JP2006325529A - Method for detecting organism species, microarray used therefor and kit for detecting organism species - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting an organism species, with which the organism species is judged positive or false positive. <P>SOLUTION: The method for detecting an organism species comprises preparing a microarray in which spots for a plurality of kinds of detection probes specific to the organism species of a detection target are arranged, preparing the detection target labeled by a first labeled compound from nucleic acids in a test sample, preparing a detection reference labeled by a second labeled compound from the standard sample of the organism species of the detection target, mixing the target with the reference to hybridize the microarray, measuring the label signals of the first and the second labeled compounds in the spots and a judgment process for calculating and assaying a correlation coefficient r in the correlation between the two label signals in the spots, judging the organism species positive when a correlation is confirmed at a fixed significant level and the value of the correlation coefficient r exceeds a fixed value and judging the organism species false positive in a case except that. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a biological species detection method, a microarray used therefor, and a biological species detection kit.

  As a method for rapidly detecting a specific species, a method using PCR and a DNA probe has been conventionally performed. In this method, for example, first of all, a probe having a sequence specific to the biological species among the genomic base sequences of a specific biological species is prepared, and then complementation of the probe is performed by PCR from a nucleic acid contained in a test sample. A target containing a sequence and labeled with a detectable labeling compound is prepared, and the probe and the target are hybridized to measure the label signal of the target hybridized with the probe to detect the species. It is a method to do. In this method, a microarray (also referred to as a DNA chip) in which DNA probes are immobilized on a substrate is used, and it is possible to detect a plurality of types of biological species by one hybridization. .

  As a problem of the above-mentioned species detection method, for example, there is a false positive problem. That is, even though the genome base sequences of many biological species are being determined, it can be said that most of the genome base sequences remain unclear at this stage when viewed from all the species on the earth. Then, for example, even if a target signal is detected for a probe of a certain biological species when the detection method is attempted on a sample outside the laboratory, the signal may be a false positive due to cross-hybridization. It always exists as a technical limit. As a method for solving this problem, for example, a competitive hybridization method (see Non-Patent Documents 1 and 2) and a method using a blocking agent containing a modified nucleotide (see Patent Document 1) are disclosed. However, even with these methods, there is a problem that it is difficult to eliminate cross-hybridization of a false positive target including a sequence that is identical or highly homologous to the probe sequence.

  As another problem of the above-described species detection method, for example, there is a problem of quantitativeness. That is, as a quantification method using a microarray, for example, a method using an internal standard or an external standard is disclosed (see Patent Document 2), but the cross-hybridization that occurs when a large number of biological species are detected simultaneously is disclosed. The resulting quantitative uncertainty is not resolved.

On the other hand, a method for detecting a large number of biological species is expected to be used, for example, for detecting pollutant-degrading bacteria in a contaminated environment. For example, contamination of groundwater and soil by various organic chlorinated compounds such as tetrachlorethylene (PCE) and trichlorethylene (TCE) is a serious problem common to the world, and is often taken up in detail in the media such as newspapers. There is a strong social demand for technological development to repair environmental pollution caused by these substances. As a biological method (bioremediation) of remediation technology for contaminated environment, a method that enhances the ability to decompose and remove bacteria originally inhabiting contaminated soil and groundwater (biostimulation) and the ability to decompose and remove environmental pollutants There is a method (bioaugmentation) that directly introduces bacteria having a contamination into the contaminated environment. Therefore, in applying bioremediation, if the contaminated environment can be monitored quickly by detecting and quantifying pollutant-degrading species present in the contaminated environment, the contaminated site can be biostimulated, or It can quickly determine whether bioaugmentation has to be applied.
JP 2005-502346 A JP 2004-28695 A Oliveira, R et al. (2002) “Competitive hybridisation on spotted microarrays as a tool to conduct competitive analysis of Xylella festidosa strains EM. Let. 216: 15-21. Abecassis, V.M. et al. (2003) “Microarray-Based Method for Combinatorial Library Sequencing and Charac- terization” BioTechniques 34: 1272-1279.

  An object of this invention is to provide the detection method of the biological species which can determine positive or false positive.

  In order to achieve the above object, a method for detecting a biological species of the present invention is a method for detecting a biological species in a test sample, wherein a plurality of types of detection probe spots specific to the target biological species are detected. From the microarray preparation step of preparing the arranged microarray, the target preparation step of preparing the detection target labeled with the first labeling compound from the nucleic acid in the test sample, and the standard sample of the species to be detected Preparing a reference for detection labeled with a second labeling compound, mixing the target and the reference and hybridizing them to the microarray, and the first and second labeling compounds in the spot A competitive hybridization step for measuring each of the labeled signals, and the two labeled signals in the spot For the correlation, the correlation coefficient r is calculated and tested. If the correlation is affirmed at a predetermined significance level and the value of the correlation coefficient r exceeds a predetermined value, it is determined to be positive. It is the detection method of the biological species including the determination process which determines a case as a false positive.

  The present inventors have conducted extensive research on a method for detecting a species in an environmental sample using a microarray, and focused on the advantage of arranging a plurality of types of detection probe spots for detecting one species of organism. did. Even in the prior art, from the viewpoint of improving the reliability of detection, for example, two to three types of probes were arranged for one type of biological species, but there was no specific method for eliminating false positives. The inventors of the present invention are able to obtain a measured target by paying attention to the correlation of the distribution of labeled signals obtained by competitive hybridization of the target and the reference to a microarray in which more types and numbers of probe spots are arranged. The present invention has been found that it can be determined whether or not the signal is false positive.

  According to the method for detecting a biological species of the present invention, even if the target label signal is strongly measured in some probe spots, the presence or absence of correlation with the reference label signal is tested. By calculating the correlation coefficient r indicating the strength of the positive correlation and comparing it with a predetermined value, it can be determined whether the target label signal is positive or false positive. Therefore, according to the present invention, a method for detecting a species that is simple, quick, and more reliable is possible.

  Further, even when the target label signal is weak, it is possible to determine whether it is positive or false positive based on the correlation with the reference label signal, and to eliminate false positives. As described above, according to the present invention, since positive / false positive can be determined regardless of the strength of the target label signal, a simple and quick method for detecting a species with improved detection sensitivity and reliability is possible. It becomes.

  Furthermore, in competitive hybridization, the target / reference ratio of the labeled signal at each spot reflects the concentration ratio of both, so for example, the slope of the regression line when the target and reference signals are scatter-plotted (regression coefficient) Using this, the target can be quantified. Therefore, according to the method for detecting a species of the present invention, it is possible to easily and quickly quantify the species.

  Furthermore, since the present invention uses a microarray, the species can be detected and quantified at once for a plurality of species.

  If the method for detecting a biological species of the present invention is applied to, for example, detection or quantification of organochlorine-decomposing bacteria present in an environment contaminated with organochlorine compounds such as tetrachloroethylene (PCE) and trichlorethylene (TCE). In addition, it is possible to easily and quickly monitor the type and abundance of bacteria in a contaminated environment with improved reliability.

  The species detection method of the present invention includes a quantification step of calculating the abundance of the species to be detected in the test sample, and the quantification step includes a slope of a regression line (regression of the two labeled signals in the spot). It is preferable to include calculating the coefficient b).

