WO2002052034A1 - Methodes de bioscopie et d'amplification d'acides nucleiques - Google Patents

Methodes de bioscopie et d'amplification d'acides nucleiques Download PDF

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WO2002052034A1
WO2002052034A1 PCT/JP2001/011489 JP0111489W WO02052034A1 WO 2002052034 A1 WO2002052034 A1 WO 2002052034A1 JP 0111489 W JP0111489 W JP 0111489W WO 02052034 A1 WO02052034 A1 WO 02052034A1
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dna
pcr
rna
bacteria
viability
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PCT/JP2001/011489
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WO2002052034A8 (fr
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Joji Oshima
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Joji Oshima
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Publication of WO2002052034A8 publication Critical patent/WO2002052034A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • the present invention relates to a method for determining the life and death of cells and / or bacteria by applying nucleic acid quantification, and efficiently and directly amplify genes of cells and / or nucleic acids And a method for amplifying nucleic acid.
  • Background Art Conventionally, as a method for determining the viability of single cells, which are the smallest organisms, or fungi such as bacteria and fungi, fungi collected from a specimen by an appropriate means are adjusted to a predetermined number of bacteria, and an agar medium or the like is used.
  • Japanese Patent Application Laid-Open No. 9-27599 / 98 discloses a fluorescent dye comprising fluorescein or a derivative thereof and a fluorescent dye comprising propidium iodide.
  • the fungus thus obtained is mixed with a staining solution containing a salt, chitines or a cellulase, which is then mixed with a saline solution, and the fungus cells are stained by heating.
  • a method of discriminating is disclosed ( Japanese Patent Application Laid-Open No. H10-2485997 discloses a method in which a bacterial solution stained with a fluorescent dye is injected into a transparent cell, and a predetermined wavelength is applied from one direction.
  • a photodiode detector tube Irradiate a pulse light with a pulse interval of at least 75 seconds or more, and on one side orthogonal to the axis light of this pulse light, a photodiode detector tube passes through a band pass filter that transmits fluorescent light from live bacteria. Detect, on the other hand In through the bandpass Sufiru evening one that transmits the fluorescence light from the killed cells was detected by Fotodaiodo detector tube, sterile Senshokukin solution A method and a device for discriminating between live and dead bacteria of a fungus by computer processing a difference between the detection voltage and the detection voltage of a stained bacterial solution containing a fungus are disclosed.
  • a fluorescent dye which is obtained by penetrating and staining fluorescently stained bacterial liquid into living cells and dead cells in a chemically stable state, has a strong fluorescence emission intensity and a clear fluorescence.
  • Japanese Patent Laid-Open No. 11-17885-68 discloses a method in which after detecting an image with a photodiode, electrical processing is performed to immediately determine the bacterial species, the number of bacteria, the number of viable bacteria and the number of dead bacteria with high accuracy. No. 6,086,045.
  • the determination of the survival of multicellular organisms, including humans is made by measuring stiffness and rectal temperature after death and observing changes in appearance.
  • the method of distinguishing live cells from dead cells by staining bacterial cells with a fluorescent dye has the advantage of being able to distinguish quickly, it requires specialized knowledge and technology, and is easy to distinguish. Not a law.
  • the bacteria when testing bacterial genes to identify bacteria, for example, drug-resistant or toxin-producing bacteria, the bacteria are detected after culturing the collected specimens, and nucleic acid extraction from the bacteria is performed.
  • a method of performing PCR as a nucleic acid amplification method through a quantification step, performing electrophoresis, staining the obtained gel with a reagent, and observing a gene band is generally employed. This method usually required one to two weeks of inspection time and labor, and also required highly skilled technical skills.
  • JP-A-9-194100 discloses that DNA having a specific base sequence present in the 16S ribosomal RNA (rRNA) gene of bacteria is primed.
