Technical field
[0001]
　The present invention relates to a method for rapid identification of bacteria in a sample and a kit for that purpose.
Background technology
[0002]
　The number of patients with sepsis, which is a serious systemic infectious disease and requires detection and identification of the causative microorganism in the blood for definitive diagnosis, has been increasing in recent years with the advancement of medical treatment such as cancer treatment and organ transplantation. .. From the viewpoint of nosocomial infections, methicillin-resistant Staphylococcus aureus (MRSA) and other multidrug-resistant bacteria are often the causative agents of sepsis, so to select appropriate antibiotics and save lives for patients. It is clinically important to detect and identify the causative microorganism in blood as quickly as possible (Patent Document 1).
　Against this background, a testing system using PCR (polymerase chain reaction) has been studied as a rapid testing method for sepsis. As such a test method, DNA is extracted from a microorganism, the gene is amplified by PCR or the like using a specific primer pair using this as a template, and then the melting temperature (Tm value) specific to the microorganism is determined. A PCR method is known that rapidly detects and identifies the causative organism by combining or analyzing the difference between each Tm value (Patent Document 2).
　In the above PCR method, 1st PCR is performed using the extracted DNA as a template, the total length of the target DNA fragment is amplified, and 2nd PCR (nested PCR) is performed using the amplified DNA fragment as a template for highly sensitive detection. doing. However, the need for two-step PCR is a problem in terms of test time and workability. Especially in terms of workability, the two PCRs are complicated, and improvements are required for reducing the burden on the operator and fully automating the work.
　Bacterial RNA in various clinical, food, environmental, or other experimental samples has also been detected to identify contaminants or pathogens. Specific probes have been developed that enable detection even in the presence of undetected bacterial species by targeting several important bacterial species and detecting RNA molecules specific to the detected bacterial species. ing. In the reverse transcription PCR assay method, a method of synthesizing cDNA by a reverse transcription reaction using the target RNA as a template and then detecting the target RNA molecule through amplification of the cDNA in the subsequent PCR is also performed (Patent Document 3).
　Regarding the bacterial identification method based on the Tm value, Patent Document 1 states that RNA can be extracted instead of DNA, and cDNA prepared based on the obtained RNA can also be used, but detailed studies have not been conducted. It was.
　Patent Document 3 describes a method for directly detecting diagnostic RNA from bacteria.
　Patent Document 4 describes a specific method for detecting RNA species in a biological sample.
Prior art literature
Patent documents
[0003]
Patent Document 1: International Publication No. 2010/082640
Patent Document 2: International Publication No. 2007/097323
Patent Document 3: Japanese Patent Application Laid-Open No. 2012-34705
Patent Document 4: International Publication No. 2010/054819
Outline of the invention
Problems to be solved by the invention
[0004]
　Based on the technique disclosed in Patent Document 2, it is possible to detect and identify microorganisms with high sensitivity in a short time. However, as described above, the above method requires PCR to be performed twice, which is considered to be complicated and burdensome to the operator, and may hinder the full automation of the work. ..
　Further, Patent Document 4 describes a method of detecting a cDNA amplified by PCR after reverse transcribing RNA into cDNA using reverse transcriptase.
　RNA is more abundant in bacteria than DNA. It is logically better to extract RNA from the bacteria in the sample, prepare the cDNA by reverse transcription reaction using RNA instead of the first PCR, and perform PCR using the prepared cDNA as a template. It can be expected to improve the detection sensitivity of.
　However, according to the study by the present inventors, when the reaction as described in Patent Document 4 is carried out, an extra reaction occurs due to the reverse transcription reaction during PCR, and unnecessary DNA fragments other than the target DNA fragment are generated. It was found that the bacteria could not be identified because many of them were obtained.
　The present invention is a bacterial identification method capable of identifying many bacteria, not only a limited number of bacteria, by targeting the RNA of the bacteria in the sample without changing the reagents and conditions for identifying the bacteria. And to provide a kit for that purpose. Another object of the present invention is to provide an identification method capable of detecting and identifying with high sensitivity by only one PCR and reducing complicated operations and burden on the operator, and a kit used therefor. ..
　Another object of the present invention is to find a method for identifying bacteria necessary for solving the above-mentioned problems.
　As described above, it is clear from the examination by the inventors that the detection and identification of bacteria may not be achieved only by the conventional idea of ​​using RNA in a sample. In the case where the concentration of the primer for reverse transcription brought into the PCR step for detecting the target cDNA is too high, the bacteria cannot be substantially identified by the method described in Patent Document 4. I found out that it was the cause.
　The present inventors have repeatedly studied using this experimental fact as a starting point, and have found for the first time that there is a new solution problem that has not been recognized by those skilled in the art. That is, it was found that when cDNA is prepared from RNA extracted from a sample by reverse transcriptase and PCR is performed using the prepared cDNA as a template, it is necessary to suppress the production of DNA fragments other than the intended one generated during PCR. It was. This problem itself is an unknown problem that is not recognized by those skilled in the art in the prior art.
Means to solve problems
[0005]
　As described above, the present inventors have made extensive studies to solve a completely new problem.
　As a result, if the concentration of the reverse transcription primer brought in from the reverse transcription reaction in the reaction system during the PCR reaction is adjusted, and the concentration is adjusted to be extremely lower than normally thought, unnecessary DNA is unnecessary. It was found that the formation of fragments was remarkably suppressed, and that bacteria in the sample could be detected and identified by appropriately adjusting the Tm mapping conditions.
　Furthermore, there is an optimum range for the concentration of the reverse transcription primer present in the reaction system during the PCR reaction, and if the concentration of the reverse transcription primer is appropriately controlled so as to be within that range, even if the concentration in the sample is controlled. It was found that even if the number of bacteria is extremely low, it can be detected and identified with high sensitivity.
　The present invention has been completed based on the above-mentioned completely new findings.
　That is, the present invention is as follows.
[1] A method for identifying a bacterium, which comprises the following steps (1) to (3).
(1) First, which contains a primer for reverse transcription for preparing a cDNA containing a nucleotide sequence for identifying the bacterium to be identified, RNA extracted from the bacterium in the sample, and an enzyme having RNA-dependent DNA polymerase activity. A step of performing a reverse transcription reaction using the reaction system of the above to obtain a reaction mixture containing the synthesized cDNA and the primer for reverse transcription.
(2) Using a second reaction system containing the reaction mixture, a primer pair for synthesizing double-stranded DNA containing a base sequence for identifying the identification target bacterium, and an enzyme having DNA-dependent DNA polymerase activity. In the step of performing PCR, however, in the second reaction system, the concentration of the primer for reverse transcription supplied from the reaction mixture is 0.08 nM or more and 20 nM or less, and
(3) the second reaction after PCR. A step of detecting the generation of double-stranded DNA containing a base sequence for identifying the identification target bacterium from the reaction system of.
[2] The method for identifying bacteria according to the above item [1], wherein the steps (1) and (2) are sequentially performed in another reaction vessel or in the same reaction vessel.
[3] Item [1] above, wherein the base sequence for identifying the bacterium to be identified is a base sequence contained in the 16S rDNA of the bacterium, and the RNA contains the 16S rRNA of the bacterium in the sample. Alternatively, the method for identifying bacteria according to item [2].
[4] The method for identifying a bacterium according to [3], wherein the reverse transcription primer comprises the nucleotide sequence of SEQ ID NO: 1.
[5] The bacterium according to the above [1] or [4], wherein the primer pair is at least one primer pair selected from the group consisting of the following (group A) to (group C). Identification method.
(Group A)
-Primer pair 1a: Combined with a
forward primer consisting of the nucleotide sequence of SEQ ID NO: 2 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 3. Primer pair 2a: Forward primer and sequence consisting of the nucleotide sequence of SEQ ID NO: 4. Combined with a reverse primer consisting of the base sequence of No. 5,
-Primer pair 3a: Combined with a forward primer consisting of the nucleotide sequence of SEQ ID NO: 6 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 7.
