JP4878263B2 - Nucleic acid concentration determination method and nucleic acid analysis method - Google Patents

Description

  The present invention relates to a method for determining a nucleic acid concentration in a test sample using an absorbance measurement method, a test method, and an apparatus therefor.

  When genetic testing is performed using nucleic acid extracted from a biological sample, the amount of nucleic acid is greatly related to testing accuracy and testing success rate. For example, in the case of the LOH test in which the test region is amplified by the PCR-SSCP method and the quantity ratio of the amplified allele is examined, an accurate test result cannot be obtained if the amount of genomic DNA decreases. Therefore, in order to maintain the reliability of the test result, concentration measurement with high quantitativeness is required. Further, in a fragment analysis using a DNA sequencer, when a test sample having a nucleic acid concentration of a certain value or more is used, the measured value exceeds the detection range, so the test sample must be diluted and analyzed again. In addition, in the STR test for personal identification, in order to increase the certainty of the STR reaction, the nucleic acid sample solution concentration in a narrow range of about 0.25 ng to 2 ng is required for human genomic DNA (Non-patent Document 1).

  As shown in the above example, when the nucleic acid concentration of the test sample affects the test result, it is necessary to measure the nucleic acid concentration in the test sample. Conventional methods for measuring the concentration of nucleic acid include a fluorescence measurement method using a fluorescent substance that specifically binds to a double-stranded nucleic acid or a single-stranded nucleic acid, and a method of hybridizing a probe to which a fluorescent molecule is bound to a nucleic acid. Nucleic acid concentration measurement methods, methods of hybridizing probes, amplification reaction, pyrophosphoric acid as a byproduct of amplification reaction, chemiluminescence method, electrochemical detection, and the like are used. These measurement methods can measure the nucleic acid concentration with extremely high accuracy even if proteins or the like are mixed in the test sample. However, the measurement cost is high because the addition of reagents is necessary for the measurement, and if the test sample does not enter the measurement range, it is necessary to change the concentration of the test sample and perform the measurement again. Is the method. In addition, the inspection sample used for concentration measurement after measuring the concentration cannot be used for inspection, and there is a problem that leads to loss of a rare inspection sample.

  On the other hand, the absorbance measurement method can measure the absorbance of a test sample and convert the nucleic acid concentration from the absorbance at a wavelength of 260 nm (hereinafter, A260). Moreover, the nucleic acid purity can be evaluated from the absorbance ratio (hereinafter referred to as A260 / A280) between the wavelength of 260 nm and the wavelength of 280 nm (Non-patent Document 2). When a protein component is mixed in the sample, the absorbance at a wavelength of 280 nm increases, and when a carbohydrate, peptide, or low molecular component is mixed, the absorbance at a wavelength of 230 nm increases (Non-patent Document 3). Although this measurement method is simple and does not require addition of reagents, it is an inexpensive method. However, contamination with protein components, carbohydrates, peptides, aromatic compounds such as phenol, etc. in the test sample also affects the absorbance. Measurement of nucleic acid concentration may be difficult.

J. et al. Forensic Sci. 2001 May; 46 (3): 647-60. BioTechniques 22: 474-481 Spectrophotometry of DNA or RNA. Moleclar Cloning. Third Edition 3: A8.20-A8.21

  As a problem in the conventional genetic test, there is a problem that the nucleic acid concentration in the test sample cannot be accurately measured when protein components, carbohydrates, peptides, small molecules, etc. are mixed in the test sample extracted from the biological sample. In particular, it has been found that when the nucleic acid concentration in the test sample is low, the conventional method of measuring the nucleic acid concentration using the absorbance at a wavelength of 260 nm causes a problem that the measured value is higher than the actual nucleic acid concentration. Therefore, with the conventional method alone, there is a problem in that even if the test sample concentration is adjusted so as to be in the concentration range required for the inspection according to the measured concentration, the inspection cannot be performed within the measurement range and the inspection becomes defective.