  • rRNA ribosomal RNA
  • Japanese Patent Publication No. 6-1113899 discloses that genomic DNA was extracted from butyric acid bacteria, rRNA gene was amplified by PCR using a specific primer, and amplified. A method for identifying butyric acid bacteria by analyzing and comparing their lengths by electrophoresis is disclosed.
  • Japanese Patent Application Laid-Open No. Hei 7-231197 discloses a cDNA synthesized from mRNA to be detected derived from an intron-free portion of a gene. Is amplified with the genomic DNA corresponding to the cDNA.
  • the PCR reaction solution is added to the PCR reaction solution with a polyamine or surfactant to suppress the action of substances that inhibit the nucleic acid amplification enzyme reaction.
  • a polyamine or surfactant to suppress the action of substances that inhibit the nucleic acid amplification enzyme reaction.
  • the present invention is proposed in view of the above-mentioned circumstances, and is a method for determining the viability of cells such as fungi by applying nucleic acid quantification, and a method for determining the viability of cells and / or bacteria. It is an object of the present invention to provide a nucleic acid amplification method for directly and efficiently amplifying a gene.
  • a viability determining method is a viability determining method for determining the viability of cells and / or bacteria serving as a specimen.
  • the method is characterized in that at least a part of the sample is quantified, and the death is determined by comparing the quantitative ratio of the DNA and the RNA in the sample.
  • a viability determining method is a viability determining method for determining the viability of cells and / or bacteria as specimens, wherein DNA and RNA are extracted from the specimens. And amplifying the amplified product, quantifying the amplified product, and comparing the quantitative ratio of the DNA and the RNA in the sample to determine the viability.
  • the DNA and RNA can be amplified by the quantitative PCR method and the quantitative reverse transcription PCR method, respectively.
  • a viability determining method is a viability determining method for determining the viability of cells and Z or bacteria as specimens,
  • the relative DNA amount and relative RNA amount are the cycle threshold obtained from the relationship between the number of PCR cycles and the amount of amplified DNA or amplified cDNA. It can be determined based on the value.
  • a viability determining method is a viability determining method for determining the viability of cells and / or bacteria as a specimen, wherein DNA is extracted from the specimen, It is characterized by amplifying the unstable part of, and quantifying the amplification product to determine whether it is alive or not.
  • a viability determining method is a viability determining method for determining the viability of cells and / or bacteria serving as a specimen, wherein the method comprises the steps of: Extracting multiple samples, extracting DNA from the above multiple samples and real-time: Amplifying by PCR method, comparing the amplified DNA amount between the above multiple samples to determine whether it is alive or not here c is intended, wherein, the DNA amount is obtained based on the cycle thread Uz Chaux-de-value is obtained, et al from the relationship between the amplified DNA amount P CR cycles. Further, the plurality of samples may be extracted from the sample at different culture times.
  • the 138 or RNA was directly amplified in real-time P 01 using a PCR buffer without extracting DNA from the plurality of samples, and the amount of amplified DNA was reduced between the plurality of samples. It is also possible to judge whether a person is alive or not by making a comparison.
  • a viability determining method is a viability determining method for determining the viability of cells and / or bacteria serving as a specimen, wherein the method comprises the steps of: Extracting multiple samples, extracting RNA from the multiple samples, amplifying the RNA by real-time reverse transcription PCR, and comparing the amount of amplified RNA among the multiple samples to determine viability. It is a characteristic.
  • the RNA amount is determined based on a cycle threshold value obtained from the relationship between the number of PCR cycles and the amount of amplified RNA.
  • the plurality The RNA is directly amplified using the real-time reverse transcription PCR method using a PCR buffer without extracting RNA from the sample, and the amount of amplified RNA is compared between the multiple samples to determine viability. You may make it.
  • a nucleic acid amplification method is a nucleic acid amplification method for amplifying a nucleic acid of a cell and / or a bacteria as a specimen, wherein the nucleic acid is exposed by destroying the specimen. It is characterized in that the disrupted product is filtered to remove or suppress the reaction inhibitor, thereby amplifying the nucleic acid.