Primer pair 4a: A forward primer consisting of the nucleotide sequence of SEQ ID NO: 8 and the base of SEQ ID NO: 9. Combined with a reverse primer consisting of a sequence,
・ Primer pair 5a: Combined with a forward primer consisting of the base sequence of SEQ ID NO: 10 and a reverse primer consisting of the base sequence of SEQ ID NO: 11
・ Primer pair 6a: Consisting of the base sequence of SEQ ID NO: 12. Combined with a forward primer and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 13,
・ Primer pair 7a: Combined with a forward primer consisting of the nucleotide sequence of SEQ ID NO: 14 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 15.
(Group B) A primer pair in which one or two bases are added, deleted or substituted as long as a part of the base sequence of one or both of the primer pairs of the above (Group A) is not impaired as a primer. , And
(Group C) A primer pair consisting of a complementary sequence corresponding to the base sequence of one or both of the above-mentioned (Group A) or (Group B) primer pairs.
[6] In the step (3), the Tm value of the generated double-stranded DNA was measured, and the double-stranded DNA containing the obtained Tm value and the previously measured base sequence for identification of the identification target bacterium was included. The bacterial identification according to any one of the above [1] to [5], which comprises identifying the bacteria in the sample by comparing the Tm value of DNA or the data obtained secondarily from them. Method.
[7] The item according to any one of [1] to [6] above, wherein after the step (1), the reaction mixture is used alone or diluted directly or in the step (2). Bacterial identification method.
[8] A kit for identifying the bacteria in the sample by PCR using the cDNA contained in the reaction mixture by the reverse transcription reaction of RNA extracted from the bacteria in the sample as a template, and the following
　(a) to (
A primer for reverse transcription , which has c) and (a) prepares a cDNA containing a base sequence for identifying the bacterium to be identified for preparation of the first reaction system for the reverse transcription reaction.
(B) A primer pair for synthesizing a double-stranded DNA containing a base sequence for identifying the bacterium to be identified for preparation of a second reaction system for the PCR, and
(c) an RNA-dependent type. An enzyme having DNA polymerase activity,
　the second reaction system contained a reaction mixture by the reverse transcription reaction, and an amount of the primer for the reverse transcription was supplied to the second reaction system via the reaction mixture. The
kit is characterized in that the concentration of the primer for reverse transcription is adjusted to 0.08 nM or more and 20 nM or less .
[9] The kit according to the above [8], further comprising an instruction manual describing the reverse transcription reaction and the PCR procedure.
[10] The above-mentioned instruction manual is characterized in that the procedure is described so that the concentration of the primer for reverse transcription contained in the second reaction system is 0.08 nM or more and 20 nM or less. 9] The kit described in.
[11] The base sequence for identifying the bacterium to be identified is the base sequence contained in the 16S rDNA of the bacterium, and the RNA contains the 16S rRNA of the bacterium in the sample. The kit according to any one of [10].
[12] The kit according to the above [11], wherein the reverse transcription primer comprises the base sequence of SEQ ID NO: 1.
[13] The kit according to the above [11] or [12], wherein the primer pair is at least one primer pair selected from the group consisting of the following (group A) to (group C). ..
(Group A)
-Primer pair 1a: Combined with a
forward primer consisting of the nucleotide sequence of SEQ ID NO: 2 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 3. Primer pair 2a: Forward primer and sequence consisting of the nucleotide sequence of SEQ ID NO: 4. Combined with a reverse primer consisting of the base sequence of No. 5,
・ Primer pair 3a: Combined with a forward primer consisting of the base sequence of SEQ ID NO: 6 and a reverse primer consisting of the base sequence of SEQ ID NO: 7
・ Primer pair 4a: of SEQ ID NO: 8. reverse primer combination consisting of the nucleotide sequence of the forward primer and SEQ ID NO: 9 comprising the nucleotide sequence,
primer pairs 5a: reverse primer combination consisting of the nucleotide sequence of the forward primer and SEQ ID NO: 11 comprising the nucleotide sequence of SEQ ID NO: 10,
& Primer pair 6a: Combined with a forward primer consisting of the nucleotide sequence of SEQ ID NO: 12 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 13.
-Primer pair 7a: A combination of a forward primer consisting of the nucleotide sequence of SEQ ID NO: 14 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 15.
(Group B) A primer pair in which one or two bases are added, deleted or substituted as long as a part of the base sequence of one or both of the primer pairs of the above (Group A) is not impaired as a primer. , And
(Group C) A primer pair consisting of a complementary sequence corresponding to the base sequence of one or both of the above-mentioned (Group A) or (Group B) primer pairs.
The invention's effect
[0006]
　According to the present invention, bacteria can be detected and identified by a single PCR while maintaining the detection sensitivity. Therefore, the burden on the operator can be reduced and the system can be automated.
　The identification method of the present invention detects bacteria with high sensitivity even for a sample containing dilute bacteria having a bacterial count of about 100 CFU / sample by appropriately adjusting the concentration of the reverse transcription primer in the second reaction system. Identification is possible.
A brief description of the drawing
[0007]
FIG. 1 is a diagram showing the results of agarose gel electrophoresis of the PCR product in Example 1.
FIG. 2 is a diagram showing the results of agarose gel electrophoresis of the PCR product in Example 2.
FIG. 3 is a diagram showing the results of agarose gel electrophoresis of the PCR product in Example 3.
Mode for carrying out the invention
[0008]
　The bacterial identification method according to the present invention has the following steps.
(I) A step of obtaining a reaction mixture containing cDNA by a reverse transcription reaction using RNA extracted from bacteria in a sample.
(II) A step of performing PCR using a reaction mixture containing cDNA and a primer pair for bacterial identification.
(III) A step of detecting the production of double-stranded DNA amplified by PCR using a primer pair for bacterial identification.
　For the reverse transcription reaction in step (I), a primer for reverse transcription for preparing a cDNA containing a base sequence for identifying the bacterium to be identified, RNA extracted from the bacterium in the sample, and an RNA-dependent DNA polymerase A first reaction system containing an active enzyme is used.
　The reaction mixture in the present invention means a mixture containing a reaction mixture obtained by performing a reverse transcription reaction in the first reaction system and an unreacted product that remains unreacted without participating in the reaction.
　The primer for reverse transcription used in the first reaction system is consumed during the synthesis of cDNA, but is usually used in an excess amount larger than the amount required for the synthesis amount of the target cDNA. Therefore, the reaction mixture by the reverse transcription reaction by the first reaction system contains the synthesized cDNA as a reaction product and the remaining primer for reverse transcription as an unreacted product.
　The PCR in step (II) includes the reaction mixture obtained in step (I), a primer pair for synthesizing double-stranded DNA containing a base sequence for identifying the bacterium to be identified, and DNA-dependent DNA polymerase activity. A second reaction system containing the enzyme is used.
　As described above, the reaction mixture obtained in step (I) contains a primer for reverse transcription that was not consumed in the reverse transcription reaction, and the amount of this primer for reverse transcription in step (II) was adjusted. By doing so, high-sensitivity detection can be performed. Therefore, the concentration of the primer for reverse transcription during the preparation of the second reaction system is selected from 0.08 nM to 20 nM, more preferably 0.4 nM to 20 nM, further preferably 0.8 nM to 20 nM, still more preferably 2 nM to 20 nM. Will be done.
　When the amount of primer for reverse transcription carried into the second reaction system is large, the amplification product by PCR contains an undesired amplification product, and the identification accuracy decreases due to the increase in background in bacterial identification. May occur. Therefore, the concentration of the primer for reverse transcription in the second reaction system is set in the above range.
　Steps (I) and (II) can be carried out in sequence in different reaction vessels or in the same reaction vessel.
　The kit for identifying bacteria according to the present invention has the following components (a) to (c).
(A) Primer for reverse transcription for preparation of cDNA containing the nucleotide sequence for identification of the bacterium to be identified for preparation of the first reaction system for reverse transcription reaction,
(b) For PCR. A primer pair for synthesizing a double-stranded DNA containing a base sequence for identifying a bacterium to be identified for preparation of a second reaction system, and
(c) RNA for preparation of a first reaction system. An enzyme with dependent DNA polymerase activity.
　The kit for identifying bacteria according to the present invention is further desired.
