WO2019007251A1 - 检测寄生虫感染的方法和试剂盒 - Google Patents

检测寄生虫感染的方法和试剂盒 Download PDF

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WO2019007251A1
WO2019007251A1 PCT/CN2018/093254 CN2018093254W WO2019007251A1 WO 2019007251 A1 WO2019007251 A1 WO 2019007251A1 CN 2018093254 W CN2018093254 W CN 2018093254W WO 2019007251 A1 WO2019007251 A1 WO 2019007251A1
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dna
sample
parasite
derived
sequence
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PCT/CN2018/093254
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English (en)
French (fr)
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张灼华
万正卿
凌捷
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中南大学湘雅医院
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Priority to US16/628,259 priority Critical patent/US11702705B2/en
Priority to CN201880004078.7A priority patent/CN109890985A/zh
Publication of WO2019007251A1 publication Critical patent/WO2019007251A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa

Definitions

  • the present invention relates to the detection of parasitic infections, in particular to determining the parasitic infection status by detecting parasite free DNA in a subject's body fluids, such as blood.
  • the invention also relates to kits for such detection and their use for detecting parasitic infections.
  • Parasitic diseases are diseases caused by parasites invading the human body. Pathological changes and clinical manifestations vary depending on the species and parasitic parts. This type of disease is widely distributed and can be seen all over the world, especially in tropical and subtropical regions. Parasitic infections are often asymptomatic or at least aseptic in the early stages of infection. In order to determine early whether the human body is infected with parasites and timely intervention, effective early detection means are needed.
  • Hydatid disease also known as hydatid disease
  • hydatid disease is a serious zoonotic parasitic disease.
  • echinococcosis There are two main types of echinococcosis, namely, cystic echinococcosis caused by Echinococcus granulosus infection and echinococcosis caused by infection of Echinococcus multilocularis.
  • cystic echinococcosis caused by Echinococcus granulosus infection
  • echinococcosis caused by infection of Echinococcus multilocularis.
  • the echinococcosis is scattered in the host tissue and the vesicle wall is very thin, which is difficult to cleanly and completely removed. Therefore, the echinococcosis is also called worm cancer.
  • the main parasitic part of the hydatid is the liver, and it can also be parasitic on tissues such as the lungs and the brain. Its pathogenic effect is mainly manifested in mechanical compression, toxin action and allergic reaction. In the late stage of the disease, a large cyst is formed in the parasitic part, which compresses the surrounding normal tissue and causes irreversible damage. In recent years, with the development of tourism, the flow of population and the rapid increase of domestic dogs, echinococcosis has become a pandemic disease in the world, seriously jeopardizing world public health and economic development. In China, echinococcosis is mainly prevalent in western provinces such as Georgia, Xinjiang, Qinghai, Sichuan, Inner Mongolia, etc. With the implementation of the national “Belt and Road” program, the prevention and treatment of echinococcosis has received increasing attention and new effective screening has been established. And diagnostic tools are urgent.
  • the diagnosis of existing echinococcosis is based on the results of epidemiological history, clinical manifestations, imaging features, tissue biopsy and laboratory tests (mainly serum immunological tests).
  • Imaging examination can visually observe the capsule or bubble formed by infection, which can better support clinical diagnosis and is the preferred method for the diagnosis of echinococcosis.
  • imaging examinations are susceptible to local tissue conditions, and sometimes it is difficult to make a clear judgment. The requirements for instruments and equipment are high and are not suitable for screening.
  • Pathogen examination tissue biopsy
  • etiological examination is difficult to sample, and direct puncture may cause the hydatid to spread from the ruptured vesicle into the abdominal cavity.
  • imaging examinations and pathogen examinations are required to be performed after significant vesicles are formed, and it is difficult to detect echinococcosis infections before clinical symptoms appear.
  • Immunological examination methods can detect echinococcosis earlier than clinical manifestations and imaging studies.
  • Currently commonly used immunological tests include enzyme-linked immunosorbent assay (ELISA), indirect hemagglutination test (IHA), complement fixation test (CFT), and indirect fluorescent antibody test (IFA).
  • ELISA enzyme-linked immunosorbent assay
  • IHA indirect hemagglutination test
  • CFT complement fixation test
  • IFA indirect fluorescent antibody test
  • Chinese Patent Application Publication No. CN103374615A discloses a PCR detection kit for detecting canine cysticercosis, which is PCR-amplified to detect 12S rDNA of Echinococcus granulosus contained in feces of a test animal to determine whether it is infected with a hydatid disease.
  • Chinese Patent Application Publication Nos. CN105018613A and CN105925724A respectively disclose a method of PCR amplification to detect Echinococcus specific DNA in a test animal such as fox feces to determine whether the test animal is infected with echinococcosis.
  • Animals such as dogs are the ultimate hosts of Echinococcus, and there are adults in the intestines, and there are a large number of eggs in the feces. It is a pity that humans are the intermediate hosts of the echinococcosis, there are no adults in the intestines, and there are no eggs in the feces, so these methods cannot be applied to humans.
  • the inventors have unexpectedly discovered that free DNA derived from parasites such as hydatid is present in host body fluids (such as blood, saliva, urine) infected with parasites such as hydatid.
  • host body fluids such as blood, saliva, urine
  • parasites such as hydatid
  • it can be judged whether or not the host has a parasitic infection.
  • the parasite-derived free DNA in the host body fluid can be detected by the method of the present invention in the host body fluid in which the parasite is infected. Therefore, parasitic infections can be detected early in the infection, thereby guiding therapeutic interventions for parasitic infections; and monitoring the effects of parasitic diseases.
  • the present invention has thus been completed on the basis of these work.
  • the invention provides a method of detecting parasite-derived DNA in a sample, comprising
  • step 3 analyzing the sequencing result of step 3); wherein if the sequencing result contains a DNA sequence of parasite origin, it indicates that the sample contains free DNA derived from a parasite.
  • the sample is a body fluid sample, preferably a sample of blood, saliva and/or urine, in particular a sample of serum and/or plasma.
  • the body fluid sample can be from a mammal, such as a human.
  • the parasite is selected from the group consisting of a protozoan, a nematode, a trematode, and a mites, specifically selected from the group consisting of hookworms, aphids, schistosomes, aphids, aphids, toxoplasma, amoeba, and more particularly aphids.
  • Echinococcus granulosus for example, Echinococcus granulosus and/or Echinococcus multilocularis.
  • step 2) selective enrichment uses independent DNA that does not participate in the enrichment amplification process nor affects the enrichment amplification process, as a reference for homogenization comparisons of different samples;
  • the independent DNA is, for example, lambda phage genomic DNA, and it is also conceivable to use human genomic DNA in the sample as a reference for homogenization comparison.
  • the invention provides a method of diagnosing whether a host subject is undergoing a parasitic infection, comprising
  • step 2) If it is determined according to step 2) that parasite-derived DNA is present in the sample, then the host subject is indicated to be undergoing parasitic infection.
  • the present invention provides a method of treating a parasitic infection in a host subject, comprising: performing the method of the second aspect of the invention to diagnose whether the host subject is undergoing a parasitic infection; if the host object is undergoing parasitic Insect infection, treatment of parasitic infections.
  • the invention provides a method of determining the efficacy of a treatment for a parasitic infection in a host subject, comprising
  • step 2) If it is determined according to step 2) that there is no or only a small amount of parasite-derived DNA in the sample compared to before treatment, then the parasite infection in the host subject is indicated to be treated.
  • the invention provides a method of screening for treatment of a parasitic infection, comprising
  • step 2) If it is determined according to step 2) that there is no or only a small amount of parasite-derived DNA in the sample compared to the pre-treatment, the candidate treatment indicating the parasitic infection is effective.
  • the present invention provides a kit for detecting the presence or absence of a parasite-derived DNA in a sample, comprising: an agent for selectively enriching parasite-derived DNA in a sample, and instructions for use .
  • the kit is for use in the method of any one of the first to fourth aspects of the invention. Accordingly, in another aspect, the invention provides a kit for use in the method of any one of the first to fourth aspects of the invention.
  • the sample to be tested is a body fluid sample, preferably a sample of blood, saliva and/or urine, in particular a sample of serum and/or plasma.
  • the body fluid sample can be from a mammal, such as a human.
  • the kit of the present invention may further comprise a parasitic infection therapeutic agent for treating a parasitic infection in a host subject using the kit.
  • the kit further comprises at least one reagent selected from the group consisting of a DNA extraction reagent, a selective enrichment reagent (such as a PCR reagent), and a high throughput sequencing reagent.
  • the technical solution of the present invention provides at least one of the following advantages or other aspects: no need to extract DNA from parasites; only need to extract the host
  • the free DNA in the body fluid sample can detect trace amounts of free DNA from the parasite, making the collection of samples at the time of testing simplified to the need to collect a small amount of body fluid samples such as peripheral blood or urine.
  • beneficial effects of the present invention are manifested in at least one of the following aspects or others: high level of evidence: direct detection of parasite DNA, with diagnostic significance consistent with pathogenic examination; sensitivity is extremely high: based on selective enrichment reactions such as PCR reactions, Single molecule sensitivity, suitable for early detection of infection; high specificity: can be designed to avoid cross-reaction with other pathogens, can track changes in the disease in real time; non-invasive: only need to collect a tube of peripheral blood, no tissue biopsy or surgery Such operations that cause substantial trauma greatly improve patient compliance and/or feasibility; simplicity: as a screening tool, large instrument support is not required on site.
  • selected from means selecting from among a plurality of candidate targets, and may be any one or more of them.
  • base pair can be used when describing the length of a double-stranded polynucleotide such as DNA.
  • base pair can be used when describing the length of a double-stranded polynucleotide such as DNA.
  • double-stranded polynucleotide such as DNA
  • the present invention provides a method of detecting DNA derived from a parasite in a sample, comprising
  • step 3 analyzing the sequencing result of step 3); wherein if the sequencing result contains a DNA sequence of parasite origin, it indicates that the sample contains free DNA derived from a parasite.
  • free DNA refers to extracellular DNA present in body fluids, usually fragmented DNA that is detached from its naturally occurring state.
  • genomic DNA includes not only chromosomal DNA but also mitochondrial DNA other than chromosomes.
  • the sample is a sample of a bodily fluid of a mammal, preferably a sample of blood, saliva and/or urine, in particular a serum and/or plasma.
  • a mammal preferably a human.
  • the selective enrichment of step 2) is carried out using an amplicon-based capture method and/or a hybridization-based capture method.
  • the amplicon-based capture method refers to a method of amplifying a target nucleic acid fragment from a sample using a nucleic acid amplification technique such as polymerase chain reaction PCR.
