JP2005168408A - Method for determining target microorganism or gene by quantitative pcr method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for determining a microorganism or a gene by a quantitative PCR method by which the accuracy of the determination is secured even for a sample in which the recovery of the DNA is low and a large amount of impurities is contained. <P>SOLUTION: The method for determining the target microorganism or gene present in the test sample by collecting a nucleic acid sample from the test sample, and carrying out the quantitative PCR by using the nucleic acid sample as a template is characterized by the use of two or more kinds of standard materials in a step for collecting the nucleic acid sample and a step for carrying out the quantitative PCR. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for quantifying a microorganism or gene by a quantitative PCR method, a microorganism or gene quantification kit based on this technique, a biological treatment control unit, and the like. More specifically, the recovery rate of DNA is low and contaminants are low. The present invention relates to a quantitative method that is effective even for a sample that is difficult to perform highly accurate quantitative evaluation.

  Various methods using an external standard or an internal standard are used in the quantitative evaluation of microorganisms by the PCR method. In the external standard method (Heid et al .: Genome Res .: 6: 986-994 (1996): Non-Patent Document 1), a calibration curve is prepared by amplifying the purified DNA of the target microorganism or the available standard DNA alone. A technique for quantitative analysis is widely used. This method is effective for samples with a high or constant DNA recovery rate and a small amount of inhibitors to the PCR reaction, but for samples that have high DNA adsorptive properties such as soil and contain many contaminants. In this case, the DNA recovery rate is not evaluated, and the difference in amplification efficiency between the standard substance and the target gene cannot be corrected, so that accurate quantitative evaluation is difficult.

  In addition, in the internal standard method (Gilliland et al .: Proc. Natl. Acad. Sci. USA: 87: 2725-2729 (1990): Non-Patent Document 2), the by-product after PCR cannot be distinguished from the by-product of the target DNA. Quantitative evaluation that can avoid the influence of contaminants derived from the sample is possible by adding possible template DNA to the sample DNA as a standard substance and amplifying both DNAs simultaneously. Furthermore, in this method, it was possible to carry out quantitative evaluation with the DNA recovery rate corrected by adding the standard substance directly to the sample before DNA extraction.

Heid et al .: Genome Res .: 6: 986-994 (1996) Gilliland et al .: Proc. Natl. Acad. Sci. USA: 87: 2725-2729 (1990)

However, in the method of adding a standard substance to a sample before DNA extraction, it is necessary to extract DNA from many tubes in order to prepare a calibration curve for subjecting a sample of unknown concentration to quantitative evaluation. For example, when it is expected that about 10 ³ to 10 ⁸ target microorganisms are present, it is necessary to extract DNA from 7 tubes per specimen even when preparing a calibration curve of an order level. In the process of quantitative DNA evaluation by PCR method, extraction and purification of DNA from a sample is the most complicated and time-consuming process. It was a method that lacked versatility. In this way, the recovery rate of DNA from the sample is determined, and quantitative evaluation by PCR reaction is ensured to be quantitative without being affected by contaminants, and the establishment of a quantitative method that reduces the workload of the complicated DNA extraction process. Was desired.

  In view of the circumstances as described above, the present invention provides a method for quantitatively evaluating microorganisms or genes by PCR reaction, which is usually difficult to perform quantitative evaluation with high DNA recovery rate and a large amount of impurities. However, it is possible to ensure the accuracy of quantification, and in particular to provide a quantification method capable of evaluating even one sample of DNA extraction treatment per sample. It is an object to provide a biological treatment control unit and the like.

  In the quantitative evaluation of target microorganisms or nucleic acids by the PCR method, the present inventors have conducted an improved study on the most versatile external standard method and the quantitative evaluation method by the internal standard method. By using multiple different reference materials in the process of determining the recovery rate of the target and in the process of quantitative evaluation of the target gene by PCR, it is possible to ensure quantitativeness without being affected by contaminants and the workload of the DNA extraction process The inventors have found a few techniques and have completed the present invention.

That is, the present invention is a method for recovering a nucleic acid sample from a test sample, performing quantitative PCR using the nucleic acid sample as a template, and quantifying a target microorganism or gene present in the test sample,
There is provided a method characterized in that at least two or more different standard substances are used in the step of recovering the nucleic acid sample and the step of quantitative PCR.

One aspect of the method of the present invention is a quantitative PCR method using a primer for amplifying a target nucleic acid serving as an indicator of the target microorganism or gene.
The present invention provides a method comprising using a nucleic acid fragment to which a third primer target site different from the target site of the primer is added in the nucleic acid sample as an amplification factor standard substance for the PCR method.

  In another aspect of the method of the present invention, PCR is carried out using a nucleic acid sample containing the amplification factor standard substance at different concentrations as a template, and using a third primer for the third primer target site for each sample. Including.

The present invention is also a method for producing an amplification factor standard used in the above method,
PCR is performed using a nucleic acid sample derived from a target microorganism or gene as a template, and primers for amplifying the target nucleic acid serving as an index thereof, and further, the PCR amplification product is used as a template, and the primer is used as a template. Provides a method comprising performing a PCR method using a linked primer in which different third primers are linked.

  In another aspect of the present method, a third microorganism or gene different from the target microorganism or gene is added to the test sample as a recovery rate standard substance in the recovery step, and these are used as indicators. Performing PCR using primers for amplifying the three target nucleic acids.

  The above aspect of the present method further includes a third primer target different from the primer for amplifying the third target nucleic acid as an amplification factor standard substance for the PCR method for amplifying the third target nucleic acid. It may include using a nucleic acid fragment to which a site has been added.

