JPH11276176A - DNA fragment amplification method, microorganism existence state estimation method, and organic waste state estimation method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a D capable of amplifying an analyzable amount of a DNA fragment from a plurality of different DNAs without information on a base sequence.
An object of the present invention is to provide a method for amplifying an NA fragment. SOLUTION: SEQ ID NOS: 41 to 43, 45 to 54, 56
~ 58, 60 ~ 64, 66 ~ 69, 71 ~ 78, 80 ~
82, 84-86, 89-91, 93, 95-97, 1
Using any one of primers 00, 103, 106, 107 and 109, a random PC was applied to a plurality of microorganisms collected from the treatment tank of the garbage disposal machine or from the soil.
By applying the R method, DNA fragments of a plurality of microorganisms are amplified. The amplified DNA fragment is analyzed using an electrophoresis method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for amplifying a DNA fragment, a method for estimating the presence of microorganisms, and a method for estimating the state of organic waste.

[0002]

2. Description of the Related Art In recent years, garbage disposers for composting organic wastes (so-called garbage) discharged from households and the like have been actively researched and developed. In a garbage disposer, fertilizer is produced by microorganisms such as bacteria and protozoa decomposing organic matter.

In such composting using a garbage disposer, the degree of composting is evaluated by monitoring the temperature and the like in the composting process (organic matter decomposition process). Then, based on the evaluation, the state of the garbage disposer is adjusted so that high-quality fertilizer is produced.

[0004] However, in order to produce a higher quality fertilizer, information on the state of microorganisms (at least the type of microorganisms) that functions inside the garbage disposal machine is required. This information on the presence state of microorganisms is also necessary for controlling the decomposition of garbage by microorganisms. In addition, in order to improve the soil by adding the produced fertilizer, it is also important to know the state of microorganisms in the soil.

[0005]

Conventionally, as a method for obtaining information on the state of existence of microorganisms, for example, the state of existence of bacteria,
A method of isolating individual bacteria and conducting a biochemical test is used. However, this method is time-consuming and difficult to examine for bacteria that are difficult to isolate.

On the other hand, PCR is used as a method for amplifying DNA.
(Polymerase Chain Reaction)
Method is used. This PCR method uses a primer having a base sequence complementary to the base sequences at both ends of the DNA to be amplified (template DNA) and a heat-resistant DNA polymerase, and comprises a heat denaturation step, an annealing (heat treatment) step and an extension reaction step. By repeating a cycle consisting of steps, it is possible to amplify a DNA fragment substantially the same as the template DNA. By using this PCR method, it is possible to amplify, for example, 100,000 to 1,000,000 times a predetermined fragment in one DNA of a bacterium which exists only in a small amount.

However, in order to use this PCR method, it is necessary that the nucleotide sequences of at least both ends of the template DNA are known. Therefore, in the conventional PCR method, if the type and base sequence of the microorganisms that function inside the garbage disposer and the microorganisms that exist in the soil are not known,
The DNA fragments of those microorganisms cannot be amplified.

[0008] Therefore, RAPD (Random Amplified Polymorphic D) which amplifies a large amount of DNA fragments simultaneously from one kind of DNA with a single primer without base sequence information.
NA) method or AP-PCR (Arbitrarily Primed-P)
olymerase Chain Reaction) method has been proposed. In these methods, the sequence specificity at the time of binding of the primer is reduced by lowering the annealing temperature of the primer during the PCR reaction and further increasing the magnesium ion concentration in the reaction solution. Then, the primer binds to the chromosomal DNA of the microorganism with a mismatch, and the DNA fragment is replicated.

[0009] These RAPD method or AP-PCR
According to the method, even if there is no information on the base sequence of the DNA to be amplified, any DNA fragment is amplified in a large amount by a single primer. A DNA fingerprint is obtained by separating the amplified DNA fragments by gel electrophoresis. By analyzing this DNA fingerprint, it is possible to analyze the state of the microorganism.

[0010] However, in the conventional RAPD method or AP-PCR method, even if conditions are appropriately selected, a very large number of DNA fragments are amplified depending on the type of primer. Therefore, it becomes difficult to analyze the existence state of each microorganism in garbage or soil where a plurality of microorganisms exist.

An object of the present invention is to provide a DNA fragment amplification method capable of amplifying an analyzable amount of a DNA fragment from a plurality of different DNAs without knowledge of the base sequence, a method for estimating the presence of microorganisms using the same, and an organic system. It is to provide a waste state estimation method.

[0012]

Means for Solving the Problems and Effects of the Invention The method for amplifying a DNA fragment according to the present invention comprises the steps of:
5-54, 56-58, 60-64, 66-69, 71
~ 78, 80-82, 84-86, 89-91, 93,
95-97, 100, 103, 106, 107 and 1
09, using a polymerase chain reaction method in which a heat denaturation step, a primer annealing step, and an elongation reaction step with a polymerase are repeated in this order on a plurality of different DNAs at a time, a plurality of different DNAs are obtained. Wherein the DNA fragment is amplified. The DNA includes not only biological DNA but also DNA fragments.

In the method for amplifying a DNA fragment according to the present invention, by applying the polymerase chain reaction to a plurality of different DNAs at once, some of the plurality of different DNAs can be obtained without knowledge of the DNA base sequence. Different DNA
Can be amplified. In particular, the use of the above-selected primers enables amplification of an analyzable amount of a DNA fragment. in this case,
In the electrophoresis image of the amplified DNA fragment, 1 to 10 clear bands appear. Therefore, it is possible to easily estimate the presence state of the microorganism from the electrophoresis image.

In addition, a DNA probe is prepared by cloning the amplified DNA fragment, and the presence of a microorganism can be confirmed by a hybridization method, and the amount of the microorganism can be measured more accurately.

In particular, it is preferable that the conditions of the polymerase chain reaction are set such that a band corresponding to at least one of a plurality of different DNAs appears in an electrophoretic image of a DNA fragment amplified by application of the polymerase chain reaction. Features.

