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WO2023181694A1 - Procédé de calcul d'efficacité de traitement d'acide nucléique, procédé de correction de valeur quantitative d'acide nucléique l'utilisant, et dispositif de mesure d'arn cible - Google Patents

Procédé de calcul d'efficacité de traitement d'acide nucléique, procédé de correction de valeur quantitative d'acide nucléique l'utilisant, et dispositif de mesure d'arn cible Download PDF

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WO2023181694A1
WO2023181694A1 PCT/JP2023/004626 JP2023004626W WO2023181694A1 WO 2023181694 A1 WO2023181694 A1 WO 2023181694A1 JP 2023004626 W JP2023004626 W JP 2023004626W WO 2023181694 A1 WO2023181694 A1 WO 2023181694A1
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nucleic acid
standard
sample
rna
target
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和紀 渡邊
洋敬 海野
聡 中澤
侑希 米川
雨農 季
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Ricoh Co Ltd
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Definitions

  • the present invention relates to a method for calculating processing efficiency in nucleic acid processing, a method for correcting a quantitative value of a target nucleic acid obtained from a calibration curve using the calculation method, and a target RNA measuring device.
  • Nucleic acid analysis methods in which a trace amount of target nucleic acid present in a sample is amplified and then the resulting amplification product is analyzed in detail, are widely used in various fields such as infectious disease diagnosis, food testing, blood testing, and DNA identification. ing.
  • Typical nucleic acid analysis techniques include, for example, real-time polymerase chain reaction (PCR) in PCR testing.
  • PCR real-time polymerase chain reaction
  • Real-time PCR refers to the timely detection of fluorescence according to the amount of amplified nucleic acid during the PCR process, and relative comparison of the detected value of the target nucleic acid in the target sample with a standard sample series whose copy number has been specified. This is a method for quantifying the nucleic acid in the sample.
  • target nucleic acids have been quantified based on a calibration curve calculated from a standard nucleic acid group defined by several thousand copies.
  • the target nucleic acid is extracted from the sample, or if the extracted target nucleic acid is RNA, reverse transcription of DNA from RNA is performed. This is done after going through multiple processing steps. Therefore, in PCR tests using conventional methods, it is inevitable that a portion of the target nucleic acid is lost in each processing step, and only a composite quantitative result can be obtained through multiple processing steps.
  • Patent Document 1 discloses a device in which a predetermined number of cells into which a target base sequence has been introduced by genetic recombination is dispensed. Using this device, it is possible to control the amplification accuracy in PCR, but the amount of target nucleic acid lost in the RNA extraction process or reverse transcription reaction process cannot be determined.
  • Patent Document 2 discloses the use of virus-like particles (VLPs) as internal standards. According to this method, the target nucleic acid present in the sample can be quantified, but the processing efficiency of each step cannot be calculated.
  • VLPs virus-like particles
  • Patent Document 3 discloses a method for producing armored RNA. Armored RNA with a specified copy number is used as an RNA standard material, processed according to a known operating procedure, and measured to confirm the presence or absence of detection. However, as with conventional methods, the amount of target nucleic acid lost during the manipulation process is unknown.
  • the purpose of the present invention is to obtain more accurate quantitative values of target nucleic acids present in a sample.
  • the present inventors have conducted intensive research and development, and have calculated the processing efficiency of nucleic acids actually processed in the nucleic acid processing steps required for quantifying target nucleic acids. By correcting the quantitative values obtained in the process based on the processing efficiency, we were able to correct the amount of target nucleic acid lost during the treatment process and succeeded in obtaining more accurate quantitative values.
  • the present invention is based on the research and development results and provides the following.
  • a method for calculating nucleic acid processing efficiency which includes a sample processing step in which a standard sample containing a known amount of standard nucleic acid is subjected to nucleic acid extraction treatment or reverse transcription treatment, a standard nucleic acid obtained from the standard sample, and a plurality of different standard nucleic acids. a measurement step of measuring a known amount of a standard nucleic acid group to obtain the measured value; a calibration curve creation step of creating a calibration curve from each measurement value of the standard nucleic acid group; and an amount of the standard nucleic acid calculated from the calibration curve.
  • the method includes a nucleic acid processing efficiency calculation step of calculating the nucleic acid processing efficiency of the standard nucleic acid in the sample processing step by dividing by a known amount.
  • This specification includes the disclosure content of Japanese Patent Application Nos. 2022-046500 and 2022-127284, which are the basis of the priority of this application.
  • the treatment efficiency of the actually treated nucleic acids can be calculated.
  • FIG. 1 is a conceptual diagram of a 96-well microtiter plate type target RNA measurement device prepared in Example 1 of the present invention.
  • Each column is a diagram showing the arrangement of samples or nucleic acids in each well of the device.
  • (A) to (C) represent standard samples (A) to (C) in Examples and test No. 1 to 3 are test sample No. 1 to 3 are shown.
  • the numerical value in each column indicates the copy number of the sample.
  • 1 is a DNA standard curve and an RNA standard curve showing the relationship between Cq value and copy number obtained in Example 1 of the present invention.
  • FIG. 2 is a conceptual diagram of a 96-well microtiter plate type target RNA measurement device prepared in Example 2 of the present invention.
  • Each column is a diagram showing the arrangement of samples or nucleic acids in each well of the device. Similarly to Example 1, in the figure, (A) to (C) represent the standard samples (A) to (C) in Example 2, and test sample No. 1 to 3 are test sample No. 1 to 3 are shown. Further, the numerical value in each column indicates the copy number of the sample. 2 is a DNA calibration curve and an RNA calibration curve showing the relationship between Cq value and copy number obtained in Example 2 of the present invention.
  • Nucleic acid treatment efficiency calculation method 1-1 Overview
  • the first aspect of the present invention is a method for calculating nucleic acid processing efficiency.
  • the standard nucleic acid substantially treated by one or more nucleic acid treatments performed on a standard sample is calculated as the nucleic acid treatment efficiency. This makes it possible to estimate the amount of nucleic acid lost during the nucleic acid treatment process.
  • nucleic acid refers to a biopolymer whose constituent units are, in principle, nucleotides linked by phosphodiester bonds.
  • Nucleic acids include natural nucleic acids such as DNA (including cDNA), RNA, or a combination thereof, which are linked only by natural nucleotides. Also includes natural nucleic acids.
  • a "natural nucleotide” is a nucleotide that exists in nature.
  • deoxyribonucleotides that constitute DNA and have any of the bases adenine, guanine, cytosine, and thymine and ribonucleotides that constitute RNA and have any of the bases adenine, guanine, cytosine, and uracil.