  • the nucleic acid can be amplified by the PCR method or the real-time PCR method.
  • FIG. 1 is a diagram illustrating an electrophoresis pattern after amplification of a DNA stable portion and a DNA unstable portion for samples A to H having different mixing ratios of live and dead bacteria.
  • FIG. 2 is a diagram illustrating a conceptual configuration of a nucleic acid amplification apparatus provided with a description of the present invention, comprising a destruction chamber, a filtration chamber, a PCR reaction chamber, and a PCR reaction solution reservoir.
  • FIG. 3 is a diagram illustrating an electrophoresis pattern obtained by electrophoresing a PCR product after PCR reaction on an agarose gel.
  • DNA and RNA collected from a sample were collected using a mechanism in which a certain amount of specific RNA was synthesized from a certain amount of specific DNA as long as the cell was alive. Amplification is performed, the DNA and RNA in the amplification product are quantified, and the ratio of the number of moles of the RNA to the DNA (RNA / DNA) is measured to determine cell viability.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • RNA is relatively stable and hard to break, and while live cells constantly decompose and produce RNA, dead cells Since RNA is not generated and is degraded early, the RNA / DNA molar ratio relative to living cells is relatively reduced. Therefore, by measuring the RNA / DNA molar ratio, it is possible to determine the viability of cells.
  • any nucleic acid amplification method such as a DNA cloning method using a DNA fragment and a PCR method can be used.
  • the DNA cloning method has the disadvantage that the amplification operation is troublesome, and the PCR method has the advantage that the target DNA can be amplified in a short time, but the target DNA is exponentially amplified. Therefore, the amount of amplification product is greatly affected by a slight difference in amplification efficiency, and there is a problem in quantitative analysis.
  • the nucleic acid amplification method quantitative reverse transcription PCR (quantitative RT-PCR) of RNA extracted from a sample and quantitative PCR of DNA are used. It is needless to say that the method is not limited to the quantitative RT-PCR method. Furthermore, there is no restriction on the presence or absence of nucleic acid amplification. That is, if the amounts of DNA and RNA extracted from the cells / bacteria are large, the viability of cells / bacteria can be determined directly from the DNAZRNA amount ratio without amplification.
  • Example 1 first, a competition for quantitative RT-PCR is prepared. Specifically, a Competitive DNA construction kit (manufactured by Takara Shuzo Co., Ltd.) was used according to the operating procedure. Using the first nucleotide sequence (5'-CAGCAGC CGCGGTAATAC-3,) and the second nucleotide sequence (5'-ACGACACGAGTCGACGAC-3 '), which encodes a part of the bacterial 16S ribosomal RNA To make a competition. The produced competition evening was quantitatively determined to have a known concentration.
  • a Competitive DNA construction kit manufactured by Takara Shuzo Co., Ltd.
  • IS0GEN manufactured by Nippon Gene Co., Ltd.
  • genomic DNA and total RNA derived from bacteria were extracted according to the usual operation procedure.
  • the extracted total RNA was quantified, and a reverse transcription reaction was performed by adding a random hexamer as a reverse transcription primer to 2 g of the total RNA.
  • quantification is performed using the first primer (5, -CAGCAGCCGCGGT A AT AC-3 ') and the second primer (5, -ACGACACGAGCTGAC GAC-3,) using 5 ⁇ 1 of the reverse transcript.
  • RT PCR reactions were performed.
  • the PCR product after the completion of the reaction was electrophoresed on an agarose gel, and after staining with ethidium / methylene chloride, the difference in band concentration was measured with a densitometer or the like.
  • a calibration curve was prepared with the competition concentration on the X axis and the ethidium bromide staining concentration on the Y axis, and the ethidium promide staining concentration of the sample with a competition concentration of 0 was applied to the calibration curve, and the mRNA concentration of mRNA was determined. The molarity was determined.
  • a PCR reaction was performed using the 2 zg, and the first and second primers were added to 5/1 of the completed PCR product. To perform a quantitative PCR reaction.
