus of a pregnant woman, or a combination thereof.
  • the exosomal nucleic acid comprises fetal-derived exosomal DNA.
  • the exosomal nucleic acid is explant DNA derived from the placenta and the gestational tissue.
  • the microorganism is selected from the group consisting of a virus, a bacterium, a fungus, a parasite, a Chlamydia, a Mycoplasma, or a combination thereof.
  • the sample to be tested is selected from the group consisting of a blood sample, or a body fluid sample.
  • the blood sample is selected from the group consisting of plasma, serum or a combination thereof.
  • the body fluid sample is selected from the group consisting of urine, saliva, pleural fluid, cerebrospinal fluid, sweat, amniotic fluid, cell culture fluid, or a combination thereof.
  • the nucleic acid separation device comprises:
  • An exosomal separation unit that separates exosomes from the sample to be tested.
  • nucleic acid extraction unit connected to the exosomal separation unit, the nucleic acid extraction unit extracting a nucleic acid from the isolated exosomes.
  • the exosomal separation unit separates the exosomes from the sample to be tested by the following method: magnetic bead separation, affinity separation, or a combination thereof.
  • the isolating comprises sorting or capturing using a specific antibody against at least one of the following antigens: PLAP, CD9, CD63, or CD81.
  • the magnetic bead separation method is performed using magnetic beads having a surface labeled with a CD63 antibody or a PLAPA antibody.
  • the sequencing device comprises a high throughput sequencing device; preferably, the sequencing device comprises a BGISEQ series or a MGISEQ series sequencing device.
  • the data screening device comprises:
  • the species data removal unit compares the sequencing result with the nucleic acid sequence information of the first species according to a predetermined alignment parameter, and removes the sequence on the alignment, thereby obtaining an unaligned a sequence, thereby obtaining first pre-processed sequence data;
  • the joint data removing unit is connected to the species data removing unit, and the joint data removing unit compares the first pre-processed sequence data with a sequencing joint sequence according to a predetermined fault tolerance ratio, thereby Truncating the linker sequence in the sequence to obtain the second preprocessed sequence data;
  • the short sequence removing unit removes a sequence having a length less than L from the second pre-processed sequence data according to a length L, thereby obtaining the sequence data of the removed processing, wherein L is 18- A positive integer of 25.
  • the predetermined alignment parameter in the species data removal unit is a maximum fault tolerance of 3 bases, or a maximum of 3 base insertions or deletions.
  • the predetermined fault tolerance rate in the joint data removal unit is 15%-25%.
  • the nucleic acid sequence information of the first species is the Hg19 genomic sequence.
  • the Kraken method is used for microbial species classification.
  • the Kraken method is used to classify microbial species, including the following units:
  • a database construction unit that constructs a K-mer database based on known genomic data
  • segment generating unit being connected to the K-mer database building unit, the segment generating unit interrupting each of the sequences based on the removed processed sequence data obtained by the data filtering unit a fragment of a predetermined length K-mer;
  • the segment aligning unit is connected to the segment generating unit, the segment aligning unit compares the segment of the predetermined length K-mer with a K-mer database, and further according to a recent common ancestor
  • the species is classified by species in principle.
  • the K-mer database includes sequence data for the following species: human genome, bacteria/archaea, virus, fungus, protozoa, plasmid.
  • 1 is a flow diagram of exosomal non-human sequence analysis provided in accordance with an embodiment of the present invention.
  • Figure 2 is a schematic illustration of the reverse mixing provided in accordance with an embodiment of the present invention.
  • FIG. 3 is a schematic diagram of a system for determining microorganisms in a sample to be tested, in accordance with an embodiment of the present invention.
  • FIG. 4 is a schematic diagram of the data screening device provided in accordance with an embodiment of the present invention.
  • Fig. 5 is a schematic view showing the structure of a microbial species classification by the Kraken method according to an embodiment of the present invention.
  • the terms “installation”, “connected”, “connected”, “fixed” and the like shall be understood broadly, and may be either a fixed connection or a detachable connection, unless explicitly stated and defined otherwise. Or in one piece; it may be a mechanical connection, or it may be an electrical connection or a communication with each other; it may be directly connected or indirectly connected through an intermediate medium, and may be an internal connection of two elements or an interaction relationship between two elements. Unless otherwise expressly defined. For those skilled in the art, the specific meanings of the above terms in the present invention can be understood on a case-by-case basis.
  • first and second are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated.
  • features defining “first” or “second” may include at least one of the features, either explicitly or implicitly.
  • the meaning of "a plurality” is at least two, such as two, three, etc., unless specifically defined otherwise.
  • the first feature "on” or “under” the second feature may be a direct contact of the first and second features, or the first and second features may be indirectly through an intermediate medium, unless otherwise explicitly stated and defined. contact.
  • the first feature "above”, “above” and “above” the second feature may be that the first feature is directly above or above the second feature, or merely that the first feature level is higher than the second feature.
  • the first feature “below”, “below” and “below” the second feature may be that the first feature is directly below or obliquely below the second feature, or merely that the first feature level is less than the second feature.
  • the present invention has been completed on the basis of the following findings of the inventors:
  • the inventors have extensively and intensively studied, for the first time, surprisingly found that various pathogenic microorganisms (bacteria, fungi, viruses) can be detected by isolating or enriching exogenous DNA of pregnancy. , parasites, etc.), compared to conventional plasma cfDNA (cell free DNA) methods, the present invention based on exosome DNA methods to detect more species, higher abundance.
  • Information for the detection of unknown pathogenic microorganisms in clinical patients eg, febrile populations or pregnant populations
  • the present invention has been completed on this basis.
  • the present invention utilizes exosomal nucleic acid to achieve microbial information enrichment in an individual, and provides a method of confirming microorganisms in a biological individual, comprising the steps of: (a) from said Ex vivo nucleic acid containing the individual genetic information and microbial genetic information is isolated from the biological individual; (b) sequencing the exosomal nucleic acid to obtain sequencing results consisting of nucleic acid sequence data; (c) based on the Nucleic acid sequence information of the biological individual, excluding the nucleic acid sequence corresponding to the individual from the sequencing result, thereby obtaining the removed processed sequence data; and (d) nucleic acid of the removed processed sequence data and the microorganism database The sequences are aligned and microbial species are classified to obtain microbial detection results in the sample.
  • the present invention whether expressed as a first species or as a biological individual, or a biological sample, or an individual, etc., it is indicated that the present invention can diagnose and confirm microorganisms present in a living body.
  • exosomes refers to extracellular nanoscale vesicles (30-100 nm) formed by a series of regulatory processes such as “endocytosis-fusion-exclusion” of living cell secondary endosomes, carried. Inclusions such as RNA, proteins, and lipids have the functions of regulating gene transcription and expression, and can realize signal transduction between cells and between individuals. Almost all types of cells secrete exosomes, including: serum, plasma, urine, cell culture supernatants (immune system cells, tumor cells), milk, saliva, ascites, amniotic fluid, tracheal alveolar lavage fluid, joint slip liquid. Separation of exosomes can be achieved by density gradient centrifugation, and a cup-like structure unique to exosomes can be observed under a transmission electron microscope.
  • the change in exosome content during pregnancy is related to gestational age: compared with non-pregnant women, the exogenous secretion of peripheral blood in pregnant women with normal pregnancy is significantly increased by about 20-50 times, and the exogenous content increases with gestational age. rise.
  • the fetal exosomes in the peripheral blood of pregnant women can be detected at 5-6 weeks of gestation, and the number also increases rapidly with the increase of gestational age, suggesting that the placenta matures and exchanges between maternal and fetal. Active, the release of placental-specific exosomes into the maternal circulation is also increased.
  • Total exosomes separation method SBI company based on the principle of precipitation centrifugation, Thermo Fisher based on immunomagnetic beads separation; the present invention combines the two, using SBI company reagent to obtain a large amount of exosomes, and through the immunomagnetic beads method The former is not pure enough.
  • enrichment of plasma total vesicles ie microvesicels (or other undefined types of extracellular vesicles, as well as platelet nucleic acids) and exosomes, may also be achieved and excreted.
  • a similar pathogenic microbial enrichment effect may also be achieved.
  • the detection of plasma exogenous pathogenic microorganisms can be useful in assessing the efficacy of tumor immunotherapy.
  • plasma exocytogenic pathogen detection can be used for pathogen monitoring after transplantation.
  • exosomes are an intercellular communication method
  • the detection of pathogenic microorganisms in the DNA contents reflects the infection of pathogenic microorganisms in the human body. This information can be used for the detection of unknown pathogenic microorganisms in clinical patients (such as fever) and during pregnancy. Common disease screening and risk prediction in the population.
  • capture agent As used herein, the terms “capture agent”, “sorting agent” are used interchangeably and refer to an agent that captures or enriches or sorts exosomes from a blood or body fluid sample.
  • a typical capture agent comprises (a) a specific antibody to at least one of the following antigens: PLAP, CD9, CD63, or CD81; or (b) magnetic beads coupled to the antibody.
  • a specific antibody to at least one of the following antigens: PLAP, CD9, CD63, or CD81
  • magnetic beads coupled to the antibody.
  • Exoquick reagents and/or immunomagnetic beads can be employed that are capable of specifically recognizing CD63 and/or PLAP (placental alkaline phosphatase, placental alkaline phosphatase).
  • Immunomagnetic beads are spherical magnetic particles that are conjugated with monoclonal antibodies on the surface of magnetic beads (for example, can be coupled by a streptomycin-avidin system), by antibodies and exosome surface antigens (eg CD9, CD63, CD81) Interacting specifically captures plasma total exosomes; it is also possible to isolate tissue- and organ-specific exosomes by a certain type of tissue-organized antigen (such as PLAP), and to detect its contents by detecting its contents. Improve the sensitivity and specificity of disease detection.
  • exosomal nucleic acid refers to a nucleic acid component in an exosomes.
  • exosomal DNA refers to the DNA component of the exosomes
  • exosomal RNA refers to the RNA component of the exosomes.
  • the present inventors have found through research that the total exosomal DNA extracted from the peripheral blood of pregnant women contains both maternal-derived (maternal) DNA and fetal-derived (fetal-derived) DNA, and the fetal DNA is It exists as a free DNA fragment.
  • placenta extracted from maternal peripheral blood and exosomes derived from pregnant tissues contain both maternal-derived (maternal) DNA and fetal sources (fetals). Source) DNA.
  • exosomes released from placental trophoblast cells in maternal blood during pregnancy which contain fetal-derived free DNA fragments; are detected by high-throughput sequencing for pregnancy diseases Screening and risk prediction.
