JP2003259879A - Quantitative analysis method for compound microorganism group - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analysis method utilizing PCR (polymerase chain reaction) product and added quantitativity by determining a copy number of the objective microorganisms in a compound microorganism group. <P>SOLUTION: This quantitative analysis method for the compound microorganism group is characterized by amplifying an indicator sequence by PCR method using a primer suitable for amplifying the indicator sequence based on mixed DNA collected from a sample containing the compound microorganism group and, in the obtained PCR product, qualitatively specifying microbial species derived from the indicator sequence and evaluating the quantitative value of the indicator sequence concentration based on the repetition count n of the specified microbial species inherent to the DNA of the specified microbial species. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to, for example, activated sludge,
Quantitative analysis method of complex microorganism group enabling quantitative monitoring of microorganisms in a sample composed of various microorganisms such as soil, and DN usable for the method
It relates to a method for estimating the number of repeats of the target sequence in A.

[0002]

2. Description of the Related Art At present, the PCR method is used in all kinds of molecular biological experiments. For example, TRFLP (Terminal Restriction Fragmen) is also used in dynamic analysis of complex microbial community samples obtained from activated sludge or soil.
t Length Polymorphism) method, DGGE (Denaturing Gr)
A method of analyzing an amplified DNA (PCR product) by combining an adient gel electrophoresis) method and the like with a PCR method is becoming widespread. In these analysis methods, a gene corresponding to 16SrRNA (hereinafter sometimes referred to as “16SrDNA”) contained in the microbial genome is grown by the PCR method and subjected to analysis. This 16SrDN
In A, since the variable region base sequence differs depending on the microorganism species,
It is used as a classification indicator. However, 16Sr
The number of DNAs contained in the genome varies depending on the type of microorganism. For example, for example, Bacillus subtilis (Bacillus s)
ubtilis), 10 in the genome, Pseudomonas
The number is 4 in Pseudomonas aeruginosa and 7 in Escherichia coli. That is, the number of repeats of 16S rDNA in the genome (hereinafter sometimes referred to as “copy number”) differs depending on the bacterial species. Generally, in the PCR method, replication is performed according to the number of genes of the target sequence and the affinity with the primer,
Although the design of the primer is important, when 16S rDNA is amplified as an index for evaluating the kinetics of microorganisms, an effective primer is often used for all bacteria.
Affinity of the primer does not matter much. Rather, as described above, the variation in the copy number of the target gene becomes a problem.

As described above, the copy number of 16S rDNA is unique to each bacterium and is not the same.
The higher the copy number of 6SrDNA, the higher the number of copies, and naturally the higher the PCR product of the bacterial copy with a high copy number. As a result, the quantification result using the PCR product will not accurately reflect the population of the original sample (distribution of the number of bacteria by type). For example, for a microorganism having a large copy number of 16S rDNA in the genome, PCR is performed even if the proportion of the population is small.
Even in a microorganism having a large amount of product and conversely a large proportion of the population, when the copy number of 16SrDNA in the genome is small, the reversal phenomenon that the amount of PCR product is small may occur. For this reason, there is a drawback in that the concentration of the PCR product does not reflect the microbial population of the original sample. Therefore, the above-mentioned TRFLP method and DGGE
There is a limit to the quantitative analysis in the analysis method using the method, etc., and it is only used as a relative evaluation tool by pattern analysis of the distribution of microorganisms in the population.

On the other hand, recently, a quantitative PCR method has been developed which is capable of carrying out quantification together with amplification of a target sequence. However, it is very difficult to design a primer, and the equipment used is compared to ordinary PCR equipment. There is a problem that it is expensive.

[0005]

If the copy number of the target gene to be amplified is known by the PCR method, it is possible to quantitatively discuss the number of bacteria from the intensity of the band and the copy number by, for example, electrophoresis. . Taking the case of the TRFLP method as an example, when 27F (see Examples below) is used as a primer, 16S rDNA fragmented with Hha I is used.
In, Bacillus subtilis is 240base,
Pseudomonas aeruginosa shows a fragment unique to 154base. Therefore, quantitative comparison should be possible by dividing the intensity (concentration) of the fragment by the copy number.

The above-mentioned copy number can be searched from the database for bacteria for which the analysis of the entire genome has been completed. However, the number of bacteria that can be searched from the database is extremely small, for example, about several tens of 16SrDNA, and the copy number is grasped by some means in order to carry out a quantitative evaluation by analysis of a complex microorganism group using the PCR method. Will be required. If a method of grasping the copy number of the target gene such as 16S rDNA of the bacterium of interest in the population can be established, quantitative evaluation by PCR products can be realized.