  • the hybridization-based capture method refers to a method of specifically capturing/enriching a target nucleic acid fragment using a nucleic acid hybridization technique.
  • the amplicon-based capture method is selected from the group consisting of: multiplex PCR, circular PCR, circular single molecule amplification, and resequencing technology (Circulating Single-Molecule Amplification and Resequencing Technology, cSMART) ), bridge PCR, drop PCR, and DNA isothermal amplification techniques, for example, using multiplex PCR to perform the selective enrichment of step 2).
  • the hybridization-based capture method is selected from the group consisting of: solid phase (eg, chip) hybrid capture, liquid phase hybrid capture, and inverted probe based capture methods such as Molecular inversion probes (MIP) )capture.
  • solid phase hybrid capture eg, chip
  • liquid phase hybrid capture e.g., liquid phase hybrid capture
  • inverted probe based capture methods such as Molecular inversion probes (MIP)
  • one or more specific target sequences from parasite genomic DNA are selectively enriched, for example 1-100, 2-50, 3-40, 4-30 or 5- 20, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 49 or 50, preferably the specific target sequence is a repeat sequence in the parasite genomic DNA.
  • At least one of said specific target sequences is derived from genomic DNA of one or more parasitic species/variants/serotypes, eg, from two or more parasitic species/variants/serotypes Genomic DNA, and thus useful for determining species/variants/serotypes of parasites that may be present in a sample; in particular, at least one of said specific target sequences is two or more parasitic species/variants/serotypes A sequence shared in the genomic DNA, or a non-consensus unique sequence; and/or at least one of the specific target sequences is a repeat in the parasite genome.
  • the parasite is selected from the group consisting of a protozoan, a nematode, a trematode, and a mites, specifically selected from the group consisting of Leishmania, Plasmodium, M. elegans (hookworm), aphids, schistosomiasis, aphids, aphids, arches. Insects, amoeba, more specifically aphids, such as Echinococcus, such as Echinococcus granulosus and/or Echinococcus multilocularis. More specifically, it is a schistosomiasis.
  • one or more sets of primers or probes are used in step 2) to selectively enrich parasite-derived DNA, eg, selectively enrich one or more from parasite genomic DNA Specific target sequences; for example, 1-100, 2-50, 3-40, 4-30 or 5-20 primers or probes, specifically 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 sets of primers or probes, or 60, 70, 80, Primers or probes of groups of any value between 90, 100, 150, 200, 250, 300, 400, 500 and any two values.
  • two or more primer sets can share primers.
  • the selectively enriched specific target sequence in step 2) is 50-500 base pairs in length, for example 60-400 bp, 70-300 bp, 80-200 bp, 100-200 bp. 100-150 bp, 150-200 bp, or 50, 60, 70, 80, 90, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270
  • parasite-derived DNA derived from one or more of the following target sequences is selectively enriched in step 2): a hydatid corresponding to the amplified target sequences of SEQ ID NOS.
  • Genomic DNA preferably, selectively enriched for parasite-derived DNA selected from the genomic DNA corresponding to the target sequences amplified by SEQ ID NOS. 1 to 14 or SEQ ID NOS. 1 to 8; and/or Selective enrichment is carried out in step 2) using one or more sets of primers selected from the group consisting of SEQ ID NO. 1 to 26, for example selected from SEQ ID NO. 1 to 14 or SEQ ID NO. 1 to 8.
  • the DNA of each sample is selectively enriched in step 2) using a labeled primer or probe combination, and the labeled plurality of sample DNA selectively enriched in step 2) Combining, then performing high-throughput sequencing of step 3), enabling detection of multiple samples in a single high-throughput sequencing, which may be 2-10000, 3-5000, 4-3000, 5-1000, 6-500, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 Any value between 49, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more; preferably said
  • the label is selected from the group consisting of a fluorescent label, a short sequence label for encoding, and more preferably a short sequence label for encoding.
  • short sequence markers used as encodings are used to uniquely identify different samples and/or different libraries.
  • the source of the sample and/or the source of the library for a particular readout sequence can be identified.
  • the short sequence marker used for encoding can be added during the selective enrichment step, or can also be added during the database construction process prior to high throughput sequencing.
  • the selective enrichment in step 2) uses an internal control fragment (ICF) enriched with parasite-derived DNA for indicating selective enrichment amplification. Whether the process was successful.
  • ICF internal control fragment
  • step 2) selective enrichment uses independent DNA that does not participate in the enrichment amplification process nor affects the enrichment amplification process, as a reference for homogenization comparisons of different samples;
  • the independent DNA is, for example, lambda phage genomic DNA, and it is also conceivable to use human genomic DNA in the sample as a reference for homogenization comparison.
  • the high throughput sequencing of step 3) is performed using a technique selected from the group consisting of: Roche/454 FLX, illumina/Xten/Hiseq/Miseq/Nextseq, Applied Biosystems/SOLID, Thermo Fisher Scientific/Ion Torrent/Proton, And BGI/BGISEQ-500.
  • sequence fragments that match the host genomic sequence are first filtered from the sequencing results by sequence alignment and/or the additional DNA added as a reference is additionally filtered ( Sequence fragments such as lambda phage genomic DNA are matched to reduce background interference.
  • short recognition coding sequences such as barcode sequences
  • step 4 if the number of reads of the parasite-derived DNA sequence is at least 1%, 2%, 3%, 4%, 5%, 6 in the total number of sequence reads. %, 7%, 8%, 9% or 10% indicates that the sample contains parasite-derived DNA.
  • the invention provides a method of diagnosing whether a host subject is experiencing a parasitic infection, comprising
  • step 2) If it is determined according to step 2) that parasite-derived DNA is present in the sample, then the host subject is indicated to be undergoing parasitic infection.
  • the present invention provides a method of treating a parasitic infection in a host subject, comprising: performing the method of the second aspect of the invention to diagnose whether the host subject is undergoing a parasitic infection; and if the host subject is diagnosed A parasitic infection is treated, and a parasitic infection is treated.
  • the invention provides a method of determining the efficacy of a treatment for a parasitic infection in a host subject, comprising
  • step 2) If it is determined according to step 2) that there is no or only a small amount of parasite-derived DNA in the sample compared to before treatment, then the parasite infection in the host subject is indicated to be treated.
  • the invention provides a method of screening for treatment of a parasitic infection, comprising
  • step 2) If it is determined according to step 2) that there is no or only a small amount of parasite-derived DNA in the sample compared to the pre-treatment, the candidate treatment indicating the parasitic infection is effective.
  • the sample is a body fluid sample, preferably a sample of blood, saliva and/or urine, in particular a sample of serum and/or plasma.
  • the body fluid sample can be from a mammal, such as a human.
  • the present invention provides a kit for detecting the presence or absence of a parasite-derived DNA in a sample, comprising: an agent for selectively enriching parasite-derived DNA in a sample, and instructions for use .
  • the kit is for performing the methods described in any of the foregoing aspects of the invention.
  • the reagent for selectively enriching the parasite-derived DNA in the sample is one or more sets of primers for selectively enriching one or more specific target sequences of the parasite source.
  • And/or probes preferably one or more sets of primers; for example 1-100 sets, 2-50 sets, 3-40 sets, 4-30 sets or 5-20 sets of primers or probes, in particular 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 primers
  • the one or more sets of primers are selected from one or more of the following groups or all: SEQ ID NOs. 1 to 26, for example selected from SEQ ID NO. 1 to 14 or SEQ ID NO. 1 to 8. .
  • the specific target sequence is 50-500 base pairs in length, such as 60-400 bp, 70-300 bp, 80-200 bp, 100-200 bp, 100-150 bp, 150-200 bp, Or 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 Any value between 300 bp or above.
  • the primer or probe is labeled to enable detection of multiple samples in a single high throughput sequencing, which may be 2-10000, 3-5000, 4- 3000, 5-1000, 6-500, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 Any of 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more
  • said label is selected from the group consisting of a fluorescent label, a short sequence label for encoding, and more preferably a short sequence label for encoding.
  • the short sequence tag used as a coding may be 3-10 nucleotides in length, such as 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
  • the specific target sequence is derived from genomic DNA of one or more parasitic species/variants/serotypes, eg, genomic DNA from two or more parasitic species/variants/serotypes And thus can be used to determine the species/variant/serotype of parasites that may be present in the sample; in particular, at least a portion of said specific target sequences are genomes of two or more parasitic species/variants/serotypes Sequences shared in DNA, or unique sequences that are not shared.
  • At least one of said specific target sequences is a repeat of a parasite genomic DNA. Multiple copies of the repeat sequence in the genomic DNA increase the probability of detection, thereby increasing detection sensitivity.
  • the parasite is a echinococcosis, such as Echinococcus granulosus and/or Echinococcus multilocularis.
  • Echinococcus granulosus is the causative agent of echinococcosis, in which echinococcosis causes cystic echinococcosis
  • Echinococcus multilocularis is the causative agent of echinococcosis.
  • the kit further comprises an internal control fragment (ICF) for indicating whether the selective amplification process is successful, wherein each of the internal control fragments can be used for a specific target sequence
  • ICF internal control fragment
  • the internal control fragment typically has properties comparable to a specific target sequence, for example, 50 to 170 bp in length, wherein each fragment contains a recognition region of a pair of corresponding primers in a primer panel, and the GC content of the amplified product is And the length of the product is comparable to the target amplification product of the pair of primers on the hydatid genome.
  • the sequence of the ICF amplification product is known to be different from the target amplification product sequence on the hydatid genome, and can be easily distinguished by subsequent data analysis.
  • the kit further comprises at least one reagent selected from the group consisting of a DNA extraction reagent, a PCR reagent, and a high throughput sequencing reagent.
  • the sample to be tested is a body fluid sample, preferably a sample of blood, saliva and/or urine, in particular a sample of serum and/or plasma.
  • the body fluid sample can be from a mammal, such as a human.
  • the kit of the present invention may further comprise a parasitic infection therapeutic agent for treating a parasitic infection in a host subject using the kit.
  • the invention also provides the use of an agent for selectively enriching parasite-derived DNA in a sample for use in the preparation of a kit according to any of the preceding aspects of the invention.
  • Peripheral blood was collected from subjects with a needle No. 7 or above, and blood samples were collected into a Streck free DNA blood collection tube (Streck, 218997) in an amount of 10 mL.
  • the collected blood samples were stored at room temperature for at least 30 minutes, then transferred to 4 ° C for storage and transport, and plasma was separated within 96 hours.