  • exosomes derived from lymphocytes can be extracted by chromatographic analysis and immunoadsorption in peripheral blood of non-pregnant women, and The placenta-specific antibody PLAP quantification can be found in the peripheral blood of pregnant women, except for exosomes derived from lymphoids, which can also detect fetal exosomes, which prove that exosomes in maternal blood during pregnancy are mixed with maternal and fetal sources. .
  • the inventors have demonstrated in vitro and in vivo experiments that the fetal exosomes in maternal blood are mainly synthesized and released into maternal blood by placental trophoblast cells.
  • placental trophoblast cells the primary endosomes develop into mature nucleus through the invagination of the membrane, and then some enter the lysosome, and the other part carries a large number of signal molecules (DNA, mRNA, miRNA, protein, etc.).
  • the exosomes are formed and released into the extracellular matrix into the maternal blood by membrane fusion.
  • exocytogen can participate in immunity by actively wrapping tissue-specific inclusions and releasing them into the maternal circulation. Regulation of important processes during pregnancy, such as tolerance, maternal-fetal remodeling, and inflammatory response. In addition, since the exosomes have a stable bilayer lipid membrane, it seems to be more helpful in maintaining the stability of the fetal DNA.
  • the present invention provides a method for microbial detection based on exosomes in pregnant women.
  • the method comprises the steps of: (a) isolating exosomal nucleic acid from the pregnant woman; (b) sequencing the exosomal nucleic acid to obtain a nucleic acid data (c) removing nucleic acid sequences from pregnant women and fetuses from the sequencing results to obtain removed processed sequence data; (d) removing the processed sequence data from the nucleic acid sequence of the microbial database The alignment is performed to obtain microbial detection results.
  • the method of the present invention can be used not only to determine the type and proportion of pathogenic microorganisms in a pregnant woman, but also to determine the type of beneficial microorganisms in the pregnant woman, and thus can be used to indicate the physical condition of the pregnant woman. Therefore, it can be used to indicate the diagnosis of clinical medication or clinical disease, and can also carry out nutritional health care or enhance the body's disease resistance or improve physical fitness.
  • the microorganisms in the pregnant woman determined by the method can be used for assisting clinical diagnosis, providing reference for doctors or dietitians, etc., and importantly, can also be used for other non-diagnostic purposes.
  • the present invention provides a method for detecting pathogenic microorganisms based on exosomes in peripheral blood of pregnant women.
  • the method of the invention comprises the steps of:
  • peripheral blood of pregnant women can be collected by a conventional method, and then plasma or serum is separated.
  • Peripheral blood e.g., about 2-20 ml, preferably 3-10 ml
  • Plasma or serum separation can be performed using a two-step centrifugation method.
  • the isolate containing the total exosomes can be obtained by centrifugation or the like.
  • purification can be carried out, for example by magnetic bead separation, by immunomagnetic beads loaded with CD63 antibody.
  • the DNA can be extracted and the DNA detected.
  • a high-throughput sequencing library can be constructed, then sequenced and analyzed to detect the type of pathogenic microorganisms.
  • a typical method of the invention includes the steps of:
  • the first step separation of plasma total exosomes from blood samples.
  • total plasma exosomes can be separated using known methods or reagents, such as the commercially available SBI company SBI quick reagent;
  • the second step purification of the plasma total exosomes.
  • the exosomal precipitate produced in the first step is dissolved in PBS buffer, it is purified with anti-CD63 immunomagnetic beads, for example, at a certain temperature (eg, 4-8 degrees) for a period of time (eg, 2-24 hours, or Incubate overnight) to form a "magnetic bead-exosome binary complex".
  • Anti-CD63 immunomagnetic beads can be prepared by conventional methods or commercially available, for example, Thermo Fisher CD63 immunomagnetic beads;
  • Step 3 Exosome DNA extraction of the "magnetic bead-exosome binary complex" formed in the second step, followed by database construction (enhanced PCR cycle number), and/or sequencing to obtain the nucleic acid sequence Sequencing data composed of information;
  • Step 4 Analyze the information of the sequencing data to obtain the corresponding analysis results.
  • the results of the sequencing are subject to quality control, and after the human sequence is removed, the species classification is performed on the basis of the existing microbial database.
  • Species classification can be achieved, for example, by the Kraken method. Based on the specific k-mer database, the sequence data can be quickly classified and calculated. Based on the Kraken classification results, the microbial species level can be sorted and statistically analyzed. The Kraken results can also be processed by Krona to obtain more detailed results. figure 1.
  • a typical method of the invention includes the steps of:
  • the first step separation of plasma total exosomes from blood samples.
  • total plasma exosomes can be separated using known methods or reagents, such as the commercially available SBI company SBI quick reagent;
  • the second step enrichment or capture of placenta and fetal tissue-derived plasma exosomes from the plasma total exosomes.
  • the total exosome precipitate generated in the first step is dissolved in PBS buffer, it is captured or enriched with anti-PLAP immunomagnetic beads, for example, incubation at a certain temperature (eg, 4-8 degrees) for a period of time (eg, 2- 24 hours, or overnight incubation) to form a "magnetic bead-exosome binary complex.”
  • Anti-PLAP immunomagnetic beads can be prepared by conventional methods or commercially available, for example, Thermo Fisher PLAP immunomagnetic beads;
  • Step 3 Exosome DNA extraction of the "magnetic bead-exosome binary complex" formed in the second step, followed by database construction (enhanced PCR cycle number), and/or sequencing to obtain the nucleic acid sequence Sequencing data composed of information;
  • Step 4 Analyze the information of the sequencing data to obtain the corresponding analysis results.
  • the results of the sequencing are subject to quality control, and after the human sequence is removed, the species classification is performed on the basis of the existing microbial database.
  • Species classification can be achieved, for example, by Kraken software. Based on the specific k-mer database, the sequence data can be quickly classified and calculated. Based on the Kraken classification results, the microbial species level can be sorted and statistically analyzed. Kraken's results can also be processed with Krona to obtain more detailed results.
  • a typical Kraken method consists of steps:
  • the K-mer database required for Kraken classification needs to be constructed based on genomic data.
  • the genomic data used includes: human genome (GRCh38), bacteria/ Egyptian bacteria, viruses, fungi, protozoa, plasmids, except for human data.
  • the data is from the Refseq database of NCBI;
  • each sequence of reads needs to be broken into segments of K-mer of a specific length, and then all K-mers of the sequence of reads are compared back to the K-mer database, according to the most recent common ancestor (LCA) classifies the sequence of reads; and
  • read sequence is also referred to as “reads” and refers to the nucleic acid sequence generated at the end of each round of sequencing reactions.
  • the original sequencing data obtained after high-throughput sequencing is the original read sequence, and those skilled in the art can filter the original read sequence as needed, for example, remove the low-quality read sequence, remove the linker sequence, and the like. It can be understood that for a sequencing library, a large number of read sequences are generated after sequencing, and those skilled in the art can assemble the genome according to the overlapping relationship between the read sequences.
  • the “Kraken” described herein is a microbial metagenomic data analysis software that enables high-sensitivity and high-speed short DNA sequence classification by using k-mers' precise alignment and new classification algorithms. For more information about Kraken, see http://ccb.jhu.edu/software/kraken/.
  • K-mer refers to dividing reads into strings containing k bases.
  • a pair of reads of length m can be divided into (m-k+1) k-mers, and m and k are both greater than A positive integer of 1 and m is related to the sequencing platform and sequencing strategy, for example SE90 sequencing using the BGISEQ500 sequencer, m is 90.
  • a reads sequence is: ATCGTTGCTTAATGACGTCAGTCGAATGCGATGACGTGACTGACTG, which needs to be divided into a 13-base string, 13-mer, which can be cut into the following fragment forms:
  • K-mer database refers to a database composed of K-mer data.
  • the K-mer data can be from any open source database, such as NCBI's Refseq database, which includes bacterial/paleobacterial data, viral data, fungal data, protozoan data, and the like.
  • the present invention provides a system for determining microorganisms in a sample to be tested, as shown in FIG.
  • the system includes: a nucleic acid separation device, a sequencing device, a data screening device, and a data analysis device, wherein the nucleic acid separation device separates the sample to be tested to obtain an exosomal nucleic acid, wherein the sample to be tested is from a first species; the sequencing device is coupled to the exosome nucleic acid separation device, the sequencing device sequencing the exosomal nucleic acid to obtain a sequencing result consisting of nucleic acid sequence data; the data screening device and the Connected to a sequencing device, the data screening device excluding nucleic acid sequences corresponding to the first species from the sequencing results based on nucleic acid sequence information of the first species, thereby obtaining removed sequence data; a data analysis device is coupled to the data screening device, the data analysis device comparing the removed processed sequence data with a nucleic acid sequence of a microbial database,
  • the data screening apparatus of the present invention further includes: a species data removing unit, a joint data removing unit, and a short sequence removing unit, wherein the species data removing unit performs the sequencing according to a predetermined alignment parameter Comparing with the nucleic acid sequence information of the first species, removing the aligned sequences, thereby obtaining unaligned sequences, thereby obtaining first pre-processed sequence data; the linker data removal unit and the species The data removal unit is connected, and the linker data removing unit compares the first pre-processed sequence data with the sequencing linker sequence according to a predetermined fault tolerance rate, thereby truncating the linker sequence in the sequence from the first pre-processed sequence data, and further Obtaining second pre-processed sequence data; the short sequence removing unit removes a sequence of length less than L from the second pre-processed sequence data according to a length L, thereby obtaining the sequence data of the removed process, wherein L A positive integer of
  • the kraken method is used for microbial species classification
  • the data analysis device may further include the following units: a database construction unit, a segment generation a unit and a segment aligning unit, wherein the database building unit constructs a K-mer database based on known genomic data; the segment generating unit is coupled to the K-mer database building unit, and the segment generating unit filters based on the data Destroying the sequence data obtained by the unit, interrupting each of the sequences into segments of a predetermined length K-mer; the segment alignment unit is connected to the segment generation unit, and the segment comparison unit will The fragments of the predetermined length K-mer are compared with the K-mer database, and the sequences are species classified according to the principle of the recent common ancestor.
  • the method of the present invention does not require microbial culture and can detect unknown pathogens
  • the method for detecting pathogenic microorganisms based on exosomal DNA of the present invention has higher sensitivity and wider detection range than conventional plasma cfDNA method;
  • the invention is based on the enrichment of pathogenic microorganisms in exogenous DNA during pregnancy, and greatly improves the sensitivity and detection range of detecting pathogenic microorganisms based on plasma metagenomics;
  • the invention can detect intrauterine infection by separating the placenta and the explants of the pregnant tissue. Compared with the traditional amniocentesis, the invention is a non-invasive detection method, and is simple and easy to perform without abortion risk.