Therefore, the object of the present invention is to clarify the copy number of the target microorganism from the complex microorganism group,
It is to provide an analysis method in which quantitativeness is added to the analysis using a CR product.

[0008]

In order to solve the above-mentioned problems, the invention of the method for quantitatively analyzing a complex microorganism group according to claim 1 is based on DNA in a mixed state collected from a sample containing the complex microorganism group. Amplifying the indicator sequence by the PCR method, qualitatively identifying the microorganism species from which the indicator sequence is derived in the obtained PCR product, quantifying the concentration of the indicator sequence, and quantifying the concentration of the indicator sequence. The value is evaluated based on the number of repetitions n (n is a natural number; the same applies hereinafter) of the indicator sequence unique to the DNA of the identified microbial species. According to this feature, the molecular biology method is evaluated by evaluating the quantitative value of the concentration of the indicator sequence for the sample containing the complex microorganism group based on the number of repetitions n of the indicator sequence peculiar to the DNA of the microbial species. It is possible to rapidly perform a quantitative analysis in consideration of the number of repetitions n of the index sequence. In addition, compared with the quantitative PCR method, it is advantageous because general-purpose primers can be used and can be performed with a simple device. And since it is a molecular biological analysis method that can quantitatively analyze samples in which various microbial species coexist, it can be used advantageously as a monitoring method for observing the dynamics of microorganisms in the environment such as activated sludge and soil. it can.

The invention of the method for quantitatively analyzing a complex microorganism group according to claim 2 is the method according to claim 1, wherein the number of repetitions n of the indicator sequence is such that a DNA containing the indicator sequence is isolated from the sample. DNA containing the isolated indicator sequence
With DNA containing a similar index sequence similar to the above-mentioned index sequence with a known repeat number m (m is a natural number; the same applies hereinafter), isolated from a comparative microorganism, and m times when amplified by the PCR method under the same conditions. The concentration is compared with the quantitative value of the similar index sequence, and is compared with the number m of repetitions of the similar index sequence based on the ratio of the amount of the isolated DNA containing the index sequence before amplification to the amount of the isolated DNA containing the similar index sequence. It is characterized by being estimated by According to this feature, by estimating the copy number of the indicator sequence by comparison with a similar index sequence with a known copy number, a microorganism whose copy number of the indicator sequence is unknown can be identified if it can be qualitatively specified. Quantitative analysis is also possible for species.

The invention of a method for quantitatively analyzing a complex microorganism group according to claim 3 is the method according to claim 1 or 2, wherein the indicator sequence is a nucleotide sequence of a DNA corresponding to 16S rRNA. . In the method of the present invention, since the quantification is performed in consideration of the copy number of the indicator sequence, the DNA corresponding to 16SrRNA having a different copy number depending on the microorganism
Accurate quantitative analysis is possible using (16SrDNA). Therefore, qualitative and quantitative analysis can be realized using 16S rRNA widely used as a classification index of microorganisms.

The invention of the method of estimating the number of repeats of a target sequence in DNA according to claim 4 is a method of estimating the number of repeats n of a target sequence in a certain DNA, wherein the target sequence is defined by the number of repeats n. The test DNA containing the target DNA was amplified by the PCR method, the target sequence at an n-fold concentration was quantified from the obtained PCR product, and a comparative DNA containing a control sequence similar to the target sequence at a known repeat number m was PCR-treated under the same conditions. By comparing with the quantitative value of the control sequence at an m-fold concentration in the case of amplification by the method, and comparing with the repeat number m of the control sequence based on the ratio between the amount of test DNA before amplification and the amount of comparative DNA.
It is characterized by estimating the number of repeats n of the target sequence in A. The invention of this method for estimating the number of repeats of a target sequence is capable of easily and rapidly estimating the number of copies from a DNA of which the number of repeats of a target sequence is unknown, by a molecular biology experimental method. Not only medicine,
It can be used in a wide range of fields such as biology.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The method for quantitatively analyzing a complex microorganism group according to the present invention can be carried out according to the following procedures (a) to (d).