  In addition, the aspect of the present method described above is the PCR method for amplifying the third target nucleic acid, wherein a nucleic acid sample containing the amplification factor standard substance at a different concentration is used as a template, and each of them is directed to the third primer target site. It may also include performing PCR using a third primer.

The present invention is also a method for producing an amplification factor reference material for a third microorganism or gene used in the above method,
Using the nucleic acid sample derived from the third microorganism or gene as a template, PCR is performed using a primer for amplifying the third target nucleic acid serving as an index thereof, and the PCR amplification product is used as a template. And a method comprising PCR using a linked primer obtained by linking a third primer different from the primer to the primer.

  Further, the present invention relates to a third target nucleic acid obtained by any one of the above third target microorganisms or gene quantification methods with respect to the target nucleic acid quantitative value obtained by any one of the above target microorganisms or genes quantification method. The recovery rate of the target microorganism or gene is determined by applying the recovery rate of the target microorganism.

The test sample can be a sample derived from soil, groundwater, rivers, lakes, seawater, drainage, sludge, sludge, or activated sludge.
Furthermore, the present invention is a quantitative evaluation kit for carrying out any of the above methods, the amplification rate standard substance, the recovery rate standard substance, a reagent for preparing these standard substances, and the like. A kit including primers and the like is provided.

  Furthermore, the present invention is a biological treatment control apparatus for controlling a biological treatment process, wherein a sampling unit that automatically samples a sample from a biological treatment tank, and any of the above-described quantitative methods is applied to the sampled sample. A device comprising a DNA quantification unit for quantifying a target microorganism or gene, a condition control unit for controlling the conditions in the biological treatment tank, and a control unit for controlling the condition control unit based on the quantification value obtained by the DNA quantification unit I will provide a.

  FIG. 1 is a process diagram showing a preferred embodiment of the quantitative method of the present invention. In this method, first, purified DNA is obtained from a test sample containing target cells through a DNA extraction step and a DNA purification step, and then a quantitative PCR method is performed using the purified DNA as a template. This is a method for quantitative evaluation of numbers, using a non-target microorganism (third microorganism) as a standard substance for determining the recovery rate of DNA, and a non-target nucleic acid as a standard substance for quantitative evaluation by PCR. Synthetic DNA (nucleic acid amplified by a third primer different from the detection primer) is used.

  The third microorganism to be used as the standard sample is a microorganism that is similar to the target microorganism but can be specifically detected by PCR, but is preferably selected from microorganisms that are not originally present in the test sample. In addition, as a synthetic DNA for quantitative evaluation (hereinafter also referred to as amplification factor reference material), preferably a nucleic acid fragment having the same sequence as the target nucleic acid but having a primer target sequence that can be specifically detected separately from the target nucleic acid is used. To do.

  A specific quantitative evaluation method is to calculate the target cell number (concentration) from the PCR quantitative value for the target nucleic acid in reverse. That is, the amount before amplification, that is, the amount of target nucleic acid in the template nucleic acid is obtained by multiplying the amount of target nucleic acid amplified by PCR by the amplification rate by PCR. Next, the target nucleic acid amount in the initial sample is obtained by multiplying the target nucleic acid amount by the recovery rate, and the value obtained by dividing the number of the target nucleic acid by the number of copies of the target nucleic acid per microbial cell is obtained. It can be the number of cells.

The PCR amplification rate is evaluated as follows.
First, a detection target region (target nucleic acid) that serves as an index of a target microorganism is selected. This is a region selectively amplified by the PCR, and is preferably determined by selecting a primer pair targeting a partial sequence specific to the target microorganism. The sequence to be targeted by the primer can be selected from nucleic acid sequences such as 16SrRNA and 23SrRNA according to conventional methods in the art, and is preferably specific even in other microbial communities present in the test sample. This is a site that enables easy detection.

On the other hand, as the amplification factor standard substance, a nucleic acid fragment including the detection target region and having a third primer target site different from the detection primer target site at both ends thereof is used.
As described above, the amplification factor standard substance containing the target nucleic acid region but having the third primer target site can be easily produced using a linked primer in which the third primer is linked to the detection primer. (See Figure 2). That is, first, purified DNA derived from a target microorganism is used as a template, PCR (I) is performed using a detection primer pair, and an amplified nucleic acid fragment of the target nucleic acid is obtained. PCR (II) is performed using a linking primer having a configuration in which a third primer is added to the non-opposing ends (usually on the 5 ′ end side) of the primer pair for use. Thus, a nucleic acid fragment having the same sequence as the target nucleic acid and having a third primer target site at each end is amplified. The third primer may be a synthetic DNA that does not hybridize to the nucleic acid from the organism group in the sample. The standard substance having the third primer target site is selectively amplified by PCR (III) using the third primer, and a calibration curve can be prepared using this PCR reaction.

  The evaluation with the amplification factor standard substance can be performed as follows using one purified DNA sample. A purified DNA sample extracted from the sample is divided, and an amplification factor standard substance is added to each at various concentrations to prepare a plurality of template samples. Quantitative PCR using the third primer is performed on each template sample, and a calibration curve of amplification rate with respect to the concentration is created from the results. In practice, the concentration of the target nucleic acid contained in the template sample is unknown, so by applying the amplification result by PCR to the calibration curve for the test sample, an appropriate amplification factor is applied, and the exact target before amplification is applied. The amount (concentration) of nucleic acid can be determined. According to this method, since the quantitative accuracy can be ensured by internally evaluating the amplification rate of the target nucleic acid using a standard substance having the same sequence structure, and a calibration curve can be obtained from one purified DNA sample, it is unknown. Quantitative evaluation can be performed with the minimum necessary purification work for the target microorganisms at a concentration.

