CN110938701A - Standard substance for metagenome sequencing quantification - Google Patents
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- CN110938701A CN110938701A CN201911175446.7A CN201911175446A CN110938701A CN 110938701 A CN110938701 A CN 110938701A CN 201911175446 A CN201911175446 A CN 201911175446A CN 110938701 A CN110938701 A CN 110938701A
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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
- 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
Abstract
The invention discloses a standard substance for metagenome sequencing quantification. The microorganisms in the standard are all derived from human intestinal contents or feces. In addition, the standard may be presented in both lyophilized bacterial form or genomic DNA form. For example, the standard contains 20 species of intestinal bacteria, all isolated from human feces, close to the actual condition of intestinal microorganisms. The standard substance can be applied to the development of metagenomic analysis methods, can be used for evaluating the accuracy and the difference of different sequencing platforms, and can also be used for modeling and testing bioinformatics.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a standard substance for metagenomic sequencing quantification.
Background
The intestines and stomach of the human body are colonized by a wide variety of microorganisms, which are known as the intestinal flora. The intestinal flora is composed according to a certain proportion, and different intestinal flora constitutions have different influences on the health of human bodies. Sequencing-based intestinal flora studies have described these states of gut flora dysregulation from the perspective of proportional changes in microbial composition. While these relative proportions enable detection of variations in disease-associated microorganisms, they have limited ability to reveal interactions between the microorganisms and the host's health. Comparative analysis of microbiology relative data does not provide information about the degree or direction of changes in microbial population abundance or metabolic potential. If the total microbial load varies greatly between samples, then the relative analysis will actually link the microbiological characteristics to quantitative data (e.g., physiological parameters or metabolite concentrations). Most of the current studies on the structure of the intestinal microbial flora do not reflect the influence of the change of the specific microbial population on the host, and the relative proportion studies ignore the possibility that the change of the abundance of the total microbes is a main reason which may cause the disease of the host. Therefore, in order to actually reflect the host-microbial interaction, the microbial population must be studied in absolute proportion.
Disclosure of Invention
The invention discloses a standard substance for simulating an intestinal microflora, which can be applied to the field of microbial metagenome research, is from method optimization to data interpretation and is used as quality control of the whole process and daily operation of microbial information analysis. The method is suitable for confirmation test and test development on any platform. The method is used for metagenomic research, and the consistency and reproducibility of the data after operation are improved.
The invention provides a standard substance for metagenome sequencing quantification, wherein microorganisms in the standard substance are completely derived from human intestinal contents or feces.
In the above-described standard for metagenomic sequencing quantification, the standard comprises 20 different species of enteric bacteria.
The invention also provides a standard substance for metagenomic sequencing quantification, wherein the standard substance is genome DNA of microorganisms which are completely derived from human intestinal contents or feces.
The invention discloses a standard substance for metagenome sequencing quantification. The microorganisms in the standard are all derived from human intestinal contents or feces. For example, the standard contains 20 species of intestinal bacteria, all isolated from human feces, close to the actual condition of intestinal microorganisms. In addition, the standard may be presented in both lyophilized bacterial form or genomic DNA form. The standard substance can be applied to the development of metagenomic analysis methods, can be used for evaluating the accuracy and the difference of different sequencing platforms, and can also be used for modeling and testing bioinformatics.
Drawings
Figure 1 shows the population abundance after sequencing of a simulated intestinal microflora standard.
Detailed Description
The following examples are presented to enable those skilled in the art to more fully understand the present invention and are not intended to limit the invention in any way. The starting materials used in the present invention are all commercially available conventional materials and reagents, unless otherwise specified.
The invention provides a standard substance for simulating an intestinal microflora, wherein microorganisms in the standard substance are all derived from human intestinal contents or feces, and the standard substance is closer to the real situation. The separation method from fresh excrement is a conventional microorganism separation culture method, a BHI or MRS culture medium is sampled to carry out anaerobic culture on a sample, and a single colony is picked, streaked, purified and identified.
