JP2015057958A - Quantitative method of nucleic acid, primer set used for the same, dna chip and assay kit, and judging method of normal flora using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide means for easily quantifying nucleic acids in a sample for grasping the amount of hepatitis C virus or the like.SOLUTION: A method of the embodiment comprises: preparing a sample comprising nucleic acids, a primer set comprising a first primer and a second primer, and a concentration standard nucleic acid; subjecting the sample, the primer set, and the first concentration standard nucleic acid of known concentration to amplification reaction by the LAMP method in one reaction environment to obtain amplification products; reacting the first nucleic acid probe and the second nucleic acid probe immobilized on the substrate with the amplification products; and detecting and comparing the amount of hybridization to quantify the nucleic acids in the sample.

Description

  Embodiments of the present invention relate to a nucleic acid quantification method, a primer set used therefor, a DNA chip and an assay kit, and a method for determining resident bacteria using the same.

  In general, blood virus concentration is an important indicator of the degree of viremia. For example, in the case of hepatitis C virus (HCV), patients with low blood virus concentration have low values of AST and ALT indicating liver function and liver damage is minor. Conversely, patients with high blood virus levels often have severe liver damage and impaired liver function. Furthermore, blood virus concentration also affects the effectiveness of antiviral therapy. In the case of the same HCV, there is a high possibility that interferon therapy will be effective in cases with low blood viral load, but there is a tendency that a therapeutic effect is difficult to obtain in cases with high blood viral load.

  There are about 2 million people infected with hepatitis C virus in Japan. About 70% of them develop chronic hepatitis. These patients are known to develop liver cancer at a high rate after the lapse of 10 to 20 years, and it is strongly desired to completely eliminate the virus in chronic hepatitis. Interferon therapy is considered to be an effective method for eliminating viruses, but in practice, monotherapy is effective only in about 30% of patients. On the other hand, interferon therapy has serious side effects and treatment is expensive. Therefore, measuring the amount of viral virus in the blood before the start of treatment and predicting the effect of interferon therapy to some extent, and selecting patients who are likely to respond to treatment are important for reducing the burden on patients.

  The effect of interferon therapy for chronic hepatitis C is often defined mainly by viral factors. Factors that are considered to be particularly influential are virus type (ie genotype) and viral load. These two factors are related to each other. That is, there is a tendency that the viral load tends to increase in the genotype 1b, and conversely, the viral load does not increase so much in the 2a and 2b types. In addition, there is a report that how the viral load changes after the start of treatment is reflected in the therapeutic effect (Chayama K et al., Viral Hepatitis and Liver Disease. 1994, 617-620). Measuring the viral load is an important judgment material in determining the treatment policy or proceeding with the treatment.

  In addition, measuring the amount of mRNA expressed in a tissue is considered to be an important indicator for observing an individual's reactivity to a certain drug. For example, when considering the effect of interferon therapy for chronic hepatitis C, hepatic tissues are collected before and after the start of treatment, and the amount of mRNA expressed in each is compared with several genes. It becomes possible to examine the reactivity of the individual to interferon. For this purpose, first, the expression patterns of several genes are compared before and after the start of treatment, and the pattern of meaningful expression changes of genes is specified in order to define the presence or absence of the effect of interferon. From the results, the change in the expression level of a specific gene before and after the start of treatment is measured. Thereby, the therapeutic effect can be predicted in advance.

  The amount of these viruses is measured using Competitive PCR method, Branched DNA probe assay (manufactured by Bayer), Amplicore monitor method (manufactured by Roche Diagnostics), TaqMan PCR method (manufactured by Applied Biosystems), etc. It has been broken. The Competitive PCR method calculates viral load by competitive PCR using a concentration standard nucleic acid. Electrophoresis is used for detection, and some experience is required to read the electrophoretic image. Moreover, it is necessary to run several PCRs per sample, and the procedure is complicated. In order to perform Branched DNA probe assay, a special technique for synthesizing Branched DNA probe is required. Amplicon and TaqMan methods require a special measuring device.

  Conventional methods for measuring the amount of mRNA in tissues include TaqMan method (Applied Biosystems Co., Ltd.) that requires special equipment and nucleic acid labeling, methods using various microarrays, etc. Can be mentioned.

  On the other hand, a bacterial culture method has been conventionally employed to examine the amount of infectious cells in an infection caused by bacteria. In this technology, a sample is applied to a medium suitable for each bacterium, and kept at an appropriate temperature for about 1 to 3 days to visualize one cell (colony formation) and count the number of colonies. By doing this, the amount of bacterial cells in the applied sample amount is calculated. In this method, it takes time to form colonies, and in order to form colonies, it is necessary to select a medium suitable for the bacteria to be tested, and the amount of specimen that can be applied is limited. In addition, some bacterial species are originally unsuitable for culture.

Kaneko S et al. , J. et al. Med. Virol. 37, 278-282, 1992 Chayama K et al. , J Gastroenterol. Hepatol. 8, S40-44, 1993

  An object of the present invention is to provide a means for easily quantifying a nucleic acid in a sample.

  A method for quantifying a nucleic acid contained in a sample for solving the above-mentioned problem is to prepare a sample containing a nucleic acid, a primer set containing a first primer and a second primer, and a concentration standard nucleic acid, An amplification product is obtained by performing an amplification reaction by the LAMP method in a single reaction environment with the sample, the primer set, and the first concentration standard nucleic acid having a known concentration, and the first immobilized on the substrate. Reacting the nucleic acid probe and the second nucleic acid probe with the amplification product, detecting and comparing the amount of hybridization, and quantifying the nucleic acid in the sample. The target template sequence to be amplified by the primer set includes F3 region, F2 region, first probe binding region and F1 region from the 5 ′ end side, and B3c region, B2c region, LBc region and B1c region from the 3 ′ end side. including. The concentration standard nucleic acid includes an F3 region from the 5 ′ end side, an F2 region, a second probe binding region having a sequence different from that of the first probe binding region, and an F1 region, and a B3c region from the 3 ′ end side. A B2c region, an LBc region, and a B1c region.

The figure which shows a target template nucleic acid and a primer set. The figure which shows the principle of one example of embodiment. The figure which shows an example of embodiment. The figure which shows an example of embodiment. The figure which shows an example of embodiment. The figure which shows an example of embodiment. The figure which shows an example of the result of having quantified Escherichia coli from the sample whose bacterial culture result is known. The figure which shows the result of Example 1. FIG. The figure which shows the result of Example 2. FIG. The figure which showed the DNA chip used in Example 3 typically. The figure which shows the result of Example 3. The figure which shows the result of Example 4. The enlarged view about a part of result of Example 4 shown in Drawing 12A.

  Hereinafter, embodiments of the present invention will be described in detail.

  By this method, it is possible to measure the amount of a specific nucleic acid in a specimen. In order to know the abundance of a specific nucleic acid of interest in a sample, the method amplifies with a primer set using the nucleic acid to be detected as a target template nucleic acid, and uses the same primer set for competitive concentration. Amplify using standard nucleic acid as template. These competitive amplifications are performed in one reaction environment. At this time, the amount of the concentration standard nucleic acid is known. The obtained amplification product is reacted with a nucleic acid probe, and the resulting hybridization is detected. By comparing the amount of hybridization based on the target template nucleic acid and the amount of hybridization based on the concentration standard nucleic acid, the amount of the target template nucleic acid in the sample is determined. More types by increasing the types of concentration standard nucleic acids and amplifying them with the same primer set in a single reaction environment with different amounts of each concentration standard nucleic acid, for example, stepwise different amounts It is possible to determine the amount of target template nucleic acid.

  In other words, the accuracy of the quantification method can be adjusted as desired according to the type and number of concentration standard nucleic acids used and the concentration. When it is desired to know a rough abundance, a concentration standard nucleic acid of a smaller kind, for example, one kind or two kinds, may be used. It is also possible to use a specific reference, for example, a threshold value in advance as desired, and to use the method as a method for determining whether the amount of bacteria in the sample is larger or smaller than the reference value. .

  The nucleic acid contained in the sample is referred to as “sample nucleic acid”. Among the sample nucleic acids, a nucleic acid to be quantified by amplification with a primer is referred to as a “target template nucleic acid”. The nucleic acid to be amplified by the primer is called “template sequence”. A nucleic acid containing a template sequence is referred to as a “template nucleic acid” or “template”.

  The target template nucleic acid 1 and the primer set will be described with reference to FIG.

  The target template nucleic acid 1 includes F3 region, F2 region, first probe binding region 2 and F1 region in this order from the 5 ′ end side, and B3c region, B2c region, LBc region and B1c from the 3 ′ end side. Includes areas in this order. When the target template nucleic acid 1 is present in a double strand, the complementary sequence of the target template nucleic acid 3 includes the B3 region, the B2 region, the LB region, and the B1 region in this order from the 5 ′ end side, and From the 3 ′ end side, the F3c region, the F2c region, the complementary region of the first probe binding region, and the F1c region are included in this order. The F3 region, the F2 region, the F1 region, the B3 region, the B2 region, the B1 region, and the LB region have an F3 array, an F2 array, an F1 array, a B3 array, a B2 array, a B1 array, and an LB array, respectively. The F3c region, F2c region, F1c region, B3c region, B2c region, B1c region, and LBc region have sequences complementary to the F3 region, F2 region, F1 region, B3 region, B2 region, B1 region, and LB region, respectively. .

  The primer set includes a first primer 4 and a second primer 5. The first primer 4 is an FIP primer having an F1c sequence complementary to F1 on the 5 ′ end side and having the same F2 sequence on F2 on the 3 ′ end side. The second primer 5 is a BIP primer having the same B1c sequence as B1c on the 5 ′ end side and a B2 sequence complementary to B2c on the 3 ′ end side.

  The concentration standard nucleic acid 6 includes the F3 region, the F2 region, the second probe binding region 7 and the F1 region in this order from the 5 ′ end side, and the B3c region, the B2c region, the LBc region and the B1c from the 3 ′ end side. Includes areas in this order. The sequence of the second probe binding region 7 is different from the sequence of the first probe binding region 2. That is, the concentration standard nucleic acid 6 is the same as the configuration of the target template nucleic acid 1 except for the sequence of the first probe binding region 2 in the target template nucleic acid 1. Although not shown in FIG. 1, when the concentration standard nucleic acid 6 is present in a double strand, its complementary sequence is the B3 region, B2 region, LB region and B1 region from the 5 ′ end side. The F3c region, the F2c region, the complementary region of the second probe binding region, and the F1c region are included in this order from the 3 ′ end side.

  The specimen, the primer set, and the first concentration standard nucleic acid having a known concentration are brought into one reaction environment, and an amplification reaction is performed by the LAMP method to obtain an amplification product.

  The sequences of the target template nucleic acid 1 and the concentration standard nucleic acid 6 differ only in the sequence of the probe binding region, and are amplified by the same primer set. When the target template nucleic acid 1 and the concentration standard nucleic acid 6 are competitively co-amplified in one reaction environment with the same primer set, the more existing one in the reaction environment is preferentially amplified.

