1. A method for identifying microbial populations based on nucleotide synthesis sequencing maps,

performing trinucleotide and mononucleotide cycle sequencing reaction on a target fragment of the microorganism to obtain a sequencing map, drawing a ratio color band according to the sequencing map, and determining the type of the microorganism by analyzing the ratio color band or/and the sequencing map;

the trinucleotide is any three nucleotides of dATP alpha S, dCTP, dTTP and dGTP;

the single nucleotide is another nucleotide not included in the trinucleotide.

2. The method for identifying a microbial population based on a nucleotide sequencing by synthesis map according to claim 1, comprising the steps of:

(1) extracting a DNA template of the microorganism, and carrying out PCR amplification on a part of variable regions under the action of polymerase;

(2) combining the amplification product with a sequencing primer;

(3) adding trinucleotide and mononucleotide alternately and circularly to perform pyrosequencing to obtain a sequencing map;

(4) drawing a comparison color band according to a sequencing map;

(5) the microbial population is analyzed by colorimetric bands or/and sequencing maps.

3. The method for identifying a microbial population according to claim 1, wherein said sequencing map comprises all information consisting of nucleotide number, nucleotide type, signal intensity and positional arrangement of trinucleotides and mononucleotides.

4. The method for identifying a microbial population according to claim 1, wherein said method for analyzing a sequencing map comprises:

the type of microorganism is determined by analyzing the site of the single nucleotide.

5. The method of claim 1, wherein the colorimetric band comprises a plurality of units arranged in sequence, one unit corresponding to a nucleotide site; the unit site occupied by the trinucleotide is designated as region A, and the unit site occupied by the mononucleotide is designated as region B.

6. The method for identifying a microbial population according to claim 5, wherein the analysis of the specific color band comprises:

and overlapping the colorimetric bands of the microorganisms and the similar colorimetric bands, and determining the types of the microorganisms according to the overlapping degree.

7. Use of a method for identifying a microbial population based on a nucleotide sequencing map, wherein the method for identifying a microbial population according to any one of claims 1 to 6 is used for sequencing a site of a directed mutation.

Background

The identification and sequencing of the microbial population is based on a second generation high-throughput technology, and the technology identifies the microbial population by selectively sequencing gene sequences of 16SrRNA/18SrRNA/ITS and the like of microorganisms and comparing the gene sequences with a gene library. With the continuous development and refinement of microbial population identification and sequencing technology, the application of the technology in the fields of disease prevention and treatment and the like is increasingly wide. Particularly, in clinic, the species of pathogenic microorganisms can be rapidly and accurately identified, the diagnosis speed can be increased, and doctors can conveniently carry out targeted medication treatment on patients. Meanwhile, the application in this direction also puts higher use requirements on the microbial population identification sequencing technology.

The specific method for identifying and sequencing the microbial population comprises the following steps: extracting total DNA of microorganisms in a sample, amplifying 16SrDNA in a bacterial genome and 18SrDNA or ITS regions in a fungal genome by using a PCR technology to obtain amplification products of most 16SrDNA, 18SrDNA or ITS high variable regions of the microorganisms, and constructing a library of the amplification products; then, extracting the DNA of the microorganism to be detected, amplifying by using a PCR technology, sequencing the amplified product by using a sequencing platform, and identifying the type of the microorganism by comparing and analyzing a sequencing result and a gene library.

At present, Sanger sequencing and pyrosequencing are commonly used as methods for identifying the microbial population based on a PCR amplification technology, the two methods have high accuracy, and the microbial species is determined by completely determining the base information of the sequence of a PCR product and comparing the base information with the base information of a characteristic sequence of a strain. However, in practical application, the problems of tedious detection process, complicated analysis process of detection results and the like exist, which greatly limits the application of microorganism population identification sequencing in disease diagnosis and treatment.

Disclosure of Invention

In view of the above problems, it is an object of the present invention to provide a method for identifying a microbial population based on a nucleotide synthesis sequencing map, which has a short detection period, high detection accuracy, and a simple analysis process.

In order to achieve the aim, the invention provides a method for identifying a microbial population based on a nucleotide synthesis sequencing map, which comprises the steps of carrying out trinucleotide and mononucleotide cycle sequencing reaction on a target fragment of a microorganism to obtain a sequencing map, drawing a ratio color band according to the sequencing map, and determining the type of the microorganism by analyzing the ratio color band or/and the sequencing map; the trinucleotide is any three nucleotides of dATP alpha S, dCTP, dTTP and dGTP; the single nucleotide is a fixed nucleotide not included in the trinucleotide.