Next, evaluation based on the DNA recovery rate will be described.
In this method, in order to examine the recovery rate, a third microorganism different from the target microorganism is added to the test sample as a recovery rate standard substance at a specific concentration, and quantitative PCR is performed. That is, the recovery rate of the third microorganism from the same sample by the same process is obtained, and this recovery rate is applied as the recovery rate of the standard microorganism, so that the number of target microorganisms in the initial sample can be accurately determined.

  The recovery rate is determined by quantitative PCR using a primer that can selectively amplify the third target nucleic acid, which is an indicator of the third microorganism, using the nucleic acid sample obtained from the sample added with the third microorganism as a template. This is obtained by comparing the calculated quantitative number of bacteria for the third target nucleic acid with the actual addition amount. The third target nucleic acid region is a region that is selectively amplified by the PCR, and is preferably determined by selecting a primer pair that targets a partial sequence specific to the third microorganism. The sequence to be targeted by the primer can be selected from nucleic acid sequences such as 16SrRNA and 23SrRNA according to conventional methods in the art, and is preferably detected specifically in other microbial communities present in the test sample. It is a part that makes possible.

  Furthermore, when determining the recovery rate of the third microorganism, a calibration curve for the amplification factor may be determined for the quantitative PCR method in the same manner as the target microorganism. In this case, a nucleic acid fragment containing the third target nucleic acid and having a third primer target site different from the detection primer target site at both ends is used as the amplification factor standard substance.

  Amplification rate reference material for the third target nucleic acid can also be produced in the same manner as the above-mentioned target microorganism, using a linked primer in which the third primer is linked to the detection primer for the third target nucleic acid. Manufactured (see FIG. 2). That is, using purified DNA derived from the third microorganism as a template, first performing PCR (I) using a detection primer pair to obtain an amplified nucleic acid fragment of the third target nucleic acid, and then using this amplified nucleic acid fragment PCR (II) is performed using a linking primer having a configuration in which a third primer is added to the non-opposing ends (usually on the 5 ′ end side) of the detection primer pair as a template. The nucleic acid fragment thus amplified has the same sequence as the third target nucleic acid and has a third primer target site at each end. The third primer is preferably a synthetic DNA that does not hybridize to a nucleic acid derived from a biological group in the sample. In addition, you may use the 3rd primer utilized here with the same arrangement | sequence as the case of the above-mentioned target microorganism. The standard substance having the third primer target site is selectively amplified by PCR (III) using the third primer, and a calibration curve can be prepared using this PCR reaction.

  In the quantitative evaluation of the third microorganism, the amplification factor can be obtained using one purified DNA sample. In other words, separate the purified DNA, add the amplification factor standard substance at various concentrations, prepare multiple template samples, and perform quantitative PCR using the third primer for each template sample. From these results, a calibration curve of the amplification rate can be created for the third target nucleic acid in the PCR. Thus, the amount of the third target nucleic acid before amplification can be determined easily and accurately, and the recovery rate can be accurately calculated.

Below, the one aspect | mode of this method to which both amplification rate evaluation and recovery rate evaluation were applied is shown.
First, a third microorganism at a specific concentration is added to the test sample, DNA extraction and purification steps are performed, and PCR is performed using the purified DNA as a template sample and a primer that targets the third target nucleic acid. In parallel, prepare a system in which amplification factor standards (nucleic acid fragments having a third primer target site to the third target nucleic acid) are added to the same template sample at various concentrations, and PCR is performed under the same conditions for each. . Based on the calibration curve obtained as a result, the amount of the third target nucleic acid before amplification (in the purified DNA sample) is determined. Compare the calculated number of bacteria estimated from this nucleic acid quantitative value with the actual number of added cells, and if necessary, take into account the number of copies of the nucleic acid, and the ratio for the third microorganism. DNA recovery rate.

  Separately, quantitative PCR specific for the target microorganism is performed. In parallel, prepare a system in which amplification rate standard substances (nucleic acid fragments having a third primer target site to the target nucleic acid) are added to the purified DNA sample at various concentrations, and PCR is performed under the same conditions for each. Based on the calibration curve obtained as a result, the amount of target nucleic acid before amplification (in the purified DNA sample) is determined. The recovery rate obtained above is applied to this nucleic acid quantitative value, and the number of bacterial cells in the initial sample of the target microorganism can be calculated in consideration of the copy number of the nucleic acid if necessary.

In the above description, the method for quantifying a specific microorganism is exemplified. However, if a gene is targeted instead of a microorganism, the specific gene can be quantified in the same manner as described above.
In addition, in order to easily carry out the above quantification method, a set of necessary detection primers, each standard substance or reagents for preparing standard substances, and a corresponding third primer, etc. A kit for quantitative evaluation can be provided.

Next, an aspect of an apparatus for carrying out the quantitative method of the present invention will be described.
A preferred embodiment of the apparatus of the present invention is directed to monitoring or controlling a biological treatment process such as a sludge fermenter. Such a biological treatment control apparatus includes a sampling unit that automatically samples a sample from a biological treatment tank, a DNA quantification unit that quantifies a target microorganism or gene by applying the quantification method of the present invention to the sampled sample, and DNA quantification. A condition control unit that changes the operating parameters of the biological treatment based on a quantitative value obtained by the unit, a control unit that controls each unit, and the like are configured (see FIG. 4). This type of apparatus can be constructed by combining various automatic analysis means commercially available in the technical field with general-purpose computer control means. By computer control, DNA extraction, PCR, data storage, calculation for quantification and its Output can be processed automatically.

  In the biological treatment control apparatus, the control unit monitors the environmental conditions in the biological treatment tank, such as the number of cells of a specific microorganism, via the DNA quantification unit, and activates the condition control unit based on the monitoring result. Then, a process for optimizing the operation parameters, for example, addition of microorganisms is performed. The apparatus of the present invention may be combined with other analysis means for detecting environmental factors other than the number of bacterial cells, and is effective for all control and diagnosis of biological treatment processes.