  In determining whether the nucleic acid concentration in the test sample is higher or lower than the predetermined value, the present invention measures the absorbance of the test sample at least at a wavelength of 220 nm to 260 nm, In addition to the conventional determination method that determines that the nucleic acid concentration of the test sample is lower than the predetermined nucleic acid concentration when the nucleic acid concentration is lower than the converted value converted to the absorbance at a wavelength of 260 nm, the nucleic acid of the test sample is determined by the conventional determination method. When it is determined that the concentration is high, the nucleic acid concentration of the test sample is higher than the predetermined nucleic acid concentration when all the absorbance in the wavelength range of 220 nm to 260 nm of the test sample is higher than the absorbance of the test sample at the wavelength of 260 nm. A step of determining that the concentration is low, and a measured wavelength 260 nm of absorbance at a wavelength of 260 nm converted from a predetermined nucleic acid concentration Higher than the absorbance of, and if any of the absorbance at a wavelength less than 220nm or 260nm of the test sample is less than 0, the nucleic acid concentration in the test sample is carried out low, determining than the predetermined nucleic acid concentration.

  According to the present invention, it is possible to determine whether a test sample is higher or lower than a predetermined concentration with a predetermined concentration as a threshold with higher accuracy than the conventional method. In particular, when the nucleic acid concentration in the test sample is low, it is possible to avoid the problem that the conventional method determines that the measured value is higher than the actual nucleic acid concentration. Therefore, since the concentration of the inspection sample group outside the measurement range can be adjusted in advance, the number of inspections can be reduced and the inspection cost can be reduced.

  In one preferred embodiment of the present invention, the absorbance of the test sample is measured in the range of at least a wavelength of 220 nm or more and 260 nm or less by an absorbance measurement method, and (A) the absorbance of the test sample at a wavelength of 260 nm and a predetermined nucleic acid concentration are 260 nm. In addition to the determination condition for determining that the nucleic acid concentration of the test sample is lower than the predetermined concentration when the absorbance at a wavelength of 260 nm of the test sample is lower than the conversion value from the predetermined concentration. (B) Determination that the nucleic acid concentration of the test sample is lower than the predetermined nucleic acid concentration when all the absorbance in the wavelength range of 220 nm to 260 nm of the test sample is higher than the absorbance of the test sample at the wavelength of 260 nm. In addition, (C) if any absorbance at a wavelength of less than 260 nm is less than 0, Determining the nucleic acid concentration in the lower than the predetermined nucleic acid concentration. In any determination condition, when it is not determined that the nucleic acid concentration of the test sample is lower than the predetermined nucleic acid concentration, the nucleic acid concentration of the test sample is determined to be higher than the predetermined nucleic acid concentration.

  In addition, in the determination condition A, the absorbance at a wavelength of 260 nm of the test sample is converted into the nucleic acid concentration, and even if the nucleic acid concentration of the test sample is lower than the predetermined nucleic acid concentration as compared with the predetermined nucleic acid concentration, It can be determined that the density is lower than the predetermined density. In the determination condition B, more preferably, all absorbances at wavelengths from 250 nm to 230 nm are compared with absorbances at a wavelength of 260 nm converted from a predetermined nucleic acid concentration, and all absorbances at wavelengths from 250 nm to 230 nm are compared with a wavelength of 260 nm of the test sample. When the absorbance is higher than the absorbance, the nucleic acid concentration in the test sample is determined to be lower than the predetermined nucleic acid concentration.

  The predetermined concentration can be set to a value of 1 to 100 ng / μL, and particularly preferably set to a value of 3 to 50 ng / μL. The test sample may be an extracted nucleic acid derived from urine, and the nucleic acid to be measured may be genomic DNA.

  Yet another embodiment of the present invention is a method for analyzing nucleic acid in a test sample, comprising the step of adjusting the input amount of the test sample after determining whether the nucleic acid concentration in the test sample is higher or lower than a predetermined concentration. . Furthermore, after the determination of the nucleic acid concentration in the test sample, it can be determined whether or not the test result of the test sample lower than the predetermined nucleic acid concentration is accepted. In addition, after the method for determining the nucleic acid concentration in the test sample, a step of diluting or concentrating the determined test sample can be included.