(A) DNA in a mixed state is collected from a sample containing a complex microorganism group. Examples of samples containing complex microorganism groups include, in addition to activated sludge and soil, microbiologically treated food waste, anaerobic digestion liquid of livestock wastewater, and other samples in which various microorganisms such as bacteria are alive. You can Collection of DNA from a sample can be performed by a known method. For example, as in Examples described later, Dne
DNA extraction may be carried out using asy Tissue Kit (manufactured by QIAGEN) or the like.

(B) The indicator sequence is amplified by the PCR method using the primers. As the index sequence, 16Sr which is generally used as a molecular biological classification index of microorganisms
It is preferable to select the RNA gene (16S rDNA).

It is important to use a primer suitable for amplification of the indicator sequence. A primer corresponding to the indicator sequence is designed so that all the copies of the indicator sequence are included in the amplified region. When 16S rDNA is used as an indicator sequence, 27F and 1492 used in Examples described later.
It is preferable to use a primer such as R or 907R.

(C) In the obtained PCR product, the microbial species from which the indicator sequence is derived is qualitatively specified, and
Quantify the concentration of the indicator sequence. The qualitative identification of the microorganism containing the indicator sequence and the quantification of the concentration thereof can be carried out using a known method, and for example, the TRFLP method utilizing restriction enzyme fragment polymorphism is preferably used.

(D) The quantitative value of the concentration of the index sequence is evaluated based on the copy number n of the index sequence unique to the DNA of the specified microbial species. For example, the approximate amount of the microorganism containing the indicator sequence in the sample can be quantified by dividing the quantitative value of the concentration of the indicator sequence by the copy number. In other words, the concentration of the indicator array to be quantified is the copy number of the indicator array n
When the concentration of the initial indicator sequence is S and the number of PCR cycles is x, it should be possible to express it as S × n × 2 ^x until the steady state (plateau) in PCR is reached. The divided value will always reflect the population of microorganisms in the sample. Therefore, by standardizing the above operations (a) to (c) and creating a calibration curve based on the data of another sample whose population has been quantitatively analyzed in advance, even an unknown sample can be obtained. The target microorganism can be quantified.

In addition, a plurality of microbial species in the sample are identified by the methods (a) to (c) using the TRFLP method or the like, and the concentration is quantified as the intensity of the electrophoretic band, and the By dividing the quantification result by the copy number, it becomes possible to quantitatively compare the microbial species in the population.

The copy number n of the index sequence is known for a microorganism whose whole genome has been decoded and can be known from a database. The microbial species whose copy number is unknown can be estimated using the estimation method of the present invention described below. The outline thereof is, for example, (a) to (c) above.
As a separate operation from the above, DNA containing an indicator sequence is isolated from a sample containing the complex microorganism group of the above (a), and the isolated DNA containing the indicator sequence is replaced with a similar indicator sequence similar to the indicator sequence. Amplification is carried out by the PCR method under the same conditions, together with DNA isolated from a comparative microorganism containing a known repeat number m. At this time, isolated DN containing the indicator sequence before amplification
The ratio between the amount of A and the amount of isolated DNA containing the similar index sequence is defined in advance, for example, by making it 1: 1.

Next, by comparing the quantitative value of the m-fold concentration of the similar index sequence obtained by the above operation with the quantitative value of the index sequence, the amount of isolated DNA containing the index sequence before amplification and the similar index sequence are compared. It is possible to estimate the copy number of the indicator sequence, taking into consideration the ratio with the amount of isolated DNA containing (in the case where the ratio is 1: 1).

According to the method for quantitatively analyzing a complex microbial group of the present invention as described above, it is possible to identify a complex microbial sample such as activated sludge or soil by using an index such as 16SrDNA as a clue. Since microorganisms can be roughly quantified and the proportion of population can be evaluated, not only qualitative results but also quantitative analysis can be performed. Therefore, it can be advantageously used for periodic monitoring surveys of microorganisms in the environment.

Next, the method for estimating the number of repeats of the target sequence in DNA according to the present invention will be described. This estimation method is a method that can be used for the quantitative analysis method of the above-mentioned complex microorganism group, and also the copy number of the target sequence is present in all DNA (including fragmented ones). It can be used when estimating.

The estimation method of the present invention is based on, for example, the following (1)
To (5) are included.

(1) A test DNA containing a target sequence with a repeat number n is amplified by the PCR method. Here, the “target sequence” may be a sequence that can be detected by a technique such as electrophoresis and has a molecular biological function (for example,
It does not matter whether it is a structural gene or not. The term “detectable” is not limited to the case where the target sequence itself is directly detected, but also includes the case where a fragment of the target sequence cleaved by a restriction enzyme is detected based on the label. In this step, it is necessary to design the primers used for amplification so that the n target sequences can be amplified without exception.