  In the following examples, examples by the external standard method are shown, but the present invention is not limited to this, and can be applied to an evaluation method by the internal standard method.

Example 1
[Subject]
5 × 10 ⁶ cultured cells of Escherichia coli B strain were added to three types of soil, sandy soil, silty soil and clay, respectively, and used as specimens.

[DNA extraction]
On ice, 0.5 g of a sample was placed in a 2.0 ml micro test tube, and 1 × 10 ⁶ cultured cells of Bachillus subtilis IAM 12118 strain was added as a standard substance for DNA recovery rate evaluation, followed by DNA extraction. 0.5 ml of 100 mM phosphate buffer (pH 8.0), 0.5 g of zirconia / silica beads with a diameter of 0.1 mm, TNSE buffer (0.5 M Tris-HCl pH 8.0, 0.1 M NaCl, 10% SDS, 2 mM EDTA) After adding 250 μl, a mini bead beater (BIOSPEC PRODUCTS) treatment was performed for 3 min. After centrifugation at 15,000 rpm × 3 min, the supernatant was transferred to a new tube, mixed with 40% amount of 7.5 M ammonium acetate, and allowed to stand at 4 ° C. for 10 min. The supernatant after centrifugation was collected in a new tube, mixed with 2 volumes of isopropanol, and allowed to stand for 10 min. After centrifugation at 15,000 rpm × 10 min at room temperature, the supernatant was removed, and the precipitate was washed twice with 80% ethanol. The precipitate was dried with a centrifugal concentrator and then suspended in 50 μl of TE buffer supplemented with 1 mg / ml RNase and treated for 1 hour at 40 ° C. to obtain extracted DNA.

[DNA purification]
The extracted DNA was electrophoresed at an agarose concentration of 1.2%. As the electrophoresis tank, Mupid (Cosmo Bio) was used, and the entire amount of the extracted DNA was subjected to electrophoresis. After energizing for 20 min at 100 V, insert RECOCHIP (Takara) into the tip of the chromosomal DNA (corresponding to a size of about 4 kb) while observing the fluorescent image of the DNA, and energize again for 40 min. It was. After electrophoresis, RECO CHIP was transferred to an attached test tube, and DNA was collected by centrifugation for 10 seconds, and prepared to 50 μl with sterile water. For the PCR reaction, a 1/10 dilution of this purified DNA solution was used.

[PCR primer]
Primers with the following sequences were used in the PCR reaction.

[Preparation of standard substance]
FIG. 2 shows a method for preparing a standard substance. Prepare 50 μl of PCR reaction solution (primer Ecl62f and Ecl238r) containing 50 ng of chromosomal DNA of E. coli, heat denaturation at 95 ° C for 2 minutes with DNA Thermal Cycler (manufactured by PerkinElmer-Citas), 94 ° C-15 seconds, 59 ° C A cycle of -30 seconds, 72 ° C-60 seconds was performed 30 times. Furthermore, 50 μl of a PCR reaction solution (primers ST-Ecl62f and ST-Ecl238r) containing 5 μl of a 100,000-fold diluted solution of the by-product was prepared, and the same PCR cycle as described above was performed 30 times. The PCR product by-product obtained by simple purification using Wizard SV Gel and PCR Clean-up System (Promega) was used as standard substance E for quantitative evaluation of E. coli. The concentration of the standard substance was determined from the absorbance at 260 nm.

  A standard substance B for standard microorganism quantitative evaluation was prepared from the chromosomal DNA of Bachillus subtilis by the same procedure as that for E. coli. However, Bsb79f and Bsb230r were used as the first PCR primer, and ST-Bsb79f and ST-Bsb230r were used as the second primer.

[Evaluation of DNA recovery rate]
For the PCR reaction, FastStart DNA Mster SYBR Green I (Roche) was used as an enzyme kit, and LightCycler ^™ (Roche) was used as an amplification device. Add ₂ μl of the master mix solution supplied with the kit to the glass capillary of the reaction vessel, 5 μl of the test DNA as template DNA, and add 3.5 mM MgCl ₂ and 0.5 μM primers Bsb79f and Bsb230r at the final concentration, and then use the sterile water supplied with the kit. The solution volume was adjusted to 20 μl, and after heat denaturation at 95 ° C. for 10 minutes, a cycle of 95 ° C.-15 seconds, 59 ° C.-10 seconds, 72 ° C.-20 seconds, 84 ° C.-0 seconds was performed 45 times. Further, a standard substance B for quantitative evaluation of a standard microorganism prepared from a PCR product of chromosomal DNA of Bachillus subtilis under the PCR conditions described above is 1 × 10 ¹ , 1 × 10 ² , 1 × 10 ³ , 1 × 10 ⁴ per PCR reaction. A system with 1 × 10 ⁵ and 1 × 10 ⁶ copies added was prepared, and a calibration curve was prepared. In this case, ST-f and ST-r were used as primers.

From the analysis results by LightCycler ^TM, the target gene copy numbers of Bachillus subtilis in sandy soil, silty soil and clay are 1.4 × 10 ⁷ , 5.0 × 10 ⁶ , 4.4 × 10 ⁵ copies / g-, respectively. It became soil. Since the target gene copy number of Bachillus subtilis was 10, the DNA recovery rates in sandy soil, silty soil and clay during DNA extraction and purification processes were 68%, 25% and 2.2%, respectively.

