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WO2019114049A1 - 一种用于分析肠道微生物的引物组合物及其应用 - Google Patents

一种用于分析肠道微生物的引物组合物及其应用 Download PDF

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WO2019114049A1
WO2019114049A1 PCT/CN2017/120443 CN2017120443W WO2019114049A1 WO 2019114049 A1 WO2019114049 A1 WO 2019114049A1 CN 2017120443 W CN2017120443 W CN 2017120443W WO 2019114049 A1 WO2019114049 A1 WO 2019114049A1
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sequence
primer
sequencing
seq
analysis
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French (fr)
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朱永亮
穆延召
周燕
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Suzhou Precision Gene Co Ltd
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Suzhou Precision Gene Co Ltd
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Priority to EP17934588.9A priority Critical patent/EP3604557A4/en
Priority to US16/611,279 priority patent/US11401561B2/en
Publication of WO2019114049A1 publication Critical patent/WO2019114049A1/zh
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • 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
    • C12Q1/6869—Methods for sequencing
    • 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
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples

Definitions

  • the present application belongs to the field of molecular biology, and relates to a primer composition for analyzing intestinal microorganisms and an application thereof, and particularly relates to a detection kit for analyzing a primer composition for classifying intestinal microorganisms by constructing a library by two rounds of PCR. And application.
  • the culture-dependent method can obtain the corresponding strains while identifying the strains, which is convenient for subsequent research, but the culture method itself is time-consuming and laborious, and the intestinal microorganisms are anaerobic bacteria and The facultative anaerobic bacteria are mainly difficult to cultivate, and the proportion of the bacteria will change during the cultivation process, which makes the application bottleneck.
  • the traditional molecular biology method that is not culture-dependent can extract the gut microbial genome from the sample without culture, and use molecular biological means to separate, identify and quantify, so that the results can accurately reflect the high-abundance bacteria in intestinal microbes. The composition and true proportion of the species.
  • 16SrDNA is a "molecular clock" of bacterial taxonomy research because of its high diversity among species. It has 9 variable regions and 10 conserved regions. The variable regions vary from bacteria to bacteria. It is closely related to the phylogeny of bacteria. Therefore, the taxonomic characteristics of each bacterium can be obtained by analyzing the sequence of the variable region.
  • CN 105937053 A discloses a method for establishing a fecal microbial gene library based on high-throughput gene sequencing, which adopts a "nested PCR" method for enrichment and amplification of 16S rDNA, thereby minimizing host and food residues. Genomic contamination, combined with V3 and V6, for specific amplification and large-scale parallel sequencing, to obtain a library of target gene sequences of the flora.
  • CN 107058490 A discloses a method for analyzing the diversity and difference of intestinal and oral flora based on a new generation of high-throughput sequencing technology, which uses the Illumina MiSeq sequencing system for sequencing to analyze the composition and function of the flora. However, the constructed library coverage is not high enough and the detection sensitivity is low.
  • the present application provides a primer composition for analyzing intestinal microorganisms and an application thereof, and the primer composition of the present application adopts a two-step amplification method to obtain a sequencing library, which comprises a random base and an added library.
  • the complexity of the sequence reduces the proportion of phix, ensures more sequencing quality, and improves library coverage.
  • the present application provides a primer composition for analyzing gut microbes comprising:
  • first set of primers comprising a first forward primer and a first reverse primer
  • first forward primer comprises four portions connected in sequence from the 5' to the 3' end: first Sequencing universal primer, 2 bp random base sequence, linker sequence and enteric microbial 16S rRNA V3-V4 region-specific forward primer
  • first reverse primer includes four portions connected in sequence from the 5' to the 3' end : a second sequencing universal primer, a 2 bp random base sequence, a linker sequence, and a gut microbial 16S rRNA V3-V4 region-specific reverse primer
  • the second set of primers comprising a second forward primer and a second reverse primer
  • the second forward primer comprises three portions connected in sequence from the 5' to the 3' end: P5 end a linker sequence, a first tag sequence, and a partial sequence of a first sequencing universal primer
  • the second reverse primer comprising three portions in sequence from the 5' to the 3' end: a P7 end linker sequence, a second tag sequence, and a Second sequencing of partial sequences of universal primers.
  • the inventors have found that a sequencing library is obtained by using a two-step amplification method, the first set of primers comprising a V3-V4 site-specific degenerate primer, which improves the coverage of the amplified fragment, detection specificity and sensitivity. In combination with data analysis, more abundant microbiome information can be obtained.
  • the first set of primers contains a random base of 2 bp, which increases the complexity of the library sequence, reduces the proportion of phix, and ensures more sequencing quality.
  • One set of primers also contained a 2 bp Linker sequence that was not homologous to the 16S genomic sequence, reducing PCR amplification preferences; the second set of primers contained a double index (label) design that allowed for more sample mixing.
  • the first sequencing universal primer and the second sequencing universal primer are independently selected from the sequencing universal primers of any one of the second generation sequencing platforms, and the present application uses the sequencing universal primer of the illumina platform.
  • the linker sequence shown is 2-5 bp in length, for example 2 bp, 3 bp, 4 bp or 5 bp, preferably 2 bp.
  • nucleic acid sequence of the first forward primer is as shown in SEQ ID NO. 1
  • nucleic acid sequence of the first reverse primer is as shown in SEQ ID NO. 2
  • nucleic acid sequence is as follows:
  • First forward primer (SEQ ID NO. 1): TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NN TGCCTACGGRRBGCASCAGKVRVGAAT;
  • First reverse primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG NN GGGGACTACNVGGGTWTCTAATCC;
  • N is expressed as any base in A ⁇ T ⁇ G ⁇ C.
  • nucleic acid sequence of the P5 end linker sequence is set forth in SEQ ID NO. 3-12, and the nucleic acid sequence is as follows:
  • SEQ ID NO. 4 TATCCTCT S503 SEQ ID NO. 5 GTAAGGAG S505 SEQ ID NO.6 ACTGCATA S506 SEQ ID NO.7 AAGGAGTA S507 SEQ ID NO.8 CTAAGCCT S508 SEQ ID NO.9 CGTCTAAT S510 SEQ ID NO.10 TCTCTCCG S511 SEQ ID NO.11 TCGACTAG S513 SEQ ID NO.12 TTCTAGCT S515
  • nucleic acid sequence of the P7 end linker sequence is set forth in SEQ ID NO. 13-22, and the nucleic acid sequence is as follows:
  • the first tag sequence of the second forward primer is set forth in SEQ ID NO. 23, and the nucleic acid sequence is as follows:
  • First tag sequence (SEQ ID NO. 23): AATGATAGCGCACCCACCGAGATCTACAC.
  • the partial sequence of the first sequencing universal primer of the second forward primer is as shown in SEQ ID NO. 24, and the nucleic acid sequence is as follows:
  • Partial sequence of the first sequencing universal primer (SEQ ID NO. 24): TCGTCGGCAGCGTC.
  • the tag sequence of the second reverse primer is set forth in SEQ ID NO. 25, and the nucleic acid sequence is as follows:
  • Second tag sequence (SEQ ID NO. 25): CAAGCAGAAGACGGCATACGAGAT.
  • the partial sequence of the second sequencing universal primer of the second reverse primer is as shown in SEQ ID NO. 26, and the nucleic acid sequence is as follows:
  • Partial sequence of the second sequencing universal primer (SEQ ID NO. 26): GTCTCGTGGGCTCGG.
  • the second forward primer is obtained by combining the partial sequence of the first sequencing universal primer represented by the first tag sequence + P5 linker + SEQ ID NO. 24 shown in SEQ ID NO. 23, for example, :AATGATACGGCGACCACCGAGATCTACAC-CTCTCTAT-TCGTCGGCAGCGTC;
  • the second reverse primer is obtained by a partial sequence combination of a second tag universal primer represented by SEQ ID NO. 25 and a second sequencing universal primer represented by SEQ ID NO.
  • it can be CAAGCAGAAGACGGCATACGAGAT-TCGCCTTA-GTCTCGTGGGCTCGG.
  • the present application provides a kit for analyzing gut microbes comprising the primer composition of the first aspect.
  • the present application provides a method for analyzing an intestinal microorganism, comprising the primer composition according to the first aspect, comprising the steps of:
  • the sample is derived from any one or a combination of at least two of feces, soil, urine or oral cells, preferably derived from feces.
  • the extraction of the sample genomic DNA described in the step (1) comprises the following specific steps:
  • step (1') adding the sample resuspended in step (1') to a remover and mixing, and centrifuging;
  • step (2') adding the sample after centrifugation in step (2') to the binding solution, mixing, adding to the adsorption column, centrifuging, rinsing, adding 75% absolute ethanol, and centrifuging;
  • the lysate of step (1') comprises Tris-Cl buffer, Tween-40, EDTA and SDS.
  • the Tris-Cl buffer has a molar concentration of 0.5-2 M, and may be, for example, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.8 M or 2 M, preferably 0.8-1.2 M.
  • the Tween-40 has a volume fraction of 8-15%, for example 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%, preferably 10-13% .
  • the EDTA has a molar concentration of from 0.1 to 1 M, for example 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M or 1 M, preferably 0.1- 0.5M.
  • the SDS has a volume fraction of 5-15%, and may be, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%. It is preferably 8-12%.
  • the remover of step (2') comprises Al 2 (SO 4 ) 3 , CTAB and NaCl.
  • the molar concentration of the Al 2 (SO 4 ) 3 is 0.5-2 M, and may be, for example, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M, 1.2 M, 1.3 M, 1.5 M, 1.6. M, 1.7 M, 1.8 M, 1.9 M or 2 M, preferably 0.8-1.2 M.
  • the CTAB has a mass concentration of 200-300 g/L, and may be, for example, 200 g/L, 210 g/L, 220 g/L, 230 g/L, 250 g/L, 260 g/L, 270 g/L, 280 g/L. 290 g/L or 300 g/L, preferably 230-260 g/L.
  • the molar concentration of the NaCl is 0.8-2M, for example 0.8M, 0.9M, 1M, 1.2M, 1.3M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M or 2M, preferably 1.2-1.8M.