(D) It may have an enzyme having DNA-dependent DNA polymerase activity for preparation of a second reaction system.
　The amount of the primer for reverse transcription included in the kit is 0.08 nM to 20 nM, more preferably 0.4 nM to 20 nM, when the primer for reverse transcription is supplied to the second reaction system via the reaction mixture obtained by the reverse transfer reaction. It is further adjusted to be 0.8 nM to 20 nM, more preferably 2 nM to 20 nM.
　The bacterium identification method according to the present invention and the kit for that purpose can also be used as a detection method for detecting the presence or absence of a bacterium to be detected in a sample.
[0009]
　Hereinafter, a method for identifying bacteria according to the present invention and a preferred form of a kit for that purpose will be described.
[0010]
As
　RNA extraction methods, alkali dissolution method, boiling method, phenol extraction and the like are known, and special RNA extraction kits are also sold by manufacturers.
　The method for extracting RNA from bacteria in a sample in the present invention is not particularly limited, and a known method can be used. Since the optimum method differs depending on the sample, it is desirable to select a method suitable for the target sample. The Roche High Pure RNA Isolation Kit used in the examples of the present application is an example of an RNA extraction method that can be preferably used.

　RNA obtained from bacteria in a sample needs to be converted into cDNA by reverse transcription reaction. The reverse transcription reaction method in the present invention is not particularly limited, and a known method can be used. Promega's M-MLV Reverse Transcriptase, RNase (H-), and Point Mutant used in Examples described later are examples of preferably usable reverse transcriptase methods.
　The amount of RNA and RNA-dependent DNA polymerase added to the first reaction system is not particularly limited, and an amount that enables the synthesis of the desired cDNA may be selected.

　The primer for reverse transcription is used for synthesis of cDNA containing a base sequence used for bacterial identification by a reverse transcription reaction. The primer for reverse transcription is not particularly limited as long as it is a primer capable of synthesizing a cDNA (fragment) containing a base sequence complementary to the base sequence required for bacterial identification in RNA extracted from the bacteria in the sample. Known or newly prepared primers capable of synthesizing DNA containing a base sequence for bacterial identification can be used. Also, commercially available primers can be used.
　As a base sequence for bacterial identification, many base sequences are known in 16S rDNA, and it is preferable to use 16S rRNA corresponding to 16S rDNA as an RNA for bacterial identification.
　When targeting bacterial 16S rRNA, as a primer for reverse transcription, use a known primer complementary to the starting position of cDNA synthesis containing a region of 16S rRNA that can be used for bacterial identification, or a newly prepared primer. Can be used. Also, commercially available primers can be used.
　It is preferable to select the reverse transcription primer so that the cDNA synthesized by the reverse transcription primer has a region that can be amplified by the primer pair for bacterial identification described later.
　An example of a particularly suitable reverse transcription primer is a primer consisting of the following nucleotide sequence of SEQ ID NO: 1.
　SEQ ID NO: 1: AGACCCGGGA ACGTATTC
[0011]
　The amount of the reverse transcription primer added to the first reaction system is an amount required for the desired reverse transcription reaction, and the reaction mixture of the reverse transcription reaction using the first reaction system is added. The concentration of the reverse transcription primer in the reaction system is adjusted to 20 nM or less. The lower limit of the concentration of the primer for reverse transcription in the second reaction system is such that cDNA can be synthesized from RNA to the extent that bacteria present in the sample can be detected and identified. From this point of view, the concentration of the primer for reverse transcription of the second reaction system is preferably 0.08 nM to 20 nM, more preferably 0.4 nM to 20 nM, still more preferably 0.8 nM to 20 nM, and even more preferably 2 nM to. Adjust so that it is in the range of 20nM.
　The following method can be used to adjust the amount of the reverse transcription primer.
(A) When the reaction mixture obtained by the reverse transcription reaction in
　the first reaction system is used alone for PCR by the second reaction system The amount of the reaction mixture obtained in the first reaction system and this reaction Calculate the carry-on concentration of primers for reverse transcription by the reaction mixture relative to the total amount of the second reaction system containing the mixture. However, the amount of reverse transcription primer consumed in the reverse transcription reaction is not considered in the calculation of this concentration. Therefore, the amount of reverse transcription primer used in calculating this concentration is determined based on the amount of reverse transcription primer added to the first reaction system. The concentration of the reverse transfer primer added to the first reaction system is set so that the concentration of the reverse transfer primer brought into the second reaction system calculated in this manner is within the above range.
　The composition of the first reaction system and the reverse transcription reaction so that the desired reverse transcription reaction can be carried out based on the concentration of the primer for reverse transcription of the first reaction system set as described above. It is preferable to set the conditions.
　For example, when the amount of the second reaction system is 10 times the amount of the reaction mixture obtained from the first reaction system, the concentration of the primer for reverse transcription added to the first reaction system. May be set to 10 times the concentration of the primer for reverse transcription contained in the second reaction system. When the concentration of the primer for reverse transcription in the second reaction system is set to 0.08 nM to 20 nM as the above-mentioned preferable range, the concentration of the primer for reverse transcription in the first reaction system is in the range of 0.8 nM to 200 nM. Set to.
　By setting the concentration of the primer for reverse transcription to be sufficiently excessive than the amount required for the reverse transcription reaction, the above-mentioned amount of the primer for reverse transcription brought into the second rebellious system can be calculated. This carry-in amount can be approximately calculated without considering the amount of primer consumed in the reverse transcription reaction.
　Further, the present invention is characterized in that the upper limit of the amount of the primer for reverse transcription brought into the second reaction system is set as a threshold value for obtaining the effect of the present invention. By setting the concentration of the primer for reverse transcription added to the first reaction system without considering the amount of primer consumed in the reverse transcription reaction as described above, the amount consumed in the reverse transcription reaction can be reduced. The amount of the subtracted reverse transcription primer brought into the second reaction system is automatically equal to or less than the above-mentioned threshold.
　In the above-mentioned preparation method (A), the reaction mixture obtained by the reverse transcription reaction is directly second as it is, that is, without adding anything and separating nothing from the reaction mixture. It is used to prepare the reaction system.
(B) When the reaction mixture obtained by the reverse transcription reaction in the first reaction system is diluted and used
　for PCR in the second reaction system , the reverse transcription primer necessary for carrying out the desired reverse transcription reaction When the concentration is used and the reaction mixture by the reverse transcription reaction is used for the preparation of the second reaction solution according to the above preparation method (A), the concentration of the primer for reverse transcription in the second reaction system is 20 nM. If it exceeds, the reactants obtained from the first reaction system are diluted to the concentration of the primer for reverse transcription in the second reaction system described above. The dilution rate in this dilution can be set based on the concentration of the primer for reverse transcription in the first reaction system, the amount of the reaction mixture obtained from the first reaction system, and the amount of the second reaction system.
　Even in the calculation of the concentration of the primer for reverse transcription in this adjustment method (B), the amount of primer consumed in the reverse transcription reaction in the first reaction system can be ignored.
　Further, for diluting the reaction mixture, a liquid medium such as a buffer solution for diluting the primer solution or a liquid medium such as a buffer solution used for preparing a second reaction system for PCR can be used.
[0012]

　The primer pair for PCR, that is, the combination of the forward primer and the reverse primer is not limited as long as it can amplify double-stranded DNA containing a base sequence for bacterial identification. The primer pair can be selected according to the bacterial species to be identified.
　When targeting bacterial 16S rDNA, a primer pair capable of amplifying the bacterial identification base sequence in 16S rDNA can be used for bacterial identification. Various base sequences for bacterial identification are known in 16S rDNA, and as a primer pair, a primer pair capable of amplifying double-stranded DNA containing a known base sequence for bacterial identification can be used.
　As will be described later, when multiple DNA fragments are amplified by PCR from cDNA and bacteria are identified based on the Tm value of those DNA fragments, the variable region existing on 16S rDNA (S. Chakravorty, et al.,, It is preferable to use a primer pair in which double-stranded DNA containing each of them (see Journal of Microbiological Methods 2007, 69, 330-339) is amplified.
