JP7040562B2 - Bacterial flora analysis method and device for bacterial flora analysis - Google Patents

Description

The present invention relates to a device for analyzing a bacterial flora and a method for analyzing a bacterial flora using the device.

The identification and quantification results of bacterial species contained in the sample are useful as an index for evaluating the environment, the health condition of animals and plants, etc., and there are various application examples including infectious disease diagnosis today.
As such an evaluation method, there is a method of actually observing the bacteria contained in the sample individually with a microscope or the like, identifying the bacterial species from the morphology, etc., and directly counting the number of each bacterium. .. In some cases, the identification efficiency may be improved by screening the bacteria using a selective medium or the like.

Further, there are a method of detecting a protein synthesized by a bacterium by using an antibody specific to the protein, and a method of detecting a metabolite by a mass spectrometer.
The most rapid and widely used techniques are molecular biological nucleic acid amplification techniques such as PCR and LAMP method, and signal amplification techniques such as invader method. Methods for selectively amplifying and detecting nucleic acids having a specific sequence of a detection target bacterium by these methods are widely used (Patent Documents 1 and 2).

On the other hand, many studies have been conducted with the aim of applying the technology for comprehensive analysis of bacterial groups contained in samples, so-called bacterial flora, in fields such as environmental evaluation and diagnosis. For example, the nucleic acid is non-selectively amplified based on the nucleic acid sequence common to the bacteria to be detected, and then fragment analysis is performed by restriction enzyme treatment, cloning is performed and the sequence is determined by a sequencer or the like, or the amplification is performed. Identification by hybridization with a specific sequence in the sequence may be mentioned. For these purposes, an electrophoresis device or a high-throughput device such as a DNA chip may be used (Patent Documents 3 and 4).

Japanese Unexamined Patent Publication No. 2004-57059 JP-A-2007-244349A Japanese Unexamined Patent Publication No. 2004-290171 Japanese Patent No. 5139620

Bacteria present in the environment, such as intestinal bacteria, indigenous bacteria on the skin, oral bacteria, bacteria contained in soil, bacteria contained in activated sludge, etc., are affected by various types of bacteria while interacting with each other. It also has a great influence on the environment in which it exists.
When analyzing these bacterial flora, the method using culture takes time, and there is a possibility that only some bacteria composed of many kinds of bacteria can be detected. It is also possible that the flora differs before and after culturing. The method using antigen-antibody reaction or mass spectrometry can be detected because it is difficult to apply if there are bacteria without specific antibodies or bacteria for which proteins and metabolites characteristic of the bacteria are not clear in advance. The types of bacteria are limited.

For example, in order to collectively evaluate various types of bacteria contained in the flora, the nucleic acid contained in the flora is collectively amplified by an amplification means such as PCR, and the specificity of hybridization with a probe is based on a DNA chip or the like. Although it is possible to select individual bacteria and evaluate them qualitatively, even if it is an exhaustive array, considering the possibility of unknown bacteria in the flora, as a whole Quantitative evaluation of the amount of bacteria It was difficult to evaluate. In addition, it was difficult to evaluate quantitatively because the hybridization efficiency of the probe was different for each individual.

Therefore, an object of the present invention is to provide a bacterial flora analysis method for quantitatively evaluating a bacterial flora and a device used for the method.

As a result of diligent research to solve the above problems, the present inventor has made a probe consisting of a nucleic acid that hybridizes to 16S rRNA specific to each of the bacteria, a total amount index probe, and /.
Alternatively, they have found that the bacterial flora can be analyzed by using an absolute amount index probe, and have completed the present invention.

That is, the present invention is a device equipped with the following probe (a) and at least one of the probes (b) and (c).
(A) Probe consisting of nucleic acid that hybridizes to 16S rRNA specific to each of one or more types of bacteria to be detected (b) Total amount index probe (c) One or more types of absolute amount index probe

In the device of the present invention, the probe (b) is one or two to be detected as described above.
It is preferable that it contains a base sequence common to more than one species of bacteria. Further, the probe (c) is a mixture of a predetermined external addition control 1 and at least one type of external control 2 different from the external addition control 1, and is the external addition control 1.
It is preferable that the externally added control 2 is contained in the mixture in which the external addition control 2 is mixed at a concentration ratio of 2 to the nth root (n is an arbitrary integer).

Here, examples of the bacteria to be detected include intestinal bacteria, indigenous bacteria on the skin, and oral bacteria.
Examples of intestinal bacteria include Lactobacillus and Streptococ.
Included are at least one of the genus us, the genus Vionella, the genus Bacteroides, the genus Eubacterium, the genus Bifidobacterium, and the genus Clostridium.
Examples of indigenous bacteria on the skin include Propionibacterium and Sta.
At least one of the bacteria of the genus phylococcus can be mentioned.
Examples of oral bacteria include Porphyromonas, Tannerella, Treponema, Campylobacter, and Fusobacterium.
Genus, Parabimonas, Streptococcus, Aggregataba
Genus cter, Capnocytophaga, Eikenella, Actinom
Genus yces, genus Veillonella, genus Serenomonas, lactobac
Genus illus, Pseudomonas, Haemophilus, Klebsie
Included are at least one of the bacteria of the genera lla, Serratia, Moraxella, and Candida.
Further, as the oral bacteria, more specifically, for example, Porphyromonas g.
ingivalis, Tannerella forsythia, Treponema
detentola, Campylobacter gracilis, Campyl
obactor rectus, Campylobacter showae, Fuso
Bacteria nucleatum subsp. Vincentii, Fus
obacterium nucleatum subsp. polymorphum,
Fusobacterium nucleatum subsp. animalis,
Fusobacterium nucleatum subsp. nucleatum
, Fusobacterium periodonicum, Parvimonas
micra, Prevotella intermedia, Prevotella n
igrescens, Streptococcus constellatus, Agg
regatibacter actinomycetemcomitans, Campy
lobacter concisus, Capnocytophaga gingiva
lis, Capnocytophaga ochracea, Capnocytophaga
ga sputigna, Eikenella corrodons, Strepto
coccus gordonii, Streptococcus intermediau
s, Streptococcus mitis, Streptococcus mitis
s bv 2, Actinomyces odorolyticus, Veillon
ella parvula, Actinomyces naeslundii II, S
At least one of elenomonas noxia and Streptococcus mutans can be mentioned.