  In the method for detecting a species of the present invention, the microarray preparation step, the target preparation step, and the reference preparation step each arrange a spot of an internal standard probe, and prepare an internal standard target labeled with a first labeling compound. And preparing an internal standard reference labeled with a second labeling compound, wherein the determining step comprises modifying the detection target and the reference label signal based on the signal of the internal standard It is preferable.

  In the microarray of the microarray preparation step, the number of detection probes is three or more for one type of detection target species, and the number of spots of the detection probe is six or more for one type of detection target species. It is preferable that

  In the determination step, it is preferable that the predetermined significance level is in a range of 20% to 0.1%, and the predetermined value of the correlation coefficient is in a range of 0.80 to 0.99.

  In the target preparation step and the reference preparation step, it is preferable that the target and the reference are prepared by a LATE-PCR method.

  In the method for detecting a biological species of the present invention, the detection target biological species may be a plurality of types. Further, the species to be detected may be bacteria, and the detection probe may be a probe derived from the ITS region of the bacteria.

  In the method for detecting a biological species of the present invention, the test sample is collected from an environment contaminated with an organic chlorine compound selected from the group consisting of tetrachlorethylene (PCE), trichlorethylene (TCE), and their dehalogenated products. It may be a sample.

  The microarray of the present invention is a microarray used in the method for detecting a species of the present invention. The biological species detection kit of the present invention includes the microarray of the present invention and is a kit used for the biological species detection method of the present invention.

  In the present invention, the “species” to be detected is not particularly limited, and includes animals, plants, fungi, and specifically, for example, bacteria, fungi, slime molds, unicellular algae, protozoa, viruses, Examples include fungi.

  In the present invention, the “detection probe” is a probe containing a polynucleotide that can specifically hybridize to the genome region of the species to be detected. Although it does not restrict | limit especially as length of the said probe for a detection, For example, it is 10-200 nucleotides, 20-100 nucleotides are preferable, More preferably, it is 40-80 nucleotides. The probe length may be the same or different between the plurality of probes. In general, the shorter the length of a probe, the greater the sequence specificity and the higher the reliability. The polynucleotide of the probe is not particularly limited, and DNA, RNA, or a conventionally known derivative or analog thereof can be used. The method for producing the detection probe is not particularly limited, and for example, it may be produced by chemical synthesis by a conventionally known method, or may be produced enzymatically in vivo or in vitro. In the present invention, since the detection probe is used as a microarray, the detection probe may be modified to be fixed to the substrate of the microarray. As the modification, a conventionally known modification can be selected depending on the type of the microarray substrate to be used, and examples thereof include C (3-7) amination on the 5 'or 3' side of the polynucleotide of the probe.

The sequence of the detection probe may be a conventionally known sequence that is specific to the species to be detected, or may be designed based on the genome sequence of the species. The method for designing the probe sequence is not particularly limited, and for example, a conventionally known algorithm or software can be used. Examples of the software include PRIMOROS ver. 1.1.7 (Kevin E. Ashelford, Andrew J. Weightman andJohn C. Fry (2002) "PRIMROSE: a computer program for generating and estimating the phylogenetic range of 16S rRNA oligonucleotide probes and primers in conjunction with the RDP-II database ”
Nucleic Acids Research, Vol. 30, no. 15 3481-3489) and the like. General preferred requirements in probe design include, for example, GC content (eg 50%), length (eg 40-80 nt), no hairpin structure, the same base, A + T, or G + C in succession No alignment of 5 or more, NCBI-BLAST homology search, no hits that show the same or 90%, 92.5%, or 95% homology, mismatch near the probe center, sense Examples of the sequence include a chain sequence, a terminal portion of a gene, and when there are a plurality of species to be detected at the same time, at least no cross-hybridization is performed between these species. When the species to be detected is a bacterium, the detection probe can use, for example, a sequence derived from 16S rDNA or an ITS region. The ITS region described here refers to a region that receives transcription between 16S ribosomal DNA and 23S ribosomal DNA of the bacterial genome (Internal Transscribed Spacer). Bacterial ribosomal RNA (rRNA) 16SrRNA, 23SrRNA and 5SrRNA are usually transcribed as one transcription unit (operon), and therefore the 16SrRNA gene and the 23SrRNA gene are arranged side by side on the genome. A region between the 16SrRNA gene and the 23SrRNA gene is a region called 16S-23S Internal Transcribed Spacer (ITS).

  In the present invention, “plural types of detection probes” are a plurality of types of probes specific to one type of detection target species. As the number of types, for example, 3 types, 4 types, 5 types, 6 types, 7 types, 8 types, 9 types, 10 types, 11 types, 12 types or more, and 5 types or more are preferable. The more types, the more preferable. This is because the reliability of eliminating false positives in the determination step is improved. Moreover, the spots of the same type of detection probe may be arranged on the same microarray so as to overlap, for example, 2, 3, 4, 5, or more. For example, when there are five types of detection probes for a certain species to be detected and three probes are overlapped, the number of detection probe spots for the species is 15. The number of detection probe spots used for one type of detection target species is, for example, 6, 9, 10, 12, 15, 20, 25, 30, or more. 9 to 60 is preferable, and the larger the number of spots, the more preferable.

  In the present invention, the “microarray in which probe spots are arranged” refers to a microarray including a base material and the probe spots regularly arranged on the surface of the base material. The method for producing the microarray is not particularly limited, and for example, a conventionally known method such as an inkjet method in which a probe solution is finely dropped on a substrate or a method using a photolithography method used in a semiconductor manufacturing technology is used. Can be manufactured. The substrate is not particularly limited, and examples thereof include glass, metal, and plastic.

  In the present invention, a “detection target” is a polynucleotide labeled with a first labeling compound prepared based on a nucleic acid in a test sample. When the species to be detected is present in the test sample, a polynucleotide containing a complementary sequence of the detection probe is prepared as a detection target, hybridized with the probe on the microarray, and labeled positive A signal will be shown. The method for preparing the nucleic acid is not particularly limited, and a conventionally known method can be used. The method for preparing the detection target is not particularly limited, and examples thereof include a random priming method using the genomic DNA of the species to be detected as a template, a PCR method, and other gene amplification methods. Among these, from the viewpoint of quantification in the quantification step, it is preferable that the polynucleotide contained in the detection target is proportional to the concentration in the test sample. Furthermore, from the viewpoint of hybridization efficiency and sensitivity, the detection target preferably occupies more single strands complementary to the detection probe than double strands. In consideration of these, the detection target is preferably prepared by a random priming method or an asymmetric PCR method. The method for labeling the first labeling compound is not particularly limited, and examples thereof include a method in which a primer is labeled in advance and a method in which a labeled nucleotide is used. Therefore, a method using a labeled primer is preferable.

  In the method for detecting a biological species of the present invention, the present inventors used the LATE-PCR method (Linear-After-The-Exponential-PCR; Sanchez et al. (2004) PNAS, 101 (7); 1933-1938). It has been found that when the target and reference are prepared, the labeling signal is increased by about 1000 times compared to the case where the conventional PCR method is used. Furthermore, since the final amplification product of the LATE-PCR method is proportional to the concentration in the test sample, the quantitative property is also good. That is, if it is the detection method of the biological species of this invention using a LATE-PCR method, detection sensitivity, quantitative property, and simplicity can be improved more.