  • the inventors have found that the addition of the Tween-40 improves the efficiency of sample lysis, does not require a specific oscillating breaker, and can be operated by a conventional vortexer, which is efficient and convenient, and single sample extraction can generally be completed within 45 minutes;
  • the addition of Al 2 (SO4) 3 can effectively adsorb the corrosion quality of the sample.
  • the CTAB has a certain ability to adsorb the corrosion quality, and at high salt concentration, the polysaccharide in the sample can be effectively removed, and the three reagents are
  • the extracted DNA has high purity and can be directly used in subsequent molecular experiments.
  • the conditions of the first round of PCR described in the step (2) are: pre-denaturation at 93-96 ° C for 1-5 min; pre-denaturation at 93-96 ° C for 20-40 s, 50-55 ° C for 20-40 s, 70-75 ° C. 20-40s for a total of 25-35 cycles; 70-75 °C for 3-6 minutes.
  • the conditions of the first round of PCR in step (2) are: pre-denaturation at 95 ° C for 3 min; pre-denaturation at 95 ° C for 30 s, 53 ° C for 30 s, and 72 ° C for 30 s for 28 cycles; 72 ° C for 5 min.
  • the conditions of the second round of PCR described in the step (2) are: pre-denaturation at 93-96 ° C for 1-5 min; pre-denaturation at 93-96 ° C for 20-40 s, 54-58 ° C for 20-40 s, 70-75 ° C. 20-40s for 6-12 cycles; 70-75 °C for 3-6 minutes.
  • the conditions of the second round of PCR in step (2) are: pre-denaturation at 95 ° C for 3 min; pre-denaturation at 95 ° C for 30 s, extension at 55.5 ° C for 30 s, 72 ° C for 30 s, 8 cycles; 72 ° C for 5 min, 1 cycle.
  • step (1) further comprises a step of purifying, preferably using magnetic beads.
  • the volume ratio of the magnetic beads to the amplification product is (0.4-0.7):1, which may be, for example, 0.4:1, 0.5:1, 0.6:1 or 0.7:1, preferably 0.5:1.
  • step (3) the data analysis described in step (3) is performed by sequencing using an Illumina Miseq sequencer.
  • the sequencing analysis specifically includes the following steps: sequencing by the Illumina Miseq sequencer to obtain a sequencing read length according to the label, and assembling the full length sequence of the high variable region V3-V4 by using the overlap relationship of the read length, and then The full-length sequences were subjected to classification analysis to obtain a classification of the microbial population.
  • the classification analysis of the full-length sequence specifically includes calculating the full-length sequence difference, performing the classification of the operational taxonomy unit OTU according to the sequence difference degree, assigning the full-length sequence to the OTU, and then classifying each OTU.
  • the full-length sequence was aligned into the V6 database of 16S rRNA, and the alignment results were compared to OTU for species annotation based on the principle of the majority.
  • the present application provides a primer composition according to the first aspect, a kit according to the second aspect, or an analytical method according to the third aspect for analyzing a classified intestinal microorganism.
  • the application provides an analytical method according to the third aspect for immunohistochemical analysis.
  • the present application provides the primer composition of the first aspect, the kit of the second aspect, or the analytical method of the third aspect, for use in the diagnosis of cancer.
  • the cancer is selected from, but not limited to, any one or a combination of at least two of intestinal cancer, adenocarcinoma or bladder cancer.
  • a sequencing library is obtained by using a two-step amplification method, and the primer composition can mix more samples to a greater extent, thereby improving amplification fragment coverage, detection specificity, and sensitivity, and combining data.
  • Analysis can obtain more abundant microbiome information, increase the complexity of the library sequence, reduce the proportion of phix, ensure more sequencing quality, and reduce PCR amplification preference;
  • the data processing and analysis method provided by the present application realizes identification and diversity analysis of more microbial species in taxonomic genus and species, and obtains relative abundance values of related microbial populations;
  • the method of the present application performs sequencing and data analysis on intestinal microorganisms, and realizes identification of more species in taxonomic and even species, that is, obtaining more information on intestinal microbiology, which can be used for immunohistochemical research and clinical research. Provide certain guiding significance in medication;
  • the method of the present application can be used for detecting cancer, and the detection rate of cancer can reach more than 90%, especially the detection rate of intestinal cancer can reach 93.65%, and the detection rate of adenoma can reach 91.53%.
  • FIG. 3 is a diagram showing the result of correlation analysis after sample C data analysis in the embodiment of the present application.
  • the nucleic acid sequence of the first forward primer is shown in SEQ ID NO. 1
  • the nucleic acid sequence of the first reverse primer is shown in SEQ ID NO. 2
  • the nucleic acid sequence is as follows:
  • First forward primer (SEQ ID NO. 1): TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NN TGCCTACGGRRBGCASCAGKVRVGAAT;
  • First reverse primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG NN GGGGACTACNVGGGTWTCTAATCC;
  • N is expressed as any base in A ⁇ T ⁇ G ⁇ C.
  • the nucleic acid sequence of the P5 end linker sequence is set forth in SEQ ID NO. 3-12, and the nucleic acid sequence is as follows:
  • the nucleic acid sequence of the P7 end linker sequence is set forth in SEQ ID NO. 13-22, and the nucleic acid sequence is as follows:
  • the first tag sequence of the second forward primer is set forth in SEQ ID NO. 23, and the nucleic acid sequence is as follows:
  • First tag sequence (SEQ ID NO. 23): AATGATAGCGCACCCACCGAGATCTACAC.
  • a partial sequence of the first sequencing universal primer of the second forward primer is set forth in SEQ ID NO. 24, and the nucleic acid sequence is as follows:
  • Partial sequence of the first sequencing universal primer (SEQ ID NO. 24): TCGTCGGCAGCGTC.
  • the tag sequence of the second reverse primer is set forth in SEQ ID NO. 25, and the nucleic acid sequence is as follows:
  • Second tag sequence (SEQ ID NO. 25): CAAGCAGAAGACGGCATACGAGAT.
  • a partial sequence of the second sequencing universal primer of the second reverse primer is set forth in SEQ ID NO. 26, and the nucleic acid sequence is as follows:
  • Partial sequence of the second sequencing universal primer (SEQ ID NO. 26): GTCTCGTGGGCTCGG.
  • the second forward primer is: AATGATAGCGCACCCACCGAGATCTACAC-CTCTCTAT-TCGTCGGCAGCGTC;
  • the second reverse primer is CAAGCAGAAGACGGCATACGAGAT-TCGCCTTA-GTCTCGTGGGCTCGG.
  • Buffer A 0.5 M Tris-Cl buffer (pH 8.0), 1.0 M NaCl;
  • Lysate B 1 M Tris-Cl buffer (pH 8.0), 0.2 M EDTA, 10% SDS, 12% Tween-40;
  • Remover D 1.0M Al 2 (SO4) 3 , 250 g / L CTAB, 1.5 M NaCl;
  • Binding solution E 0.5 M Tris-Cl buffer (pH 8.0), 5 M isothiocyanate;
  • Rinsing solution F 5M Tris-Cl buffer (pH 8.0), 25% (by volume) absolute ethanol;
  • the primer composition and the detection probe are assembled with a reagent related to the kit to prepare the detection kit.
  • A, B, and C were divided into two equal parts, labeled as: A1/A2, B1/B2, C1/C2, and three samples of A1, B1, and C1 were processed and obtained by the method described in this patent.
  • Three samples of A2, B2 and C2 were sent directly to Jinweizhi for sequencing.
  • the specific DNA extraction method includes the following steps:
  • DNA can be stored at 0 ⁇ 10 ° C, if you want to store for a long time, you can place at -20 ° C.
  • the extracted DNA was subjected to purity test, and the ratio of OD 260 /OD 280 was between 1.7 and 1.9, which can be directly used for subsequent PCR construction.
  • the lysate B contained no Tween-40, the other components were the same as in Example 1, and the DNA extraction method was the same as in Example 2.
  • the lysate B contained no Al 2 (SO 4 ) 3 , the other components were the same as in Example 1, and the DNA extraction method was the same as in Example 2.
  • the lysate B contained no CTAB, the other components were the same as in Example 1, and the DNA extraction method was the same as in Example 2.
  • reaction solution was prepared on ice.
  • premixed Mix was prepared according to the following components according to the amount of reaction + ⁇ , and the prepared premixed Mix was dispensed.
  • the reaction system is as follows:
  • Reagent Usage amount 2 ⁇ PCR mixture (Mg 2+ , dNTPs) 12.5 ⁇ l Upstream primer (1 ⁇ M) 5 ⁇ l Downstream primer (1 ⁇ M) 5 ⁇ l Sample DNA 5ng/ ⁇ l (or ddH2O) 2.5 ⁇ l
  • the reaction conditions are as follows:
  • the first round of PCR amplification products were purified using Agencourt AMPure XP (Beckman Coulter) as follows:
  • the first cleaning the reaction tube is placed on the magnetic stand, and 200 ⁇ l of new 80% ethanol is added to stand at room temperature (>30 s) to remove 80% of the ethanol;
  • second cleaning repeat the above 4) first cleaning operation, gently centrifuge to remove residual 80% ethanol;
  • Agarose electrophoresis determines whether the amplification is accurate, and the amplified product is about 550 bp in length.
  • the PCR product purified in the step (2) is used as a template, and PCR is introduced into the sequencing link.
  • the specific steps are as follows:
  • Reagent Usage amount 2 ⁇ PCR mixture (Mg 2+ , dNTPs) 12.5 ⁇ l Upstream primer (1 ⁇ M) 5 ⁇ l Downstream primer (1 ⁇ M) 5 ⁇ l DNA (first round of PCR purification) 2.5 ⁇ l Total 25 ⁇ l
  • the reaction conditions are as follows:
  • step (2) wherein the volume ratio of the PCR product to the Agencourt AMPure XP magnetic beads is 2:1;
  • Agarose electrophoresis determines whether the amplification is accurate, and the length of the amplified product is about 600 bp;
  • the sequenced and constructed library was sequenced by Ilisea's Miseq sequencing, and the sequencing side rate was PE300 sequencing.
  • Quality control of the raw data obtained by sequencing high quality data is screened by information such as Barcode and primers.
  • the screening criteria are as follows: complete sequence; complete pre-primer sequence; removal of Barcode, primer, linker, sequence length Must be greater than 400 bp, does not contain ambiguous bases; there can be no more than 20 single-base repeats, and the resulting sequences correspond to each sample according to the information;
  • the detection rate of intestinal cancer is 93.65%
  • the detection rate of adenoma is 91.53%
  • the accuracy rate of healthy people is 87.88%. It can be seen that the method of the present application is for detecting cancer. The accuracy rate can reach more than 90%.
  • a sequencing library is obtained by using a two-step amplification method, and the primer composition can mix more samples to a greater extent, thereby improving amplification fragment coverage, detection specificity, and sensitivity.
  • the data processing analysis method provided by the present application, Achieve more taxonomic genus, species identification and diversity analysis of microbial species, and obtain relative abundance values of relevant microbial populations.