　As the primer pair for PCR, one kind or a combination of two kinds can be used. When a plurality of types of plumer sets are used, it is preferable to individually use 4 to 7 types of primer pairs in order to increase the number of identified bacterial species or to improve the identification accuracy.
　Preferred primer pairs include at least one primer pair selected from the following groups (Group A) to (Group C).
(Group A)
-Primer pair 1a: Combined with a
forward primer consisting of the nucleotide sequence of SEQ ID NO: 2 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 3. Primer pair 2a: Forward primer consisting of the nucleotide sequence of SEQ ID NO: 4 and the base of SEQ ID NO: 5. Combined with a reverse primer consisting of a sequence,
・ Primer pair 3a: Combined with a forward primer consisting of the base sequence of SEQ ID NO: 6 and a reverse primer consisting of the base sequence of SEQ ID NO: 7
・ Primer pair 4a: Consisting of the base sequence of SEQ ID NO: 8. Combined with a forward primer and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 9,
・ Primer pair 5a: Combined with a forward primer consisting of the nucleotide sequence of SEQ ID NO: 10 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 11
・ Primer pair 6a: Combined with a forward primer consisting of the nucleotide sequence of SEQ ID NO: 12 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 13.
Primer pair 7a: A forward primer consisting of the nucleotide sequence of SEQ ID NO: 14 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 15. In combination with.
(Group B) A modified forward in which one or two bases are added, deleted or substituted as long as a part of the base sequence of one or both of the primer pairs of the above (Group A) is not impaired as a primer.
A primer pair consisting of a primer pair containing a primer and / or a modified reverse primer, and a primer pair consisting of a complementary sequence corresponding to the base sequence of one or both of the (group C) and (group A) or (group B) primer pairs. ..
[0013]
　Specific examples of the above-mentioned PCR primers include the primer pairs described in the following groups 1 to 7. It is more preferable to individually use 4 to 7 primer pairs out of 7 primer pairs selected from each of these 7 groups.
(1) Primer pair 1st group:
　(1a) Combined with a forward primer consisting of the nucleotide sequence of SEQ ID NO : 2 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 3,
　(1b-1) Consisting of the modified nucleotide sequence of SEQ ID NO: 2. A primer pair consisting of a combination of a modified forward primer and a reverse primer consisting of SEQ ID NO: 3.
　(1b-2) A primer pair composed of a combination of a forward primer consisting of SEQ ID NO: 2 and a modified reverse primer consisting of the modified base sequence of SEQ ID NO: 3.
　(1b-3) A primer pair composed of a combination of a modified forward primer consisting of the modified base sequence of SEQ ID NO: 2 and a modified reverse primer consisting of the modified base sequence of SEQ ID NO: 3.
　(1c-1) A primer pair composed of a combination of a forward primer composed of the complementary sequence of SEQ ID NO: 2 and a reverse primer composed of the complementary sequence of SEQ ID NO: 3.
　(1c-2) A primer pair consisting of a combination of a modified forward primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 2 and a reverse primer consisting of a complementary sequence of SEQ ID NO: 3.
　(1c-3) A primer pair composed of a combination of a forward primer consisting of the complementary sequence of SEQ ID NO: 2 and a modified reverse primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 3.
　(1c-4) A primer pair composed of a combination of a modified forward primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 2 and a modified reverse primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 3.
(2) Primer pair second group:
　(2a) Combined with a forward primer consisting of the nucleotide sequence of SEQ ID NO : 4 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 5,
　(2b-1) Consisting of the modified nucleotide sequence of SEQ ID NO: 4. A primer pair consisting of a combination of a modified forward primer and a reverse primer consisting of SEQ ID NO: 5.
　(2b-2) A primer pair composed of a combination of a forward primer consisting of SEQ ID NO: 4 and a modified reverse primer consisting of the modified base sequence of SEQ ID NO: 5.
　(2b-3) A primer pair composed of a combination of a modified forward primer consisting of the modified base sequence of SEQ ID NO: 4 and a modified reverse primer consisting of the modified base sequence of SEQ ID NO: 5.
　(2c-1) A primer pair composed of a combination of a forward primer composed of the complementary sequence of SEQ ID NO: 4 and a reverse primer composed of the complementary sequence of SEQ ID NO: 5.
　(2c-2) A primer pair consisting of a combination of a modified forward primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 4 and a reverse primer consisting of a complementary sequence of SEQ ID NO: 5.
　(2c-3) A primer pair consisting of a combination of a forward primer consisting of the complementary sequence of SEQ ID NO: 4 and a modified reverse primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 5.
　(2c-4) A primer pair consisting of a combination of a modified forward primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 4 and a modified reverse primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 5.
(3) Primer pair 3rd group:
　(3a) Combined with a forward primer consisting of the nucleotide sequence of SEQ ID NO : 6 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 7,
　(3b-1) Consisting of the modified nucleotide sequence of SEQ ID NO: 6. A primer pair consisting of a combination of a modified forward primer and a reverse primer consisting of SEQ ID NO: 7.
　(3b-2) A primer pair composed of a combination of a forward primer consisting of SEQ ID NO: 6 and a modified reverse primer consisting of the modified base sequence of SEQ ID NO: 7.
　(3b-3) A primer pair composed of a combination of a modified forward primer consisting of the modified base sequence of SEQ ID NO: 6 and a modified reverse primer consisting of the modified base sequence of SEQ ID NO: 7.
　(3c-1) A primer pair composed of a combination of a forward primer composed of the complementary sequence of SEQ ID NO: 6 and a reverse primer composed of the complementary sequence of SEQ ID NO: 7.
　(3c-2) A primer pair consisting of a combination of a modified forward primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 6 and a reverse primer consisting of a complementary sequence of SEQ ID NO: 7.
　(3c-3) A primer pair consisting of a combination of a forward primer consisting of the complementary sequence of SEQ ID NO: 6 and a modified reverse primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 7.
　(3c-4) A primer pair composed of a combination of a modified forward primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 6 and a modified reverse primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 7.
(4) Primer pair 4th group:
　(4a) Combined with a forward primer consisting of the base sequence of SEQ ID NO : 8 and a reverse primer consisting of the base sequence of SEQ ID NO: 9, and
　(4b-1) consisting of a modified base sequence of SEQ ID NO: 8. A primer pair consisting of a combination of a modified forward primer and a reverse primer consisting of SEQ ID NO: 9.
　(4b-2) A primer pair composed of a combination of a forward primer consisting of SEQ ID NO: 8 and a modified reverse primer consisting of the modified base sequence of SEQ ID NO: 9.
　(4b-3) A primer pair composed of a combination of a modified forward primer consisting of the modified base sequence of SEQ ID NO: 8 and a modified reverse primer consisting of the modified base sequence of SEQ ID NO: 9.
　(4c-1) A primer pair composed of a combination of a forward primer composed of the complementary sequence of SEQ ID NO: 8 and a reverse primer composed of the complementary sequence of SEQ ID NO: 9.
　(4c-2) A primer pair consisting of a combination of a modified forward primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 8 and a reverse primer consisting of a complementary sequence of SEQ ID NO: 9.
　(4c-3) A primer pair consisting of a combination of a forward primer consisting of the complementary sequence of SEQ ID NO: 8 and a modified reverse primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 9.
　(4c-4) A primer pair consisting of a combination of a modified forward primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 8 and a modified reverse primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 9.
[0014]
(5) Primer pair 5th group:
　(5a) Combined with a forward primer consisting of the base sequence of SEQ ID NO : 10 and a reverse primer consisting of the base sequence of SEQ ID NO: 11, and
　(5b-1) consisting of a modified base sequence of SEQ ID NO: 10. A primer pair consisting of a combination of a modified forward primer and a reverse primer consisting of SEQ ID NO: 11.
　(5b-2) A primer pair composed of a combination of a forward primer consisting of SEQ ID NO: 10 and a modified reverse primer consisting of the modified base sequence of SEQ ID NO: 11.
　(5b-3) A primer pair composed of a combination of a modified forward primer consisting of the modified base sequence of SEQ ID NO: 10 and a modified reverse primer consisting of the modified base sequence of SEQ ID NO: 11.