  Still another preferred embodiment of the present invention includes a mechanism for measuring the absorbance of a test sample, a function of storing all the absorbances of the obtained test sample, and a predetermined threshold value for determining the concentration of the test sample. Using the function to set the nucleic acid concentration and the database function of the nucleic acid concentration corresponding to the absorbance, or the calculation function to convert the absorbance to the nucleic acid concentration, or the nucleic acid concentration to the absorbance, and the database or the calculation function, A function that compares the nucleic acid concentration of the test sample with a predetermined nucleic acid concentration and determines that the nucleic acid concentration of the test sample is lower than the predetermined nucleic acid concentration when the nucleic acid concentration of the test sample is lower than the predetermined nucleic acid concentration; When it is determined that the concentration of the sample is higher than the predetermined concentration, all absorbances of the test sample having a wavelength of 220 nm to 260 nm are absorbed by the test sample at a wavelength of 260 nm. A function of determining that the nucleic acid concentration in the test sample is lower than the predetermined nucleic acid concentration when the test sample is higher than the predetermined temperature, and a wavelength of 220 nm or more of the test sample when the test sample concentration is determined to be higher than the predetermined concentration. This is a nucleic acid concentration determination apparatus for a test sample having a function of determining that the nucleic acid concentration in a test sample is lower than a predetermined nucleic acid concentration when any absorbance below 260 nm is lower than 0.

  As a result of examining the absorbance curve in a wavelength range of 220 nm to 350 nm with respect to 185 types of genomic DNA solutions extracted from biological samples, the present invention discovered a pattern peculiar to low-concentration test samples. It has come.

  That is, the present invention is a test sample containing nucleic acid extracted from a biological sample, and the nucleic acid concentration range of the sample is different from the nucleic acid concentration range required for the next step, as shown in FIG. The present invention provides a method that can more accurately determine whether a predetermined concentration is higher or lower than a predetermined concentration using a predetermined concentration as a threshold, and reduce inspection defects in the next process or improve inspection reliability. Hereinafter, the procedure of the present invention will be described with reference to the drawings.

  In FIG. 1, 1 is a step of measuring absorbance, 2 is a step of converting absorbance to nucleic acid concentration, 3 is a step of comparing the converted value with a predetermined concentration, 4 is an absorbance comparison step, 5 is an absorbance evaluation step, and 6 is a predetermined concentration. A step of determining a higher sample, 7 is a step of determining a sample lower than a predetermined concentration.

  Step 1: Using an absorbance measurement method, measure the wavelength of the test sample in a range of at least 220 nm to 260 nm.

  Step 2: Using the determination conditions A, B, and C shown in FIG. 1, it is determined whether the test sample is higher or lower than a predetermined concentration with a predetermined concentration as a threshold value.

  (A) When the nucleic acid concentration conversion value in A260 of the test sample is lower than a predetermined concentration, it is determined that the nucleic acid concentration in the test sample is lower than the predetermined nucleic acid concentration.

  (B) All values of absorbance at wavelengths of 220 nm or more and 260 nm or less are higher than absorbance at wavelength of 260 nm converted from a predetermined nucleic acid concentration, or more preferably, all values of absorbance measurement values at wavelengths of 230 nm or more and 250 nm or less are When the absorbance at a wavelength of 260 nm converted from the predetermined nucleic acid concentration is higher, it is determined that the nucleic acid concentration in the test sample is lower than the predetermined nucleic acid concentration.

  (C) When the measured absorbance at a wavelength of 260 nm is higher than the absorbance at a wavelength of 260 nm converted from a predetermined nucleic acid concentration and the absorbance at a wavelength of less than 260 nm is lower than 0, the nucleic acid concentration in the test sample is higher than the predetermined nucleic acid concentration Judge as low.

  Further, if it is not determined that the nucleic acid concentration in the test sample is lower than the predetermined concentration under any of the determination conditions, it is determined that the nucleic acid concentration in the test sample is higher than the predetermined concentration.

  Step 3: The test sample whose nucleic acid concentration is determined by the above-described determination method is diluted or concentrated according to the next process, or the test sample input amount to the next process is adjusted. In the case of a threshold value, it is determined whether or not inspection is possible.

  Furthermore, an outline of an apparatus for determining a nucleic acid concentration in a test sample according to the present invention will be described with reference to FIG. In the diagram shown in FIG. 2, 10 is an absorptiometer, 11 is a function for converting absorbance and nucleic acid concentration, 12 is a first comparison / determination function, 13 is a second comparison / determination function, 14 is an absorbance evaluation / determination function, and 15 is an output. A device, 16 is a database, and 17 is a storage device. Each function performs each step shown in FIG.