  • non-natural nucleotide refers to a nucleotide that does not exist in nature and has an artificially constructed structure, or a nucleotide that has been artificially chemically modified.
  • non-natural nucleic acid refers to an artificially constructed nucleic acid analog having a structure and/or properties similar to natural nucleic acids. Examples include bridged nucleic acid/locked nucleic acid (BNA/LNA), peptide nucleic acid (PNA), peptide nucleic acid having a phosphate group (PHONA), morpholino nucleic acid, and the like.
  • nucleic acid of the present specification may also contain chemically modified nucleic acids or nucleic acid analogs such as methylphosphonate type DNA/RNA, phosphorothioate type DNA/RNA, phosphoramidate type DNA/RNA, and 2'-O-methyl type DNA/RNA.
  • the phosphate group, sugar and/or base may be labeled with a labeling substance, if necessary.
  • the labeling position of the labeling substance may be determined as appropriate depending on the characteristics of the labeling substance and the purpose of use, and although not limited, the 5' end and/or 3' end is usually preferred.
  • the labeling substance any substance known in the art can be used.
  • radioactive isotopes examples include radioactive isotopes, fluorescent substances, quenchers, chemiluminescent substances, DIG, biotin, and magnetic beads.
  • radioactive isotope refers to an element that emits radiation among isotopes with different mass numbers. Examples include 32 P, 33 P, or 35 S.
  • the shape of the nucleic acid is not limited herein. For example, it may be a linear nucleic acid or a circular nucleic acid.
  • target nucleic acid refers to a nucleic acid of interest in the present invention, which is present in a sample and is a target to be quantified and/or a target to be detected.
  • the target nucleic acid is, in principle, a nucleic acid that can be amplified by a nucleic acid amplification method.
  • the target nucleic acid may be either DNA or RNA.
  • target DNA when the target nucleic acid is DNA, it is referred to as “target DNA”, and when it is RNA, it is referred to as "target RNA”.
  • the base length of the target nucleic acid is not limited as long as it can be amplified by the nucleic acid amplification method, and a desired base length can be appropriately selected depending on the purpose. For example, if it is short, it may be a few bases (for example, 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, or 10 bases), or if it is long, it can be a few bases such as genomic DNA. It may range from 5 million bases to 3 billion bases. Usually, a range of 20 bases to 10,000 bases, 100 bases to 8,000 bases, or 500 bases to 5,000 bases is appropriate.
  • the origin of the target nucleic acid is not limited either, but it may be derived from natural products that exist in nature, or it may be derived from non-natural products obtained by chemical synthesis or genetic recombination, that is, it may be derived from artificial products. If it is derived from a natural product, it may be derived from a virus, a bacteria, an animal, or a plant.
  • loss of target nucleic acid refers to the loss of part of the target nucleic acid during the operation process performed on a sample in order to quantify the target sequence contained in the sample. For example, when performing nucleic acid extraction from biological tissue, cells remaining unbroken during cell crushing treatment are removed in a subsequent operation. At this time, the target nucleic acid contained in the undisrupted cells is lost along with the cells and is therefore counted as a loss.
  • standard nucleic acid refers to a standard substance composed of a nucleic acid consisting of a specific base sequence with a defined copy number.
  • a “reference material” is a reference material for determining a measured value in the measurement of a chemical substance.
  • the standard nucleic acid particularly refers to a nucleic acid consisting of the same base sequence as a target nucleic acid.
  • standard nucleic acid group refers to a group consisting of a plurality of standard nucleic acids with the same base sequence and different copy numbers.
  • the standard nucleic acid group is used when creating a standard nucleic acid calibration curve.
  • copy refers to a replica of a nucleic acid as a basic unit consisting of a predetermined base sequence (herein often referred to as “unit nucleic acid”).
  • copy number refers to the number of unit nucleic acids.
  • Quantification means determining the amount of components contained in a sample.
  • quantification particularly refers to determining the amount of nucleic acid contained in a sample based on a calibration curve.
  • quantitative value refers to the amount of nucleic acid obtained by quantification, that is, the copy number of nucleic acid.
  • measured value refers to a value obtained by measuring a nucleic acid. It may be an absolute value or a relative value.
  • a measured value in real-time PCR may be a Cq value (or Ct value).
  • the Cq value is the number of cycles (Threshold Cycle) when the amplified nucleic acid reaches the detection limit (threshold value) in a nucleic acid amplification reaction.
  • test sample refers to a sample that contains a target nucleic acid and is the subject of a test for quantifying the target nucleic acid.
  • the form of the test sample does not matter. It may be a biological sample containing the target nucleic acid, an artificial sample in which nucleic acid is artificially packaged such as armored RNA or liposome, or a total RNA solution dissolved in a buffer. It may also be a naked nucleic acid that is not included in a shell such as a protein.
  • biological sample refers to a sample directly collected from a living body, etc.
  • a biological sample usually exists in a state in which the target nucleic acid is encapsulated in other components such as proteins and lipids.
  • body fluid refers to a liquid biological sample. Examples include blood (including serum, plasma, and interstitial fluid), cerebrospinal fluid (cerebrospinal fluid), urine, lymph fluid, digestive fluid, ascites, pleural effusion, periradicular fluid, extracts of various tissues or cells, etc.
  • a "virus particle” is a virus main body that includes at least a virus genome and a capsid (outer shell) protein that includes the virus genome.
  • the viral genome may be either DNA or RNA.
  • standard sample refers to a sample of the same type as the test sample, which contains a known amount of a standard nucleic acid having the same base sequence as the target nucleic acid.
  • the difference between a standard sample and a test sample is whether they each contain a known amount or an unknown amount of standard nucleic acid or target nucleic acid.
  • nucleic acid treatment efficiency refers to the amount of nucleic acid actually measured in the nucleic acid treatment described below relative to the amount of nucleic acid actually contained in the sample when a standard sample or test sample is subjected to nucleic acid treatment. Expressed as a percentage of quantity.
  • the method of the present invention includes a sample treatment step, a measurement step, a calibration curve creation step, and a processing efficiency calculation step as essential steps. Each process will be explained below.
  • sample processing step is a step in which a standard sample is subjected to nucleic acid treatment.
  • the standard sample in this step is a sample of the same type as the test sample, as described above, and may be, for example, a biological sample, an artificial sample, or a naked nucleic acid.
  • the standard sample used in this step contains a standard nucleic acid.