  • peripheral blood standard collection operation 10 cases of pregnant women's peripheral blood were collected, and 10 mL of blood was collected for each case. After blood collection, please immediately reverse the inversion 10 times to mix the blood and the components in the tube (Fig. 2). Delaying the mixing time may cause The test failed. After mixing, place the blood collection tube upright on the test tube rack (6-35 ° C).
  • step 3.4 The precipitate produced in step 3.3 was thawed in 100 ⁇ L of PBS buffer at 37 ° C, and the obtained solution was labeled as "SBI exosomes" (Note: The SBI exosomes obtained here were obtained using the SBI exosome preparation kit). The total exosomes were distinguished from the exosomes obtained in the subsequent Examples 2 and 3 and the comparative examples, which the inventors labeled as SBI exosomes).
  • the "SBI exosomes" solution obtained in step 3.4 was subjected to total exosomal DNA extraction using the Magen MagPure Circulating DNA Mini KF Kit, and the procedure was carried out in accordance with the kit instructions.
  • the obtained DNA was labeled as "SBI exosomal DNA”.
  • the SBI exosomal DNA obtained in the step 4 was subjected to library construction according to the method disclosed in the patent application "Method for constructing a sequencing library based on blood samples and its use in determining fetal genetic abnormality" (Application Publication No. CN105400864 A) High-throughput sequencing (where the number of PCR cycles at the time of library construction was 19, and other steps and parameters were consistent with those disclosed in the above patent application), and sequencing data was obtained.
  • Kraken Kraken-0.10.5-beta
  • the genomic data used include: human genome (GRCh38), bacteria/archaea, virus , fungi, protozoa, plasmids, except for human data, the rest of the data comes from NCBI's Refseq database.
  • GRCh38 human genome
  • bacteria/archaea bacteria/archaea
  • virus virus
  • fungi fungi
  • protozoa plasmids
  • plasmids except for human data
  • the rest of the data comes from NCBI's Refseq database.
  • each reads need to be broken into fragments of K-mer of specific length, and then all K-mers of the reads are compared back to the K-mer database, and according to the recent common ancestor (LCA)
  • LCA recent common ancestor
  • the classification of microorganisms was statistically compiled, and the results of the classification can also be visualized using Krona (KronaTools-2.5) tools to obtain more detailed results.
  • Count the number of readouts of various types of microorganisms in each sample and the number of microbial detection reads (the number of microbes detected reads is the sum of bacteria, viruses, archaea, and fungus detected reads), and calculate the microbial coefficient, microbial coefficient microorganism
  • M number of reads/sequence total reads
  • Results The results of microbial detection based on the SBI exosome DNA method are shown in Table 1. It can be seen from Table 1 that the corrected microbial coefficient based on the SBI exosome DNA method was 148.50 after being corrected by the sequencing data.
  • Example 1 10 pregnant women's peripheral blood were collected (samples are the same as in Example 1), plasma separation was performed using a two-step method, and plasma total exosomes were separated using SBI System Biosciences ExoQuick Exosome Precipitation Solution reagent, the same procedure as in Example 1.
  • the "SBI exosomes” obtained in step 3.4 were purified using Thermo Fisher Exosome-Human CD63 magnetic beads. Specific steps are as follows:
  • step 3.7 and the SBI exosome mixture 3000g were briefly centrifuged for 5s, 500 ⁇ L of separation buffer was added, placed in a magnetic stand for 2min, and the supernatant was aspirated;
  • CD63 exosomes obtained here are first Total exosomes obtained by purification of total exosomes using CD63 magnetic beads after total exosomes were obtained using the SBI exosome preparation kit, exogenously obtained for Examples 1, 3 and Comparative Examples The body was distinguished and the inventors labeled it as a CD63 exosome).
  • CD63 exosomes obtained in the step 3.9 were subjected to total exosomal DNA extraction using a Magen MagPure Circulating DNA Mini KF Kit, and the operation was carried out in accordance with the kit instructions.
  • the obtained DNA was labeled as "CD63 exosome DNA”.
  • results of microbial detection based on the CD63 exosome DNA method are shown in Table 1. As can be seen from Table 1, the corrected microbial coefficient based on the CD63 exosome DNA method was corrected to 6658.87 after being corrected by the sequencing data. It is indicated that the CD63-based exosomal DNA method can enrich microorganisms to a large extent compared with the plasma free nucleic acid method and the SBI exosome method, and the enrichment effect is extremely remarkable.
  • Example 2 10 cases of pregnant women's peripheral blood were collected (samples are the same as in Example 1), and the method disclosed in the patent application "Method for constructing a sequencing library based on blood samples and its use in determining fetal genetic abnormality" (Application Publication No. CN105400864A) Plasma free nucleic acid library construction and high throughput sequencing were performed. Finally, the obtained sequencing data is analyzed, and the specific analysis steps are the same as those in the first embodiment.
  • results and analysis The results of microbial detection based on the plasma free nucleic acid method are shown in Table 1 (the sample in the sample of Table 1 is "sample 1 plasma - sample 10 plasma” represents the microbial detection result using this comparative method) .
  • the corrected microbial coefficient based on the plasma free nucleic acid method was 68.10 after being corrected by the sequencing data.
  • sample 1 a sample of pregnant women with peripheral blood and amniotic fluid who had been diagnosed with hepatitis B virus infection (which has been diagnosed by the existing hepatitis B antigen immunoassay) was collected as sample 1; 3 cases of undiagnosed hepatitis B virus were collected (received now) Some pregnant women with peripheral blood and amniotic fluid samples confirmed by hepatitis B antigen immunoassay were used as negative controls and labeled as samples 2-4.
  • Each sample was simultaneously tested for pathogenic microorganisms based on the SBI exosome method, the PLAP exosome method, the conventional plasma free nucleic acid method, and the conventional amniotic fluid cell method.
  • the amniotic fluid cells are roughly centrifuged first, then the amniotic fluid DNA is extracted, and finally the DNA library construction and high-throughput sequencing are performed, and the obtained sequencing data is obtained.
  • the analysis method is the same as in the first embodiment.
  • Plasma separation was performed using a two-step method, and then plasma total exosomes were separated using SBI System Biosciences ExoQuick Exosome Precipitation Solution reagent, the same procedure as in Example 1.
  • the "SBI exosomes" obtained in step 3.4 were purified using Thermo Fisher Exosome-Human PLAP magnetic beads. Specific steps are as follows:
  • step 3.7 and the SBI exosome mixture 3000g were briefly centrifuged for 5s, 500 ⁇ L of separation buffer was added, placed in a magnetic stand for 2min, and the supernatant was aspirated;
  • PLAP exosomes obtained here are first Obtained by obtaining the total exosomes using the SBI exosome preparation kit, and then using the PLAP magnetic beads to capture the explants of the placenta and the pregnancy tissue, for exogenous obtained with Examples 1, 2 and Comparative Examples The body is distinguished and the inventors mark it as a PLAP exosomes).
  • the "PLAP exosomes" obtained in step 3.9 were subjected to placental and excised tissue-derived exosomal DNA extraction, and the operation was carried out in accordance with the kit instructions.
  • the obtained DNA was labeled as "PLAP exosomal DNA”.
  • results and analysis The microbial detection results of each sample are shown in Table 2. It can be seen from Table 2 that hepatitis B virus can be detected in both plasma and amniotic fluid of sample 1, indicating that hepatitis B virus is infected in pregnant women and their uterus; in sample 1 based on PLAP exosomes, a large amount of hepatitis B virus can be detected. Intrauterine infection is indirectly reflected by detecting microbes from the placenta and exocrine-derived exosomes. In the negative control sample 2-3, no hepatitis B virus was detected in plasma and amniotic fluid, and no hepatitis B virus was detected in the method based on PLAP exosomes, indicating that no false positive was detected.

