JP2008278783A - Device for detecting abnormal value in gene measurement by using fluorescent intensity as index and method for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve the accuracy of the measurements by detecting non-normal fluorescent intensity values in a device for determining the amount of a gene and detecting abnormality of the gene by measuring the amplification, etc., of the gene as a fluorescent intensity value. <P>SOLUTION: This gene measurement device capable of measuring the amount of the gene quantitatively by using the fluorescent intensity as an index is characterized by having: a gene-amplifying part for amplifying 1 or ≥2 genes desired to be measured; a measuring part for measuring the amount of the gene of the amplified gene over time as the fluorescent intensity; a storing part for storing the measured data of the amount of the gene over time; further an extracting part extracting the amplified pattern information capable of corresponding to the condition of the measurement by storing the information of an amplified patterns under specific conditions in the storing part; a data-watching part performing a comparative judgement of the fluorescent intensity values obtained one by one in the process of amplifying the genes for measurements, with the amplified pattern information and watching whether the value is an abnormal fluorescent intensity value deviated from the amplified pattern or not; and a signaling part for sending an alarm signal when there is the abnormal fluorescent intensity value. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an apparatus for detecting an abnormal value in measurement of a quantitative gene amount or the like using a change in fluorescence intensity as an index, a method for detecting the abnormal value, and a program for causing a computer to execute the method.

  In recent years, quantitative or semi-quantitative measurement of gene amount using a change in fluorescence intensity as an index, detection of gene polymorphism, and the like are widely performed. In some cases, the current value is used as an index using an intercalator instead of the fluorescence intensity change. In these measurements, a PCR method for amplifying a gene is used, and in the detection of gene polymorphism, an invader method is sometimes used. In either case, the fluorescence intensity is used as an index for measurement.

  Real-time PCR is a typical example of quantitative gene amount measurement. Real-time PCR is a technology that monitors and analyzes the increase in PCR amplification products in real time. Compared with the conventional PCR method for confirming PCR amplification products at the endpoint, DNA and RNA can be accurately quantified, and analysis can be performed in simple steps without requiring electrophoresis. In this method, since quantification is performed in an exponential amplification region where genes are amplified, accurate quantification based on PCR amplification kinetics is possible.

  Using a standard in which the gene to be quantified is diluted step by step, a real-time PCR reaction is performed, and an amplification curve arranged at equal intervals in the descending order of gene amount is obtained. A point (Ct value) where the curves intersect is calculated. There is a linear relationship between the Ct value and the initial template amount, and a calibration curve can be obtained to determine the initial template amount.

  In real-time PCR, PCR amplification products are detected by fluorescence. The detection method includes a method using an intercalator method and a method using a fluorescently labeled probe. Intercalators such as SYBR Green1 bind to double-stranded DNA synthesized by the PCR reaction and emit fluorescence when irradiated with excitation light. By detecting the fluorescence intensity, the amount of amplification product produced is monitored.

  Next, as a method for detecting gene polymorphism, there is a method using a fluorescent probe. Since fluorescent probes have high detection specificity, they can be detected by distinguishing even sequences with high gene homology. As a method using a fluorescently labeled probe, there is a method using a TaqMan probe. An oligonucleotide having a 5 ′ end modified with a fluorescent substance and a 3 ′ end modified with a quencher substance is used as a probe. Since this probe has a quencher substance nearby together with a fluorescent substance, the generation of fluorescence is suppressed. When the Taq Man probe hybridized to the gene strand to be measured is degraded by the 5 ′ → 3 ′ exonuclease activity of Taq DNA polymerase during the PCR extension step, the fluorescent dye is released from the probe, The suppression by the quencher is released and fluorescence is generated.

  As an application example, two types of allele-specific oligos are simultaneously mixed in a PCR reagent, and a Taq Man PCR reaction is performed with a gene whose gene polymorphism is to be detected. Thereafter, the intensity of two types of fluorescence (eg, FAM and VIC) is measured with a fluorescence detector. As a result, if the template is a homozygote of allele 1 or allele 2, strong fluorescence intensity of either FAM or VIC is recognized, and the fluorescence intensity of the paired fluorescence is not recognized, and the template is allele 1 And allele 2 are detected as both FAM and VIC fluorescence (FIG. 1).