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Abstract

本发明提供了一种用于分析肠道微生物的引物组合物、其组成的检测试剂盒及其应用，其中采用两步法扩增获得测序文库，其中包含了随机碱基。

Description

一种用于分析肠道微生物的引物组合物及其应用 技术领域
本申请属于分子生物学领域，涉及一种用于分析肠道微生物的引物组合物及其应用，具体涉及通过两轮PCR构建文库，分析分类肠道微生物的引物组合物，其组成的检测试剂盒及应用。
背景技术
动物胃肠道内庞大多样的微生物群落与动物的食性、机体的免疫功能、疾病与健康等有着密切联系，越来越引起重视并成为研究热点。然而采用传统方法破环性的采集胃肠道样品对其进行研究受到很大制约，且对于野生保护动物并不可取，因此很多研究都以动物的粪便作为研究样品。
科学家们还发现，不同国家人群中的肠道微生物存在很多差异，差异最显著的是微生物的多样性程度。例如：印第安人和马拉维人的肠道微生物组比美国人的肠道微生物具有更大的多样性。有观点认为，微生物多样性程度越高，人体越健康。该研究还发现，尽管来自三种不同地域人群的肠道微生物组存在很多差异，但它们之间也存在惊人的相似性。如，三个不同国家的婴儿微生物组形成过程具有共同的模式，即婴儿需要6-9个月的时间来获得第一组6-700个细菌，然后再经过几年的时间才能获得成人的微生物组。该研究还发现了一个非常有趣的现象，即肠道微生物组的构成会随年龄增长而发生改变，而这一变化恰恰适应了不同年龄段人体的需求。
肠道微生物的研究大体经历了培养依赖的方法，非培养依赖的传统分子生物学方法，基于测序的高通量组学方法3个阶段。
通过比较3个阶段研究方法的特点与应用，人们可以发现培养依赖的方法 在鉴定菌种的同时即可获得相应菌株，方便后续研究，但培养法本身费时费力，肠道微生物以厌氧菌和兼性厌氧菌为主，培养起来更加困难，并且在培养的过程中菌种比例会发生改变，使得其应用存在瓶颈。非培养依赖的传统分子生物学方法可以不经培养直接从样品中提取肠道微生物基因组，利用分子生物学手段进行分离、鉴定和定量，使其结果可以比较准确的反应肠道微生物中高丰度菌种的组成和真实比例。
但是传统分子生物学方法存在着通量低的缺陷，靶向方法如实时定量PCR每次只能研究一种或一类肠道微生物，而非靶向方法如最常用的梯度变形凝胶电泳(Denaturinggradient gel electrophoresis，DGGE)受限于灵敏度，往往只能研究肠道中高丰度的微生物。肠道微生物群落组成复杂，每种细菌都与其他细菌形成复杂的相互关系网络，低丰度的菌种同样扮演者重要的角色，所以传统分子生物学研究结果往往具有片面性。
16SrDNA因其序列在物种间的高度多样性，成为细菌分类学研究的“分子钟”，具9个可变区和10个保守区间隔排列的特征，可变区因细菌而异，且变异程度与细菌的系统发育密切相关。因此通过分析可变区的序列即可得到各细菌的分类学特征。
CN 105937053 A公开了一种基于高通量基因测序建立粪便菌群基因文库的方法，该方法采用“巢式PCR”的方法对以16S rDNA进行富集扩增，最大限度减少了宿主和食物残渣基因组污染，同时将V3和V6组合，进行特异扩增并进行大规模并行测序，获得菌群的靶标基因序列库。CN 107058490 A公开了一种基于新一代高通量测序技术的肠道及口腔菌群多样性及差异性的分析方法，该方法利用illumina MiSeq测序系统进行测序，从而分析菌群的组成与功能。 但构建的文库覆盖率不够高，检测灵敏度较低。
基于上述问题，如何设计引物，能够尽可能全面覆盖所述样本的微生物，提高检测的特异性和灵敏度，就成为亟待解决的问题。
发明内容
针对现有技术的不足，本申请提供了一种分析肠道微生物的引物组合物及其应用，本申请引物组合物采用两步法扩增获得测序文库，其中包含了随机碱基，增加了文库序列的复杂度，降低了phix的比例，保证更多测序质量，提高了文库的覆盖度。
第一方面，本申请提供了一种用于分析肠道微生物的引物组合物，其包含：
第一组引物，所述第一组引物包含第一正向引物和第一反向引物，其中，所述第一正向引物从5’到3’端包括依次相连的四个部分：第一测序通用引物、2bp的随机碱基序列、linker序列和肠道微生物16S rRNA V3-V4区特异性正向引物；所述第一反向引物从5’到3’端包括依次相连的四个部分：第二测序通用引物、2bp的随机碱基序列、linker序列和肠道微生物16S rRNA V3-V4区特异性反向引物；
第二组引物，所述第二组引物包含第二正向引物和第二反向引物，其中，所述第二正向引物从5’到3’端包括依次相连的三个部分：P5端接头序列、第一标签序列和第一测序通用引物的部分序列；所述第二反向引物从5’到3’端包括依次相连的三个部分：P7端接头序列、第二标签序列和第二测序通用引物的部分序列。
本申请中，发明人发现，通过采用两步法扩增获得测序文库，所述第一组引物包含V3-V4位点特异性简并引物，提高了扩增片段覆盖率、检测特异性及 灵敏度，结合数据分析，可获得更丰富的微生物组信息，此外，所述第一组引物包含2bp的随机碱基，增加了文库序列的复杂度，降低了phix的比例，保证更多测序质量，第一组引物还包含2bp不与16S基因组序列同源的Linker序列，降低了PCR扩增偏好性；第二组引物包含双index(标签)的设计，可以更大程度地混合更多的样本。
根据本申请，所述第一测序通用引物和所述第二测序通用引物独立地选自任意一种二代测序平台的测序通用引物，本申请采用illumina平台的测序通用引物。
根据本申请，所示linker序列的长度为2-5bp，例如可以是2bp、3bp、4bp或5bp，优选为2bp。
根据本申请，所述第一正向引物的核酸序列如SEQ ID NO.1所示，所述第一反向引物的核酸序列如SEQ ID NO.2所示，所述核酸序列如下：
第一正向引物(SEQ ID NO.1)：TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNTGCCTACGGRRBGCASCAGKVRVGAAT；
第一反向引物(SEQ ID NO.2)：GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG NNGGGGACTACNVGGGTWTCTAATCC；
其中，N表示为A\T\G\C中的任意一个碱基。
根据本申请，所述P5端接头序列的核酸序列如SEQ ID NO.3-12所示，所述核酸序列如下：
  序列 接头名称
SEQ ID NO.3 CTCTCTAT S502
SEQ ID NO.4 TATCCTCT S503
SEQ ID NO.5 GTAAGGAG S505
SEQ ID NO.6 ACTGCATA S506
SEQ ID NO.7 AAGGAGTA S507
SEQ ID NO.8 CTAAGCCT S508
SEQ ID NO.9 CGTCTAAT S510
SEQ ID NO.10 TCTCTCCG S511
SEQ ID NO.11 TCGACTAG S513
SEQ ID NO.12 TTCTAGCT S515
根据本申请，所述P7端接头序列的核酸序列如SEQ ID NO.13-22所示，所述核酸序列如下：