  • the specificity can be increased by performing the PCR in two stages, but the first stage PCR may be omitted.
  • the primer used in the first-stage PCR is located outside the first primer and the second primer used in the second-stage quantitative PCR.
  • the quantitative PCR product was subjected to agarose gel electrophoresis, and after staining with ethidium bromide, the band concentration difference was measured using a densitometer or the like.
  • the quantitative RT-PCR and the quantitative PCR provide the molar concentrations of the mRNA and genomic DNA of the 16S ribosomal RNA derived from bacteria in the sample. Since the ratio of RNA / DNA was high for live bacteria and low for dead bacteria, it was determined from the results whether or not the test sample contained live or dead bacteria.
  • Example 2 visceral parts such as liver, kidney, spleen, heart, lung, etc., muscle, skin and eyeball of a dead rat were taken out, and quantitative RT-PCR was performed under the same conditions as in Example 1. As a result, a decrease in RNA over time was observed.
  • DNA and RNA extracted from a sample are amplified and their molar ratios are determined to accurately and quickly determine the viability of cells in the sample. It is possible.
  • the first experiment is based on the detection of bacterial genomic DNA, even if the bacterium is dead, it is positive as long as the bacterium retains the genomic DNA, ie There is an inherent risk of making a decision.
  • RNA extracted from the sample were amplified using the real-time PCR method and the real-time reverse transcription PCR (RTPCR) method, respectively, and the number of PCR cycles was measured. (Cycle threshold) value obtained from the relationship between the amount of DNA and the amount of amplified RNA Is determined.
  • the genomic DNA extracted from cells and bacteria was used to amplify nucleic acids by PCR, etc., based on the instability of the genomic DNA depending on the site. Is determined.
  • the PCR method is a technique for amplifying a specific target site of DNA, but it is essential that the PCR target site has continuity. Thus, if there is a discontinuity at this site or if the DNA exhibits extreme conformational changes due to instability, the PCR will not be completed and the target site will not be amplified. In particular, when a dramatic change in cells, such as cell death, occurs in the cells, the probability of a change occurring at the target site is extremely high. In other words, target sites with high stability are amplified in both live and dead cells, whereas target sites with high instability are amplified in live cells but not in dead cells. In addition, it is possible to determine the viability of bacteria and cells.
  • the method using a DNA unstable site it is useful to use the PCR method.
  • the method is not limited to the PCR method, and any nucleic acid amplification method can be used.
  • Example 3 cell viability was determined by comparing the relative amount of RNA and the relative amount of RNA determined using the real-time PCR method and the real-time RT-PCR method.
  • the real-time PCR method is a double-stranded DNA (PCR product) that increases with the progress of the PCR cycle by allowing a fluorochrome that emits fluorescence when incorporated into the double-stranded DNA to be present in the reaction solution.
  • This is a method of detecting the increase in PCR product by regarding the increase in fluorescence intensity.
  • the PCR product is increased in the early PCR cycle if the original type I DNA is large, and the fluorescence intensity is also enhanced. Therefore, a certain fluorescence intensity is determined in advance and this intensity Assuming that the number of PCR cycles to reach the Ct value is Ct value, if the type DN DNA is large or the PCR efficiency is high, the Ct value will be required to reach this fluorescence intensity at the early PCR cycle number. If the value is low and the type I DNA is low or the PCR efficiency is low, the C7 value will increase to reach this fluorescence intensity at a corresponding number of PCR cycles. Then, the C7 value is compared with a C7 value previously determined using a known amount of DNA, and the DNA amount corresponding to the Ct value is defined as a relative DNA amount. The same applies to RNA.
  • Example 3 extraction of bacterial genomic DNA and total RNA from the sample was performed by adding IS OGEN (manufactured by Nippon Gene Co., Ltd.) to the collected sample and collecting the sample by an ordinary method.
  • IS OGEN manufactured by Nippon Gene Co., Ltd.
  • the relative number of moles of DNA was estimated by the real-time PCR method by preparing PCR reaction solutions shown in Table 1 below.