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Abstract

本发明提供了基于外泌体核酸进行微生物检测的方法及其应用。所述方法包括：(a)从待测样品中分离获得外泌体核酸，所述待测样品来自于第一物种；(b)对所述外泌体核酸进行测序，获得测序结果；(c)从所述测序结果中排除对应于所述第一物种的核酸序列，从而获得经去除处理的序列数据；和(d)将所述经去除处理的序列数据与微生物数据库的核酸序列进行比对，获得所述样品中的微生物检测结果。

Description

基于外泌体核酸进行微生物检测的方法及其应用
本申请要求于2017年04月18日提交的申请号为201710254374.X的中国专利申请的权益，并将其全部引入本文。
技术领域
本发明涉及微生物检测技术领域，具体地，涉及基于外泌体核酸进行微生物检测的方法。
背景技术
人类发生感染，其中一个重要原因在于被病原微生物侵入人体组织，直接或间接造成对人体的伤害；这些病原微生物包括细菌、真菌、病毒、寄生虫等。当前，由未知、新发病原体引发的疾病负担在日益加重，这对人类的生命健康安全造成了越来越严重的威胁。实现对未知病原微生物的快速准确检测是临床上病原微生物的准确鉴定对于临床正确诊断与有效防治至关重要，孕期感染(如我国孕前阶段广泛开展的TORCH筛查)是导致早产、流产、妊娠期高血压等常见疾病的重要因素。
传统的病原微生物鉴定技术主要分为两类:基于细胞培养的方法例如形态学观察、细胞生理生化特征、细菌培养分型、基因芯片、自动化微生物分析系统等,和基于特异引物/探针/抗体的方法例如抗原抗体反应、PCR反应检测以及各种特异性的病原微生物快速检测系统等。这些技术在日常病原微生物确证中发挥了重要作用，但也存在一定的不足，如前者需要依赖培养、周期长、鉴定精度低，后者需要一定的微生物序列先验知识、无法应对未知或突变病原体等。
传统对于病原微生物的鉴定主要依靠分离培养、PCR等技术实现的，但是这些技术都存在花费时间长以及鉴定数目有限等局限性，而通过高通量测序进行病原鉴定却一定程度上克服了这些局限。随着测序技术的提高以及成本的下降，通过高通量测序技术对临床样本进行病原微生物宏基因组学鉴定逐渐流行起来。宏基因组学，是对环境中所有微生物基因组集合的研究技术和方法。根据研究方法的不同，宏基因组学可以分为核糖体rDNA的分类和鉴定，以及全部宏基因组DNA序列的分析鉴定。
进行病原鉴定的样本种类繁多，比如鼻咽拭子，痰液，尿液，血液(全血，血清，血浆)等，而相对鼻咽拭子、痰液、尿液，血液由于受外界环境干扰较小，存在污染的可能性较小，更适合作为病原鉴定的样本。用血液进行病原鉴定的方法通常是提取血浆中的游离DNA，经过PCR扩增，然后通过高通量测序的方法对这些游离DNA进行测序。由于数据 中存在很多人的序列，所以需要对人的序列进行去除，减少对下一步微生物分类的干扰，处理后的序列结果再和已有的微生物数据库进行比对(比如细菌，病毒，真菌等)，针对比对的注释结果再进行微生物水平的鉴定，以及病原微生物的判断。
因此，对于个体中的微生物的检测手段，仍然需要进一步改进。
发明内容
本发明的发明人在研究过程中发现：借助于高通量测序技术，利用血液样本进行病原鉴定，由于血液中微生物序列的比例远比检测个体(如人)的序列比例要低的多，所以常常会出现测序结果中人的比例较高而微生物比例较低，测序饱和度远远不够的问题。基于此，本发明的目的在于提供一种基于外泌体DNA的高准确性和高灵敏度的病原微生物检测方法。该方法可以使得微生物的序列在一定程度上得到富集，从而可以显著提高测序过程中微生物测序信息的准确性，从而使得微生物检测结果的准确性有很大程度的提升。而且，通过借助于外泌体就可以实现这种富集作用，简单方便。
在本发明的第一方面，提供了一种检测待测样品中的微生物的方法，所述方法包括如下步骤：
(a)对所述待测样品进行分离，从而获得外泌体核酸，其中所述待测样品来自于第一物种；
(b)对所述外泌体核酸进行测序，获得由核酸序列数据组成的测序结果；
(c)基于所述第一物种的核酸序列信息，从所述测序结果中排除对应于所述第一物种的核酸序列，从而获得经去除处理的序列数据；和
(d)将所述经去除处理的序列数据与微生物数据库的核酸序列进行比对，并进行微生物物种分类，从而获得所述样品中的微生物检测结果。
本发明中所述微生物数据库来源于已有的微生物数据库，且这些数据库并不是一成不变的，可以随着技术发展不断补充和完善。根据需要，微生物数据库可以使用来自于NCBI的Refseq数据，其包括细菌/古细菌数据、病毒数据、真菌数据、原虫数据、质粒数据等。
利用本发明检测待测样品中的微生物的方法可以用来确定某一个体或者某一物种体内的微生物。而且该方法不仅可以用来检测病原微生物，还可以用来检测个体或者物种中的有益微生物，为个体改善或者调整身体状况或者体质提供借鉴和参考。
在另一优选例中，所述外泌体核酸选自下组：外泌体DNA、外泌体RNA、或其组合；优选为外泌体DNA。
在另一优选例中，所述第一物种选自下组：哺乳动物、鸟类、或爬行动物，更佳地所述哺乳动物为人。当所述第一物种为人时，可以参照hg19，UCSC中的refMrna数据库，和/或炎黄数据库等将人的核酸序列信息去除。
在另一优选例中，所述样品为来自人(包括男性和女性)的样品。
在另一优选例中，所述待测样品为来自正常个体的样品。
在另一优选例中，所述待测样品为来自发热个体的样品。
在另一优选例中，所述待测样品为来自孕期个体的样品。
在另一优选例中，所述外泌体核酸包括孕妇自身来源的外泌体核酸、所述孕妇的胎儿来源的外泌体核酸、或其组合。当所述待测样品为来自孕期个体的样品时，所获得的外泌体核酸中不仅含有孕妇自身来源的外泌体核酸，而且还有孕妇体内的胎儿来源的外泌体核酸，这样通过对孕妇体内的外泌体核酸进行分析，就可以实现对于孕妇和胎儿的病原感染情况的判断。
在另一优选例中，所述外泌体核酸包括胎儿来源外泌体DNA。
在另一优选例中，所述外泌体核酸为胎盘及妊娠组织来源的外泌体DNA。
在另一优选例中，所述微生物选自下组：病毒、细菌、真菌、寄生虫、衣原体、支原体、或其组合。
在另一优选例中，所述微生物检测结果包括微生物的种类、以及所述微生物的数量或丰度。
在另一优选例中，所述待测样品选自下组：血液样品、或体液样品。
在另一优选例中，所述血液样品选自下组：血浆、血清或其组合。
在另一优选例中，所述体液样品选自下组：尿液、唾液、胸腹水、脑脊液，汗液，羊水、细胞培养液、或其组合。
在另一优选例中，所述血液样品是经预处理的血液样品。
在另一优选例中，所述血液样品是将血样离心后，收集得到的上清。
在另一优选例中，所述上清是用两步法制备得到的：
(1)使用收集装置收集血液样品，其中，所述收集装置中含有抗凝血剂，和
(2)对所述样品进行高速离心，从而获得所述上清。
在另一优选例中，在步骤(a)中，包括从所述待测样品中先分离出外泌体，然后从所述外泌体获得或制备外泌体核酸。
在另一优选例中，在步骤(a)中，所述分离包括如下步骤：
(a1)从所述待测样品中分离外泌体；和
(a2)从所述分离的外泌体中提取核酸。
在另一优选例中，所述分离采用磁珠分离法、亲和分离法、或其组合。
在另一优选例中，所述磁珠分离法采用表面标记有CD63抗体或PLAP抗体的磁珠进行分离。
在另一优选例中，所述分离包括使用针对以下至少一种抗原的特异性抗体进行分选或捕获：PLAP、CD9、CD63、或CD81。
在另一优选例中，在步骤(b)中，所述测序包括高通量测序，更佳地，包括使用BGISEQ系列或MGISEQ系列测序平台进行测序。
在另一优选例中，在步骤(c)中，包括以下子步骤：
(c1)按照预定比对参数，将所述测序结果与所述第一物种的核酸序列信息进行比对，去除比对上的序列，从而获得未比对上的序列，进而获得第一预处理序列数据；
(c2)按照预定容错率，将所述第一预处理序列数据与测序接头序列比对，从而从第一预处理序列数据中截去序列中的接头序列，进而获得第二预处理序列数据；和
(c3)按长度L为标准，从所述第二预处理序列数据中去除长度小于L的序列，从而获得所述经去除处理的序列数据，其中L为18-25的正整数。
在另一优选例中，在步骤(c1)中，所述预定比对参数为最大容错3个碱基，或最大允许3个碱基的插入或缺失。
在另一优选例中，在步骤(c2)中，所述预定容错率为15％-25％。
在另一优选例中，在步骤(c)中，当第一物种为人时，所述第一物种的核酸序列信息为Hg19基因组序列。
在另一优选例中，在步骤(d)中，采用Kraken法(如Kraken-0.10.5-beta版本)进行微生物物种分类。
在另一优选例中，在步骤(d)，采用Kraken法进行微生物物种分类包括：
(d1)根据已知基因组数据构建K-mer数据库；
(d2)基于步骤(c)获得的所述经去除处理的序列数据，将每一条所述序列打断为预定长度K-mer的片段；
(d3)将所述预定长度K-mer的片段与K-mer数据库进行比对，进而根据最近共同祖先的原则对所述序列进行物种分类。
在另一优选例中，所述K-mer数据库包括以下物种的序列数据：人基因组、细菌/古细菌、病毒、真菌、原虫、质粒。
在另一优选例中，所述方法还包括：对于步骤(c)中，对于所述被排除的所述对应于所 述第一物种的核酸序列，进一步进行基因检测分析，从而获得对应于第一物种的基因检测结果。
在另一优选例中，所述对应于第一物种的基因检测结果包括：无创产前基因检测结果。所述无创产前基因检测结果可以是反应孕妇腹中胎儿染色体遗传异常的情况，例如胎儿染色体非整倍性、胎儿染色体微缺失、胎儿染色体微重复等。
根据本发明的第二方面，本发明提供了一种确定待测样品中的微生物的系统。所述系统包括：核酸分离装置，所述核酸分离装置对所述待测样品进行分离，获得外泌体核酸，其中，所述待测样品来自于第一物种；测序装置，所述测序装置与所述外泌体核酸分离装置相连，所述测序装置对所述外泌体核酸进行测序，获得由核酸序列数据组成的测序结果；数据筛选装置，所述数据筛选装置与所述测序装置相连，所述数据筛选装置基于所述第一物种的核酸序列信息，从所述测序结果中排除对应于所述第一物种的核酸序列，从而获得经去除处理的序列数据；和数据分析装置，所述数据分析装置与所述数据筛选装置相连，所述数据分析装置将所述经去除处理的序列数据与微生物数据库的核酸序列进行比对，并进行微生物物种分类，从而获得所述样品中的微生物检测结果。
在一优选例中，所述外泌体核酸选自下组：外泌体DNA、外泌体RNA、或其组合；优选为外泌体DNA。
在一优选例中，所述第一物种选自下组：哺乳动物、鸟类或爬行动物。所述哺乳动物优选为人。
在一优选例中，所述待测样品为来自正常个体的样品、发热个体的样品或者是来自孕妇个体的样品。
在一优选例中，所述外泌体核酸包括孕妇自身来源的外泌体核酸、孕妇的胎儿来源的外泌体核酸、或其组合。