  序列 接头名称
SEQ ID NO.13 TCGCCTTA N701
SEQ ID NO.14 CTAGTACG N702
SEQ ID NO.15 TTCTGCCT N703
SEQ ID NO.16 GCTCAGGA N704
SEQ ID NO.17 AGGAGTCC N705
SEQ ID NO.18 CATGCCTA N706
SEQ ID NO.19 GTAGAGAG N707
SEQ ID NO.20 CAGCCTCG N710
SEQ ID NO.21 TGCCTCTT N711
SEQ ID NO.22 TCCTCTAC N712
根据本申请，所述第二正向引物的第一标签序列如SEQ ID NO.23所示，所述核酸序列如下：
第一标签序列(SEQ ID NO.23)：AATGATACGGCGACCACCGAGATCTACAC.
根据本申请，所述第二正向引物的第一测序通用引物的部分序列如SEQ ID  NO.24所示，所述核酸序列如下：
第一测序通用引物的部分序列(SEQ ID NO.24)：TCGTCGGCAGCGTC.
根据本申请，所述第二反向引物的标签序列如SEQ ID NO.25所示，所述核酸序列如下：
第二标签序列(SEQ ID NO.25)：CAAGCAGAAGACGGCATACGAGAT.
根据本申请，所述第二反向引物的第二测序通用引物的部分序列如SEQ ID NO.26所示，所述核酸序列如下：
第二测序通用引物的部分序列(SEQ ID NO.26)：GTCTCGTGGGCTCGG.
本申请中，所述第二正向引物通过SEQ ID NO.23所示的第一标签序列+P5接头+SEQ ID NO.24所示的第一测序通用引物的部分序列组合得到，例如可以是：AATGATACGGCGACCACCGAGATCTACAC-CTCTCTAT-TCGTCGGCAGCGTC；所述第二反向引物通过SEQ ID NO.25所示的第二标签序列+P7接头+SEQ ID NO.26所示的第二测序通用引物的部分序列组合得到，例如可以是CAAGCAGAAGACGGCATACGAGAT-TCGCCTTA-GTCTCGTGGGCTCGG.
第二方面，本申请提供一种分析肠道微生物的试剂盒，其包括如第一方面所述的引物组合物。
第三方面，本申请提供一种肠道微生物的分析方法，采用如第一方面所述的引物组合物，包括如下步骤：
(1)样本基因组DNA的提取；
(2)扩增子文库的构建：进行两轮PCR扩增，第一轮PCR扩增采用第一组引物组，第二轮PCR扩增采用第二组引物组，构建高通量测序文库；
(3)数据分析。
根据本申请，所述样本来源于粪便、土壤、尿液或口腔细胞中的任意一种或至少两种的组合，优选为来源于粪便。
根据本申请，步骤(1)所述样本基因组DNA的提取包括如下具体步骤：
(1’)将样本与裂解液混合后，重悬；
(2’)将步骤(1’)重悬后的样本加入去除剂混匀，离心；
(3’)将步骤(2’)离心后的样本加入结合液混匀，加入吸附柱中，离心，漂洗，加入75％的无水乙醇，离心；
(4’)将步骤(3’)离心后的样本加入洗脱液，离心得到所述基因组DNA。
根据本申请，步骤(1’)所述裂解液包括Tris-Cl缓冲液、Tween-40、EDTA和SDS。
优选地，所述Tris-Cl缓冲液的摩尔浓度为0.5-2M，例如可以是0.5M、0.6M、0.7M、0.8M、0.9M、1M、1.1M、1.2M、1.3M、1.4M、1.5M、1.6M、1.8M或2M，优选为0.8-1.2M。
优选地，所述Tween-40的体积分数为8-15％，例如可以是8％、9％、10％、11％、12％、13％、14％或15％，优选为10-13％。
优选地，所述EDTA的摩尔浓度为0.1-1M，例如可以是0.1M、0.2M、0.3M、0.4M、0.5M、0.6M、0.7M、0.8M、0.9M或1M，优选为0.1-0.5M。
优选地，所述SDS的体积分数为5-15％，例如可以是5％、6％、7％、8％、9％、10％、11％、12％、13％、14％或15％，优选为8-12％。
根据本申请，步骤(2’)所述去除剂包括Al 2(SO 4) 3、CTAB和NaCl。
优选地，所述Al 2(SO 4) 3的摩尔浓度为0.5-2M，例如可以是0.5M、0.6M、 0.7M、0.8M、0.9M、1M、1.2M、1.3M、1.5M、1.6M、1.7M、1.8M、1.9M或2M，优选为0.8-1.2M。
优选地，所述CTAB的质量浓度为200-300g/L，例如可以是200g/L、210g/L、220g/L、230g/L、250g/L、260g/L、270g/L、280g/L、290g/L或300g/L，优选为230-260g/L。
优选地，所述NaCl的摩尔浓度为0.8-2M，例如可以是0.8M、0.9M、1M、1.2M、1.3M、1.5M、1.6M、1.7M、1.8M、1.9M或2M，优选为1.2-1.8M。
本申请中，发明人发现，所述Tween-40的添加提高了样本裂解效率，无需特定震荡破碎仪，普通涡旋仪即可操作，高效便捷，单个样品提取一般可在45min内完成；所述Al 2(SO4) 3的添加可有效吸附样本腐蚀质，所述CTAB对腐蚀质的吸附有一定能力，且在高盐浓度下，可有效去除样本中多糖类物质，通过这三个试剂的配合，提取得到的DNA纯度高，可直接用于后续分子实验。
根据本申请，步骤(2)所述第一轮PCR的条件为：93-96℃预变性1-5min；93-96℃预变性20-40s，50-55℃20-40s，70-75℃20-40s，共进行25-35个循环；70-75℃延伸3-6min。
优选地，步骤(2)所述第一轮PCR的条件为：95℃预变性3min；95℃预变性30s，53℃30s，72℃30s，共进行28个循环；72℃延伸5min。
根据本申请，步骤(2)所述第二轮PCR的条件为：93-96℃预变性1-5min；93-96℃预变性20-40s，54-58℃20-40s，70-75℃20-40s，共进行6-12个循环；70-75℃延伸3-6min。
优选地，步骤(2)所述第二轮PCR的条件为：95℃预变性3min；95℃预变性30s，55.5℃延伸30s，72℃30s，8个循环；72℃5min，1个循环。
根据本申请，步骤(1)之后还包括纯化的步骤，优选采用磁珠进行纯化。
根据本申请，所述磁珠与扩增产物的体积比为(0.4-0.7)∶1，例如可以是0.4∶1、0.5∶1、0.6∶1或0.7∶1，优选为0.5∶1。
根据本申请，步骤(3)所述数据分析为采用Illumina Miseq测序仪进行测序分析。
根据本申请，所述测序分析具体包括如下步骤：通过Illumina Miseq测序仪测序得到按照标签区分的测序读长，利用读长的重叠关系组装得到高可变区V3-V4的全长序列，再对全长序列进行分类分析，得到所述微生物群体的分类。
根据本申请，所述全长序列进行分类分析具体包括计算全长序列差异度，根据序列差异度执行操作分类学单元OTU的分类，将全长序列分配到OTU中，再将每一个OTU分类中的全长序列比对到16S rRNA的V6数据库中，将比对结果根据众数原则对OTU进行物种注释。
第四方面，本申请提供一种如第一方面所述的引物组合物、如第二方面所述的试剂盒或如第三方面所述的分析方法用于分析分类肠道微生物。