　(5c-1) A primer pair composed of a combination of a forward primer composed of the complementary sequence of SEQ ID NO: 10 and a reverse primer composed of the complementary sequence of SEQ ID NO: 11.
　(5c-2) A primer pair consisting of a combination of a modified forward primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 10 and a reverse primer consisting of a complementary sequence of SEQ ID NO: 11.
　(5c-3) A primer pair consisting of a combination of a forward primer consisting of the complementary sequence of SEQ ID NO: 10 and a modified reverse primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 11.
　(5c-4) A primer pair consisting of a combination of a modified forward primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 10 and a modified reverse primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 11.
(6) Primer pair 6th group:
　(6a) Combined with a forward primer consisting of the base sequence of SEQ ID NO : 12 and a reverse primer consisting of the base sequence of SEQ ID NO: 13, and
　(6b-1) consisting of a modified base sequence of SEQ ID NO: 12. A primer pair consisting of a combination of a modified forward primer and a reverse primer consisting of SEQ ID NO: 13.
　(6b-2) A primer pair composed of a combination of a forward primer consisting of SEQ ID NO: 12 and a modified reverse primer consisting of the modified base sequence of SEQ ID NO: 13.
　(6b-3) A primer pair composed of a combination of a modified forward primer consisting of the modified base sequence of SEQ ID NO: 12 and a modified reverse primer consisting of the modified base sequence of SEQ ID NO: 13.
　(6c-1) A primer pair composed of a combination of a forward primer composed of the complementary sequence of SEQ ID NO: 12 and a reverse primer composed of the complementary sequence of SEQ ID NO: 13.
　(6c-2) A primer pair consisting of a combination of a modified forward primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 12 and a reverse primer consisting of a complementary sequence of SEQ ID NO: 13.
　(6c-3) A primer pair consisting of a combination of a forward primer consisting of the complementary sequence of SEQ ID NO: 12 and a modified reverse primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 13.
　(6c-4) A primer pair composed of a combination of a modified forward primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 12 and a modified reverse primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 13.
(7) Primer pair 7th group:
　(7a) Combined with a forward primer consisting of the base sequence of SEQ ID NO : 14 and a reverse primer consisting of the base sequence of SEQ ID NO: 15, and
　(7b-1) consisting of a modified base sequence of SEQ ID NO: 14. A primer pair consisting of a combination of a modified forward primer and a reverse primer consisting of SEQ ID NO: 15.
　(7b-2) A primer pair composed of a combination of a forward primer consisting of SEQ ID NO: 14 and a modified reverse primer consisting of the modified base sequence of SEQ ID NO: 15.
　(7b-3) A primer pair composed of a combination of a modified forward primer consisting of the modified base sequence of SEQ ID NO: 14 and a modified reverse primer consisting of the modified base sequence of SEQ ID NO: 15.
　(7c-1) A primer pair composed of a combination of a forward primer composed of the complementary sequence of SEQ ID NO: 14 and a reverse primer composed of the complementary sequence of SEQ ID NO: 15.
　(7c-2) A primer pair consisting of a combination of a modified forward primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 14 and a reverse primer consisting of a complementary sequence of SEQ ID NO: 15.
　(7c-3) A primer pair consisting of a combination of a forward primer consisting of the complementary sequence of SEQ ID NO: 14 and a modified reverse primer consisting of the complementary sequence of the modified base sequence of SEQ ID NO: 15.
　(7c-4) A primer pair composed of a combination of a modified forward primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 14 and a modified reverse primer consisting of a complementary sequence of the modified base sequence of SEQ ID NO: 15.
[0015]
　The nucleotide sequences of SEQ ID NOs: 2 to 15 are shown below.
SEQ ID NO: 2: GCAGGCTTAA CACATGCAAG TCG
SEQ ID NO: 3: CGTAGGAGTC TGGACCGT
SEQ ID NO: 4: GTCCAGACTC CTACGGGAG
SEQ ID NO: 5: CCTACGTATT ACCGCGG
SEQ ID NO: 6: AGCAGCGCG GTAATA
SEQ ID NO: 7: GGACTACCAG GGTATCTAAT CCT
SEQ ID NO: 8
: AACAGGATTA CATGCTCCAC C
SEQ ID NO: 10: TGGTTTAATT CGATGCAACG C
SEQ ID NO: 11: GAGCTGACGA CAGCCAT
SEQ ID NO: 12: GTTAAGTCCC GCAACGAG
SEQ ID NO: 13: CCATTGTAGC ACGTGTGTAG CC
SEQ ID NO: 14: GGCTACACAC GTGCTACAAT GG
SEQ ID NO: 15: AGACCCGGGA ACGTATTC
[0016]
As the reagents
　other than the primer pair used for PCR analysis in the present invention, known reagents can be used in known combinations. Reagents required for measurement include enzymes for PCR, pH buffer, dNTP, Mg 2+ source, sterilized water purified by filtration and the like. In addition to the above, a fluorescent dye is required for real-time PCR analysis.

　The enzyme for PCR used in the present invention is a DNA-dependent DNA polymerase, and various commercially available ones can be used, but a test for a sample that is particularly negative. Therefore, it is preferable to use a DNA-dependent heat-resistant DNA polymerase that minimizes the amount of DNA contamination derived from bacteria that causes false positives. Specific examples thereof include those produced in eukaryotes, those treated with high purification, and those treated with EMA (ethidium monoazide) or PMA (propidium monoazide), which are selective membrane-permeable dyes. , Not limited to this.

　The cDNA and primer pair as the reaction product obtained by the reverse transcription reaction are used in the PCR method. As the PCR method, various PCR methods can be used as long as it is a PCR method for amplifying double-stranded DNA containing a base sequence used for identification of bacteria. A preferred analytical method is real-time PCR, more preferably a combination of real-time PCR and melting curve analysis for Tm value measurement.
　The conditions of the temperature cycle in PCR are not particularly limited, but the conditions implemented in the examples described later, "95 ° C, 5 minutes → 94 ° C, 10 seconds / 60 ° C, 20 seconds / 72 ° C, 10 seconds / 85". 40 cycles of ℃, 2 seconds → 95 ℃, 10 seconds ”is an example.
　In real-time PCR, after the DNA amplification step is completed, the Tm value of the amplified DNA can be measured by melting curve analysis. Such measurements are possible with many models of real-time PCR devices and can be performed according to how the device is used.
[0017]
In
　order to identify bacteria in a sample, the Tm value of a double-stranded DNA fragment contained in an amplification product obtained from a reaction mixture containing cDNA can be used using the primer pair according to the present invention. ..
　The following methods can be mentioned as preferable identification methods.
　First, using 16S rRNA or 16S rDNA of a plurality of species, preferably a large number of bacteria, which may be contained in a sample, by the identification method according to the present invention, or by utilizing a part of the method according to the present invention. The double-stranded DNA fragment and its Tm value are acquired in advance by the method described above, and the Tm value (measured value itself) or the data secondarily obtained from the measured Tm value is acquired and stored. Keep it. For example, the “relative value of Tm value” described later is saved as comparison data or a database. This comparative data is data obtained in advance from the "relative value of Tm value" of a double-stranded DNA fragment obtained from a known bacterium. In addition, this database of comparative data stores data obtained by comprehensively acquiring the above-mentioned comparative data for double-stranded DNA fragments from a plurality of bacteria.
　Next, the Tm value or "relative value of the Tm value" obtained from the bacteria in the sample can be compared with this stored comparative data or database to identify the bacteria of unknown species in the sample. ..
　As an algorithm for identification, not only the combination of the Tm values ​​themselves described above but also the combination of the differences between the Tm values ​​is used for identification, so that the influence of the measurement error such as the measurement error for each trial of the device is used. It is possible to add a process that minimizes.