  The obtained amplification product is reacted with a group of nucleic acid probes immobilized on a substrate, and the amount of the target template nucleic acid present in the sample is determined based on the amount of hybridization produced.

In this method, a plurality of concentration standard nucleic acids having different concentrations may be used. For example, when a plurality of concentration standard nucleic acids from 1 to n are used, each of the probe binding regions of the _first to nth concentration standard nucleic acids includes 2 _1st to _2n probe binding regions, respectively. Here, n is an integer of 2 or more. The 2 ₁ to 2 _n probe binding regions are all different from the sequence of the first probe binding region 2 contained in the target template nucleic acid 1 and are the other 2 ₁ to 2 _n probe binding regions. The sequences have different sequences from each other. The primer binding region of each concentration standard nucleic acid has the same sequence as the target template nucleic acid 1 except for the probe binding region. In the case of using a plurality of concentration standard nucleic acids, a specimen, any one of the first to nth concentration standard nucleic acids, and a primer set are brought in one reaction environment. The prepared reaction environments are the first to nth reaction environments, and the amplification reaction is performed competitively for all of the prepared first to nth concentration standard nucleic acids. After the amplification reaction, the first to nth amplification products respectively obtained in the first to nth reaction environments are combined into one. The combined amplification products include the first to nth amplification products, that is, when n is 3, for example, the first amplification product, the second amplification product, and the third amplification product. . Thereafter, the combined amplification product is reacted with a group of nucleic acid probes, the resulting hybridization signal is detected, and the presence / absence or magnitude of the obtained hybridization signal is compared with each other, whereby the target template nucleic acid 1 in the sample is detected. Determine the concentration.

  The principle of this method will be described with reference to FIG. Prepare the specimen. A target template nucleic acid 1 to be detected is selected. The target template nucleic acid 1 includes a first probe binding region 2. A primer set for amplifying the target template nucleic acid 1 with LAMP and a concentration standard nucleic acid 6 are prepared. The primer set includes a first primer 4 and a second primer 5. The concentration standard nucleic acid 6 has a second probe binding region 7 having a sequence different from that of the first probe binding region 2. Next, the specimen, the concentration standard nucleic acid 6 and the probe set are brought into one reaction environment, and the LAMP amplification reaction is performed competitively.

  As shown in (a-1), for example, when the abundance ratio of the target template nucleic acid 1 and the concentration standard nucleic acid 6 in the reaction environment is 10: 1, that is, the amount of the target template nucleic acid present in the reaction environment If the amount of concentration standard nucleic acid is biased, the template with the larger abundance is preferentially amplified. An image of the amount of amplification product obtained by amplification is shown in FIG. As shown in FIG. 2 (a-2), there are more amplification products from the template with the larger abundance than the amplification product from the other template.

  As shown in (b-1), for example, when the abundance ratio of the target template nucleic acid 1 and the concentration standard nucleic acid 6 in the reaction environment is 1: 1, that is, the amount of the target template nucleic acid present in the reaction environment And the amount of concentration standard nucleic acid are equal, the amplification reaction proceeds equally for the two templates. That is, as shown in FIG. 2 (b-2), the amplification products from the target template nucleic acid 1 and the concentration standard nucleic acid 6 are present in equal amounts.

  Therefore, after performing a LAMP amplification reaction competitively between a concentration standard nucleic acid of a known concentration and a sample, the concentration of the target template nucleic acid in the sample is determined using the concentration standard nucleic acid concentration as an index by detecting the amplification product. Is possible.

  The reaction between the nucleic acid probe and the amplification product may be performed using a DNA chip.

  A “nucleic acid probe” is a nucleic acid comprising a sequence that is complementary to a target sequence. The nucleic acid probe may be immobilized on a known solid phase. A preferred nucleic acid probe is used by being immobilized on the surface of a substrate. An apparatus including such a substrate and a nucleic acid probe immobilized on the substrate may be generally performed by a DNA chip. A “DNA chip” is an apparatus that analyzes a nucleic acid using a hybridization reaction between a nucleic acid probe having a sequence complementary to the nucleic acid to be detected and the nucleic acid to be detected. The term DNA chip is synonymous with commonly used terms such as “nucleic acid chip”, “microarray” and “DNA array”, and is used interchangeably.

  The nucleic acid probe is prepared so that an amplification product derived from the target template nucleic acid and all amplification products derived from all the concentration standard nucleic acids used at the time of amplification can be detected. For example, when a target template nucleic acid and one kind of concentration standard nucleic acid are co-amplified, the DNA chip is immobilized on the first nucleic acid probe 8 immobilized on the first immobilization region and on the second immobilization region. The second nucleic acid probe 9 is provided. As shown in FIG. 1, the first nucleic acid probe 8 has a sequence complementary to a first target sequence including an F2 region on the 5 ′ side and a first probe binding region 2 on the 3 ′ side. The second nucleic acid probe 9 has a sequence complementary to the second target sequence including the 5'-side F2 region and the 3'-side second probe binding region 7. Furthermore, the DNA chip may further include a third nucleic acid probe and / or a fourth nucleic acid probe that are complementary to the first nucleic acid probe 8 and the second nucleic acid probe 9, respectively.

When the first to nth concentration standard nucleic acids are used, 2 _1st to _2nth nucleic acid probes, that is, n types of nucleic acid probes having different sequences are prepared. Here, n is an integer of 2 or more. Each nucleic acid probe is complementary to a second _1-target sequences of the 2 _n include respective concentrations F2 region standard nucleic acid comprises a second _{1 through} 2 _n of one of the probe-binding region sequences, respectively Has an array. That is, the second _first nucleic acid probe has a sequence complementary to a second ₁ target sequence including and 'F2 region and 3' side side 5 second _first probe-binding region. The 2 _n nucleic acid probe has a sequence complementary to the 2 _n target sequence including a 5 ′ F2 region and a 3 _n 2 _n probe binding region. The second _{1 through} 2 _n of the nucleic acid probe may be respectively fixed to each type immobilized region of the second _{1 through} 2 _n of the DNA chip.

Alternatively, the first nucleic acid probe 8 may have the same sequence as the first target sequence including the F2 region and the first probe binding region 2. The second nucleic acid probe 9 may have the same sequence as the second target sequence including the F2 region and the second probe binding region 7. Likewise the nucleic acid probe of the n may have the same sequence as the target sequence of the n including F2 region and a probe binding region of the 2 _n.

  A “target sequence” is a sequence to be detected by binding a nucleic acid probe to a sequence of amplification products obtained by amplifying a template nucleic acid using a primer set. A nucleic acid containing a target sequence is called a target nucleic acid. The target sequence includes the F2 region and the probe binding region of the template nucleic acid sequence, or consists of the F2 region and the probe binding region. The target sequence is used to detect the target nucleic acid using a nucleic acid probe.

  The nucleic acid probe may be used by being immobilized on a DNA chip. Preferably, all the nucleic acid probes to be used for detection are contained in one DNA chip. Preferably, the nucleic acid probe contained in one DNA chip includes an amplification product derived from the target template nucleic acid and amplification products derived from all concentration standard nucleic acids used for quantification thereof, and detects them simultaneously. To do. However, the nucleic acid probe required for the target quantification may be immobilized on a plurality of substrates and used.

  It is also possible to simultaneously quantify a plurality of types of specific nucleic acids contained in a specimen. In that case, for each specific nucleic acid to be detected, a target template sequence is determined, and a primer set for amplifying all of the target template sequences, respectively, and amplifying in competition with each target template sequence A nucleic acid probe for detecting the concentration standard nucleic acid to be detected and all the amplification products to be detected obtained is prepared. These sequences and nucleic acids may be prepared in the same configuration as described above. In addition, nucleic acid probes prepared for a plurality of types of specific nucleic acids may be immobilized on one substrate or may be immobilized on a plurality of substrates.

  A plurality of types of specific nucleic acids are subjected to an amplification reaction by the LAMP method in competition with each concentration standard nucleic acid in each reaction environment. Thereafter, all the amplification products obtained are combined into one amplification product mixture. This amplification product mixture is brought into, for example, a DNA chip including all nucleic acid probes and reacted with the nucleic acid probes. A plurality of types of specific nucleic acids can be simultaneously quantified from the obtained hybridization signal.

  The sample may be any substance containing nucleic acid, such as body fluid, tissue, organ, urine, stool, milk, blood, serum, plasma, vomit, tissue scraping sample, etc. It is sufficient if the sample is present in a typical concentration that is worth quantifying. “Nucleic acid” is a term that collectively indicates substances that can represent a part of the structure by a base sequence, such as DNA, RNA, PNA, LNA, S-oligo, and methylphosphonate oligo. For example, the nucleic acid may be a partially or completely artificially synthesized and / or designed artificial nucleic acid, such as naturally occurring DNA and RNA, and mixtures thereof. The “specimen” is a target on which the method of the embodiment is to be performed, and may be a sample that may contain a nucleic acid. The specimen is preferably in a state that does not interfere with the amplification reaction and / or the hybridization reaction. For example, in order to use a material obtained from a living body or the like as a specimen according to the present embodiment, pretreatment may be performed by any means known per se. For example, the specimen may be a liquid.

  The nucleic acid to be quantified may be, for example, an exogenous nucleic acid, for example, a nucleic acid such as a microorganism, bacteria, virus, mycoplasma, algae, fungi and the like. Further, the nucleic acid to be detected may be any nucleic acid present in the individual, for example, mRNA expressed in the body of the individual, for example, blood or tissue, urine, saliva or milk. Good.

  “In one reaction environment” indicates, for example, one reaction field in which a reaction can be performed. For example, what is necessary is just to understand the inside of reaction containers, such as a test tube, a cup, and a well, as reaction environment.

  According to the embodiment, it is possible to measure in vivo concentration, blood concentration, tissue concentration, urine concentration, saliva concentration or milk concentration of a pathogen.

  Two sequences are “identical” or “same” refer to sequences in which the two sequences are equal to each other by 90% or more, 95% or more, 98% or more, or 100% over their entire length. Two sequences are “complementary” when the two sequences are complementary to each other by 90%, 95%, 98% or 100% over their entire length.

  In the above description, the DNA chip is described. However, when the nucleic acid probe is immobilized and used, the substrate is not limited to the substrate for the DNA chip. For example, the substrate may be an insoluble support such as magnetic beads, microtiter plates and tubes.

  According to the embodiment, when a sample nucleic acid contained in a sample is LAMP amplified as a target template nucleic acid, a concentration standard nucleic acid of a known concentration is mixed in the same reaction environment, and the resulting amplification products are compared, Thereby, it is determined which of the sample nucleic acid and the concentration standard nucleic acid is amplified. Thereby, it is possible to estimate the amount of the target template nucleic acid. By such a method, the in vivo pathogen concentration and the amount of mRNA can be easily measured. In addition, it helps to understand the pathology of bacterial and viral infectious diseases and to determine the treatment policy.