  • the supernatant (plasma) was collected by centrifugation at 1 600 g for 10 min at 4 ° C, and dispensed into a plurality of 1.5 mL centrifuge tubes. Be careful not to inhale the white blood cells in the middle layer and the red blood cells on the tube wall during the process of taking up the plasma. When turning the tube, pay attention to the operation, and avoid splashing the plasma on the tube cover and the nozzle.
  • a commercially available blood nucleic acid extraction kit (QIAGEN Circulating Nucleic Acid Extraction Kit) was used for extraction.
  • Other methods based on silica gel column adsorption, magnetic bead capture or DNA precipitation can also be used.
  • the centrifuge tube was taken out, 5.4 mL of buffer ACB was added, shaken and mixed, and placed on ice for 5 min.
  • the DNA-buffer mixture in the centrifuge tube was poured into the extension tube, the vacuum pump was turned on, and the mixture was sequentially aspirated through the extension tube-binding column-vacuum base, and the free DNA was bound to the binding column.
  • the extension tube is taken off, the vacuum pump is turned off, 600 ⁇ L of ACW1 is added to the binding column, and the vacuum pump is turned on, which is the first step of cleaning;
  • the vacuum pump is turned off, 750 ⁇ L of ACW2 is added to the binding column, and the vacuum pump is turned on, which is the second step of cleaning;
  • the vacuum pump was turned off, the binding column was taken off, placed in a collection tube matched with the binding column, and centrifuged at 16 000 g for 3 min at room temperature to completely discard the remaining absolute ethanol in the binding column.
  • the binding column was placed in a new 1.5 mL centrifuge tube and dried at 56 ° C for 10 min.
  • the dsDNA HS Assay Kit (Qubit, Q32854) accurately quantifies the extracted free DNA. Samples with free DNA concentrations below 0.2 ng/ ⁇ L were excluded and returned to the ingestor to schedule a heavy draw. The free DNA extraction step was completed, and the extracted free DNA was frozen at -80 ° C for use.
  • the enrichment method used in this example is that multiple pairs of primers amplify multiple sites of the hydatid genome repeat.
  • the free DNA of the subject was amplified and enriched using a self-developed hydatid-specific amplification primer.
  • ICF fragment was added to the amplification system, and the ICF fragment was used as a template to participate in the amplification process along with the genomic DNA of the genomic DNA in the free DNA, as a monitoring system for successful single sample amplification.
  • a certain amount of exogenous lambda phage DNA is added to the amplification system as an internal reference, which is in the amplification system but does not participate in the amplification process, and is used for homogenizing the positive sequence in the sequencing result.
  • the PCR amplification system is as follows:
  • Premix Ex Taq DNA Polymerase HS Version (Cat. No. RR030A) is a pre-mixed PCR reaction solution commercialized by TAKARA, containing TaKaRa Ex Taq HS (1.25 U/25 ⁇ L), PCR Buffer (containing 4 mM Mg 2+ ), dNTP (each 0.4 mM), anti-Taq antibody.
  • sequences of the enriched primer combinations are shown in SEQ ID NOS. 1 to 26.
  • 1F and 1R constitute a pair of primers that specifically amplify corresponding repeats on the genome of the hydatid when using the E. granulosus genome as a template.
  • 2F/2R, 3F/3R, and the like respectively constitute respective primer pairs.
  • ICF internal control fragment
  • the PCR amplification conditions are:
  • the product was purified using commercial AMPure XP Beads (Beckman Coulter, A63881) and self-prepared polyethylene glycol buffer (PEG buffer).
  • AMPure XP Beads 50 ⁇ L of AMPure XP Beads, 20 ⁇ L of PCR amplification product, and 5 ⁇ L of PEG buffer were sequentially added to a 1.5 mL centrifuge tube, and the mixture was thoroughly mixed, and allowed to stand at room temperature for 5 minutes.
  • the centrifuge tube After about 5 minutes, the centrifuge tube is fully dried, the centrifuge tube is removed from the magnetic stand, 22 ⁇ L of 0.1 ⁇ TE is added, and the mixture is gently shaken, and allowed to stand at room temperature for 5 min;
  • the centrifuge tube was placed on a magnetic stand and allowed to stand for 5 min. After the magnetic beads were fully adsorbed to the side, 20 ⁇ L of the supernatant was pipetted to a new PCR tube for use.
  • the dsDNA HS Assay Kit accurately quantifies the purified PCR product. Take 10 ng of PCR product to Ultra TM II DNA Library Prep Kit for (NEB, E7645S) to build the library. Unless otherwise specified, the procedures used below, as well as reagents, oligonucleotides such as primers and linkers, are described in the manufacturer's manual, including but not limited to Ultra TM II DNA Library Prep Kit for with Multiplex Oligos for
  • the product was made up to 25 ⁇ L with 0.1 ⁇ TE buffer, and the following components were sequentially added to the PCR tube:
  • NEBNext Ultra II End Prep Reaction Buffer 3.5 ⁇ L
  • NEBNext Ultra II End Prep Enzyme Mix 1.5 ⁇ L
  • Purified PCR product 25 ⁇ L total capacity 30 ⁇ L
  • the mixture was pipetted with a 100 ⁇ L pipette and centrifuged to return the liquid to the bottom of the tube.
  • the PCR tube was taken out, 2 ⁇ L of USER Enzyme was added, and the mixture was mixed by blowing, and the liquid was returned to the bottom of the tube by instantaneous centrifugation.
  • the centrifuge tube was placed on a magnetic stand and allowed to stand for 5 min. After the magnetic beads were fully adsorbed to the side, the supernatant was transferred to a new 1.5 mL centrifuge tube.
  • AMPure XP Beads 25 ⁇ L was added to the tube, mixed well, and allowed to stand at room temperature for 5 min.
  • the tube was allowed to dry sufficiently, the tube was removed from the magnetic stand, 17 ⁇ L of 0.1 ⁇ TE buffer was added, and the mixture was gently shaken and allowed to stand at room temperature for 5 min.
  • the centrifuge tube was placed on a magnetic stand and allowed to stand for 5 min. After the magnetic beads were fully adsorbed to the side, 15 ⁇ L of the supernatant was aspirated to a new PCR tube for use.
  • the constructed library was subjected to high-throughput sequencing.
  • the selected sequencing platform was Illumina Miseq 500. Each sample was measured for 2M reads, and different amounts of readouts could be measured as needed.
  • Data analysis can be performed manually or assisted by various bioinformatics analysis software.
  • the data analysis of this embodiment uses commercially available analysis software and self-developed analysis software. Although not required, these analysis software can significantly improve the efficiency of the analysis. Download high-throughput sequencing raw data, file in fastq format. The sequencing data was imported into the self-developed bioinformatics analysis software.
  • Each short segment sequence measured in high throughput sequencing is often referred to as a read.
  • the short segment sequence information of multiple samples measured by high-throughput sequencing in the previous step is input into the analysis software; first, these sequences are compared with the genomic sequence of ⁇ -phage, and the number of successfully matched reads is calculated as uniformity.
  • the denominator of the ratio ( ⁇ -DNA is in the amplification system but not involved in the amplification process, which can be used as a reference); the unaligned sequence is then further aligned with the human reference genome sequence, giving up the success ratio with the human reference genome sequence.
  • the sequence of the pair, the remaining sequence was used for subsequent analysis; the sequences were aligned with the two genomic repeats of the hydatid (Echinococcus granulosus and Echinococcus multilocularis), and the short films of the repeat region of the hydatid genome were successfully matched.
  • the segment sequence readout is considered to be the sequence from the echinococcosis sample, and the positive rate and the homogenization ratio of the two reference worm reference genomes are calculated (for the positive rate, the denominator is the total number of reads of the sample, ie, the short segment sequence data. Set R3; for the homogenization ratio, the denominator is the number of reads matching the upper ⁇ -phage genome, ie, the short fragment sequence data set RC).
  • the number of reads successfully aligned with the human genome can also be used as a basis for subsequent homogenization treatment, replacing the additionally added ⁇ -DNA.
  • the pre-processed short segment sequence data set is labeled as R1.
  • Step 1 Comparison to the lambda-phage reference genome: using the bowtie2 alignment tool
  • the segment sequence data set R1 is aligned to the lambda-phage reference genome. Record the number of successfully matched reads, recorded as the short segment sequence data set RC. Based on the alignment results, these short sequences that successfully align to the lambda phage reference genome were discarded, and the short fragment sequences unsuccessfully aligned to the lambda phage reference genome were collected, and these short fragment sequence data were labeled as short fragment sequences. Data set R2.
  • Step 2 (Comparative to the Human Reference Genome): The short segment sequence data set R2 was aligned to the human reference genome using the bowtie2 alignment tool. Based on the alignment results, these short sequence sequences successfully aligned to the human reference genome were discarded, short sequence sequences unsynchronized to the human reference genome were collected, and these short fragment sequence data were labeled as short fragment sequence data set R3.
  • Step 3 (Comparing to the repeat region of the hydatid genome):
  • the short segment sequence data set R3 is aligned to the repeat region of the two hydatid genomes using the bowtie2 alignment tool, and the data set on the successful alignment is recorded as R4.
  • each sample was successfully compared to the positive rate of the hydatid genome repeat region, and the homogenization ratio was calculated.
  • the positive rate is calculated as the percentage of the number of sequences of the sample A in the short segment sequence data set R4 to the number of short segment sequences in R3.
  • the normalization ratio is calculated as the number of sequences of the sample A in the short segment sequence data set R4 divided by the number of sequences of the short segment sequence data set RC.
  • the cystic fluid free DNA of the patient diagnosed with echinococcosis was extracted and added to the amplification system of Example 4 as a simulated sample of the hydatid; the corresponding free DNA was not added to the cystic fluid.
  • the sample was used as a control sample.
  • high-throughput sequencing was followed by selective enrichment and the sequencing results were analyzed, wherein the hydatid simulated samples were selectively plated using 2fg of the cyst fluid free DNA sample as a template. The test was performed three times using the echinococcosis simulation sample and the control sample was performed once, and the analysis results of the four samples were as shown in the following table.
  • the proportion of the hydatid sequence reads in the total sequence reads is referred to herein as the positive rate. It is thus seen that the positive mock sample and the control sample without the hydatid DNA can be significantly distinguished using the method of the present invention.