　As a method of correcting the measurement error for each trial of the device, the "mean value of the combination of Tm values" obtained for each of a plurality of primer pairs is calculated, and the "relative" of each Tm value from the average value is calculated. "Combination of values" can be used. As this relative value, a value obtained by taking the difference between each Tm value from the average value can be used, and this relative value is the above-mentioned “relative value of Tm value”. In other words, it is a method of identifying the arrangement of combinations of Tm values ​​as "shape". The "shape" that shows the arrangement of the combination of Tm values ​​in two dimensions is not affected by the measurement error. For example, the combination of Tm values ​​specific to the detected bacteria (n (n is an integer of 4 or more and 7 or less)) is T1db to Tndb (db is database), and the relative values ​​from the average value are d1db to dndb, respectively. And. Similarly, the combination of Tm values ​​(n (n is an integer of 4 or more and 7 or less)) of unknown detection target organisms obtained from the sample is T1ref to Tnref (ref is reference), and the relative value from the average value. Let d1ref to dnref, respectively. Then, it is compared with the database, and "the one in which the combination of relative values ​​is approximate = the one in which the arrangement of the combination of Tm values ​​is close in shape" is used as the identification algorithm.
　Specific calculation methods include, for example, a method of calculating the distance between two points in Euclidean space (Equation 1), but the present invention is not limited to this.
[0018]
[Number 1]

[0019]
　If the calculation method is based on the formula 1, the one having the Dist. Value closest to 0 obtained by this formula is identified as the detected bacterial species. However, due to the measurement error of the PCR equipment used, the allowable range of the Dist. Value is 0 to 0.37, preferably 0 to 0.30, although it depends on the temperature control specifications of the equipment and the number of primers.
　The above algorithm can be used as database-type identification software on a computer.
　Bacterial species identification using the relative values ​​of the above Tm values, that is, bacterial species identification by the Tm mapping method can be described, for example, in Patent Document 2, Paragraph [0237] of International Publication No. 2010/082640 or International Publication No. 2015/053293. Paragraphs [0111] to [0116] may be carried out according to the method of disclosure, or the method of disclosure may be appropriately modified thereto. In an example of bacterial species identification by the Tm mapping method, a combination of Tm values ​​of a plurality of amplification products amplified by a plurality of specific primer pairs obtained from a bacterium of a known bacterial species, or a difference between a plurality of Tm values. Identification data for the combination of is used. The combination of Tm values ​​of amplification products obtained by the same multiple primer pairs from unknown bacterial cell samples collected from the sample, or the combination of differences between Tm values ​​is compared with the identification database, and the consistency is judged. , Identify unknown bacteria in the sample.
[0020]

　The kit for bacterial identification according to the present invention has the following components (a) to (c) described above.
(A) A primer for reverse transcription for preparing a cDNA containing a base sequence for identifying a bacterium to be identified, for preparing a first reaction system for a reverse transcription reaction.
(B) A primer pair for synthesizing double-stranded DNA containing a base sequence for identification of a bacterium to be identified for preparation of a second reaction system for PCR.
(C) An enzyme having RNA-dependent DN A polymerase activity for the preparation of the first reaction system.
　In addition to these constituents, it may have the following (d).
(D) An enzyme having DNA-dependent DNA polymerase activity for the preparation of a second reaction system.
　Further, an instruction manual describing the procedures for the reverse transcription reaction and PCR, for example, the procedures for steps (I) to (III) mentioned above, may be attached to the kit.
　Further, in this instruction manual, the concentration of the primer for reverse transcription contained in the second reaction system is 20 nM or less, preferably 0.08 nM to 20 nM, more preferably 0.4 nM to 20 nM, and further preferably 0.8 nM to 20. It is preferable that the procedure, for example, the adjustment methods (A) and (B) mentioned above, is described so as to be nM, more preferably 2nM to 20nM.
　The instruction manual states that the reaction using the first reaction system and the reaction using the second reaction system can be carried out in different reaction vessels or in the same reaction vessel in this order. A description may be included.
　The kit for bacterial identification includes each reagent listed above in the section , for example, PCR enzyme, pH buffer, dNTP, Mg 2+ source, sterile water purified by filtration, etc. In addition, it may have at least one kind such as a fluorescent dye for real-time PCR analysis. Furthermore, when identifying a bacterial species using the Tm values ​​listed above, a medium containing Tm values ​​obtained from known bacterial species or comparative data or databases may be added to the kit. In addition, these stored data may be provided to the user via the Internet, and the instruction manual explains this point and the database-type identification software that can be used on the computer mentioned above. May include.
　In addition, the kit may further include a positive control and a negative control for identification.
[0021]

　If the identifiable microorganism corresponds to a bacterium in terms of classification, it can be mechanically detected and the bacterial species can be identified. Specific bacterial species include Achromobacter denitrificans, Achromobacter xylosoxidans, Acinetobacter baumannii, Acinetobacter calcoaceticus, Actinomyces israelii, Aerococcus christensenii, Aeromonas hydrophila, Aeromonas sobria, Aggregatibacter actinomycetemcomitans, Alcalige Arcanobacterium pyogenes, Arthrobacter cumminsii, Atopobium vaginae, Bacillus anthracis, Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillides sphaericus, Bacillus subtilis, Bacillus subtilis , Bacteroides uniformis, Bacteroides vulgatus, Bartonella henselae, Bartonella quintana, Bifidobacterium
Example
[0022]
(Production Example 1)
　The DNA polymerase used in the present invention was produced by the method described in paragraphs 0195 to 0205 of Patent Document 1.
(Production Example 2)
　The database manufactured by Mitsui Chemicals used in the present invention was created by the method described in paragraphs 0022 to 0030 of Patent Document 2.
(Microbial strain)
　MRSA (Methicillin-resistant Staphylococcus aureus): JMC16555 strain (catalog number), Escherichia coli: JCM1649T (catalog number), Pseudomonas aeruginosa: JCM5962T strain (catalog number) used in the following examples are all RIKEN. It is publicly available from the Japan Collection of Microorganisms (JCM) (Address: 3-1-1 Takanodai, Tsukuba City, Ibaraki Prefecture, 305-0074).
[0023]
(Example 1)
　MRSA (Methicillin-resistant Staphylococcus aureus) was inoculated on LB agar medium, cultured at 30 ° C. overnight, and stored at 4 ° C. after colony formation. The colonies were inoculated into 2 ml of LB liquid medium and cultured with shaking at 30 ° C. and 180 rpm for 16 hours. For the culture solution, the number of bacteria was measured using CytoFLEX (Beckman Coulter), and the bacterial concentration of the undiluted solution was determined.
　The stock solution of the MRSA culture solution obtained above was diluted with 1 × PBS so that the number of bacteria per 200 μl was 100 CFU, 500 CFU, 1,000 CFU, 5,000 CFU, and 10,000 CFU. 200 μl of each diluted culture was added to Pathogen Lysis Tubes (Qiagen). Furthermore, 100 μl of Reagent DX included with the product and 400 μl of Lysis /-Binding Buffer included with the High Pure RNA Isolation Kit (Roche) were added, set in the Delta Mixer Se-08 (Tietech), and vortexed for 10 minutes.
　Set the column attached to the High Pure RNA Isolation Kit (Roche) in the reservoir, transfer 550 μl of the supernatant of the above bacterial crushed solution to the column, and use a desktop micro high-speed centrifuge CT15E (Hitachi Koki) to make 8,000 x g. Centrifuge for 1 minute. After setting the column in a new reservoir, 500 μl of Wash Buffer I attached to the High Pure RNA Isolation Kit (Roche) was added, and the mixture was centrifuged at 8,000 × g for 1 minute. The column was set in a new reservoir again, 500 μl of Wash Buffer II included in the High Pure RNA Isolation Kit (Roche) was added, and the mixture was centrifuged at 8,000 × g for 1 minute. The column was set in a new reservoir, 200 μl of Wash Buffer II included in the High Pure RNA Isolation Kit (Roche) was added, and the mixture was centrifuged at 13,000 × g for 2 minutes. The column was set in a new 1.5 ml Eppendorf tube, 50 μl of the Elution Buffer attached to the High Pure RNA Isolation Kit (Roche) was added, and the mixture was centrifuged at 8,000 × g for 1 minute to collect the RNA solution.