  The entire structure of the nucleic acid of the amplification product may be a double-stranded linear DNA such as a PCR product or a circular DNA. The length of the amplification product is preferably 200 base pairs or more and 5000 base pairs or less if it is linear DNA. If it is cyclic, a length of 200 base pairs to 10,000 base pairs is preferred. It is preferably used after confirming that there is no sequence homologous or similar to the detection portion and the primer region in a region other than the sequence used for detection. Further, it is also preferable to devise not to include mutually complementary sequences so as not to include a region having a high GC content so that the amplification product does not take an extremely three-dimensional structure. The synthesis method of each primer and concentration standard nucleic acid contained in the primer set can be obtained as a double-stranded DNA synthesis method such as a PCR method, or a cloning product incorporated into a plasmid or the like. The vector species used for cloning is not particularly limited.

  When the target template nucleic acid is RNA, the concentration standard nucleic acid is also preferably RNA. In that case, a DNA fragment in which the probe portion is replaced with another sequence is once incorporated into a transcription vector containing SP6 and T7 RNA polymerase promoter sequences, and RNA transcription can be performed therefrom to obtain the necessary RNA. There are no particular restrictions on the vector or enzyme used for transcription.

  The sequence of the probe binding region on the concentration standard nucleic acid is preferably similar in Tm value and length to the probe binding region on the target template nucleic acid. It is also necessary to select a sequence that is not homologous or non-complementary to the sequence of other parts of the whole nucleic acid used as the concentration standard nucleic acid or the target template nucleic acid. As a precaution, select the base sequence of the pathogen that does not infect the species to be tested, or a sequence that is different from the sequence on the genome of the species to be tested, as the probe binding region sequence of the concentration standard nucleic acid. It is also preferable to do.

Table 1 shows the sequence for the probe-binding region 2 of a concentration standard that can be used for testing to measure Escherichia coli (Enterobacteriaceae), streptococci, and staphylococci as causative agents of bovine mastitis. An example of

  The sequence for the probe binding region 2 of the concentration standard nucleic acid is preferably selected from a biological species not related to the nucleic acid to be detected. In the example of Table 1, 20 types of probe binding region sequence candidates prepared based on the genome sequence of human hepatitis B virus are shown. In addition, it is necessary to select a probe binding region that does not change much in length and Tm value compared to the region set as the probe binding region in the target template sequence. The sequences shown in Table 1 cannot be used when designing a quantitative system for human infectious agents such as hepatitis virus.

  In addition, a part of the genome of human hepatitis B virus that does not infect cattle is used. The length of the probe sequence is preferably about 15 to 35 bases, and the Tm value is preferably selected between 62 ° C and 78 ° C.

  FIG. 3 shows an example of measurement when two types of concentration standard nucleic acid are used. The two kinds of concentration standard nucleic acids added to the two tubes, which are reaction environments, are different from each other. In the example of FIG. 3A, 10 copies (ie, 1E + 01 copy) of a low concentration standard nucleic acid and 100 copies (ie, 1E + 02 copy) of a high concentration standard nucleic acid are added. The concentration of the target template nucleic acid to be quantified is to compare the amount of amplification product of the target template nucleic acid with the amount of amplification product for the two concentration standard nucleic acids, ie the first and second concentration standard nucleic acids. Is obtained. For example, the results when the concentration of the target template nucleic acid in the reaction environment is lower than the low concentration first concentration standard nucleic acid are shown in (b) and (c). In this case, the amplification products of the first and second concentration standard nucleic acids are both larger than the amount of the amplification product of the target template nucleic acid, and the amplification of the target template nucleic acid is low. The results when the concentration of the target template nucleic acid in the reaction environment is higher than the low concentration first concentration standard nucleic acid and lower than the high concentration second concentration standard nucleic acid are shown in (d) and (e). Indicated. In this case, there are many amplification products of the target template nucleic acid and the second concentration standard nucleic acid, and there are few amplification products of the first concentration standard nucleic acid. The results when the concentration of the target template nucleic acid is higher than the high concentration of the second concentration standard nucleic acid are shown in (f) and (g). In this case, only the target template nucleic acid is amplified, and the amplification of the first and second concentration standard nucleic acids is low. Thus, the concentration of the standard template nucleic acid in the reaction environment can be measured, for example, in three stages from the relationship between the amount of amplification product present between the two types of concentration standard nucleic acid and the target template nucleic acid that is the sample nucleic acid. it can.

FIG. 4 shows the target template nucleic acid A at three different concentrations, namely 1 × 10 ¹ (1.00E + 01), 1 × 10 ³ (1.00E + 03) and 1 × 10 ⁵ (1.00E + 05), respectively. An example of quantifying a sample containing 5 using five kinds of concentration standard nucleic acids is shown. For each sample, five reaction environments, for example, reaction containers are prepared, and the amplification reaction is performed competitively. The five kinds of concentration standard nucleic acids are added in different addition amounts. As a result of each competitive amplification reaction, FIGS. 4 (A) to (O) and examples of hybridization signals detected by the DNA chip for each specimen are shown in FIGS. 4 (P) to (R).

First, the reaction between each concentration standard nucleic acid brought in at each concentration in the reaction environment 1 and the target template nucleic acid present in the reaction environment at a concentration of 1 × 10 ¹ (that is, 1.00E + 01) will be described.

For the sample 1, in the reaction environment 1, the amplification reaction competes between the target template nucleic acid A and the first concentration standard nucleic acid B present at a concentration of 1 × 10 ¹ . In the reaction environment 2, the amplification reaction competes between the target template nucleic acid A and the second concentration standard nucleic acid C present at a concentration of 1 × 10 ² (ie, 1E + 02). In the reaction environment 3, the amplification reaction competes between the target template nucleic acid A and the third concentration standard nucleic acid D present at a concentration of 1 × 10 ³ (ie, 1.00E + 03). In the reaction environment 4, the amplification reaction competes between the target template nucleic acid A and the fourth concentration standard nucleic acid E present at a concentration of 1 × 10 ⁴ (ie, 1.00E + 04). In the reaction environment 5, the amplification reaction competes between the target template nucleic acid A and the fifth concentration standard nucleic acid F present at a concentration of 1 × 10 ⁵ (ie, 1.00E + 05). In the reaction environment 1, since two nucleic acids are present in equal amounts, both nucleic acids are similarly amplified as shown in (A). In the reaction environments 2 to 5, since the second concentration standard nucleic acid B to the fifth concentration standard nucleic acid F are more often present in any case, the second concentration standard nucleic acid B to the fifth concentration standard nucleic acid F are present. Is amplified in preference to the target template nucleic acid A (see (D), (G), (J) and (M), respectively). The results of detection with a DNA chip for the mixture 1 obtained by mixing the amplification products obtained for the specimen 1 are shown in (P). From this result, it is determined that the target template nucleic acid A has a concentration of 1 × 10 ¹ .

In the sample 2, the target template nucleic acid A is present at a concentration of 1 × 10 ³ . In this case, in the reaction environments 1 and 2, the target template nucleic acid A is present more than the concentration standard nucleic acids B and C. Therefore, in the reaction environments 1 and 2, the target template nucleic acid A is preferentially amplified as shown in (B) and (E). In the reaction environment 3, since the two nucleic acids are present in equal amounts, both nucleic acids are similarly amplified as shown in (H). In the reaction environment 4 and the environment 5, the fourth concentration standard nucleic acid E to the fifth concentration standard nucleic acid F are more often present in any case. Therefore, the second concentration standard nucleic acid E to the fifth concentration standard nucleic acid are present. F is amplified in preference to the target template nucleic acid A (see (K) and (N), respectively). The results of detection with a DNA chip for the mixture 2 obtained by mixing the amplification products obtained for the specimen 2 are shown in (Q). From this result, it is determined that the target template nucleic acid A has a concentration of 1 × 10 ³ .

In the sample 3, the target template nucleic acid A is present at a concentration of 1 × 10 ⁵ . In this case, in the reaction environments 1 to 4, the target template nucleic acid A is present more than the concentration standard nucleic acids B to F. Therefore, in the reaction environments 1 to 4, the target template nucleic acid A is preferentially amplified as shown in (C), (F), (I) and (L). In the reaction environment 5, since the two nucleic acids are present in equal amounts, both nucleic acids are similarly amplified as shown in (O). (R) shows the result of the sample 3 detected with the DNA chip for the mixture 3 obtained by mixing the amplification products obtained in the respective reaction environments. From this result, it is determined that the target template nucleic acid A has a concentration of 1 × 10 ⁵ .

  Thus, when the concentration standard nucleic acid is present at a concentration lower than the target template nucleic acid concentration, a hybridization signal derived from the concentration standard nucleic acid at such a concentration cannot be obtained. Therefore, the amount of the target template nucleic acid can be determined by preparing the concentration of the concentration standard nucleic acid stepwise as many as desired. Further, when it is desired to know the amount of the target template nucleic acid in general, the number of concentrations of the concentration standard nucleic acid may be reduced. The concentration of the standard nucleic acid can be made in any number of steps as required. The concentration standard nucleic acids used at different concentrations may be different from each other in the sequence of the probe binding region.

  The addition amount of the concentration standard nucleic acid may be, for example, an equal dilution or an arbitrary different multiple. For example, it may be diluted by 2 times, may be diluted by a necessary integer multiple, and may be diluted by chopping at a necessary pitch. What is necessary is just to set the density | concentration of a density | concentration standard nucleic acid as desired.

  For example, an example of determining the concentration of the target template nucleic acid is shown in FIG. In the case of FIG. 5A, the concentration standard nucleic acid at any concentration is amplified in preference to the target template nucleic acid. Therefore, in this case, it is determined that the target template nucleic acid has a concentration equal to or lower than the lowest concentration standard nucleic acid. Furthermore, by immobilizing a nucleic acid probe for qualitative testing on the same substrate, it is possible to determine whether it is positive or negative even at a low concentration.

  Although not shown in the figure, when only the target template nucleic acid is preferentially amplified and no amplification of any standard nucleic acid is detected, the target template nucleic acid is present at the highest concentration of the standard nucleic acid. It determines with it being above.

In the case of FIGS. 5B to 5D, it is a graph when there is a result in which the hybridized signal shows an intermediate value. In this case, it is considered that the concentrations of the target template nucleic acid and the concentration standard nucleic acid were in competition within the reaction environment. In this case, as in (b) or (c), if a signal that is twice or more the lowest hybridization signal is obtained, it can be determined that the amplification of the concentration standard nucleic acid has won in the reaction. When the hybridization signal is less than twice the lowest hybridization signal as in (d), it is determined that the concentration of the standard nucleic acid and the target template nucleic acid are contained at the same concentration. In the case of the example of FIG. 5 (c), if the concentration standard nucleic acid E is added at a concentration of 1 × 10 ⁴ (ie, 1E + 04) copies, the target template nucleic acid of (c) is 1 × 10 ⁴ copies (ie, 1E + 04) or more and 1 × 10 ⁵ (ie, 1E + 05) copies or less, and the specimen (D) is determined that the target template nucleic acid was 1 × 10 ⁴ (ie, 1E + 04) copies.