Further, the method specifically comprises the following steps: (1) extracting a DNA template of the microorganism, and carrying out PCR amplification on a part of variable regions under the action of polymerase; (2) combining the amplification product with a sequencing primer; (3) adding trinucleotide and mononucleotide alternately and circularly to perform pyrosequencing to obtain a sequencing map; (4) drawing a comparison color band according to a sequencing map; (5) the microbial population is analyzed by colorimetric bands or/and sequencing maps.

Further, the sequencing map includes all information consisting of the number of nucleotides, the kind of nucleotides, the signal intensity, and the positional arrangement of the trinucleotides and the mononucleotides.

Further, the analysis method of the sequencing map comprises the following steps: the type of microorganism is determined by analyzing the site of the single nucleotide.

Furthermore, the colorimetric band comprises a plurality of units which are arranged in sequence, wherein one unit corresponds to one nucleotide site; the unit site occupied by the trinucleotide is designated as region A, and the unit site occupied by the mononucleotide is designated as region B.

Further, the analysis method of the color bar comprises the following steps: and overlapping the colorimetric bands of the microorganisms and the similar colorimetric bands, and determining the types of the microorganisms according to the overlapping degree.

The method for identifying the microbial population based on the nucleotide sequencing map has the beneficial effects that:

(1) according to the method for identifying the microbial population, the target fragments of the microbes are sequenced in a mode of alternately and circularly adding trinucleotide and mononucleotide, when the target fragments with fixed lengths are sequenced, compared with the traditional cyclic sequencing method of mononucleotide or dinucleotide, the sequencing process is simplified, the sequencing time is effectively shortened, and meanwhile, the microbial population can be accurately identified according to the sequencing result;

(2) according to the microbial population identification method, a sequencing map can be obtained through sequencing reaction, the sequencing map contains information such as the number of nucleotides, the types of nucleotides and signal intensity, the types of microorganisms can be determined by determining the sites of a plurality of mononucleotides and comparing the sites with corresponding gene libraries, the determination accuracy is high, and the map analysis process is simple;

(3) in the microbial population identification method, the single nucleotide can be any one of dATP alpha S, dCTP, dTTP and dGTP, and in practical application, the common mutant base type of the microorganism to be detected can be avoided, and the influence of a mutant site on a sequencing result is reduced.

(4) The microorganism population identification method comprises the step of drawing a ratio color band through a sequencing map, wherein the color band consists of an area A and an area B which are alternately arranged, the area A corresponds to the unit position occupied by trinucleotide in a sequencing reaction, the area B corresponds to the unit position occupied by mononucleotide, namely, different microorganism strains have different characteristic ratio color bands, in the practical application, the types of microorganisms can be analyzed through directly overlapping the ratio color band, the process is simple, and the result is clear.

Another object of the present invention is to provide a use of the method for identifying a microorganism population based on a nucleotide sequencing map, wherein the method for identifying a microorganism population according to any one of claims 1 to 6 is also applicable to sequencing a mutation site.

Drawings

FIG. 1 is a sequencing map of an artificially synthesized sequence in example 1 of the present invention;

FIG. 2 is a colorimetric band of the artificially synthesized sequence in example 1 of the present invention;

FIG. 3 is a predicted sequencing map of the artificially synthesized sequence in example 1 of the present invention;

FIG. 4 is a color bar of the prediction ratio of the artificially synthesized sequence in example 1 of the present invention;

FIG. 5 is a comparison of the stack of FIGS. 2 and 4 in accordance with the present invention;

FIG. 6 is a sequencing map of the artificially synthesized sequence in example 2 of the present invention;

FIG. 7 is a colorimetric band of the artificially synthesized sequence in example 2 of the present invention;

FIG. 8 is a comparison of the stack of FIGS. 2 and 7 in accordance with the present invention;

FIG. 9 is another sequencing map of the artificially synthesized sequence in example 1 of the present invention.

Detailed Description

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Referring to FIG. 1, a sequencing map of the artificially synthesized sequence of example 1 is shown, in which the abscissa represents the time taken for sequencing and the ordinate represents the signal intensity (the signal intensity is understood as the number of base pairs occurring), each column represents a sequencing reaction in which trinucleotides and mononucleotides are alternately added, and each column is labeled with the base type of the corresponding nucleotide and the corresponding signal intensity.