第五方面，本申请提供一种如第三方面所述的分析方法用于免疫组学分析。
第六方面，本申请提供一种如第一方面所述的引物组合物、如第二方面所述的试剂盒或如第三方面所述的分析方法用于癌症的诊断。
优选地，所述癌症选自但不限于肠癌、腺癌或膀胱癌中的任意一种或至少两种的组合。
与现有技术相比，本申请具有如下有益效果：
(1)本申请中，通过采用两步法扩增获得测序文库，所述引物组合物可以更大程度地混合更多的样本，提高了扩增片段覆盖率、检测特异性及灵敏度， 结合数据分析，可获得更丰富的微生物组信息，增加了文库序列的复杂度，降低了phix的比例，保证更多测序质量，降低了PCR扩增偏好性；
(2)本申请提供的数据处理分析方法，实现更多微生物物种在分类学属、种上的鉴别及多样性分析，并得到相关微生物群体的相对丰度值；
(3)本申请方法对肠道微生物进行测序及数据分析，实现了更多物种在分类学属甚至种上的鉴别，即获得更多肠道微生物生物学信息，可为免疫组学研究以及临床用药上提供一定指导意义；
(4)本申请方法可用于检测癌症，癌症的检出率可达90％以上，尤其针对肠癌的检出率可达93.65％，针对腺瘤的检出率可达91.53％。
附图说明
图1为本申请实施例中样本A数据分析后相关性分析的结果图；
图2为本申请实施例中样本B数据分析后相关性分析的结果图；
图3为本申请实施例中样本C数据分析后相关性分析的结果图。
具体实施方式
下面结合附图和实施例对本申请作进一步的详细说明。可以理解的是，此处所描述的具体实施例仅仅用于解释本申请，而非对本申请的限定。另外还需要说明的是，为了便于描述，附图中仅示出了与本申请相关的部分而非全部结构。
实施例1检测试剂盒的制备
(1)引物组合物的制备
所述第一正向引物的核酸序列如SEQ ID NO.1所示，所述第一反向引物的核酸序列如SEQ ID NO.2所示，所述核酸序列如下：
第一正向引物(SEQ ID NO.1)：TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNTGCCTACGGRRBGCASCAGKVRVGAAT；
第一反向引物(SEQ ID NO.2)：GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG NNGGGGACTACNVGGGTWTCTAATCC；
其中，N表示为A\T\G\C中的任意一个碱基。
所述P5端接头序列的核酸序列如SEQ ID NO.3-12所示，所述核酸序列如下：
  序列 接头名称
SEQ ID NO.3 CTCTCTAT S502
SEQ ID NO.4 TATCCTCT S503
SEQ ID NO.5 GTAAGGAG S505
SEQ ID NO.6 ACTGCATA S506
SEQ ID NO.7 AAGGAGTA S507
SEQ ID NO.8 CTAAGCCT S508
SEQ ID NO.9 CGTCTAAT S510
SEQ ID NO.10 TCTCTCCG S511
SEQ ID NO.11 TCGACTAG S513
SEQ ID NO.12 TTCTAGCT S515
所述P7端接头序列的核酸序列如SEQ ID NO.13-22所示，所述核酸序列如下：
  序列 接头名称
SEQ ID NO.13 TCGCCTTA N701
SEQ ID NO.14 CTAGTACG N702
SEQ ID NO.15 TTCTGCCT N703
SEQ ID NO.16 GCTCAGGA N704
SEQ ID NO.17 AGGAGTCC N705
SEQ ID NO.18 CATGCCTA N706
SEQ ID NO.19 GTAGAGAG N707
SEQ ID NO.20 CAGCCTCG N710
SEQ ID NO.21 TGCCTCTT N711
SEQ ID NO.22 TCCTCTAC N712
所述第二正向引物的第一标签序列如SEQ ID NO.23所示，所述核酸序列如下：
第一标签序列(SEQ ID NO.23)：AATGATACGGCGACCACCGAGATCTACAC.
所述第二正向引物的第一测序通用引物的部分序列如SEQ ID NO.24所示，所述核酸序列如下：
第一测序通用引物的部分序列(SEQ ID NO.24)：TCGTCGGCAGCGTC.
所述第二反向引物的标签序列如SEQ ID NO.25所示，所述核酸序列如下：
第二标签序列(SEQ ID NO.25)：CAAGCAGAAGACGGCATACGAGAT.
所述第二反向引物的第二测序通用引物的部分序列如SEQ ID NO.26所示，所述核酸序列如下：
第二测序通用引物的部分序列(SEQ ID NO.26)：GTCTCGTGGGCTCGG.
所述第二正向引物为：AATGATACGGCGACCACCGAGATCTACAC-CTCTCTAT-TCGTCGGCAGCGTC；
所述第二反向引物为CAAGCAGAAGACGGCATACGAGAT-TCGCCTTA-GTCTCGTGGGCTCGG.
(2)其他试剂的制备
缓冲液A：0.5M Tris-Cl缓冲液(pH8.0)，1.0M NaCl；
裂解液B：1M Tris-Cl缓冲液(pH 8.0)，0.2M EDTA，10％SDS，12％Tween-40；
悬浮液C：30mg/ml RNAaseA
去除剂D：1.0M Al 2(SO4) 3，250g/L CTAB，1.5M NaCl；
结合液E：0.5M Tris-Cl缓冲液(pH 8.0)，5M异硫腈酸胍；
漂洗液F：5M Tris-Cl缓冲液(pH 8.0)，25％(体积比)无水乙醇；
洗脱液G：无菌去离子水
(3)试剂盒的组装
将所述引物组合物和检测探针与试剂盒相关的试剂进行组装，制备成所述检测试剂盒。
实施例2样本DNA的提取
选取3个粪便样本：A、B、C，平均分成2份，标记为：A1/A2，B1/B2，C1/C2，A1、B1、C1三个样本用本专利说明的方法处理并获得数据，A2、B2、C2三个样本直接送去金唯智测序，具体的DNA提取方法包括如下步骤：
(1)取700～800μl缓冲液A和250mg玻璃珠至2ml离心管中；
(2)在上述2ml离心管中加入250mg样本，涡旋混匀10s；
(3)向样本中加入70μl裂解液B，涡旋振荡7min混匀样本，12,000rpm(～13,400×g)，离心60s。转移上清液(500μl)至新的2ml离心管；
(4)加入250μl悬浮液C，涡旋振荡10s，室温放置2min，12,000rpm(～13,400×g)，离心60s，沉淀样本颗粒；
(5)转移上清至新的2ml离心管，加入300μl去除剂D混匀，4℃放置2～5min；
(6)12,000rpm(～13,400×g)，离心60s。转移上清液至新的2ml离心管，加入1000μl结合液E颠倒混匀；
(7)取上一步所得溶液700μl加入到一个吸附柱中(吸附柱放入收集管中)，12,000rpm(～13,400×g)，离心30s，倒掉废液，将吸附柱放入收集管中；
(8)向吸附柱中，加入700μl漂洗液F，12,000rpm(～13,400×g)，离心30s，倒掉废液，将吸附柱放入收集管中；