  In addition, according to the embodiment, using the above-described quantification method, a test method for quantifying three bacterial groups of coliform bacteria, staphylococci, and streptococci that are causative bacteria of bovine mastitis, and probes necessary for the same Also provided are primers, concentration standard nucleic acids.

  Bovine mastitis is a dairy cow disease that causes significant economic losses each year for the dairy industry. Aiming at the eradication, the development of the inspection method is planned, but so far only the inspection method by bacterial culture exists. If a genetic test represented by a DNA chip is about to enter, the high sensitivity becomes a problem. Unlike the strains such as Streptococcus agalactiae that require immediate countermeasures, even the presence of a few is confirmed, and the aforementioned coliforms (ie, Enterobacteriaceae), staphylococci, linkage Cocci are resident bacteria that are often not a problem if the infection is small. Therefore, in a highly sensitive genetic test, it is necessary to adjust the sensitivity (cut off) by discarding a certain amount of bacteria and determining a positive concentration above a necessary concentration.

  The nucleic acid quantification method can also be applied to the three causative species of bovine mastitis. In the case of bovine mastitis, the target numerical value can only be referred to the numerical value of CFU (colony forming unit) / mL, which is the result of the bacterial culture method that is an existing test method. This number is that how many colonies are formed when 1 mL of a sample is cultured. However, one actual colony is not always derived from one cell, and the specimen includes a living bacterium that cannot form a colony. Therefore, CFU / mL may be considered as a guideline number. Therefore, it is also preferable to use 16S rRNA, which is easy to observe as a target template nucleic acid, because it is contained not only in the bacterial genome but also in several copies per cell. For example, it is possible to perform the quantification method by selecting 16S rRNA of these three types of bacteria as a target template nucleic acid and designing primers and concentration standard nucleic acid. Furthermore, it is possible to determine the cut-off line in light of actual clinical symptoms.

Examples of primers that can be used for quantification of coliforms, staphylococci and streptococci are shown in Table 2.

  For quantification using coliform genomes, the results show the total number of total coliforms. Detection of Escherichia coli, staphylococci, and streptococci using one 16S rRNA as a target template nucleic acid does not result in the number of bacteria detected because several copies of 16S rRNA are present in one cell. In order to obtain the number of bacteria, the detected amount should be divided by the number of copies in one cell.

Table 3 shows examples of nucleic acid probes for detecting the above three bacteria.

Table 4 shows examples of target template nucleic acids and concentration standard nucleic acids for quantifying the above three bacteria.

  Table 4-1 (1) (a) shows an example of the target template nucleic acid represented by SEQ ID NO: 60 for 16S rRNA of Enterobacteriaceae. In addition, the F1, F2, F3, B1, B2, B3, and LBc regions for the LAMP primer set in the sequence are indicated by squares. In Table 4-1, (1) (b) and (c) show concentration standard nucleic acid 1 (SEQ ID NO: 61) and concentration standard nucleic acid 2 (SEQ ID NO: 62) for use with SEQ ID NO: 60, respectively. Further, each region for the LAMP primer set is indicated by a square.

  Examples of the target template nucleic acid represented by SEQ ID NO: 63 for rpoA of Enterobacteriaceae are shown in Table 4-2 (2) (a). In addition, the F1, F2, F3, B1, B2, B3, and LBc regions for the LAMP primer set in the sequence are indicated by squares. In Table 4-2, (2) (b) and (c) show concentration standard nucleic acid 1 (SEQ ID NO: 64) and concentration standard nucleic acid 2 (SEQ ID NO: 65) for use with SEQ ID NO: 63, respectively. Further, each region for the LAMP primer set is indicated by a square.

  Examples of the target template nucleic acid represented by SEQ ID NO: 66 for staphylococcal 16S rRNA are shown in Table 4-3 (3) (a). In addition, the F1, F2, F3, B1, B2, B3, and LBc regions for the LAMP primer set in the sequence are indicated by squares. Table 4-3 (3) (b) and (c) and Table 4-4 (d) include concentration standard nucleic acid 1 (SEQ ID NO: 67) and concentration standard nucleic acid for use together with SEQ ID NO: 66. 2 (SEQ ID NO: 68) and concentration standard nucleic acid 3 (SEQ ID NO: 69) are shown, and each region for the LAMP primer set is indicated by a square.

  Table 4-4 (4) (a) shows an example of the target template nucleic acid represented by SEQ ID NO: 70 for Streptococcus 16S rRNA. In addition, the F1, F2, F3, B1, B2, B3, and LBc regions for the LAMP primer set in the sequence are indicated by squares. (4) (b) in Table 4-4 and (c) and (d) in Table 4-5 include concentration standard nucleic acid 1 (SEQ ID NO: 71) and concentration standard nucleic acid for use together with SEQ ID NO: 70. 2 (SEQ ID NO: 72) and concentration standard nucleic acid 3 (SEQ ID NO: 73) are shown, and each region for the LAMP primer set is shown by a square.

  Combinations of these target template nucleic acids and concentration standard nucleic acids can be used for quantification of each bacterium. In addition, these target template nucleic acid and concentration standard nucleic acid may be designed so as to include each region for the LAMP primer set shown in each table above and the above-described probe binding region. Therefore, the sequence may be longer or shorter than the sequences shown in each table. In addition, the sequence of the probe binding region is not limited to the concentration standard nucleic acids shown in Tables 4-1 to 4-5, and the probe binding region candidate sequences shown in Table 1 may be used. Is possible. Further, those skilled in the art can refer to the candidate sequence of the probe binding region shown in Table 1 to have an appropriate length and an appropriate Tm value and can be discriminated from the original sequence by hybridization. Can be designed as appropriate, and such a sequence may also be used as a probe binding region sequence in this embodiment.

  For example, a test for mastitis in cattle can be performed as follows. The sample to be examined may be cow's milk (milk). As a method for extracting a pathogen gene from collected milk, for example, a commercially available kit such as FAST-ID DNA extraction kit (manufactured by Japan Authentication Service Co., Ltd.) can be selected. However, the method is not particularly limited thereto. Absent. The extracted genome of the pathogen is obtained in a state dissolved in a general buffer such as TE buffer. If not immediately subjected to inspection, it should be stored in an environment of -20 ° C or lower.

  The extracted genome is then subjected to the necessary amplification by the LAMP reaction. At this time, not only a competitive reaction necessary for quantification, but also a normal fungus-specific LAMP reaction may be performed simultaneously. Thereby, it is possible to perform the qualitative test of specific bacteria simultaneously. For example, when a specific bacterium is present, the result is positive, and when the specific bacterium is not present, the result is negative. This test is also called a positive-negative test. FIG. 6 shows an example of a container set including a plurality of containers for performing LAMP amplification for examining the above-mentioned three causative bacteria related to bovine mastitis. FIG. 6A shows reaction vessels for coliform bacteria and staphylococci, that is, reaction environments. The container (1) is a qualitative test container for coliform bacteria, and the containers (2), (3) and (4) are containers for quantitative determination of coliform bacteria. The container (5) is a staphylococcal qualitative test container, and the containers (6), (7) and (8) are staphylococcal quantification containers. FIG. 6B includes a reaction vessel for streptococci, a reaction vessel for qualitative testing of other bacteria, and a reaction vessel for control. The container (9) is a qualitative test container for streptococci, and the containers (10), (11) and (12) are containers for quantification of streptococci. The containers (13) and (14) are reaction containers for amplifying one or a plurality of arbitrary bacteria, respectively. Containers (15) and (16) are control containers. Three kinds of concentration standard nucleic acids prepared at three different concentrations are added to each quantitative container. A container set including a plurality of containers is preferable because it is ready to perform an amplification reaction under the same conditions. It is preferable to design the primer set used simultaneously in such a container set so that the optimal temperature of the reaction is the same.

  The primer set for the qualitative test may be a primer set that can specifically amplify the nucleic acid of a specific bacterial species to be detected. The same temperature as the quantitative inspection is the optimum temperature for the reaction, and it is preferable to employ an amplification reaction with substantially the same reaction start time.

  Bacteria other than the three types of bacteria may also be amplified for qualitative use in the containers (13) and (14). Furthermore, a further reaction vessel may be prepared and included in the vessel set. For example, qualitative tests such as mycoplasma, corynebacterium, Pseudomonas aeruginosa, yeast-like fungi and / or Candida may be performed simultaneously with quantification and qualitative testing of coliforms, staphylococci and streptococci. If necessary, a container for performing a quantitative test may be mounted on the container set for these additional bacterial species.

  Amplification in the container set may be performed as follows. A LAMP reaction solution prepared in advance and a genomic nucleic acid extracted from milk are added to each container as a target template nucleic acid. At this time, a concentration standard nucleic acid is also added to the quantitative container. The genomic nucleic acid extracted from the milk may be preheated if necessary. The heat denaturation condition may be about 1 to 5 minutes at a temperature of 80 ° C. to 95 ° C., but is not limited thereto. After heat denaturation, it is also preferable to add a quenching step by transferring the sample onto ice.

  Examples of the composition of the reaction solution are shown below.

・ LAMP reaction solution composition 20 mM Tris-HCl (pH 8.8)
10 mM KCl
8 mM MgSO ₄
10 mM (NH ₄ ) ₂ SO ₄
0.1% Tween20
0.8M Betaine
1.4mMeach dNTPs.

  When the reaction is carried out with a reaction solution of this composition in a total volume of 25 μL, each primer may be brought in at the following concentration.

・ Primer addition amount FIP 40pmoL
BIP 40pmoL
F3 5pmoL
B3 5pmoL
Loop 20 pmoL.

  Furthermore, when the reaction is performed in a total amount of 25 μL, for example, Bst DNA Polymerase is added at 1 to 2 μL (8 units / uL) as an enzyme solution. The enzyme for amplification is not limited to Bst DNA polymerase, and other enzymes may be used as long as they have strand displacement activity. When the target template nucleic acid is RNA, reverse transcriptase may be added to the enzyme solution.

  The reaction mixture is mixed with the template and placed in a PCR tube to start the reaction. The reaction temperature varies depending on the enzyme used and the characteristics of the target template nucleic acid, and may be appropriately selected. For example, it is often selected within the range of about 59 ° C. to 66 ° C., and this range may be used here. The reaction apparatus may be a turbidity measurement apparatus used for detecting a normal LAMP reaction, or may be a thermal cycler used for normal PCR. Since the reaction time varies depending on the characteristics of the primer used, it may be appropriately selected. For example, it is often selected between 30 minutes and 60 minutes, and this range may also be used here. The competitive LAMP reaction is performed for the first time in each individual by mixing two types of templates having different concentrations. However, even under such reaction conditions, a phenomenon in which the template with the smaller amount mixed is amplified by increasing the reaction time was not confirmed.