In addition, there is an invader method as a method for detecting a gene polymorphism without using a PCR reaction. In the invader method, the target DNA for which the presence or absence of a single gene polymorphism (SNP) is to be determined, the invader oligo and the signal probe are hybridized. This invader oligo and signal probe are designed to be complementary sequences to the target DNA fragment containing the SNP site, and the invader oligo and signal probe have a structure in which one base overlaps. It is characterized. Next, the Cleavase enzyme recognizes that the invader oligo and the signal probe are completely matched, and then cleaves the Flap. The cleaved Flap is detected by the fluorescent probe (FRET).
Probe) and the Cleavase enzyme acts again here. In this FRET Probe, when a fluorescent dye (F) and a quencher (Q) are arranged adjacent to each other, emission of a fluorescent signal is suppressed. Here, when the Cleavase enzyme acts, the fluorescent dye (F) and the quencher (Q) are separated to emit a fluorescent signal. In this way, a method for detecting a gene polymorphism can be detected without using a PCR reaction (Patent Document 1).

JP-T-2002-515737 Publication Invasive cleavage of nucleic acid

  However, when quantifying the amount of a gene with conventional gene amplification, etc., or detecting a gene mutation, etc., it is only necessary to determine the amount of a gene, detect a gene mutation, etc. based on the value of the amount of amplification. It has not been determined whether the value of the amplification amount is a normal value or an abnormal value. Even if the apparent amplification amount shows a certain value, if it is not a normal value, it may cause an error in the determination of gene amount, detection of gene mutation, etc., or obtain incorrect data It will be.

  When the value of gene amplification etc. is not normal, foreign matter etc. is mixed in the solution to amplify the gene, the gene is not normally amplified, and the primer to amplify the gene is foreign matter etc. In other words, unintended genes are amplified and apparent amplification occurs. In addition, foreign substances and the like impede normal gene amplification, resulting in partial gene amplification, primer sequence design is not appropriate, and gene amplification process is impaired.

  Therefore, the present invention aims to improve the accuracy of these measurements by detecting abnormal values with respect to quantification of gene amounts, detection of gene mutations, and the like.

  As a result of intensive efforts to solve the above problems, the present inventor has found that, in the gene amplification process and the invader method, the fluorescence intensity curve in the process in which the gene is cleaved and the fluorescence is enhanced, or a certain period of time elapses. However, paying attention to an experimental example without gene amplification, the present invention revealed that abnormal gene amplification or the like can be detected by evaluating the entire curve or a specific range.

  The present invention is a gene measuring apparatus capable of quantitatively measuring the amount of gene using fluorescence intensity as an index, a gene amplification unit for amplifying one or more genes to be measured, and the amount of gene of the amplified gene is fluorescent A measurement unit that measures the intensity over time as a strength, and a storage unit that stores measurement data of the gene amount over time, the amplification pattern information of genes under specific conditions is stored in the storage unit in advance, An extraction unit for extracting amplification pattern information that can correspond to the measurement conditions, the fluorescence intensity value sequentially obtained in the process of amplifying the gene for measurement is compared with the amplification pattern information, and the amplification pattern A data monitoring unit for monitoring whether the abnormal fluorescence intensity value deviates from the signal, and a signal transmission unit for transmitting a warning signal when there is an abnormal fluorescence intensity value. There is provided a gene measuring device. FIG. 2 is a flowchart for explaining the processing steps of the present apparatus.

Here, the gene refers to a nucleic acid such as DNA or RNA. A gene measuring apparatus capable of quantitatively measuring gene amount refers to an apparatus capable of performing real-time PCR, and includes a real-time PCR dedicated apparatus in which a thermal cycler and a spectrofluorometer are integrated. While amplifying the DNA with a thermal cycler, the amplified product is monitored with a spectrophotometer. Various types of dedicated real-time PCR devices are available, one that can react in units of 96-well plates (such as the Thermal Cycler Dice Real Time System), and one that can perform PCR reactions at high speed (Smart
Cycler System etc.). In addition, when a warning signal is generated, the data value corresponding to the abnormal amplification pattern may be highlighted with a specific color when the data is displayed on a monitor or the like.