(9)向吸附柱中，加入500μl 75％无水乙醇，12,000rpm(～13,400×g)，离心30s，倒掉废液，将吸附柱放入收集管中；
(10)将吸附柱放回空收集管中，12,000rpm(～13,400×g)，离心60s，尽量除去漂洗液，以免漂洗液中残留乙醇抑制下游反应；
(11)取出吸附柱，放入一个干净的离心管中，在吸附膜的中间部位加50～100μl洗脱液G(洗脱液可事先在60～70℃水浴中预热)，室温放置2min，12,000rpm(～13,400×g)，离心60s，将得到的溶液重新加入离心吸附柱中，室温放置2min，12,000rpm(～13,400×g)，离心1min；
(12)使用分光光度计或Qubit对样品进行精确定量，DNA可以存放在0～10℃，如果要长时间存放，可以放置在-20℃。
将提取后的DNA进行纯度测试，OD 260/OD 280的比值在1.7～1.9之间，可以直接用于后续的PCR建库。
对比例1
所述裂解液B中不含有Tween-40，其他组分同实施例1，DNA提取方法同 实施例2。
结果证明，将提取后的DNA进行纯度测试，OD 260/OD 280的比值在1.3-1.6之间，纯度相比于实施例2降低。
对比例2
所述裂解液B中不含有Al 2(SO 4) 3，其他组分同实施例1，DNA提取方法同实施例2。
结果证明，将提取后的DNA进行纯度测试，OD 260/OD 280的比值在1.3-1.6之间，纯度相比于实施例2降低。
对比例3
所述裂解液B中不含有CTAB，其他组分同实施例1，DNA提取方法同实施例2。
结果证明，将提取后的DNA进行纯度测试，OD 260/OD 280的比值在1.3-1.6之间，纯度相比于实施例2降低。
实施例3文库构建
(1)在冰上配制以下反应液，除DNA提取液和H 2O以外，依照如下组份先按反应数+α的量配制扩增预混Mix，取配制好的预混Mix分装到PCR反应管中，反应体系如下：
试剂 使用量
2×PCR混合液(Mg 2+，dNTPs) 12.5μl
上游引物(1μM) 5μl
下游引物(1μM) 5μl
样本DNA 5ng/μl(或者ddH2O) 2.5μl
Total 25μl
设置一组以水为样本的阴性对照。
反应条件如下：
Figure PCTCN2017120443-appb-000001
(2)第一轮PCR产物的纯化
使用Agencourt AMPure XP(Beckman Coulter公司)对第一轮PCR扩增产物进行纯化，具体步骤如下：
1)4℃冰箱取出，室温放置25min，将Agencourt AMPure XP磁珠充分打散，混匀；
2)扩增产物与Agencourt AMPure XP磁珠的结合，所述扩增产物与Agencourt AMPure XP磁珠的体积比为5∶4；
3)充分混匀，室温孵育15min，瞬离，磁力架上静置5min，去除上清液；
4)第一次清洗：反应管放置在磁力架上不动，加入200μl新配的80％的乙醇室温静置(＞30s)，去除80％的乙醇；
5)第二次清洗：重复一次上述4)第一次清洗操作，轻轻离心，去除残留80％的乙醇；
6)磁珠的干燥：室温干燥1min；
7)洗脱：加入30μl的洗脱液，轻轻地上下吸打，充分混匀，室温孵育15min，瞬离，磁力架上静置5min，回收上清液；
(3)片段大小确定
琼脂糖电泳确定扩增是否准确，扩增产物长度约550bp。
(4)第二轮PCR
将步骤(2)中纯化得到的PCR产物为模板，进行PCR导入测序接头，具体步骤如下：
1)在冰上配制以下反应液，除第一轮PCR纯化产物以外，依照如下组份先按反应数+α的量配制扩增预混Mix，取配制好的预混Mix分装到PCR反应管中，反应体系如下：
试剂 使用量
2×PCR混合液(Mg 2+，dNTPs) 12.5μl
上游引物(1μM) 5μl
下游引物(1μM) 5μl
DNA(第一轮PCR纯化产物) 2.5μl
Total 25μl
设置一组以水为样本的阴性对照。
反应条件如下：
Figure PCTCN2017120443-appb-000002
Figure PCTCN2017120443-appb-000003
(5)第二轮PCR产物的纯化
具体步骤同第一轮PCR产物纯化，即步骤(2)，其中PCR产物与Agencourt AMPure XP磁珠的体积比为2∶1；
(6)片段大小及浓度测定
琼脂糖电泳确定扩增是否准确，扩增产物长度约600bp；
(7)使用Qubit dsDNA HS Assay Kit(Thermo Fisher)等测定PCR产物的浓度；
(8)测序建好的文库用illumina公司的Miseq测序进行测序，测序侧率为PE300测序。
实施例4数据分析
(1)对测序得到的原始数据进行质控：通过Barcode和引物等信息筛选出高质量的数据，筛选标准如下：含有完整的序列；具有完整前引物序列；去除Barcode、引物、接头，序列长度必须大于400bp，不含有模糊碱基；不能存在超过20个的单碱基重复，将得到的序列根据信息分别与各个样品相对应；
(2)通过数据库比对工具进行比对，将序列聚类生成OUT；
(3)将得到的OTU表，按照A1/A2，B1/B2，C1/C2进行相关性分析，结果如图1-3所示。
从图1可以看出，样本A，分成两份后，分别用本专利方法和金唯智公司方法建库测序，得到的数据分析后进行相关性分析发现，两种方法得到的OUT相关性达到0.9988；从图2可以看出，样本B，分成两份后，分别用本专利方法和金唯智公司方法建库测序，得到的数据分析后进行相关性分析发现，两种方法得到的OUT相关性达到0.9403；从图3可以看出，样本C，分成两份后，分别用本专利方法和金唯智公司方法建库测序，得到的数据分析后进行相关性分析发现，两种方法得到的OUT相关性达到0.9428。
实施例5分析验证
选取63例肠癌患者粪便样本，59例腺瘤患者粪便样本和66例健康患者粪便样本，用本专利所述的方法提取建库测序获得DNA序列信息，根据序列信息预测肠癌风险，具体试验方法同实施例2-实施例4，结果如表1所示。
表1
  肠癌 腺瘤 健康人
实际数目 63 59 66
肠癌检出数目 59 5 0
腺瘤检出数目 4 54 8
健康检出数目 0 0 58
准确率 93.65％ 91.53％ 87.88％
从表1可以看出，本申请检测中，肠癌的检出率为93.65％，腺瘤的检出率为91.53％，健康人的准确率为87.88％，可见，本申请方法用于检测癌症的准确 率可达90％以上。
综上所述，本申请中，通过采用两步法扩增获得测序文库，所述引物组合物可以更大程度地混合更多的样本，提高了扩增片段覆盖率、检测特异性及灵敏度，结合数据分析，可获得更丰富的微生物组信息，增加了文库序列的复杂度，降低了phix的比例，保证更多测序质量，降低了PCR扩增偏好性；本申请提供的数据处理分析方法，实现更多微生物物种在分类学属、种上的鉴别及多样性分析，并得到相关微生物群体的相对丰度值。
注意，上述仅为本申请的较佳实施例及所运用技术原理。本领域技术人员会理解，本申请不限于这里所述的特定实施例，对本领域技术人员来说能够进行各种明显的变化、重新调整和替代而不会脱离本申请的保护范围。因此，虽然通过以上实施例对本申请进行了较为详细的说明，但是本申请不仅仅限于以上实施例，在不脱离本申请构思的情况下，还可以包括更多其他等效实施例，而本申请的范围由所附的权利要求范围决定。