1. Operation flow for separating, culturing and identifying fecal microorganism
(1) Specific operation process
Sample pretreatment:
a. weighing 0.2g of a feces sample, adding 1.8mL of Phosphate Buffer Solution (PBS) or normal saline, placing in a metal bath at 300rpm, and shaking for 5 min;
b.50g, centrifuging for 15min, and taking 1mL of upper suspension;
c, 8000g, centrifuging for 5min, sucking an upper layer liquid, and adding 1mL of PBS into the precipitate for resuspension;
d. repeating step c twice;
e. diluting the bacterial suspension by 10 times, 100 times and 1000 times step by step;
f. dipping the bacterial suspension with cotton swab, coating the plate with fresh feces 10-3,10-4Coating the diluent;
g. the plates were inverted and incubated in an anaerobic incubator at 37 ℃ for 48 h.
Separation and purification
a. Picking single bacterial colony with inoculating loop, separating on plate, inverting plate in 37 deg.C anaerobic incubator for 48 hr;
b. the streaking separation was repeated 2 times until colonies of a single morphology were obtained;
c. inoculating the purified single colony into 5mL of liquid culture medium, and carrying out anaerobic culture at 37 ℃ for 48 h;
d. taking 2mL of a freezing tube, adding 1mL of 20% glycerol, scraping the purified bacterial colony, and suspending in the glycerol; placing in a programmed cooling box, and freezing and storing at-80 ℃.
16s rRNA gene amplification and quality control
Universal primer 27F for bacterial sequencing by using colony DNA as template
(5'-AGAGTTTGATCCTGGCTCAG-3' (upstream primer)) and 1492R (1492R:
5'-TACGGCTACCTTGTTACGACTT-3' (downstream primer)) was used for the amplification of the 16S rRNA gene fragment. The PCR amplification reaction system and the amplification procedure are shown in tables 1.1 and 1.2.
TABLE 1.1PCR reaction System
TABLE 1.2PCR reaction procedure
Quality control: taking 5 mu L of PCR reaction final solution, carrying out agarose gel electrophoresis or detecting a target band by using a LabChip, and storing a positive PCR product at-20 ℃.
Amplicon sequence alignment
The PCR product is sent to a company for sequencing a 16s rRNA gene amplicon, the base sequence in the determination result is input into an NCBI (http:// blast. NCBI. nlm. nih. gov/blast. cgi) database for comparison, and the evolutionary relationship of the strain is analyzed.
The microorganisms in the standard were isolated from human fresh feces and consisted of and in the following proportions:
| strain name | Ratio 1 (%) | Ratio 2 (%) |
| |
18 | 5 |
| Escherichia coli | 18 | 5 |
| |
18 | 5 |
| |
18 | 5 |
| |
18 | 5 |
| Streptococcus gallolyticus | 1.8 | 5 |
| Bifidobacterium pseudocatenulatum | 1.8 | 5 |
| Lactococcus lactis | 1.8 | 5 |
| Staphylococcus pasteuri | 1.8 | 5 |
| Lactobacillus rhamnosus | 1.8 | 5 |
| Lactobacillus plantarum | 0.18 | 5 |
| Bifidobacterium animalis | 0.18 | 5 |
| Clostridium butyricum | 0.18 | 5 |
| Streptococcus mutans | 0.18 | 5 |
| Weissella confusa | 0.18 | 5 |
| Weissella cibaria | 0.02 | 5 |
| Klebsiella pneumoniae | 0.02 | 5 |
| Enterococcus gallinarum | 0.02 | 5 |
| Propionibacterium acnes | 0.02 | 5 |
| Lactobacillus paracasei | 0.02 | 5 |
The preparation method of the standard substance for simulating the intestinal microflora comprises the following steps:
(1) after the microorganisms were recovered and cultured to a passable solubility in a specific medium, the subculture was continued 3 times at an inoculum size of 1%. After counting by flow cytometry, the culture broth of 20 bacteria was diluted to the power of 9 of 10, respectively. And combining the mixed bacteria liquid according to the corresponding percentage to form mixed bacteria liquid, and then freezing and drying the mixed bacteria liquid to obtain the simulated intestinal microorganism bacteria standard product. The above percentages refer to the percentage of the number of cells of the respective bacteria.