  “The gene amplification pattern information in a specific condition is stored in advance in the storage unit” means that the gene amplification pattern may differ depending on primer sequence conditions, PCR reaction conditions, etc., but one or more gene amplification patterns Is stored in a storage unit of a computer, and can be compared with a gene amplification pattern under conditions similar to those under which a gene is actually amplified by a PCR reaction. Further, as a difference from the invention of claim 1, what is detected when the fluorescence intensity value sequentially obtained in the process of amplifying the gene for measurement is different from the gene amplification pattern to be compared It is characteristic that abnormal measurement data can be captured in the amplification process at the same time as the gene amplification is completed.

  The present invention also relates to a gene measuring apparatus capable of quantitatively measuring a gene amount using fluorescence intensity as an index, a gene amplification unit for amplifying one or more genes to be measured, and a gene amount of the amplified gene Has a measurement unit that measures the fluorescence intensity over time and a storage unit that stores the measurement data of the gene amount over time, and the fluorescence intensity value at any measurement time is the measurement time before the measurement time And a difference between the two points is larger than a value corresponding to 0.1 obtained by subtracting the measurement baseline value from the fluorescence intensity value at the time of the measurement. Provided is a gene measuring apparatus comprising a data monitoring unit that determines that an abnormal fluorescence intensity value is a difference and a signal transmission unit that transmits a warning signal when there is an abnormal fluorescence intensity value. is there.

  This is characterized by means for detecting an abnormal fluorescence intensity value based only on gene amplification data for measurement, regardless of the gene amplification pattern. In addition, it is determined that it is an abnormal fluorescence intensity value when the difference is larger than a value corresponding to 0.1 of the value obtained by subtracting the measurement baseline value from the fluorescence intensity value at the time of measurement. That is why. Usually, due to measurement fluctuations, the fluorescence intensity value at any measurement time may be lower than the fluorescence intensity value at the measurement time before the measurement time. In order to distinguish that measurement is impossible, it is characterized by having a certain margin from experience values. FIG. 3 is a flowchart for explaining the processing steps of the present apparatus.

  Further, in the present invention, even when there is no amplification pattern to be compared, even if the number of cycles reaches from 0 to the arbitrarily determined number of PCR reaction cycles, the fluorescence intensity value does not improve, or the PCR reaction Even when the latter stage is reached, the fact that the improved fluorescence intensity value does not reach the plateau may be used as a reference for determining that the data includes the abnormal fluorescence intensity value.

  Further, the present invention is characterized in that the gene amplification section is a temperature variable constant temperature section capable of performing PCR by controlling the temperature of the solution containing the gene according to a temperature variable condition specified in advance. Good.

  The present invention also relates to a gene measuring apparatus capable of quantitatively measuring a gene amount using fluorescence intensity as an index, a gene amplification unit for amplifying one or more genes to be measured, and a gene amount of the amplified gene Has a measurement unit that measures the fluorescence intensity over time and a storage unit that stores measurement data of the gene amount over time, and the number of processes for amplifying the gene is selected from 15 to 25 times Even if the number of times is above or after the reaction time for amplifying the gene has passed a time selected from 45 minutes to 75 minutes, the fluorescence intensity value at the time of the measurement is compared with the measurement baseline value. And a data monitoring unit for determining that measurement is impossible when the fluorescence intensity value is not high, and a signal transmission unit for transmitting a warning signal when there is an abnormal fluorescence intensity value. It is intended to provide.

  Here, the number of processes for amplifying a gene means that the number of processes is 1 as a cycle process in which three kinds of temperature conditions are changed in order as in PCR to amplify a gene. In the case of PCR, the number of cycles, that is, the number of processes is usually about 20-45 times. Even if the number of treatments to amplify the gene is more than the number selected from 15 to 25, if it does not rise from the measurement baseline to draw a sigmoid curve, there is a problem with the amplification conditions and the sample. It is judged that there is. From the inventor's experience, the number of times selected from the 15th to the 25th was found that there is a problem in the gene amplification process if the number of times is the same number or more but does not rise from the measurement baseline. This is an automated version. FIG. 4 is a flowchart illustrating the processing steps of the present apparatus.

