JP2008022732A - Method for inspecting nucleic acid sample, method for judging the inspected result, and method for managing the inspection of the nucleic acid sample based on the judged result - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for inspecting a nucleic acid sample, suitable for a sample containing a trace of the nucleic acid, and capable of being carried out readily at high accuracy at a low cost in high throughput; to provide a method for judging the inspected result; and to provide a method for managing the inspection of the nucleic acid sample based on the judged result. <P>SOLUTION: The method for inspecting the nucleic acid sample includes subjecting a sample having possibility of containing a nucleic acid of an analysis object, and a sample containing a standard nucleic acid to a nucleic acid-extracting step without mixing them under the nearly same condition at the nearly same time, subjecting the extracts to an amplification step without mixing them under the nearly same condition at the nearly same time, subjecting the amplification products to a detection step without mixing them under the nearly same condition at the nearly same time, and ascertaining the detected values of the amplified products of the nucleic acids of the analysis object and the standard nucleic acid. The method for judging the inspected result of the nucleic acid sample includes judging the presence or absence of error in the steps from the inspected result, and when the error is not recognized, judging the presence or absence of the nucleic acid of the analysis object in the sample having the possibility of containing the nucleic acid of the analysis object. The method for managing the inspection of the nucleic acid sample includes carrying out again the inspection of the nucleic acid sample wherein the error in the steps is recognized from the judged result, and carrying out the judgement of the inspected result again. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a nucleic acid sample test method, a test result determination method, and a nucleic acid sample test management method based on the determination result, which are suitable for a sample containing a small amount of nucleic acid.

  In recent years, global human genome analysis has been completed, and as post-genomic research, attention has been paid to the association of gene mutations such as single nucleotide polymorphisms and microsatellite polymorphisms with diseases or drug reactions. In particular, research on malignant neoplasms (cancer) with the highest mortality rate has been conducted widely, and it has recently been clarified that DNA of cancer cells is slightly leaked into the blood (non-) Patent Document 1). A large amount of DNA derived from cancer cells in blood is present in plasma and serum excluding blood cell components, and a plasma sample may be analyzed as a cancer research target (see Non-Patent Document 1). The amount of DNA derived from cancer cells in plasma is 2.8 ng of total DNA in 1 μl of plasma of ovarian cancer (see Non-Patent Document 2), and 211 pg of total DNA in 1 μl of plasma of breast cancer (see Non-Patent Document 3). ), It is reported that the amount of total DNA in 1 μl of plasma in small cell lung cancer is very small at 39 pg (see Non-Patent Document 4).

  Such detection of a trace amount of DNA is generally performed after the DNA is amplified. As a gene amplification method, a polymerase chain reaction method (hereinafter sometimes abbreviated as a PCR method) is widely used (see Non-Patent Document 5). Since a gene with a very small amount can be isolated and amplified by a gene amplification method such as the PCR method, detection of such a gene can be facilitated.

A typical gene analysis step includes a step of extracting a nucleic acid to be analyzed from a sample, a step of amplifying the extract, and a step of detecting an amplification product. Nucleic acid extraction methods include the protease K / phenol method (see Non-Patent Document 6) and nucleic acids equipped with spin columns, microplates, etc. that apply the property that nucleic acids bind to silicon oxide (silica) under specific conditions. Methods using extraction kits and nucleic acid extraction automation apparatuses are widely known (see Patent Documents 1 to 5).
As an amplification method, a PCR method (see Patent Documents 6 and 7), a nucleic acid sequence-based amplification method (hereinafter abbreviated as NASBA method) (see Patent Document 8), and the like are known, and frequently used in the bio field and the medical field. It's being used. Furthermore, an amplification method using competitive amplification or multiplex amplification is also well known.
Known detection methods include nucleic acid fragment length analysis by gel electrophoresis, DNA microarray analysis, sequencing method, high performance liquid chromatography analysis, mass spectrometry and the like. In particular, nucleic acid fragment length analysis by capillary electrophoresis is often used for microsatellite analysis, which is a kind of gene polymorphism.