  The absorptiometer 10 measures the absorbance of the test sample, stores the measurement result in the storage device 17, and converts the absorbance at 260 nm into the nucleic acid concentration by the conversion function 11. The nucleic acid concentration obtained by the conversion is compared with a predetermined concentration given from the database 16, and the first comparison determination function 12 determines the determination condition A in FIG. As a result of the determination, if the nucleic acid concentration of the test sample obtained by conversion is higher than a predetermined concentration, the second comparison determination function 13 determines the determination condition B in FIG. More preferably, the function compares the absorbance in the entire region extending from 230 nm to 250 nm with the absorbance at a wavelength of 260 nm of the test sample. As a result of the determination, if the nucleic acid concentration of the test sample is higher than a predetermined concentration, the determination condition C determination of FIG. If it is determined that the concentration is higher than the predetermined concentration under any of the determination conditions, it is determined that the concentration of the test sample is higher than the predetermined concentration. All the results of the above comparison and determination are stored in the storage device 17, and necessary data is output to the output device 15.

  The biological sample in the present invention is particularly limited to screening samples such as group screening, health checkup, dock screening, mail screening, blood, tissues, urine of outpatients and hospitalized patients in hospitals, animals, plants, microorganisms, etc. The target is a biological sample containing all nucleic acids and a substance to which the biological sample is attached. The nucleic acid in the present invention is all nucleic acids including DNA and RNA extracted from the sample.

  A method for extracting the nucleic acid from the biological sample can be performed by a known method. More preferably, a method is used in which the nucleic acid is adsorbed on a silica column and washed, and then the nucleic acid is eluted using a nucleic acid solution.

  Further, the test sample in the present invention may be any sample as long as it contains a nucleic acid to be used for the test, for example. In addition, the test in the present invention is a test for analyzing nucleic acid, which is applied subsequent to the determination method of the present invention, and any analysis method may be used as long as the amount of nucleic acid to be used for analysis affects the measurement result. . Specifically, quantitative tests that accurately measure nucleic acid concentrations, SNP tests that detect single nucleotide polymorphisms, methylation tests that detect genomic methylation sites, LOH tests that detect chromosome deletions, microsatellite polymorphisms Examples include STR inspection (personal identification inspection) for identifying an individual using a mold, but applicable inspections are not limited to these.

  Further, the test sample group in the present invention means a plurality of test samples obtained from a plurality of individuals or a plurality of test samples collected over time from a certain individual. The sample does not need to be used for the present invention at the same time, and only one test sample may be used for the determination method of the present invention at a certain time point.

  The predetermined concentration in the present invention means a threshold concentration for determining the test sample, preferably at least 1 ng / μL to 100 ng / μL, and can be determined with a higher determination effect than the conventional method. However, when the predetermined concentration is low, the appropriateness of the inspection sample higher than the predetermined concentration is decreased, and when the predetermined concentration is high, the appropriateness of the inspection sample lower than the predetermined concentration is decreased. From the results of Examples, when the predetermined concentration is less than 1 ng / μL, the appropriateness and specificity of the test sample higher than the predetermined concentration are reduced by about 40%, and when the predetermined concentration exceeds 100 ng / μL, it is lower than the predetermined concentration. The appropriateness of the test sample is less than half. More preferably, the predetermined concentration is set to 3 ng / μL to 50 ng / μL.

  The apparatus used in the present invention may be any apparatus as long as it can measure absorbance at a wavelength of 350 nm to 180 nm, at least a wavelength of 260 nm to 220 nm. Specific examples include an absorbance measuring device and a spectrophotometer. More specifically, Hitachi diode array type biophotometer (manufactured by Hitachi High-Technologies Corporation), 1 μL spectrophotometer (manufactured by NanoDrop Technologies), etc. can be mentioned. In the present invention, the absorbance of nucleic acids in a test sample is measured. What is necessary is just to be able to do and it is not limited to the above-mentioned analyzer.

  A test sample is generally inspected as follows using the sample and the conventional apparatus. That is, not only in the nucleic acid test, the test instrument is provided with a measurement range. When the concentration range of the test sample group is different from the measurement range of the test instrument, the test sample is prepared with a plurality of concentrations from the beginning. Then, all of the samples having the prepared concentration are inspected, or the test sample that has not been inspected within the measurement range at the initial concentration is subjected to re-measurement by changing the concentration. Alternatively, when a low-concentration inspection sample cannot be measured with an inspection device, inspection below the measurement lower limit of the inspection device is wasted. Moreover, when it is desired to measure the amount ratio of alleles in the genomic nucleic acid, the amount of nucleic acid in the test sample is related to the test accuracy. As in these examples, when the inspection sample is inspected without measuring the concentration, the inspection accuracy decreases, the cost increases, and the throughput decreases.