Further, in the device of the present invention, the probe (a) preferably has any of the following sequences.
(A) A sequence substantially the same as the complementary sequence (c) (a) or (b) of at least two sequences (b) and (a) selected from the base sequences shown in SEQ ID NOs: 3 to 59. The device of the present invention can be a fiber type microarray.

Furthermore, the present invention is a method for estimating the absolute amount of bacterial species to be detected, including the following steps.
(1) A step of calculating a coefficient from the signal intensity ratio of the probe for detecting the detection target bacterium for each of the detection target bacteria isolated in advance (2) The calculated value calculated in the above step (1). Step of calculating the number of copies of each detection target bacterial species in the data obtained from the test sample using the hybridization efficiency coefficient of the probe (3) After the calculation calculated in the step (2) above. The process of comparing the signal strength of the absolute quantity index probe with the signal strength of the absolute quantity index probe.

By using a device equipped with a bacterial species-specific probe and one or both of a total amount index probe and an absolute amount index probe in the sequence of the amplification product, a plurality of bacteria in the flora can be aggregated at once. Quantification is possible. In addition, the total amount of bacteria contained in the sample can be quantified.

Hereinafter, the present invention will be described in detail. The scope of the invention is not bound by these explanations.
Other than the following examples, the present invention can be appropriately modified and implemented as long as the gist of the present invention is not impaired. In addition, this specification includes the whole specification of Japanese Patent Application No. 2014-098719 (filed on May 12, 2014) which is the basis of the priority claim of the present application. In addition, all publications cited herein, such as prior art documents, and publications, patent gazettes and other patent documents, are incorporated herein by reference.

The method for estimating the absolute amount of the bacterial species to be detected (absolute amount estimation method) of the present invention includes the following steps.
(1) A step of calculating a coefficient from the signal intensity ratio of the probe for detecting the detection target bacterium for each of the detection target bacteria isolated in advance (2) The calculated value calculated in the above step (1). Step of calculating the number of copies of each detection target bacterial species in the data obtained from the test sample using the hybridization efficiency coefficient of the probe (3) After the calculation calculated in the step (2) above. The process of comparing the signal strength of the absolute quantity index probe with the signal strength of the absolute quantity index probe.

Here, the device that can be used for the absolute quantity estimation method of the present invention is
It is a device equipped with the following probe (a) and at least one of the probes (b) and (c).
(A) A probe consisting of nucleic acid that hybridizes to 16S rRNA specific to each of a plurality of types of bacteria to be detected (b) Total amount index probe (c) One or more types of absolute amount index probe

Any type of device can be used as long as the device probe is an array type device that is fixed so that it can be located independently and can be located. The type of the array is not particularly limited, but for the purpose of stably acquiring the signal with the total amount index probe described later, an array having a larger amount of probe immobilization than a probe simply immobilized on a flat substrate is available. Are suitable. As such an array, a DNA microarray is preferable. Especially,
Examples thereof include a fibrous DNA microarray that three-dimensionally immobilizes a probe via a gel. The form of the support of the device is not particularly limited, and any form such as a flat plate, a rod, or beads can be used. When a flat plate is used as a support, predetermined probes can be fixed to the flat plate at predetermined intervals for each type (spotting method, etc.;
See Science 270, 467-470 (1995), etc.). Further, predetermined probes can be sequentially synthesized for each type at a specific position on a flat plate (see Photolithography method, etc .; Science 251, 767-773 (1991), etc.).

Other preferred support forms include those using hollow fibers. When using hollow fibers as a support, a device obtained by fixing an oligonucleotide probe to each hollow fiber for each type, focusing and fixing all the hollow fibers, and then repeating cutting in the longitudinal direction of the fibers is obtained. It can be preferably exemplified. This device can be described as a type in which an oligonucleotide probe is immobilized on a through-hole substrate (Patent No. 35108).
See Publication No. 82, etc., FIG. 1).

The method of immobilizing the probe on the support is not particularly limited, and any binding mode may be used.
Further, the present invention is not limited to direct immobilization on the support, and for example, the support may be previously coated with a polymer such as polylysine, and the probe may be immobilized on the treated support.
Further, when a tubular body such as a hollow fiber is used as the support, the tubular body can hold a gel-like substance, and the probe can be immobilized on the gel-like substance.

Probes In the present specification, nucleic acids derived from bacteria are captured by a hybridization reaction, and the captured nucleic acids are measured by fluorescence, chemiluminescence, coloring, RI, etc. to determine the presence or absence of bacteria and to measure the amount. Called detection.
The probe captures the nucleic acid of the bacterium to be detected by hybridization, and oligo DNA composed of a complementary sequence having a sequence specific to the nucleic acid to be captured is often used, but the nucleic acid to be captured is the nucleic acid to be captured. It may be DNA or RNA as long as it hybridizes with a specific sequence. It may contain PNA, LNA and the like. The cDNA may be immobilized depending on the application.