[Quantitative evaluation of target microorganisms]
In the PCR reaction, FastStart DNA Mster SYBR Green I was used as an enzyme kit, and LightCycler ^™ was used as an amplification device. All were carried out in the same manner as the PCR procedure described above except that the final concentration of MgCl ₂ was 3.8 mM, and Ecl62f and Ecl238r were used as primers. In addition, a standard substance E for quantitative evaluation of E. coli prepared from a PCR product of chromosomal DNA of E. coli was 1 × 10 ¹ , 1 × 10 ² , 1 × 10 ³ , 1 × 10 ⁴ , 1 × 10 ⁵ , 1 per PCR reaction. A system with 10 ⁶ copies added was prepared and a calibration curve was prepared. In this case, ST-f and ST-r were used as primers.

As a result of multiplying the analysis result by LightCycler ^TM with the amplification efficiency ratio of the target gene and standard substance, the dilution factor of the sample, etc., the target gene copy number of Escherichia coli in sandy soil, silty soil and clay is 2.5 respectively. × 10 ⁷ , 8.2 × 10 ⁶ , and 7.4 × 10 ⁵ copies / g-soil. The number of E. coli target gene copies is 7, and the numbers of E. coli in the initial sample are 3.5 × 10 ⁶ , 1.2 × 10 ⁶ , and 1.1 × 10 ⁵ cells / g-soil, respectively. As described above, the quantitative value of the target gene or microorganism varies greatly depending on the soil, but it is 5.1 × 10 ⁶ , 4.7 × 10 ⁶ , and 4.8 × 10 ⁶ cells / percent when corrected by the previously obtained DNA recovery rate. It became g-soil. As described above, in the method for quantifying a target microorganism according to the present invention, a high-accuracy result is obtained with respect to an initial concentration of 5.0 × 10 ⁶ cells / g-soil in a sample by DNA extraction treatment of one specimen per sample. I was able to do things.

Comparative example 1 (conventional method 1)
[Subject]
5 × 10 ⁶ cultured cells of Escherichia coli B strain were added to three types of soil, sandy soil, silty soil and clay, respectively, and used as specimens.

[DNA extraction]
On ice, 0.5 g of the sample was placed in a 2.0 ml micro test tube, and DNA extraction was performed. 0.5 ml of 100 mM phosphate buffer (pH 8.0), 0.5 g of zirconia / silica beads with a diameter of 0.1 mm, TNSE buffer (0.5 M Tris-HCl pH 8.0, 0.1 M NaCl, 10% SDS, 2 mM EDTA) After adding 250 μl, a mini bead beater (BIOSPEC PRODUCTS) treatment was performed for 3 min. After centrifugation at 15,000 rpm × 3 min, the supernatant was transferred to a new tube, mixed with 40% amount of 7.5 M ammonium acetate, and allowed to stand at 4 ° C. for 10 min. The supernatant after centrifugation was collected in a new tube, mixed with 2 volumes of isopropanol, and allowed to stand for 10 min. After centrifugation at 15,000 rpm × 10 min at room temperature, the supernatant was removed, and the precipitate was washed twice with 80% ethanol. After the precipitate was dried with a centrifugal concentrator, a sample suspended in 50 μl of TE buffer supplemented with 1 mg / ml RNase was treated at 40 ° C. for 1 hour to obtain extracted DNA.

[DNA purification]
The extracted DNA was electrophoresed at an agarose concentration of 1.2%. As the electrophoresis tank, Mupid (Cosmo Bio) was used, and the entire amount of the extracted DNA was subjected to electrophoresis. After energizing for 20 min at 100 V, insert RECOCHIP (Takara) into the tip of the chromosomal DNA (corresponding to a size of about 4 kb) while observing the fluorescent image of the DNA, and energize again for 40 min. It was. After electrophoresis, RECO CHIP was transferred to an attached test tube, and DNA was collected by centrifugation for 10 seconds, and prepared to 50 μl with sterile water. For the PCR reaction, a 1/10 dilution of this purified DNA solution was used.

[PCR primer]
Primers with the following sequences were used in the PCR reaction.

[Preparation of standard substance]
Chromosomal DNA of Bachillus subtilis IAM 12118 strain was extracted and purified according to a conventional method (method described in Molecular Cloning), and the DNA was used as template DNA. For the PCR reaction, EX-Taq HotStart Ver. (Takara Shuzo) was used as an enzyme kit. Add 5 μl of the master mix solution supplied with the kit, 50 ng of template DNA, and 0.5 μM primers Bsb79f and Bsb230r to a 0.2 ml micro test tube, and then adjust the volume to 50 μl with the sterile water supplied with the kit. After heat denaturation at 95 ° C. for 2 minutes using Perkin Elma-Citas, a cycle of 94 ° C.-15 seconds, 59 ° C.-30 seconds, 72 ° C.-60 seconds was performed 30 times. The PCR by-product was immediately subjected to simple purification using Wizard SV Gel and PCR Clean-up System (Promega) and used as a standard substance for quantitative evaluation. The concentration of the standard substance was determined from the absorbance at 260 nm.

[Quantitative evaluation of target microorganisms]
For the PCR reaction, FastStart DNA Mster SYBR Green I (Roche) was used as an enzyme kit, and LightCycler ^™ (Roche) was used as an amplification device. Add 2 μl of the master mix solution supplied with the kit to the glass capillary of the reaction vessel, 5 μl of the test DNA as template DNA, and add 3.8 mM MgCl ₂ and 0.5 μM primers Ecl62f and Ecl238r at the final concentration, then use the sterile water supplied with the kit. The solution volume was adjusted to 20 μl, and after heat denaturation at 95 ° C. for 10 minutes, a cycle of 95 ° C.-15 seconds, 59 ° C.-10 seconds, 72 ° C.-20 seconds, 84 ° C.-0 seconds was performed 45 times. In addition, a standard substance prepared from a PCR product of Bachillus subtilis chromosomal DNA under the PCR conditions described above was 1 × 10 ¹ , 1 × 10 ² , 1 × 10 ³ , 1 × 10 ⁴ , 1 × 10 ⁵ , 1 A system to which x10 ⁶ and 1 x 10 ⁷ copies were added was prepared, and a calibration curve was prepared. In this case, Bsb79f and Bsb230r were used as primers, and the final concentration of MgCl ₂ was 3.8 mM.