  • the amount of standard nucleic acid contained in the standard sample is a known amount and can be predefined to a specific amount. Although not limited, the range may be, for example, 5 to 1000 copies, 10 to 500 copies, 20 to 200 copies, or 50 to 100 copies.
  • nucleic acid treatment in this step includes nucleic acid extraction treatment, reverse transcription treatment, and nucleic acid amplification treatment.
  • nucleic acid extraction process and the reverse transcription process can be performed alone, but the nucleic acid amplification process is performed in combination with the nucleic acid extraction process and/or the reverse transcription process.
  • Each nucleic acid treatment will be specifically explained below.
  • Nucleic acid extraction process is a process of extracting nucleic acids including standard nucleic acids from a standard sample.
  • the nucleic acid to be extracted may be DNA, RNA, or a mixture thereof.
  • DNA includes all kinds of DNA such as genomic DNA and plasmid DNA.
  • RNA includes all RNAs such as total RNA, mRNA, tRNA, rRNA, miRNA (including precursors), and snRNA.
  • any nucleic acid extraction method known in the art may be used.
  • the standard sample is a cell
  • it is homogenized as necessary to prepare a suspension.
  • the sample is a nucleus, virus particle, armored RNA, etc.
  • Tris/EDTA pH 8.0
  • Nucleic acids can be extracted from the suspension by methods known in the art, such as the alkaline method and the boiling method. A specific extraction method is described, for example, by Green, M. R. and Sambrook, J.
  • nucleic acid extraction kits are commercially available from various life science manufacturers, and these kits may be used. When using a kit, the specific extraction method may be performed according to or in accordance with the attached protocol.
  • (1-2) Reverse Transcription Process is performed when the standard sample is RNA and cDNA needs to be prepared for use as a template in the nucleic acid amplification process described below.
  • the reverse transcription process is achieved by a reverse transcription reaction using RNA as a template and dNTP as a substrate, primers, and reverse transcriptase.
  • methods known in the art can be used. For example, Green, M. R. and Sambrook, J. It may be performed according to the reverse transcription method described in , 2012.
  • reverse transcriptase for example, M-MLV RTase, PrimeScript TM RTase (TaKaRa), Super Script (registered trademark) III RTase (Thermo Fisher Scientific), etc.
  • reverse transcription kits are commercially available from various life science manufacturers, and these kits may be used. When using a kit, the specific reverse transcription method may be performed according to or in accordance with the attached protocol.
  • the cDNA obtained by reverse transcription can be used in the nucleic acid amplification process described below.
  • Nucleic acid amplification treatment is achieved by a nucleic acid amplification method that amplifies a specific region of a target nucleic acid.
  • the nucleic acid amplification method refers to a method in which the specific region sandwiched between forward/reverse primers is amplified by repeating a nucleic acid elongation reaction using a nucleic acid polymerase.
  • the standard sample is DNA
  • examples include the PCR method using DNA polymerase, the ICAN method, and the LAMP (registered trademark) method.
  • the test/standard sample is RNA
  • the NASBA method using RNA polymerase may be used.
  • the PCR method is used.
  • PCR reaction conditions can be set appropriately. For example, using a thermostable DNA polymerase such as Taq polymerase or Pfu polymerase, dNTP, and Mg 2+ -containing PCR buffer, the steps consisting of, for example, denaturation reaction, annealing reaction, and extension reaction are repeated as one cycle, and then incubated at 72°C for 5 days. Conditions for reacting for more than a minute can be mentioned. Specific reaction conditions include, for example, the denaturation reaction at 94 to 97°C for 20 to 30 seconds, the annealing reaction at 55 to 60°C for 30 seconds to 1 minute, and the extension reaction at 72°C for 1 to 2 minutes. Examples include, but are not limited to.
  • PCR kits are available from each life science manufacturer. These kits are commercially available and can also be used. When using a kit, the specific PCR method may be performed according to or in accordance with the attached protocol.
  • nucleic acid amplification process is a nucleic acid process that is performed secondary to the nucleic acid extraction process and/or the reverse transcription process in the nucleic acid processing efficiency calculation method of the present invention.
  • nucleic acid extraction processing and nucleic acid amplification processing combinations of nucleic acid extraction processing and nucleic acid amplification processing, combinations of reverse transcription processing and nucleic acid amplification processing, and nucleic acid A combination of extraction treatment, reverse transcription treatment, and nucleic acid amplification treatment may also be mentioned.
  • a measurement step and a treatment efficiency calculation step are carried out in the nucleic acid extraction treatment and the reverse transcription treatment, and a treatment efficiency calculation step is carried out in the nucleic acid amplification treatment.
  • Nucleic acid extraction/reverse transcription processing is a sample processing step that combines two nucleic acid treatments: nucleic acid extraction processing and reverse transcription processing. This process is used when the first test sample is a biological sample or an artificial sample, the standard nucleic acid is RNA contained in the sample, and the standard nucleic acid, which is cDNA after reverse transcription, is to be finally quantified. will be implemented.
  • Nucleic acid extraction/nucleic acid amplification processing is a sample processing process that combines two nucleic acid treatments: nucleic acid extraction processing and nucleic acid amplification processing. This process is used when the first test sample is an artificial biological sample or a naked nucleic acid, and the standard nucleic acid is DNA or RNA contained therein, and the extracted standard nucleic acid is quantified by a nucleic acid amplification reaction without reverse transcription. Implemented when necessary.
  • Reverse transcription/nucleic acid amplification treatment is a sample processing process that combines two nucleic acid treatments: reverse transcription treatment and nucleic acid amplification treatment.
  • the first test sample is, for example, a total RNA solution
  • the standard nucleic acid is a specific RNA contained therein
  • the cDNA of the standard nucleic acid obtained through reverse transcription reaction is quantified by nucleic acid amplification reaction.
  • RNA which is a standard nucleic acid
  • Nucleic acid extraction/reverse transcription/nucleic acid amplification processing is a sample processing process that combines three nucleic acid treatments: nucleic acid extraction processing, reverse transcription processing, and nucleic acid amplification processing. It is. Since three different nucleic acid treatments are performed, the amount of nucleic acid lost during the treatment process is the largest, and the relative error in the quantitative value after quantification is also the largest. Therefore, the combination of these three nucleic acid treatments is the combination of sample treatments that can exhibit the most effect in the nucleic acid treatment efficiency calculation method described in the first aspect and the quantitative value correction method described in the second aspect.
  • This process is performed when the first test sample is a biological sample or an artificial sample, the standard nucleic acid is RNA, and the cDNA of the standard nucleic acid obtained through a reverse transcription reaction is quantified by a nucleic acid amplification reaction. be done.