  • RNA 2 g was used for reverse transcription after quantification.
  • the reaction was performed using anti-You—prime first strand beads (manufactured by Amersham phariacia biotech).
  • the reverse transcription primer a random hexamer was used.
  • the ratio of relative RNA amount / relative DNA amount in dead cells is smaller than that in live cells, and when both are mixed, the ratio is about the middle value. Therefore, by determining the reference value experimentally in advance, it is possible to accurately and easily determine the viability of bacteria.
  • Example 4 the stable and unstable portions of bacterial DNA were amplified by PCR, and the amplified product was subjected to gel electrophoresis to determine the viability of the bacteria.
  • a PCR reaction solution having the formulation shown in Table 3 below was prepared.
  • Second or fourth primer (10 mol / ⁇ 1)
  • the first and second primers described above are used as primers for detecting a DNA stable portion, and the third primer (5, one AACTGGAGGAAGGT GGGGAY-3,) is used as a primer for detecting a DNA unstable portion. And the fourth primer (5, -AGGAGGTGAT C CAACCGCA-3 ') was used.
  • the PCR was performed for 30 cycles using a protocol of 98 ° C./1 second, 55 ° C./10 seconds, and ⁇ 2 V / 10 seconds. After completion of the PCR, the PCR product was electrophoresed on a 100% gel at 100 V for 30 minutes using a 1% agarose gel according to a standard method, and the PCR product was stained with ethidium amide. I checked the band.
  • PCR products are confirmed because both live and dead bacteria are amplified by PCR, but in the primer for detecting an unstable DNA portion, only the PCR product of live bacteria is detected. Confirmed, no PCR product of dead bacteria was observed or extremely low width.
  • Figure 1 shows the results of an examination of eight groups with different mixing ratios of live and dead bacteria.
  • the ratio of live bacteria to dead bacteria in each group is as shown in Table 4 below.
  • the alphabet corresponds to the sample groups in Table 4.
  • Dead bacteria were prepared by heat treatment in autoclave at 121 ° C for 20 minutes.
  • amplification is observed in all samples in the DNA stable region, but in the DNA unstable region, all bacteria in the sample are dead (sample A), or a majority of the cells are dead In the case of bacteria (samples B-D), it can be seen that they were not amplified by PCR. In addition, in the case where some live bacteria were mixed with dead bacteria (samples E-G), only low amplification was performed. Is observed. However, in the case of live bacteria (specimen H), PCR is normally amplified and detected even in the DNA unstable part.
  • Example 5 bacteria-derived DNA was extracted from the specimen using IS0GEN (manufactured by Nippon Gene Co., Ltd.) as in Example 4, and live and dead bacteria were discriminated by the real-time PCR method.
  • IS0GEN manufactured by Nippon Gene Co., Ltd.
  • Example 5 real-time PCR was performed using a PCR reaction solution having the formulation shown in Table 5 below. (Table 5)
  • the first primer and the second primer used in Example 3 were used as primers for detecting a DNA stable portion, and the primers used in Example 4 were used as primers for detecting a DNA unstable portion.
  • a fourth primer was used.
  • the PCR was performed for 40 cycles with a protocol of 98 ° C / 1 second, 55 V / 10 seconds, 72 ° C / 10 seconds.
  • Real-time PCR was performed using GeneAmp 5700 (manufactured by Applied Bio).
  • the primer for detection of the DNA stable portion is used to amplify both live and dead bacteria by PCR: PCR products can be confirmed, whereas the primer for detection of the DNA unstable portion can Only PCR products of live bacteria are confirmed, and PCR products of dead bacteria are not confirmed or extremely low amplification is confirmed.
  • the primer for detecting the stable portion of DNA shows almost constant Ct value for both live and dead cells, but the primer for detecting the unstable portion of DNA is Since the Ct value becomes higher than that of live bacteria or a PCR reaction does not occur, it is possible to distinguish live bacteria from dead bacteria.