Thus, by setting the conditions of the polymerase chain reaction so that a band corresponding to at least one DNA appears in the electrophoresis image of the amplified DNA fragment, the presence of at least one microorganism can be determined from the electrophoresis image. The state can be easily estimated.

The method for amplifying a DNA fragment according to the present invention employs a polymerase chain reaction method in which a heat denaturation step, a primer annealing step and a polymerase extension reaction step are repeated for a plurality of different DNAs in this order at a time. By so doing, when amplifying the DNA fragments of a plurality of different DNAs, the number of bands corresponding to each DNA appearing in the electrophoresis image of the DNA fragments amplified by the application of the polymerase chain reaction is reduced to several or less. And the conditions for primer and polymerase chain reaction are set.

In this case, since at most a few bands corresponding to each DNA appear in the electrophoresis image of the amplified DNA fragment, the presence state of the microorganism can be easily estimated from the electrophoresis image.

In addition, a DNA probe is prepared by cloning the amplified DNA fragment, the presence of a microorganism can be confirmed by a hybridization method, and the amount of the microorganism can be measured more accurately.

In particular, as the conditions for the polymerase chain reaction, the magnesium concentration of the reaction solution is 1.5 mM, Taq
It is preferable that the polymerase concentration is 0.025 unit / μL, the annealing temperature in the annealing step is 45 ° C., and the number of cycles is 35. Thereby, it becomes possible to amplify an analyzable amount of the DNA fragment.

In particular, when the discrimination method is the electrophoresis method, a discernable number of bands appears in the electrophoresis image of the amplified DNA fragment, so that the presence of microorganisms in the object can be easily determined from the electrophoresis image. Can be estimated.

The method for estimating the presence of microorganisms according to the present invention comprises:
A method for estimating the presence of microorganisms in an object, comprising the steps of:
A, using the above-described DNA fragment amplification method, amplifying the DNA fragments of the DNAs of a plurality of different microorganisms, classifying the amplified DNA fragments by an identification method, and estimating the presence state of the microorganisms in the object. Features. In particular, the identification method may be an electrophoresis method.

In the method for estimating the presence of microorganisms according to the present invention, the DNA fragments of a plurality of different microorganisms in the subject are amplified in an amount that can be analyzed without knowledge of the base sequence. Is classified by the identification method, it is possible to easily estimate the presence state of the microorganism.

An organic waste state estimating method according to the present invention is an organic waste state estimating method for estimating an organic waste state in an organic matter decomposition process in which organic matter in an organic waste in which a plurality of microorganisms are mixed is decomposed by the microorganisms. A method for estimating a waste state, comprising: amplifying DNA fragments of DNAs of a plurality of different microorganisms using the above DNA fragment amplification method with respect to DNAs of a plurality of microorganisms in an organic waste;
The method is characterized in that the fragment A is classified by an identification method, the state of the microorganisms in the organic waste is estimated, and the state of the organic waste is estimated from the estimation result. In particular, the identification method may be an electrophoresis method.

In the method for estimating the state of an organic waste according to the present invention, a DNA fragment of a plurality of microorganisms in the organic waste that can be analyzed without a base sequence information is amplified. By classifying the DNA fragments according to the identification method, it is possible to estimate the state of the microorganisms in the organic waste, and to easily estimate the state of the organic waste from the estimation result.

Further, a DNA probe is prepared by cloning the amplified DNA fragment, and the presence of the microorganism can be confirmed by the hybridization method, and the amount of the microorganism can be measured more accurately.

In particular, when the discrimination method is the electrophoresis method, a discernable number of bands appears in the electrophoresis image of the amplified DNA fragment, so that the state of the organic waste can be easily estimated from the electrophoresis image. can do.

[0028]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, an example of a DNA fragment amplification method, a microorganism existence state estimation method and an organic waste state estimation method according to the present invention will be described.

In the method for amplifying a DNA fragment according to the present invention, a reaction method is used in which a DNA fragment is amplified in a chain reaction from a plurality of different microorganisms having an unknown base sequence using a primer having a specific base sequence. Hereinafter, this reaction method
It is called a random PCR (Polymerase Chain Reaction) method.

The bacterial chromosomal DNA is 8 × 10^Five-10⁷
Since it consists of bases, the probability is 10 ⁷One pair about base
If primers of sufficient length are used,
One DNA fragment can be amplified from a bacterium. Only
And the number of DNA fragments amplified from one bacterium is probability
Not determined by the base sequence of the primer
Greatly affected. In the present invention, it has a specific base sequence.
Primers can be used to identify unknown base sequences.
Analytical amounts of DNA fragments from multiple different bacteria
Can be amplified.

In the random PCR method used in the DNA fragment amplification method of the present invention, the following three steps are repeated similarly to the conventional PCR method. However, in the random PCR method, as will be described later, an analyzable amount of a DNA fragment is amplified by using a primer having a specific base sequence for a plurality of different microorganisms having an unknown base sequence.

(1) Heat denaturation step The DNA (at the initial stage) or the DNA fragment is denatured by heating to form a single strand (DNA strand).

(2) Step of annealing primer (primer binding step) Heat treatment is performed so that the primer binds to the end of the amplification region of the DNA strand.

(3) Elongation reaction step by polymerase (replication step by polymerase) A complementary strand is synthesized by a polymerase with a primer as a starting point, and double-stranded.

The above steps (1) to (3) are repeated as one cycle. Next, random PC
The R method will be specifically described. First, several different microorganisms,
A polymerase chain reaction buffer solution, primers, a thermostable thermophilic bacterium DNA polymerase, and 5 ′ deoxyribonucleotide triphosphate (dAT)
A reaction solution containing (P, dGTP, dCTP, dTTP) is prepared.

Here, the DNA polymerase is an enzyme which uses four types of 5 'deoxyribonucleotide triphosphates as substrates and catalyzes a polymerization reaction of a DNA chain having a base sequence complementary to the template DNA. DN by DNA polymerase
The directionality of the polymerization of the A chain is from 5 ′ to 3 ′. The primer is a DNA fragment (short-chain oligonucleotide) having a 3′-OH group at the terminal which is essential for the action of DNA polymerase. In the present invention, a primer having a specific base sequence and base length is used.