Generally, when a device (for example, a DNA microarray) on which many types of probes are collectively mounted is used, since the various types of probes are subjected to the hybridization reaction under the same conditions, the hybridization efficiency between the probes should be uniform. Is important. For this purpose, the probe needs to adjust the GC content and the sequence length at the time of design to make the Tm value as constant as possible. The type and number of probes mounted on the device are not particularly limited.

The bacterium-specific probe (the probe (a)) contains a base sequence specific to the type of bacterium in the base sequence serving as a template or the base sequence of the probe.

When hybridizing a sample to a device (eg, a DNA microarray), the PC is often used to improve detection sensitivity and add a detectable label.
Nucleic acid amplification methods such as R and LAMP methods are applied. In the amplification reaction, it is important to amplify the range required for hybridization with the device in the subsequent step, and it is desirable not to amplify the portion not required for hybridization as much as possible from the viewpoint of reagent cost and the like.

Since the above-mentioned PCR method amplifies only the range sandwiched between the primer pairs, it is suitable for performing the amplification reaction after excluding the sequences unnecessary for hybridization. Here "pair"
Is the minimum set of primers required for the amplification reaction, and the type may differ depending on the method of the amplification reaction. In the case of general PCR, a primer pair is composed of two types of oligo DNA, a forward primer and a reverse primer.

The specific primer means that the sequence to be amplified is limited, and the primer pair does not necessarily have to be one pair. If necessary, a multiplex method using two or more pairs of primers can also be applied. For example, a primer pair for bacterial amplification (SEQ ID NOs: 1 and 2) and a primer pair for external addition control (SEQ ID NOs: 91 and 92) can be used.

When performing bacterial flora analysis using a device as described above (for example, a DNA microarray), the application is not particularly limited. For health condition inspection / diagnostic applications, intestinal bacterial flora, oral bacterial flora, indigenous bacteria on the skin, etc. can be considered. In addition, if it is used for environmental inspection, it can be used for bacterial flora in soil, activated sludge in water treatment, and the like.

Among these, when evaluating the intestinal bacterial flora, for example, Lactobacillus
Genus, Streptococcus, Veionella, Bacteroides, Eubacterium, Bifidobacterium, and Clostri
Bacteria of the genus dium can be used as the species to be detected.

When assessing indigenous bacteria on the skin, for example, Propionibacterium genus bacteria (eg, Propionibacterium acnes), and Staphyl.
Bacteria belonging to the genus ococcus can be used as the species to be detected.

When assessing the oral bacterial plexus, for example, Porphyromonas, Tanne
Genus rella, genus Treponema, genus Campylobacter, genus Fusobac
Genus terium, genus Parvimonas, genus Streptococcus, Aggre
Genus gatibacter, genus Capnocytophaga, genus Eikenella, A
Genus ctinomyces, genus Veillonella, genus Serenomonas, La
Genus ctobacillus, genus Pseudomonas, genus Haemophilus, K
Genus lebsiella, genus Serratia, genus Moraxella, and Candid
Bacteria of the genus a can be detected as a target bacterial species. More specifically, for example, Porphyromonas g, which is currently thought to be associated with periodontal disease, tooth decay and opportunistic infections.
ingivalis, Tannerella forsythia, Treponema
detentola, Prevotella intermedia, Aggrega
tibacter actinomycetemcomitans, Fusobacte
rium nucleatum subsp. Vincentii, Fusobact
erium nucleatum subsp. polymorphum, Fusob
acterium nucleatum subsp. animalis, Fusob
acterium nucleatum subsp. nucleatum, Stre
ptococcus mutans, Streptococcus salivariu
s, Streptococcus sanguis, Streptococcus mi
ris, Actinomyces viscosus, Lactobacillus g
asseri, Lactobacillus phage, Lactobacillus
casei, Lactobacillus delbruecchii subsp.
bulgaricus, Streptococcus aureus, Pseudomo
nas aeruginosa, Streptococcus pyogenes, St
reptococcus pneumoniae, Haemophilus influ
enzae, Klebsiella pneumoniae, Serratia mar
cesces, Serratia macces, Moraxella cat
arrhalis, Candida albicans, Campylobacter
gracilis, Campylobacter rectus, Campylobacter
ter shower, Fusobacterium periodonticum, P
arvimonas micra, Prevotella nigrescens, St
reptococcus constellatus, Campylobacter c
oncisus, Capnocytophaga gingivalis, Capnoc
ytophaga ochracea, Capnocytophaga sputige
na, Eikenella corrodons, Streptococcus gor
donii, Streptococcus intermedius, Streptoc
occus mitis bv 2, Actinomyces odonictric
us, Veillonella parvula, Actinomyces naesl
Bacteria such as undii II and Serenomonas noxia are preferably the species to be detected, and more preferably Porphyromonas gingiva.
lis, Tannerella forsythia, and Treponema den
Ticola, particularly preferably Porphyromonas gingival
is.

For these, for example, when amplifying using the primers (SEQ ID NOs: 1 and 2) shown in Table 1 described later, the sequences shown in SEQ ID NOs: 3 to 59 can be used as a probe. It is preferable to use at least two sequences selected from the base sequences shown in SEQ ID NOs: 3 to 59. It may be a complementary sequence of at least two sequences selected from the base sequences shown in SEQ ID NOs: 3 to 59, and is substantially the same sequence as at least two sequences selected from the base sequences shown in SEQ ID NOs: 3 to 59. Alternatively, the sequence may be substantially the same as the complementary sequence of at least two sequences selected from the base sequences shown in SEQ ID NOs: 3 to 59.