  • Table 6 shows the results of performing real-time PCR using the same eight samples as in Example 4 and calculating the Ct value.
  • Example 6 the same test as in Example 5 was performed on a sample that was treated with ultraviolet irradiation, sodium hypochlorite, strong acid, and strong alkali and placed in an environment in which DNA was randomly destroyed. Test was carried out. As a result, all ct values showed high values. This was presumed to be due to the fact that in such an environment, the DNA stable part was also destroyed and killed.
  • Example 7 the viability of cells and bacteria was determined using a Tm value or a Tm pattern for detecting a temperature dissociation change of a double-stranded DNA.
  • bacteria and DNA derived from bacteria are extracted from the specimen, and PCR is performed in real time using the reaction solution shown in Table 7 below. went.
  • the change was examined by measuring the Tm value or Tm pattern of the double-stranded DNA at a temperature from 65 ° C to 95 ° C.
  • Table 8 shows the Tm values measured 10 times for each of 100% killed E. coli and 100% E. coli.
  • the dead bacteria were prepared by treating them with autoclaves at 121 ° C for 20 minutes.
  • the Tm value or Tm pattern of the PCR product is The value or pattern differs from the Tm value or Tm pattern. Therefore, by examining the Tm value or Tm pattern, live bacteria and dead bacteria can be distinguished.
  • the Tm value and the Tm pattern derived from the higher-order structure of DNA are peculiar to each bacterial species, and by analyzing live and dead bacteria for each bacterial species, the bacterial species can be easily determined. Life and death can be determined for each.
  • DNA and RNA were extracted multiple times from the sample with a time lag, amplified by the real-time PCR method and real-time reverse transcription PCR method, respectively, and the number of PCR cycles and the amount of amplified DNA or amplified RNA were determined.
  • Cell viability is determined by comparing the Ct values obtained from the relationship with the amount.
  • the cell is a living cell or a living bacterium.
  • real-time In the case of using the PCR method or the like, if the Ct value decreases after a lapse of time (meaning an increase in the amount of DNA or RNA), it means that live cells-viable bacteria However, if the CT value does not change after a lapse of time, it can be estimated that dead cells and dead bacteria. In particular, in the nucleic acid quantification method using the real-time PCR method or the like, the Ct value before reaching the plateau can be used as a criterion, so that even small changes in the amount of DNA or RNA can be detected. Even if the time difference between sample collection is short, it is possible to distinguish between live and dead bacteria.
  • a direct PCR buffer represented by Ampdirecct manufactured by Shimadzu Corporation
  • Ampdirecct manufactured by Shimadzu Corporation
  • Example 8 using the real-time PCR method, samples were collected multiple times at different time intervals, and the viability of cells was determined by comparing the amount of DNA.
  • the specimen was prepared by mixing E. coli at a concentration of 10 8 / ml in physiological saline.
  • both live bacteria and dead bacteria autoclave sterilization
  • PCR was performed for 40 cycles using a protocol of 98 ° C / 1 second, 55 ° C / 10 seconds, and 72 ° C / 10 seconds, and the Ct value was applied to a previously prepared calibration curve to evaluate the evaluation DNA. The amount was calculated.
  • samples were placed under appropriate conditions, samples were taken multiple times with a time lag, the DNA or RNA in the samples was quantified, and the changes were compared. By doing so, it is possible to accurately and quickly determine the viability of cells and bacteria.
  • the specimen was destroyed so that the collected cells and bacterial nucleic acids were exposed, and the disrupted product was filtered to remove or control the nucleic acid amplification enzyme reaction inhibitor and the physical-optical reaction inhibitor. Nucleic acid amplification is performed using the obtained nucleic acid amplification reaction solution to detect bacterial genes in the sample.
  • a method of exfoliating the nucleic acids of cells and bacteria from the specimen physical treatment methods such as heat treatment, osmotic treatment, ultrasonic treatment or electric treatment, surfactant treatment, enzyme treatment, etc.