The following random PCR was performed on the above reaction solution.
Apply the law. As a first cycle, a heat denaturation step of holding the reaction solution at, for example, 94 ° C. for 2 minutes, a primer annealing step of holding the reaction solution at, for example, 35 ° C. for 2 minutes, and a step of holding the reaction solution at, for example, 72 ° C. for 3 minutes The extension reaction step using the retained polymerase is performed in this order. The heat denaturation step of this cycle is a long complete D
In order to completely separate NA into single strands, the length is set longer than the heat denaturation step in the following cycle.

Subsequently, as a second cycle, a heat denaturation step in which the reaction solution is kept at, for example, 94 ° C. for 1 minute, a primer annealing step in which the reaction solution is kept at, for example, 35 ° C. for 2 minutes, and a step in which the reaction solution is kept at, for example, 72 minutes The extension reaction step using a polymerase maintained at 3 ° C. for 3 minutes is repeated in this order, for example, 33 times.

Finally, as a third cycle, a heat denaturation step in which the reaction solution is kept at, for example, 94 ° C. for 1 minute, a primer annealing step in which the reaction solution is kept at, for example, 35 ° C. for 2 minutes, and a step in which the reaction solution is kept at 72 ° C. An extension reaction step using a polymerase kept at 10 ° C. for 10 minutes is performed in this order. The extension reaction step using the polymerase in the present cycle is set longer than the extension reaction step using the polymerase in the first and second cycles in order to complete replication at the end.

Here, the first cycle, the first cycle of the second cycle, and the second cycle of the second cycle will be described with reference to FIGS. Note that the figure is schematically shown, and a single strand of DNA or the like indicates a base sequence of a portion binding to a primer.

In FIGS. 1 to 3, GGCTTCGAATC
A primer having the base sequence of G (SEQ ID NO: 69) is used. T represents thymine, A represents adenine, G represents guanine, and C represents cytosine.

As shown in FIG. 1A, initially, the reaction solution contained long DNA contained in a plurality of different microorganisms.
(Complete DNA) 1 is present. Here, one complete D
A description will be given focusing on NA.

First, as shown in FIG. 1 (b), in the first cycle, the long DNA denaturation step is performed longer than the heat denaturation steps of the second and third cycles, whereby
1 is heated and denatured, the double strands are separated from each other, and two single strands (DNA strands) 2a and 2b are formed.

Next, as shown in FIG. 1 (c), in the primer annealing step, the primer 11a is arranged at a suitable position (complementary sequence) of each of the single strands 2a and 2b that match the base sequence. Join. Here, the matching position is the position of the base sequence to be combined with the base sequence of the primer and the position of the base sequence similar to the base sequence to be combined with the base sequence of the primer. The above random PC
In the R method, the primer also binds to a portion of a DNA strand having a base sequence similar to the base sequence by setting the annealing temperature in the primer annealing step low. That is, the primer can not only bind to a single-stranded position having a nucleotide sequence completely complementary to its nucleotide sequence, but also bind to a single strand with some mismatch. FIGS. 1 to 3 show, for simplicity, the case where the primer binds to the position of the base sequence to be bound when viewed from the base sequence.

Subsequently, as shown in FIG. 1 (d), an elongation reaction occurs by the polymerase in the elongation reaction step by the polymerase, and the single strands 2c and 2d are extended along the single strands 2a and 2b, respectively. Main chains 3a and 3b are formed.

In the first cycle of the second cycle,
The chains 3a and 3b that have been double-stranded in the first cycle become single-stranded (DNA strands) 2a and 2c and single-stranded (DNA strands) 2b and 2d in the thermal denaturation step, respectively.
The following description focuses on the single-strand 2c that is separated from the first strand.

As shown in FIG. 2, in the subsequent primer annealing step, the primer 11b is bonded to the single strand 2c separated in the heat denaturation step so as to be located at a suitable position.
Thereafter, an elongation reaction is caused by the polymerase in an elongation reaction step by the polymerase, whereby the single strand 2e is extended along the single strand 2c to become a double-stranded chain 3d.

Thereafter, as shown in FIG. 3, in the second cycle of the second cycle, the double-stranded chain 3d
Become single strands 2c and 2e, respectively, in the heat denaturation step. Here, the description will focus on the single strand 2e separated from the chain 3d.

As shown in FIG. 3, in the next primer annealing step, the primer 11c is similarly bound to the single strand 2e separated in the heat denaturation step so as to be located at a suitable position. Thereafter, an elongation reaction occurs by the polymerase in an elongation reaction step by the polymerase.
e, the single-stranded 2f is extended to form a double-stranded chain (DN
A fragment) 3e is formed.

Thus, a DNA fragment is formed.
Since a DNA fragment is formed from the NA fragment and a DNA fragment is also formed from other similar DNAs, and a similar reaction continues in a chain reaction, the DNA fragment is amplified by this method.

The D amplified by the above random PCR
The NA fragments are subjected to gel electrophoresis to classify the different microorganisms. Then, by analyzing the band (band) of the electrophoresis image, the presence state of the microorganism can be estimated. In addition, a DNA fragment is amplified by applying the above-mentioned random PCR method to microorganisms collected at a later time, and the amplified DNA fragment is similarly subjected to gel electrophoresis. Then, by analyzing the change in the state of the band in the electrophoretic image, it is possible to estimate the change in the state of the microorganism over time.

By applying the above-described DNA fragment amplification method to a plurality of microorganisms collected from the processing tank of the garbage disposer or from the soil, the presence state of the microorganisms in the processing tank of the garbage disposer or in the soil can be determined. Can be estimated. Also,
By examining the state of microorganisms in the processing tank of the garbage disposal machine or in the soil that was similarly collected at a later time, changes in the state of microorganisms over time in the processing tank of the garbage processing machine or in the soil can be determined. Can be estimated. Further, it is possible to estimate the decomposition state of the organic matter in the process of decomposing the organic matter in the garbage.

By changing the conditions of the processing tank of the garbage disposer according to the results, garbage can be satisfactorily treated and a good fertilizer can be produced.