  • Hydatid simulation sample 1 Positive rate Hydatid reads/total reads Hydatid simulation sample 1 (2fg) 95.46% 384432/402720 Hydatid simulation sample 2 (2fg) 95.76% 416071/434492 Hydatid simulation sample 3 (2fg) 95.72% 510087/532877 Control sample (0fg) 0.06% 151/243889
  • Example 7 With reference to the procedure of Example 7, the amount of the cystic liquid free DNA sample used as a template at the time of selective enrichment was changed (see the table below for the specific amount), and the specimen was simulated using the hydatid DNA simulation sample several times. The results of the analysis of the addition of different amounts of free DNA from the cystic fluid as a template are shown in the table below. It can be noted that there is a difference in the proportion of positive readings of samples with the same amount of DNA added.
  • the concentration of the DNA sample used as a template is very low (1fg/ul), and for samples with very little (1fg/2fg/4fg), there is virtually no guarantee that exactly the sample will be inhaled when the DNA is aspirated, thus resulting in There is a difference in the proportion of positive readings in different samples.
  • the method of the present invention can detect as little as 1 fg of free DNA sample; as the amount of free DNA in the sample increases, the positive rate in the analysis increases.
  • patients with echinococcosis had significantly higher positive detection rates of hydatid DNA, except for patients with P4, P5, P10, P11 and P12. Further through imaging and tissue biopsy, patients with P4 had cysts instead of echinococcosis; patients with P11 had calcified cysts, but no live echinococcosis. It can be speculated that the patient P4 is misdiagnosed; the patient P11 was infected with echinococcosis, but there was no more echinococcosis infection at the time of sampling. Patients with P5, P10 and P12 lost contact and could not be further confirmed.
  • the method of the present invention can not only significantly distinguish between echinococcosis patients and healthy control subjects, but also distinguish between current infection status and past infection status.
  • Example 7 different amounts of hydatid cystic free DNA were added to the amplification system of Example 4 as a model for the echinococcosis simulation, and a corresponding sample without cystic free DNA was used as a control sample.
  • high-throughput sequencing was performed after selective enrichment and the sequencing results were analyzed.
  • the proportion of the hydatid sequence reads in the total sequence reads is referred to herein as the positive rate.
  • the ratio of the hydatid sequence read number to the lambda phage DNA sequence read number is referred to herein as the homogenization ratio.

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Abstract

本发明提供一种检测样品中寄生虫来源的DNA的方法，以及利用该检测方法诊断宿主对象是否正经受寄生虫感染的方法，确定寄生虫感染治疗功效的方法，以及筛选寄生虫感染候选治疗的方法，还提供用于上述方法的试剂盒。

Description

检测寄生虫感染的方法和试剂盒
本申请要求于2017年07月02日提交中国专利局、申请号为CN 201710554315.4、发明名称为“检测寄生虫感染的方法和试剂盒”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及寄生虫感染的检测，具体地涉及通过检测受试者体液(如血液)中寄生虫游离DNA来确定所述寄生虫感染状态。本发明还涉及用于这种检测的试剂盒及其用于检测寄生虫感染的用途。
背景技术
寄生虫病是寄生虫侵入人体而引起的疾病。因虫种和寄生部位不同，引起的病理变化和临床表现各异。本类疾病分布广泛，世界各地均可见到，尤以热带和亚热带地区更多。寄生虫感染经常是无症状的或者至少在感染早期是无症状的。为了早期确定人体是否感染寄生虫以及时介入治疗，需要有效的早期检测手段。
包虫病，又名棘球蚴病，是一种严重的人畜共患性寄生虫病。包虫病主要有两种类型，即细粒棘球绦虫感染所致的囊型包虫病和多房棘球绦虫感染所致的泡型包虫病。对于泡型包虫病，棘球蚴呈多个泡散布于宿主组织中且囊泡壁很薄，难以干净彻底地手术去除，因此泡型包虫病又被称为虫癌。棘球蚴主要寄生部位为肝脏，亦可寄生于肺部及脑部等组织。其致病作用主要表现在机械性压迫、毒素作用和过敏反应等几个方面，疾病晚期在寄生部位形成巨大囊肿，压迫周围正常组织，造成不可逆转的损害。近年来随着旅游业的发展、人口的流动和家犬的急剧增多，包虫病已成为世界流行性疾病，严重危害世界公共卫生和经济发展。在我国，包虫病主要在西藏、新疆、青海、四川、内蒙古等西部省份流行，随着国家“一带一路”计划的实施，包虫病的防治工作日益受到重视，建立新的有效的筛查和诊断手段刻不容 缓。
现有包虫病的诊断是根据流行病学史、临床表现、影像学特征、组织活检和实验室检查(主要是血清免疫学检查)的结果进行综合诊断。
影像学检查可以直观地观察到感染形成的囊或泡，能较好地支持临床诊断，为当前诊断包虫病的首选方法。然而，影像学检查易受局部组织情况影响，有时难以明确判断，对仪器设备要求较高，不适于筛查。
病原学检查(组织活检)是包虫病检测的“金标准”，能直接看到虫体，具有确诊意义。但是病原学检查取样困难，直接穿刺可能导致棘球蚴从破裂的囊泡中扩散到腹腔中。
然而，影像学检查和病原学检查均需要在形成显著囊泡以后才能进行，难以在临床症状出现之前早期发现包虫病感染。
免疫学检查方法可早于临床表现及影像学检查发现包虫病。目前常用的免疫学检测包括酶联免疫吸附试验(ELISA)、间接血凝试验(IHA)、补体结合试验(CFT)和间接荧光抗体试验(IFA)等。这些方法在检测包虫病上各有优劣，但主要缺点是均存在非特异反应及交叉免疫，有一定的假阳性率；并且难以与既往感染区分，因而不适用于病情实时监测。
中国专利申请公开CN103374615A披露了一种检测犬囊性包虫病的PCR检测试剂盒，通过PCR扩增以检测受检动物粪便中含有的细粒棘球绦虫的12S rDNA来确定其是否感染包虫病。类似地，中国专利申请公开CN105018613A和CN105925724A分别披露了PCR扩增检测受试动物如狐狸粪便中的棘球绦虫特异性DNA来确定受试动物是否感染包虫病的方法。犬类等动物是棘球绦虫的最终宿主，其肠道中也有成虫，粪便中有大量虫卵。可惜的是，人是棘球绦虫的中间宿主，肠道中无成虫，粪便中也无虫卵，因此这些方法无法适用于人。
寄生虫病(如包虫病)患者往往由于缺乏有效的筛查和诊断手段，错过了最佳治疗时机。仍然需要更有效地早期检测寄生虫病感染的方法，以及时发现寄生虫感染并尽早实施治疗性介入。
发明内容
发明人意外地发现，感染寄生虫(如棘球蚴)的宿主体液(如血液、唾液、尿液)中存在源自寄生虫(如棘球蚴)的游离DNA。通过本发明方法选择性富集和特异性检测宿主体液中这些寄生虫来源的游离DNA，可以判断宿主是否存在寄生虫感染。即便是在未发现临床症状之前，感染寄生虫的宿主体液中也能通过本发明方法检测到宿主体液中寄生虫来源的游离DNA。因此，可以在感染早期就发现寄生虫感染，进而指导针对寄生虫感染实施治疗性介入；而且可以监测寄生虫病治疗的效果。从而在这些工作的基础上完成了本发明。
在一个方面中，本发明提供一种检测样品中寄生虫来源的DNA的方法，其包括
1)提取样品中的DNA；
2)选择性富集步骤1)所得样品DNA中的寄生虫来源的DNA；
3)将步骤2)经富集的DNA进行高通量测序；和
4)分析步骤3)的测序结果；其中如果测序结果中包含寄生虫来源的DNA序列，则指示所述样品包含寄生虫来源的游离DNA。
在一个实施方案中，所述样品是体液样品，优选地是血液、唾液和/或尿液的样品，特别地是血清和/或血浆的样品。所述体液样品可以来自哺乳动物，例如人。