  • a chemical treatment method such as virus infection lysis treatment, and an effective and powerful method can be used for cells and bacteria to be examined.
  • methods for removing the nucleic acid amplification enzyme reaction inhibitor and the physical-optical reaction inhibitor from the disrupted product by filtration include a method using a column or a filter, a method using magnetic adsorption, and the like. Is also good.
  • the purity of the nucleic acid amplification reaction solution is improved, and the nuclei for PCR and other methods
  • the acid amplification enzyme reaction can be smoothly performed.
  • the polyamine is an oligomer or a polymer-polyvalent amine having one NH- group in the main chain of the molecule, and the polyamine added to the filtration layer to be used depends on the type of the specimen. Can be used.
  • the PCR method and real-time quantification using the PCR method can be used to amplify the sample sample that has been destroyed and removed by filtration to remove the reaction inhibitor, and the PCR method can be used, but is not limited thereto. .
  • Example 9 bacterial gene detection was performed using the nucleic acid amplification device shown in FIG. 2.
  • the nucleic acid amplification device was composed of a destruction chamber 1, a filtration chamber 2, and a filtration layer 3. And a PCR reaction chamber 4 and a PCR reaction liquid reservoir 5.
  • FIG. 2 shows a conceptual configuration of the nucleic acid amplifying apparatus, and it is a matter of course that the shape and configuration of each chamber are not limited to this example.
  • the filtration chamber 2 has a filtration layer 3 having a pore diameter impregnated with Sephadex G-50 (manufactured by Pharmacia), which is a powder ion exchanger using dextran as a carrier. It consists of a filter medium consisting of three layers of 0.22 ⁇ nitrocellulose membrane.
  • the PCR reaction chamber 4 was filled with a PCR reaction solution having the formulation shown in Table 12 below, and was mixed with the filtered filtrate to perform a PCR reaction.
  • the PCR cycle was 30 cycles with a protocol of 98 ° C / 1 s, 55 V / 10 s, 72 ° C / 10 s. Kuru went.
  • the first primer and the second primer described above were used as primers.
  • TAK ARAz-Taq DNA Polymerase (Takara Shuzo) is a DNA polymerase for performing high-sensitivity, high-speed PCR, and PCR that enables PCR without extracting DNA from bacteria.
  • Amp direct (manufactured by Shimadzu Corporation) is suitable, but not limited thereto.
  • the PCR product 101 after the completion of the PCR was subjected to the conventional method at 100 V with 1% agarose gel according to a standard method. After electrophoresis for 30 minutes, the PCR product was stained with ethidium bromide, and bacterial genes were confirmed by ultraviolet irradiation.
  • Electrophoresis photographs of an example containing no Escherichia coli (sample A) as a control, an example using the normal PCR method (samples B and C), and an example using the PCR method of the present invention (samples D and E) are shown in FIG. Shown in Also confirmed by the sample formulation and the electrophoresis photograph in Figure 3. The results obtained are shown in Table 13 below.
  • the nucleic acid of the sample was exposed in the destruction chamber, and the reaction inhibitor was removed in the filtration chamber to efficiently amplify the nucleic acid of the sample and detect the gene. can do.
  • the PCR reaction solution was made to contain a fluorescent interlator such as Cyber Green, and the real-time quantitative PCR method was used. Even when bacteria are detected in combination, accurate and reliable detection can be achieved.
  • nucleic acids of cells and Z or bacteria can be directly and quickly amplified efficiently to detect the gene.

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Abstract

L'invention porte sur une méthode de bioscopie selon laquelle on juge de l'état vivant ou mort de cellules et/ou de bactéries en recourant à une quantification des acides nucléiques, et sur une méthode d'amplification d'acides nucléiques permettant d'amplifier efficacement et directement les gènes de cellules et/ou d'acides nucléiques. On peut par exemple amplifier par PCR les parties stables et instables de l'ADN dans des échantillons contenant des cellules vivantes ou mortes en proportions variables. On observe ainsi: des produits PCR dans un échantillon A dont les parties instables d'ADN contiennent exclusivement des cellules vivantes, et pas de produits PCR dans un échantillon H dont les parties instables d'ADN contiennent exclusivement des cellules mortes. On peut ainsi comprendre les rapports entre cellules vivantes et mortes et les proportions du mélange dans des échantillons.