[0054]

EXAMPLE Good primers for use in the DNA fragment amplification method according to the present invention were selected by the following method.

Primers and Standard Samples (Samples) As primers to be examined, Nippon Gene 216
Different types of DNA Oligomer were used. To select primers, chromosomal DNAs of seven bacteria shown in Table 1 were used as standard samples.

[0056]

[Table 1]

As shown in Table 1, typical bacteria are Escherichia coli K12 strain (E. coli) and Bacillus.
subtilis natto I2 strain (natto) was used. As the other five bacteria, bacteria that decompose garbage in the treatment tank of the garbage disposal machine were used. For these five bacteria,
The numbers No. 10, No. 30, No. 38, No. 46 and No. 103 are given respectively.

Operating method of garbage disposal machine SNS- manufactured by Sanyo Electric Co., Ltd.
Using T1 (external dimensions: 580 x 450 x 795 mm), an improvement was made to connect an air pump and an air flow controller to a drain port attached to the garbage disposal machine.
The garbage disposer was installed in a prefabricated laboratory at a temperature of 30 ° C. and a relative humidity of 60%.

25 kg of wood chips (water content 70%) as a processing carrier in the processing tank of the garbage processing machine (average particle size 1.
5 mm cedar). 1 kg of garbage consisting of 450 g of vegetables, 300 g of fruits, 40 g of fish, 30 g of meat, and 180 g of cooked rice is put into the treatment tank once a day five times a week,
After putting the garbage, the inside of the processing tank was stirred by the stirring blades of the garbage processing machine.

The water content of the wood chips in the treatment tank is 35 to 4
A good processing condition was maintained by adjusting to 5%. The water content was finely adjusted by adjusting the amount of ventilation from the air pump with an air amount regulator.

Isolation of Garbage-Treated Bacteria An agar medium for bacterial culture was prepared with the composition shown in Table 2.

[0062]

[Table 2]

About 9 months after the operation of the garbage disposer, 10 g of wood chips in the treatment tank were collected, and 90 mL of sterilized 0.85% saline was added to sufficiently remove bacteria from the wood chips. Until suspended. The suspension was further diluted to 10 ⁻⁶ and 100 μL of the diluted suspension was uniformly inoculated on an agar medium. After culturing at 37 ° C. for 3 days, all the colonies were transferred to a new agar medium to isolate bacteria.

Five bacteria having different colony shapes among the isolated colonies were used as PCR standard samples. The isolated bacteria include No. 10, No. 30, No. 38, No. 46 and No. 103 described above.
Numbered. Bacterial No. 103 showed that Pr
It is known to be oteus mirabilis .

Chromosome DN of bacteria used as standard sample
Preparation method of A Escherichia coli K12 strain and Bacillus subtilis nat
The toI2 strain was cultured for 16 hours in an 18 g / L normal broth medium (Eiken E-MC35). Five bacteria isolated from the garbage disposer were cultured for 16 hours in the medium shown in Table 3.

[0066]

[Table 3]

Bacterial chromosomal DNA is obtained from Current Protocols
in Molecular Biology (publishedby Greene Publishin
g Associates and Wiley-Interscience) 2.4.1-2.
“Preparation of Genomic DNA from Bacte” described in 4.2
ria ”.

Setting of PCR Conditions In the random PCR method used in the present invention, when many DNA fragments are amplified from one kind of microorganism, it becomes difficult to associate each of a plurality of microorganisms with a DNA fragment. Therefore, it is necessary to increase the selectivity of amplification and reduce the number of DNA fragments to be amplified. In order to improve the selectivity, the length of the primer, the magnesium concentration of the reaction solution composition, the annealing temperature of the reaction cycle, and the number of reaction cycles are important factors.

The PCR System 9700 from PE Applied Biosystem
And DNA amplifier MIR-D40 manufactured by Sanyo Electric Co., Ltd.
Was used to set PCR conditions.

The PCR conditions were examined using DNA Oligomer H81 manufactured by Nippon Gene as a primer having a base length of 12 bp. In this study, the composition of the reaction solution was such that the Tris-HCl concentration was 10 mM and the KCl concentration was 5 mM.
0 mM, dATP, dCTP, dGTP and dT
The concentration of TP was 200 μM each. As a result, the optimal value of the magnesium concentration was 1.5 mM and the optimal value of the Taq polymerase concentration was 0.025 unit / μL.
It was found that the optimal value of the cycle annealing temperature was 55 ° C. and the optimal value of the cycle number was 35 cycles. The efficiency of the PCR reaction slightly changes depending on the type of primer and the setting conditions. In consideration of this, in the following primer selection experiment, the annealing temperature was set to 45 ° C. to slightly lower the selectivity.

Table 4 shows the composition of the reaction solution used for random PCR in the following primer selection experiments.

[0072]

[Table 4]

In Table 4, the Tris-HCl Tri
s is an abbreviation for Tris (hydroxymethyl) aminometane. d
NTPmix is dATP (2'-deoxyadenosine-5'-triphosphate), dCTP (2'-deoxycytidine-5'-triphosphate),
dGTP (2'-deoxyguanosine-5'-triphosphate), dTT
It is an equal concentration mixed solution of P (2'-deoxythymidine-5'-triphosphate). The reaction solution volume was 20 μL.

Random P in the primer selection experiment
Table 5 shows the CR cycle.

[0075]

[Table 5]

Primer selection experiment One primer was subjected to seven types of random PCR using the above seven bacteria as standard samples, and 5 μL of the reaction solution after the random PCR was analyzed by 1.5% agarose gel electrophoresis. . The electrophoresis conditions are 3.6 V / c
m constant voltage. After the electrophoresis, the gel was stained with ethidium bromide, and a fluorescent image of ethidium bromide when irradiated with ultraviolet light having a wavelength of 254 nm was taken with an instant film.

Selection of primers The number of bands (bands) observed from the electrophoresis image was counted. Tables 6 to 13 show the 216 types of primers (SEQ ID NOs: 1 to 216) used in the primer selection experiment and the number of bands (total number of bands) observed from the electrophoresis image.