  In the present invention, the “LATE-PCR method” refers to a method obtained by improving the asymmetric PCR method. The asymmetric PCR method is a PCR method mainly intended to produce a single-stranded DNA product by making the molar ratio of a set of primers unequal, for example, 100: 1. The LATE-PCR method, as the name “Linear-After-The-Exponential” indicates, is a PCR method that allows linear amplification after logarithmic amplification, that is, does not become a plateau (Sanchez et al. (2004) PNAS, 101 (7); 1933-1938). The point of the LATE-PCR method is primer design, and the Tm value of the smaller primer is set to be equal to or higher than the Tm value of the excess primer. The calculation of the Tm value is preferably performed by, for example, an adjacent method (Nearest Neighbor Method), and can be calculated by software such as the PRIMOROSE, for example. However, the “LATE-PCR method” in the present invention is not limited to the above method as long as it can be linearly amplified after logarithmic amplification and is mainly intended to generate a single-stranded DNA product. Further improved PCR methods are included.

  In the present invention, the “reference for detection” is a polynucleotide labeled with a second labeling compound prepared from a standard sample of a species to be detected. The preparation method is the same as the detection target except that the standard sample is used instead of the nucleic acid in the test sample, and the second label compound is used instead of the first label compound. Preferably there is. The detection reference is used for competitive hybridization to the microarray together with the detection target. This competitive hybridization not only serves to block the cross-hybridization of the target for detection, but also provides a target / reference ratio of the labeled signal at a plurality of probe spots. False positives that could not be excluded by the hybridization method are excluded, and further, the species to be detected can be quantified. The standard sample of the species to be detected refers to a species to be detected having a known number of cells isolated and cultured, or a nucleic acid prepared therefrom. In the quantification step, as described later, the number of cells of the species to be detected can be quantified from the number of cells of the standard sample corresponding to the reference for detection and the target / reference ratio. When detecting a plurality of biological species, a reference for detection may be prepared by mixing the standard samples, or may be prepared for each biological species to be detected.

  In the present invention, the “label compound” is not particularly limited, and examples thereof include a luminescent label and a radioactive label compound. Examples of the luminescent label include a fluorescent label, a bioluminescent label, a chemiluminescent label, and a colorimetric label, and a fluorescent label compound such as fluorescein, phosphor, rhodamine, and polymethine dye derivative is preferable. Examples of commercially available fluorescent labeling compounds include fluorescence such as fluorprime (FluorePrime, manufactured by Amersham Pharmacia), fluoredite (Fluoredite, manufactured by Millipore), FAM (manufactured by ABI), Cy3 and Cy5 (manufactured by Amersham Pharmacia), and the like. Phosphoramidites are mentioned. The first and second labeling compounds are preferably labeling compounds that can measure the labeling signal independently of each other even if they are mixed in the same probe spot. For example, in the case of a fluorescent labeling compound, it is preferable to select the first and second labeling compounds having different excitation wavelengths and fluorescence wavelengths.

  In the present invention, the “internal standard probe” is for correcting the label signal of the first labeled compound and the second labeled compound, and can be used by being arranged in a microarray together with the detection probe. When competitive hybridization is performed, for example, even if the detection target and the detection reference are combined at a ratio of 1: 1 at a certain spot, the measured label signal may not be 1: 1. . In preparation for such a case, by performing competitive hybridization on the same microarray together with the detection target and the detection reference, a known quantity of the internal standard target and the internal standard reference prepared from the same internal standard sample, The measured label signal can be corrected to improve the quantitativeness. The sequence of the internal standard probe preferably does not cause cross-hybridization with the detection target and detection reference contained in the microarray. For example, it is derived from a biological species that is clearly not present in the test sample such as human or mouse. These sequences can be selected and used. The length and preparation method of the internal standard probe are not particularly limited, and are the same as, for example, the detection probe. The preparation and labeling methods of the internal standard target and the internal standard reference are not particularly limited, and are the same as, for example, the detection target and the detection reference. The type and the number of spots of the internal standard probe in the microarray are not particularly limited, and are, for example, the same type and the number of spots as the detection probe for one type of microorganism to be detected.

  In the present invention, the “test sample” is not particularly limited, and may be, for example, a sample collected from food or a living body in addition to a sample collected from an environment such as soil, groundwater, pond, seawater or the like. Further, as will be described later, the test sample is a sample taken from an environment contaminated with an organic chlorine compound selected from the group consisting of tetrachlorethylene (PCE), trichlorethylene (TCE) and their dehalogenated compounds. Also good.

  In the present invention, “false positive” means that a detection target derived from a biological species that is not the detection target biological species cross-hybridizes with the detection probe, and a labeled signal of the target is observed. In the biological species detection method of the present invention, the step of determining whether the label signal of the detection target is positive or false positive is the determination step.

  Next, the biological species detection method of the present invention will be described.

(Microarray preparation process)
First, in the microarray preparation step, a microarray in which probes for detection with respect to one or a plurality of types of species to be detected and, if necessary, the internal standard probes are arranged on a substrate is prepared. The preparation method, type, and number of spots of the detection probe and internal standard probe are as described above. The microarray may be manufactured before the biological species detection method of the present invention is performed, or the microarray included in the microarray of the present invention or the biological species detection kit of the present invention may be used.

(Target preparation process)
Next, in the target preparation step, a detection target labeled with the first labeling compound is prepared from the nucleic acid in the test sample. Moreover, if necessary, an internal standard target labeled with the first labeling compound is prepared. The method for preparing these targets is as described above. There is a biological species other than the detection target in the test sample and has a sequence that is identical or highly homologous to the detection probe of the detection target biological species, and this sequence is prepared as a detection target. And a false positive signal due to cross-hybridization. In the method for detecting a species of the present invention, the cross hybridization and false positive can be suppressed and excluded by a competitive hybridization step and a determination step described later.

(Reference preparation process)
Simultaneously or separately from the target preparation step, a detection reference labeled with a second labeling compound is prepared from a standard sample of the species to be detected. Further, if necessary, an internal standard reference labeled with a second labeling compound is prepared. The reference preparation method is preferably the same as the target preparation method. As described above, the standard sample is preferably a known number of cells of the species to be detected or a nucleic acid obtained therefrom.

(Competitive hybridization process)
Next, the detection target and reference prepared in the three preparation steps, and the internal standard target and reference, if necessary, are mixed, and the mixture is hybridized to the microarray. Each second label signal is measured. The target and reference contained in the mixture hybridize to each spot in proportion to their concentration, and the distribution of the target and reference label signals in each spot shows a positive correlation, and the label signal The target / reference ratio shows a constant value. By utilizing this relationship, as described later, it becomes possible to eliminate false positives and quantify the species to be detected by correlation analysis or the like. In addition, when utilizing the above-mentioned internal standard, it is preferable to use equal amounts of the internal standard target and the reference. This is because it is easy to calculate the correction value of the label signal if the amount is equal.

  The method and conditions for competitive hybridization are not particularly limited, and can be performed by a conventionally known method. Those skilled in the art can appropriately adjust depending on the type of probe, target, polynucleotide, and the like. As a general hybridization method and conditions, for example, the target and reference are thermally denatured in 5 × SSC + 0.3% SDS, and then added to the microarray, followed by hybridization at 65 ° C. for 4 to 16 hours. Examples include washing with 2 × SSC + 0.1% SDS and 2 × SSC for 5 minutes and rinsing with 0.05 × SSC.