(2) The concentration of the target sequence is quantified from the obtained PCR product. This concentration is 1 copy of the target sequence
It is supposed to be n times as large as the case (no repetition). The concentration can be quantified by a known method, for example, by measuring the intensity of the electrophoretic band according to a conventional method.

(3) A comparative DNA containing a control sequence similar to the target sequence at a known repeat number m is amplified by the PCR method under the same conditions as in (1) above. Here, the “control sequence similar to the target sequence” means a sequence having the same function as the target sequence. For example, if the target sequence is microbial 16SrD
If NA, 16SrDN derived from different microbial species
When the target sequence is a gene encoding a certain human protein, it means that the gene has a relationship such as a gene encoding a mouse protein having a similar function. In this case, the repeat number m of the control sequence in the comparison DNA needs to be known. The “same condition” in PCR means conditions related to the amplification amount of the control sequence, such as the primer and DNA polymerase used, the number of cycles, and the temperature. Here, it is necessary to confirm the ratio of the initial amount (number of molecules, number of moles, etc.) before amplification by the PCR method for each of the target sequence and the control sequence. The initial amount is calculated from the total size of DNA including the target sequence and control sequence (for example, genome size in the case of bacteria),
It can be calculated. The total size of DNA can be measured by a technique such as pulse field electrophoresis. To facilitate the copy number comparison described later,
It is preferable that the initial amount is the same.

(4) The concentration of the amplified control sequence is quantified by the same method as in (2) above. The quantitative value of the control sequence is
The number of copies of the control target sequence should be m times as compared with the case where the copy number is 1 (no repeat). This quantification is performed by the same method as in (2) above.

(5) Based on the ratio between the amount of test DNA before amplification and the amount of comparative DNA confirmed in (3) above, the quantitative value of the target sequence and the quantitative value of the control sequence are compared to obtain the control sequence. The number n of repetitions of the target sequence in the test DNA is estimated from the number m of repetitions (known). When the initial amounts of the target sequence and the control sequence are the same, the relationship (ratio) between the quantitative value of the target sequence and the quantitative value of the control sequence should be the same as the ratio between the copy numbers n and m. The copy number n of the target sequence can be estimated.

Amplification and detection of the control sequences in (3) and (4) above may be carried out at the same time as amplification and detection of the target sequence, provided that the PCR conditions and the detection method (preferably also the apparatus used) are the same. Alternatively, it can be implemented regardless of time and space. That is, it is possible to carry out amplification and detection of a control sequence under predetermined conditions, and to evaluate the results of amplification and detection of the target sequence with reference to the results. For example, 16Sr of several typical microorganisms
It is also possible to prepare a detection result obtained when the DNA is amplified under specific conditions as a table (evaluation table) showing the relationship between the number of PCRs and the detected concentration. From this, an evaluation table, a protocol for performing amplification and detection under specific conditions, a reagent, etc. are provided,
It can be a kit for carrying out the estimation method of the present invention. Also, instead of the evaluation table, it is possible to prepare a PCR kit for evaluation of the copy number, which contains the control sequence in a storable form so that the control sequence and the target sequence can be detected (or amplified and detected) at the same time. Is.

[0030]

EXAMPLES Next, the present invention will be described in more detail by way of examples, which should not be construed as limiting the invention.

Example 1 (I) Forward primer [27] for obtaining a PCR product using bacterial 16S rDNA as a target sequence
F: 5'-agagttttgatcmtggctca
g: m is a or c (SEQ ID NO: 1)] and a reverse primer [1492R: 5'-acggytacctgtgt.
tacgactt: y is t or c (SEQ ID NO: 2)] 2.5 μl of each 4 pmol solution and Taq PCR Mas
The ter Kit (manufactured by QIAGEN) was adjusted according to the protocol. The template DNA is 5 × 10 ⁵ pieces /
Prediluted to give a volume of 1 μl and used 1 μl. Gene Amp 9700 manufactured by Perkin Elmer Co., Ltd. was used for amplification by the PCR method at 94 ° C. for 1 minute, 5 minutes.
Up to 22 cycles of 0 ° C.-1 minute and 72 ° C.-1 minute 30 seconds were performed.