[0078]

[Table 6]

[0079]

[Table 7]

[0080]

[Table 8]

[0081]

[Table 9]

[0082]

[Table 10]

[0083]

[Table 11]

[0084]

[Table 12]

[0085]

[Table 13]

The number of bands differs for each primer,
117. FIG. 4 shows the distribution of the number of primers for every six bands. Many of the primers were biased toward the smaller number of bands.

In the method for amplifying a DNA fragment of the present invention, it is necessary to use a primer having a band that appears as little as possible in an electrophoretic image. FIG. 5 shows the number of primers per band number for primers having 15 or less bands. From FIG. 5, there are 73 primers (SEQ ID NOs: 41 to 113) in the range of 1 to 10 bands (34%),
These 73 primers are considered to be useful in the DNA fragment amplification method of the present invention. There were 40 primers (19%) from which no band was obtained in the electrophoresis image from the seven bacteria.

Although the length of the primer is the same as 12 bp (base pairs), the frequency of band appearance varies greatly depending on the type of primer. This is due to the difference in affinity (affinity) between the primer and the template DNA. One index for determining the affinity is GC content (GC content). The GC content is defined as C based on the total number of four bases A, C, G, and T.
And the ratio of the total number of G.

FIG. 6 shows the number of bands (total number of bands) for GC content of 216 primers. Tables 6 to 13 above also show the GC content of each primer. As shown in FIG. 6, a positive correlation is found between the GC content and the number of bands (correlation coefficient = 0.54).
It was confirmed that the higher the C content, the higher the number of bands.

However, even for the same GC content, the number of bands was widely dispersed, and it was also found that the appearance frequency of the band of the electrophoretic image was not determined only by the GC content.
This indicates that the affinity for the template DNA greatly differs depending on the base sequence of the primer. Therefore, in order to find primers having a desired amplification probability, it is necessary to confirm individual primers by random PCR.

In the present example, a primer which gave a clear amplification result in an electrophoretic image was selected as a good quality primer. Specifically, 51 primers having 1 to 10 bands and a clear band appeared in the electrophoresis image were selected as good quality primers.

The sequence numbers of the high quality primers are 41 to 4
3, 45 to 54, 56 to 58, 60 to 64, 66 to 6
9, 71-78, 80-82, 84-86, 89-9
1, 93, 95 to 97, 100, 103, 106, 10
7 and 109. In addition, these good quality primers are shown by * mark in Table 7 and Table 8.

FIGS. 7 to 32 show the electrophoresis images of the high quality primers. 7 to 32, M is a molecular weight marker (λ / Hind III and φx174 / Hae III), and 1 to 7 are electrophoresis images corresponding to the seven bacteria shown in Table 1.
8 is an electrophoresis image without bacteria (control experiment).

As shown in FIGS. 7 to 32, when the random PCR method is applied using the selected good quality primers,
1 to 9 clear bands appear in the electrophoresis image. Therefore, by analyzing this electrophoretic image, the presence state of the microorganism can be easily estimated.

Further, it is possible to easily estimate the presence state of the microorganisms collected in the processing tank of the garbage processing machine or in the soil, and to detect the presence of microorganisms over time in the processing tank of the garbage processing machine or in the soil. Changes in state can also be estimated.
Further, it is possible to estimate the decomposition state of the organic matter in the process of decomposing the organic matter in the garbage.

Next, a method for detecting bacteria using the DNA fragment obtained by the random PCR method of the present example as a DNA probe will be described. The DNA fragment amplified by the random PCR method is separated by electrophoresis, and the DNA
After extracting and purifying the fragment, it is cloned into a vector cut into blunt ends (blunt ends) by treatment with a restriction enzyme. The DNA thus obtained can be used as a DNA probe for a colony hybridization method, an in situ hybridization method, a southern hybridization method, or the like, and a bacterium serving as a base of the amplified DNA fragment can be found.

In particular, it is considered that many soil bacteria are difficult to isolate, but the random PCR method of the present invention can detect a plurality of bacteria present in soil. Then, by using the above-mentioned hybridization method, the presence of bacteria can be confirmed, which can be useful for finding culture conditions for isolation.

[0098]

[Sequence list]