在另一个实施方案中，所述寄生虫选自原虫、线虫、吸虫和绦虫，具体地选自钩虫、蛔虫、血吸虫、绦虫、蛲虫、弓形虫、阿米巴原虫，更具体地是绦虫，例如棘球绦虫，例如是细粒棘球绦虫和/或多房棘球绦虫。
在另一个实施方案中，在步骤2)选择性富集中使用不参与富集性扩增过程也不影响富集性扩增过程的独立DNA，作为对不同样品进行均一化比较的参照；所述独立DNA例如是λ噬菌体基因组DNA，还可以考虑使用样品中人基因组DNA作为均一化比较的参照。
在第二个方面中，本发明提供一种诊断宿主对象是否正经受寄生 虫感染的方法，其包括
1)提供来自宿主对象的样品；
2)使用本发明第一方面中所述的方法检测所述样品是否存在寄生虫来源的DNA；
3)如果根据步骤2)确定所述样品中存在寄生虫来源的DNA，则指示所述宿主对象正经受寄生虫感染。
此外，本发明还提供一种治疗宿主对象中寄生虫感染的方法，其包括：实施本发明第二个方面的方法诊断所述宿主对象是否正经受寄生虫感染；如果所述宿主对象正经受寄生虫感染，对其实施寄生虫感染的治疗。
在第三个方面中，本发明提供一种确定宿主对象中寄生虫感染治疗的功效的方法，其包括
1)提供来自已接受寄生虫感染治疗的宿主对象的样品；
2)使用本发明第一方面中所述的方法检测所述样品是否存在寄生虫来源的DNA；
3)如果根据步骤2)确定所述样品中不存在或仅存在与治疗前相比更少量的寄生虫来源的DNA，则指示所述宿主对象中的寄生虫感染得到治疗。
在第四个方面中，本发明提供一种筛选寄生虫感染治疗的方法，其包括
1)向宿主对象实施寄生虫感染的候选治疗；
2)使用本发明第一方面中所述的方法检测来自所述宿主对象的样品是否存在寄生虫来源的DNA；
3)如果根据步骤2)确定所述样品中不存在或仅存在与所述治疗前相比更少量的寄生虫来源的DNA，则指示所述寄生虫感染的候选治疗是有效的。
在第五个方面中，本发明提供一种用于检测样品中是否存在寄生虫来源的DNA的试剂盒，其包含：用于选择性富集样品中的寄生虫 来源DNA的试剂，和使用说明。
在一个实施方案中，所述试剂盒用于本发明第一至第四个方面中任一项所述的方法中。相应地在另一个方面，本发明还提供用于本发明第一至第四个方面中任一项所述方法的试剂盒。在这些试剂盒的一个实施方案中，所述待检测样品是体液样品，优选地是血液、唾液和/或尿液的样品，特别地是血清和/或血浆的样品。所述体液样品可以来自哺乳动物，例如人。此外，本发明的试剂盒还可以包含寄生虫感染治疗药，以使用该试剂盒治疗宿主对象中的寄生虫感染。
在一个具体实施方案中，所述试剂盒还包含选自以下的至少一种试剂：DNA提取试剂、选择性富集试剂(如PCR试剂)和高通量测序试剂。
与现有技术中已知的寄生虫病(如包虫病)检测方法相比，本发明的技术方案提供以下至少一种优势或其他方面：无需从寄生虫虫体提取DNA；只需要提取宿主体液样品中的游离DNA，就可以检测出痕量的寄生虫来源的游离DNA，使得检测时收集样本的工作简化到只需采集少量外周血或尿液等体液样品。
本发明的有益效果体现在以下至少一个方面或其他：证据级别高：直接检测寄生虫的DNA，具有与病原学检查一致的确诊意义；灵敏度极高：基于选择性富集反应如PCR反应，可达到单分子灵敏度，适于感染早期检出；特异性极高：可设计成不存在与其他病原体的交叉反应，可实时追踪病情变化；无创：只需采集一管外周血，无需组织活检或手术等造成实质性创伤的操作，大大提高患者依从性和/或可行性；简便：作为筛查手段时，现场不需大型仪器支持。
具体实施方式
除非特别指明，本文所用术语具有所属技术领域一般技术人员理解的通常含义。以下列出了本文所用一些术语的解释，除非特别指明，这些术语的解释以本文定义为准。
术语“选自”表示从众多候选目标中进行选择，并且可以是其中的任何一种或更多种。
在本发明中，当描述双链多核苷酸如DNA的长度时，可以使用“碱基对”。例如，当记载某双链多核苷酸如DNA的长度为n个碱基对，这意味着该双链多核苷酸如DNA的一条链有n个核苷酸。
在第一个方面中，本发明提供一种检测样品中寄生虫来源的DNA的方法，其包括
1)提取样品中的DNA；
2)选择性富集步骤1)所得样品DNA中的寄生虫来源的DNA；
3)将步骤2)经富集的DNA进行高通量测序；和
4)分析步骤3)的测序结果；其中如果测序结果中包含寄生虫来源的DNA序列，则指示所述样品包含寄生虫来源的游离DNA。
术语“游离DNA”是指体液中存在的细胞外DNA，通常是脱离其天然存在状态的片段化的DNA。
在本发明中，术语“基因组DNA”不仅包括染色体DNA，也包括染色体以外的线粒体DNA。
在一个实施方案中，所述样品是哺乳动物的体液样品，优选地是血液、唾液和/或尿液的样品，特别地是血清和/或血浆的样品。所述哺乳动物例如是人。
在另一个实施方案中，使用基于扩增子的捕获方法和/或基于杂交的捕获方法来实施步骤2)的选择性富集。所述基于扩增子的捕获方法是指利用核酸扩增技术(如聚合酶链式反应PCR)从样品中扩增目标核酸片段的方法。所述基于杂交的捕获方法是指利用核酸杂交技术来特异性捕获/富集目标核酸片段的方法。
在一个具体实施方案中，所述基于扩增子的捕获方法选自：多重PCR(multiplex PCR)、环式PCR、循环单分子扩增及重测序技术(Circulating Single-Molecule Amplification and Resequencing Technology，cSMART)、桥式PCR、液滴PCR(droplet PCR)和DNA等温扩增技术，例如使用多重PCR来实施步骤2)的选择性富集。
在另一个具体实施方案中，所述基于杂交的捕获方法选自：固相(如芯片)杂交捕获、液相杂交捕获和基于倒置探针的捕获方法如分 子倒置探针(Molecular inversion probes，MIP)捕获。
在另一个实施方案中，选择性富集来自寄生虫基因组DNA的一个或更多个特异性靶标序列，例如1-100个、2-50个、3-40个、4-30个或5-20个，具体地1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50个，优选地所述特异性靶标序列是寄生虫基因组DNA中的重复序列。
在一个具体实施方案中，至少一个所述特异性靶标序列来自一个或更多个寄生虫物种/变种/血清型的基因组DNA，例如来自两个或更多个寄生虫物种/变种/血清型的基因组DNA，并由此可用于确定样品中可能存在的寄生虫的物种/变种/血清型；特别地，至少一个所述特异性靶标序列是两个或更多个寄生虫物种/变种/血清型的基因组DNA中共有的序列、或者是非共有的独特序列；和/或至少一个所述特异性靶标序列是寄生虫基因组中的重复序列。
在另一个实施方案中，所述寄生虫选自原虫、线虫、吸虫和绦虫，具体地选自利什曼原虫、疟原虫、沟口线虫(钩虫)、蛔虫、血吸虫、绦虫、蛲虫、弓形虫、阿米巴原虫，更具体地是绦虫，例如棘球绦虫，例如是细粒棘球绦虫和/或多房棘球绦虫。另外地更具体地是血吸虫。
在另一个实施方案中，在步骤2)中使用一组或更多组引物或探针来选择性富集寄生虫来源的DNA，例如选择性富集来自寄生虫基因组DNA的一个或更多个特异性靶标序列；例如1-100组、2-50组、3-40组、4-30组或5-20组引物或探针，具体地1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50组引物或探针，或者60、70、80、90、100、150、200、250、300、400、500以及任意两个数值之间的任意值的组的引物或探针。特别地，两个或更多个引物组可共享引物。
在一个具体实施方案中，在步骤2)中经选择性富集的特异性靶标序列的长度是50-500个碱基对，例如是60-400bp、70-300bp、 80-200bp、100-200bp、100-150bp、150-200bp、或者50、60、70、80、90、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300bp或者以上任意两个数值之间的任意值。
在另一个实施方案中，在步骤2)中选择性富集源自一个或更多个以下靶序列的寄生虫来源的DNA：与SEQ ID NO.1至26所扩增靶序列对应的包虫基因组DNA，优选地，选择性富集选自与SEQ ID NO.1至14或SEQ ID NO.1至8所扩增靶序列对应之包虫基因组DNA的寄生虫来源的DNA；和/或在步骤2)中使用选自以下的一组或更多组引物进行选择性富集：SEQ ID NO.1至26，例如选自SEQ ID NO.1至14或SEQ ID NO.1至8。
在另一个实施方案中，在步骤2)中使用带标记的引物或探针组合分别选择性富集各种样品的DNA，并将经步骤2)选择性富集的带标记的多个样品DNA合并，然后进行步骤3)的高通量测序，使得能够在一次高通量测序中检测多个样品，所述多个样品可以是2-10000、3-5000、4-3000、5-1000、6-500个，例如是2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、60、70、80、90、100、150、200、300、400、500、600、700、800、900、1000个或者以上任意两者之间的任意值；优选地所述标记选自荧光标记、用作编码的短序列标记，更优选地是用作编码的短序列标记。这些用作编码的短序列标记用于独特地标识不同的样品和/或不同的文库。具体地，通过在测序结果中识别这些用作编码的短序列标记，可以识别出特定读出序列的样品来源和/或文库来源。在一个具体实施方案中，所述用作编码的短序列标记可以在选择性富集步骤中加入，或者也可以在高通量测序前的建库过程中加入。
在另一个实施方案中，在步骤2)的选择性富集中使用随寄生虫来源的DNA一起富集性扩增的内对照片段(Internal Control Fragment,ICF)，用于指示选择性富集扩增过程是否成功进行。
在另一个实施方案中，在步骤2)选择性富集中使用不参与富集性扩增过程也不影响富集性扩增过程的独立DNA，作为对不同样品进行均一化比较的参照；所述独立DNA例如是λ噬菌体基因组DNA，还可以考虑使用样品中人基因组DNA作为均一化比较的参照。
在另一个实施方案中，使用选自以下的技术进行步骤3)的高通量测序：Roche/454FLX、illumina/Xten/Hiseq/Miseq/Nextseq、Applied Biosystems/SOLID、ThermorFisher Scientific/Ion Torrent/Proton、和BGI/BGISEQ-500。
在另一个实施方案中，在步骤4)的分析中，先通过序列比对从测序结果中滤除与宿主基因组序列匹配的序列片段和/或滤除与额外添加之作为参照物的独立DNA(如λ-噬菌体基因组DNA)匹配的序列片段，以减少背景干扰。另外，还可以预先从高通量测序结果中去掉每个测序读出中的短识别编码序列(如barcode序列)。
在另一个实施方案中，在步骤4)中如果寄生虫来源的DNA序列的读出数在测序结果总序列读出数中占至少1％，2％，3％、4％、5％、6％、7％、8％、9％或10％，则指示所述样品包含寄生虫来源的DNA。
在第二个方面中，本发明提供一种诊断宿主对象是否正经受寄生虫感染的方法，其包括
1)提供来自宿主对象的样品；
2)使用本发明第一方面中的方法检测所述样品是否存在寄生虫来源的DNA；
3)如果根据步骤2)确定所述样品中存在寄生虫来源的DNA，则指示所述宿主对象正经受寄生虫感染。
此外，本发明还提供一种治疗宿主对象中寄生虫感染的方法，其包括：实施本发明第二个方面的方法诊断所述宿主对象是否正经受寄 生虫感染；以及如果所述宿主对象经诊断正经受寄生虫感染，则对其实施寄生虫感染的治疗。