  The detection means detects whether or not a DNA fragment is immobilized as a nucleic acid probe on an insoluble substrate such as a DNA chip, and the sequence of nucleic acid fragments present in a solution is detected by detecting whether or not their complementary sequences bind to each other. Any measurement method based on the principle of reading a part of the image may be used. For example, on a platform where the hybridization reaction environment needs to be changed for each probe, such as a microtiter plate or a bead, the qualitative amplification product is reacted with a qualitative nucleic acid probe adapted thereto to form a complementary strand. You can check whether or not. As for the amplification product for quantification, it is preferable to react the amplification product obtained by one competing amplification reaction simultaneously with the nucleic acid probe for detecting the target template nucleic acid and the nucleic acid probe for detecting the concentration standard nucleic acid. In addition, all the reaction products obtained by each reaction performed in different containers are combined into one, and a plurality of types of nucleic acid probes configured to correspond to the target nucleic acid to be detected are combined into one substrate. Detection may be performed by adding to a DNA chip immobilized on the DNA chip.

  In bovine mastitis, resident bacteria and pathogens coexist in milk. Therefore, in order to identify and quantify resident bacteria and pathogenic bacteria, it is also preferable to determine a cut-off line using the amount of bacteria as an index.

  The cut-off line is determined by first measuring samples containing coliforms, staphylococci and streptococci as resident bacteria and samples containing coliforms, staphylococci and streptococci as pathogens as resident bacteria or pathogens. It is necessary to determine the amount of concentration standard nucleic acid to be divided.

  FIG. 7 shows an example of the result of the quantification method performed on the extract from milk, which was a positive specimen of the coliform group and showed a numerical value of 700 cfu / mL by the culture method. In this case, the concentration standard nucleic acid brings 2e + 01 copy / reaction at the lower side and 2e + 02 copy / reaction at the higher side. The result of 700 cfu / mL in the culture method means that in the present invention, the number of brought-in genomes is smaller than the concentration standard nucleic acid of 2e + 01 copy / reaction. If the amount of bacteria is low enough so that it does not appear to be a problem as a mastitis-causing bacterium, this bacterium is determined to have a concentration standard nucleic acid of 2e + 01 copy / reaction as a cutoff line, and to determine that it is negative be able to. However, in the culture method, a numerical value of 100 cfu / mL is also obtained. For bacteria that should be cut at 100 cfu / mL, it is necessary to draw a cut line in an area smaller than 2e + 01 copy / reaction. In that case, there is a possibility that the amount of the mold is too small to cause a sampling error. Therefore, it is preferable to use 16s rRNA present in several copies as a target template nucleic acid, instead of using a genomic nucleic acid having only one genome per cell as a target template nucleic acid. If this nucleic acid is used, even if 1 cfu / mL is a cutoff line, the concentration standard nucleic acid may not be 1 copy / mL. However, a more clinically meaningful cut-off line can be set for each bacterial species by experiencing many specimens.

  Embodiments also provide a reference for determining whether the bacteria present in a specimen are resident or pathogenic, i.e., a method for creating a cut-off line. This method can prevent false positive determination. Moreover, it is possible to adjust the detection sensitivity in the means for detecting a specific microbe, for example, a detection apparatus or a detection method, by the method for producing the cut line.

Still further, embodiments provide a method for determining whether a particular bacterium contained in a specimen is a resident or a pathogenic bacterium. For example, a method for determining whether a specific bacterium contained in a sample is a resident bacterium is as follows:
(A) preparing a sample containing nucleic acid;
(B) F3 region, F2 region, first probe binding region and F1 region are included in this order from the 5 ′ end side, and B3c region, B2c region, LBc region and B1c region are included in this order from the 3 ′ end side. A primer set comprising a first primer and a second primer for amplifying the target template sequence contained in the first primer, wherein the first primer has a F1c sequence complementary to F1 on the 5 ′ end side; and FIP primer having the same F2 sequence as F2 on the 3 ′ end side, the second primer having the same B1c sequence on B1c on the 5 ′ end side, and a B2 sequence complementary to B2c on the 3 ′ end side Preparing a primer set that is a BIP primer having
(C) preparing a first concentration standard nucleic acid to a third concentration standard nucleic acid; wherein the first concentration standard nucleic acid comprises an F3 region, an F2 region, and other probe bindings from the 5 ′ end side. A second probe-binding region having a sequence different from that of the region, and an F1 region in this order, and a B3c region, a B2c region, an LBc region, and a B1c region in this order from the 3 ′ end side, respectively. The second concentration standard nucleic acid includes, from the 5 ′ end side, an F3 region, an F2 region, a third probe binding region different in sequence from the other probe binding regions, and an F1 region in this order, respectively. And the B3c region, the B2c region, the LBc region, and the B1c region in this order from the 3 ′ end side, respectively, and the third concentration standard nucleic acid includes the F3 region and the F2 region from the 5 ′ end side. And other probe binding regions Each containing a different fourth probe binding region of sequence and F1 region contains respectively in this order, and the B3c region from 3 'terminal side, and B2c region, and LBc region, and B1c region this order,
(D) a first concentration standard nucleic acid having a concentration previously determined to be a low concentration, a second concentration standard nucleic acid having a concentration predetermined to be a medium concentration, and a first concentration standard nucleic acid having a concentration predetermined to be a high concentration. 3 concentration standard nucleic acids are brought into the first to third reaction environments for each type of concentration standard nucleic acid, together with the sample and the primer set, respectively, and amplification reaction is performed by the LAMP method. Getting each,
(E) combining the first to third amplification products obtained in the first to third reaction environments into one;
(F) reacting the first to fourth nucleic acid probes immobilized on the substrate with the combined amplification product obtained in (e); wherein the first nucleic acid probe comprises It is immobilized on a first immobilization region of a substrate and is complementary to a first target sequence including the F2 region and the first probe binding region or a complementary sequence thereof, and the second nucleic acid probe is the substrate The second nucleic acid probe is complementary to a second target sequence including the F2 region and the second probe binding region or a complementary sequence thereof, and the third nucleic acid probe is immobilized on the second immobilization region of the substrate. 3 and is complementary to a third target sequence including the F2 region and the third probe binding region or a complementary sequence thereof, and the fourth nucleic acid probe is a fourth nucleic acid probe of the substrate. Immobilized in the immobilization region, the F2 It is complementary to the fourth target sequence or its complementary sequence, including the frequency and the fourth probe binding region,
(G) quantifying a specific nucleic acid contained in the specimen by detecting the amount of hybridization for each of the first to fourth nucleic acid probes generated in (f) and comparing them with each other;
including.

  The determination of whether the bacterium contained in the specimen is a resident or a pathogenic bacterium is, for example, when the amount of the bacterium quantified in (g) is determined to be a low bacterium, It may be determined that there is. Alternatively, when the amount of bacteria quantified in (g) is determined to be a low or moderate amount, it may be determined to be a resident bacteria. In this case, a low or moderate amount of bacteria may be set as a predetermined reference value, that is, a cut-off line.

  As a method for determining a specific concentration as a low concentration in advance, a determination as a medium concentration in advance, or a determination as a high concentration in advance, for example, a method known per se, for example, a culture method, Real time, A PCR method, a FISH method (Fluorescence in situ Hybridization method), a method using various bacterial counters, and the like may be used. The culture method is a method of determining the amount of bacteria by applying a sample to a medium and culturing, and converting the number of colonies generated to the original sample concentration. The Real time PCR method is a nucleic acid quantification method using the PCR method, and is a method for determining the amount of bacteria by comparison with an amplification rise time with a concentration standard nucleic acid. The FISH method is a kind of nucleic acid quantification method, and is a method in which the amount of bacteria is determined by counting signals by hybridization of oligonucleotide probes fluorescently labeled with FITC or CY3. The method using other various bacterial counters is a method of determining the amount of bacteria by counting signals indicated by the markers using various markers uniquely developed by each manufacturer.

Or, if a specific bacterium contained in a specific sample is within this range, the numerical value for judging that it is a resident bacterium is specifically shown, such a numerical value is used as a cutoff line. Also good. In that case, the amount of bacteria represented by such a numerical value may be determined as the low or medium amount of bacteria in the determination method, and a concentration-standard nucleic acid may be prepared and used in a competitive amplification reaction. Such numerical values are known, for example, for the amount of coliform bacteria as resident bacteria in feces excreted by humans per day, and the average amount of bacteria is 10 ¹¹ to 10 ^13. It has been broken.

  The method for determining whether the bacteria contained in such specimens are resident or pathogenic is combined with a qualitative test method known per se and / or a more accurate qualitative and / or quantitative test method. May be used. Thereby, it is possible to identify the bacteria to be tested more precisely.

[Example]
[Example 1]
A DNA chip was prepared by using a cloned rpoA gene (SEQ ID NO: 63) of a standard strain of E. coli (ATCC: 25922) as a positive control, and performing a competitive reaction with one type of standard nucleic acid (SEQ ID NO: 64). Detected with. The result is shown in FIG. The graphs of FIGS. 8A to 8C in the upper row illustrate the copy number of the positive control and the concentration standard nucleic acid added to each reaction. The graphs of FIGS. 8D to 8F in the lower stage show the results of measuring the amount of amplification product obtained by each competitive amplification reaction using a DNA chip. From these results, it was confirmed that an amplification product was generated according to the size of the copy number of the added concentration standard nucleic acid.

[Example 2]
(1) Target template nucleic acids for quantification of coliforms, staphylococci, streptococci Target template nucleic acids for coliforms (SEQ ID NO: 60), target template nucleic acids for staphylococci (SEQ ID NO: 66), streptococcal A target template nucleic acid (SEQ ID NO: 70) was selected for.

(2) Design of nucleic acid probes and primers for quantification of coliform bacteria, staphylococci and streptococci Primer sets and nucleic acid probes for quantification of coliform bacteria, staphylococci and streptococci were designed. The designed primer sets are shown in Table 2, and the nucleic acid probes are shown in Table 3. All nucleic acid probes used were immobilized on one DNA chip.

(3) Concentration standard nucleic acid for quantification of coliform bacteria, staphylococci and streptococci Based on the sequences (1) and (2) above, concentration standard nucleic acid 1 (SEQ ID NO: 61) and concentration standard for coliform bacteria Nucleic acid 2 (SEQ ID NO: 62), concentration standard nucleic acid 1 (SEQ ID NO: 67) and concentration standard nucleic acid 2 (SEQ ID NO: 68) for staphylococci, concentration standard nucleic acid 1 (SEQ ID NO: 71) and concentration standards for streptococci Nucleic acid 2 (SEQ ID NO: 72) was designed and prepared.

(4) Comparison of bacterial culture test results in the milk of cows with mastitis and quantification results using DNA chips in E. coli, staphylococci, and streptococci that are bovine mastitis-causing bacteria A quantitative method of morphology was performed. Furthermore, the results obtained thereby were compared with the results obtained by a quantitative method using bacterial culture.

Materials Milk squeezed from cattle that developed mastitis at farm A in Tokachi district, Hokkaido was transported to an experimental facility near Tokyo in a refrigerated state as it was, and bacterial culture inspection was started the day after it was collected. The same milk was stored in a refrigerated state for one more day after the start of the culture test, and then subjected to a DNA chip test.