  For this reason, measurement of the test sample concentration by a simple and low-cost method is required before the test. Examples of such a concentration measuring method include a method of measuring the absorbance and measuring the nucleic acid concentration in the test sample. However, in this method, when the test sample contains not only the nucleic acid but also a biological sample component, more specifically, an organic compound such as a protein component, a peptide, an aromatic amino acid, etc., the wavelength at which the nucleic acid is detected (260 nm). Since it is detected in the vicinity, there has been a problem that the exact concentration of nucleic acid cannot be measured.

  According to the nucleic acid concentration determination method of the present invention, it is possible to more accurately determine whether the test sample is higher or lower than a predetermined concentration. This makes it possible to determine in advance a test sample that is higher or lower than the measurement range and dilute or concentrate it within the measurement range. Alternatively, it is possible to determine an inspection sample that is lower than the measurement range and is not to be inspected, thereby preventing unnecessary inspection.

  According to the present invention, (1) a step of measuring the wavelength of a test sample group from 220 nm to 260 nm using an absorbance measurement method, and (2) a test sample group having a predetermined concentration according to the determination method shown in FIG. A step of determining a test sample group higher than a predetermined concentration and a test sample group lower than a predetermined concentration as threshold values; and (3) the test sample group determined in step 2 in accordance with the measurement conditions of the test sample test. By three steps of diluting or concentrating, determining whether test is possible, or adjusting the test sample input amount to the next step, it is possible to improve the test accuracy of nucleic acid test, or to reduce cost and improve throughput.

  Hereinafter, conditions for applying the present invention will be described in detail according to each step, but the sample, apparatus, and the like to be described are not limited to the methods described below.

  In step 1, the test sample is irradiated with light, and the absorbance at a wavelength of 220 nm to 260 nm is measured. A spectrophotometer can be used as an analyzer using a conventional absorbance measurement method for the method, but in this step, it is only necessary to measure the absorbance of a test sample at a wavelength of 220 nm or more and 260 nm or less. It is not limited to a measurement procedure, a measurement method, or a measurement apparatus.

  In step 2, the test sample A260 is converted into a nucleic acid concentration, compared with a predetermined concentration, and a determination condition A for determining that a test sample whose converted concentration is lower than the predetermined concentration is lower than the predetermined concentration. Determination condition B for comparing the absorbance obtained in the wavelength band of less than A260 of the test sample and determining that the test sample having all absorbances in the wavelength band higher than A260 of the test sample is lower than the predetermined concentration Thus, the accuracy of determining the concentration of the test sample can be increased by setting at least 1 ng / μL to 100 ng / μL as the predetermined concentration. Moreover, as will be clear from the results of the examples described later, by using the determination condition C in which any one of the test samples having a wavelength of 220 nm or more and less than 260 nm has an absorbance lower than 0, the test sample is lower than a predetermined concentration. The determination accuracy can be further increased at a predetermined concentration of 5 ng or less.

  That is, the determination method in the present invention determines that a test sample satisfying at least one of the determination conditions A and B is a test sample lower than a predetermined concentration, and more preferably satisfies any of the determination conditions A, B, and C. The test sample is determined to be a test sample lower than a predetermined concentration. Note that the determination conditions A, B, and C need not be used in the order of the determination conditions described above, and can be used in any order. Hereinafter, the determination conditions A and B will be described in more detail.

  As a method for converting the absorbance at a wavelength of 260 nm of the measured test sample into the nucleic acid concentration under the determination condition A, a known method can be used. More specifically, when the solvent of the test sample solution is neutral to weakly alkaline and the measurement optical path length is 1 cm and A260 = 1.0, the double-stranded DNA nucleic acid is 50 μg / mL, and the single-stranded RNA is 40 μg / mL, single-stranded DNA is known to be 33 μg / mL, and the nucleic acid concentration can be converted from here, but this method only needs to convert the absorbance at a wavelength of 260 nm to the nucleic acid concentration. It is not limited to the said conditions. Moreover, it goes without saying that a predetermined concentration can be converted to absorbance and compared with A260 of the test sample, contrary to the method of converting the absorbance to the nucleic acid concentration.