  On the other hand, the processing time for gene amplification is based on the detection of the fluorescence that occurs when a certain gene is cleaved, as in the invader method used for SNP typing. This is because more gene fragments are generated and the fluorescence intensity increases depending on the reaction time. Here, the reaction time to amplify the gene is selected from 45 minutes to 75 minutes, as in the previous case, from the inventor's experience, even if the number is the same number or more, it does not rise from the measurement baseline Found that there was a problem in the gene amplification process and automated it.

  Further, the present invention is characterized in that the gene amplification section is a temperature variable constant temperature section capable of performing a PCR reaction by controlling the temperature of the solution containing the gene according to a temperature variable condition specified in advance. It may be a gene measuring device.

  Further, the present invention is a method for evaluating by computer the data obtained by quantitatively measuring the gene amount using the fluorescence intensity or the current value as an index, the gene amplification step for amplifying one or more genes to be measured, A step of measuring the gene amount of the amplified gene as fluorescence intensity over time, a step of storing the measurement data of the gene amount over time by the storage unit, and the fluorescence intensity value at an arbitrary measurement time before the measurement time A value that is lower than the fluorescence intensity value at the time of measurement, and the difference between the two points is a value corresponding to 0.1 of the value obtained by subtracting the measurement baseline value from the fluorescence intensity value at the measurement time And a step of determining that the value is an abnormal fluorescence intensity value when the difference is larger than the above. Here, in the “step of extracting stored amplification pattern information from the extraction unit”, the computer selects from the amplification patterns stored in advance based on the input signals for the operator to select and instruct several amplification patterns. Extraction may be performed, and specific amplification patterns may be extracted based on instruction information to the apparatus regarding solution conditions for gene amplification, PCR reaction conditions, primer sequence conditions, and the like. In this case, information in which the condition information and the amplification pattern are associated with each other may be stored in advance.

  Here, the “current value” was cited as the reason for measuring the amplification of a gene fragment by PCR, not only the fluorescence intensity but also the reaction with a conductive substance that specifically binds to double strands. This is because it is possible to measure by a change in electric current or the like due to the local accumulation in the liquid. In the present invention, when a gene is amplified while generating a double-stranded gene, it may be measured by a change in current or the like together with measurement by fluorescence intensity. In the present invention, gene amplification can be performed by FRAT (fluorescence energy transfer) as in the invader method, in addition to those in which a copy is generated exponentially from a template gene as in PCR. Also obtained are those in which the gene for fluorescence detection is cleaved and gene fragments increase.

  In the present invention, the fluorescence intensity value determined as abnormal data may be based on a single fluorescence intensity value or may be based on a plurality of fluorescence intensity values. Even if the fluorescence intensity value of only one point is an abnormal value due to fluctuations in the measurement process, there may be no particular problem, so it may be based on a plurality of fluorescence intensity values.

  According to the present invention, it is possible to improve the accuracy of these measurements by detecting an abnormal value with respect to a detection apparatus or method for quantification of gene amount, gene mutation, or the like. As a result, even if the amplification amount shows a certain value, if it is not a normal value, it may cause an error in the determination of the gene amount, detection of the gene mutation, etc., or erroneous data may be obtained. Can be prevented.

  Hereinafter, a configuration of a gene measuring apparatus, an evaluation method, and the like that are characterized by detecting abnormal data of the present invention will be described with reference to examples. The technical scope of the present invention is not limited by these examples.

(Normal PCR reaction and S-curve)
The lower diagram of FIG. 8 shows a gene amplification pattern by PCR. The horizontal axis represents time (Time) and the number of PCR rotations, and the vertical axis represents the fluorescence intensity value (Signal). When the amount of change in the fluorescence intensity value is plotted on a normal graph as a function of time (number of cycles in the case of the PCR method), it is shown that an S-shaped (sigmoid) curve is obtained (Signal 2). In the PCR reaction, a primer and a target gene are annealed, a part of the target gene is copied, and again, a part of the copied target gene is repeatedly annealed with the primer, so that gene amplification is exponentially increased. However, this curve shows this, and at the beginning of the PCR reaction, the number of target genes that can be aligned is small and the probability is low, so the genes are not amplified or only a little if amplified. . Then, when the number of copied target genes reaches a certain number, gene amplification increases exponentially. In the latter stage of the PCR reaction, gene amplification is suppressed and reaches a plateau due to consumption of materials and nucleic acids for gene amplification.