Claims (15)

  1. 一种用于分析肠道微生物的引物组合物，其包含：
    第一组引物，所述第一组引物包含第一正向引物和第一反向引物，其中，所述第一正向引物从5’到3’端包括依次相连的四个部分：第一测序通用引物、2bp的随机碱基序列、linker序列和肠道微生物16S rRNA V3-V4区特异性正向引物；所述第一反向引物从5’到3’端包括依次相连的四个部分：第二测序通用引物、2bp的随机碱基序列、linker序列和肠道微生物16S rRNA V3-V4区特异性反向引物；
    第二组引物，所述第二组引物包含第二正向引物和第二反向引物，其中，所述第二正向引物从5’到3’端包括依次相连的三个部分：P5端接头序列、第一标签序列和第一测序通用引物的部分序列；所述第二反向引物从5’到3’端包括依次相连的三个部分：P7端接头序列、第二标签序列和第二测序通用引物的部分序列。
  2. 根据权利要求1所述的引物组合物，其中，所述第一测序通用引物和所述第二测序通用引物独立地选自任意一种二代测序平台的测序通用引物。
  3. 根据权利要求1所述的引物组合物，其中，所述linker序列的长度为2-5bp，优选为2bp；
    优选地，所述第一正向引物的核酸序列如SEQ ID NO.1所示，所述第一反向引物的核酸序列如SEQ ID NO.2所示；
    优选地，所述P5端接头序列的核酸序列如SEQ ID NO.3-12所示；
    优选地，所述P7端接头序列的核酸序列如SEQ ID NO.13-22所示；
    优选地，所述第二正向引物的第一标签序列如SEQ ID NO.23所示；
    优选地，所述第二正向引物的第一测序通用引物的部分序列如SEQ ID  NO.24所示；
    优选地，所述第二反向引物的第二标签序列如SEQ ID NO.25所示；
    优选地，所述第二反向引物的第二测序通用引物的部分序列如SEQ ID NO.26所示。
  4. 一种分析肠道微生物的试剂盒，其包括如权利要求1-3任一项所述的引物组合物。
  5. 一种肠道微生物的分析方法，其采用如权利要求1-3任一项所述的引物组合物，包括如下步骤：
    (1)样本基因组DNA的提取；
    (2)扩增子文库的构建：进行两轮PCR扩增，第一轮PCR扩增采用第一组引物组，第二轮PCR扩增采用第二组引物组，构建高通量测序文库；
    (3)数据分析。
  6. 根据权利要求5所述的分析方法，其中，步骤(1)所述样本基因组DNA的提取包括如下具体步骤：
    (1’)将样本与裂解液混合后，重悬；
    (2’)将步骤(1’)重悬后的样本加入去除剂混匀，离心；
    (3’)将步骤(2’)离心后的样本加入结合液混匀，加入吸附柱中，离心，漂洗，加入75％的无水乙醇，离心；
    (4’)将步骤(3’)离心后的样本加入洗脱液，离心得到所述基因组DNA。
  7. 根据权利要求6所述的分析方法，其中，步骤(1’)所述裂解液包括Tris-Cl缓冲液、Tween-40、EDTA和SDS；
    优选地，所述Tris-Cl缓冲液的摩尔浓度为0.5-2M，优选为0.8-1.2M；
    优选地，所述Tween-40的体积分数为8-15％，优选为10-13％；
    优选地，所述EDTA的摩尔浓度为0.1-1M，优选为0.1-0.5M；
    优选地，所述SDS的体积分数为5-15％，优选为8-12％。
  8. 根据权利要求6或7所述的分析方法，其中，步骤(2’)所述去除剂包括Al 2(SO 4) 3、CTAB和NaCl；
    优选地，所述Al 2(SO 4) 3的摩尔浓度为0.5-2M，优选为0.8-1.2M；
    优选地，所述CTAB的质量浓度为200-300g/L，优选为230-260g/L；
    优选地，所述NaCl的摩尔浓度为0.8-2M，优选为1.2-1.8M。
  9. 根据权利要求5-8中任一项所述的分析方法，其中，步骤(2)所述第一轮PCR的条件为：93-96℃预变性1-5min；93-96℃预变性20-40s，50-55℃ 20-40s，70-75℃ 20-40s，共进行25-35个循环；70-75℃延伸3-6min；
    优选地，步骤(2)所述第一轮PCR的条件为：95℃预变性3min；95℃预变性30s，53℃ 30s，72℃ 30s，共进行28个循环；72℃延伸5min；
    优选地，步骤(2)所述第二轮PCR的条件为：93-96℃预变性1-5min；93-96℃预变性20-40s，54-58℃ 20-40s，70-75℃ 20-40s，共进行6-12个循环；70-75℃延伸3-6min；
    优选地，步骤(2)所述第二轮PCR的条件为：95℃预变性3min；95℃预变性30s，55.5℃延伸30s，72℃ 30s，8个循环；72℃ 5min，1个循环。
  10. 根据权利要求5-9中任一项所述的分析方法，其中，步骤(1)中所述样本来源于粪便、土壤、尿液或口腔细胞中的任意一种或至少两种的组合，优选来源于粪便；
    优选地，步骤(1)之后还包括纯化的步骤，优选采用磁珠进行纯化；
    优选地，所述磁珠与扩增产物的体积比为(0.4-0.7)∶1，优选为0.5∶1。
  11. 根据权利要求5-10中任一项所述的分析方法，其中，步骤(3)所述数据分析为采用Illumina Miseq测序仪进行测序分析；
    优选地，所述测序分析具体包括如下步骤：通过Illumina Miseq测序仪测序得到按照标签区分的测序读长，利用读长的重叠关系组装得到高可变区V3-V4的全长序列，再对全长序列进行分类分析，得到所述微生物群体的分类；
    优选地，所述全长序列进行分类分析具体包括计算全长序列差异度，根据序列差异度执行操作分类学单元OTU的分类，将全长序列分配到OTU中，再将每一个OTU分类中的全长序列比对到16S rRNA的V6数据库中，将比对结果根据众数原则对OTU进行物种注释。
  12. 一种如权利要求1-3任一项所述的引物组合物、如权利要求4所述的试剂盒或如权利要求5-11中任一项所述的分析方法用于分析分类肠道微生物的用途。
  13. 一种如权利要求5-11中任一项所述的分析方法用于免疫组学分析的用途。
  14. 一种如权利要求1-3任一项所述的引物组合物、如权利要求4所述的试剂盒或如权利要求5-11中任一项所述的分析方法用于癌症的诊断的用途。
  15. 根据权利要求14所述的用途，其中，所述癌症为肠癌、腺癌或膀胱癌中的任意一种或至少两种的组合。
PCT/CN2017/120443 2017-12-12 2017-12-31 一种用于分析肠道微生物的引物组合物及其应用 Ceased WO2019114049A1 (zh)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111304344A (zh) * 2020-02-20 2020-06-19 佛山科学技术学院 一种活体脂肪量沉积的无损检测方法
CN111986732A (zh) * 2020-07-24 2020-11-24 贵州师范大学 一种利用鸳鸯粪便分析鸳鸯食性的方法
CN113046418A (zh) * 2021-04-02 2021-06-29 武汉博越致和生物科技有限公司 一种用于妊娠期糖尿病肠道菌群检测16S rDNA扩增子建库方法