  • the “measurement step” is a step of measuring a standard nucleic acid and a plurality of different groups of known standard nucleic acids to obtain the measured values.
  • the nucleic acids to be measured in this step are the standard nucleic acid obtained from the standard sample in the sample processing step, and a group of different known amounts of standard nucleic acids.
  • the plural here is not limited to 2 or more, 3 or more, or 4 or more, but in view of creating a calibration curve described later, at least 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, It is preferably 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more.
  • Each standard nucleic acid constituting the standard nucleic acid group is a nucleic acid consisting of the same base sequence as the standard nucleic acid contained in the standard sample. However, the type thereof may be the same as or different from the standard nucleic acid.
  • the standard nucleic acid contained in the standard sample is RNA
  • the standard nucleic acid constituting the standard nucleic acid group in this step may be RNA or DNA.
  • the different known amounts of standard nucleic acids constituting the standard nucleic acid group differ only in the amount of nucleic acid.
  • This amount of nucleic acid can be defined by, but is not limited to, the number of copies of a standard nucleic acid.
  • the amount of nucleic acid of each standard nucleic acid is not particularly limited, but considering that a group of standard nucleic acids can contribute to the creation of a calibration curve, the amount of nucleic acid of each standard nucleic acid is distributed so that it increases stepwise. It is preferable to keep it.
  • each standard nucleic acid constituting the standard nucleic acid group exists as a nucleic acid solution containing no protein or the like, in which a known amount of the standard nucleic acid is dissolved in water or a buffer.
  • the standard nucleic acid group may include multiple standard nucleic acids of the same known amount.
  • the measured value can be the average value.
  • the method for measuring nucleic acids in this step is not particularly limited as long as it can measure the standard nucleic acid and each standard nucleic acid that constitutes the standard nucleic acid group. However, each nucleic acid is measured using the same method.
  • the measured value obtained as a result of the measurement may be an absolute value or a relative value of the amount of nucleic acid.
  • dPCR method When measuring as an absolute value, for example, measurement using a digital PCR method (in this specification, often referred to as "dPCR method") can be mentioned.
  • a digital PCR method in this specification, often referred to as "dPCR method”
  • dPCR method a limiting dilution sample is dispersed so that the target DNA is 1 or 0 copies in a microcompartment, and then PCR is performed to collect the microcompartment in which the nucleic acid has been amplified, that is, to contain 1 copy of the target DNA.
  • This method measures the concentration of the target DNA in a sample by counting the number of microcompartments that are present.
  • Real-time PCR is a reaction system in which the amplified products are specifically fluorescently labeled during PCR using DNA in a sample as a template, and the amount of amplified products of the target nucleic acid during the reaction is monitored in real time as fluorescence intensity.
  • the measured value at this time may be a Cq value (Ct value). Since the Cq value and the initial template nucleic acid amount have an inverse correlation, the amount of each nucleic acid can be relatively quantified based on the Cq value.
  • the real-time PCR method is a method known in the art, and may be performed according to a conventional method.
  • methods for fluorescently labeling amplified products include methods using fluorescently labeled probes such as TaqMan (registered trademark) PCR method.
  • the probe used in the TaqMan PCR method is modified with a quencher at one end and a fluorescent substance at the other end. Normally, a quencher on a probe suppresses the adjacent fluorescent substance, but during the nucleic acid elongation reaction in PCR, the fluorescent substance is degraded by the 5' ⁇ 3' exonuclease activity of Taq polymerase. It becomes free and begins to fluoresce. Therefore, the fluorescence intensity indicating the amount of fluorescence reflects the amount of amplified product.
  • the reaction conditions for real-time PCR are based on known PCR methods, and include the base length of the nucleic acid fragment to be amplified, the amount of template nucleic acid, the base length and Tm value of the primers used, the optimal reaction temperature of the nucleic acid polymerase used, and Since it varies depending on the optimum pH, etc., it may be determined appropriately by taking these conditions into consideration.
  • the denaturation reaction is usually carried out at 94-95°C for 5 seconds to 5 minutes
  • the annealing reaction is carried out at 50-70°C for 10 seconds to 1 minute
  • the extension reaction is carried out at 68-72°C for 30 seconds to 3 minutes.
  • the elongation reaction can be repeated for about 15 to 40 cycles as one cycle.
  • the protocol attached to the kit may be followed.
  • this step is performed on a standard nucleic acid in a standard sample and a group of standard nucleic acids in a nucleic acid extraction process and a reverse transcription process.
  • the “calibration curve creation step” is a step of creating a calibration curve from each measurement value of the standard nucleic acid group after the measurement step.
  • the amounts (copy numbers) of a plurality of different standard nucleic acids constituting the standard nucleic acid group are known.
  • the calibration curve may be created using a known method. In this step, for example, it can be created by comparing each known amount of the standard nucleic acid in the standard nucleic acid group with its measured value.
  • the calibration curve may be a DNA calibration curve or an RNA calibration curve. From this calibration curve, it becomes possible to calculate the measured value measured as an absolute value or a relative value as a copy number.
  • a calibration curve can be used for both target nucleic acids and standard nucleic acids.
  • Nucleic acid processing efficiency calculation step is a step of calculating the nucleic acid processing efficiency in the sample processing step, and is a central step in the nucleic acid processing efficiency calculation method of the present invention. Through this step, it is possible to calculate the percentage of nucleic acids that have been processed without being lost during the operation process in the sample processing step.
  • nucleic acid treatment efficiency differs depending on the type of nucleic acid treatment.
  • the respective nucleic acid treatment efficiency is the amount of standard nucleic acid calculated from the measured value of the standard nucleic acid based on the calibration curve with respect to the known amount of the standard nucleic acid in the standard sample. Calculated as a percentage of This is determined by dividing the amount of the standard nucleic acid calculated based on the calibration curve from the measured value of the standard nucleic acid by the known amount of the standard nucleic acid. The amount (copy number) of the standard nucleic acid in the standard sample before the sample processing step is known.
  • the amount (copy number) of the standard nucleic acid in the standard sample after the measurement step can be obtained based on the calibration curve created in the calibration curve creation step from the measured values of the standard nucleic acid in the standard sample after the measurement step.
  • Nucleic acid treatment efficiency can be expressed as a relative value of the amount of standard nucleic acid in the standard sample after the measurement step, when the known amount of the standard nucleic acid contained in the standard sample before the sample treatment step is taken as 1. Therefore, nucleic acid processing efficiency is expressed as a numerical value of 0 or more and 1 or less.