  Quantitative PCR is performed by using a conventionally known method using a nucleic acid probe labeled with dATP, dGTP, dCT and dTTP or dUTP, a target gene (DNA or RNA), Taq polymerase, a primer, and a fluorescent dye. In the presence of ions, the target gene is amplified while repeating low and high temperatures, and the increase in emission of the fluorescent dye in the amplification process is monitored in real time.

  In the upper diagram of FIG. 5, the PCR reaction is performed under the same conditions as in the lower diagram, but only the target gene is changed. Thus, it can be seen that the primer used in this example cannot be annealed with the target gene, and the gene is not amplified (Signal 1). That is, the presence or absence of a target gene complementary to this primer sequence can be known, and this can be used for detection of genetic polymorphism such that only a part of the gene sequence is different. The left figure shows the fluorescence intensity value detected in the PCR reaction without the time axis for the S-shaped (sigmoid) curve when plotted on a normal graph. In Signal 1, since no gene amplification is observed, the fluorescence intensity shows almost 0, whereas in Signal 2, gene amplification is shown. Thus, the fluorescence intensity value shows various values of the number of each cycle.

  In the data analysis for detecting the gene polymorphism, instead of using the fluorescence intensity values at all the number of cycles in the PCR reaction, as shown in FIG. 6, the gene is amplified exponentially in the later stage of the PCR reaction. It covers the number of cycles that cover the part that is in progress and the part where gene amplification has reached a plateau. Here, the latter 15 cycles of the PCR reaction indicated by shading are targeted.

(Abnormal PCR reaction and S-curve)
7 and 8, an abnormal PCR reaction (Signal 1) is shown together with a normal PCR reaction (Signal 2). In the upper diagram of FIG. 7 (Signal 1), there is essentially no amplification of the gene and no fluorescence intensity is shown, but the fluorescence intensity value is improved in the later stage of the PCR reaction. Or, originally, the gene should be amplified and considered to exhibit its fluorescence intensity. When normal PCR reaction is not performed, there is a foreign substance in the PCR reaction solution, which has reacted with a fluorescent probe, etc., or there is a mistake in the design of the primer sequence that anneals with the target gene. It is considered that the gene amplification reaction is observed in the second half of the PCR reaction without long. If data of only the end point of PCR is evaluated, it is assumed that there is a signal 1 reaction.

  In the upper diagram of FIG. 8 (Signal 1), there is originally no amplification of the gene and no fluorescence intensity is shown, but a fluorescence intensity value unrelated to the PCR reaction is observed in the later stage of the PCR reaction. . This may be because there is a foreign substance in the PCR reaction solution, and this has reacted with the fluorescent probe or the like. 7 and 8, focusing only on the end point of the PCR reaction, it is unclear whether or not these fluorescence intensity values are due to the result of gene amplification, and accuracy in quantitative measurement of genes and detection of gene polymorphisms is unknown. It will be lower.

  FIG. 9 shows the result of data analysis when an abnormal fluorescence intensity value is included. When genotypes are measured with different alleles, signal 1 resulting from reacting only to allele 1, signal 2 resulting from reacting only to allele 1, and signal resulting from reacting to both alleles are: It can be seen that the signal is detected between signal1 and signal2. Abnormal fluorescence intensity values may be in a remote region from these signal groups, reducing measurement accuracy.

  The gene measuring apparatus and the measurement evaluation method of the present invention focus on the amplification curve and amplification pattern when amplifying the gene amount by PCR reaction in order to measure the gene amount of the target gene. Therefore, abnormal data (fluorescence intensity value) can be detected. According to the present invention, it is possible to improve the accuracy of these measurements by detecting an abnormal value with respect to a detection apparatus or method for quantification of gene amount, gene mutation, or the like. As a result, even if the amplification amount shows a certain value, if it is not a normal value, it may cause an error in the determination of the gene amount, detection of the gene mutation, etc., or erroneous data may be obtained. Can be prevented. The present invention can also be used in measurement that does not involve gene amplification and involves changes in fluorescence intensity values in the invader method.