  In the determination condition B, as the absorbance compared with A260, an absorbance obtained from a wavelength band of at least a wavelength of 220 nm or more and less than 260 nm is used. More preferably, the determination accuracy can be improved by using absorbance in a wavelength band of 230 nm or more and less than 250 nm. As a determination method of this condition, compared to the absorbance in the wavelength band and A260 of the test sample, it is determined that a test sample whose absorbance in the wavelength band is higher than A260 of the test sample is a test sample lower than a predetermined concentration. To do.

  In step 3, the concentration of the test sample and the amount to be sampled are adjusted according to the need for the next process in the present invention. To give a more specific example, when the inspection in the next step is an inspection in a measurement range of a nucleic acid concentration of 20 ng / μL to 0.2 ng / μL, a test sample group is determined at a predetermined concentration of 20 ng / μL, and 20 ng / A dilution operation is uniformly performed on a test sample group having a concentration higher than μL. In this example, when 50-fold dilution is performed, it is possible to measure up to a maximum test sample concentration of 1000 ng / μL within the measurement range of the next process. However, in this process, it is only necessary to dilute and concentrate the test sample, adjust the amount of separation, and determine whether or not the test can be performed according to the test in the next process.

  Hereinafter, although the Example which determined the selection conditions of this invention and the typical use which uses this invention are shown, this invention is not limited to these examples.

(Example) Determination of nucleic acid concentration of human genomic nucleic acid extracted from urine Preparation of test sample In this experiment, genomic nucleic acid extracted from 185 natural urine samples was used as a test sample. The collected natural urine was stored at 4 ° C. immediately after collection, and then centrifuged at 3000 rpm for 10 minutes within 2 days. The supernatant was removed, washed with PBS, and suspended in 1 ml of PBS. QIAamp DNA Blood Mini Kit (manufactured by QIAGEN) was used for nucleic acid extraction of this suspension, and a known method was used as the extraction method. More specifically, the clinical specimen was dissolved with a lysis reagent and adsorbed onto a silica column. Next, the sample was washed with a washing reagent and eluted from a silica column with a nucleic acid elution reagent, which was used as a test sample for concentration measurement.

2. Quantitative Measurement of Nucleic Acid Concentration with PicoGreen In order to quantify the exact double-stranded nucleic acid concentration of 185 kinds of test sample groups extracted from urine, the present inventors are PicoGreen, a fluorescent reagent that binds only to double-stranded nucleic acid. Nucleic acid concentration was quantified using dsDNA Quantitation Reagent (Molecular Probes). This quantification method used a known method. More specifically, a dilution series was prepared using a standard nucleic acid, and the test sample was diluted so as to fall within the concentration range of the prepared dilution series. Next, a fluorescent reagent that binds only to the double-stranded nucleic acid was added, and the amount of fluorescence was measured. As a result, the nucleic acid concentration of this test sample group was in the concentration range of 1500 ng / μL to 0.01 ng / μL.

3. Concentration measurement by absorbance measurement method The present inventors measured the nucleic acid concentration in the test sample using the absorbance measurement method. Using a 1 μL spectrophotometer (manufactured by NanoDrop Technologies) as the measuring apparatus, 1 μμ of the sample solution was taken from the test sample, and the absorbance was measured. As a result, absorbance curves were obtained in the range of wavelengths from 350 nm to 220 nm for 185 types of test samples, converted from the absorbance at a wavelength of 260 nm to the concentration of double-stranded nucleic acid, and a concentration range of about 1900 ng / μL to 0.09 ng / μL. The measurement result of was obtained.

4). Determination of test sample Seven predetermined concentrations (1.0 ng / μL, 3.0 ng / μL, 5.0 ng / μL, 10 ng / μL, 20 ng / μL, 50 ng / μL, 100 ng / μL) for the test sample group Was determined using a plurality of determination conditions shown in Table 1, and the appropriate number of each determination means was examined using the result determined by PicoGreen as a reference value. The determination means in the method of the present invention uses a plurality of the determination conditions shown in Table 1, and the notations representing the determination conditions are shown in parallel.

  From Table 2, in the conventional method, when the predetermined concentration was determined to be 10 ng / μL, it was determined that 60 samples out of 148 types of test samples having a concentration lower than the predetermined concentration were 10 ng / μL or more. On the other hand, in the method of the present invention, it was determined that 14 types of the samples out of 148 types of test samples having a concentration lower than the predetermined concentration were 10 ng / μL or more.