What is important in carrying out such complicated multiple steps is to manage the series of nucleic acid extraction, amplification and detection steps so that they are normally performed. Can be confirmed using appropriate standards.
As a conventional technique for confirming the presence or absence of such process abnormalities, each step of nucleic acid extraction, amplification and detection is carried out with different types of internal standard nucleic acids coexisting with the nucleic acid to be analyzed for each step, thereby detecting the internal standard nucleic acids. The method of confirming the success or failure of the method is mentioned (see Patent Document 9).
Japanese National Patent Publication No. 10-504834 JP-T 9-505724 JP-A-8-320274 JP-A-9-304385 Japanese Patent No. 2807090 US Pat. No. 4,683,195 US Pat. No. 4,683,202 European Patent 0,329,822 International Publication No. 2002/024949 Pamphlet Clin Chem, 51: 1, 113-118, 2005. Clin Cancer Res. 2004 Jul 1; 10 (13): 4420-6. Ann Surg Oncol. 2002 Jan-Feb; 9 (1): 71-6. Nat Med. 1996 Sep; 2 (9): 1033-5. Science: Vol. 239, pp. 487-491 (1988) Molecular Cloning: A Laboratory Manual Appendix E3-E4. New York Cold Spring Harbor Laboratory, 1989

However, the method described in Patent Document 9 has a problem that the operation is complicated. This will be described in detail with reference to FIGS.
FIG. 5 is a flowchart illustrating a procedure of a conventional method including the method described in Patent Document 9. FIG. 6 is an explanatory diagram for explaining the procedure.
In the conventional method, since it is necessary to use an internal standard nucleic acid of a different type from the nucleic acid to be analyzed for each step of nucleic acid extraction, amplification and detection, at least three types of internal standard nucleic acids must be prepared. Moreover, since it is necessary to add the internal standard nucleic acid to all samples of the nucleic acid to be analyzed, a considerable amount must be prepared. Furthermore, after each internal standard nucleic acid is separated and detected, the detection result must be determined. Accordingly, it takes time and labor to prepare the internal standard nucleic acid, and it takes time to add the internal standard nucleic acid to all the nucleic acids to be analyzed even at the implementation stage, and it also takes time to determine the detection result. That is, there is a problem that it is difficult to perform analysis easily at a low throughput, at a low cost. Patent Document 9 does not specifically describe what kind of internal standard nucleic acid can be used.

  Furthermore, the conventional methods including the method described in Patent Document 9 are based on the premise that the concentration of the nucleic acid sample to be used is relatively high. That is, it is not considered to use a nucleic acid sample having a very small concentration such as several pg / l to several μg / l, such as plasma containing the aforementioned cancer cell-derived DNA. For example, when an amplification step is performed in the presence of an internal standard nucleic acid using such a small amount of nucleic acid as an analysis target nucleic acid, if the internal standard nucleic acid is present in a relatively larger amount than the analysis target nucleic acid, the internal standard In some cases, the nucleic acid is first amplified and it becomes difficult to amplify the nucleic acid to be analyzed. Therefore, although the process abnormality that the nucleic acid to be analyzed is not detected is detected but the internal standard nucleic acid is normally detected, the extraction, amplification, and detection processes are normally performed. There was a problem that it was mistakenly determined.

  The present invention has been made in view of the above circumstances, and is suitable for a sample containing a small amount of nucleic acid, and can be performed with high accuracy, simply and at low cost and with high throughput, and the method for testing a nucleic acid sample It is an object of the present invention to provide a test result determination method and a nucleic acid sample test management method based on the determination result.