Analysis example: as can be analyzed from FIG. 1, the bases at the first six nucleotide positions of the artificially synthesized sequence in example 1 can be paired with the base (A, T, G) in the trinucleotide; the base at the seventh nucleotide position can be C-paired and should be G; in this way, sequencing maps can be analyzed to identify populations of microorganisms.

Referring to FIG. 2, the colorimetric band for the artificially synthesized sequence in example 1 is composed of a number of rectangular units, one rectangular unit represents one nucleotide site, and the number of rectangular units is equal to the sum of signal intensities in the corresponding sequencing spectra. Each rectangular unit characterizes its sequencing by filling in a different pattern or color.

Example 1

The microbial population identification method based on nucleotide synthesis sequencing map determines artificially synthesized sequence 3 '-AAAAAAGGAGTTAGCCGGTGCTTCT-5', and the specific sequencing process includes the following steps:

(1) mixing the artificially synthesized sequence modified with biotin at the 5' end with the magnetic beads modified with streptavidin, and shaking for 1 hour at room temperature; then, after washing with PBS five times, magnetic beads on which artificially synthesized sequences were immobilized on the outer surface were obtained by magnetic separation.

(2) And (3) preserving the heat of the separated magnetic beads and the sequencing primer for 5 minutes at 75 ℃, naturally cooling to room temperature, and separating the magnetic beads from the liquid, wherein the artificially synthesized sequence fixed on the outer surfaces of the magnetic beads is combined with the sequencing primer for later use.

(3) Placing the magnetic beads separated in the step (2) into a pyrosequencer for sequencing: in the sequencing process, trinucleotide and mononucleotide are alternately and circularly added to carry out pyrosequencing; wherein, the trinucleotide is selected from dATP alpha S, dTTP and dGTP, the content of the three is 1:1:1, and the mononucleotide is selected from dCTP; the sequencing map shown in FIG. 1 was obtained by a sequencing reaction.

(4) Drawing a colorimetric band according to the sequencing map, wherein the colorimetric band is shown in figure 2; wherein, the region A corresponding to the trinucleotide is filled with a transverse line, and the region B corresponding to the mononucleotide is filled with an oblique line.

(5) Results and analysis

In order to characterize the utility and the specific analysis process of the microbial population identification method of the present invention in the identification of the microbial population, a predictive sequencing map as shown in fig. 3 was prepared based on the known synthetic sequence 3 '-AAAAAAGGAGTTAGCCGGTGCTTCT-5', and a predictive ratio band was drawn as shown in fig. 4.

Setting the artificial synthesis sequence unknown, overlapping and comparing the specific color bands shown in fig. 2 and fig. 4, wherein the overlapping result is shown in fig. 5; from the stacking, the colorimetric bands plotted in example 1 completely overlapped with the predicted colorimetric bands. Therefore, the method for identifying the microbial population has application value.

Besides the method for identifying the microbial population by the stacked colorimetric card, the method can also directly analyze a sequencing map to identify the microbial population, and the specific identification process comprises the following steps:

the single nucleotide on the sequencing map of FIG. 1 is positioned, and the information of the number, the position, the intensity and the like of the single nucleotide sites on the two sequencing maps is completely the same by comparing a plurality of single nucleotide sites on FIGS. 1 and 3. Therefore, the invention can identify the microbial population by comparing the sequencing maps.

Example 2

The invention can also be applied to sequencing of the directed mutation sites.

An artificially synthesized sequence 3 '-AAAGAAGGAGTTAGCCGGTGCTTCT-5' was selected, which differs from the artificially synthesized sequence of example 1 in that: the fourth nucleotide site is changed from A to G, namely the mutation type of the directional mutation to G. The present embodiment is designed to prove that the identification method of the present invention can be used for determining and further sequencing a directional mutation site, and therefore, the sequencing process specifically comprises the following steps:

(1) mixing the artificially synthesized sequence modified with biotin at the 5' end with the magnetic beads modified with streptavidin, and shaking for 1 hour at room temperature; then, washing the magnetic beads with PBS for five times, and obtaining magnetic beads with artificially synthesized sequences fixed on the surfaces through magnetic separation;