在一优选例中，所述外泌体核酸包括胎儿来源外泌体DNA。
在一优选例中，所述外泌体核酸为胎盘及妊娠组织来源的外泌体DNA。
在一优选例中，所述微生物选自下组：病毒、细菌、真菌、寄生虫、衣原体、支原体、或其组合。
在一优选例中，所述待测样品选自下组：血液样品、或体液样品。
在一优选例，所述血液样品选自下组：血浆、血清或其组合。
在一优选例中，所述体液样品选自下组：尿液、唾液、胸腹水、脑脊液，汗液，羊水、细胞培养液、或其组合。
在一优选例中，所述核酸分离装置包括：
外泌体分离单元，所述外泌体分离单元从所述待测样品中分离外泌体；和
核酸提取单元，所述核酸提取单元与所述外泌体分离单元相连，所述核酸提取单元从所述分离的外泌体中提取核酸。
在一优选例中，所述外泌体分离单元采用下组方式从所述待测样品中分离外泌体：磁珠分离法、亲和分离法、或其组合。
在一优选例中，所述分离包括使用针对以下至少一种抗原的特异性抗体进行分选或捕获：PLAP、CD9、CD63、或CD81。
在一优选例中，所述磁珠分离法采用表面标记有CD63抗体或PLAP抗体的磁珠进行分离。
在一优选例中，所述测序装置包括高通量测序装置；优选地，所述测序装置包括BGISEQ系列或MGISEQ系列测序装置。
在一优选例中，所述数据筛选装置包括：
物种数据去除单元，所述物种数据去除单元按照预定比对参数，将所述测序结果与所述第一物种的核酸序列信息进行比对，去除比对上的序列，从而获得未比对上的序列，进而获得第一预处理序列数据；
接头数据去除单元，所述接头数据去除单元与所述物种数据去除单元相连，所述接头数据去除单元按照预定容错率，将所述第一预处理序列数据与测序接头序列比对，从而从第一预处理序列数据中截去序列中的接头序列，进而获得第二预处理序列数据；和
短序列去除单元，所述短序列去除单元按长度L为标准，从所述第二预处理序列数据中去除长度小于L的序列，从而获得所述经去除处理的序列数据，其中L为18-25的正整数。
在一优选例中，所述物种数据去除单元中所述预定比对参数为最大容错3个碱基，或最大允许3个碱基的插入或缺失。
在一优选例中，所述接头数据去除单元中所述预定容错率为15％-25％。
在一优选例中，当第一物种为人时，所述第一物种的核酸序列信息为Hg19基因组序列。
在一优选例中，在所述数据分析装置中，采用Kraken法进行微生物物种分类。
在一优选例中，所述采用Kraken法进行微生物物种分类，包括以下单元：
数据库构建单元，所述数据库构建单元根据已知基因组数据构建K-mer数据库；
片段生成单元，所述片段生成单元与所述K-mer数据库构建单元相连，所述片段生成单元基于所述数据筛选单元获得的所述经去除处理的序列数据，将每一条所述序列打断为预定长度K-mer的片段；
片段比对单元，所述片段比对单元与所述片段生成单元相连，所述片段比对单元将所述预定长度K-mer的片段与K-mer数据库进行比对，进而根据最近共同祖先的原则对所述 序列进行物种分类。
在一优选例中，所述K-mer数据库包括以下物种的序列数据：人基因组、细菌/古细菌、病毒、真菌、原虫、质粒。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1根据本发明的实施例提供的外泌体非人序列分析流程图。
图2根据本发明的实施例提供的颠倒混匀示意图。
图3为根据本发明的实施例提供的一种确定待测样品中的微生物的系统的示意图。
图4为根据本发明的实施例提供的所述数据筛选装置的示意图。
图5为根据本发明的实施例提供的利用Kraken法进行微生物物种分类时的结构示意图。
具体实施方式
在本发明中，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”、“固定”等术语应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或成一体；可以是机械连接，也可以是电连接或彼此可通讯；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通或两个元件的相互作用关系，除非另有明确的限定。对于本领域的普通技术人员而言，可以根据具体情况理解上述术语在本发明中的具体含义。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
在本发明中，除非另有明确的规定和限定，第一特征在第二特征“上”或“下”可以是第一和第二特征直接接触，或第一和第二特征通过中间媒介间接接触。而且，第一特征在第二特征“之上”、“上方”和“上面”可是第一特征在第二特征正上方或斜上方，或仅仅表示第一特征水平高度高于第二特征。第一特征在第二特征“之下”、“下方”和“下面”可以是第一特征在第二特征正下方或斜下方，或仅仅表示第一特征水平高度小于第二特征。
本发明基于发明人的下列发现而完成的：本发明人经过广泛而深入的研究，首次意外地发现，通过分离或富集妊娠期外泌体DNA可检测各类病原微生物(细菌、真菌、病毒、 寄生虫等)，相对于常规血浆cfDNA(cell free DNA)的方法，本发明基于外泌体DNA的方法检出种类更多，丰度更高。可以通过例如高通量测序准确提供用于临床病患(如发热人群或孕期人群)未知病原微生物检测的信息。在此基础上完成了本发明。
根据本发明的一个实施例，本发明利用外泌体核酸可以实现个体中的微生物信息富集的功能，提供了一种确认生物个体中的微生物的方法，包括如下步骤：(a)从所述生物个体中分离获得含有所述个体遗传信息和微生物遗传信息的外泌体核酸；(b)对所述外泌体核酸进行测序，获得由核酸序列数据组成的测序结果；(c)基于所述生物个体的核酸序列信息，从所述测序结果中排除对应于所述个体的核酸序列，从而获得经去除处理的序列数据；和(d)将所述经去除处理的序列数据与微生物数据库的核酸序列进行比对，并进行微生物物种分类，从而获得所述样品中的微生物检测结果。
在本发明中，无论是表述成第一物种还是表示成生物个体，或者生物样本，或者个体等，均表示本发明可以对于存在于生物体内的微生物进行诊断和确认。
外泌体
如本文所用，术语“外泌体”是指活细胞次级内体经过“内吞-融合-外排”等一系列调控过程而形成的细胞外纳米级小囊泡(30-100nm)，携带RNA、蛋白质、脂质等内含物，具有调节基因转录和表达等作用，可以实现细胞间和个体间的信号转导。几乎所有类型的细胞都能分泌外泌体，包括：血清、血浆、尿液、细胞培养上清(免疫系统细胞、肿瘤细胞)、乳汁、唾液、腹水、羊水、气管肺泡灌洗液、关节滑液。采用密度梯度离心可以实现外泌体的分离，在透射电子显微镜下可观察到外泌体特有的杯状结构。
外泌体在妊娠期间的含量变化与孕周相关：与未妊娠女性对照相比，正常妊娠孕妇外周血中外泌体含量显著增加约20-50倍，且随孕周增大外泌体含量持续上升。通过对外泌体表面PLAP定量发现，孕妇外周血中胎儿来源外泌体在孕5-6周时可以检出，数量亦随孕周增大而迅速上升，提示随着胎盘成熟和母胎间交换的活跃，胎盘特异性外泌体向母体循环中的释放也增多。
总外泌体分离方法：SBI公司基于沉淀离心原理、Thermo Fisher公司基于免疫磁珠分离；本发明将二者结合起来，利用SBI公司试剂获得外泌体总量多，而通过免疫磁珠法解决前者纯度不够高的问题。
外泌体分离纯度较高的其他方法，如超速离心、密度梯度离心，也可能可以达到和免疫磁珠类似的病原微生物富集效应。
在另一优选例中，富集血浆总囊泡，即微泡(microvesicel)(或其他尚未明确界定种类的细胞外囊泡，另如血小板核酸)和外泌体，也可能可以达到和外泌体类似的病原微生物富集 效应。
在另一优选例中，血浆外泌体病原微生物检测可以有助于评估肿瘤免疫治疗疗效。
在另一优选例中，血浆外泌体病原微生物检测可以用于移植后病原监测。
由于外泌体是一种细胞间的通讯方式，其DNA内含物中病原微生物检测反映了人体内病原微生物的感染情况，该信息可用于临床病患(如发热人群)未知病原微生物检测及孕期人群常见疾病筛查和风险预测。
捕获剂或分选剂
如本文所用，术语“捕获剂”、“分选剂”可互换使用，指将外泌体从血液或体液样品中捕获或富集或分选出的试剂。
一种典型的捕获剂包括(a)针对以下至少一种抗原的特异性抗体：PLAP、CD9、CD63、或CD81；或(b)偶联有所述抗体的磁珠。例如，可以采用Exoquick试剂和/或免疫磁珠，所述免疫磁珠能够特异性识别CD63和/或PLAP(placental alkaline phosphatase,胎盘碱性磷酸酶)。
免疫磁珠是在磁珠表面偶联单克隆抗体的球形磁性微粒(例如，可以通过链霉素-亲和素系统偶联)，通过抗体与外泌体表面抗原(如CD9，CD63，CD81)相互作用而特异性地捕获血浆总外泌体；也可以通过某一类组织、器官特异表达的抗原(如PLAP)而分离具有组织、器官特异性的外泌体，进行通过检测其内含物，提高对疾病检测的灵敏度、特异性。
需要说明的是，本文所述“外泌体核酸”是指外泌体中的核酸组分。同样的，“外泌体DNA”是指外泌体中的DNA组分，“外泌体RNA”是指外泌体中的RNA组分。本发明人经过研究发现，从孕妇外周血中提取的总外泌体DNA中，既包含母亲来源(母源)的DNA，又包含胎儿来源(胎源)的DNA，且所述胎源DNA是以游离DNA片段形式存在。同样的，从孕妇外周血中提取的胎盘及妊娠组织来源外泌体(如使用PLAP磁珠分离获得的外泌体)中，既包含母亲来源(母源)的DNA，又包含胎儿来源(胎源)的DNA。
机理
为了便于理解本发明，提供了以下发明机理供参考。应理解，本发明的保护范围并不受上述发明机理的限制。
本发明人提出如下科学机理和解释：妊娠期间母血中存在来自胎盘滋养层细胞释放的外泌体，其中含有胎儿来源游离DNA片段；通过高通量测序的方法进行检测，用于妊娠期疾病筛查和风险预测。
具体地，妊娠期母血中存在母源外泌体和胎源外泌体：在未妊娠女性外周血中通过色 谱分析法和免疫吸附法可以提取到来源于淋巴细胞的外泌体，而通过胎盘特异性抗体PLAP定量可以发现在妊娠孕妇外周血中除了来源于淋巴的外泌体还可以检测到胎源外泌体，证明妊娠期母血中的外泌体是母源和胎源混合的。
本发明人通过体外和体内实验证明，母血中的胎源外泌体主要是由胎盘滋养层细胞合成和释放入母血的。在胎盘滋养层细胞中，初级核内体经过膜的内陷作用发育为成熟核内体，之后一部分进入溶酶体，另一部分经过包裹作用携带大量信号分子(DNA、mRNA、miRNA、蛋白质等)形成外泌体，通过膜融合作用被释放到细胞外基质进入母血中。
虽然外泌体的生物合成、转运、内含物包裹、作用尚不完全清楚，但是本发明的研究认为通过主动包裹组织特异性内含物并释放进母体循环，胎源外泌体可以参与免疫耐受、母胎界面重塑、炎症反应等妊娠期间重要过程的调节。此外，由于外泌体具有稳定的双层脂质膜，似乎更有助于保持胎源DNA的稳定。