[0024]
　10.75 μl of the recovered RNA solution and 1 μl of a primer for reverse transcription (consisting of the nucleotide sequence of SEQ ID NO: 1) (invitrogen) at each concentration of 0.005 μM to 100 μM are mixed and incubated at 70 ° C. for 5 minutes, and then 4 ° C. Incubated for 5 minutes. The final concentration of the primer for reverse transcription in the first reaction system for reverse transcription reaction is 0.2 nM to 4000 nM. After incubation, M-MLV RT (H-) Point Mutant 1 μl and M-MLV included with M-MLV Reverse Transcriptase, RNase (H-), Point Mutant (Promega) are added to the mixture of RNA and reverse transcription primers. RT 5 × Reaction Buffer 5 μl, dNTP (Nippon Gene) 6.25 μl, RNase Inhibitor Recombinant type (ToYoBo) 0.2 μl, 1 × PBS 0.8 μl were added to obtain the first reaction system (solution). The obtained first reaction system was incubated at 50 ° C. for 20 minutes to obtain a liquid reaction mixture containing the synthesized cDNA.
　PCR was performed using the cDNA (2 μl) in the reaction mixture thus obtained as a template. The final concentration of the reverse transcription primer, which was 0.2 nM to 4000 nM during the reverse transcription reaction, in the second reaction system (solution) for PCR is 0.02 nM to 400 nM. The PCR solution as the second reaction system for PCR is 10 × Buffer (Nippon Gene) 2 μl, 2.0 mM CleanAMP TM Hot Start PCR (TriLink) 2 μl, 25 mM MgCl 2(Nippongene) 2 μl, Primer pair for 5 μM PCR (invitrogen) 1.2 μl, 1 U / μl DNA polymerase (produced by the method described in paragraphs 0195 to 0205 of Patent Document 1) 1 μl, Evagreen (Biotium) 1 μl, DW (Nacalai Tesque) ) Consists of 8.8 μl.
[0025]
　The following primer pairs were individually used as the primer pairs for PCR.
-Primer pair 1a: Combined with a
forward primer consisting of the nucleotide sequence of SEQ ID NO: 2 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 3. Primer pair 2a: Forward primer consisting of the nucleotide sequence of SEQ ID NO: 4 and the base of SEQ ID NO: 5. Combined with a reverse primer consisting of a sequence,
・ Primer pair 3a: Combined with a forward primer consisting of the base sequence of SEQ ID NO: 6 and a reverse primer consisting of the base sequence of SEQ ID NO: 7
・ Primer pair 4a: Consisting of the base sequence of SEQ ID NO: 8. Combined with a forward primer and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 9,
・ Primer pair 5a: Combined with a forward primer consisting of the nucleotide sequence of SEQ ID NO: 10 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 11
・ Primer pair 6a: Combined with a forward primer consisting of the nucleotide sequence of SEQ ID NO: 12 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 13.
Primer pair 7a: A forward primer consisting of the nucleotide sequence of SEQ ID NO: 14 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 15. In combination with.
[0026]
　After preparing the PCR solution, the PCR tube was set in Rotor-Gene Q (Qiagen) and real-time PCR was performed. The reaction conditions for PCR were 95 ° C, 5 minutes → 94 ° C, 10 seconds / 60 ° C, 20 seconds / 72 ° C, 10 seconds / 85 ° C, 2 seconds for 40 cycles → 95 ° C, 10 seconds. Bacteria were identified by collating the Tm value (melting temperature) of each PCR product obtained from the analysis data with the database manufactured by Mitsui Chemicals (prepared by the method described in paragraphs 0022 to 0030 of Patent Document 2).
　The results identified by the method described above are shown in Table 1. The evaluation in Table 1 was performed as follows.
〇: Identifiable
Δ: Identifiable but peak dirty
×: Unidentifiable
―: Test not performed In
　addition, the results of agarose gel electrophoresis on the PCR product of 100 CFU with the added primer concentration of 2, 5, 10 μM, It is shown in FIG. FIG. 1 (A) shows the results of agarose gel electrophoresis on the added PCR product when the primer concentration for reverse transcription was 2 μM. FIG. 1B shows the results of agarose gel electrophoresis on the added PCR product when the primer concentration for reverse transcription was 5 μM. FIG. 1C shows the results of agarose gel electrophoresis on the added PCR product when the primer concentration for reverse transcription was 10 μM. Each lane indicated by the numbers in FIGS. 1 (A) to 1 (C) shows the results of electrophoresis of the following products and compounds.
(1) PCR product of primer pair 1a
(2) PCR product of primer pair 2a
(3) PCR product of primer pair 3a
(4) PCR product of primer pair 4a
(5) PCR product of primer pair 5a
(6) PCR product of primer pair 6a
(7) PCR product of primer pair 7a
(8) marker
[0027]
[table 1]

[0028]
　As shown in Table 1, it was found that whether or not the bacteria could be identified depends on the concentration of the reverse transcription primer for each number of bacteria.
　It was also found that by appropriately adjusting the reaction conditions, identification can be performed with sufficient sensitivity even with a small number of bacteria.
[0029]
(Example 2) Using
　E. coli, the primer concentration and the number of bacteria were examined. This is the same as in Example 1 except that the bacterium is E. coli. The identification results are shown in Table 2. The results of agarose gel electrophoresis are shown in FIG. FIGS. 2 (A) to 2 (C) show the results of agarose gel electrophoresis at primer concentrations of 2 μM, 5 μM, and 10 μM of the reverse transcription primer solution added in the same manner as in FIG. Each lane indicated by the numbers in FIGS. 2 (A) to 2 (C) shows the results of electrophoresis of the following products and compounds.
FIG. 2 (A)
(1) Marker
(2) PCR product of primer pair 1a
(3) PCR product of primer pair 2a
(4) PCR product of primer pair 3a
(5) PCR product of primer pair 4a
(6) Primer PCR product of pair 5a
(7) PCR product of primer pair 6a
(8) PCR product of primer pair 7a
・ FIG. 2 (B)
(1) Marker
(2) PCR product of primer pair 1a
(3) PCR of primer pair 2a Product
(4) PCR product of primer pair 3a
(5) PCR product of primer pair 4a
(6) PCR product of primer pair 5a
(7) PCR product of primer pair 6a
(8) PCR product of primer pair 7a
(9) Marker
-Fig. 2 (C)
(1) PCR product of primer pair 1a
(2) PCR product of primer pair 2a
(3) Primer PCR product of pair 3a
(4) PCR product of primer pair 4a
(5) PCR product of primer pair 5a
(6) PCR product of primer pair 6a
(7) PCR product of primer pair 7a
(8) Marker
[0030]
[Table 2]

[0031]
　As shown in Table 2, it was found that whether or not the bacteria could be identified depends on the concentration of the reverse transcription primer for each number of bacteria.
　It was also found that by appropriately adjusting the reaction conditions, identification can be performed with sufficient sensitivity even with a small number of bacteria.
[0032]
(Example 3) The
　primer concentration and the number of bacteria were examined using P. aeruginosa. This is the same as in Example 1 except that the bacterium was P. aeruginosa. The identification results are shown in Table 3. The results of agarose gel electrophoresis are shown in FIG. 3 (A) to 3 (C) show the results of agarose gel electrophoresis at primer concentrations of 2 μM, 5 μM, and 10 μM of the primer solution for reverse transcription added in the same manner as in FIG. Each lane indicated by the numbers in FIGS. 3 (A) to 3 (C) shows the results of electrophoresis of the following products and compounds.
FIG. 3 (A)
(1) Marker
(2) PCR product of primer pair 1a
(3) PCR product of primer pair 2a
(4) PCR product of primer pair 3a
(5) PCR product of primer pair 4a
(6) Primer PCR product of pair 5a
(7) PCR product of primer pair 6a
(8) PCR product of primer pair 7a
(9) Markers
-FIGS. 3 (B) and (C)
(1) PCR product of primer pair 1a
(2) Primer PCR product of pair 2a
(3) PCR product of primer pair 3a
(4) PCR product of primer pair 4a
(5) PCR product of primer pair 5a
(6) PCR product of primer pair 6a
(7) PCR product of primer pair 7a
(8) marker
[0033]
[Table 3]

[0034]
　As shown in Table 3, it was found that whether or not the bacteria can be identified depends on the concentration of the reverse transcription primer for each number of bacteria.