  The method for detecting and measuring the target and reference label signals hybridized with the probe is not particularly limited, and can be measured by a conventionally known method or apparatus according to the type of label. For example, when a fluorescent label is used as the label, it can be automatically detected and measured using a microscope or a scanner. Further, when the internal standard probe is used, for example, as described above, when the internal standard target and the internal standard reference are mixed in equal amounts, the first label obtained by the internal standard is used. The detection target and reference label signals can be corrected so that the signal and the second label signal are equal.

(Judgment process)
Finally, the detection target species is detected by the determination step. That is, in order to evaluate the correlation with respect to the distribution of the labeled signal of the detection target and the detection reference, the correlation coefficient r is calculated and tested, the correlation is affirmed at a predetermined significance level, and the phase If the value of the relationship number r exceeds a predetermined value, it is determined as positive, otherwise it is determined as false positive, and if it is positive, the detection target species is detected. Specifically, the determination step can be performed as follows.

First, the correlation coefficient r is a Pearson product-moment correlation coefficient with respect to two variables x and y: r = Cov (x, y) / S _x S _y (where Cov (x, y) represents the covariance, and S _x and S _y each represent a standard deviation. Further, for example, it can be easily calculated using spreadsheet software such as Microsoft Excel (trade name; manufactured by Microsoft Corporation). The correlation coefficient r takes a value of −1 to 1, and the closer to 1, the stronger the positive correlation. When r is 0, it indicates that there is no correlation between both variables. The positive correlation is, for example, a relationship in which, when a scatter plot is performed with the detection target label signal as the vertical axis and the detection reference label signal as the horizontal axis, the plot is distributed along a straight line. The larger the correlation coefficient r between the two variables, the reference label signal and the target label signal, indicates that there is no cross-hybridization by a target derived from a species other than the detection target species, that is, no false positive. Therefore, if the calculated correlation coefficient r is greater than a predetermined value, it is positive, and if it is equal to or less than the predetermined value, it can be determined that it is false positive. Examples of the predetermined value of the correlation coefficient used for the determination include 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0. 88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98 and 0.99 are preferable, Is 0.95-0.99.

Next, the presence or absence of correlation between the reference label signal and the target label signal is tested. This is because even if the correlation coefficient r exceeds the predetermined value, it may not be said that there is a correlation between the two. In the test, for example, when there is no correlation between the two, the formula: t = | r | (n−2) ^1/2 / (1−r ² ) ^1/2 is a t distribution with n−2 degrees of freedom. Can be performed by t-test using the following. In the above equation, r is the correlation coefficient r, and n is the type of detection probe or the number of spots. If the P value calculated by the t test is less than the predetermined significance level, the correlation between the target and the reference can be recognized at the significance level. Examples of the predetermined significance level include 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.5, 0.4, 0.3, 0.2, 0.1%, preferably 10% or less, more preferably 5 % Or less, and more preferably 1% or less. Then, when the correlation at the predetermined significance level is significant and the correlation coefficient r exceeds the predetermined value, it is determined as positive, and other cases are determined as false positive. The statistical calculation in this determination step can be easily performed with spreadsheet software such as Microsoft Excel (trade name; manufactured by Microsoft Corporation).

Further, in the determination step, the determination coefficient (contribution rate) r ² of the regression line of the scatter plot may be used instead of the correlation coefficient r. The predetermined value when using the determination coefficient r ² is, for example, a value obtained by squaring the predetermined value of the correlation coefficient r. When the determination coefficient is used, the presence or absence of regression may be tested by, for example, F test instead of the t test as the test. Examples of the predetermined significance level include those of the t test. When the regression is significant and the determination coefficient r ² exceeds the predetermined value, it can be determined as positive and the others are determined as false positives.

(Quantitative process)
The species detected and detected as positive in the determination step can be quantified as follows. First, the regression coefficient b, which is the slope of the regression line of the scatter plot, with the detection target label signal on the vertical axis and the reference label signal for detection on the horizontal axis, is calculated. Next, the concentration of the detection target is obtained by multiplying the regression coefficient b by the concentration of the detection reference, and finally, the abundance of the detection target organism in the test sample is calculated. Conventionally, a method using a microarray and a PCR method has a problem that it lacks quantitativeness due to a plateau effect in the PCR method. However, the present inventors have found that in the LATE-PCR method, the concentration ratio is maintained even when competitive hybridization is performed using the final amplification product. An example is shown in FIG. FIG. 1 shows the ratio of labeled signals when competitive hybridization is performed using a target prepared from standard sample DNA corresponding to various cell numbers shown on the horizontal axis and a reference prepared from a certain amount of standard sample DNA ( It is the logarithmic graph which took the target / reference ratio on the vertical axis. As shown in the figure, when the LATE-PCR method is used, the number of cells in the sample used for target preparation is proportional to the target / reference ratio. Therefore, the target sample concentration, that is, the abundance of the detection target organism in the test sample can be quantified from the regression coefficient b and the reference standard sample concentration. For example, when the regression coefficient b is 2, it can be seen that in the test sample used for the detection target, there is a detection species corresponding to twice the standard sample used for the detection reference. When performing this quantification step, it is preferable to correct the first and second labeled signals using an internal standard probe as described above.

  The biological species detection method of the present invention can detect and quantify a plurality of biological species at the same time by using a microarray in which probes for detecting a plurality of types of biological species are arranged on a single substrate. Therefore, the biological species detection method of the present invention, for example, as described later, in the field of environmental remediation of contaminated soil such as organochlorine compounds by bioremediation or the like, rapidly detects the biological species present in the soil. This is useful for monitoring and detection purposes. In addition, for example, in the fields of food production and food distribution, it is useful for applications for detecting and quantifying biological species contaminated with food.

  The microarray of the present invention is a microarray used in the method for detecting a biological species of the present invention, in which spots of the above-described detection probes and internal standard probes are regularly arranged on a substrate. The method for producing the microarray of the present invention is not particularly limited, and can be produced using a conventionally known method as described above.

  The biological species detection kit of the present invention is a kit for the method of detecting a biological species of the present invention including the microarray of the present invention. The biological species detection kit of the present invention may further include a primer for preparing a detection target and a reference, an enzyme, a reagent, a standard sample of the biological species to be detected, and the like. Furthermore, a primer for preparing an internal standard target and a reference, an internal standard sample, and the like may be included. As the enzyme and the reagent, conventionally known reagents can be used, and examples thereof include a polymerase, a nucleotide, a labeled compound, and a buffer. The detection kit of the present invention may contain a nucleic acid extraction reagent for preparing a target or a reference, a filter, a chip, or the like, if necessary.

  In the following, an environment contaminated with an organochlorine compound selected from the group consisting of tetrachlorethylene (PCE), trichlorethylene (TCE) and their dehalogenated compounds is used as a test sample, and an organochlorine-decomposing bacterium present in the environment is selected. One mode of the method for detecting a species of the present invention to be detected will be described.