基于孕妇外周血中外泌体进行微生物检测的方法
本发明提供了一种基于孕妇体内外泌体进行微生物检测的方法。根据本发明的一种具体实施方式，所述方法包括如下步骤：(a)从所述孕妇体内分离得到外泌体核酸；(b)对所述外泌体核酸进行测序，获得由核酸数据组成的测序结果；(c)从所述测序结果中去除来自于孕妇和胎儿的核酸序列，从而获得经去除处理的序列数据；(d)将所述经去除处理的序列数据与微生物数据库的核酸序列进行比对，从而获得微生物检测结果。
需要说明的是，利用本发明的方法，不仅可以用来确定孕妇体内病原微生物的种类以及比例，还可以用来确定孕妇体内有益微生物的种类，从而可以用来指示孕妇的身体状态。由此，可以用来指示临床用药或者临床疾病的诊断，也可以进行营养保健或者增强机体的抗病能力或者是改善体质。通过该方法确定的孕妇体内的微生物，可以用于辅助临床诊断，为医生或者营养师等提供参考，重要的是还可以用于其他非诊断目的。例如包括利用这种快速无痛的确定孕妇体内微生物的方法，实现孕期妈妈对于自身体质和自身状态的监控，或者是可以利用该方法，用作科研或者其他目的，如快速实现孕妇体内微生物的确定，实现样本的采集，实现孕妇体内优势微生物种类分析，实现在整个孕期追踪胎盘与妊娠组织中微生物种类的变化情况等等。而且，即便是通过该方法确定孕妇体内的为微生物，可以用于辅助临床诊断，也并非利用该微生物信息就可以直接诊断出孕妇的疾病状态，还需要结合每种微生物的特性等进行具体分析。
根据本发明的一种具体实施方式，本发明提供了一种基于孕妇外周血中外泌体进行病原微生物检测的方法。
典型地，本发明方法包括步骤：
(a)提供一血液样品，所述血液样品为来自孕妇的外周血的血液、血浆、血清、或其组合；
(b)从所述血液样品中，分离外泌体；
(c)从所述分离的外泌体中，提取DNA(其中包含有胎儿来源DNA)；
(d)对所述经提取的(c)中所得的DNA进行检测(如PCR扩增或测序)，从而获得相应的分析结果或检测结果；和
(e)信息分析，得出检测样本外泌体中病原微生物的种类、比例及与对应血浆的比较关系。
在本发明方法中，可用常规方法采集孕妇外周血，然后分离血浆或血清。例如采用市售streck管采集外周血(如约2-20ml，较佳地3-10ml)。血浆或血清分离可采用两步离心法。
对于分离后的血浆或血清，可通过采用离心等方式获得含有总外泌体的分离物。优选地，对于所获得的总外泌体，可以进行纯化，例如采用磁珠分离法，通过负载有CD63抗体的免疫磁珠进行纯化。也可以对特定组织来源的外泌体进行富集，例如通过负载有PLAP抗体的免疫磁珠进行胎盘及妊娠组织来源外泌体的捕获/富集，以便获得含有丰富胎源DNA的外泌体。
对于分离的总外泌体或胎盘及妊娠组织来源外泌体，可提取其DNA，然后对DNA进行检测。例如，可先构建高通量测序文库，然后测序并进行分析，检测病原微生物类型等。
在另一优选例中，一种典型的本发明方法包括步骤：
第一步：从血液样品中分离血浆总外泌体。其中可用已知的方法或试剂，例如市售的SBI公司SBI quick试剂对血浆总外泌体进行分离；
第二步：对所述血浆总外泌体进行纯化。例如，对第一步生成的外泌体沉淀用PBS缓冲液溶解后，用抗CD63免疫磁珠进行纯化，例如在一定温度(如4-8度)孵育一段时间(如2-24小时，或过夜孵育)，从而形成“磁珠-外泌体二元复合物”。抗CD63免疫磁珠可用常规方法制备或市售获得，例如Thermo Fisher公司CD63免疫磁珠；
第三步：对第二步形成的“磁珠-外泌体二元复合物”进行外泌体DNA提取，然后进行建库(加大PCR循环数)、和/或测序，获得由核酸序列信息组成的测序数据；
第四步：对于测序数据进行信息分析，从而得出相应的分析结果，例如测序的结果在进行质控，并去除人的序列后，在已有微生物数据库的基础上进行物种分类。物种分类可以通过例如Kraken法实现。基于特定k-mer数据库可以对序列数据进行快速分类计算，基于Kraken的分类结果可以进行微生物物种水平的整理统计，也可以对Kraken的结果用Krona处理，得到更加详细的结果展示，具体分析流程见图1。
在另一优选例中，一种典型的本发明方法包括步骤：
第一步：从血液样品中分离血浆总外泌体。其中可用已知的方法或试剂，例如市售的SBI公司SBI quick试剂对血浆总外泌体进行分离；
第二步：从所述血浆总外泌体中富集或捕获胎盘及妊娠组织来源的血浆外泌体。例如，对第一步生成的总外泌体沉淀用PBS缓冲液溶解后，用抗PLAP免疫磁珠进行捕获或富集，例如在一定温度(如4-8度)孵育一段时间(如2-24小时，或过夜孵育)，从而形成“磁珠-外泌体二元复合物”。抗PLAP免疫磁珠可用常规方法制备或市售获得，例如Thermo Fisher公司PLAP免疫磁珠；
第三步：对第二步形成的“磁珠-外泌体二元复合物”进行外泌体DNA提取，然后进行建库(加大PCR循环数)、和/或测序，获得由核酸序列信息组成的测序数据；
第四步：对于测序数据进行信息分析，从而得出相应的分析结果，例如测序的结果在进行质控，并去除人的序列后，在已有微生物数据库的基础上进行物种分类。物种分类可以通过例如Kraken软件实现。基于特定k-mer数据库可以对序列数据进行快速分类计算，基于Kraken的分类结果可以进行微生物物种水平的整理统计，也可以对Kraken的结果用Krona处理，得到更加详细的结果展示。
一种典型的Kraken法包括步骤：
(1)首先需要根据基因组数据构建Kraken分类所需的K-mer数据库，所用的基因组数据包括：人基因组(GRCh38)、细菌/古细菌、病毒、真菌、原虫、质粒，除了人的数据其余的数据都来自NCBI的Refseq数据库；
(2)进行分类时需要将每一条读段序列打断成特定长度K-mer的片段，然后将该条读段序列的所有K-mer比对回K-mer数据库，并根据最近共同祖先(lowest common ancestor，LCA)的原则对该条读段序列进行物种分类；和
(3)基于Kraken物种分类的结果对微生物的分类进行统计整理，分类的结果也可以使用Krona(KronaTools-2.5)工具进行可视化处理，得到更为详细的展示结果。
需要说明的是，术语“读段序列”也称“reads”，指每一轮测序反应结束时产生的核酸序列。高通量测序后获得的原始测序数据即为原始读段序列，本领域技术人员根据需要，可以对原始读段序列进行过滤处理，例如去除低质量值读段序列、去除接头序列等。可以理解，对于一个测序文库，在测序后会产生众多读段序列，本领域技术人员可以根据读段序列之间的重叠关系从而将基因组组装起来。
本文所述“Kraken”，是一种微生物宏基因组数据分析软件，它可以通过使用k-mers的 精确对齐和新的分类算法来实现高灵敏度和高速度的短DNA序列分类。更多有关于Kraken的介绍，详见http://ccb.jhu.edu/software/kraken/。
本文所述“K-mer”，是指将reads分割成包含k个碱基的字符串，一条长度为m的reads可以分成(m-k+1)个k-mers，m与k均为大于1的正整数，且m与测序平台以及测序策略有关，例如使用BGISEQ500测序仪进行SE90测序，m为90。再例如一条reads序列为：ATCGTTGCTTAATGACGTCAGTCGAATGCGATGACGTGACTGACTG，需要将它分割成包含13个碱基的字符串，即13-mer，那么可以切割为以下多种片段形式：
ATCGTTGCTTAAT；
TCGTTGCTTAATG；
CGTTGCTTAATGA；
GTTGCTTAATGAC；等等。
本文所述“K-mer数据库”是指由K-mer数据构成的数据库。所述K-mer数据可以是来自于任何开源数据库，例如NCBI的Refseq数据库，其包括细菌/古细菌数据、病毒数据、真菌数据、原虫数据等。
确定待测样品中的微生物的系统
根据本发明的另一方面，本发明提供了一种确定待测样品中的微生物的系统，如图3所示。所述系统包括：核酸分离装置，测序装置，数据筛选装置和数据分析装置，其中所述核酸分离装置对所述待测样品进行分离，获得外泌体核酸，其中，所述待测样品来自于第一物种；所述测序装置与所述外泌体核酸分离装置相连，所述测序装置对所述外泌体核酸进行测序，获得由核酸序列数据组成的测序结果；所述数据筛选装置与所述测序装置相连，所述数据筛选装置基于所述第一物种的核酸序列信息，从所述测序结果中排除对应于所述第一物种的核酸序列，从而获得经去除处理的序列数据；所述数据分析装置与所述数据筛选装置相连，所述数据分析装置将所述经去除处理的序列数据与微生物数据库的核酸序列进行比对，并进行微生物物种分类，从而获得所述样品中的微生物检测结果。利用本发明提供的系统，可以实现个体上微生物的快速高效检测，灵敏度更高，检测范围更光。
根据本发明的实施例，本发明中所述数据筛选装置进一步包括：物种数据去除单元、接头数据去除单元和短序列去除单元，其中所述物种数据去除单元按照预定比对参数，将所述测序结果与所述第一物种的核酸序列信息进行比对，去除比对上的序列，从而获得未比对上的序列，进而获得第一预处理序列数据；所述接头数据去除单元与所述物种数据去除单元相连，所述接头数据去除单元按照预定容错率，将所述第一预处理序列数据与测序接头序列比对，从而从第一预处理序列数据中截去序列中的接头序列，进而获得第二预处 理序列数据；所述短序列去除单元按长度L为标准，从所述第二预处理序列数据中去除长度小于L的序列，从而获得所述经去除处理的序列数据，其中L为18-25的正整数。
根据本发明的实施例，在所述数据分析装置中，采用kraken法进行微生物物种分类，在利用kraken法进行微生物物种分类时，所述数据分析装置可以进一步包括如下单元：数据库构建单元、片段生成单元和片段比对单元，其中所述数据库构建单元根据已知基因组数据构建K-mer数据库；所述片段生成单元与所述K-mer数据库构建单元相连，所述片段生成单元基于所述数据筛选单元获得的所述经去除处理的序列数据，将每一条所述序列打断为预定长度K-mer的片段；所述片段比对单元与所述片段生成单元相连，所述片段比对单元将所述预定长度K-mer的片段与K-mer数据库进行比对，进而根据最近共同祖先的原则对所述序列进行物种分类。
本发明的主要优点在于：
(1)本发明方法不需要进行微生物培养，且能检测到未知病原体；
(2)本发明基于外泌体DNA进行病原微生物检测的方法相对常规血浆cfDNA的方法，灵敏度更高，检测范围更广；
(3)本发明基于妊娠期外泌体DNA富集病原微生物，大大提高了基于血浆宏基因组检测病原微生物的灵敏度与检测范围；
(4)本发明可通过分离胎盘及妊娠组织来源外泌体检测宫内感染，相对于传统羊水穿刺检测，本发明是无创的检测手段，且简单易行，无流产风险。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