Nucleic acid extraction method For nucleic acid extraction from milk, bead beating treatment is added to the recommended method of FAST-ID DNA extraction kit (manufactured by Japan Authentication Service Co., Ltd.) for the purpose of destroying solids, fats, and outer shells of bacteria. In order to further remove proteins and lipids, a precipitation treatment with ammonium sulfate and acetic acid was additionally performed. The milk used was 200 μL to 400 μL per test, and the nucleic acid after extraction was dissolved in 60 μL of TE buffer. The sample after extraction was stored at −20 ° C. until subsequent amplification reaction.

LAMP amplification reaction The LAMP reaction composition was as follows.

・ LAMP reaction solution composition 20 mM Tris-HCl (pH 8.8)
10 mM KCl
8 mM MgSO ₄
10 mM (NH ₄ ) ₂ SO ₄
0.1% Tween20
0.8M Betaine
1.4mMeach dNTPs
Bst DNA Polymerase 8 unit.

  Each primer was brought into the reaction solution having this composition at the following concentration.

・ Primer addition amount FIP 40pmoL
BIP 40pmoL
F3 5pmoL
B3 5pmoL
Loop 20 pmoL.

  With the above composition, the reaction was performed on a reaction scale of 25 μL. 2 μL of the extracted product that had been heat-treated was added to each reaction tube, and 2 μL each of the concentration standard nucleic acid was diluted to 1e + 01 copy / μL and 1e + 02 copy / μL. The reaction was carried out at 61 ° C. for 1 hour.

DNA chip measurement The DNA chip was prepared by adding 2 μL of amplification product per reaction using a nucleic acid probe listed in Table 3 that was solid-phased for each type.

The measuring device used was a generator 2C600. The reaction conditions are shown in Table 5.

Results The measurement results are shown in FIG. For each bacterial species, one sample was selected from the samples determined to be positive by the DNA chip, one from which the result of the culture method was determined to be a high bacterial concentration and one from which the negative was determined to be negative. For each bacterial species, those that were culture-negative showed that the current value of the probe corresponding to the target template nucleic acid did not increase even in the DNA chip, and the current value of both the two concentration standard nucleic acids increased. These were positive in the qualitative DNA chip test, but are determined to be cut off because the amount of bacteria is determined to be small. On the other hand, as a result of the DNA chip in the sample determined to have a high bacterial mass by the culture method, only the current value of the probe corresponding to the target template nucleic acid increased, and the current value of both the two concentration standard nucleic acids did not increase. . These specimens were judged to have a large amount of bacteria, consistent with the results of the culture method.

[Example 3]
Qualitative inspection of bovine mastitis causative bacteria and qualitative inspection of staphylococci and streptococci on one chip. In many qualitative tests to check for the presence or absence of other causative bacteria in bovine mastitis milk, It is necessary to collect LAMP products at a time and test on one chip. Further, whether or not the LAMP product for quantification was added to such a chip would hinder the results of both the qualitative inspection and the quantitative inspection.

Materials and Nucleic Acid Extraction Methods The same materials and nucleic acid extraction methods as in Example 2 were used.

LAMP amplification Amplification for quantification was performed under the same conditions as in Example 2. Qualitative primers include staphylococci, Staphylococcus aureus, Streptocpccus, Enterococcus, Streptococcus agalactiae, Streptococcus uberis, Enterobacteriaceae, E. E. coli, Klebsiella spp, Corynebacterium bovis, Arcanobacterium pyogenes, Mycoplasma, Mycoplasma bovis, Prototheca, Pseudomonas aeruginosa, and Candida The primers used are as shown in Table 2 above.

-DNA chip measurement As a DNA chip, an apparatus provided with the flow path shown in Fig. 10 as a reaction part was used. 10A is a plan view of the DNA chip 80, and FIG. 10B is a cross-sectional view taken along line BB of the DNA chip 80 in FIG. 10A. The DNA chip 80 includes a support body 81, a covering body 82, a flow path 83, and a nucleic acid probe 85. The flow path 83 is defined by one surface of the support body 81 and a recess formed on one surface of the covering body 82. The flow path 83 has a wavy shape in which opposing portions are parallel to each other. The flow path 83 has a flow direction from one end to the other end. The covering 82 is disposed in close contact with the support 81 so as to seal the liquid in the flow path 83.

  The plurality of probe immobilization regions 84 are arranged on the bottom surface of the channel 83 along the flow direction of the channel 83. In each of the plurality of probe immobilization regions 5, a plurality of types of probe nucleic acids 85 configured to hybridize with different target sequences are immobilized. The probe immobilization region 5 is arranged so that hybridization between the probe nucleic acid 85 immobilized on each of the plurality of probe immobilization regions 84 and the amplification product is performed independently. The sample was added to the flow path 83 from the inlet 86. The sample was discharged from the outlet 87. Therefore, the inflow port 86 is upstream of the flow path 83 and the outflow port 87 is downstream of the flow path 83.

  Table 3 shows nucleic acid probes for quantifying staphylococci and streptococci immobilized on the probe immobilization region 84. In addition, in order to qualify Escherichia coli, Corynebacterium, Mycoplasma, algae, Pseudomonas aeruginosa and yeast-like fungi, nucleic acid probes designed to have specific sequences were prepared. Furthermore, in order to qualify staphylococci and streptococci, nucleic acid probes for specifically detecting these bacteria were prepared. Nucleic acid probes for specifically detecting sequences from M. tuberculosis, hepatitis B virus (HBV) chimera and HPV were prepared for control.

  A nucleic acid probe for qualitative testing was immobilized upstream of the channel 83. Downstream thereof, staphylococcal and streptococcal quantitative probes were immobilized. Downstream thereof, nucleic acid probes for positive control (M. tuberculosis + HBV chimera; indicated by “p” in FIG. 11) and negative control (HBV; indicated by “n” in FIG. 11) were immobilized. The positive control was added for the purpose of confirming the normality of LAMP amplification in the case of positive and the normality of the DNA chip measurement, and the negative control was added for the purpose of confirming the presence or absence of non-specific reaction in the DNA chip measurement.

-Results The results of two cases measured in the same manner are shown in FIG. The result about the sample used in the test for obtaining the graph of (A) will be described. In the results of the qualitative test shown in the graph of (A), staphylococci were positive and streptococci were also positive (see the columns of “Grape” and “Streptococcus” in FIG. 11A). From the results of the quantitative test shown in the graph of (A), in staphylococci, both the concentration standard nucleic acids (C1, C3) did not increase in current value, and only the target template nucleic acid (T) increased (FIG. 11 ( (See “CNS comp” in A). Therefore, it was determined that the amount of bacteria was high. Streptococcus was determined to have a moderate amount of bacteria because the current values of the target template nucleic acid (T) and the high concentration standard nucleic acid (C10) increased (see “Strept” in FIG. 11A). comp "). Therefore, it was determined that the specimen used in the test for obtaining the graph of (A) is not a target for cutting off.

  On the other hand, the specimen used in the test for obtaining the graph of (B) was positive for staphylococci and streptococci as a result of the qualitative test. In the results of the quantitative test shown in the graph of (B), staphylococci increased the current values of the target template nucleic acid (T) and the high concentration standard nucleic acid (C3) (see “CNS comp” in FIG. 11B). ). Therefore, it was determined that staphylococci had a moderate amount of bacteria. Streptococcus was determined to have a low bacterial load because only the current value of the concentration standard nucleic acids (C9, C10) increased and the target template nucleic acid (T) did not increase (see FIG. 11B). (See “Strept comp”). Therefore, it was determined that the sample of (B) should be a target for streptococcus.

[Example 4] Effect of foot cut In the examination of bovine mastitis-causing bacteria, the significance of adjusting the sensitivity of the detection means by cutting the test result was examined by counting the results on the actual specimen.

  Details about materials, nucleic acid extraction methods and LAMP amplification are similar to those described in Examples 2 and 3.

Results From the milk collected from each of the four quarters, the results of the DNA chip test (field test 2-1; FT2-1) using the same sample as the results of the culture test, and the same cow again after 3 months The result of the DNA chip test | inspection (Field test 2-2; FT2-2) from the test | inspected milk was shown (Table 6-1-Table 6-6).

  Of the notation of the detection result of the DNA chip test, “white circle” indicates a positive result. In each table showing the results obtained by FT2-1 and FT2-2, the results of the quantitative test are indicated by the symbols “L”, “M” and “H” in the two rightmost items. “L” indicates a low bacterial load, “M” indicates a moderate bacterial load, and “H” indicates a high bacterial load. In both FT2-1 and 2-2, the positive rate was high in the columns of CNS (staphylococci), Streptococcus, and Enterococcus (both streptococci). On the other hand, the results of the culturing method were particularly positive for streptococci, and it was found that the culturing results and the results of the DNA chip test differed. This was thought to be due to the sensitivity of the DNA chip being excessively high, while the culture method is sensitive enough to not judge positive bacteria such as resident bacteria. . In addition to these results, further considering the results obtained by the quantification method according to the embodiment, the bacteria present at a low bacterial quantity indicated as “L” should be determined to be resident bacteria. Therefore, it can be determined that the subject is a cut-off target. By cutting off in this way, the positive rate can be kept low, and false positive results can be prevented. Furthermore, it was considered that bacteria present in a moderate amount of bacteria indicated as “M” may be included in the target of staphylococci. About one streptococcus, it turned out that about a microbe which exists in the low bacterial quantity shown as "L", a positive rate is restrained low by performing a truncation and determining object as negative.

  Furthermore, using a DNA chip similar to the DNA chip used in FT2-1, a plurality of bacteria were detected by the same method as described above. At the same time, Escherichia coli, staphylococci and streptococci were quantified by the same method as in Example 2. About the detection result obtained by that, the data before cutting off and after cutting off were obtained, and they were compared. The result is shown in FIG. 12A. 12B is an enlarged view of a part of FIG. 12A.

  In FIG. 12B, for Escherichia coli, bacteria present in a low bacterial amount were targeted for cutting off. As for staphylococci, bacteria present in medium amounts and bacteria present in low amounts were targeted for cutting off. About Streptococcus, it was set as the object of cutting off about the microbe which exists in a low amount of bacteria.

  In FIGS. 12A and B, positive results are indicated by black circles. As shown in FIG. 12A, before the cut-off, in most of the examined specimens, Escherichia coli, staphylococci and streptococci were positive. However, by quantifying the amount of bacteria by the quantification method of the embodiment and cutting off with a preset amount of bacteria as a threshold (or reference value), it is possible to determine that only a sample with a truly large amount of bacteria is positive Became.

  From the above, it was confirmed that quantification is indispensable in a highly sensitive genetic test for diseases in which resident bacteria exist. By making it possible to eliminate false positives, it is possible to determine whether or not the bacterium is a resident bacterium and detect only the pathogenic bacterium more accurately.