As a result of multiplying the analysis result by LightCycler ^TM with a coefficient such as the dilution ratio of the sample, the target gene copy numbers of E. coli in sandy soil, silty soil and clay were 2.9 × 10 ⁷ , 7.3 × 10 ⁶ , respectively. And 6.7 × 10 ⁵ copies / g-soil. The number of E. coli target gene copies was 7, and the number of E. coli in the initial sample was estimated to be 4.1 × 10 ⁶ , 1.0 × 10 ⁶ , and 9.6 × 10 ⁴ cells / g-soil, respectively. It was. In this example, compared with the initial concentration of the target microorganism in the sample of 5.0 × 10 ⁶ cells / g-soil, a large error was caused depending on the properties of the sample.

Comparative example 2 (conventional method 2)
[Subject]
5 × 10 ⁶ cultured cells of Escherichia coli B strain were added to three types of soil, sandy soil, silty soil and clay, respectively, and used as specimens.

[DNA extraction]
Prepare a total of 7 samples of 0.5 g sample in a 2.0 ml micro test tube on ice, and 1 × 10 ³ cultured cells of Bachillus subtilis IAM 12118 as a standard substance for quantitative evaluation. DNAs were extracted by adding 10-fold concentrations of ˜1 × 10 ^{8 in a} stepwise manner and those without addition. In each test tube, 0.5 ml of 100 mM phosphate buffer (pH 8.0), 0.5 g of zirconia / silica beads with a diameter of 0.1 mm, TNSE buffer (0.5 M Tris-HCl pH 8.0, 0.1 M NaCl, 10% SDS After adding 250 μl of 2 mM EDTA), a mini bead beater (BIOSPEC PRODUCTS) treatment was performed for 3 min. After centrifugation at 15,000 rpm × 3 min, the supernatant was transferred to a new tube, mixed with 40% amount of 7.5 M ammonium acetate, and allowed to stand at 4 ° C. for 10 min. The supernatant after centrifugation was collected in a new tube, mixed with 2 volumes of isopropanol, and allowed to stand for 10 min. After centrifugation at 15,000 rpm × 10 min at room temperature, the supernatant was removed, and the precipitate was washed twice with 80% ethanol. After the precipitate was dried with a centrifugal concentrator, a sample suspended in 50 μl of TE buffer supplemented with 1 mg / ml RNase was treated at 40 ° C. for 1 hour to obtain extracted DNA.

[DNA purification]
The extracted DNA was electrophoresed at an agarose concentration of 1.2%. As the electrophoresis tank, Mupid (Cosmo Bio) was used, and the entire amount of the extracted DNA was subjected to electrophoresis. After energizing for 20 min at 100 V, insert RECOCHIP (Takara) into the tip of the chromosomal DNA (corresponding to a size of about 4 kb) while observing the fluorescent image of the DNA, and energize again for 40 min. It was. After electrophoresis, RECO CHIP was transferred to an attached test tube, and DNA was collected by centrifugation for 10 seconds, and prepared to 50 μl with sterile water. For the PCR reaction, a 1/10 dilution of this purified DNA solution was used.

[PCR primer]
Primers with the following sequences were used in the PCR reaction.

[Quantitative evaluation of target microorganisms]
For the PCR reaction, FastStart DNA Mster SYBR Green I (Roche) was used as an enzyme kit, and LightCycler ^™ (Roche) was used as an amplification device. For samples that were extracted without adding a calibration curve microorganism, 2 μl of the master mix solution attached to the kit to the glass capillary of the reaction vessel, 5 μl of the test DNA as template DNA, and a final concentration of 3.8 mM MgCl ₂ 、 After adding 0.5μM primers Ecl62f and Ecl238r, adjust the volume to 20μl with sterilized water supplied with the kit, heat denaturation at 95 ℃ for 10 minutes, 95 ℃ -15 seconds, 59 ℃ -10 seconds, 72 The cycle of ℃ -20 seconds and 84 ℃ -0 seconds was performed 45 times. In addition, in the case of a sample to which a microbe for preparing a calibration curve was added, a calibration curve was prepared by performing a PCR reaction under the condition that Bsb79f and Bsb230r were used as primers and the final concentration of MgCl ₂ was changed to 3.5 mM. As a result of multiplying the analysis result by LightCycler ^TM with the dilution factor of the sample, the numbers of E. coli in sandy soil, silty soil and clay were 3.3 × 10 ⁶ , 3.7 × 10 ⁶ and 3.1 × 10 ^{6, respectively.} Cells / g-soil. Since the number of copies of the target gene of Bachillus subtilis IAM 12118 used for the calibration curve was 10 and the number of copies of the target gene of E. coli was 7, the number of E. coli in the initial sample was 4.7 × 10. It was evaluated as ⁶ , 5.3 × 10 ⁶ , and 4.4 × 10 ⁶ cells / g-soil. As described above, the quantitative evaluation method in which the standard substance is added to the initial sample enables accurate evaluation, but it is necessary to perform a large amount of sample processing for DNA extraction / purification, which is the most complicated operation in a series of steps.