Here, substantially the same is long enough to specifically hybridize to the sequences set forth in SEQ ID NOs: 3 to 59 under stringent conditions.
The length of the probe is, for example, a sequence of 15 bases or more, preferably 17 bases or more, and more preferably 20 bases or more. Here, the stringent condition means a condition in which a specific hybrid is formed and a non-specific hybrid is not formed. That is, a pair of polynucleotides having high homology (homology or identity of 95% or more, preferably 96% or more, more preferably 97% or more, still more preferably 98% or more, most preferably 99% or more). The condition for hybridization. More specifically, such conditions are adopted in methods well known and commonly used in the art, such as Northern blotting, dot blotting, colony hybridization, plaque hybridization, Southern blot hybridization, and the like. Conditions can be set. Specifically, 0.7 to 1 M of N using a polynucleotide-immobilized membrane.
After hybridization at 65 ° C. in the presence of aCl, SSC at a concentration of 0.1 to 2 times.
(Saline Sodium Citrate; 150 mM sodium chloride, 15 m
This can be achieved by washing the membrane at 65 ° C. with a solution of M sodium citrate).

Under these conditions, it can be expected that polynucleotides having higher homology as the temperature is raised can be efficiently obtained. However, multiple factors such as temperature and salt concentration can be considered as factors that affect the stringency of hybridization, and those skilled in the art can realize the same stringency by appropriately selecting these factors. ..

Total amount index probe (probe (b))
No matter how many types of probes are mounted on a device (eg, DNA microarray), there is a high possibility that unidentified bacteria will be mixed in the test sample, and some of the amplified nucleic acids are specific. There is a nucleic acid derived from a non-detection target bacterium (referred to as "non-detection target bacterium") that does not carry a specific probe.
Therefore, it is difficult to detect all bacteria as detection target bacteria, and in analyzing the bacterial flora, the proportion of the detection target bacteria in the entire bacterial flora including the non-detection target bacteria. It is also extremely important to evaluate the total amount of bacteria from the viewpoint of how much bacteria are present in the sample in the first place.

Bacteria to be undetected are (i) bacteria whose identity is known but do not have to be detected, and (ii).
) It can be understood as the sum of bacteria whose existence and type are unknown. On the other hand, since the existence of (ii) is unknown and the specific sequence is unknown, it is not always possible to actually detect it. Therefore, among (ii), the bacteria that could be amplified are all the detection target bacteria including the non-detection target bacteria. Since the amplified fungus always has a complementary sequence to the primer sequence, it can be hybridized to a probe having the same sequence as the primer.

Therefore, in the present invention, all the detection target bacteria mean bacteria that can be amplified by a specific primer pair.
When evaluating the total amount of such bacteria, for example, it is possible to measure the number of bacteria independently of the device (DNA microarray). In this case, it is advantageous to mount a probe, which is an index of the total amount of bacteria, in the device from the viewpoint of improving the convenience of operation. As for the probe, a base sequence common to many kinds of bacterial species may be used from the base sequences amplified by the primer pair. If such a sequence cannot be found, a plurality of relatively common sequences may be designed and comprehensively judged to serve as a total amount index probe. The total amount index probe is preferably a probe that hybridizes to a nucleic acid derived from a bacterium contained in a test sample, specifically, a plurality of types to be detected among the base sequences amplified by the specific primer pair. It is a probe containing a base sequence common to bacteria.

Since the total amount index represents the total amount of amplification products specific to each bacterial species, the amount is generally large, so that the signal intensity reaches a plateau (a signal exceeding the permissible range of the detectable signal intensity is output). ).

In order to prevent such a situation, it is possible to take measures by lowering the hybridization efficiency as compared with a probe that captures an amplification product specific to each bacterial species. When performing PCR, two types of oligo DNA are used as one primer pair, one of which is fluorescently labeled, and the other is basically unlabeled. For example, if the same unlabeled oligo DNA is used as a probe, all the amplified PCR products will hybridize, and this hybridization signal is measured. When designing a probe, for example, the Tm value of the probe is lowered. Specifically, it is conceivable to reduce the GC content or shorten the probe sequence length itself.

Further, at the time of hybridization, it is possible to reduce the signal intensity by adding a nucleic acid that acts competitively with the hybridization between the amplified nucleic acid and the total amount index probe. Examples of such nucleic acids include nucleic acids having (partially) the same sequence as the total amount index probe, nucleic acids having (partially) the complementary sequence of the total amount index probe, and the like.

On the other hand, it is also possible to use the same or partially the same sequence as the primer pair as the total amount index probe. For example, when the forward primer is fluorescently labeled, it is possible to evaluate the total amount of the amplification product by using a probe containing all or a part of the sequence constituting the reverse primer.

When the forward primer is fluorescently labeled, the strand on the side extended from the forward primer is subjected to hybridization to detect a signal. In this case, as above
There is a concern that the signal intensity will peak due to the large number of amplification products to be captured by the probe, but the effect can be suppressed by adjusting the probe length and GC content in the same manner as above. ..

Hybridization efficiency coefficient In the present specification, the hybridization efficiency coefficient is calculated from the signal intensity of the probe in each bacterial species. For probes that form hybridizations to multiple bacteria, the average value is used as the hybridization efficiency coefficient.

External addition control In the present specification, the external addition control is the DN of bacteria contained in the test sample.
It means a nucleic acid artificially designed so that A is not amplified. A nucleic acid that is added in a fixed amount to a sample before an amplification reaction or a hybridization reaction. The external addition control is a nucleic acid in which the amplification reaction is surely carried out by performing a normal amplification reaction, and serves as a so-called positive control. Therefore, if a probe specific to the external addition control is mounted on a device (for example, a DNA microarray), it is possible to evaluate whether the amplification reaction, hybridization, or the like is appropriately performed from the detection result.