  • the nucleic acid processing efficiency multiplied by 100 is the percentage (%) of the standard nucleic acid that was processed, recovered, and/or measured without being lost in the sample processing step.
  • the efficiency of the nucleic acid treatment is calculated from the slope of the calibration curve obtained in the calibration curve creation step.
  • the calculation formula for amplification efficiency is not limited to, for example, https://www.
  • the following formula regarding PCR amplification efficiency described in thermofisher.com/blog/learning-at-the-bench/qpcr-basic7/ can be used.
  • the slope of the calibration curve can be determined by, for example, the method of least squares.
  • the nucleic acid treatment efficiency is calculated from each nucleic acid treatment, and the value multiplied by each nucleic acid treatment efficiency becomes the nucleic acid treatment efficiency of the entire combined nucleic acid treatment.
  • nucleic acid extraction efficiency is calculated from the nucleic acid extraction processing, reverse transcription efficiency from the reverse transcription processing, and nucleic acid amplification efficiency from the nucleic acid amplification processing.
  • the value obtained by multiplying these three nucleic acid treatment efficiencies becomes the nucleic acid treatment efficiency after passing through all three nucleic acid treatments.
  • the second aspect of the present invention is a method for correcting quantitative values of target nucleic acids.
  • the nucleic acid treatment efficiency calculation method described in the first aspect is used. That is, after calculating the quantitative value of the target nucleic acid in the test sample in the same manner as the conventional method based on the standard nucleic acid calibration curve obtained by the nucleic acid treatment efficiency calculation method described in the first aspect, the quantitative value is calculated. Correction is made using the nucleic acid processing efficiency obtained by the nucleic acid processing efficiency calculation method described in the first aspect.
  • the method of the present invention also includes a sample processing step, a measurement step, a calibration curve creation step, a processing efficiency calculation step, a quantitative value acquisition step, and a correction step.
  • the quantitative value acquisition step and the correction step are steps unique to this embodiment.
  • the sample processing step, measurement step, calibration curve creation step, and treatment efficiency calculation step basically follow each step described in the nucleic acid treatment efficiency calculation method. Therefore, a detailed explanation of these steps that overlap with the nucleic acid treatment efficiency calculation method will be omitted.
  • the sample processing step and the measurement step include points that are characteristic of the quantitative value correction method of this embodiment. Therefore, in the following, only the characteristic points of the quantitative value correction method in the sample processing step and the measurement step will be explained, as well as the quantitative value acquisition step and the correction step, which are steps unique to the quantitative value correction method.
  • a feature of the quantitative value correction method of this embodiment is that nucleic acid treatment is performed on the test sample together with the standard sample.
  • the test sample used in this step is the same type of sample as the standard sample. For example, if the standard sample is a virus particle, the test sample used will also be a virus particle of the same type as the virus, and if the standard sample is a total RNA solution, the target sample will also be a total RNA solution.
  • the test sample is, in principle, subjected to the same nucleic acid treatment under the same conditions as the standard sample.
  • the test sample may contain a target nucleic acid.
  • This target nucleic acid is also a nucleic acid consisting of the same base sequence as the standard nucleic acid.
  • the basic composition of the test sample and target nucleic acid is the same as that of the standard sample and standard nucleic acid, respectively, but the biggest difference is that the standard nucleic acid contained in the standard sample is in a known amount, whereas The amount of target nucleic acid contained in the test sample is unknown.
  • the amount of target nucleic acid contained in the test sample is not limited.
  • the amount of target nucleic acid can be defined by its copy number, for example, 10,000 copies or less, 8,000 copies or less, 5,000 copies or less, 4,000 copies or less, 3,000 copies or less, 2,000 copies or less, 1,000 copies or less, 900 copies or less. , 800 copies or less, 700 copies or less, 600 copies or less, 500 copies or less, 400 copies or less, 300 copies or less, 200 copies or less, 100 copies or less, 90 copies or less, 80 copies or less, 70 copies or less, 60 copies or less, or It is sufficient if it is included in 50 copies or less. Preferably, it is a test sample containing a low copy of the target nucleic acid.
  • 40 copies or less for example, 40 copies or less, 30 copies or less, 20 copies or less, 10 copies or less, 9 copies or less, 8 copies or less, 7 copies or less, 6 copies or less, 5 copies or less, 4 copies or less, 3 copies or less, 2 copies or less, Or one copy.
  • the characteristic point of the quantitative value correction method of this embodiment is that, like the nucleic acid treatment step, the target nucleic acid obtained from the test sample is measured to obtain the measured value.
  • the measurement method in this step is the same as the measurement method for standard nucleic acids.
  • Quantitative value acquisition step obtains a quantitative value of the target nucleic acid contained in the test sample from the measured value of the target nucleic acid after the measurement step, based on the calibration curve obtained after the calibration curve creation step. It is a process. Through this step, the target nucleic acid contained in the test sample can be quantified as its copy number.
  • the quantitative value acquisition step is performed by a method similar to a conventional nucleic acid quantification method in which the target nucleic acid is quantified from relative values based on a calibration curve.
  • correction step is a step of correcting the quantitative value of the target nucleic acid obtained in the quantitative value acquisition step, and is the central step in the quantitative value correction method of the present invention. This step makes it possible to obtain more accurate quantitative values by estimating and correcting the amount of target nucleic acid lost during the sample processing process.
  • this step is corrected by dividing the quantitative value of the target nucleic acid obtained in the final nucleic acid treatment by the value obtained by multiplying the treatment efficiency obtained in each nucleic acid treatment.
  • "Final nucleic acid treatment” refers to the last nucleic acid treatment performed in two or more different nucleic acid treatments. For example, when nucleic acid extraction/reverse transcription/nucleic acid amplification processing is performed, the last nucleic acid processing performed is nucleic acid amplification processing.
  • the reason for using the quantitative value of the target nucleic acid obtained in the final nucleic acid treatment is that it is the value obtained after multiple nucleic acid treatments, and the combined effects of losing a portion of the target sample in each nucleic acid treatment are the most cumulative. This is because it is a result. Therefore, quantitative values for nucleic acid treatments other than the final nucleic acid treatment are not required.
  • the quantitative value of the target nucleic acid obtained after the nucleic acid amplification treatment is multiplied by the treatment efficiency obtained in each of the nucleic acid extraction treatment, reverse transcription treatment, and nucleic acid amplification treatment. The quantitative value is corrected by dividing.