(2) preserving the heat of the magnetic beads with the artificially synthesized sequences fixed on the surfaces and the sequencing primer at 75 ℃ for 5 minutes, naturally cooling to room temperature, and separating the magnetic beads from the liquid, wherein the artificially synthesized sequences fixed on the surfaces of the magnetic beads are combined with the sequencing primer for later use;

(3) placing the magnetic beads separated in the step (2) into a pyrosequencer for sequencing: in the sequencing process, trinucleotide and mononucleotide are alternately and circularly added to carry out pyrosequencing, wherein the trinucleotide is dATP alpha S, dTTP and dGTP, the content of the three is 1:1:1, and the mononucleotide is dCTP; obtaining a sequencing map as shown in FIG. 6 through a sequencing reaction;

(4) drawing a colorimetric band according to the sequencing map, wherein the colorimetric band is shown in figure 7; wherein, the region A corresponding to the trinucleotide is a region filled with transverse lines, and the region B corresponding to the mononucleotide is a region filled with oblique lines.

(5) Analytical ratio bands and sequencing maps

Setting the artificially synthesized sequence in example 1 as an unmutated sequence; the artificially synthesized sequence in example 2 was a mutated sequence.

The colorimetric bands obtained in example 1 and example 2 were stacked and compared, and the results are shown in fig. 8; the non-overlapping site is the 4 th nucleotide site, which is a mutation site. From the information on the two color bands, it was found that the nucleotide information of the mutation site was identified for G in the base mutation having a mutation site of A, T, C, and the method specifically included the following steps:

pyrosequencing was repeated on the artificially synthesized sequence (3 '-AAAAAAGGAGTTAGCCGGTGCTTCT-5') in example 1, and the sequence was mapped as shown in FIG. 9 by replacing the trinucleotide with dATP. alpha.S, dCTP, and dGTP and changing the single nucleotide to dTTP.

When the bands in FIG. 7 and the sequencing map in FIG. 9 are combined, the mutation site is from A to G. Therefore, the nucleotide sequencing map-based microbial population identification method can be applied to sequencing of the directional mutation sites.

Example 3

The microorganism population identification method can carry out complete sequence detection on a target fragment of a microorganism to be detected, and comprises the following steps:

(1) extracting a DNA template of the microorganism, amplifying the variable region under the action of DNA polymerase, and marking biotin on an amplification product;

(2) mixing the amplification product modified with biotin at the 5' end with the streptavidin-modified magnetic beads, and shaking for 1 hour at room temperature; then, after washing with PBS five times, magnetic beads on which amplification products (single-stranded DNA) are immobilized on the outer surface are obtained by magnetic separation;

(3) preserving the heat of the magnetic beads with the amplification products fixed on the surfaces and the sequencing primer at 75 ℃ for 5 minutes, naturally cooling to room temperature, separating the magnetic beads from the liquid, and combining the amplification products and the sequencing primer for later use;

(4) placing the magnetic beads separated in the step (2) in a pyrosequencer for three groups of sequencing:

performing pyrosequencing by alternately and circularly adding trinucleotides and mononucleotides in the first group of sequencing, wherein the trinucleotides are dATP alpha S, dTTP and dGTP, the content of the three is 1:1:1, and the mononucleotides are dCTP; obtaining the site of the base G in the DNA template through a sequencing result;

a second set of sequencing, identical to the first set of sequencing procedures, except that: the trinucleotide is dATP alpha S, dCTP and dGTP with the content of 1:1:1, and the mononucleotide is dTTP; obtaining the site of the base A in the DNA template through a sequencing result;

and the third group of sequencing is the same as the first and second groups of sequencing processes, except that: the trinucleotide is dATP alpha S, dCTP and dTTP with the content of 1:1:1, and the mononucleotide is dGTP; obtaining the site of the base C in the DNA template through a sequencing result;

(5) combining the three groups of sequencing results, each site occupied by the base G, A, C in the DNA template can be obtained, and the rest sites are T, so that the sequence of the DNA template can be obtained.

In addition to examples 1-3 above, the method for identifying a microbial population of the present invention can select the types of trinucleotide and mononucleotide based on the types of mutation common to a specific strain to reduce the influence of mutation on the identification result. Meanwhile, when population tests are carried out on microorganisms with highly similar target fragments, pyrosequencing can be carried out twice or more, different trinucleotides and mononucleotides can be selected in different pyrosequencing, and the population of the microorganisms is analyzed and identified by integrating and comparing the sequencing results of two times or more.

It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.