在第三个方面中，本发明提供一种确定宿主对象中寄生虫感染治疗的功效的方法，其包括
1)提供来自已接受寄生虫感染治疗的宿主对象的样品；
2)使用本发明第一方面中的方法检测所述样品是否存在寄生虫来源的DNA；
3)如果根据步骤2)确定所述样品中不存在或仅存在与治疗前相比更少量的寄生虫来源的DNA，则指示所述宿主对象中的寄生虫感染得到治疗。
在第四个方面中，本发明提供一种筛选寄生虫感染治疗的方法，其包括
1)向宿主对象实施寄生虫感染的候选治疗；
2)使用本发明第一方面中的方法检测来自所述宿主对象的样品是否存在寄生虫来源的DNA；
3)如果根据步骤2)确定所述样品中不存在或仅存在与所述治疗前相比更少量的寄生虫来源的DNA，则指示所述寄生虫感染的候选治疗是有效的。
在上述第一至三方面的一个实施方案中，所述样品是体液样品，优选地是血液、唾液和/或尿液的样品，特别地是血清和/或血浆的样品。所述体液样品可以来自哺乳动物，例如人。
在第五个方面中，本发明提供一种用于检测样品中是否存在寄生虫来源的DNA的试剂盒，其包含：用于选择性富集样品中的寄生虫来源DNA的试剂，和使用说明。
在一个实施方案中，所述试剂盒用于执行本发明前述任一方面中所述的方法。
在另一个实施方案中，用于选择性富集样品中的寄生虫来源DNA的试剂是用于选择性富集寄生虫来源的一个或更多个特异性靶标序列的一组或更多组引物和/或探针，优选地是一组或更多组引物； 例如1-100组、2-50组、3-40组、4-30组或5-20组引物或探针，具体地1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50组引物；特别地所述一组或更多组引物选自以下一组或更多组或者全部：SEQ ID NO.1至26，例如选自SEQ ID NO.1至14或SEQ ID NO.1至8。
在一个具体实施方案中，所述特异性靶标序列的长度是50-500个碱基对，例如是60-400bp、70-300bp、80-200bp、100-200bp、100-150bp、150-200bp、或者50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300bp或者以上任意两者之间的任意值。
在另一个实施方案中，所述引物或探针是带标记的，以使得能够在一次高通量测序中检测多个样品，所述多个样品可以是2-10000、3-5000、4-3000、5-1000、6-500个，例如是2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、60、70、80、90、100、150、200、300、400、500、600、700、800、900、1000个或者以上任意两者之间的任意值；优选地所述标记选自荧光标记、用作编码的短序列标记，更优选地是用作编码的短序列标记。在一个具体实施方案中，所述用作编码的短序列标记的长度可以是3-10个核苷酸，例如3、4、5、6、7、8、9或10个核苷酸。
在另一个实施方案中，所述特异性靶标序列来自一个或更多个寄生虫物种/变种/血清型的基因组DNA，例如来自两个或更多个寄生虫物种/变种/血清型的基因组DNA，并由此可用于确定样品中可能存在的寄生虫的物种/变种/血清型；特别地，至少一部分所述特异性靶标序列是两个或更多个寄生虫物种/变种/血清型的基因组DNA中共有的序列、或者是非共有的独特序列。
在另一个实施方案中，至少一个所述特异性靶标序列是寄生虫基因组DNA的重复序列。重复序列在基因组DNA中的多个拷贝增加 了检测几率，从而增加了检测灵敏度。
在另一个实施方案中，所述寄生虫是棘球绦虫，例如是细粒棘球绦虫和/或多房棘球绦虫。棘球绦虫是引发包虫病的病原体，其中细粒棘球绦虫引起囊型包虫病，多房棘球绦虫是引发泡型包虫病的病原体。
在另一个实施方案中，所述试剂盒还包含内对照片段(ICF)，用于指示选择性扩增过程是否成功进行，其中每个所述内对照片段可使用用于特异性靶标序列的其中一对引物和/或探针扩增或捕获。所述内对照片段通常具有与特异性靶标序列相当的性质，例如长度为50～170bp，其中每一片段含有引物组合(primer panel)中一对相应引物的识别区域，且其扩增产物GC含量及产物长度与该对引物在包虫基因组上的目标扩增产物相当。该ICF扩增产物的序列已知，并与包虫基因组上目标扩增产物序列差别较大，后续数据分析时能轻易区分。
在另一个实施方案中，所述试剂盒还包含选自以下的至少一种试剂：DNA提取试剂、PCR试剂和高通量测序试剂。
在所述试剂盒的一个实施方案中，所述待检测样品是体液样品，优选地是血液、唾液和/或尿液的样品，特别地是血清和/或血浆的样品。所述体液样品可以来自哺乳动物，例如人。此外，本发明的试剂盒还可以包含寄生虫感染治疗药，以使用该试剂盒治疗宿主对象中的寄生虫感染。
在第六个方面中，本发明还提供选择性富集样品中的寄生虫来源DNA的试剂在制备试剂盒中的用途，所述试剂盒用于实施本发明前述任一方面所述的方法。
实施例
1.外周血采集
7号或以上针头采集受试者外周血，血样采集至Streck游离DNA采血管(Streck，218997)中，采集量为10mL。采集的血样在室温存放至少30分钟后转至4℃冷藏保存及运输，96h内分离血浆。
2.血浆分离
在4℃条件下，以1 600g离心10min，收集上清(血浆)，将其分装到多个1.5mL离心管中。在吸取血浆过程中注意不要吸到中间层的白细胞以及管壁上的红细胞。转管时，注意操作轻柔，避免将血浆溅到管盖、管口上造成污染。
在4℃条件下，以16 000g离心10min，去除残余细胞及细胞碎片，将上清转入新的1.5mL离心管中，即得所需的血浆。吸取上清时枪头勿触到管底的细胞残余，留约50μL血浆在管底。
3.游离DNA提取
本例中采用商业供应的血液核酸提取试剂盒(QIAGEN Circulating Nucleic Acid Extraction Kit)抽提。亦可采用其他基于硅胶结合柱吸附、磁珠捕获或DNA沉淀的方法抽提。
取3mL分离后的血浆，用QIAGEN Circulating Nucleic Acid Extraction Kit(QIAGEN，55114)抽提游离DNA。
3.1血浆蛋白消化
在50mL离心管中，依次加入300μL Proteinase K(20mg/mL)、3mL血浆(可酌情调整，起始血浆量改变时，抽提buffer及洗脱液体积相应改变)、2.4mL buffer ACL及5.6μL carrier RNA(0.2μg/μL)，震荡混匀后，置于恒温水浴箱中，60℃温浴60min。
3.2沉淀DNA
温浴完成后，取出离心管，加入5.4mL buffer ACB，震荡混匀，置于冰上反应5min。此时可连接好试剂盒配套的真空泵(提供-800至-900mbar负压)、真空底座(QIAGEN，19413)以及试剂盒中提供的结合柱、延伸管，保留结合柱配套的收集管。
3.3结合DNA
将离心管中的DNA-buffer混合物倒入延伸管，打开真空泵，混 合物依次通过延伸管-结合柱-真空底座吸走，而游离DNA被结合在结合柱上。
3.4清洗
待混合物全部吸走后，取走延伸管，关闭真空泵，向结合柱中加入600μL ACW1，打开真空泵，此为第一步清洗；
待ACW1全部吸走后，关闭真空泵，向结合柱中加入750μL ACW2，打开真空泵，此为第二步清洗；
待ACW2全部吸走后，关闭真空泵，向结合柱中加入750μL无水乙醇，打开真空泵，此为第三步清洗。
3.5离心去溶剂
待无水乙醇全部吸走后，关闭真空泵，将结合柱取下，置入结合柱配套的收集管中，室温，16 000g离心3min，以充分弃去结合柱中剩余的无水乙醇。
3.6干燥
离心完成后，将结合柱置入新的1.5mL离心管，56℃干燥10min。
3.7溶解DNA
干燥步骤完成后，向结合柱中心滤膜上加入50μL AVE(可酌情调整)，静置3min，待DNA充分溶解，随后以室温16 000g离心1min，将溶解的DNA收集至1.5mL离心管。
3.8质控
以商品试剂盒
Figure PCTCN2018093254-appb-000001
dsDNA HS Assay Kit(Qubit，Q32854)对抽提的游离DNA进行精确定量。排除游离DNA浓度低于0.2ng/μL的样品，并反馈给收样员，安排重抽血。游离DNA抽提步骤完成，将抽提的游离DNA冻存于-80℃备用。
4.包虫特异性片段扩增富集
本例中采用的富集方法是多对引物对包虫基因组重复序列的多个位点进行扩增。采用自行研发的包虫特异性扩增引物对受试者游离 DNA进行扩增富集。
扩增体系中加入极少量的ICF片段，ICF片段随游离DNA中的包虫基因组DNA一起作为模板参与扩增过程，作为单个样品扩增是否成功的监测系统。
扩增体系中加入一定量的外源的λ噬菌体DNA作为内参，其处于扩增体系中但不参与扩增过程，用于对测序结果中的阳性序列进行均一化处理。
PCR扩增体系如下：
Premix Ex Taq DNA Polymerase HS Version 10μL
富集引物组合(40μM) 1μL
ICF mix(10ag/μL each) 0.5μL
λ-DNA(10ng/uL) 0.5μL
游离DNA模板 8μL
总体积 20μL
注：Premix Ex Taq DNA Polymerase HS Version(货号RR030A)为TAKARA公司商品化的预混PCR反应液，含有TaKaRa Ex Taq HS(1.25U/25μL)、PCR Buffer(含4mM Mg 2+)、dNTP(各0.4mM)、抗Taq抗体。
富集引物组合的序列如SEQ ID NO.1至26所示。作为举例，1F和1R(SEQ ID NO.1和2)构成一对引物，在以棘球绦虫基因组为模板时可特异性扩增出包虫基因组上对应的重复序列。相应地，2F/2R、3F/3R等分别构成各自的引物对。
Figure PCTCN2018093254-appb-000002
Figure PCTCN2018093254-appb-000003
Figure PCTCN2018093254-appb-000004
扩增体系中加入合成的内部质控片段(Internal Control Fragment,ICF)作为样品扩增是否成功的监测系统。ICF的序列信息参见下表，其中ICF-1至ICF-6分别对应于1F/1R、3F/3R、4F/4R、5F/5R、6F/6R和7F/7R的扩增产物。
Figure PCTCN2018093254-appb-000005
Figure PCTCN2018093254-appb-000006
PCR扩增条件为：
PCR扩增完成后，以商品AMPure XP Beads(Beckman Coulter，A63881)及自行配制的聚乙二醇缓冲液(PEG buffer)进行产物纯化。
PEG buffer配方：
PEG8000(Sigma，89510) 40g
1M EDTA(pH 8.0) 1mL
双蒸水 补至100mL
依次向1.5mL离心管中加入50μL AMPure XP Beads、20μL PCR扩增产物、5μL PEG buffer，充分混匀，室温静置5min。
将离心管放至磁力架上，静置5min，待磁珠充分吸附至侧面，将上清液弃去。
向离心管中加入200μL新配制的80％乙醇，保持离心管在磁力架上，前后左右各颠倒2次，垂直静置30s，弃去乙醇。
重复一次上一步骤。
将离心管中的乙醇吸净，保持离心管在磁力架上，打开管盖，晾干。
约5min后，待离心管充分晾干，将离心管从磁力架上取下，加入22μL 0.1×TE，轻轻弹匀，室温静置5min；