In addition, if multiple external addition controls are designed and a mixture with different concentrations is used, the bacteria contained in the sample and the total amount thereof can be quantified by associating the detection result with respect to the concentration, for example, the fluorescence intensity. Is possible.
If the external addition control is added before the amplification reaction, the nucleic acid is amplified by the above specific primer pair, that is, it has a base sequence complementary to the primer pair, and it is detected by hybridization. In order to do so, it is necessary to possess a base sequence that neither the detection target bacterium nor the non-detection target bacterium possesses.

Design of external addition control For example, RNDBETWEE of Microsoft software "EXCEL"
Using the N function, X integers from 1 to 4 are randomly generated (X is an arbitrary number), and they are connected to form an X-digit number consisting only of numbers 1 to 4. A, 2 to T, 3
By replacing C and 4 with G, a large number of random sequences of ATGC based on X bases can be obtained. For these sequences, only the sequences in which the sum of G and T is the same as the sum of A and T are extracted, and the extracted sequences are referred to the database such as GenBank of NCBI.
It can be designed by searching t, selecting those having few similar sequences, and adding primer sequences to both ends of the sequence. It is also possible to connect the designed sequences as appropriate to make them longer, or to partially remove them to make them shorter. When quantifying the bacteria to be detected using a plurality of external addition controls, in order to make the reaction efficiency at the time of the amplification reaction as constant as possible, it is necessary to make sure that there is no large difference from the base length amplified by the bacteria to be detected. desirable. For example, if the amplification product of the bacteria to be detected is about 500 bp, the amplification product of the external addition control is 3.
It is desirable to set it from 00 bp to 1000 bp. On the other hand, when confirming the amplification chain length by electrophoresis or the like after amplification, the amplification product derived from the externally added control is used as the detection target bacterium after designing the amplification product to have a length different from that of the detection target bacterium. It is also possible to detect at a position different from that of the band and confirm the success or failure of the amplification reaction before hybridization.

Further, when a plurality of external addition controls are used, it is desirable that each external addition control is composed of the same base sequence except for the sequence captured by the probe and its vicinity.

When the amplification reaction is carried out by PCR or the like after adding an external addition control One or more kinds of absolute amount index probes (the probe (c)) are probes amplified using a specific primer pair. It is a probe that does not hybridize to any of the nucleic acids derived from bacteria contained in the test sample, but hybridizes to the externally added control nucleic acid prepared as a control.
The absolute amount index probe is contained in a mixture of a predetermined external addition control 1 and at least one kind of external addition control 2 different from the external addition control 1. In this case, the external addition control 2 is mixed with the external addition control 1 at a concentration ratio of 2 to the nth root (n is an arbitrary integer). This is because PCR itself has a 2n-fold amplification product amount after n cycles. There are two or more types of external addition controls, but it is preferably 50 or less.

Considering that the dynamic range of the device ⁽ for example, DNA microarray) is about 104, if 15 types of concentration steps are prepared in 2 ⁿ times steps, 2 ¹⁵ > 10
Since it is ⁴ , it can be a sufficient index within the dynamic range of the device (for example, DNA microarray). However, when a plurality of external addition controls are prepared for each of the 15 concentration steps, it is possible to prepare more types of external addition controls. Therefore, for example, three types of external addition controls are prepared at each concentration step. It is desirable to mix about 45 kinds of external addition controls in a concentration step of 2 to the nth power in consideration of such cases.

In the present invention, when a plurality of types of absolute amount index probes are used, a plurality of types of externally added control nucleic acids are amplified and a plurality of types of externally added controls are detected by each probe. When using k-type (No. 1 to No. k) external addition controls, the concentration ratio of No. k-1 seen from No. 1 and the concentration ratio of No. k seen from No. 1 are 2 n. It can be expressed as a power (n is an arbitrary integer).

For example, let n be 3 when the number is 2 as seen from the number 1, and n when the number is 3 as seen from the number 1.
Assuming that 3 is also set, the concentration ratio of No. 2 seen from No. 1 and the concentration ratio of No. 3 seen from No. 1 are both 2.
3 = 8 times, and the same concentration ratio can be obtained. Further, another concentration ratio can be set by setting n at the time of No. 2 viewed from No. 1 to 2 and n at the time of No. 3 viewed from No. 1 to 3.
That is, a plurality of types of absolute quantity index probes may be used at the same concentration or at different concentrations.

Basically, a plurality of types of absolute quantity index probes may be inserted in any combination.
In the case of 15 types, 7 types of concentration 1 and 8 types of concentration 2 (concentration ratio to concentration 1; the same applies hereinafter) may be added, 7 types of concentration 1 and concentration 4 You may put 8 kinds of things. Two types with a concentration of 1, three types with a concentration of 4, and one type with a concentration of 8 ・
・ ・ It may be. As in the examples described later, one type has a concentration of 1, one type has a concentration of 2, one type has a concentration of 4, one type has a concentration of 8, and one type has a concentration of 15. But it may be.

In order to draw a calibration curve, it is necessary that the concentration has two or more steps, and it is sufficient that the two steps have a concentration ratio of 2 to the nth root (n is an integer).
On the other hand, if the concentration of the externally added control contained in the sample is too high, the competition between the bacteria to be detected and the amplification reaction becomes fierce, and the bacteria to be detected that should be originally detectable may not be detected. It is necessary to adjust the concentration appropriately according to the above.

In order to detect a plurality of externally added controls by hybridization, it is necessary to individually mount a complementary sequence of a sequence specific to each of the plurality of externally added controls on a device (for example, a DNA microarray) as a probe.
When analyzing the presence or absence and amount of bacterial species to be detected contained in the bacterial flora of an unknown sample (actual sample) using the above device (DNA microarray), each probe for the bacterial species to be detected contained in the above device By processing with a coefficient corrected based on the signal of the absolute amount index probe and the signal intensity of the total amount index probe, it becomes possible to absolutely quantify the nucleic acid derived from the detection target bacterium.