The DNA used as the template is Bacillus subtilis ATCC6633 strain and Pseudomonas aerugi.
nosa) The one derived from the JCM4090 strain was used. These DNAs were previously prepared by QIAGEN Dneasy Tis.
DNA extraction was performed according to the protocol of sue Kit (manufactured by QIAGEN), and the concentration was determined to be Gene Spe.
It was measured with c III (manufactured by Hitachi Ltd.).

The genome sizes of these two types of bacteria are 4.21 k base pa for Bacillus subtilis.
ir and Pseudomonas aeruginosa are 6.3k
It is a base pair. From this number, extract DNA
The number of moles of can be calculated. That is, the extracted DNA of Bacillus subtilis has a concentration of 34.26 ng / μl,
1.2525 × 10 ⁻¹⁷ mol / μl, 7.54 ×
10 ⁶ cells / μl. The extracted DNA of Pseudomonas aeruginosa was 1.289 × 10 ⁻¹⁷ mol / μl at a concentration of 49.38 ng / μl and 7.54.
× 10 ⁶ cells / μl.

As described above, the concentration of the DNA serving as the template was adjusted so that the number of genomes was the same, and then the number of PCRs was changed to perform amplification by the PCR method. The result is shown in FIG. The amount of PCR product increased as the number of cycles increased. In the case of 16S rDNA, Bacillus subtilis has 1
It is known that 0 and 4 of Pseudomonas aeruginosa exist on the genome. 20 times PCR
The PCR product concentration at the time of performing
The amount of PCR product was confirmed to be almost proportional to the above copy number, with 1.5 ng / μl for Aeruginosa and 3.2 ng / μl for Bacillus subtilis.

(II) PC obtained by amplification in the same manner as above
Regarding the R product, a primer in which the 5'end of the primer 27F was fluorescently labeled with Texas Red was used, subjected to restriction enzyme treatment with Hhal I to fragment the PCR product, and then the Hitachi SQ550
Fragment analysis was performed with a 0 DNA sequencer.
In the fragment analysis by the TRFLP method using 27F as a primer, Bacillus subtilis showed 240
Near base, Pseudomonas aeruginosa
It is known to show fragmentation near 154 bases. As a result of this fragment analysis,
The area occupancy (%) of both fragments was about 70% for Bacillus subtilis and about 30% for Pseudomonas aeruginosa, and the area ratio of the fragments was 2.3: 1, which is almost proportional to the copy number. Was given. that's all
From the results of (I) and (II), it was shown that quantitative analysis is possible in the analysis using the PCR product of the complex microorganism.

Example 2 The bacteria shown in Table 1 were cultivated in a predetermined culture solution to obtain the QIAGENDneasy Tissue Kit.
DNA extraction was performed according to the protocol (manufactured by QIAGEN), and the concentration was measured by Gene Spec III (manufactured by Hitachi, Ltd.). This concentration is also shown in Table 1.

[0037]

[Table 1]

From the genome sizes of the bacteria A to D, the molar concentration of the DNA solution was calculated, and then 5
Diluted to a concentration of × 10 ⁵ cells / μl. Amplification by the PCR method was performed from 13 cycles to 25 cycles using this DNA solution as a template in the same manner as in Example 1, and the concentration of the resulting PCR product was determined by a microchip electrophoresis apparatus Agilent 2100 Bioanalyzer (Ag.
ilent Technologies). The results of each concentration at the 15th cycle are shown in FIG.

Further, the results of each PCR were divided by the copy number peculiar to each bacterium and plotted, and an approximate value curve was obtained. As a result, almost coincident curves were obtained. The result is shown in FIG. From this result, it was shown that there is a certain correlation between the copy number and the amount of PCR product.

Example 3 The DNAs of the three types of bacteria shown in Table 2 were adjusted in the same manner as in Example 2 so that the DNA concentrations (numbers) were equal, and mixed in equal amounts. Mixed DN of these
Forward primer 27F [27F: 5'-agag] with 5'end modified with Texas red using A as a template
tttgatcmtggctcag: m is a or c
(SEQ ID NO: 1)] and reverse primer 907R
[5'-ccgtcaattcattttgagttt-
3 ′ (SEQ ID NO: 3)] and the temperature condition is 95 ° C.−1.
PCR at 15 minutes, 47 ° C. for 1 minute, and 72 ° C. for 1.5 minutes was performed, and the resulting PCR product was used as QIAGEN Purificat.
The contaminants were removed with an ion Kit (manufactured by QIAGEN) and collected. The fragment obtained by cleaving this PCR product with Hha I was subjected to electrophoresis using a fluorescent image analyzer for sequencer (HITACHI SQ5500: manufactured by Hitachi, Ltd.) to determine the position of the fragment modified with Texas Red and its band. The strength was measured. The results are also shown in Table 2.