  The features and results of Example 1 and Comparative Examples 1 and 2 are summarized in Table 1 and FIG. As is clear from these results, according to the method of Example 1, the test microorganism can be quantified easily and accurately from one extracted sample.

Example 2 (Phosphorus removal test of wastewater using a genetically modified product)
[Configuration of biological treatment control device]
FIG. 4 schematically shows a schematic configuration of the biological treatment control apparatus of the present embodiment. This equipment is a sampling unit (1) that automatically collects a certain amount of activated sludge from the phosphorus removal tank that constitutes the biological treatment process, and a standard microorganism for correcting the DNA recovery rate is added to the activated sludge to extract and purify DNA. Next, an automatic quantification unit (2) for performing quantitative PCR by adding a postscript standard substance (synthetic DNA) for quantitative evaluation to purified DNA, and a processing condition control unit (3) for optimizing the processing conditions And a control unit (4) for automatically controlling each of the units (1) to (3).

[test]
Test microorganism: E. coli MV1184 (pBC29 + pEP02.2) (Hardoyo et al., (1994) Appl Environ Microbiol 60, 3485-3490) with enhanced phosphorus accumulation ability was used as the phosphorus removal microorganism.
Raw water: Artificial drainage to which glucose was added at a final concentration of 0.1% was used. Table 2 shows the composition of artificial waste water.

  Wastewater treatment apparatus: FIG. 5 shows the structure of a phosphorus removal treatment process apparatus. Raw water was supplied to the 0.5 L phosphorus removal tank (recombinant dephosphorization tank) at a rate of 100 mL / h. A sludge concentration tank was provided after the phosphorus removal tank, and solid-liquid separation was performed using a hollow fiber membrane. The sludge return rate was set at 25%.

  In this example, a sampling unit (not shown) is installed in the phosphorus removal tank, and the concentration of E. coli MV1184 (recombinant microorganism; pBC29 + pEP02.2), which is a phosphorus removal microorganism, is automatically monitored in the tank. As a condition control unit, an apparatus for automatically supplying the phosphorus-removing microorganism from the cultured cell storage tank into the phosphorus removal tank by a pump was installed. A control unit (not shown) automatically monitors the concentration of the phosphorus-removing microorganism obtained from the sampling unit, and based on the monitoring result of the concentration of the microorganism, activates the automatic supply device if necessary. The microorganism is automatically supplied to the phosphorus removal tank.

[Test conditions]
Test system 1: The initial phosphorus removal microorganism concentration in the phosphorus removal tank was set to 5 × 10 ⁸ cells / mL, and a continuous treatment test was performed. The microorganisms were automatically monitored once a day, and the phosphorus removal microorganism concentration was recorded. The supply of phosphorus-removing microorganisms was not performed.

Test system 2: The initial phosphorus removal microorganism concentration in the phosphorus removal tank was set to 5 × 10 ⁸ cells / mL, and a continuous treatment test was performed. The microorganisms were automatically monitored once a day, and when the concentration of the phosphorus-removing microorganisms became 1 × 10 ⁸ cells / mL or less, control was performed so that 25 g of phosphorus-removing microorganisms corresponding to the dry weight were automatically supplied.

[Monitoring conditions]
[Subject]
The sample was 0.5 mL of automatically sampled sludge.

[DNA extraction and purification]
DNA was extracted from the specimen and purified by an automatic DNA extraction device.

[PCR primer]
Primers with the following sequences were used in the PCR reaction.

[Preparation of standard substance]
50 μl of PCR reaction solution (primer pBC29-Lf1 and pBC29-Lr3) containing 0.1 ng of plasmid DNA of the phosphorus-removed microorganism was prepared, and heat denaturation at 95 ° C. for 2 minutes with DNA Thermal Cycler (manufactured by PerkinElmer-Citas) A cycle of 94 ° C-15 seconds, 59 ° C-30 seconds, 72 ° C-60 seconds was performed 30 times. Furthermore, 50 μl of a PCR reaction solution (primers ST-pBCLf1 and ST-pBCLr3) containing 5 μl of a 100,000-fold diluted solution of the byproduct was prepared, and the same PCR cycle as described above was performed 30 times. A PCR product by-product obtained by simple purification using Wizard SV Gel and PCR Clean-up System (Promega) was used as a standard substance E for quantitative evaluation of phosphorus-removing microorganisms. The concentration of the standard substance was determined from the absorbance at 260 nm.

  Standard substance B for quantitative evaluation was prepared from 50 ng of chromosomal DNA of Bachillus subtilis in the same manner as the above-mentioned phosphorus-removed microorganism (see FIG. 2). However, Bsb79f and Bsb230r were used as the first PCR primer, and ST-Bsb79f and ST-Bsb230r were used as the second primer.

[Test results]
6 and 7 show the results of automatic monitoring.
Test system 1: As shown in FIG. 6, the result of automatic monitoring of phosphorus-removed microorganisms was 5.4 × 10 ⁸ cells / mL at the start of the test, and the theoretical value of the microorganism concentration was 5 × 10 ⁸ cells / mL. Good results were obtained. Then the concentration of phosphorus removal microorganisms gradually decreases, 1 × 10 ⁸ cells / mL next to 6 days after the start of the test, how the drops to 1 × 10 ⁷ cells / mL was observed at 15 days of monitoring . As shown in FIG. 7, the orthophosphoric acid concentration of the raw water in this test was about 6.0 mg / L. From the first day after the start of the test, the orthophosphoric acid concentration of the treated water became 0.1 mg / L, then 7 days. Stable water quality was obtained. After the 8th day when the concentration of microorganisms removing phosphorus was 8.5 × 10 ⁷ cells / mL or less, the quality of treated water gradually deteriorated, and the concentration of treated water orthophosphoric acid became 3.0 mg / L on the 15th day. Water quality was maintained.