Method for estimating the absolute amount of the bacterial species to be detected It cannot be denied that there is an unknown bacterium in the bacterial flora to be detected. Therefore, the device (eg, D)
It is difficult to detect all bacterial species using NA microarray). Bacteria suitable for detection are targeted when evaluating a phenomenon from previously identified bacteria. For example, there are hundreds of types of oral bacteria, and many have been identified. However, when evaluating periodontal disease, these identified bacteria are not always suitable as detection targets.

Therefore, when assessing periodontal disease, it is present in the periodontal pocket, eg, Porph.
hiromonas gingivalis, Tannerella forsythia
, Treponema denticola, Prevotella intermed
ia, Aggregatibacter actinomycetemcomitans
, Fusobacterium nucleatum subsp. Vincenti
i, Fusobacterium nucleatum subsp. polymor
phum, Fusobacterium nucleatum subsp. anim
alis, Fusobacterium nucleatum subsp. nucl
It can be said that eatum and the like are suitable bacteria for evaluating periodontal disease. More preferably, Porphyromonas gingivalis, Tannerella fo
rsysia and Treponema denticola are particularly preferred.
The hiromonas gingivalis is suitable for assessing periodontal disease.

For each of these bacteria, a sequence specific to the bacterium is selected as a probe from the base sequences amplified by the specific primer pair. That is, since the base sequences amplified by a specific primer pair include those that can be used as probes and those that cannot be used, the probes that can be used are selected. Basically, one type of primer pair is used to amplify many types of bacteria, and a probe is prepared in the amplified sequence where the sequence diversity is recognized depending on the bacterial species.

A probe having the same action and effect as a sequence that can be used as an original probe can also be used as a probe having substantially the same sequence.
Therefore, these detection target bacteria need to have a nucleic acid sequence amplified by the above specific primer pair, and in order to detect the bacterium based on these nucleic acids, in the amplified sequence, It must contain a strain-specific sequence that is different from other bacteria.

On the other hand, the specificity may be one that collectively detects bacteria of the same genus based on the specificity at the genus level, or may be a specificity that can be detected at the individual species level, and the flora. It is possible to make an appropriate judgment according to the purpose of the analysis.
When amplifying multiple bacteria at once and detecting based on a specific sequence, it is necessary to design the primer pair from the sequence conserved among the bacterial species, and the template (the range amplified by the primer). ), It is necessary to use a gene having a bacterial-specific sequence. Examples of such a template gene include 16S rRNA. In the present invention, 16S rRNA itself may be a detection target, or genomic DNA as a transcription source.
It is also possible to detect 16S rRNA in the above.

For example, first, the bacteria to be detected are isolated. At this time, those already sold as strains may be used. In addition, the bacterium may be alive or dead, and may be genomic DNA extracted from the bacterium rather than the bacterium itself.

(1) For each of the previously isolated detection target bacteria, a step of calculating a coefficient from the signal intensity ratio of the probe for detecting the detection target bacterium. The present invention relates to one of these isolated bacteria or nucleic acids. Process by the device of. As a result, a signal A detected by the bacterium-specific probe and a signal B detected by the total amount index probe are obtained.

Separately, the device of the present invention is similarly treated with an isolated bacterium or nucleic acid different from the above. As a result, the signal C detected by the bacterium-specific probe and
It is assumed that the signal D detected by the total amount index probe is obtained.

(2) The value calculated in the step (1) above is used as the hybridization efficiency coefficient of the probe, and the number of copies of each detection target bacterial species in the data obtained from the test sample is calculated using the hybridization efficiency coefficient. Step Here, since the signals B and D are signals obtained from the total amount index probe of the same sequence, it can be said that the efficiency of hybridization is theoretically the same. On the other hand, although the probe sequences themselves are different between signal A and signal B, the nucleic acids to be detected are the same, and the amount and experimental conditions subjected to hybridization are also the same. The same can be said for signal C and signal D.

Therefore, the intensity ratio of signal A and signal C when the values of signal B and signal D match is the difference in the hybridization efficiency of each probe. Therefore, A / B
The value of and the value of C / D are values that can be compared after correcting for the difference in hybridization efficiency in each probe. For example, when data is acquired by a DNA microarray using an actual sample, the reciprocal of these individual values acquired in advance is multiplied by the signal intensity of each probe to achieve hybridization efficiency between the probes. It is possible to compare the signal intensities after correcting.

(3) Step of comparing the signal intensity after the calculation calculated in the above step (2) with the signal intensity of the absolute quantity index probe By comparing these signal intensities with the signal intensity of the absolute quantity index probe, each It is possible to evaluate the absolute amount of bacterial species.
In this step, it is possible to calculate a more accurate absolute amount value by comparing the signal intensities after correcting the difference in hybridization efficiency with the external addition control whose number of molecules to be detected is known. .. That is, since the number of molecules of the external control is known, the signal intensity of the probe specific to each strain can be more accurately measured by the calibration curve created by the correspondence between the signal intensity and the number of molecules of the added external control. It becomes possible to convert.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited to these examples.

<Making devices and primers>
16SrRNA sequence data was downloaded from NCBI, and oligo DNA synthesis (Life Technologies) was performed using the two highly conserved sequences among the bacterial species as the forward primer (SEQ ID NO: 1) and the reverse primer (SEQ ID NO: 2) shown in Table 1.
Company) I went. At this time, the forward primer was fluorescently labeled with Cy5. For these primers, the forward primer was prepared at a concentration of 50 pmol / μL, and the reverse primer was prepared at a concentration of 10 pmol / μL.