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009022558A1 (fr) 2007-08-16 2009-02-19 Kyushu University Procédé de détection de microorganismes et kit de détection des microorganismes
JP2010046016A (ja) * 2008-08-21 2010-03-04 Toyo Seikan Kaisha Ltd 微生物の検出方法
WO2010082640A1 (fr) 2009-01-15 2010-07-22 北海道三井化学株式会社 Preparation enzymatique contenant de l'adn polymerase thermostable, processus de production associe et procede de detection d'organisme d'analyte
EP2659001A1 (fr) * 2010-12-31 2013-11-06 Zeus Scientific, Inc. Procédés améliorés pour déterminer la viabilité des cellules à l'aide de techniques basées sur des acides nucléiques moléculaires
WO2014015925A1 (fr) 2012-07-26 2014-01-30 Sartorius Stedim Biotech Gmbh Procédé de différenciation entre cellules vivantes et mortes
US9139866B2 (en) 2006-02-17 2015-09-22 Morinaga Milk Industry Co., Ltd. Method for detection of microorganism and kit for detection of microorganism
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CN109207616A (zh) * 2018-11-14 2019-01-15 北京工业大学 一种检测活性污泥中死菌或休眠菌的方法

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US9139866B2 (en) 2006-02-17 2015-09-22 Morinaga Milk Industry Co., Ltd. Method for detection of microorganism and kit for detection of microorganism
US9567625B2 (en) 2006-02-17 2017-02-14 Morinaga Milk Industry Co., Ltd. Method for detection of microorganism and kit for detection of microorganism
US8221975B2 (en) 2007-08-16 2012-07-17 Morinaga Milk Industry Co., Ltd. Method for detection of microorganism
WO2009022558A1 (fr) 2007-08-16 2009-02-19 Kyushu University Procédé de détection de microorganismes et kit de détection des microorganismes
JP2010046016A (ja) * 2008-08-21 2010-03-04 Toyo Seikan Kaisha Ltd 微生物の検出方法
WO2010082640A1 (fr) 2009-01-15 2010-07-22 北海道三井化学株式会社 Preparation enzymatique contenant de l'adn polymerase thermostable, processus de production associe et procede de detection d'organisme d'analyte
US9394572B2 (en) 2009-07-24 2016-07-19 Morinaga Milk Industry Co., Ltd. Method and kit for detection of microorganism
US10329604B2 (en) 2009-07-24 2019-06-25 Morinaga Milk Industry Co., Ltd. Method and kit for detection of microorganism
US20140186828A1 (en) * 2010-12-31 2014-07-03 Shawn Mark O'Hara Methods for determining cell viability using molecular nucleic acid-based techniques
EP2659001A4 (fr) * 2010-12-31 2014-07-02 Zeus Scientific Inc Procédés améliorés pour déterminer la viabilité des cellules à l'aide de techniques basées sur des acides nucléiques moléculaires
AU2011352333B2 (en) * 2010-12-31 2016-12-08 Zeus Scientific, Inc. Improved methods for determining cell viability using molecular nucleic acid-based techniques
EP2659001A1 (fr) * 2010-12-31 2013-11-06 Zeus Scientific, Inc. Procédés améliorés pour déterminer la viabilité des cellules à l'aide de techniques basées sur des acides nucléiques moléculaires
WO2014015925A1 (fr) 2012-07-26 2014-01-30 Sartorius Stedim Biotech Gmbh Procédé de différenciation entre cellules vivantes et mortes
CN109207616A (zh) * 2018-11-14 2019-01-15 北京工业大学 一种检测活性污泥中死菌或休眠菌的方法

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