将离心管放至磁力架上，静置5min，待磁珠充分吸附至侧面，吸取20μL上清液至一新的PCR管备用。
5.高通量测序
5.1建库
采用
Figure PCTCN2018093254-appb-000008
dsDNA HS Assay Kit对纯化后的PCR产物进行精确定量。取10ng PCR产物，以
Figure PCTCN2018093254-appb-000009
Ultra TMII DNA Library Prep Kit for
Figure PCTCN2018093254-appb-000010
(NEB，E7645S)进行建库。除非具体说明，以下所用操作以及试剂、寡核苷酸如引物和接头参见制造商手册，其包括但不限于
Figure PCTCN2018093254-appb-000011
Ultra TM II DNA Library Prep Kit for
Figure PCTCN2018093254-appb-000012
和
Figure PCTCN2018093254-appb-000013
Multiplex Oligos for
Figure PCTCN2018093254-appb-000014
5.1.1末端修复
用0.1×TE缓冲液将产物补齐至25μL，依次向PCR管中加入如下成分：
NEBNext Ultra II End Prep Reaction Buffer 3.5μL
NEBNext Ultra II End Prep Enzyme Mix 1.5μL
纯化后的PCR产物 25μL
总体积 30μL
用100μL移液器将混合物吹打混匀，瞬时离心使液体回到管底；
在PCR仪中，按照以下条件反应：
温度 反应时间
20℃ 30min
65℃ 30min
4℃ 保持
5.1.2加接头
取出末端修复混合物，依次加入下述成分：
End Prep Reaction Mixture(Step 5.1.1) 30μL
NEBNext Ultra II Ligation Master Mix 15μL
NEBNext Ligation Enhancer 0.5μL
NEBNext Adaptor for Illumina 1.5μL
总体积 47μL
用100μL移液器将混合物吹打混匀，瞬时离心使液体回到管底。
置入PCR仪中，通过以下条件反应：
温度 反应时间
20℃ 15min
反应完成，取出PCR管，加入2μL USER Enzyme，吹打混匀，瞬时离心使液体回到管底。
置入PCR仪中，通过以下条件反应：
温度 反应时间
37℃ 15min
5.1.3连接产物纯化
另取一1.5mL离心管，分别加入连接产物及25μL AMPure XP Beads，充分混匀，室温静置5min。
将离心管放至磁力架上，静置5min，待磁珠充分吸附至侧面，将上清液转移至一新的1.5mL离心管。
向该离心管中加入25μL AMPure XP Beads，充分混匀，室温静置5min。
将离心管放至磁力架上，静置5min，待磁珠充分吸附至侧面，将上清液弃去。
向离心管中加入200μL新配制的80％乙醇，保持离心管在磁力架上，前后左右各颠倒2次，垂直静置30s，弃去乙醇。
重复一次上一步骤。
将离心管中的乙醇吸净，保持离心管在磁力架上，打开管盖，晾干。
约5min后，待离心管充分晾干，将离心管从磁力架上取下，加入17μL 0.1×TE缓冲液，轻轻弹匀，室温静置5min。
将离心管放至磁力架上，静置5min，待磁珠充分吸附至侧面，吸取15μL上清液至一新的PCR管备用。
5.1.4PCR扩增
依次向上述PCR管中加入以下成分：
经步骤5.1.3纯化的加接头DNA片段 15μL
NEBNext Ultra II Q5Master Mix 25μL
Index Primer 5μL
Universal PCR Primer 5μL
总体积 50μL
置入PCR仪中，按照以下条件反应：
Figure PCTCN2018093254-appb-000015
5.1.5PCR产物纯化
另取一1.5mL离心管，分别加入PCR扩增产物及45μL AMPure XP Beads，充分混匀，室温静置5min。将离心管放至磁力架上，依照步骤5.1.3的方法纯化PCR产物。纯化完成后，以33μL 0.1×TE溶解，并吸取30μL至新的1.5mL离心管中备用。
5.2高通量测序
按照制造商说明，将建好的文库进行高通量测序，所选测序平台为Illumina Miseq 500，每个样品测2M个读出数(reads)，也可以根据需要测不同量的读出数。
6.数据分析及结果判定
数据分析可以人工操作，也可以使用各种生物信息学分析软件辅助进行。本实施例的数据分析中使用了可商业获得的分析软件以及自行研发的分析软件。尽管并非必需，这些分析软件可以显著提高分析效率。下载高通量测序原始数据(raw data)，文件为fastq格式。将测序数据输入自行研发的生物信息学分析软件。
高通量测序中测得的每条短片段序列通常称为一个read(读出)。 将先前步骤中经高通量测序测得的多个样品的短片段序列信息输入分析软件中；首先将这些序列与λ-噬菌体的基因组序列进行比对，计算成功匹配的reads数，作为均一化比率的分母(λ-DNA处于扩增体系中但不参与扩增过程，可作为参照)；随后将未比对上的序列进一步与人参考基因组序列进行比对，放弃与人参考基因组序列成功比对的序列，剩余序列用于后续分析；将这些序列与两种包虫(细粒棘球绦虫和多房棘球绦虫)基因组重复序列进行比对，成功匹配包虫基因组的重复序列区的短片段序列读出被认作是来自包虫病样品的序列，并计算两种包虫参考基因组的阳性率及均一化比值(针对该阳性率，分母为该样本总reads数，即短片段序列数据集R3；针对该均一化比值，分母为匹配上λ-噬菌体基因组的reads数，即短片段序列数据集RC)。
另外，当扩增体系中不额外添加λ-DNA时，也可以使用与人基因组成功比对的reads数作为后续均一化处理的基础，替代额外添加的λ-DNA。
数据分析软件的处理流程：
预步骤(预处理)：将短片段序列按标识样本的barcode信息分类，并去掉短片段序列中的barcode。将预处理后的短片段序列数据集标记为R1。
步骤1(比对到λ-噬菌体参考基因组)：用bowtie2比对工具
(http://bowtie-bio.sourceforge.net/bowtie2/index.shtml,B.Langmead&S.L.Salzberg,Fast gapped-read alignment with Bowtie 2,Nature Methods,9:357-359(2012))将短片段序列数据集R1比对到λ-噬菌体参考基因组。记录成功匹配的reads数，记为短片段序列数据集RC。根据比对结果，放弃这些成功比对到λ-噬菌体参考基因组的短片段序列，收集未成功比对到λ-噬菌体参考基因组上的短片段序列，并将这些短片段序列数据标记为短片段序列数据集R2。
步骤2(比对到人类参考基因组)：用bowtie2比对工具将短片段序列数据集R2比对到人类参考基因组。根据比对结果，放弃这些成功比对到人类参考基因组的短片段序列，收集未成功比对到人类参考基因组上的短片段序列，并将这些短片段序列数据标记为短片段序列 数据集R3。
步骤3(比对到包虫基因组的重复序列区)：用bowtie2比对工具将短片段序列数据集R3比对到两种包虫基因组的重复序列区，将成功比对上的数据集记为R4。根据比对结果，统计各个样本成功比对到包虫基因组重复序列区的阳性率，并计算均一化比值。其中，对于选定的样本A，其阳性率的计算为，样本A在短片段序列数据集R4的序列数目占R3中短片段序列数目的百分比。均一化比值的计算为，样本A在短片段序列数据集R4的序列数目除以短片段序列数据集RC的序列数目。
7.包虫病模拟样品分析
根据实施例2至3的方案，提取确诊包虫病的患者的囊液游离DNA，将其加入实施例4的扩增体系中，作为包虫模拟样品；相应的不加囊液游离游离DNA的样品作为对照样品。根据实施例4至6的方案，经选择性富集之后进行高通量测序并分析测序结果，其中选择性富集时包虫模拟样品使用2fg的囊液游离DNA样品作为模板。使用包虫模拟样品进行3次、对照样品进行1次，4个样品的分析结果如下表所示。测序结果中，包虫序列读出数(reads)在总序列读出数(reads)中所占的比例在此称为阳性率。由此看出，使用本发明的方法可以显著区分阳性模拟样品和无包虫DNA的对照样品。
DNA样品编号 阳性率 包虫reads/总reads
包虫模拟样品1(2fg) 95.46％ 384432/402720
包虫模拟样品2(2fg) 95.76％ 416071/434492
包虫模拟样品3(2fg) 95.72％ 510087/532877
对照样品(0fg) 0.06％ 151/243889
8.阳性率与模板量正相关
参照实施例7的操作，改变选择性富集时作为模板所用囊液游离DNA样品的量(具体用量参见下表)，使用包虫DNA模拟样品多次进行分析。下表中示出了加入不同量包虫囊液游离DNA作为模板的分析结果。可以注意到，同样DNA加入量的样品阳性reads比例有差异。这可能是因为用作模板的DNA样品浓度很低(1fg/ul)，对于用量极少(1fg/2fg/4fg)的样品，在吸取DNA时实际上不能保证恰好吸入准确量的样品，因此导致不同样品阳性reads比例有差别。然而，可以发现总的趋势仍然是确定的：本发明的方法可以检测到低至1fg的游离DNA样品；随着样品游离DNA用量的增加，分析结果中阳性率也随之增加。
作为经验知识，关于DNA量与包虫基因组对应关系可以使用以下换算关系：100fg DNA约等于1个包虫基因组。
Figure PCTCN2018093254-appb-000016
Figure PCTCN2018093254-appb-000017
9.扩大样品量的受试者分析
选取多个确诊包虫病的患者以及健康对照，根据实施例1至6的方案，采集受试者的游离DNA，经选择性富集之后进行高通量测序并分析测序结果，其中选择性富集时使用来源于1mL血样的游离DNA样品作为模板。具体来说，纳入包虫病患者15人(P1至P15)，少数民族健康对照5人(C1至C5)，汉族健康对照19人(C6至C24)。 分析结果如下表所示。健康对照样品中检出包虫DNA的阳性率非常低，最高也不高于3％(C13)。而包虫病患者均有显著更高的包虫DNA阳性检出率，仅患者P4、P5、P10、P11、P12例外。进一步通过影像学和组织活检发现，患者P4有囊肿而非包虫病；患者P11有已钙化的包虫包囊，但已无活的包虫。由此可以推测患者P4实为误诊；患者P11曾经感染包虫病，但在取样时已经不再有包虫感染。患者P5、P10和P12失联，无法进一步确认。
由此看出，本发明的方法不仅可以显著区分包虫病患者和健康对照对象，而且还可以区分当前感染状态和既往感染状态。
Figure PCTCN2018093254-appb-000018
Figure PCTCN2018093254-appb-000019
Figure PCTCN2018093254-appb-000020
10.包虫模拟样品计算均一化比值
参照实施例7的操作，将不同量的包虫囊液游离DNA加入实施例4的扩增体系中，作为包虫模拟样品，相应的不加囊液游离DNA的样品作为对照样品。根据实施例4至6的方案，经选择性富集之后进行高通量测序并分析测序结果。测序结果中，包虫序列读出数(reads)在总序列读出数(reads)中所占的比例在此称为阳性率。包虫序列读出数(reads)与λ-噬菌体DNA序列读出数的比值在此称为均一化比值。