  • the processing efficiency in the nucleic acid extraction process is 0.9
  • the processing efficiency in the reverse transcription process is 0.9
  • the processing efficiency in the reverse transcription process is 0.9.
  • the processing efficiency (nucleic acid amplification efficiency) in the nucleic acid amplification process is 0.7
  • the third aspect of the present invention is a device for calculating nucleic acid processing efficiency.
  • the device of the present invention is a device for easily calculating the efficiency of nucleic acid treatment by one or more nucleic acid treatments using a standard nucleic acid.
  • the device for calculating nucleic acid processing efficiency of the present invention includes a standard sample containing a known amount of standard nucleic acid, a standard RNA group consisting of a plurality of different known amounts of standard RNA, and a standard DNA group consisting of a plurality of different known amounts of standard DNA. It has a configuration located on the device.
  • the nucleic acid processing efficiency calculation method described in the first aspect can be easily carried out, and the nucleic acid processing efficiency can be easily calculated.
  • the device for calculating nucleic acid processing efficiency of the present invention is a measuring device that includes known amounts of standard RNA, standard RNA group, and standard DNA group on the device.
  • the standard RNA corresponds to the standard nucleic acid in the nucleic acid processing efficiency calculation method described in the first aspect, and its basic structure is the same as the standard according to the first aspect except that the standard nucleic acid is specified as RNA. Conforms to the structure of nucleic acids.
  • the amount of standard RNA may be, for example, the number of copies of standard RNA.
  • the standard RNA may be placed on the device as multiple standard RNAs consisting of the same amount.
  • the standard RNA group is a group consisting of a plurality of different known amounts of standard RNA, and is placed on the device for creating an RNA calibration curve. Each standard RNA constituting the standard RNA group has the same base sequence as the standard RNA.
  • the standard RNA group corresponds to the standard nucleic acid group in the nucleic acid processing efficiency calculation method described in the first aspect, and its basic structure is as described in the first aspect except that the standard nucleic acid is specified as RNA. According to the composition of the standard nucleic acid group.
  • the standard DNA group is a group consisting of a plurality of different known amounts of standard DNA, and is placed on the device for creating a DNA calibration curve. Each standard DNA constituting the standard DNA group has the same base sequence as the standard RNA.
  • the standard DNA group corresponds to the standard nucleic acid group in the nucleic acid processing efficiency calculation method described in the first aspect, and its basic structure is as described in the first aspect except that the standard nucleic acid is specified as DNA. According to the composition of the standard nucleic acid group.
  • the shape, material, and size/volume of the target RNA measurement device of the present invention are not particularly limited and can be appropriately selected depending on the purpose. Each will be explained below.
  • the target RNA measurement device of the present invention may have a shape that allows the nucleic acids of the standard RNA, standard RNA group, and standard DNA group to be isolated so as not to mix with each other, and to which an amplification reagent can be added.
  • it may be a small section, more specifically, a flat plate-shaped device including a plurality of concave portions such as wells, or a concave-shaped device.
  • the bottom of the concave portion may be, for example, a flat bottom, a round bottom, a U bottom, a V bottom, etc., and is not limited thereto.
  • the number of recesses on the device may be, for example, but not limited to, 6, 12, 24, 48, 96, 384, or 1536, or more.
  • the specific structure of the flat device is not limited, examples thereof include a chip, a plate, and the like.
  • a multi-well plate such as a 24-well plate, a 48-well plate, or a 96-well plate is suitable as a flat plate-shaped device.
  • the specific configuration of the concave device is not limited, examples thereof include a microtube, a dish, and the like. Multi-microtubes are suitable as concave shaped devices.
  • the material of the main body of the target RNA measurement device is not particularly limited as long as it does not affect the various treatments performed on the nucleic acids placed on the device. Examples include natural resins and synthetic resins, and the material may be a single material made of either of them, or a composite material made of a combination thereof.
  • thermosetting resins include epoxy resins, unsaturated polyester resins, vinyl ester resins, and phenol resins.
  • thermoplastic resins include polyethylene, polypropylene, polyester, polystyrene, polyvinyl chloride, methacrylic resin, fluororesin, polycarbonate, polyurethane, polyethylene terephthalate, aromatic polyetherketone resin, polyphenylene sulfide resin, and the like.
  • examples of the synthetic rubber include butadiene rubber, chloroprene rubber, styrene-butadiene rubber, isoprene rubber, ethylene propylene rubber, nitrile rubber, silicone rubber, acrylic rubber, fluororubber, urethane rubber, and the like.
  • the size and volume of the target RNA measuring device main body are not particularly limited and can be appropriately selected depending on the purpose.
  • the size is not limited, but it may be as large as a 96-well multiwell plate such as 130 mm x 85 mm.
  • the volume is not particularly limited and can be appropriately selected depending on the purpose.
  • the amount of sample is preferably 10 ⁇ L or more and 1000 ⁇ L or less.
  • the nucleic acid is preferably in a dry state or in a suspended state in a solution.
  • a dry state the possibility that the nucleic acid will be degraded by enzymes can be reduced, and furthermore, it can be stored at room temperature.
  • suspended in a solution it is desirable to store it at a low temperature.
  • Target RNA measurement device 4-1 Overview
  • the fourth aspect of the present invention is a target RNA measurement device.
  • the target RNA measurement device of the present invention is a device for implementing the quantitative value correction method described in the first aspect when the target nucleic acid is RNA.
  • the target RNA measurement device of the present invention includes a container in which a test sample is placed.
  • the quantitative value correction method described in the second aspect can be easily performed, and the quantitative value of the target nucleic acid can be easily corrected.
  • the target RNA measurement device of the present invention further includes a container on the device in which a test sample is placed. Therefore, most of the basic configurations are similar to the device for calculating nucleic acid processing efficiency of the third aspect. Therefore, a specific description of the configuration common to the device for calculating nucleic acid processing efficiency of the third aspect will be omitted, and the description will focus on the configuration characteristic of the target RNA measurement device of the present invention.
  • the target RNA measurement device of the present invention includes, in addition to known amounts of standard RNA, standard RNA group, and standard DNA group provided in the container on the device in the device for calculating nucleic acid processing efficiency, in a container on the device; It has a test sample container in which the test sample is placed. This container is basically empty until the target RNA measurement device is used.
  • the shape of the container may be the same as that of the container containing other standard RNAs, standard RNA groups, and standard DNA groups.
  • the shape of the container is not limited. For example, it may be in the shape of a well, a tube, a petri dish, or the like.