 SEQ ID NO: Length of one array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ACTGAGAAAA TA 12 SEQ ID NO: 2 Array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ATCTTCAAAG AT12 SEQ ID NO: 3 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ACAAAGAGAT AT 12 SEQ ID NO: 4 Array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acid Synthetic DNA sequence GAGGTGATAT TA 12 SEQ ID NO: 5 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ATCTTCTCAT CT 12 SEQ ID NO: 6 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ACTCTTCTAC AA 12 SEQ ID NO: 7 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AGAGACATAG TT 12 SEQ ID NO: 8 Array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GCCAGATATA TA 12 SEQ ID NO: 9 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CACACTACTT AT 12 SEQ ID NO: 10 Array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ATGAGAAAGG AA 12 SEQ ID NO: 11 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ATCAACACTT TC 12 SEQ ID NO: 12 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GAGACTACAA TA 12 SEQ ID NO: 13 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CCATACATAT TG 12 SEQ ID NO: 14 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GATACTGATG AT12 SEQ ID NO: 15 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AGATTCTTAC TG 12 SEQ ID NO: 16 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AGCCCTTATT TA 12 SEQ ID NO: 17 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GAGACTATGA AA 12 SEQ ID NO: 18 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ATCAAGTATC CA 12 SEQ ID NO: 19 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ACTGACCTAG TT 12 SEQ ID NO: 20 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ATTTGGATAG GG 12 SEQ ID NO: 21 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GACTGCTATA CA 12 SEQ ID NO: 22 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TGGTACGGTA TA 12 SEQ ID NO: 23 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TACTACTGTG GA 12 SEQ ID NO: 24 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TGGCTGTAGA AA 12 SEQ ID NO: 25 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TCTACACGAA GT 12 SEQ ID NO: 26 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CCAGATTTTC TG 12 SEQ ID NO: 27 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ATCAACGTAC GT 12 SEQ ID NO: 28 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TTCCGTAATC AC 12 SEQ ID NO: 29 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GCTTACATAG AC 12 SEQ ID NO: 30 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ACTCCAAATG TG 12 SEQ ID NO: 31 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AAGTCGTTTG GG 12 SEQ ID NO: 32 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CTAGTATGGG AC12 SEQ ID NO: 33 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GAGTTCGAAC GA 12 SEQ ID NO: 34 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ACTTTCCTAC GG 12 SEQ ID NO: 35 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TTCCCGTCTA TC 12 SEQ ID NO: 36 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AGCCTTACGG CA 12 SEQ ID NO: 37 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GCTATGGCAA CG 12 SEQ ID NO: 38 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CCTTGGAACT CG 12 SEQ ID NO: 39 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TCTGCTGACC GG 12 SEQ ID NO: 40 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGCAACTGGC CA 12 SEQ ID NO: 41 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GTCATTAAAG CT 12 SEQ ID NO: 42 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TACACTTTTG AC 12 SEQ ID NO: 43 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TGGATCTTTG AC 12 SEQ ID NO: 44 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GCTCTTTTGG AA 12 SEQ ID NO: 45 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ATCATCGTAC GT 12 SEQ ID NO: 46 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TACGATATGG CT 12 SEQ ID NO: 47 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GATCCAGTCT TT 12 SEQ ID NO: 48 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AGAATGTCCG TA 12 SEQ ID NO: 49 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CCGCAGTTAG AT 12 SEQ ID NO: 50 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CAGTGGGAGT TT 12 SEQ ID NO: 51 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TCTATGGACC CT 12 SEQ ID NO: 52 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TTCATTCTGG GG 12 SEQ ID NO: 53 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CCGTCTTTTC TG 12 SEQ ID NO: 54 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TGGCCTATTG GC 12 SEQ ID NO: 55 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acid Synthetic DNA sequence GTCATGCCTG GA 12 SEQ ID NO: 56 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CCTTGGCGAA GC 12 SEQ ID NO: 57 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AGCCTGTGGG CT 12 SEQ ID NO: 58 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AGAATTGGAC GA 12 SEQ ID NO: 59 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CTTGTCATGT GT 12 SEQ ID NO: 60 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acid Synthetic DNA sequence TACGTGGTAA CA 12 SEQ ID NO: 61 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGACAAGTAA TG 12 SEQ ID NO: 62 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TACCCTCAAG CT 12 SEQ ID NO: 63 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TTCGAGGATC GA 12 SEQ ID NO: 64 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GACCTGCGAT CT 12 SEQ ID NO: 65 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TGGCCTCTTG GA 12 SEQ ID NO: 66 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGTTTCCCAG GA 12 SEQ ID NO: 67 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TCGTCCGGAG AT 12 SEQ ID NO: 68 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CGCTTCGTAG CA 12 SEQ ID NO: 69 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGCTTCGAAT CG 12 SEQ ID NO: 70 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GATGAGCTAA AA 12 SEQ ID NO: 71 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GAGCAGGAAT AT12 SEQ ID NO: 72 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CTTAGGTTAC GT 12 SEQ ID NO: 73 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GTGGATCTGA AT12 SEQ ID NO: 74 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CCTATCCCAA CA 12 SEQ ID NO: 75 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GAGACTACCG AA 12 SEQ ID NO: 76 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GACAGCGTCC TA 12 SEQ ID NO: 77 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CTTGAGCGTA TT 12 SEQ ID NO: 78 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ACTGAGATAG CA 12 SEQ ID NO: 79 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GAGAGACGAT TA 12 SEQ ID NO: 80 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GACGCCCATT AT 12 SEQ ID NO: 81 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GACGGTTCTA CA 12 SEQ ID NO: 82 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GATGGTCCGT TT 12 SEQ ID NO: 83 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TTCACCAACG AG 12 SEQ ID NO: 84 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CAGGTGTGGG TT 12 SEQ ID NO: 85 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ATTGGTGCAG AA 12 SEQ ID NO: 86 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AAGGCGTGTT TA 12 SEQ ID NO: 87 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TATTGGGATT GG 12 SEQ ID NO: 88 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AACATCTCCG GG 12 SEQ ID NO: 89 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ATCATTGGCG AA 12 SEQ ID NO: 90 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TTGAGTAGTT GC 12 SEQ ID NO: 91 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TACGCCGGAA TA 12 SEQ ID NO: 92 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CCTGAGGTAG CT 12 SEQ ID NO: 93 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GCCGCTTCAG CT 12 SEQ ID NO: 94 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ATCTAAACCA CG 12 SEQ ID NO: 95 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AGAGCTGAAG TA 12 SEQ ID NO: 96 Sequence length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGTGAGGATT CA 12 SEQ ID NO: 97 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CTCAAGCGTA CA 12 SEQ ID NO: 98 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CCTTTCCGAC GT 12 SEQ ID NO: 99 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TCCTTCGAGC AG 12 SEQ ID NO: 100 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CAGGCCGAAG TC 12 SEQ ID NO: Length of 101 array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AAGCCTATAC CA 12 SEQ ID NO: 102 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CGACGATATG AT 12 SEQ ID NO: 103 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GCCCTTTTGG AC12 SEQ ID NO: 104 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ACTTTCGATC CA 12 SEQ ID NO: 105 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AGCACTGAAT CT 12 SEQ ID NO: 106 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GCCATCGAAA AA 12 SEQ ID NO: 107 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GAGTACACGA AG 12 SEQ ID NO: 108 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CTTGAGGGAT GG 12 SEQ ID NO: 109 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GCCTGCCTCA CG12 SEQ ID NO: 110 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AGAGGTGTAA