The following bacteria A to T are known as bacteria involved in the decomposition of organochlorine compounds containing PCE, TCE and their dehalides.
A: Dehalospirilum multivobolans ( Dehalospirum multivorans )
B: Desulfitobacterium frapieri
C: Actinomycetales Sm-1 (Rhodococcus sp. Sm-1) ( Actinomycetales Sm-1 ( Rhodococcus sp. Sm-1))
D: Rhodococcus Rhodococcus ( Rhodococcus rhodococcus )
E: Xantobacter flavus
F: Mycobacterium L1 ( Mycobacterium L1)
G: Death Gandolfo micro-bi Umm Norubegikamu (Death Gandolfo Vie Brio Bakyuratasu) (Desulfomicrobium norvegicum (Desulfovibrio baculatus) )
H: Desulfitobacterium dehalogenans
I: Desulfitobacterium hafniense
J: Clostridium formicoaceticum ( Clostridium formicoaceticum )
K: Desulfurononas chloroethnica ( Desulfuromonas chloroethnica )
L: Acetobacterium woody DSM 1030 ( Acetobacterium woodi DSM 1030)
M: Deharobakuta Resutorikutasu (Dehalobacter restrictus)
N: Desulfitobacterium sp. PCE1 ( Desulfitobacterium sp. Strain PCE1)
O: Desulfitobacterium flapieri TCE1 ( Desulfitobacterium frapieri TCE1)
P: Acetobacterium woody DSM 2396 ( Acetobacterium woodi DSM 2396)
Q: Desurufomoniru Tide Jay DCB-1 (Desulfomonile tiedjei DCB- 1)
R: Clostridium bifamentans DPH-1 ( Clostridium bifermentans DPH-1)
S: Clostridium sp. KYT-1 ( Clostridium sp. KYT-1)
T: Clostridium sp. DC-1 ( Clostridium sp. DC-1)
The bacteria from A to Q are given the deposit numbers of ATCC or DSMZ, which are the biological resource storage organizations shown below, and can be obtained from the corresponding organizations.
A: DSM12446 B: DSM13498 C: ATCC51239
D: ATCC 21197 E: DSM10330 F: DSM 6695
G: DSM 1741 H: DSM 9161 I: DSM 10644
J: ATCC 27076 K: DSM 12431 L: DSM 1030
M: DSM 9455 N: DSM 10344 O: DSM 12704
P: DSM 2396 Q: ATCC 49306
PCE is dechlorinated to become TCE, dichloroethylene (DCE), vinyl chloride (VC), and finally decomposed into ethene or carbon dioxide. FIG. 2 shows the general degradation pathway of PCE and the degradation activity of bacteria A to T on PCE and dechlorinated products. If the bacteria in the test sample can be detected, for example, it is possible to know the ability to decompose pollutants in the sample environment, and efficient bioremediation becomes possible.

  In order to detect the bacteria A to T using the biological species detection method of the present invention, it is first necessary to design a plurality of detection probes for each of the bacteria and prepare the microarray of the present invention used for detection. is there. The detection probe is preferably selected from the ITS region of each bacterium. Plural types of detection probes for each of the bacteria A to T have a base length of 40 mer, the Tm value by the adjacent method is 85 ° C. to 105 ° C., and homology with ITS regions of other bacteria The following is an example of design based on the absence of nucleosides, the absence of 6 or more consecutive G + C and A + T bases, the absence of probes at the end of the ITS region, and the reduction in base sequence duplication between probes.

The bacterium A detection probe is a probe comprising a polynucleotide selected from the group consisting of a polynucleotide consisting of the nucleotide sequences of SEQ ID NOs: 1 to 7, a homologous polynucleotide thereof, and a complementary polynucleotide thereof; Is a probe comprising a polynucleotide selected from the group consisting of a nucleotide sequence of SEQ ID NOs: 8 to 12, a homologous polynucleotide thereof, and a complementary polynucleotide thereof, for detecting the bacterium C The probe is a probe comprising a polynucleotide selected from the group consisting of a nucleotide sequence of SEQ ID NOS: 13 to 17, a homologous polynucleotide thereof, and a complementary polynucleotide thereof, and the probe for detecting the bacterium D comprises SEQ ID NO: 18 A probe comprising a polynucleotide selected from the group consisting of a polynucleotide consisting of 22 base sequences, a homologous polynucleotide thereof and a complementary polynucleotide thereof, wherein the probe for detection of bacteria E comprises bases of SEQ ID NOs: 23 to 27 A probe comprising a polynucleotide selected from the group consisting of a polynucleotide comprising a sequence, a homologous polynucleotide thereof and a complementary polynucleotide thereof, wherein the probe for detecting bacteria F comprises a base sequence of SEQ ID NOs: 28 to 30 A probe comprising a polynucleotide selected from the group consisting of a polynucleotide, a homologous polynucleotide thereof and a complementary polynucleotide thereof, wherein the probe for detecting bacteria G comprises a nucleotide sequence of SEQ ID NOS: 31 to 35, A probe comprising a polynucleotide selected from the group consisting of the homologous polynucleotides and their complementary polynucleotides, wherein the probe for detecting bacteria H is a polynucleotide comprising the nucleotide sequence of SEQ ID NOs: 36 to 39, and its homologous poly A probe comprising a polynucleotide selected from the group consisting of nucleotides and their complementary polynucleotides, wherein the probe for detection of bacteria I is a polynucleotide comprising the nucleotide sequence of SEQ ID NOs: 40 to 44, homologous polynucleotides thereof, and the like A probe comprising a polynucleotide selected from the group consisting of complementary polynucleotides of the above, wherein the bacterial J detection probe comprises a polynucleotide comprising the nucleotide sequence of SEQ ID NOs: 45 to 50, a homologous polynucleotide thereof, and a complementary polynucleotide thereof. A probe comprising a polynucleotide selected from the group consisting of nucleotides, wherein the bacterial K detection probe comprises a polynucleotide comprising the nucleotide sequence of SEQ ID NOs: 51 to 56, a homologous polynucleotide thereof, and a complementary polynucleotide thereof. A probe comprising a polynucleotide selected from the group, wherein the detection probe for bacteria L is selected from the group consisting of a polynucleotide consisting of the nucleotide sequence of SEQ ID NOs: 57 to 61, a homologous polynucleotide thereof, and a complementary polynucleotide thereof A probe comprising a polynucleotide selected from the group consisting of a polynucleotide comprising the nucleotide sequences of SEQ ID NOS: 62 to 68, a homologous polynucleotide thereof, and a complementary polynucleotide thereof. A probe containing rheotide, wherein the probe for detecting bacteria N comprises a polynucleotide selected from the group consisting of a polynucleotide consisting of the nucleotide sequence of SEQ ID NOs: 69 to 73, a homologous polynucleotide thereof and a complementary polynucleotide thereof The probe for detecting bacteria O is a probe comprising a polynucleotide selected from the group consisting of a polynucleotide consisting of the nucleotide sequence of SEQ ID NOs: 74 to 78, a homologous polynucleotide thereof, and a complementary polynucleotide thereof. The probe for detecting bacteria P is a probe comprising a polynucleotide selected from the group consisting of a polynucleotide consisting of the nucleotide sequence of SEQ ID NOs: 79 to 81, a homologous polynucleotide thereof, and a complementary polynucleotide thereof, The probe for detecting Q is a probe comprising a polynucleotide selected from the group consisting of a nucleotide sequence of SEQ ID NOs: 82 to 87, a homologous polynucleotide thereof, and a complementary polynucleotide thereof, The detection probe is a probe comprising a polynucleotide selected from the group consisting of a nucleotide sequence of SEQ ID NOs: 88 to 92, a homologous polynucleotide thereof, and a complementary polynucleotide thereof, and the probe for detecting the bacteria S Is a probe comprising a polynucleotide selected from the group consisting of a nucleotide sequence of SEQ ID NOs: 93 to 97, a homologous polynucleotide thereof, and a complementary polynucleotide thereof, and the probe for detection of bacteria T has the sequence Number 98-10 A probe comprising a polynucleotide selected from the group consisting of a polynucleotide comprising two nucleotide sequences, a homologous polynucleotide thereof, and a complementary polynucleotide thereof. Here, “a polynucleotide comprising the nucleotide sequence of SEQ ID NO: n, a homologous polynucleotide thereof, and a complementary polynucleotide thereof” includes the following polynucleotides (1) to (4).
(1) A polynucleotide comprising the base sequence of SEQ ID NO: n.
(2) A polynucleotide consisting of a base sequence in which one to several bases of SEQ ID NO: n are deleted, substituted or added, and complementary to the polynucleotide consisting of the base sequence of SEQ ID NO: n A polynucleotide that hybridizes with a polynucleotide comprising a simple base sequence under stringent conditions.
(3) A polynucleotide comprising a nucleotide sequence in which one to several bases of the nucleotide sequence of SEQ ID NO: n have been deleted, substituted or added, and the homology with the polynucleotide of (1) above is 90 % Of a polynucleotide.
(4) A polynucleotide comprising a base sequence complementary to the base sequence of the polynucleotide of (1) to (3).