  When inspection of 10 ng / μL or more is required for the inspection of the next process, using the above result, 97 types of inspection samples are required in the conventional method, whereas 50 types of inspection samples are required in the method of the present invention. Can be inspected. Therefore, in this example, the inspection throughput can be doubled and the cost can be reduced to ½.

5. Evaluation of Judgment Result As an evaluation method of the judgment result, a screening effect index was used. Screening means determining as much as possible one state and the other. Sensitivity, specificity, moderateness, and efficacy indices were used to represent the ability of this screening. Hereinafter, each index will be described. The alphabet in each index represents the number of determination results by screening shown in Table 4. And, using this alphabet to explain each index, the calculation formula is shown.

  First, sensitivity represents the ability to select less than a predetermined concentration according to the present invention at a rate at which an inspection sample having a concentration lower than a predetermined concentration is actually less than the predetermined concentration according to the present invention. The higher the value, the better. This index can be calculated by the method of (Equation 1).

(Formula 1)
Sensitivity = number of inspection samples less than a predetermined concentration correctly determined in the present invention / number of all inspection samples less than a predetermined concentration = a / (a + c) × 100 (%)
Next, the specificity is a ratio at which a test sample having a predetermined concentration or higher is correctly set to a predetermined concentration or higher in the present invention, and represents a capability of not mistaken for a test sample having a predetermined concentration or higher to be less than a predetermined concentration. The higher the value, the better the determination method. This index can be calculated by the method of (Formula 2).

(Formula 2)
Specificity = number of test samples having a predetermined concentration or more correctly determined in the present invention / number of all test samples having a predetermined concentration or more = d / (b + d) × 100 (%)
Next, the effectiveness means a ratio that correctly reflects the presence or absence of less than a predetermined concentration. This index can be calculated by the method of (Equation 3).

(Formula 3)
Effectiveness = (a + d) / (a + b + c + d) × 100 (%)
Appropriateness levels include moderateness less than a predetermined concentration and moderateness above a predetermined concentration. Sensitivity and specificity are indicators of the effectiveness of the method of the present invention. It means the probability that the test sample determined to be less than the concentration is actually less than the predetermined concentration, and the appropriate intermediate level means the opposite. This index can be calculated by the methods of (Equation 4) and (Equation 5).

(Formula 4)
Less than a predetermined concentration moderateness = the number of inspection samples actually below a predetermined concentration / the number of all inspection samples below a predetermined concentration
= A / (a + b) × 100 (%)
(Formula 5)
Appropriate intermediate level above specified concentration = number of test samples actually above specified concentration / number of all test samples above specified concentration
= D / (c + d) × 100 (%)
Tables 5 to 10 show the results of evaluating the effect of the determination of the present invention using the above five evaluation indexes.

  As a result of evaluating the effect of the determination method according to the present invention, any of the determination methods of the present invention showed higher effectiveness than the conventional method. Specifically, the determination condition B showed the highest effectiveness when (B-2), that is, an absorbance having a wavelength of 230 nm or more and 250 nm or less was used among the four wavelength bands. It was also found that a higher effectiveness than that of the conventional method can be obtained even in the widest wavelength range (B-4), that is, in a wavelength range of 220 nm to less than 260 nm. As an effect of the determination of each predetermined concentration, it has been found that if the predetermined concentration is lowered, the appropriateness above the predetermined concentration tends to decrease, and if the predetermined concentration is increased, the appropriateness below the predetermined concentration tends to decrease. Also showed higher effectiveness than the conventional method.

  The main effects of the embodiment of the present invention will be briefly described as follows.

  (1) In order to inspect an inspection sample group in which the concentration range of the inspection sample group is different from the concentration range of the next process within the measurement range, conventionally, measurement is performed at a plurality of concentrations or at one concentration, and inspection outside the measurement range For tests that have been remeasured by changing the concentration of the sample, the concentration of the test sample group outside the measurement range can be adjusted in advance, allowing measurement at one concentration, or reducing the number of test samples to be remeasured In addition, the inspection cost can be reduced.

  (2) When the amount of nucleic acid used for the test affects the test reliability, a test sample group having a low concentration can be determined more accurately than in the conventional absorbance measurement method, and the test reliability is improved.