AT12 SEQ ID NO: 111 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AAGCTGCAGC AA 12 SEQ ID NO: 112 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GCCTTCGTTA CG 12 SEQ ID NO: 113 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AGGGCTCTAG GC 12 SEQ ID NO: 114 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TTGCATAATC GT 12 SEQ ID NO: 115 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGCAGATATC AT 12 SEQ ID NO: 116 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TCCAAGCTAC CA 12 SEQ ID NO: 117 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TAACAACCGA GC 12 SEQ ID NO: 118 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CACAAGGAAC AT12 SEQ ID NO: 119 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TCGGTGGGAA TA 12 SEQ ID NO: 120 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ATGGAGCAGG AA 12 SEQ ID NO: 121 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGTTCGGGAA TG 12 SEQ ID NO: 122 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GAGCTCCCGA CA 12 SEQ ID NO: 123 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ACTAACCTGG AC 12 SEQ ID NO: 124 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGCGTGGTTG TA 12 SEQ ID NO: 125 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGCGAGGGAG GA 12 SEQ ID NO: 126 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GCCGCCAGAG GA 12 SEQ ID NO: 127 Array Length: 12 array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TGACACACTG TC 12 SEQ ID NO: 128 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TGCACTACAA CA 12 SEQ ID NO: 129 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CACTTCAACC AG 12 SEQ ID NO: 130 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TATCCACCGC TC 12 SEQ ID NO: 131 Sequence length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TGCCCACTAC GG 12 SEQ ID NO: 132 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GAGACTGCTG AT 12 SEQ ID NO: 133 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CAGGTGGGAC CA 12 SEQ ID NO: 134 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TCCTGGGGCG TT 12 SEQ ID NO: 135 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGCAAGGGAT AT 12 SEQ ID NO: 136 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GCATTGCAAT CG 12 SEQ ID NO: 137 Sequence length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GTTTTGTCAC CG 12 SEQ ID NO: 138 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGATCCGACG GC 12 SEQ ID NO: 139 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ATGACTGTGC GA 12 SEQ ID NO: 140 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GCGTCGGTTC GA 12 SEQ ID NO: 141 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CTCCTGCTGT TG 12 SEQ ID NO: 142 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ACTGAGGGGG GA 12 SEQ ID NO: 143 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AAGGACACAA CA 12 SEQ ID NO: 144 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ACGGGTCGTA AC 12 SEQ ID NO: 145 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GTCGGACGTC CA 12 SEQ ID NO: 146 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ACTGAGCAAC AA 12 SEQ ID NO: 147 Sequence length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CACACTCGTC AT 12 SEQ ID NO: 148 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GTCGCCTTAC CA 12 SEQ ID NO: 149 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ATCGCGGAAT AT12 SEQ ID NO: 150 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AGACCTGCTT CT 12 SEQ ID NO: 151 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TTGCCGGGAC CA 12 SEQ ID NO: 152 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGCGGTTATG AA 12 SEQ ID NO: 153 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GTGACCGATC CA 12 SEQ ID NO: 154 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GCTGGCGTAT CT 12 SEQ ID NO: 155 Sequence length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGACCTCCAT CG 12 SEQ ID NO: 156 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CCGAGGGCTG TA 12 SEQ ID NO: 157 Sequence length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CCTGCGGGAG GA 12 SEQ ID NO: 158 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AAGTGGTGGT AT 12 SEQ ID NO: 159 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TATCCTACCG GC 12 SEQ ID NO: 160 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AGGCACCCTT CG 12 SEQ ID NO: 161 Sequence length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GTCGACGGAC GT 12 SEQ ID NO: 162 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GAGGAGAAAC GG 12 SEQ ID NO: 163 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GCTGGATTCG CA 12 SEQ ID NO: 164 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGTCACCGAT CC 12 SEQ ID NO: 165 Sequence length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ATCGCGGCTT AT 12 SEQ ID NO: 166 Sequence length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ACCATCAAAC GG 12 SEQ ID NO: 167 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGCCGACTTG GC 12 SEQ ID NO: 168 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GAGTGGCAAC GT 12 SEQ ID NO: 169 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CCGCAGGGAC CA 12 SEQ ID NO: 170 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGTCAGGAAC AA 12 SEQ ID NO: 171 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GTCGGTCGTG AA 12 SEQ ID NO: 172 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GTGCAATTTG GC 12 SEQ ID NO: 173 Sequence length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ACTCACCACG CA 12 SEQ ID NO: 174 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TGGCTTCATC AC 12 SEQ ID NO: 175 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CCGTGGAATG AC 12 SEQ ID NO: 176 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGAGGATGGC CC 12 SEQ ID NO: 177 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CCTTGGCATC GG 12 SEQ ID NO: 178 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GTTAGCCCCA AT12 SEQ ID NO: 179 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGCATGGCCT TT 12 SEQ ID NO: 180 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGTGACGATG CA 12 SEQ ID NO: 181 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ACTGGCCGGC AT 12 SEQ ID NO: 182 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AAGCTGGGGG GA 12 SEQ ID NO: 183 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ATGGCTACTG GC 12 SEQ ID NO: 184 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGTGCCGGAG CA 12 SEQ ID NO: 185 Sequence length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CTCAGCGATA CG 12 SEQ ID NO: 186 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGTGGTGGTA TC 12 SEQ ID NO: 187 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GTCGACGCAT CA 12 SEQ ID NO: 188 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GACGGTTCAA GC 12 SEQ ID NO: 189 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AAGCTGTGGG CT 12 SEQ ID NO: 190 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GCGGAGGAAC CA 12 SEQ ID NO: 191 Sequence length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CCAGGAGGTG GT 12 SEQ ID NO: 192 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AGCGCGGCAA AA 12 SEQ ID NO: 193 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ACCACTCCCG CA 12 SEQ ID NO: 194 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGCGGCACAG GA 12 SEQ ID NO: 195 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GCCCCGTTAG CA 12 SEQ ID NO: 196 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AAGGCGCGAA CG12 SEQ ID NO: 197 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGTGACTGGT GG 12 SEQ ID NO: 198 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CGCAGCCGAG AT12 SEQ ID NO: 199 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AAGAAGCAGG CG12 SEQ ID NO: 200 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GTGTGGAAGC CA 12 SEQ ID NO: 201 Length of array: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GATGGATTTG GG 12 SEQ ID NO: 202 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGTCAGGCAC CA 12 SEQ ID NO: 203 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TGCCTCGCAC CA 12 SEQ ID NO: 204 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence AGCAGCGCCT CA 12 SEQ ID NO: 205 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GCCAGCTGTA CG 12 SEQ ID NO: 206 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGAGGTCGAC CA 12 SEQ ID NO: 207 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence TTCGGACGAA TA 12 SEQ ID NO: 208 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGAGAGCGGA CG12 SEQ ID NO: 209 Sequence Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GGCGATTCTG CA 12 SEQ ID NO: 210 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GTGGGTGGAC AA 12 SEQ ID NO: 211 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GTGCACGTAT GG 12 SEQ ID NO: 212 Array Length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence CGACGACGAC GA 12 SEQ ID NO: 213 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ACTGGCCGAG GG 12 SEQ ID NO: 214 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence GCGGTCAGCA CA 12 SEQ ID NO: 215 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ATCAGCGCAC CA 12 SEQ ID NO: 216 array length: 12 Array types: Number of nucleic acid strands: Type of single-stranded sequence: Other nucleic acids Synthetic DNA sequence ATGGCCGGTG GG 12