Claims (15)

A method for quantifying a specific nucleic acid contained in a specimen,
(A) preparing a sample containing nucleic acid;
(B) F3 region, F2 region, first probe binding region and F1 region are included in this order from the 5 ′ end side, and B3c region, B2c region, LBc region and B1c region are included in this order from the 3 ′ end side. A primer set comprising a first primer and a second primer for amplifying the first target template sequence contained in the first primer, wherein the first primer has an F1c sequence complementary to F1 on the 5 ′ end side. And the second primer has the same B1c sequence as B1c at the 5 ′ end and is complementary to B2c at the 3 ′ end. Providing a primer set that is a BIP primer having a B2 sequence;
(C) From the 5 ′ end side, the F3 region, the F2 region, the second probe binding region having a different sequence from the first probe binding region, and the F1 region are included in this order, and the 3 ′ end side Preparing a concentration standard nucleic acid comprising a B3c region, a B2c region, an LBc region, and a B1c region in this order;
(D) bringing the specimen, the primer set, and the first concentration standard nucleic acid having a known concentration into one reaction environment, performing an amplification reaction by the LAMP method, and obtaining an amplification product;
(E) a first nucleic acid probe immobilized on a first immobilization region of a substrate and complementary to a first target sequence including the F2 region and the first probe binding region or its complementary sequence; and the substrate A second nucleic acid probe that is immobilized on the second immobilization region of the second nucleic acid probe and that is complementary to the second target sequence including the F2 region and the second probe binding region or a complementary sequence thereof, and obtained in (d) Reacting with said amplification product;
(F) quantifying a specific nucleic acid contained in the specimen by detecting the amount of hybridization for each of the first and second nucleic acid probes generated in (e) and comparing them with each other;
Including methods.   The primer set further comprises an F3 primer having the same F3 sequence in the F3 region, a B3 primer having a B3 sequence complementary to the B3c region, an LBc primer having the same LBc sequence in the LBc region, and combinations thereof. The method of claim 1, comprising a further primer selected from the group. A method for quantifying a specific nucleic acid contained in a specimen,
(A) preparing a sample containing nucleic acid;
(B) F3 region, F2 region, first probe binding region and F1 region are included in this order from the 5 ′ end side, and B3c region, B2c region, LBc region and B1c region are included in this order from the 3 ′ end side. A primer set comprising a first primer and a second primer for amplifying the target template sequence contained in the first primer, wherein the first primer has a F1c sequence complementary to F1 on the 5 ′ end side; and FIP primer having the same F2 sequence as F2 on the 3 ′ end side, the second primer having the same B1c sequence on B1c on the 5 ′ end side, and a B2 sequence complementary to B2c on the 3 ′ end side Preparing a primer set that is a BIP primer having
(C) preparing a first concentration standard nucleic acid to an nth concentration standard nucleic acid; wherein the first concentration standard nucleic acid comprises an F3 region, an F2 region, and other probe bindings from the 5 ′ end side. A _first probe binding region having a sequence different from that of the region, and an F1 region in this order, and a B3c region, a B2c region, an LBc region, and a B1c region in this order from the 3 ′ end side. Each of the nth concentration standard nucleic acids includes an F3 region, an F2 region, a _2n probe binding region having a sequence different from that of the other probe binding regions, and an F1 region in this order from the 5 ′ end side. And from the 3 ′ end side, the B3c region, the B2c region, the LBc region, and the B1c region are included in this order, respectively, where n is an integer of 2 or more.
(D) The first to nth concentration standard nucleic acids having first to nth concentrations, which are different from each other, in the first to nth reaction environment for each type of the concentration standard nucleic acid, A sample and a primer set, respectively, and carrying out an amplification reaction by the LAMP method to obtain amplification products,
(E) combining the first to n-th amplification products obtained in the first to n-th reaction environments into one;
(F) a first nucleic acid probe immobilized on a substrate, the nucleic acid probe of the second _first nucleic acid probe-second _n, reacting the amplification products were combined said obtained in (e); Here, the first nucleic acid probe is immobilized on the first immobilization region of the substrate, and is complementary to the first target sequence including the F2 region and the first probe binding region or a complementary sequence thereof. There, the second _first nucleic acid probe is immobilized to a second _first fixed region of the substrate, the second ₁ of the target sequence or its complementary sequence including the F2 region and the second _first probe-binding region are complementary, nucleic acid probes of the 2 _n is fixed to the fixing region of the 2 _n of the substrate, the target sequence or its complement of the 2 _n including the F2 region and probe binding region of the 2 _n Is complementary to the sequence,
(G) A specific nucleic acid contained in a specimen is detected by detecting the amount of hybridization for each of the first nucleic acid probe and the 2 ₁ to 2 _n nucleic acid probes generated in (f) and comparing them with each other. Quantifying
Including methods.   The primer set further comprises an F3 primer having the same F3 sequence in the F3 region, a B3 primer having a B3 sequence complementary to the B3c region, an LBc primer having the same LBc sequence in the LBc region, and combinations thereof. 4. The method of claim 3, comprising a further primer selected from the group. A method for quantifying a plurality of types of specific nucleic acids contained in a specimen,
(A) preparing a sample containing nucleic acid;
(B) designing a target template sequence for each type of the specific nucleic acid, and performing the following (i) to (iii) for all target template sequences;
Here, the target template sequence includes F3 region, F2 region, first probe binding region and F1 region in this order from the 5 ′ end side, and B3c region, B2c region, LBc region from the 3 ′ end side. And B1c region in this order,
(I) preparing a primer set including a first primer and a second primer for amplifying the target template sequence, wherein the first primer is F1c complementary to F1 on the 5 ′ end side; A FIP primer having a sequence and having the same F2 sequence as F2 at the 3 ′ end, the second primer having the same B1c sequence as B1c at the 5 ′ end, and B2c at the 3 ′ end Providing a primer set that is a BIP primer having a complementary B2c sequence;
(Ii) any one of the 2 ₁ to 2 _n probe binding regions having a sequence different from that of the F3 region, the F2 region, and the first probe binding region from the 5 ′ end side and different from each other , And F1 region in this order, and the first to nth concentration standard nucleic acids containing the B3c region, B2c region, LBc region, and B1c region in this order from the 3 ′ end side, respectively. Where n is an integer greater than or equal to 2,
(Iii) The first to nth concentration standard nucleic acids having first to nth concentrations, which are different from each other, in the first to nth reaction environment for each type of the concentration standard nucleic acid, A sample and a primer set, respectively, and carrying out an amplification reaction by the LAMP method to obtain amplification products,
(IV) a first nucleic acid probe, and providing a nucleic acid probe of the second _first nucleic acid probe-second _n; wherein the first nucleic acid probe, the F2 region and the first probe-binding region It is complementary to the first target sequence or its complementary sequence including the second _first nucleic acid probe, the second ₁ of the target sequence or its complementary sequence including the F2 region and the second _first probe-binding region The second _n nucleic acid probe is complementary to the second _n target sequence comprising the F2 region and the second _n probe binding region, or a complementary sequence thereof,
(C) preparing a DNA chip by immobilizing the nucleic acid probes prepared for all the target template sequences on a single substrate for each type;
(D) combining all the amplification products obtained by each amplification reaction performed on all the target template sequences into one to obtain an amplification product mixture;
(E) reacting the amplification product mixture obtained in (d) with the nucleic acid probe contained in the DNA chip prepared in (c);
(F) detecting each hybridization signal generated by the reaction of (e), obtaining the presence / absence and / or magnitude of the hybridization signal for each nucleic acid probe,
(G) For each of the target template sequences, the corresponding first to nth concentration standard nucleic acids are compared with the results of comparing the presence or absence or magnitude of the hybridization signal obtained in (f). Determining the amount of the specific nucleic acid in the sample for each type based on the concentration of the 1st to nth concentration standard nucleic acids;
Including methods.   For each type of the specific nucleic acid, the primer set further includes an F3 primer having the same F3 sequence in the F3 region, a B3 primer having a B3 sequence complementary to the B3c region, and the same LBc sequence in the LBc region. 6. The method of claim 5, comprising a further primer selected from the group consisting of LBc primers having and combinations thereof. The method according to any one of claims 1 to 6, wherein the specific nucleic acid includes a nucleic acid derived from Enterobacteriaceae, staphylococci, and / or streptococci, and the primer set includes:
(1) A primer set for amplifying a target template nucleic acid derived from Enterobacteriaceae, a first primer represented by SEQ ID NO: 23, a second primer represented by SEQ ID NO: 24, and SEQ ID NO: A primer set comprising: a third primer represented by 21; a fourth primer represented by SEQ ID NO: 22; and a fifth primer represented by SEQ ID NO: 25;
(2) A primer set for amplifying a target template nucleic acid derived from Enterobacteriaceae, a first primer represented by SEQ ID NO: 28, a second primer represented by SEQ ID NO: 29, and SEQ ID NO: A primer set comprising a third primer represented by 26, a fourth primer represented by SEQ ID NO: 27, and a fifth primer represented by SEQ ID NO: 30;
(3) A primer set for amplifying a target template nucleic acid derived from staphylococci, a first primer represented by SEQ ID NO: 32, a second primer represented by SEQ ID NO: 33, and a SEQ ID NO: 31 A primer set comprising a third primer, a fourth primer represented by SEQ ID NO: 34, and a fifth primer represented by SEQ ID NO: 35; and / or (4) amplifying a target template nucleic acid derived from Streptococcus A first primer represented by SEQ ID NO: 38, a second primer represented by SEQ ID NO: 39 or SEQ ID NO: 40, a third primer represented by SEQ ID NO: 36, and a SEQ ID NO: A primer group comprising a fourth primer represented by 37 and a fifth primer represented by SEQ ID NO: 41 or 42 Door;
Including
The concentration standard nucleic acid is
(1 ′) a first concentration standard nucleic acid that is a concentration standard nucleic acid for Enterobacteriaceae, and is selected from at least one first concentration standard nucleic acid set represented by SEQ ID NO: 61 and SEQ ID NO: 62, respectively;
(2 ′) a second concentration standard nucleic acid, which is a concentration standard nucleic acid for staphylococci, selected from at least one second concentration standard nucleic acid set represented by SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69, respectively And / or (3 ′) a concentration standard nucleic acid derived from Streptococcus, a third selected from at least one third concentration standard nucleic acid set represented by SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 72, respectively. Concentration of standard nucleic acids;
Including
The nucleic acid probe is
(1 ″) Nucleic acid probes for detecting amplification products derived from Enterobacteriaceae, which are represented by SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, respectively And / or nucleic acid probe sets comprising nucleic acid probes each represented by a complementary sequence thereof;
(2 ″) a nucleic acid probe for detecting an amplification product derived from Enterobacteriaceae, comprising a nucleic acid probe represented by SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively Nucleic acid probe sets comprising nucleic acid probes each represented by a set and / or their complementary sequences;