[0041]

[Table 2]

From this result, it was shown that the ratio of the intensities of the PCR product fragments derived from each microorganism corresponds to the copy number of 16S rDNA.

[0043]

EFFECTS OF THE INVENTION According to the method for analyzing a complex microorganism group according to the present invention, the quantitative value of the concentration of the indicator sequence in the sample containing the complex microorganism group is set to the repetition number n of the indicator sequence peculiar to the DNA of the microbial species. Based on the evaluation, a quantitative analysis considering the copy number n of the indicator sequence can be rapidly performed using a molecular biology method. Further, unlike the quantitative PCR method, since the equipment used can be general-purpose equipment, it is economically advantageous.

Further, the invention of the method for estimating the number of repeats of a target sequence according to the present invention is a DN in which the number of repeats of a target sequence is unknown.
Since the copy number can be easily and quickly estimated from A by a molecular biological experiment method, it can be used not only in the quantitative analysis of the complex microorganism but also in a wide range of fields such as medicine and biology.

[Sequence Listing Free Text] SEQ ID NO: 1 An oligonucleotide primer designed for PCR. SEQ ID NO: 2 Oligonucleotide primer designed for PCR. SEQ ID NO: 3 Oligonucleotide primer designed for PCR.

[Sequence list]                                SEQUENCE LISTING        <110> MITSUI ENGINEERRING & SHIPBUILDING CO., LTD. <120> Quantitative Analysis of Micropopulation <130> P97221 <140> <141> <160> 3 <170> PatentIn Ver. 2.1 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       oligonucleotide primer for PCR <400> 1 agagtttgat cmtggctcag 20    <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       oligonucleotide primer for PCR <400> 2 tacggytacc ttgttacgac tt 22    <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       oligonucleotide primer for PCR <400> 3 ccgtcaattc atttgagttt 20

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the number of PCRs and the amount of PCR products.

FIG. 2 is a graph showing the concentration of each PCR product at the 15th PCR cycle, in which the central peak is PCR.
The product concentration is shown, and the peaks on both sides show the internal standard.

FIG. 3 is a graph showing the relationship between the number of PCRs and the amount of PCR products per copy number, where A is Bacillus subtilis, B is Staphylococcus aureus, C is Streptococcus pyogenes, and D is Pseudomonas.
DNA derived from aeruginosa.

Claims (4)

[Claims] 1. A marker sequence is amplified by the PCR method based on mixed DNA collected from a sample containing a complex microbial group, and the obtained PCR product is qualitatively the microbial species from which the marker sequence is derived. In addition, the concentration of the indicator sequence is quantified, and the quantitative value of the concentration of the indicator sequence is evaluated based on the repeat number n of the indicator sequence unique to the DNA of the identified microbial species. A method for quantitative analysis of complex microbial community. 2. The repeat number n of the indicator sequence according to claim 1, wherein the DNA containing the indicator sequence is isolated from the sample, and the isolated DNA containing the indicator sequence is isolated from a comparative microorganism. PC under the same conditions together with the separated DNA containing a similar index sequence similar to the above index sequence with a known repeat number m
Compared to the quantitative value of the m-fold concentration of the similar indicator sequence in the case of amplification by the R method, the above similarity based on the ratio between the amount of isolated DNA containing the indicator sequence before amplification and the amount of isolated DNA containing the similar indicator sequence. A quantitative analysis method of a complex microorganism group, which is estimated by comparing with the number of repetitions m of the index sequence. 3. The method for quantitative analysis of a complex microbial group according to claim 1 or 2, wherein the indicator sequence is a nucleotide sequence of DNA corresponding to 16SrRNA. 4. A method for estimating the number of repetitions n of a target sequence in a certain DNA, wherein a test DNA containing the target sequence at the number of repetitions n is amplified by PCR, and the resulting PCR product is n-fold concentrated. Of the target sequence, and a comparative DNA containing a control sequence similar to the target sequence at a known repeat number m was compared with the quantitative value of the m-fold concentration of the control sequence when amplified by the PCR method under the same conditions, Based on the ratio between the amount of test DNA before amplification and the amount of comparative DNA, the number of repeats n of the target sequence in the test DNA is estimated by comparing with the number of repeats m of the control sequence. A method for estimating the number of repeats of a target sequence.