Test system 2: The result of automatic monitoring of phosphorus-removed microorganisms was 5.2 × 10 ⁸ cells / mL at the start of the test, and good results were obtained for the theoretical value of the microorganism concentration of 5 × 10 ⁸ cells / mL. (FIG. 6). Thereafter, the concentration of the phosphorus-removing microorganism gradually decreased and reached 1 × 10 ⁸ cells / mL on the 6th day after the start of the test. As a result of automatic supply of phosphorus-removed microorganisms, the result of monitoring on the seventh day was 4.6 × 10 ⁸ cells / mL. Thereafter, the concentration of the phosphorus-removing microorganism gradually decreased, and again reached 1 × 10 ⁸ cells / mL on the 14th day. Thereafter, the concentration of the phosphorus-removing microorganism was maintained at 1 × 10 ⁸ cells / mL or more until the 20th day. The concentration of orthophosphoric acid in the raw water in this test was about 6.0 mg / L, but the concentration of orthophosphoric acid in the treated water became 0.1 mg / L from the first day after the start of the test, and was stable until the 20th day after the end of the test. Water quality was maintained (Figure 7).

  As shown in the above examples, the automatic microorganism quantification apparatus using the quantification method according to the present method enables accurate evaluation even in a sample in an environment where quantitative evaluation is usually difficult. It is possible to maintain optimum conditions without waste in the process management.

  The application examples for wastewater treatment shown above do not limit the scope of application of this method, and can be applied to any treatment process as long as it is a process involving biological reactions such as contaminated soil treatment.

FIG. 1 is a process diagram showing one embodiment of the quantitative method of the present invention. FIG. 2 is a diagram showing a preparation process of an amplification factor standard substance. FIG. 3 is a table summarizing the number of samples used in Example 1 and Comparative Examples 1 and 2. FIG. 4 is a schematic diagram illustrating a schematic configuration of the biological treatment control apparatus according to the second embodiment. FIG. 5 is a diagram illustrating the configuration of the phosphorus removal processing apparatus according to the second embodiment. FIG. 6 is a diagram showing the monitoring results for the microorganism concentration. FIG. 7 is a diagram showing the monitoring results for the phosphoric acid concentration.

Explanation of symbols

1 Sampling unit 2 DNA quantification unit 3 Condition control unit 4 Control unit

Claims (12)

A method of recovering a nucleic acid sample from a test sample, performing quantitative PCR using the nucleic acid sample as a template, and quantifying a target microorganism or gene present in the test sample,
A method comprising using at least two or more different standard substances in the step of recovering the nucleic acid sample and the step of quantitative PCR. In a quantitative PCR method using a primer for amplifying a target nucleic acid serving as an indicator of the target microorganism or gene,
2. The method according to claim 1, comprising using a nucleic acid fragment to which a third primer target site different from the target site of the primer is added in the nucleic acid sample as the amplification rate standard for the PCR method.   The method according to claim 2, comprising performing PCR using a nucleic acid sample containing the amplification factor standard substance at different concentrations as a template and using a third primer for each of the third primer target sites. It is a manufacturing method of the amplification factor standard substance used for the method of any one of Claims 1-3,
PCR is performed using a nucleic acid sample derived from a target microorganism or gene as a template, and primers for amplifying the target nucleic acid serving as an index thereof, and further, the PCR amplification product is used as a template, and the primer is used as a template. Is a method comprising performing PCR using a linked primer obtained by linking different third primers.   As a recovery rate standard substance in the recovery step, a third microorganism or gene different from the target microorganism or gene is added to the test sample, and a third target nucleic acid serving as an index thereof is amplified. The method according to any one of claims 1 to 3, comprising performing PCR using a primer.   Furthermore, a nucleic acid fragment to which a third primer target site different from the primer for amplifying the third target nucleic acid is added as an amplification rate standard for the PCR method for amplifying the third target nucleic acid. 6. The method of claim 5, comprising using.   In the PCR method for amplifying the third target nucleic acid, PCR is performed using a nucleic acid sample containing the amplification factor standard substance at a different concentration as a template, and a third primer for the third primer target site, respectively. The method according to claim 6, comprising performing: A method for producing a standard substance used in the method according to claim 6 or 7,
Using the nucleic acid sample derived from the third microorganism or gene as a template, PCR is performed using a primer for amplifying the third target nucleic acid serving as an index thereof, and the PCR amplification product is used as a template. A method comprising performing PCR using a linked primer in which a third primer different from this primer is linked to the primer.   The recovery of the third target nucleic acid obtained by the method according to any one of claims 5 to 7 with respect to the quantitative value of the target nucleic acid obtained by the method according to any one of claims 1 to 3. A method for quantifying a target microorganism or gene, wherein the rate is applied to determine the abundance of the target microorganism or gene in the sample.   10. The test sample according to claim 1, wherein the test sample is a sample derived from soil, groundwater, rivers, lakes, seawater, drainage, sludge, sludge, or activated sludge. the method of.   A kit for quantitative evaluation for carrying out the method according to any one of claims 1 to 3, 5 to 7, or 9, wherein the amplification factor reference material, the recovery rate reference material, or these reference materials A kit containing reagents and the necessary primers for preparation.   A biological treatment control apparatus for controlling a biological treatment process, wherein the sampling unit automatically samples a sample from a biological treatment tank, and the sampled sample is any one of claims 1 to 3, 5 to 7, or 9. A DNA quantification unit for quantifying a target microorganism or gene by applying the quantification method described in Item 1, a condition control unit for controlling conditions in a biological treatment tank, and condition control based on a quantification value obtained by the DNA quantification unit A device comprising a control unit for controlling the unit.

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