(3 ″) a nucleic acid probe for detecting an amplification product derived from staphylococci, comprising a nucleic acid probe represented by SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55, and And / or a nucleic acid probe set comprising a nucleic acid probe represented by each of these complementary sequences; and / or (4 ″) a nucleic acid probe for detecting an amplification product derived from Streptococcus, SEQ ID NO: 56, SEQ ID NO: 67 A nucleic acid probe set comprising a nucleic acid probe represented by SEQ ID NO: 58 and SEQ ID NO: 59, respectively, and / or a nucleic acid probe set comprising a nucleic acid probe respectively represented by a complementary sequence thereof;
Including methods. An assay kit for use in the method of claim 1 comprising:
(A) F3 region, F2 region, first probe binding region and F1 region are included in this order from the 5 ′ end side, and B3c region, B2c region, LBc region and B1c region are included in this order from the 3 ′ end side. A first primer and a second primer for amplifying the first template sequence contained in the above, wherein the first primer has an F1c sequence complementary to F1 on the 5 ′ end side, and 3 ′ FIP primer having the same F2 sequence in F2 on the terminal side, the second primer having the same B1c sequence in B1c on the 5 ′ terminal side, and a B2 sequence complementary to B2c on the 3 ′ terminal side A primer set as a primer;
(B) From the 5 ′ end side, the F3 region, the F2 region, the second probe binding region having a sequence different from the first probe binding region, and the F1 region are included in this order, and the 3 ′ end side To B3c region, B2c region, LBc region, and concentration standard nucleic acid containing B1c region in this order,
(C) a first nucleic acid probe immobilized on a first immobilization region of a substrate and complementary to a first target sequence including the F2 region and the first probe binding region or a complementary sequence thereof, and the substrate A second nucleic acid probe which is immobilized on the second immobilization region of the second nucleic acid probe and which is complementary to the second target sequence including the F2 region and the second probe binding region or a complementary sequence thereof;
An assay kit comprising:   9. The assay kit according to claim 8, wherein the primer set further comprises a further primer, and the further primer is an F3 primer having the same F3 sequence in the F3 region, complementary to the B3c region. An assay kit, which is a primer selected from the group consisting of a B3 primer having a B3 sequence, an LBc primer having the same LBc sequence in the LBc region, and combinations thereof. An assay kit for use in the method of claim 3 comprising:
(A) F3 region, F2 region, first probe binding region and F1 region are included in this order from the 5 ′ end side, and B3c region, B2c region, LBc region and B1c region are included in this order from the 3 ′ end side. A primer set for amplifying the target template sequence contained in the primer set, wherein the primer set includes a first primer and a second primer, and the first primer is F1c complementary to F1 on the 5 ′ end side. A FIP primer having a sequence and having the same F2 sequence as F2 at the 3 ′ end, the second primer having the same B1c sequence as B1c at the 5 ′ end, and B2c at the 3 ′ end A primer set that is a BIP primer having a complementary B2 sequence;
(B) a first concentration standard nucleic acid to an nth concentration standard nucleic acid, wherein the F3 region, the F2 region, and the other probe binding regions are arranged from the 5 ′ end side of the first concentration standard nucleic acid. a different second _one probe binding region comprises respectively a F1 region in this order, and the B3c region from 3 'terminal side, and B2c region, and LBc region comprises respectively a B1c region in this order, the The n concentration standard nucleic acid includes an F3 region, an F2 region, a second _n probe binding region having a sequence different from that of the other probe binding regions, and an F1 region in this order from the 5 ′ end side, In addition, the first concentration standard nucleic acid to the nth concentration standard nucleic acid each including a B3c region, a B2c region, an LBc region, and a B1c region in this order from the 3 ′ end side, and n is an integer of 2 or more. When,
(C) a substrate, a first nucleic acid probe and the second ₁ of the nucleic acid probe-second _n DNA chip comprising a nucleic acid probe immobilized on the substrate, wherein the first nucleic acid probe, the is immobilized on a first fixed region of a substrate, said complementary to the first target sequence or its complementary sequence including the F2 region and the first probe-binding region, said second _first nucleic acid probe, wherein immobilized to the second _first fixed region of a substrate, said complementary to the second ₁ of the target sequence or its complementary sequence including the F2 region and the second _first probe-binding region, the nucleic acid probe of the 2 _n is A DNA chip that is immobilized on the 2 _n immobilization region of the substrate and is complementary to the 2 _n target sequence including the F2 region and the 2 _n probe binding region or a complementary sequence thereof,
An assay kit comprising: An assay kit for use in the method of claim 7, comprising:
The primer set is
(1) A primer set for amplifying a target template nucleic acid derived from Enterobacteriaceae, a first primer represented by SEQ ID NO: 23, a second primer represented by SEQ ID NO: 24, and SEQ ID NO: A primer set comprising: a third primer represented by 21; a fourth primer represented by SEQ ID NO: 22; and a fifth primer represented by SEQ ID NO: 25;
(2) A primer set for amplifying a target template nucleic acid derived from Enterobacteriaceae, a first primer represented by SEQ ID NO: 28, a second primer represented by SEQ ID NO: 29, and SEQ ID NO: A primer set comprising a third primer represented by 26, a fourth primer represented by SEQ ID NO: 27, and a fifth primer represented by SEQ ID NO: 30;
(3) A primer set for amplifying a target template nucleic acid derived from staphylococci, a first primer represented by SEQ ID NO: 32, a second primer represented by SEQ ID NO: 33, and a SEQ ID NO: 31 A primer set comprising a third primer, a fourth primer represented by SEQ ID NO: 34, and a fifth primer represented by SEQ ID NO: 35; and / or (4) amplifying a target template nucleic acid derived from Streptococcus A first primer represented by SEQ ID NO: 38, a second primer represented by SEQ ID NO: 39 or SEQ ID NO: 40, a third primer represented by SEQ ID NO: 36, and a SEQ ID NO: A primer group comprising a fourth primer represented by 37 and a fifth primer represented by SEQ ID NO: 41 or 42 Door;
Including
The nucleic acid probe is
(1 ″) Nucleic acid probes for detecting amplification products derived from Enterobacteriaceae, which are represented by SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, respectively And / or nucleic acid probe sets comprising nucleic acid probes each represented by a complementary sequence thereof;
(2 ″) a nucleic acid probe for detecting an amplification product derived from Enterobacteriaceae, comprising a nucleic acid probe represented by SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively Nucleic acid probe sets comprising nucleic acid probes each represented by a set and / or their complementary sequences;
(3 ″) a nucleic acid probe for detecting an amplification product derived from staphylococci, comprising a nucleic acid probe represented by SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55, and And / or a nucleic acid probe set comprising a nucleic acid probe represented by each of these complementary sequences; and / or (4 ″) a nucleic acid probe for detecting an amplification product derived from Streptococcus, SEQ ID NO: 56, SEQ ID NO: 67 A nucleic acid probe set comprising a nucleic acid probe represented by SEQ ID NO: 58 and SEQ ID NO: 59, respectively, and / or a nucleic acid probe set comprising a nucleic acid probe respectively represented by a complementary sequence thereof;
An assay kit comprising: 8. The assay kit of claim 7, further comprising concentration standard nucleic acids for Enterobacteriaceae, Staphylococci and Streptococcus, wherein the concentration standard nucleic acid comprises:
(1 ′) a concentration standard nucleic acid for Enterobacteriaceae, a first concentration standard nucleic acid selected from at least one first concentration standard nucleic acid set represented by SEQ ID NO: 61 and SEQ ID NO: 62, respectively; ,
(2 ′) a second concentration standard nucleic acid, which is a concentration standard nucleic acid for staphylococci, selected from at least one second concentration standard nucleic acid set represented by SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69, respectively When,
(3 ′) a third standard nucleic acid derived from streptococci and selected from at least one third standard nucleic acid set represented by SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 72, respectively An assay kit comprising: A method for determining whether or not a specific bacterium contained in a specimen is a resident bacterium,
(A) preparing a sample containing nucleic acid;
(B) F3 region, F2 region, first probe binding region and F1 region are included in this order from the 5 ′ end side, and B3c region, B2c region, LBc region and B1c region are included in this order from the 3 ′ end side. A primer set comprising a first primer and a second primer for amplifying the target template sequence contained in the first primer, wherein the first primer has a F1c sequence complementary to F1 on the 5 ′ end side; and FIP primer having the same F2 sequence as F2 on the 3 ′ end side, the second primer having the same B1c sequence on B1c on the 5 ′ end side, and a B2 sequence complementary to B2c on the 3 ′ end side Preparing a primer set that is a BIP primer having
(C) preparing a first concentration standard nucleic acid to a third concentration standard nucleic acid; wherein the first concentration standard nucleic acid comprises an F3 region, an F2 region, and other probe bindings from the 5 ′ end side. A second probe binding region having a sequence different from that of the region, and an F1 region in this order, and a B3c region, a B2c region, an LBc region, and a B1c region in this order from the 3 ′ end side, The second concentration standard nucleic acid includes an F3 region, an F2 region, a third probe binding region having a sequence different from that of the other probe binding regions, and an F1 region in this order from the 5 ′ end side, and The B3c region, the B2c region, the LBc region, and the B1c region are included in this order from the 3 ′ end side, and the third concentration standard nucleic acid includes the F3 region, the F2 region, and the other from the 5 ′ end side. A fourth probe bond having a sequence different from that of the probe binding region Includes a region, a F1 region in this order, comprising from and 3 'terminal side and the B3c region, the B2c region, and LBc region, and B1c region in this order,
(D) a first concentration standard nucleic acid having a concentration determined to be a low concentration in advance, a second concentration standard nucleic acid having a concentration determined to be a medium concentration in advance, and a concentration determined to be a high concentration in advance. The third concentration standard nucleic acid is brought into the first to third reaction environments for each type of concentration standard nucleic acid together with the specimen and the primer set, and an amplification reaction is performed by the LAMP method to obtain an amplification product. Getting each,
(E) combining the first to third amplification products obtained in the first to third reaction environments into one;
(F) reacting the first to fourth nucleic acid probes immobilized on the substrate with the combined amplification product obtained in (e); wherein the first nucleic acid probe comprises It is immobilized on a first immobilization region of a substrate and is complementary to a first target sequence including the F2 region and the first probe binding region or a complementary sequence thereof, and the second nucleic acid probe is the substrate The second nucleic acid probe is complementary to a second target sequence including the F2 region and the second probe binding region or a complementary sequence thereof, and the third nucleic acid probe is immobilized on the second immobilization region of the substrate. 3 and is complementary to a third target sequence including the F2 region and the third probe binding region or a complementary sequence thereof, and the fourth nucleic acid probe is a fourth nucleic acid probe of the substrate. Immobilized in the immobilization region, the F2 It is complementary to the fourth target sequence or its complementary sequence, including the frequency and the fourth probe binding region,
(G) Whether the specific nucleic acid contained in the sample is a resident bacterium by detecting the amount of hybridization for each of the first to fourth nucleic acid probes generated in (f) and comparing them with each other Determining whether or not
Including methods.   The method according to claim 13, wherein when the amount of the bacteria quantified in (g) is determined to be a low amount of bacteria, it is determined that the bacteria are resident.   The method according to claim 13, wherein when the amount of the bacteria quantified in (g) is determined to be a low or moderate amount of bacteria, it is determined to be a resident bacteria. Method.

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