Graph showing gene polymorphism results using fluorescence intensity as an index The flowchart which shows the process process of the detection method of abnormal data based on the amplification pattern memorize | stored beforehand. The flowchart which shows the processing process of the detection method of abnormal data which makes a parameter | index that fluorescence intensity falls The flowchart which shows the process process of the detection method of the abnormal data based on the fluorescence intensity value after passing through a fixed process process Graph showing gene amplification pattern showing normal PCR reaction Explanatory diagram showing collecting data between the number of cycles of a specific PCR reaction Graph 1 showing gene amplification pattern showing abnormal PCR reaction Graph 2 showing gene amplification pattern showing abnormal PCR reaction Graph showing the results of genetic polymorphism with abnormal fluorescence intensity values

Claims (5)

A gene measuring device capable of quantitatively measuring the amount of a gene using fluorescence intensity as an index,
A gene amplification section for amplifying one or more genes to be measured,
A measurement unit for measuring the gene amount of the amplified gene as fluorescence intensity over time,
A storage unit for storing measurement data of the gene amount over time,
Amplification pattern information of genes under specific conditions is stored in the storage unit in advance,
An extraction unit that extracts amplification pattern information that can be compatible with the measurement conditions,
Data for monitoring whether or not the fluorescence intensity values sequentially obtained in the process of amplifying a gene for measurement are abnormal fluorescence intensity values deviating from the amplification pattern by performing comparison judgment with the amplification pattern information Monitoring department,
A signal transmitter that transmits a warning signal when there is an abnormal fluorescence intensity value,
A gene measuring apparatus characterized by comprising: A gene measuring device capable of quantitatively measuring the amount of a gene using fluorescence intensity as an index,
A gene amplification section for amplifying one or more genes to be measured,
A measurement unit for measuring the gene amount of the amplified gene as fluorescence intensity over time,
A storage unit for storing measurement data of the gene amount over time,
The fluorescence intensity value at any measurement time point is lower than the fluorescence intensity value at the measurement time point before the measurement time point,
Data that is determined to be an abnormal fluorescence intensity value when the difference between the two points is greater than a value corresponding to 0.1, which is a value obtained by subtracting the measurement baseline value from the fluorescence intensity value at the time of measurement. Monitoring department,
A signal transmitter that transmits a warning signal when there is an abnormal fluorescence intensity value,
A gene measuring apparatus characterized by comprising: A gene measuring device capable of quantitatively measuring the amount of a gene using fluorescence intensity as an index,
A gene amplification section for amplifying one or more genes to be measured,
A measurement unit for measuring the gene amount of the amplified gene as fluorescence intensity over time,
A storage unit for storing measurement data of the gene amount over time,
Even if the number of treatments for amplifying the gene is more than the number selected from 15 to 25 times, or after the reaction time for amplifying the gene has passed the time selected from 45 to 75 minutes. Even
If the fluorescence intensity value at the time of the measurement is not higher than the measurement baseline value,
A data monitoring unit that determines that measurement is impossible,
A signal transmitter that transmits a warning signal when there is an abnormal fluorescence intensity value,
A gene measuring apparatus characterized by comprising: The gene amplification section is in accordance with a temperature variable condition specified in advance.
By controlling the temperature of the solution containing the gene,
It is a temperature-variable constant temperature part capable of performing a PCR reaction,
The gene measuring device according to claim 1. It is a method of evaluating by computer the data obtained by quantitatively measuring the gene amount using the fluorescence intensity or current value as an index,
A gene amplification step of amplifying one or more genes to be measured;
Measuring the gene amount of the amplified gene over time as fluorescence intensity;
A storage unit storing the measurement data of the gene amount over time,
The fluorescence intensity value at any measurement time point is lower than the fluorescence intensity value at the measurement time point before the measurement time point,
A step of determining that the difference between the two points is an abnormal fluorescence intensity value when the difference between the two points is greater than a value corresponding to 0.1 obtained by subtracting the measurement baseline value from the fluorescence intensity value at the time of the measurement. And a method for evaluating gene amplification.

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