  • the target RNA measurement device of the present invention is configured such that a test sample that can contain a target nucleic acid of the same type as a standard sample already placed on the device can be placed therein.
  • Example 1 ⁇ Correction of quantitative value of target nucleic acid contained in test sample (1)> (the purpose) The accuracy of the quantitative value of target nucleic acid in a sample obtained by the quantitative value correction method of the present invention will be verified.
  • the target nucleic acid is shown by SEQ ID NO: 1 in Table 1, which encodes the nucleocapsid of SARS-CoV-2. Nucleic acid.
  • the coating was removed by adding 0.4 U Zaymolyase and incubating at 38°C for 40 minutes and 98°C for 2 minutes, and then the copy number was measured using digital PCR to determine the specific copy number (5.5 copies). / ⁇ L).
  • the PCR conditions were as follows: reverse transcription step at 50°C for 60 minutes, enzyme activation step at 95°C for 10 minutes, DNA strand dissociation step at 95°C for 30 seconds, annealing step at 60°C for 1 minute, and 40 cycles of DNA synthesis step.
  • An enzyme inactivation step was performed at 98°C for 10 minutes. Thereafter, the copy number was measured using a QX200 droplet reader (Bio-Rad).
  • (B) Standard RNA consisting of the same base sequence as the target nucleic acid and a specific copy number (uncoated standard sample) The copy number was measured by digital PCR and adjusted to a specific copy number (20, 50, 100, 200, and 500 copies). Digital PCR was performed in the same manner as in (A).
  • (C) Standard DNA consisting of the same base sequence as the target nucleic acid and containing a specific copy number Standard DNA was prepared by the method for producing a nucleic acid standard device described in Patent Document 1. Specifically, cells into which the same base sequence as the target nucleic acid has been introduced by genetic recombination method are incubated at specific copy numbers (1, 2, 4, 8, 10, 20, 50, 100, 200, and 500 copies). Specific copies of standard DNA were prepared by ejecting with an inkjet and dispensing while counting.
  • test sample containing target nucleic acid and device dispensing Although the target nucleic acid in the test sample is originally an unknown amount, in this example, a sample containing a known amount of target nucleic acid was prepared as the test sample. Specifically, the sample (A) above was serially diluted and prepared. After preparation, the test sample was dispensed into each well at the arrangement position and copy number shown in FIG.
  • the RT-PCR conditions were a reverse transcription step at 55°C for 10 minutes, followed by a 1-minute DNA strand dissociation step at 95°C, a 10-second DNA strand dissociation step at 95°C, and a 10-second DNA strand dissociation step at 95°C. Fifty cycles of 30 second annealing/DNA synthesis steps were performed.
  • 2019-nCoV_N1-P ACCCCGCATTACGTTTGGTGGACC: SEQ ID NO: 4, reporter: FAM, quencher: BHQ
  • Cq values of each standard nucleic acid and target nucleic acid of standard samples (A) to (C) were obtained as measured values.
  • Nucleic acid extraction efficiency was calculated as the ratio of the quantitative copy number obtained from the RNA standard curve to the initial copy number in (A). Table 2 shows the average initial copy number, average quantitative copy number, and nucleic acid extraction efficiency per well of (A) on the device.
  • Quantification of target nucleic acid in test sample and its correction As a quantitative value of target nucleic acid in a test sample, the quantitative copy number was calculated in "4. Creation of calibration curve and calculation of quantitative value of standard nucleic acid" above. For this quantitative copy number, a corrected quantitative value was calculated based on the processing efficiency in each nucleic acid treatment (nucleic acid extraction treatment, reverse transcription treatment, and nucleic acid amplification treatment). The corrected quantitative value is a quantitative copy number corrected in consideration of the target nucleic acid lost during each nucleic acid treatment. This corrected quantitative copy number was compared with the initial copy number of the target nucleic acid in the test sample to confirm the quantitative accuracy.
  • Test sample No. The quantitative copy number per well of the target nucleic acid in Example 1 was 25 copies.
  • the nucleic acid extraction efficiency, reverse transcription efficiency, and nucleic acid amplification efficiency calculated in "5. Calculation of processing efficiency" above are 0.926, 0.577, and 0.976, respectively. Therefore, the corrected quantitative copy number was 48 ( ⁇ 25/(0.926 ⁇ 0.577 ⁇ 0.976)) copies.
  • Test sample No. The initial copy number in No. 1 is 51 copies. Since the quantitative copy number was 25 copies, it means that more than 50% of the target nucleic acid was lost during each treatment process. On the other hand, the difference in the initial copy number obtained by the quantitative value correction method of the present invention was 3 copies, and the loss rate (relative error) was about 6%.
  • Example 2 ⁇ Correction of quantitative value of target nucleic acid contained in test sample (2)> (the purpose) The accuracy of the quantitative value of target nucleic acid in a sample obtained by the quantitative value correction method of the present invention will be verified.
  • the standard sample was prepared by the method described in US5,677,124A.
  • the target nucleic acid was the nucleic acid shown by SEQ ID NO: 1 in Table 1, which encodes the nucleocapsid of SARS-CoV-2.
  • the PCR conditions were: 95°C for 10 minutes enzyme activation step, 94°C for 30 seconds for DNA strand dissociation step, 60°C for 1 minute annealing step, DNA synthesis step for 40 cycles, and 98°C for 10 minutes for enzyme inactivation step. did. Thereafter, the copy number was measured using a QX200 droplet reader (Bio-Rad).
  • test sample containing target nucleic acid was prepared as the test sample. Specifically, the sample (A) above was serially diluted and prepared. After preparation, the test sample was dispensed into each well at the arrangement position and copy number shown in FIG. 3.
  • RNA calibration curve and calculating the quantitative value of the standard nucleic acid From the measured values and known copy numbers of the standard RNA in (B) and the standard DNA in (C), create an RNA calibration curve and a graph showing the relationship between the Cq value and the copy number. A DNA standard curve was created. The prepared calibration curve is shown in FIG. 4.
  • Nucleic acid extraction efficiency was calculated as the ratio of the quantitative copy number obtained from the RNA standard curve to the initial copy number in (A). Table 5 shows the average initial copy number, average quantitative copy number, and nucleic acid extraction efficiency per well of (A) on the device.
  • nucleic acid amplification efficiency The nucleic acid amplification efficiency by PCR was calculated from the amplification efficiency calculation formula described in Example 1 based on the DNA standard curve created from the initial copy number and measured values in C). As a result, the nucleic acid amplification efficiency was 1.024.