Figure PCTCN2018093254-appb-000021
11.感染者血浆模拟样品分析
利用实施例7所述的包虫囊液游离DNA，将不同量的囊液游离 DNA(0fg,5fg,10fg)加入正常对照的血浆中，作为感染者血浆模拟样品，相应的不加囊液游离DNA的样品作为对照样品(NTC)。根据实施例3的方案，提取感染者血浆模拟样品中的游离DNA，经选择性富集之后进行高通量测序并分析测序结果。表格中测序结果中，包虫序列读出数(reads)在总序列读出数(reads)中所占的比例在此称为阳性率。由此看出，使用本发明的方法可以检测出低至5fg/mL浓度的感染者血浆模拟样品。
Figure PCTCN2018093254-appb-000022

Claims (30)

  1. 一种检测样品中寄生虫来源的DNA的方法，其包括
    1)提取样品中的DNA；
    2)选择性富集步骤1)所得样品DNA中的寄生虫来源的DNA；
    3)将步骤2)经富集的DNA进行高通量测序；和
    4)分析步骤3)的测序结果；其中如果测序结果中包含寄生虫来源的DNA序列，则指示所述样品包含寄生虫来源的游离DNA。
  2. 根据权利要求1的方法，其中在步骤2)的选择性富集中使用随寄生虫来源的DNA一起富集性扩增的内对照片段(internal control fragment,ICF)，用于指示选择性富集扩增过程是否成功进行。
  3. 根据前述权利要求中任一项的方法，其中使用基于扩增子的捕获方法和/或基于杂交的捕获方法来实施步骤2)的选择性富集。
  4. 根据权利要求3的方法，其中所述基于扩增子的捕获方法选自：多重PCR、环式PCR、循环单分子扩增及重测序技术、桥式PCR、液滴PCR(droplet PCR)、DNA等温扩增技术，例如使用多重PCR来实施步骤2)的选择性富集。
  5. 根据权利要求3的方法，其中所述基于杂交的捕获方法选自：固相(芯片)杂交捕获、液相杂交捕获和分子倒置探针捕获。
  6. 根据权利要求3至5中任一项的方法，其中选择性富集来自寄生虫基因组DNA的一个或更多个特异性靶标序列，例如1-100个、2-50个、3-40个、4-30个或5-20个，具体地1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50个，优选地所述特异性靶标序列是寄生虫基因组DNA中的重复序列。
  7. 根据权利要求6的方法，其中至少一个所述特异性靶标序列来自一个或更多个寄生虫物种/变种/血清型的基因组DNA，例如来自两个或更多个寄生虫物种/变种/血清型的基因组DNA，并由此可用于 确定样品中可能存在的寄生虫的物种/变种/血清型；特别地，至少一个所述特异性靶标序列是两个或更多个寄生虫物种/变种/血清型的基因组DNA中共有的序列、或者是非共有的独特序列；和/或至少一个所述特异性靶标序列是寄生虫基因组中的重复序列。
  8. 根据前述权利要求中任一项的方法，其中所述寄生虫选自原虫、线虫、吸虫和绦虫，具体地选自钩虫、蛔虫、血吸虫、绦虫、蛲虫、弓形虫、阿米巴原虫，更具体地是绦虫，例如棘球绦虫，例如是细粒棘球绦虫和/或多房棘球绦虫。
  9. 根据前述权利要求中任一项的方法，其中在步骤2)中使用一组或更多组引物或探针来选择性富集寄生虫来源的DNA，例如选择性富集来自寄生虫基因组DNA的一个或更多个特异性靶标序列；例如1-100组、2-50组、3-40组、4-30组或5-20组引物或探针，具体地1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50组引物或探针。
  10. 根据权利要求9的方法，其中在步骤2)中经选择性富集的特异性靶标序列的长度是50-500个碱基对，例如是60-400bp、70-300bp、80-200bp、100-200bp、100-150bp、150-200bp、或者50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300bp或者以上任意两个数值之间的任意值。
  11. 根据前述权利要求中任一项的方法，其中
    在步骤2)中选择性富集源自一个或更多个以下靶序列的寄生虫来源的DNA：与SEQ ID NO.1至26所扩增靶序列对应的包虫基因组DNA，优选地，选择性富集选自与SEQ ID NO.1至14或SEQ ID NO.1至8所扩增靶序列对应之包虫基因组DNA的寄生虫来源的DNA；和/或
    在步骤2)中使用选自以下的一组或更多组引物进行选择性富集：SEQ ID NO.1至26，例如选自SEQ ID NO.1至14或SEQ ID NO.1至 8。
  12. 根据前述权利要求中任一项的方法，其中在步骤2)中使用带标记的引物或探针组合分别选择性富集各种样品的DNA，并将经步骤2)选择性富集的带标记的多个样品DNA合并，然后进行步骤3)的高通量测序，使得能够在一次高通量测序中检测多个样品，所述多个样品可以是2-10000、3-5000、4-3000、5-1000、6-500个，例如是2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、60、70、80、90、100、150、200、300、400、500、600、700、800、900、1000个或者以上任意两者之间的任意值；优选地所述标记选自荧光标记、用作编码的短序列标记，更优选地是用作编码的短序列标记。
  13. 根据前述权利要求中任一项的方法，其中使用选自以下的技术进行步骤3)的高通量测序：Roche/454FLX、illumina/Xten/Hiseq/Miseq/Nextseq、Applied Biosystems/SOLID、ThermorFisher Scientific/Ion Torrent/Proton、和BGI/BGISEQ-500。
  14. 根据前述权利要求中任一项的方法，其中在步骤4)的分析中，先通过序列比对从测序结果中滤除与宿主基因组序列匹配的序列片段，以减少背景干扰。
  15. 根据前述权利要求中任一项的方法，其中在步骤4)中如果寄生虫来源的DNA序列的读出数在测序结果总序列读出数中占至少1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、15％、20％、25％、30％、35％或40％，则指示所述样品包含寄生虫来源的DNA。
  16. 根据前述权利要求中任一项的方法，其中在步骤2)选择性富集中使用不参与富集性扩增过程也不影响富集性扩增过程的独立DNA，作为对不同样品进行均一化比较的参照；所述独立DNA例如是λ噬菌体基因组DNA。
  17. 一种诊断宿主对象是否正经受寄生虫感染的方法，其包括
    1)提供来自宿主对象的样品；
    2)使用权利要求1-16中任一项的方法检测所述样品是否存在寄生虫来源的DNA；
    3)如果根据步骤2)确定所述样品中存在寄生虫来源的DNA，则指示所述宿主对象正经受寄生虫感染；和
    4)任选地，向经诊断正经受寄生虫感染的所述宿主对象实施寄生虫感染的治疗。
  18. 一种确定宿主对象中寄生虫感染治疗的功效的方法，其包括
    1)提供来自已接受寄生虫感染治疗的宿主对象的样品；
    2)使用权利要求1-16中任一项的方法检测所述样品是否存在寄生虫来源的DNA；
    3)如果根据步骤2)确定所述样品中不存在或仅存在与治疗前相比更少量的寄生虫来源的DNA，则指示所述宿主对象中的寄生虫感染得到治疗。
  19. 一种筛选寄生虫感染治疗的方法，其包括
    1)向宿主对象实施寄生虫感染的候选治疗；
    2)使用权利要求1-16中任一项的方法检测来自所述宿主对象的样品是否存在寄生虫来源的DNA；
    3)如果根据步骤2)确定所述样品中不存在或仅存在与所述治疗前相比更少量的寄生虫来源的DNA，则指示所述寄生虫感染的候选治疗是有效的。
  20. 根据权利要求17-19中任一项的方法，其中所述样品是体液样品，优选地是血液、唾液和/或尿液的样品，特别地是血清和/或血浆的样品。
  21. 一种用于检测样品中是否存在寄生虫来源的DNA的试剂盒，其包含：用于选择性富集样品中的寄生虫来源DNA的试剂，和使用说明。
  22. 根据权利要求20的试剂盒，其中所述试剂盒用于权利要求1-19中任一项所述的方法中。
  23. 根据权利要求20或21的试剂盒，其中用于选择性富集样品中的寄生虫来源DNA的试剂是用于选择性富集寄生虫来源的一个或更多个特异性靶标序列的一组或更多组引物和/或探针，优选地是一组或更多组引物；例如1-100组、2-50组、3-40组、4-30组或5-20组引物或探针，具体地1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50组引物或探针；特别地所述一组或更多组引物选自以下一组或更多组或者全部：SEQ ID NO.1至26，例如选自SEQ ID NO.1至14或SEQ ID NO.1至8。
  24. 根据权利要求22的试剂盒，其中所述特异性靶标序列的长度是50-500个碱基对，例如是60-400bp、70-300bp、80-200bp、100-200bp、100-150bp、150-200bp、或者50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300bp或者以上任意两者之间的任意值。
  25. 根据权利要求20至23中任一项的试剂盒，其中所述引物或探针是带标记的，以使得能够在一次高通量测序中检测多个样品，所述多个样品可以是2-10000、3-5000、4-3000、5-1000、6-500个，例如是2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、60、70、80、90、100、150、200、300、400、500、600、700、800、900、1000个或者以上任意两者之间的任意值；优选地所述标记选自荧光标记、用作编码的短序列标记，更优选地是用作编码的短序列标记。
  26. 根据权利要求20至24中任一项的试剂盒，其中所述特异性靶标序列来自一个或更多个寄生虫物种/变种/血清型的基因组DNA，例如来自两个或更多个寄生虫物种/变种/血清型的基因组DNA，并由此可用于确定样品中可能存在的寄生虫的物种/变种/血清型；特别地，至少一部分所述特异性靶标序列是两个或更多个寄生虫物种/变种/血 清型的基因组DNA中共有的序列、或者是非共有的独特序列。
  27. 根据权利要求20至25中任一项的试剂盒，其中所述寄生虫是棘球绦虫，例如是细粒棘球绦虫和/或多房棘球绦虫。
  28. 根据权利要求20至26中任一项的试剂盒，其还包含内对照片段(ICF)，用于指示选择性扩增过程是否成功进行。
  29. 根据权利要求20至27中任一项的试剂盒，其还包含选自以下的至少一种试剂：DNA提取试剂、PCR试剂和高通量测序试剂。
  30. 根据权利要求20-27中任一项的试剂盒，其还包含寄生虫感染治疗药，以使用该试剂盒治疗宿主对象中的寄生虫感染。
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CN113355431A (zh) * 2020-08-24 2021-09-07 四川大学华西医院 肝包虫基因片段筛选方法、扩增引物及试剂盒

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