　It was also found that by appropriately adjusting the reaction conditions, identification can be performed with sufficient sensitivity even with a small number of bacteria.
[0035]
(Example 4)
　Using the MRSA used in Example 1, the detection sensitivity in this method was confirmed. The number of bacteria per sample was 20 CFU and 40 CFU, a primer solution for reverse transcription with a primer concentration of 1 μM was used, and the final concentration of the primer for reverse transcription in the second reaction system was 4 nM. An identification test was performed in the same manner as in Example 1. Table 4 shows the obtained Tm values ​​and identification results for the primer pairs 1a to 7a.
[0036]
[Table 4]

Industrial applicability
[0037]
　According to the method of the present invention, infectious disease-causing bacteria, particularly sepsis-causing bacteria, can be rapidly detected and identified, and a rapid diagnostic method for sepsis is realized.
　That is, since the present invention makes it possible to construct a system for identifying the causative bacteria by one PCR, it is possible to reduce the work load and fully automate the inspection.
The scope of the claims
[Claim 1]
　A method for identifying a bacterium, which comprises the following steps (1) to (3).
(1) A first containing a primer for reverse transcription for preparing a cDNA containing a nucleotide sequence for identifying a target bacterium, RNA extracted from the bacterium in a sample, and an enzyme having RNA-dependent DNA polymerase activity.
(2) The reaction mixture and the base sequence for identification of the bacterium to be identified are obtained by carrying out a reverse transcription reaction using the above reaction system to obtain a reaction mixture containing the synthesized cDNA and the primer for reverse transcription. A step of performing PCR using a second reaction system containing a primer pair for synthesizing the containing double-stranded DNA and an enzyme having DNA-dependent DNA polymerase activity, provided that the reaction is carried out in the second reaction system. The concentration of the primer for reverse transcription supplied from the mixture is 0.08 nM or more and 20 nM or less, and
(3) the base sequence for identification of the bacterium to be identified is contained from the second reaction system after PCR. A step of detecting the formation of double-stranded DNA.
[Claim 2]
　The method for identifying bacteria according to claim 1, wherein the steps (1) and (2) are sequentially carried out in another reaction vessel or in the same reaction vessel.
[Claim 3]
　The invention according to claim 1 or 2, wherein the nucleotide sequence for identification of the bacterium to be identified is a nucleotide sequence contained in the 16S rDNA of the bacterium, and the RNA contains the 16S rRNA of the bacterium in the sample. Bacterial identification method.
[Claim 4]
　The method for identifying a bacterium according to claim 3, wherein the primer for reverse transcription comprises the nucleotide sequence of SEQ ID NO: 1.
[Claim 5]
　The method for identifying a bacterium according to claim 3 or 4, wherein the primer pair is at least one primer pair selected from the group consisting of the following (group A) to (group C).
(Group A)
・ Primer pair 1a: Combined with a
forward primer consisting of the nucleotide sequence of SEQ ID NO: 2 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 3 ・ Primer pair 2a: Forward primer and sequence consisting of the nucleotide sequence of SEQ ID NO: 4 Combined with a reverse primer consisting of the base sequence of No. 5,
・ Primer pair 3a: Combined with a forward primer consisting of the base sequence of SEQ ID NO: 6 and a reverse primer consisting of the base sequence of SEQ ID NO: 7
・ Primer pair 4a: of SEQ ID NO: 8. reverse primer combination consisting of the nucleotide sequence of the forward primer and SEQ ID NO: 9 comprising the nucleotide sequence,
primer pairs 5a: reverse primer combination consisting of the nucleotide sequence of the forward primer and SEQ ID NO: 11 comprising the nucleotide sequence of SEQ ID NO: 10,
& Primer pair 6a: Combined with a forward primer consisting of the nucleotide sequence of SEQ ID NO: 12 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 13.
・ Primer pair 7a: Forward primer consisting of the nucleotide sequence of SEQ ID NO: 14 and the nucleotide sequence of SEQ ID NO: 15 Combined with a reverse primer consisting of.
(Group B) A primer pair in which one or two bases are added, deleted or substituted as long as a part of the base sequence of one or both of the primer pairs of the above (Group A) is not impaired as a primer. ,as well as
(Group C) A primer pair consisting of a complementary sequence corresponding to the base sequence of one or both of the primer pairs of (Group A) or (Group B).
[Claim 6]
　In the step (3), the Tm value of the generated double-stranded DNA was measured, and the Tm of the double-stranded DNA containing the obtained Tm value and the previously measured base sequence for identification of the bacterium to be identified. The bacterium identification method according to any one of claims 1 to 5, wherein the bacterium in the sample is identified by comparing the values ​​or the data obtained secondarily from the values.
[Claim 7]
　The method for identifying a bacterium according to any one of claims 1 to 6, wherein after the step (1), the reaction mixture is used alone or diluted directly or in the step (2).
[Claim 8]
　A kit for identifying the bacteria in the sample by PCR using the cDNA contained in the reaction mixture by the reverse transcription reaction of RNA extracted from the bacteria in the sample as a template, wherein the following
　(a) to (c) are a,
(a) a first reaction system for the preparation of primers for reverse transcription to prepare a cDNA comprising the nucleotide sequence for identification of the identification target bacteria for the reverse transcription reaction,
(b) A primer pair for synthesizing double-stranded DNA containing a base sequence for identifying the bacterium to be identified for preparation of a second reaction system for PCR, and
(c) RNA-dependent DNA polymerase activity. enzyme with,
　the second reaction system comprises a reaction mixture by the reverse transcription reaction, reversal when the amount of the primer for the reverse transcription, is supplied to the second reaction system via a reaction mixture
A kit characterized in that the concentration of the primer for copying is adjusted to 0.08 nM or more and 20 nM or less .
[Claim 9]
　The kit according to claim 8, further comprising an instruction manual describing the reverse transcription reaction and the PCR procedure.
[Claim 10]
　The ninth aspect of the present invention is the instruction manual, wherein the procedure is described so that the concentration of the primer for reverse transcription contained in the second reaction system is 0.08 nM or more and 20 nM or less. Kit.
[Claim 11]
　Any of claims 8 to 10, wherein the base sequence for identifying the bacterium to be identified is a base sequence contained in the 16S rDNA of the bacterium, and the RNA contains the 16S rRNA of the bacterium in the sample. The kit according to 1.
[Claim 12]
　The kit according to claim 11, wherein the primer for reverse transcription comprises the base sequence of SEQ ID NO: 1.
[Claim 13]
　The kit according to claim 11 or 12, wherein the primer pair is at least one primer pair selected from the group consisting of the following (group A) to (group C).
(Group A)
・ Primer pair 1a: Combined with a
forward primer consisting of the nucleotide sequence of SEQ ID NO: 2 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 3 ・ Primer pair 2a: Forward primer and sequence consisting of the nucleotide sequence of SEQ ID NO: 4 Combined with a reverse primer consisting of the base sequence of No. 5,
・ Primer pair 3a: Combined with a forward primer consisting of the base sequence of SEQ ID NO: 6 and a reverse primer consisting of the base sequence of SEQ ID NO: 7
・ Primer pair 4a: of SEQ ID NO: 8. reverse primer combination consisting of the nucleotide sequence of the forward primer and SEQ ID NO: 9 comprising the nucleotide sequence,
primer pairs 5a: reverse primer combination consisting of the nucleotide sequence of the forward primer and SEQ ID NO: 11 comprising the nucleotide sequence of SEQ ID NO: 10,
& Primer pair 6a: Combined with a forward primer consisting of the nucleotide sequence of SEQ ID NO: 12 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 13.
・ Primer pair 7a: Forward primer consisting of the nucleotide sequence of SEQ ID NO: 14 and the nucleotide sequence of SEQ ID NO: 15 Combined with a reverse primer consisting of.
(Group B) A primer pair in which one or two bases are added, deleted or substituted as long as a part of the base sequence of one or both of the primer pairs of the above (Group A) is not impaired as a primer. ,as well as
(Group C) A primer pair consisting of a complementary sequence corresponding to the base sequence of one or both of the primer pairs of (Group A) or (Group B).