  In the polynucleotide (2) or (3), the number of bases that can be deleted, substituted or added is, for example, 1 to 6 by deletion / addition to 40 bases, The number is preferably from 1 to 3, more preferably from 1 to 2, and from 1 to 4 by substitution, preferably from 1 to 2 and more preferably 1. Hybridization under stringent conditions also means that two DNA fragments hybridize to each other under standard hybridization conditions as described by Sambrook J et al. (Expression of cloned genes). in E. coli (Molecular Cloning: A laboratory manual (1989)) Cold Spring harbor Laboratory Press, New York, USA, 9.47-9.62 and 11.45-11.61). More specifically, this means that hybridization and washing (for example, about 2.0 × SSC, 50 ° C.) are performed based on a Tm value of ± 10 ° C. Moreover, as said homology, it is 90% or more, for example, Preferably it is 95% or more, More preferably, it is 97.5% or more.

  The microarray of the present invention for detecting the bacteria A to T can be prepared by arranging the detection probe on a substrate. The detection target and the detection reference can be prepared by designing and using universal primers capable of amplifying the ITS region of the bacteria A to T when asymmetric PCR or LATE-PCR is used.

  The present invention will be further described below using examples. Note that the numerical values set in the embodiments do not limit the scope of the present invention.

(Competitive hybridization using standard samples)
1. Production of Detection Probes A plurality of detection probes consisting of 40 bases were designed from the ITS region using the bacteria A to T as detection target species. The design of the detection probe is a single strand of 40 bases, 48-50% GC content, no or little homology, and no hits in the international database GenBank (at least two mispairs) It was done on the basis of that. The sequence numbers (SEQ ID NOs: 1 to 87) of the base sequences of the prepared DNA probes for detecting biological species (3 to 10 types for each bacterium) are shown below. The C-terminal of each probe was C6 aminated.

  Bacteria A probes A1 to A7 are SEQ ID Nos. 1 to 7, respectively. Bacteria B probes B1 to B5 are respectively SEQ ID Nos. 8 to 12, and bacteria C probes C1 to C5 are arranged respectively. Nos. 13 to 17, probes D1 to D5 of bacteria D are respectively SEQ ID NOs: 18 to 22, probes E1 to E5 of bacteria E are SEQ ID NOs: 23 to 27, respectively, and probe F1 of bacteria F To F3 are SEQ ID NOs: 28 to 30, respectively, and bacteria G probes G1 to G5 are SEQ ID NOs: 31 to 35, respectively, and bacteria H probes H1 to H4 are SEQ ID NOs: 36 to 39, respectively. And bacteria I probes I1 to I5, respectively, from SEQ ID NO: 40 4, the probes J1 to J6 of the bacteria J are SEQ ID NOs: 45 to 50, the probes K1 to K6 of the bacteria K are respectively SEQ ID NOs: 51 to 56, and the probes L1 to L5 of the bacteria L are SEQ ID NOs: 57 to 61, bacteria M probes M1 to M7, respectively, SEQ ID NOs 62 to 68, and bacteria N probes N1 to N5, respectively, SEQ ID NOs 69 to 73, O probes O1 to O5 are SEQ ID NOs 74 to 78, bacteria P probes P1 to P3 are SEQ ID NOs 79 to 81, and bacteria Q probes Q1 to Q6 are SEQ ID NOs: 82 to 87, and bacteria R probes R1 to R5 are respectively It is 92 of SEQ ID NO: 88, from probe S1 S5 bacteria S, respectively, 97 of SEQ ID NO: 93, a probe T1 from T5 bacteria T, respectively, are 102 from SEQ ID NO: 98.

2. Preparation of DNA microarray and confirmation of specificity of DNA probe Next, the above-mentioned 102 kinds of detection probes were custom-made by Affymetrix 417 Arrayer (manufactured by Affymetrix) to each three Takara Hublide (manufactured by Takara). A microarray was produced by printing. In addition, a detection target was prepared for each bacterial standard sample, and a detection target was prepared from a standard sample containing all bacteria.

  Since the detection probe is a sense strand of the ITS region, the detection target and the detection reference were prepared by a LATE-PCR method using antisense (reverse) primers labeled with Cy5 and Cy3, respectively. The forward primer and the reverse primer are based on the nucleotide sequences of SEQ ID NO: 103 and SEQ ID NO: 104, respectively, so that the Tm value by the neighbor method (Nearest Neighbor Method) becomes Tm (forward) −Tm (reverse) ≧ 0. The modified version was used. The Tm value is calculated using software: PRIMOROSE ver. 1.1.7 (Kevin E. Ashelford, Andrew J. Weightman andJohn C. Fry (2002) "PRIMROSE: a computer program for generating and estimating the phylogenetic range of 16S rRNA oligonucleotide probes and primers in conjunction with the RDP-II database "Nucleic Acids Research, Vol. 30, No. 15 3481-3489). In the LATE-PCR method, the forward primer and the reverse primer were used at a concentration ratio of 1: 100. The amplification reaction was carried out at 94 ° C. for 10 minutes, followed by 15 cycles of 94 ° C. 30 seconds-55 ° C. 30 seconds-72 ° C. 60 seconds, and further 25 cycles of 94 ° C. 30 seconds-50 ° C. 30 seconds-72 ° C. 60 seconds. It was. The amplification product was desalted using Autoseq G-50 (manufactured by Pharmacia) and sucked and dried using SpeedVac (manufactured by Savant).

  A solution obtained by dissolving the detection target and the detection reference in a buffer of 5 × SSC, 0.2% SDS, 50% formamide was boiled at 94 ° C. for 3 minutes and ice-cooled for at least 2 minutes. Applied to microarray. A cover glass was placed on the microarray and then placed in a hybridization chamber set at 65 ° C. for 4-12 hours, after which the microarray was placed in 0.2 × SSC, 0.2% SDS for 5 minutes, 0 minutes. Washed with 2 × SSC for 5 minutes and 0.05 × SSC for a few seconds and spin dried at 1500 rpm. The result was measured by scanning with Scanary version 5 (Perkin Elmer Japan).

  A summary of the results is shown in FIG. FIG. 3 is a table showing whether targets derived from 20 types of bacteria A to T shown on the vertical axis are hybridized to spots of 102 types of probes shown on the horizontal axis. In the figure, black circles indicate that the target has hybridized to all the bacterial spots shown on the horizontal axis with a signal of 500 fluorescent units or more. As shown in the figure, it was shown that the target prepared from each of the bacteria A to T specifically hybridized only with the same bacterial spot without cross-hybridization.