  In addition, it evaluated using the parameter | index of a screening effect as an effect parameter | index of test sample determination by this invention. As a result of determining the concentration of 185 samples with a nucleic acid concentration of 10 ng / μL as a predetermined concentration, the sensitivity is improved by 32% (60% → 92%) compared to the determination result by the nucleic acid concentration converted from A260, which is a conventional method, The probability that the nucleic acid concentration is less than 10 ng / μL was improved by 37% (38% → 75%), and the effectiveness was improved by 26% (67% → 93%).

The flowchart of the nucleic acid analysis method by the Example of this invention. The diagram which shows the structure of the nucleic acid concentration determination apparatus in the test sample by this invention.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... The step which measures absorbance, 2 ... The step which converts an absorbance into a nucleic acid concentration, 3 ... The step which compares a converted value with predetermined concentration, 4 ... Absorbance comparison step, 5 ... Absorbance evaluation step, 6 ... Sample higher than predetermined concentration Step for determining: 7: Step for determining a sample lower than a predetermined concentration, 10: Absorbance photometer, 11: Conversion function of absorbance, 12: First comparison determination function, 13: Second comparison determination function, 14: Absorbance evaluation determination Function, 15 ... output device, 16 ... database, 17 ... storage device.

Claims (11)

In the method for determining whether the nucleic acid concentration of a test sample is higher or lower than a predetermined nucleic acid concentration, the absorbance of the test sample in a wavelength range of 220 nm to 260 nm is measured, and the absorbance of the test sample at a wavelength of 260 nm A step of determining that the nucleic acid concentration in the test sample is lower than a predetermined nucleic acid concentration when any absorbance at a wavelength of 260 nm less than the wavelength of 260 nm converted from the concentration is lower than 0. A method for determining a nucleic acid concentration in a test sample. Method of determining nucleic acid concentration in a test sample of the serial loading to claim 1, characterized in that for setting the predetermined nucleic acid concentration to a value of 1-100 ng / [mu] L. Method of determining nucleic acid concentration in a test sample of the serial loading to claim 1, characterized in that for setting the predetermined nucleic acid concentration to a value of 3~50ng / μL. Method of determining nucleic acid concentration in a test sample of the serial loading to claim 1, wherein the test sample is extracted nucleic acid from the urine. The method for determining a nucleic acid concentration in a test sample according to claim 1 or 2, wherein the nucleic acid is genomic DNA. In the method for determining whether the nucleic acid concentration of a test sample is higher or lower than a predetermined nucleic acid concentration, the absorbance of the test sample in a wavelength range of 220 nm to 260 nm is measured, and the absorbance of the test sample at a wavelength of 260 nm After the step of determining that the nucleic acid concentration in the test sample is lower than the predetermined nucleic acid concentration when the absorbance at a wavelength of 260 nm converted from the concentration is higher and any absorbance at a wavelength of less than 260 nm of the test sample is smaller than 0 And a method for analyzing a nucleic acid in a test sample, comprising the step of adjusting an amount of the test sample to be dispensed or input. In the method for determining whether the nucleic acid concentration of a test sample is higher or lower than a predetermined nucleic acid concentration, the absorbance of the test sample in a wavelength range of 220 nm to 260 nm is measured, and the absorbance of the test sample at a wavelength of 260 nm After the step of determining that the nucleic acid concentration in the test sample is lower than the predetermined nucleic acid concentration when the absorbance at a wavelength of 260 nm converted from the concentration is higher and any absorbance at a wavelength of less than 260 nm of the test sample is smaller than 0 A method for analyzing nucleic acid in a test sample, comprising the step of determining whether or not to accept a test result when the nucleic acid concentration in the test sample is lower than a predetermined nucleic acid concentration. The nucleic acid analysis method according to claim 7, wherein adjusting the aliquot or input amount of the test sample in accordance with the nucleic acid concentration of the determined test sample. The nucleic acid analysis method according to claim 7, characterized in that the test sample is diluted or concentrated depending on the concentration of the nucleic acid test sample is determined. The nucleic acid analysis method according to claim 7, characterized in that for setting the predetermined nucleic acid concentration to a value of 1-100 ng / [mu] L. The nucleic acid analysis method according to claim 7, characterized in that for setting the predetermined nucleic acid concentration to a value of 3~50ng / μL.

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