本发明中所涉及的实验材料如无特殊说明均可从市售渠道获得。
实施例1 基于孕妇血浆中总外泌体的病原微生物检测(SBI外泌体)
两步法进行血浆分离
1、Streck Cell-Free DNA BCT采血
按照外周血标准采集操作，采集10例孕妇外周血，每1例分别采血10mL，采血后请立即缓慢颠倒10次使血液与管内成分混匀(如图2)，拖延颠倒混匀时间可能会造成检测失 败。混匀后请将采血管直立至于试管架上放置(6-35℃)。
2、孕妇血浆分离
2.1在4℃条件下以1600g离心10分钟，在冰盒上将上清液分装到多个2.0mL离心管中；
2.2在4℃条件下以16000g离心10分钟，在冰盒上将所得上清液转入新的已编号的2.0mL离心管中，每个离心管转入600μL血浆；
2.3血浆样本保存：血浆分离后立即低温保存，一周之内-20℃条件下暂存：长期保存需置于-80℃冰箱中。
3、总外泌体分离：利用SBI System Biosciences公司ExoQuick Exosome Precipitation Solution试剂分离血浆总外泌体
3.1在分离后的血浆中加入Exoquick试剂(添加比例为250μL血浆加入63μL Exoquick试剂)，充分吸打混匀，冰浴放置30min；
3.2 1500g离心30min，吸去上清；
3.3将步骤3.2产生的沉淀1500g离心5min，仔细吸去痕量上清；
3.4用100μL PBS缓冲液于37℃融解步骤3.3产生的沉淀，获得的溶液标记为“SBI外泌体”(说明：此处获得的SBI外泌体为使用SBI公司外泌体制备试剂盒而获得的总外泌体，为了与后续的实施例2、3以及对比例获得的外泌体进行区分，发明人将其标记为SBI外泌体)。
4、总外泌体DNA提取
使用美基生物公司试剂盒(Magen MagPure Circulating DNA Mini KF Kit)将步骤3.4获得的“SBI外泌体”溶液进行总外泌体DNA提取，操作完全按照试剂盒说明书进行。获得的DNA标记为“SBI外泌体DNA”。
5、高通量建库测序
将步骤4获得的SBI外泌体DNA按照专利申请“用于基于血液样品构建测序文库的方法及其在确定胎儿遗传异常中的用途”(申请公布号CN105400864 A)所公开的方法进行文库构建与高通量测序(其中，文库构建时的PCR循环数为19，其他步骤和参数与上述专利申请公开的一致)，获得测序数据。
6、测序数据分析
6.1高通量测序数据通过bwa比对到人类基因组参考序列hg19，最大容错3碱基或者3个indel，根据比对结果过滤出未比对上的序列；然后使用软件“cutadapt”以20％的容错率与测序接头比较，截去剩下的序列中的接头序列，并过滤掉剩余序列不足20bp的序列，最终得到待分析的非人序列；
6.2采用Kraken(Kraken-0.10.5-beta)进行微生物物种分类，首先根据基因组数据构建Kraken分类所需的K-mer数据库，所用的基因组数据包括：人基因组(GRCh38)、细菌/古细菌、病毒、真菌、原虫、质粒，除了人的数据其余的数据都来自NCBI的Refseq数据库。进行分类时需要将每一条reads打断成特定长度K-mer的片段，然后将该条reads的所有K-mer比对回K-mer数据库，并根据最近共同祖先(lowest common ancestor，LCA)的原则对该条reads进行物种分类。基于Kraken物种分类的结果对微生物的分类进行统计整理，分类的结果也可以使用Krona(KronaTools-2.5)工具进行可视化处理，得到更为详细的展示结果。统计每一样本各种类型微生物检出reads数以及微生物检出reads数(微生物检出reads数为细菌、病毒、古细菌、真菌检出reads数之和)，并计算微生物系数，微生物系数＝微生物检出reads数/测序总reads数(M)。
结果：基于SBI外泌体DNA方法的微生物检出结果如表1所示。从表1可知，经测序数据校正后，基于SBI外泌体DNA方法的检出微生物系数为148.50。
实施例2 基于孕妇血浆中总外泌体的病原微生物检测(CD63外泌体)
首先采集10例孕妇外周血(样本同实施例1)，使用两步法进行血浆分离，然后利用SBI System Biosciences公司ExoQuick Exosome Precipitation Solution试剂分离血浆总外泌体，具体步骤同实施例1。
使用Thermo Fisher公司Exosome-Human CD63磁珠分离纯化步骤3.4获得的“SBI外泌体”。具体步骤如下：
3.5将CD63磁珠振荡30s以充分混匀；
3.6取出40μL磁珠加入500μL分离缓冲液(PBS+0.1％BSA)，吸打混匀后3000g短暂离心5s后，置于磁力架放置2min，吸去上清；
3.7将步骤3.4产生的100μL“SBI外泌体”加入到步骤3.6准备好的磁珠中，吸打混匀，在旋转摇床4℃孵育过夜(18-22h)；
3.8将步骤3.7的磁珠、SBI外泌体混合物3000g短暂离心5s，加入500μL分离缓冲液，置于磁力架放置2min，吸去上清；
3.9加入500μL分离缓冲液，置于磁力架放置2min，吸去上清，获得携带有外泌体的磁珠，标记为“CD63外泌体”(说明：此处获得的CD63外泌体为先使用SBI公司外泌体 制备试剂盒获得总外泌体后，再使用CD63磁珠对总外泌体进行纯化而获得的总外泌体，为了与实施例1、3以及对比例获得的外泌体进行区分，发明人将其标记为CD63外泌体)。
然后，使用美基生物公司试剂盒(Magen MagPure Circulating DNA Mini KF Kit)将步骤3.9获得的“CD63外泌体”进行总外泌体DNA提取，操作完全按照试剂盒说明书进行。获得的DNA标记为“CD63外泌体DNA”。
最后，将获得的“CD63外泌体DNA”进行文库构建、高通量测序与测序数据分析，具体步骤同实施例1。
结果：基于CD63外泌体DNA方法的微生物检出结果如表1所示。从表1可知，经测序数据校正后，基于CD63外泌体DNA方法的检出微生物系数为6586.87。说明基于CD63外泌体DNA方法相较于血浆游离核酸方法、SBI外泌体方法，可以极大程度地富集微生物，富集效果极其显著。
对比例 基于常规的血浆游离核酸的病原微生物检测
首先采集10例孕妇外周血(样本同实施例1)，按专利申请“用于基于血液样品构建测序文库的方法及其在确定胎儿遗传异常中的用途”(申请公布号CN105400864A)所公开的方法进行血浆游离核酸文库构建与高通量测序。最后对获得的测序数据进行分析，具体分析步骤同实施例1。
结果与分析：基于血浆游离核酸方法的微生物检出结果如表1所示(表1中样本注释为“样本1血浆-样本10血浆”的样本即代表使用本对比例方法的微生物检出结果)。从表1可知，经测序数据校正后，基于血浆游离核酸方法的检出微生物系数为68.10。
表1 基于CD63外泌体、血浆游离核酸与SBI外泌体方法的微生物检出结果比较
Figure PCTCN2018083230-appb-000001
Figure PCTCN2018083230-appb-000002
实施例3 基于孕妇血浆中胎盘及妊娠组织来源外泌体的病原微生物检测
首先采集1例已确诊为乙肝病毒感染(已通过现有的乙肝抗原免疫学检测方法确诊)的孕妇外周血及羊水样本，标记为样本1；采集3例已确诊未感染乙肝病毒(已通过现有的乙肝抗原免疫学检测方法确诊)的孕妇外周血及羊水样本，作为阴性对照，标记为样本2-4。
每一例样本均同时进行基于SBI外泌体方法、基于PLAP外泌体方法、基于常规的血浆游离核酸方法、基于常规的羊水细胞方法进行病原微生物检测。
基于SBI外泌体方法进行病原微生物检测具体步骤同实施例1。
基于常规的血浆游离核酸方法进行病原微生物检测具体步骤同对比例，每一个样本做两个平行试验。
基于常规的羊水细胞方法进行病原微生物检测，按照现有的常规的操作方法，大致为先离心分离羊水细胞，然后提取羊水细胞DNA，最后进行DNA文库构建与高通量测序，对于获得的测序数据的分析方法同实施例1。
基于PLAP外泌体方法进行病原微生物检测具体步骤如下：
使用两步法进行血浆分离，然后利用SBI System Biosciences公司ExoQuick Exosome Precipitation Solution试剂分离血浆总外泌体，具体步骤同实施例1。
使用Thermo Fisher公司Exosome-Human PLAP磁珠分离纯化步骤3.4获得的“SBI外泌体”。具体步骤如下：
3.5将PLAP磁珠振荡30s以充分混匀；
3.6取出40μL磁珠加入500μL分离缓冲液(PBS+0.1％BSA)，吸打混匀后3000g短暂离心5s后，置于磁力架放置2min，吸去上清；
3.7将步骤3.4产生的100μL“SBI外泌体”加入到步骤3.6准备好的磁珠中，吸打混匀，在旋转摇床4℃孵育过夜(18-22h)；
3.8将步骤3.7的磁珠、SBI外泌体混合物3000g短暂离心5s，加入500μL分离缓冲液，置于磁力架放置2min，吸去上清；
3.9加入500μL分离缓冲液，置于磁力架放置2min，吸去上清，获得携带有外泌体的磁珠，标记为“PLAP外泌体”(说明：此处获得的PLAP外泌体为先使用SBI公司外泌体制备试剂盒获得总外泌体后，再使用PLAP磁珠对胎盘及妊娠组织来源外泌体进行捕获而获得的，为了与实施例1、2以及对比例获得的外泌体进行区分，发明人将其标记为PLAP外泌体)。
然后，使用美基生物公司试剂盒(Magen MagPure Circulating DNA Mini KF Kit)将步骤3.9获得的“PLAP外泌体”进行胎盘及妊娠组织来源外泌体DNA提取，操作完全按照试剂盒说明书进行。获得的DNA标记为“PLAP外泌体DNA”。
最后，将获得的“PLAP外泌体DNA”进行文库构建、高通量测序与测序数据分析，具体步骤同实施例1。
结果与分析：各样本的微生物检出结果见表2。从表2中可知，样本1血浆、羊水中均可检出乙肝病毒，说明孕妇及其子宫内均感染乙肝病毒；基于PLAP外泌体方法的样本1中，可检测出大量乙肝病毒，说明可通过检测胎盘及妊娠组织来源外泌体的微生物来间接反映宫内感染情况。而阴性对照样本2-3，血浆与羊水中均未检出乙肝病毒，其基于PLAP外泌体的方法中也未检出乙肝病毒，说明无检测假阳性存在。而阴性对照样本4，基于血浆与基于SBI外泌体的方法中均检测出少量乙肝病毒，但是羊水样本并没有检测出乙肝病毒感染，基于PLAP外泌体的方法中也没有检测出乙肝病毒感染。这说明，相对于基于血浆与基于SBI外泌体方法来说，PLAP外泌体检测宫内感染特异性更好。表2的结果证明基于PLAP外泌体的病原微生物检测，可用于诊断宫内感染。