3. Detection of bacteria A and E Next, a target for detection prepared from standard samples of bacteria A and E is mixed with a reference for detection prepared from 16 types of standard samples of bacteria A to P, and competitive hybridization is performed. It was. Target and reference preparation methods and hybridization conditions were the same as described above. FIG. 4A shows a scatter plot of the obtained target Cy5 signal intensity and reference Cy3 signal intensity on the vertical and horizontal axes, respectively. In addition, the straight line in the figure is a regression line. As shown in the figure, strong target signals were observed for bacteria A and E. The results of correlation analysis and regression analysis for bacteria A and E are shown in Table 1 below. As shown in Table 1 below, for both bacteria A and E, a correlation between the target and the reference was observed at a significance level of 1%, and a strong correlation with a correlation coefficient r exceeding 0.90 was shown. Further, as shown in FIG. 4B and Table 1 below, the regression coefficient b representing the target / reference ratio (the slope of the regression line) was 14.9 for bacteria A and 4.4 for bacteria E. It was found that the amounts of bacteria A and E used to prepare the target were 14.9 times and 4.4 times that of the reference, respectively.

  Other than using a mixed culture sample containing bacteria capable of degrading PCE (provided by Dr. TH Lee, Korea) as a test sample and preparing a Cy5-labeled target using nucleic acid extracted therefrom In the same manner as in Example 1, bacteria present in the test sample were detected. A scatter plot of signal intensity for bacteria AP is shown in FIG. 5A. The results of correlation analysis and regression analysis are shown in Table 2 below. The right column of Table 2 below shows the determination results when the predetermined value of the correlation coefficient r is 0.9 and the predetermined significance level is 1%. As shown in FIG. 5A and Table 2 below, it can be seen that, for example, the bacteria M whose signal intensity of the target was strongly measured could be excluded as false positives. In addition, for example, bacteria B, G, H, etc. were determined to be positive although the signal intensity of the target was weak. The presence of Bacteria B was confirmed by cloning the Bacterium B genome and determining the base sequence, and presenting it in the test material. FIG. 5B shows the target / reference ratio for bacteria judged positive, ie, the relative abundance of each bacterium.

  Bacteria present in the TCE-contaminated soil were detected in the same manner as in Example 1 except that TCE-contaminated soil was used as a test sample and a Cy5-labeled target was prepared using a nucleic acid extracted therefrom. In the TCE-contaminated soil, 0.03 mg / ml TCE and 0.33 mg / ml cDCE (cisdichloroethylene) were present. A scatter plot of signal intensity for bacteria AP is shown in FIG. 6A. The results of correlation analysis and regression analysis are shown in Table 3 below. The right column of Table 3 below shows the determination results when the predetermined value of the correlation coefficient r is 0.9 and the predetermined significance level is 1%. As shown in FIG. 6A and Table 3 below, it can be seen that, for example, bacteria M and O were measured with strong target signal intensity, but could be excluded as false positives. In addition, for example, bacteria D, E, and the like were found to be positive although the signal intensity of the target was weak. FIG. 6B shows the target / reference ratio for bacteria judged positive, ie, the relative abundance of each bacterium.

  As described above, the method for detecting a species of the present invention enables determination of false positive or positive, thereby realizing higher reliability and enabling quantification of detected bacteria, By realizing good convenience and using a microarray, it is possible to realize excellent processing capabilities such as easy and rapid operation and batch processing of a large number of samples. Therefore, the present invention is useful, for example, in the fields of environmental research, environmental repair, bioremediation, food production, and food distribution.

FIG. 1 is a graph showing the relationship between the target / reference ratio of a microarray and the number of target cells. FIG. 2 is a diagram for explaining the degradation pathway of PCE and the organochlorine compound degradation activity of bacteria A to T. FIG. 3 is a table showing the specificity of each detection probe used in the examples for each bacterium. FIG. 4A is a scatter plot of the target and reference, and FIG. 4B is a histogram of the target / reference ratio (Example 1). FIG. 5A is a scatter plot of target and reference, and FIG. 5B is a histogram of target / reference ratio (Example 2). FIG. 6A is a scatter plot of the target and reference, and FIG. 6B is a histogram of the target / reference ratio (Example 3).

SEQ ID NO: 103 sense primer for PCR SEQ ID NO: 104 antisense primer for PCR

Claims (13)