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109727644B (zh) * 2018-11-12 2021-09-07 山东省医学科学院基础医学研究所 基于微生物基因组二代测序数据的Venn图制作方法及系统
CN110176305A (zh) * 2019-05-27 2019-08-27 天益健康科学研究院（镇江）有限公司 一种利用高通量基因测序评估肠道菌群健康的方法
CN113025761A (zh) * 2021-05-27 2021-06-25 广州赛哲生物科技股份有限公司 病原微生物鉴定的多重扩增配合高通量测序方法及试剂盒

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105189748A (zh) * 2013-03-15 2015-12-23 血统生物科学公司 测序免疫组库的方法
CN105506063A (zh) * 2014-09-22 2016-04-20 深圳华大基因科技有限公司 引物组合物及其用途
WO2016118719A1 (en) * 2015-01-23 2016-07-28 Qiagen Sciences, Llc High multiplex pcr with molecular barcoding
CN105937053A (zh) 2015-12-02 2016-09-14 广州赛哲生物科技股份有限公司 一种基于高通量基因测序建立粪便菌群基因文库的方法
CN106497926A (zh) * 2016-11-03 2017-03-15 承启医学（深圳）科技有限公司 一种用于构建微生物细菌16s　rDNA可变区测序文库的扩增子引物及构建方法
CN107058490A (zh) 2017-01-12 2017-08-18 杨俊杰 肠道及口腔菌群多样性及差异性的分析方法

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140171339A1 (en) 2011-06-06 2014-06-19 The University Of North Carolina At Chapel Hill Methods and kits for detecting adenomas, colorectal cancer, and uses thereof
US11060148B2 (en) 2012-10-16 2021-07-13 Dana-Farber Cancer Institute, Inc. Diagnosing and treating colorectal cancer
LT3087198T (lt) * 2013-12-24 2020-03-10 Université de Ličge Mėginių metagenominė analizė
RS61976B1 (sr) * 2014-02-18 2021-07-30 Illumina Inc Postupci i kompozicije za određivanje dnk profila
US9745611B2 (en) * 2014-05-06 2017-08-29 Genewiz Inc. Methods and kits for identifying microorganisms in a sample
US20200011873A1 (en) * 2017-03-17 2020-01-09 Second Genome, Inc. Leveraging sequence-based fecal microbial community survey data to identify a composite biomarker for colorectal cancer

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105189748A (zh) * 2013-03-15 2015-12-23 血统生物科学公司 测序免疫组库的方法
CN105506063A (zh) * 2014-09-22 2016-04-20 深圳华大基因科技有限公司 引物组合物及其用途
WO2016118719A1 (en) * 2015-01-23 2016-07-28 Qiagen Sciences, Llc High multiplex pcr with molecular barcoding
CN105937053A (zh) 2015-12-02 2016-09-14 广州赛哲生物科技股份有限公司 一种基于高通量基因测序建立粪便菌群基因文库的方法
CN106497926A (zh) * 2016-11-03 2017-03-15 承启医学（深圳）科技有限公司 一种用于构建微生物细菌16s　rDNA可变区测序文库的扩增子引物及构建方法
CN107058490A (zh) 2017-01-12 2017-08-18 杨俊杰 肠道及口腔菌群多样性及差异性的分析方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3604557A4

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111304344A (zh) * 2020-02-20 2020-06-19 佛山科学技术学院 一种活体脂肪量沉积的无损检测方法
CN111986732A (zh) * 2020-07-24 2020-11-24 贵州师范大学 一种利用鸳鸯粪便分析鸳鸯食性的方法
CN113046418A (zh) * 2021-04-02 2021-06-29 武汉博越致和生物科技有限公司 一种用于妊娠期糖尿病肠道菌群检测16S rDNA扩增子建库方法

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