The specific operation steps of the strain vacuum freeze-drying preservation method are as follows:
① treating ampoule tube with inner diameter of 8-10 mm and length not less than 100m, soaking in washing solution via capillary tube overnight, washing with distilled water, drying, plugging with cotton, wrapping with paper, sterilizing at 121 deg.C for 30min, and drying in 60 deg.C oven.
② preparation of protective agent, the protective agent can adopt low molecular and high molecular compounds, such as amino acids, organic acids, saccharides, proteins, polysaccharides, etc., skimmed milk and blood serum are commonly used, such as horse blood serum or horse blood serum with 7.5% glucose, and the protective agent is sterilized by proper method before use.
③ bacterial suspension is prepared, strains are cultured under the optimal culture conditions (such as culture medium, temperature, culture time and the like), the culture time is controlled at the later growth stage, because the resistance of cells in logarithmic phase to freeze drying is weak, the bacteria are generally cultured for 24-48 h, during operation, a small amount of protective agent is firstly added into an inclined plane under the aseptic condition, bacterial lawn or spores are lightly scraped to prepare bacterial suspension, 0.1-0.2 ml of bacterial suspension is taken by a capillary dropper and added into a sterilized ampoule tube, and a cotton plug is plugged.
④, freeze drying at-15 deg.C to-30 deg.C, freezing the ampoule tube in the bacterial suspension as soon as possible to prevent the bacterial precipitation as non-uniform bacterial suspension and the secondary growth or germination of the microorganism, starting the vacuum pump immediately after the freezing temperature is reached, raising the vacuum degree to 66.661 Pa. within 15mins to above 13.3322Pa, raising the temperature to 25-30 deg.C, stopping the vacuum pump after the drying is finished, exhausting, taking out the ampoule tube, vacuumizing again on the porous pipeline, and sealing.
⑤ storing ampoule tubes, the ampoule tubes can be stored for 5-10 years at 4-5 ℃, the storing effect at room temperature is not good, the factors influencing the storing effect are directly related to the water content in the sample besides the strain and the age of the bacteria, the storing effect is better when the water content is 1-3%, the storing effect is correspondingly reduced when the water content is 5-6%, and the sample is difficult to store when the water content is more than 10%.
(2) Extracting the genome DNA of the standard substance by using a bacterial DNA extraction kit (purchased from Takara company), and combining according to the corresponding nucleic acid mass percentage to obtain the nucleic acid standard substance simulating the intestinal bacterial community. The above percentages refer to the nucleic acid mass percentages of the respective bacteria.
1. Application of standard substance for simulating intestinal microflora
The standard substance can be applied to quantitative metagenome research and used as a known sample for evaluating the accuracy of different sequencing platforms or different data analysis methods.
Example 1: application of intestinal microflora simulation standard in evaluating difference of different sequencing platforms
Preparing intestinal microflora simulated genomic DNA, extracting genomic DNA of different bacteria, performing nucleic acid mass combination according to the proportion 1 to obtain mixed flora genomic DNA, and sequencing the DNA sample by using HiSeq X10 and Pacbio sequencing platforms respectively.
The results show that the sequencing results of the two sequencing platforms are basically similar to the abundance of the preset flora and slightly deviate.
Those skilled in the art will appreciate that the above embodiments are merely exemplary embodiments and that various changes, substitutions, and alterations can be made without departing from the spirit and scope of the application.
Sequence listing
<110> Kangmeihua Dagenetechnology Co., Ltd
<120> standard substance for metagenome sequencing quantification
<130>HP190002LZ
<141>2019-11-22
<160>2
<170>SIPOSequenceListing 1.0
<210>1
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<213> Artificial sequence ()
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tacggctacc ttgttacgac tt 22
Claims (3)
1. A standard substance for metagenomic sequencing quantification, wherein all microorganisms in the standard substance are derived from human intestinal contents or feces.
2. The standard for metagenomic sequencing quantification according to claim 1, wherein said standard comprises 20 different species of enteric bacteria.
3. A standard substance for metagenomic sequencing quantification, which is characterized in that the standard substance is genome DNA of microorganisms which are completely derived from human intestinal contents or feces.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112662795A (en) * | 2021-01-26 | 2021-04-16 | 苏州系统医学研究所 | Positive control for infectious pathogen detection and preparation method and application thereof |
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