A method for detecting a biological species in a test sample,
A microarray preparation step of preparing a microarray in which spots of a plurality of types of detection probes specific to the species to be detected are arranged;
A target preparation step of preparing a detection target labeled with a first labeling compound from the nucleic acid in the test sample;
A reference preparation step of preparing a reference for detection labeled with a second labeling compound from a standard sample of the species to be detected;
A competitive hybridization step in which the target and the reference are mixed and hybridized to the microarray, and the labeling signals of the first and second labeling compounds in the spot are measured, respectively.
When the correlation coefficient r is calculated and tested for the correlation between the two labeled signals in the spot, the correlation is affirmed at a predetermined significance level, and the value of the correlation coefficient r exceeds a predetermined value And a determination step of determining that the other cases are false positives. Furthermore, it includes a quantification step of calculating the abundance of the species to be detected in the test sample,
The method for detecting a biological species according to claim 1, wherein the quantification step includes calculating a slope (regression coefficient) b of a regression line of the two labeled signals at the spot.   The microarray preparation step, the target preparation step and the reference preparation step are respectively arranged with a spot of an internal standard probe, preparing an internal standard target labeled with a first labeling compound, and a second labeling compound. The method of claim 1, comprising preparing a labeled internal standard reference, wherein the determining step includes correcting the detection target label signal and the detection reference label signal based on the signal of the internal standard. The method for detecting a biological species as described.   In the microarray of the microarray preparation step, the number of detection probes is three or more for one type of detection target species, and the number of spots of the detection probe is six or more for one type of detection target species. The method for detecting a biological species according to any one of claims 1 to 3.   The predetermined significance level in the determination step is in a range of 20% to 0.1%, and the predetermined value of the correlation coefficient is in a range of 0.80 to 0.99. The method for detecting a biological species according to claim 1.   The method for detecting a biological species according to any one of claims 1 to 5, wherein the target and reference are prepared in the target preparation step and the reference preparation step by a LATE-PCR method.   The method of detecting a biological species according to any one of claims 1 to 6, wherein there are a plurality of types of biological species to be detected.   The method of detecting a biological species according to any one of claims 1 to 7, wherein the biological species to be detected is a bacterium, and the detection probe is derived from an ITS region of the bacterium.   The test sample is a sample taken from an environment contaminated with an organic chlorine compound selected from the group consisting of tetrachlorethylene (PCE), trichlorethylene (TCE) and their dehalogenated compounds. The method for detecting a biological species according to Item 1. The method for detecting a biological species according to any one of claims 1 to 9, wherein the biological species to be detected is a bacterium comprising at least one selected from the group consisting of A to T below.
A: Dehalospirilum multivobolans ( Dehalospirum multivorans )
B: Desulfitobacterium frapieri
C: Actinomycetales Sm-1 (Rhodococcus sp. Sm-1) ( Actinomycetales Sm-1 ( Rhodococcus sp. Sm-1))
D: Rhodococcus Rhodococcus ( Rhodococcus rhodococcus )
E: Xantobacter flavus
F: Mycobacterium L1 ( Mycobacterium L1)
G: Death Gandolfo micro-bi Umm Norubegikamu (Death Gandolfo Vie Brio Bakyuratasu) (Desulfomicrobium norvegicum (Desulfovibrio baculatus) )
H: Desulfitobacterium dehalogenans
I: Desulfitobacterium hafniense
J: Clostridium formicoaceticum ( Clostridium formicoaceticum )
K: Desulfurononas chloroethnica ( Desulfuromonas chloroethnica )
L: Acetobacterium woody DSM 1030 ( Acetobacterium woodi DSM 1030)
M: Deharobakuta Resutorikutasu (Dehalobacter restrictus)
N: Desulfitobacterium sp. PCE1 ( Desulfitobacterium sp. Strain PCE1)
O: Desulfitobacterium flapieri TCE1 ( Desulfitobacterium frapieri TCE1)
P: Acetobacterium woody DSM 2396 ( Acetobacterium woodi DSM 2396)
Q: Desurufomoniru Tide Jay DCB-1 (Desulfomonile tiedjei DCB- 1)
R: Clostridium bifamentans DPH-1 ( Clostridium bifermentans DPH-1)
S: Clostridium sp. KYT-1 ( Clostridium sp. KYT-1)
T: Clostridium sp. DC-1 ( Clostridium sp. DC-1) The method for detecting a biological species according to claim 10,
The bacterium A detection probe is a probe comprising a polynucleotide selected from the group consisting of a polynucleotide consisting of the nucleotide sequences of SEQ ID NOs: 1 to 7, a homologous polynucleotide thereof, and a complementary polynucleotide thereof; Is a probe comprising a polynucleotide selected from the group consisting of a nucleotide sequence of SEQ ID NOs: 8 to 12, a homologous polynucleotide thereof, and a complementary polynucleotide thereof, for detecting the bacterium C The probe is a probe comprising a polynucleotide selected from the group consisting of a nucleotide sequence of SEQ ID NOS: 13 to 17, a homologous polynucleotide thereof, and a complementary polynucleotide thereof, and the probe for detecting the bacterium D comprises SEQ ID NO: 18 A probe comprising a polynucleotide selected from the group consisting of a polynucleotide consisting of 22 base sequences, a homologous polynucleotide thereof and a complementary polynucleotide thereof, wherein the probe for detection of bacteria E comprises bases of SEQ ID NOs: 23 to 27 A probe comprising a polynucleotide selected from the group consisting of a polynucleotide comprising a sequence, a homologous polynucleotide thereof and a complementary polynucleotide thereof, wherein the probe for detecting bacteria F comprises a base sequence of SEQ ID NOs: 28 to 30 A probe comprising a polynucleotide selected from the group consisting of a polynucleotide, a homologous polynucleotide thereof and a complementary polynucleotide thereof, wherein the probe for detecting bacteria G comprises a nucleotide sequence of SEQ ID NOS: 31 to 35, A probe comprising a polynucleotide selected from the group consisting of the homologous polynucleotides and their complementary polynucleotides, wherein the probe for detecting bacteria H is a polynucleotide comprising the nucleotide sequence of SEQ ID NOs: 36 to 39, and its homologous poly A probe comprising a polynucleotide selected from the group consisting of nucleotides and their complementary polynucleotides, wherein the probe for detection of bacteria I is a polynucleotide comprising the nucleotide sequence of SEQ ID NOs: 40 to 44, homologous polynucleotides thereof, and the like A probe comprising a polynucleotide selected from the group consisting of complementary polynucleotides of the above, wherein the bacterial J detection probe comprises a polynucleotide comprising the nucleotide sequence of SEQ ID NOs: 45 to 50, a homologous polynucleotide thereof, and a complementary polynucleotide thereof. A probe comprising a polynucleotide selected from the group consisting of nucleotides, wherein the bacterial K detection probe comprises a polynucleotide comprising the nucleotide sequence of SEQ ID NOs: 51 to 56, a homologous polynucleotide thereof, and a complementary polynucleotide thereof. A probe comprising a polynucleotide selected from the group, wherein the detection probe for bacteria L is selected from the group consisting of a polynucleotide consisting of the nucleotide sequence of SEQ ID NOs: 57 to 61, a homologous polynucleotide thereof, and a complementary polynucleotide thereof A probe comprising a polynucleotide selected from the group consisting of a polynucleotide comprising the nucleotide sequences of SEQ ID NOS: 62 to 68, a homologous polynucleotide thereof, and a complementary polynucleotide thereof. A probe containing rheotide, wherein the probe for detecting bacteria N comprises a polynucleotide selected from the group consisting of a polynucleotide consisting of the nucleotide sequence of SEQ ID NOs: 69 to 73, a homologous polynucleotide thereof and a complementary polynucleotide thereof The probe for detecting bacteria O is a probe comprising a polynucleotide selected from the group consisting of a polynucleotide consisting of the nucleotide sequence of SEQ ID NOs: 74 to 78, a homologous polynucleotide thereof, and a complementary polynucleotide thereof. The probe for detecting bacteria P is a probe comprising a polynucleotide selected from the group consisting of a polynucleotide consisting of the nucleotide sequence of SEQ ID NOs: 79 to 81, a homologous polynucleotide thereof, and a complementary polynucleotide thereof, The probe for detecting Q is a probe comprising a polynucleotide selected from the group consisting of a nucleotide sequence of SEQ ID NOs: 82 to 87, a homologous polynucleotide thereof, and a complementary polynucleotide thereof, The detection probe is a probe comprising a polynucleotide selected from the group consisting of a nucleotide sequence of SEQ ID NOs: 88 to 92, a homologous polynucleotide thereof, and a complementary polynucleotide thereof, and the probe for detecting the bacteria S Is a probe comprising a polynucleotide selected from the group consisting of a nucleotide sequence of SEQ ID NOs: 93 to 97, a homologous polynucleotide thereof, and a complementary polynucleotide thereof, and the probe for detection of bacteria T has the sequence Number 98-10 A probe comprising a polynucleotide selected from the group consisting of a polynucleotide comprising two nucleotide sequences, a homologous polynucleotide thereof and a complementary polynucleotide thereof;
Here, the method for detecting a biological species, wherein the “polynucleotide consisting of the base sequence of SEQ ID NO: n, its homologous polynucleotide and their complementary polynucleotide” comprises the following polynucleotides (1) to (4).
(1) A polynucleotide comprising the base sequence of SEQ ID NO: n.
(2) A polynucleotide comprising a nucleotide sequence in which one to several bases of SEQ ID NO: n are deleted, substituted or added, and complementary to a polynucleotide comprising the nucleotide sequence of SEQ ID NO: n A polynucleotide that hybridizes with a polynucleotide comprising a simple base sequence under stringent conditions.
(3) A polynucleotide comprising a nucleotide sequence in which one to several bases of the nucleotide sequence of SEQ ID NO: n have been deleted, substituted or added, and has a homology of 90 with the polynucleotide of (1) above % Of a polynucleotide.
(4) A polynucleotide comprising a base sequence complementary to the base sequence of the polynucleotide of (1) to (3).   The microarray used for the detection method of the biological species of any one of Claim 1 to 11.   A biological species detection kit for the biological species detection method according to any one of claims 1 to 11, comprising the microarray according to claim 12.

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