  • Quantification of target nucleic acid in test sample and its correction As a quantitative value of target nucleic acid in a test sample, the quantitative copy number was calculated in "4. Creation of calibration curve and calculation of quantitative value of standard nucleic acid" above. For this quantitative copy number, a corrected quantitative value was calculated based on the processing efficiency in each nucleic acid treatment (nucleic acid extraction treatment, reverse transcription treatment, and nucleic acid amplification treatment). The corrected quantitative value is a quantitative copy number corrected in consideration of the target nucleic acid lost during each nucleic acid treatment. This corrected quantitative copy number was compared with the initial copy number of the target nucleic acid in the test sample to confirm the quantitative accuracy.
  • Test sample No. The quantitative copy number per well of the target nucleic acid in Example 1 was 187 copies.
  • the nucleic acid extraction efficiency, reverse transcription efficiency, and nucleic acid amplification efficiency calculated in "5. Calculation of processing efficiency" above are 0.709, 0.912, and 1.024, respectively. Therefore, the corrected quantitative copy number was 196 ( ⁇ 130/(0.709 ⁇ 0.912 ⁇ 1.024)) copies.
  • Test sample No. The initial copy number in No. 1 is 187 copies. Since the quantitative copy number was 130 copies, it means that 30% or more of the target nucleic acid was lost during each treatment process. On the other hand, the difference in the initial copy number obtained by the quantitative value correction method of the present invention was 9 copies, and the loss rate was about 5%.
  • the treatment efficiency of the actually treated nucleic acids can be calculated.
  • the quantitative value correction method of the present invention in the process of obtaining a quantitative value of a target nucleic acid present in a sample, by correcting the amount of target nucleic acid lost due to various processing operations, a highly accurate quantitative value can be obtained. can be obtained.
  • the quantitative value correction method of the present invention even if the target nucleic acid in the sample is at a low concentration of 200 copies or less, or even at an extremely low concentration of 50 copies or less, an accurate quantitative value of the target nucleic acid contained in the sample can be obtained. can be obtained.
  • the device for calculating nucleic acid processing efficiency of the present invention by using the device, it is possible to simply and easily calculate the percentage of nucleic acids actually processed by nucleic acid processing among the nucleic acids contained in a sample as the nucleic acid processing efficiency. It can be calculated as follows.
  • the device for measuring target RNA of the present invention by using the device, the target RNA contained in the sample can be calculated based on the calibration curve, and the obtained quantitative value can be corrected by the actual nucleic acid processing efficiency. , more accurate quantitative values can be obtained.
  • a method for calculating nucleic acid processing efficiency comprising: A sample processing step of subjecting a standard sample containing a known amount of standard nucleic acid to a nucleic acid extraction process or reverse transcription process, measuring the standard nucleic acid obtained from the standard sample and a plurality of different groups of standard nucleic acids with known amounts, and measuring the measured value.
  • the measurement process to obtain a calibration curve creation step of creating a calibration curve from each measurement value of the standard nucleic acid group; and dividing the amount of the standard nucleic acid calculated from the calibration curve by a known amount to determine the nucleic acid processing efficiency of the standard nucleic acid in the sample processing step.
  • the method includes a step of calculating nucleic acid processing efficiency.
  • nucleic acid treatment efficiency calculation step all nucleic acid treatment efficiencies are calculated by multiplying the nucleic acid treatment efficiencies obtained from each nucleic acid treatment.
  • the treatment efficiency step after further calculating the nucleic acid treatment efficiency in the nucleic acid amplification treatment of the standard nucleic acid from the slope of the calibration curve, all nucleic acid treatment efficiencies are calculated by multiplying by the previously calculated nucleic acid treatment efficiency. , (1) or (2).
  • test sample is of the same type as the standard sample, and the target nucleic acid has the same base sequence as the standard nucleic acid.
  • test sample in the nucleic acid extraction process is a biological sample.
  • biological sample is a cell, nucleus, or virus particle.
  • quantitative value is the copy number of the target nucleic acid.
  • the number of copies of the target nucleic acid in the test sample in the sample processing step is 50 copies or less.
  • the target nucleic acid is a nucleic acid containing an exogenous target sequence.
  • a known amount of standard RNA contained in the standard sample A plurality of different known amounts of standard RNA groups having the same base sequence as the standard RNA; and a plurality of different known amounts of standard DNA having the same or complementary base sequence as the standard RNA.
  • a device for calculating nucleic acid processing efficiency (11) The device for calculating nucleic acid processing efficiency according to (10), wherein the amounts of the standard RNA and standard DNA are respective copy numbers.
  • a device for measuring target RNA comprising the device for calculating nucleic acid processing efficiency according to any one of (10) to (13), further comprising a container for measuring a test sample.

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Abstract

La présente invention a pour but d'obtenir une valeur quantitative plus précise d'un acide nucléique cible présent dans un échantillon.　La valeur quantitative est corrigée en fonction de l'efficacité du traitement au cours de divers traitements jusqu'à ce que l'acide nucléique cible de l'échantillon soit quantifié. Il est ainsi possible d'obtenir une valeur quantitative très précise lorsqu'une correction a été apportée pour une partie de l'acide nucléique cible qui a été perdue au cours de diverses étapes de traitement.
PCT/JP2023/004626 2022-03-23 2023-02-10 Procédé de calcul d'efficacité de traitement d'acide nucléique, procédé de correction de valeur quantitative d'acide nucléique l'utilisant, et dispositif de mesure d'arn cible Ceased WO2023181694A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2021145642A (ja) * 2020-03-23 2021-09-27 株式会社リコー 担体及び検査方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NATARAJAN ARAVIND, HAN ALVIN, ZLITNI SOUMAYA, BROOKS ERIN F., VANCE SUMMER E., WOLFE MARLENE, SINGH UPINDER, JAGANNATHAN PRASANNA,: "Standardized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA", NATURE COMMUNICATIONS, vol. 12, no. 1, pages 1 - 12, XP093095742, DOI: 10.1038/s41467-021-25576-6 *
SHIN-ICHIRO FUJII, NAOHIRO NODA, SACHIE SHIBAYAMA, YUJI SEKIGUCHI, MEGUMI KATO: "Development of Nucleic Acid Certified Reference Materials and Response for Testing for Coronavirus Infection", JOURNAL OF THE JAPAN SOCIETY FOR PRECISION ENGINEERING, vol. 87, no. 1, 5 January 2021 (2021-01-05), pages 21 - 24, XP009538249, ISSN: 0912-0289, DOI: 10.2493/jjspe.87.21 *

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