Bacteria in the mouth to be detected (Porphyromonas gingivalis (P.
g. ), Tannerella forsythia (TF), Treponema
detentola (T.d.), Campylobacter gracilis (
C. gr. ), Campylobacter rectus (C.r.), Campyl
obactor showae (C. sh.), Fusobacterium nucl
eatum subsp. Vincentii (Fnv), Fusobacter
ium nucleatum subsp. polymorphum (F.n.p),
Fusobacterium nucleatum subsp. animalis (
F. n. a), Fusobacterium nucleatum subsp. nu
clearum (F.n.a), Fusobacterium periodontic
um (F.p.), Parvimonas micro (P.m.), Prevotel
la intermedia (P.i.), Prevotella nigressen
s (P.n.), Streptococcus constellatus (S.c.)
, Aggregatibacter actinomycetemcomitans (A)
.. a. ), Campylobacter concisus (C.c.), Capnoc
ytophaga gingivalis (C. gi.), Capnocytophag
a ochracea (C.o.), Capnocytophaga sputien
a (C.sp.), Eikenella corrodons (E.c.), Strep
tococcus gordonii (S.g.), Streptococcus in
Termidius (S.i.), Streptococcus mitis (S.m.).
), Streptococcus mitis bv 2 (S. mb.), Actino
myces odontolyticus (A.o.), Veillonella pa
rvula (V.p.), Actinomyces naeslundii II (A.
n. ), Serenomonas noxia (S.n.)) Based on the genomic DNA of each, a 20-base sequence was designed while shifting the range amplified by the above primers by one base.

Next, from the above-designed base sequences, those having an approximate GC content of about 50% were extracted, and all of these sequences were homologized using the NCBI BLAST program and the 16S rRNA sequences for all bacterial species as a masking database. A sex search was performed to extract the two sequences with the highest sequence specificity. The extracted sequences were compared between bacterial species, and 1 to 2 bases were added before and after the sequence for those having a higher GC content than others, and 1 to 2 bases were deleted for those with a lower GC content.

In order to use the obtained sequence as a probe, 5-terminal vinyl chloride oligo DNA was synthesized (SEQ ID NOs: 3-59). Separately, among the base sequences amplified by the specific primer pair, a 5-terminal vinyl chloride oligo DNA having a base sequence common to a plurality of types of bacteria to be detected.
Was synthesized (SEQ ID NO: 60).
Furthermore, 15 types of probes (SEQ ID NOs: 61 to 75) consisting of sequences not contained in 16S rRNA were prepared, and 5-terminal vinyl chloride oligo DNA was also synthesized for these probes.

All of these types of 5-terminal vinyl chloride oligo DNA were used and the probes were arrayed using the platform of the Mitsubishi Rayon DNA chip Genopearl. Table 2 shows all the designed probe sequences.

<Preparation of sample>
Next, the thermal cycler was turned on and the program was set as shown in Table 3.

The rubber gloves, the laboratory table, the pipetman, the tube rack, etc. were cleaned with rubbing alcohol and Kimwipe / Kim towel, etc., and the tube rack was placed on ice. Evaluation samples, primers (2), Ex Taq, external addition control, and nuclease-free water were placed on the tube rack. Reagents were dissolved, vortexed and spun down as needed. Eppen tube for making premix, Eppen tube for primer dilution,
An externally added control dilution tube and an Eppen tube (0.2 mL, for the number of samples) for carrying out the reaction were placed on a tube rack. Primers were diluted to 10 pmol / μL and placed in the primer diluting Eppen tube.

<Preparation of Premix for PCR>
A sample number x 6 μL of water was placed in an Eppen tube. A 100,000-fold diluted external addition control was added in the number of samples × 1 μL. 10 pmol / μL primer (reverse) sample size × 1 μL was added. 50 pmol / μL primer (forward) sample size × 1 μL was added. 2x Taq was added in the number of samples × 10 μL. The mixture that was stirred with a vortex and spun down was designated as Premix.

<Preparation of reaction solution>
19 μL of Premix was dispensed into a reaction Eppen tube (0.2 mL), and 1 μL of the sample shown in the table below was placed in the reaction Eppen tube. Vortex agitate and
It was spun down to prepare a reaction solution.
The sample used was a freeze-dried strain or genomic DNA purchased from ATCC. The samples used are shown in Table 4. Genomic DNA was adjusted to 10 ng / μL, and strains were dissolved in 300 μL of water to Dneasy Blo.
DNA was extracted using od & Tissue Kit (QIAGEN). The concentration was measured in the required range from 0.01 to 10 pg.

<PCR>
The reaction solution was set in the thermal cycler, the lid of the thermal cycler was closed tightly, and the start button was pressed to start the reaction.

<Preparation for hybridization>
The thermal cycler was switched on and the program [95 ° C. 5 min (leaving at 4 ° C. for any time if necessary) reaction solution volume: 100 μL, LampSpeed: Max] was set. Ice was put in a small container (large enough to hold 96 wellplates), and pure water was put in the gaps between the ice so that water could spread.

<Preparation of Premix for hybridization>
A 1.5 mL Eppen tube or an 8 mL conical tube was prepared. The tube was filled with clean water x 1.1 x 64 μL. 1M Tris-H on the tube
Cl was added in the number of samples × 1.1 × 48 μL. Number of samples of 1M NaCl in the tube ×
1.1 × 48 μL was added. 0.5% Tween 20 in a tube Number of samples x 1.1 x
20 μL was added. It was mixed well with a vortex and spun down as needed to give Premix for hybridization.

<Preparation of hybridization solution>
180 μL of Premix for hybridization was dispensed into a sample solution (0.2 mL Eppen tube containing PCR product). It was vortexed and spun down to a hybridization solution.