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a process during a first cycle in a random PCR method.

FIG. 2 shows one of the second cycles in the random PCR method.
It is a schematic diagram which shows the process in the 2nd cycle.

FIG. 3 shows the second cycle of the random PCR method.
It is a schematic diagram which shows the process in the 2nd cycle.

FIG. 4 is a diagram showing the distribution of the number of primers for every six bands.

FIG. 5 is a diagram showing the number of primers per band number for primers having 15 or less bands.

FIG. 6 is a diagram showing the number of bands for GC content of 216 primers.

FIG. 7 is a view showing an electrophoresis image of a good quality primer.

FIG. 8 is a view showing an electrophoresis image of a good quality primer.

FIG. 9 is a view showing an electrophoresis image of a good quality primer.

FIG. 10 is a view showing an electrophoresis image of a good quality primer.

FIG. 11 is a view showing an electrophoresis image of a good quality primer.

FIG. 12 is a view showing an electrophoretic image of a good quality primer.

FIG. 13 is a view showing an electrophoresis image of a good quality primer.

FIG. 14 is a view showing an electrophoresis image of a good quality primer.

FIG. 15 is a view showing an electrophoresis image of a good quality primer.

FIG. 16 is a view showing an electrophoresis image of a good quality primer.

FIG. 17 is a view showing an electrophoretic image of a good quality primer.

FIG. 18 is a view showing an electrophoresis image of a good quality primer.

FIG. 19 is a view showing an electrophoresis image of a good quality primer.

FIG. 20 is a view showing an electrophoresis image of a good quality primer.

FIG. 21 is a view showing an electrophoresis image of a good quality primer.

FIG. 22 is a view showing an electrophoresis image of a good quality primer.

FIG. 23 is a view showing an electrophoresis image of a good quality primer.

FIG. 24 is a view showing an electrophoresis image of a good quality primer.

FIG. 25 is a view showing an electrophoresis image of a good quality primer.

FIG. 26 is a view showing an electrophoresis image of a good quality primer.

FIG. 27 is a view showing an electrophoresis image of a good quality primer.

FIG. 28 is a view showing an electrophoresis image of a good quality primer.

FIG. 29 is a view showing an electrophoresis image of a good quality primer.

FIG. 30 is a view showing an electrophoresis image of a good quality primer.

FIG. 31 is a view showing an electrophoresis image of a good quality primer.

FIG. 32 is a view showing an electrophoresis image of a good quality primer.

[Explanation of symbols]

 1 DNA 2a, 2b, 2c, 2d, 2e Single strand 3a, 3b, 3c, 3d, 3e Double strand 11a, 11b, 11c Primer

Claims (7)

[Claims] 1. SEQ ID NOS: 41-43, 45-54, 56
~ 58, 60 ~ 64, 66 ~ 69, 71 ~ 78, 80 ~
82, 84-86, 89-91, 93, 95-97, 1
A polymerase chain reaction method in which a heat denaturation step, a primer annealing step and a polymerase extension reaction step are repeatedly performed in this order on a plurality of different DNAs using any one of primers 00, 103, 106, 107 and 109. A method for amplifying a DNA fragment, comprising amplifying DNA fragments of a plurality of different DNAs by applying them at a time. 2. The polymerase chain reaction conditions are set such that a band corresponding to at least one of the plurality of different DNAs appears in an electrophoretic image of a DNA fragment amplified by applying the polymerase chain reaction. The method for amplifying a DNA fragment according to claim 1, wherein: 3. A DNA polymerase of a plurality of different DNAs by applying a polymerase chain reaction method in which a heat denaturation step, a primer annealing step, and an extension reaction step with a polymerase are repeatedly performed in this order on a plurality of different DNAs at a time. When amplifying the fragment, the primer and polymerase chain reaction conditions are set so that the number of bands corresponding to each DNA appearing in the electrophoresis image of the DNA fragment amplified by application of the polymerase chain reaction method is several or less. A method for amplifying a DNA fragment, comprising: 4. A method for estimating the presence of microorganisms in a subject, the method comprising the step of estimating the presence of microorganisms in a subject, comprising: Method,
A method for estimating the presence of microorganisms, comprising: amplifying DNA fragments of DNAs of a plurality of different microorganisms; classifying the amplified DNA fragments by an identification method; and estimating the presence of microorganisms in the object. 5. The method according to claim 4, wherein the identification method is an electrophoresis method. 6. An organic waste state estimation method for estimating an organic waste state in an organic matter decomposition process in which organic matter in an organic waste in which a plurality of microorganisms are mixed is decomposed by the microorganism. A method for amplifying DNA fragments according to claim 1, 2 or 3 for DNAs of a plurality of microorganisms in a system waste,
After amplifying the DNA fragments of the DNAs of a plurality of different microorganisms, the amplified DNA fragments are classified by an identification method, the state of microorganisms in the organic waste is estimated, and the state of the organic waste is estimated from the estimation result. An organic waste state estimating method characterized by estimating. 7. The method according to claim 6, wherein the identification method is an electrophoresis method.