表2 基于PLAP外泌体检测宫内感染
Figure PCTCN2018083230-appb-000003
Figure PCTCN2018083230-appb-000004
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (46)

  1. 一种检测待测样品中的微生物的方法，其特征在于，所述方法包括如下步骤：
    (a)对所述待测样品进行分离，获得外泌体核酸，其中，所述待测样品来自于第一物种；
    (b)对所述外泌体核酸进行测序，获得由核酸序列数据组成的测序结果；
    (c)基于所述第一物种的核酸序列信息，从所述测序结果中排除对应于所述第一物种的核酸序列，从而获得经去除处理的序列数据；和
    (d)将所述经去除处理的序列数据与微生物数据库的核酸序列进行比对，并进行微生物物种分类，从而获得所述样品中的微生物检测结果。
  2. 根据权利要求1所述方法，其特征在于，所述外泌体核酸选自下组：外泌体DNA、外泌体RNA、或其组合。
  3. 根据权利要求1所述方法，其特征在于，所述第一物种选自下组：哺乳动物、鸟类、或爬行动物。
  4. 根据权利要求1所述方法，其特征在于，所述待测样品为来自正常个体的样品、发热个体的样品或者是来自孕期个体的样品。
  5. 根据权利要求2或4所述方法，其特征在于，所述外泌体核酸包括孕妇自身来源的外泌体核酸、孕妇的胎儿来源的外泌体核酸、或其组合；优选地，所述外泌体核酸包括胎儿来源外泌体DNA。
  6. 根据权利要求1所述方法，其特征在于，所述微生物选自下组：病毒、细菌、真菌、寄生虫、衣原体、支原体、或其组合。
  7. 根据权利要求1所述方法，其特征在于，所述微生物检测结果包括微生物的种类、以及所述微生物的数量或丰度。
  8. 根据权利要求1所述方法，其特征在于，所述待测样品选自下组：血液样品、或体液样品。
  9. 根据权利要求8所述方法，其特征在于，所述血液样品选自下组：血浆、血清或其组合。
  10. 根据权利要求8所述方法，其特征在于，所述体液样品选自下组：尿液、唾液、胸腹水、脑脊液，汗液，羊水、细胞培养液、或其组合。
  11. 根据权利要求8所述方法，其特征在于，所述血液样品是将血样离心后，收集得到的上清。
  12. 根据权利要求11所述方法，其特征在于，所述上清是用两步法制备得到的：
    (1)使用收集装置收集血液样品，其中，所述收集装置中含有抗凝血剂，和
    (2)对所述样品进行高速离心，从而获得所述上清。
  13. 根据权利要求1、2或5所述方法，其特征在于，在步骤(a)中，所述分离包括如下步骤：
    (a1)从所述待测样品中分离外泌体；和
    (a2)从所述分离的外泌体中提取核酸。
  14. 根据权利要求1所述方法，其特征在于，所述分离采用磁珠分离法、亲和分离法、或其组合。
  15. 根据权利要求14所述方法，其特征在于，所述磁珠分离法采用表面标记有CD63抗体或PLAP抗体的磁珠进行分离。
  16. 根据权利要求1所述方法，其特征在于，所述分离包括使用针对以下至少一种抗原的特异性抗体进行分选或捕获：PLAP、CD9、CD63、或CD81。
  17. 根据权利要求1所述方法，其特征在于，所述测序包括高通量测序。
  18. 根据权利要求1、2或5任一项所述方法，其特征在于，在步骤(c)中，包括以下子步骤：
    (c1)按照预定比对参数，将所述测序结果与所述第一物种的核酸序列信息进行比对，去除比对上的序列，从而获得未比对上的序列，进而获得第一预处理序列数据；
    (c2)按照预定容错率，将所述第一预处理序列数据与测序接头序列比对，从而从第一预处理序列数据中截去序列中的接头序列，进而获得第二预处理序列数据；和
    (c3)按长度L为标准，从所述第二预处理序列数据中去除长度小于L的序列，从而获得所述经去除处理的序列数据，其中L为18-25的正整数。
  19. 根据权利要求18所述方法，其特征在于，步骤(c1)中所述预定比对参数为最大容错3个碱基，或最大允许3个碱基的插入或缺失。
  20. 根据权利要求18所述方法，其特征在于，步骤(c2)中所述预定容错率为15％-25％。
  21. 根据权利要求18所述方法，其特征在于，当第一物种为人时，所述第一物种的核酸序列信息为Hg19基因组序列。
  22. 根据权利要求1、2、5或18任一项所述方法，其特征在于，在步骤(d)中，采用Kraken法进行微生物物种分类。
  23. 根据权利要求22所述方法，其特征在于，所述采用Kraken法进行微生物物种分类，包括以下子步骤：
    (d1)根据已知基因组数据构建K-mer数据库；
    (d2)基于步骤(c)获得的所述经去除处理的序列数据，将每一条所述序列打断为预定长度K-mer的片段；
    (d3)将所述预定长度K-mer的片段与K-mer数据库进行比对，进而根据最近共同祖先的原则对所述序列进行物种分类。
  24. 根据权利要求1所述方法，其特征在于，所述方法还包括：对于步骤(c)中，对于所述被排除的所述对应于所述第一物种的核酸序列，进一步进行基因检测分析，从而获得对应于第一物种的基因检测结果。
  25. 根据权利要求24所述方法，其特征在于，所述对应于第一物种的基因检测结果包括：无创产前基因检测结果。
  26. 一种确定待测样品中的微生物的系统，其特征在于，所述系统包括：
    核酸分离装置，所述核酸分离装置对所述待测样品进行分离，获得外泌体核酸，其中，所述待测样品来自于第一物种；
    测序装置，所述测序装置与所述外泌体核酸分离装置相连，所述测序装置对所述外泌体核酸进行测序，获得由核酸序列数据组成的测序结果；
    数据筛选装置，所述数据筛选装置与所述测序装置相连，所述数据筛选装置基于所述第一物种的核酸序列信息，从所述测序结果中排除对应于所述第一物种的核酸序列，从而获得经去除处理的序列数据；和
    数据分析装置，所述数据分析装置与所述数据筛选装置相连，所述数据分析装置将所述经去除处理的序列数据与微生物数据库的核酸序列进行比对，并进行微生物物种分类，从而获得所述样品中的微生物检测结果。
  27. 根据权利要求26所述系统，其特征在于，所述外泌体核酸选自下组：外泌体DNA、外泌体RNA、或其组合；优选为外泌体DNA。
  28. 根据权利要求26所述系统，其特征在于，所述第一物种选自下组：哺乳动物、鸟类或爬行动物。
  29. 根据权利要求26所述系统，其特征在于，所述待测样品为来自正常个体的样品、发热个体的样品或者是来自孕妇个体的样品。
  30. 根据权利要求27、或28所述系统，其特征在于，所述外泌体核酸包括孕妇自身来源的外泌体核酸、孕妇的胎儿来源的外泌体核酸、或其组合；优选地，所述外泌体核酸包括胎儿来源外泌体DNA。
  31. 根据权利要求26所述系统，其特征在于，所述微生物选自下组：病毒、细菌、真菌、寄生虫、衣原体、支原体、或其组合。
  32. 根据权利要求26所述系统，其特征在于，所述待测样品选自下组：血液样品、或体液样品。
  33. 根据权利要求32所述系统，其特征在于，所述血液样品选自下组：血浆、血清或 其组合。
  34. 根据权利要求32所述方法，其特征在于，所述体液样品选自下组：尿液、唾液、胸腹水、脑脊液，汗液，羊水、细胞培养液、或其组合。
  35. 根据权利要求26、27或30任一项所述方法，其特征在于，所述核酸分离装置包括：
    外泌体分离单元，所述外泌体分离单元从所述待测样品中分离外泌体；和
    核酸提取单元，所述核酸提取单元与所述外泌体分离单元相连，所述核酸提取单元从所述分离的外泌体中提取核酸。
  36. 根据权利要求35所述方法，其特征在于，所述外泌体分离单元采用下组方式从所述待测样品中分离外泌体：磁珠分离法、亲和分离法、或其组合。
  37. 根据权利要求36所述方法，其特征在于，所述磁珠分离法采用表面标记有CD63抗体或PLAP抗体的磁珠进行分离。
  38. 根据权利要求35所述方法，其特征在于，所述分离包括使用针对以下至少一种抗原的特异性抗体进行分选或捕获：PLAP、CD9、CD63、或CD81。
  39. 根据权利要求35所述方法，其特征在于，所述测序装置包括高通量测序装置，优选地，所述测序装置包括BGISEQ系列或MGISEQ系列测序装置。
  40. 根据权利要求26、27或30任一项所述系统，其特征在于，所述数据筛选装置包括：
    物种数据去除单元，所述物种数据去除单元按照预定比对参数，将所述测序结果与所述第一物种的核酸序列信息进行比对，去除比对上的序列，从而获得未比对上的序列，进而获得第一预处理序列数据；
    接头数据去除单元，所述接头数据去除单元与所述物种数据去除单元相连，所述接头数据去除单元按照预定容错率，将所述第一预处理序列数据与测序接头序列比对，从而从第一预处理序列数据中截去序列中的接头序列，进而获得第二预处理序列数据；和
    短序列去除单元，所述短序列去除单元按长度L为标准，从所述第二预处理序列数据中去除长度小于L的序列，从而获得所述经去除处理的序列数据，其中L为18-25的正整数。
  41. 根据权利要求40所述系统，其特征在于，所述物种数据去除单元中所述预定比对参数为最大容错3个碱基，或最大允许3个碱基的插入或缺失。
  42. 根据权利要求40所述系统，其特征在于，所述接头数据去除单元中所述预定容错率为15％-25％。
  43. 根据权利要求40所述系统，其特征在于，当第一物种为人时，所述第一物种的核酸序列信息为Hg19基因组序列。
  44. 根据权利要求26、27、30或40任一项所述系统，其特征在于，在所述数据分析装 置中，采用Kraken法进行微生物物种分类。
  45. 根据权利要求44所述系统，其特征在于，所述采用Kraken法进行微生物物种分类，包括以下单元：
    数据库构建单元，根据已知基因组数据构建K-mer数据库；
    片段生成单元，所述片段生成单元与所述K-mer数据库构建单元相连，所述片段生成单元基于所述数据筛选单元获得的所述经去除处理的序列数据，将每一条所述序列打断为预定长度K-mer的片段；
    片段比对单元，所述片段比对单元与所述片段生成单元相连，所述片段比对单元将所述预定长度K-mer的片段与K-mer数据库进行比对，进而根据最近共同祖先的原则对所述序列进行物种分类。
  46. 根据权利要求45所述系统，其特征在于，所述K-mer数据库包括以下物种的序列数据：人基因组、细菌/古细菌、病毒、真菌、原虫、质粒。
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