<Hybridization pretreatment>
The hybridization solution was set in a thermal cycler, the lid was tightly closed, and heating was started. Immediately after heating at 95 ° C for 5 minutes, open the lid, take out the sample tube inside with the plastic rack, and immerse it in a small container containing ice water.
It was left for 2 minutes. The sample tube was then removed from the ice water and placed in a rack on ice.

<Hybridization>
After confirming that the temperature of the air incubator was 50 ° C., the entire amount (200 μl) of the hybridization solution was placed in the hybrid chamber. It was immersed in the Genopearl hybrid chamber prepared above. The hybrid chamber was covered with a lid, placed in an air incubator in a sealed state, and left in a light-shielded state for 2 hours.

<Washing>
After 2 hours, the hybrid chamber was removed from the air incubator and the lid was opened. The conical tube containing the 0.24 M TNT buffer was removed from the water incubator and rotated with the conical tube covered once to remove condensation on the lid, and then the lid was opened. Remove the tip from the hybrid chamber with tweezers and
The bottom surface was brought into contact with Kimwipe or the like to absorb and remove the excess hybrid solution. The chips were placed in a conical tube, covered again, immersed in a water incubator and heated for 20 minutes. After 20 minutes, the chips were transferred to another conical tube containing the same buffer and heated for another 20 minutes in the same manner as above. After 20 minutes, the chips were then transferred to a conical tube containing 0.24 MTN buffer and heated for an additional 10 minutes in the same manner as above. After 10 minutes had passed, the chips were transferred to an 8 mL conical tube containing the same buffer by the same method as described above, and the mixture was rotated and stirred for 10 minutes or more using a rotary stand.

<Detection operation>
The chip was taken out from the conical tube and set in a detection case for Genopal Reader (manufactured by Mitsubishi Rayon).
Using a Genopal Reader (manufactured by Mitsubishi Rayon), the DNA microarray was imaged under the following exposure times of 40 seconds, 4 seconds, 1 second, and 0.1 seconds according to the device instruction manual, and the detected fluorescence signal was detected. The numerical data converted based on the above was used for the analysis.

<Analysis>
Using commercially available spreadsheet software, information on sample names, samples, chips, experimental conditions, and signal intensity values were pasted on a personal computer.
The average value of multiple background spots (spots on which the probe is not mounted) excluding outliers was used as the background value. The standard deviation value at that time was set as the minimum signal value.
The background value was subtracted from the signal value of the probe spot to calculate the net signal value.

If the net signal value was lower than the minimum signal value, they were replaced with the minimum signal value.
The results are shown in Table 5 (Tables 5-1 to 5-5; Tables 5 in which Tables 5-2 to 5-5 are connected in order to the left end side of Table 5-1 are shown in Table 5). Enclosures in the data indicate signals from bacterial species-specific hybridization. Although cross-hybridization was partially observed, strong signals were detected in the probe for detecting the bacterial species and the total amount index probe in the approximate sample.

Next, the hybridization efficiency coefficient for each probe was calculated from the signal intensity of the probe for each bacterial species. For probes that form hybridizations to multiple bacteria, the average value was used as the hybridization efficiency coefficient, and the results are shown in Table 6. In the portion where the value is not described, the hybridization efficiency coefficient could not be calculated because there was no isolated bacterium or nucleic acid.

<Preparation of external addition control>
SEQ ID NOs: 76 to 76, which can be amplified with a forward primer and a reverse primer, and can be hybridized independently using the probes of SEQ ID NOs: 61 to 75, respectively.
The double-stranded DNA described in 90 was synthesized. The synthesized sequence is shown in Table 7.

<From PCR to data acquisition>
DNA was extracted from 500 μL of saliva of one person to be inspected, which was publicly recruited in-house instead of the strain or genomic DNA solution, using Dneasy Blood & Tissue Kit (QIAGEN). The concentration was 14.8 ng / μL, and 1 μL of a 3000-fold diluted solution was used as an actual sample, and 1 μL of a 100,000-fold diluted external addition control was added. Other than that, PCR was performed by the same method as in Example 1 except that the amount of water in the reaction solution was reduced by 1 μL per sample, and then hybridization was performed with a DNA microarray to obtain fluorescence signal data for each probe. .. The results are summarized in Table 8 for probes in which signals above the detection limit were detected.

The results in Table 8 are obtained by calculating the number of copies in each probe using the hybridization efficiency coefficient calculated in Example 1.
In Table 8, the numbers in the "Number of copies" column mean the actual number of bacteria.

The value of the slope of the calibration curve created within the quantification range of the absolute quantity index probe (data excluding the data of the probe that was below the detection limit and the probe whose signal intensity reached a plateau) was calculated. The results are shown in Table 9.

From the above, it has become possible to absolutely quantify the bacteria to be detected contained in the saliva sample of the person to be inspected.

According to the present invention, each of a plurality of bacteria contained in the environment can be quantified at once, and the increase or decrease in the total amount can be evaluated. Therefore, the device of the present invention can be used, for example, for evaluation of the health condition of the intestine, skin, mouth, etc., environmental evaluation of soil, seawater, river, performance evaluation of activated sludge, and the like.

SEQ ID NOs: 1-92: Synthetic nucleic acid

Claims (1)

A device for bacterial plexus analysis equipped with the following probe (a), probe (b), and probe (c) .
(A) Complementary sequence of nucleic acid that hybridizes to 16S rRNA specific to each of two or more bacteria to be detected selected from bacteria contained in intestine, skin, mouth, soil, seawater, river or activated sludge. Probe consisting of nucleic acid
(B) Total amount index probe containing a probe that hybridizes to the base sequence common to the two or more types of bacteria (c) The test sample does not hybridize to the nucleic acid derived from the bacteria contained in the test sample. One or more absolute index probes that hybridize to an artificially designed nucleic acid so that the bacterial DNA contained therein is not amplified.