To solve the above problem,
The invention described in claim 1 provides a sample that may contain the nucleic acid to be analyzed and a sample that contains the standard nucleic acid to the nucleic acid extraction step at substantially the same time under substantially the same conditions without mixing. The obtained extract is subjected to an amplification step at about the same time under substantially the same conditions without mixing, and the obtained amplification product is subjected to a detection step at about the same time under substantially the same conditions without mixing. A method for examining a nucleic acid sample, characterized by confirming the presence or absence of an amplification product of a nucleic acid to be analyzed and a standard nucleic acid.
The invention according to claim 2 is characterized in that the size of the standard nucleic acid is substantially the same as the size of the nucleic acid to be analyzed, and the concentration of the standard nucleic acid in the sample is substantially the same as the concentration of the nucleic acid to be analyzed in the sample. The method for examining a nucleic acid sample according to claim 1.
In the invention according to claim 3, the size of the standard nucleic acid is 0.5 to 2.0 times the size of the nucleic acid to be analyzed, and the concentration of the standard nucleic acid in the sample is 0. The method for examining a nucleic acid sample according to claim 2, wherein the method is 5 to 2.0 times.
The invention according to claim 4 is characterized in that the size of the standard nucleic acid is 100 to 300 base pairs, and the concentration of the standard nucleic acid in the sample is 1 pg / μl to 1 μg / μl. 4. The method for examining a nucleic acid sample according to any one of 3 above.
In the invention according to claim 5, there are a plurality of samples that may contain the nucleic acid to be analyzed to be subjected to the nucleic acid extraction step, and at least one standard nucleic acid to be subjected to the nucleic acid extraction step is provided for the nucleic acid extraction step. The method for examining a nucleic acid sample according to any one of claims 1 to 4, wherein at least one sample contains a standard nucleic acid.
In the invention according to claim 6, after the nucleic acid sample is inspected by the method according to any one of claims 1 to 5, from the detection result of the standard nucleic acid, the extraction step, the amplification step, and the detection step are performed. In addition to determining the presence or absence of abnormality in any of them, if it is determined that there is no abnormality, the presence or absence of the target nucleic acid in the sample that may contain the target nucleic acid is determined from the detection result of the target nucleic acid. It is a method for determining a test result of a nucleic acid sample, characterized by determining.
In the invention according to claim 7, after determining the test result of the nucleic acid sample by the method according to claim 6, an abnormality in any of the extraction process, the amplification process and the detection process is recognized from the determination result. A nucleic acid sample inspection management method, wherein the inspection method performs again the inspection of the nucleic acid sample in which an abnormality is recognized, and then determines the inspection result again by the determination method. is there.

  According to the present invention, even when the content in a sample is very small, the analysis of the nucleic acid to be analyzed can be performed with high accuracy, simply and at low cost and with high throughput.

Hereinafter, the present invention will be described in detail.
(Nucleic acid sample inspection method)
In the present invention, the nucleic acid to be analyzed is not particularly limited as long as it can be amplified when subjected to the amplification step. For example, all DNA including cDNA, genomic DNA, synthetic DNA, and the like, or analogs thereof, may be derived from a living body or artificially synthesized. Of these, DNA derived from cancer cells is preferable as derived from a living body.
The sample that may contain the nucleic acid to be analyzed is not particularly limited, but preferred examples include biological tissues, blood, plasma, serum, body fluids, urine, cells, bacteria, viruses, and the like. A sample is mentioned. Moreover, what was collected by the conventionally well-known method should just be used for the sample which may contain the nucleic acid to be analyzed.

  Moreover, as a standard nucleic acid, the thing similar to the above-mentioned analysis object nucleic acid can be used, and it does not specifically limit. However, it has been confirmed that extraction, amplification and detection from a sample can be normally performed by a method intended to be applied to the inspection of a nucleic acid to be analyzed, and more preferably a known size. If it is confirmed that extraction, amplification and detection can be normally performed, the nucleic acid may be the same as the nucleic acid to be analyzed. Here, the size of the nucleic acid refers to the number of base pairs of the nucleic acid, and this also applies to the following. The sample containing the standard nucleic acid may be collected in the same manner as the sample that may contain the nucleic acid to be analyzed, or a separately isolated standard nucleic acid is prepared in a solution with an appropriate concentration. You may get by doing.

  In the nucleic acid extraction step of the present invention (hereinafter sometimes abbreviated as the extraction step), at least the standard nucleic acid is extracted from the sample containing the standard nucleic acid. If the nucleic acid to be analyzed is actually contained in a sample that may contain the nucleic acid to be analyzed, at least the nucleic acid to be analyzed is extracted. It is preferable that there are a plurality of samples per one level that may contain the nucleic acid to be analyzed to be subjected to the extraction process. For these samples, if the subsequent amplification and detection processes are performed, the test results to be described later can be determined. It becomes easy. And it is preferable to increase the number of these several samples in the range which does not impair the effect of this invention.

  In the test method of the present invention, a sample that may contain the nucleic acid to be analyzed and a sample that contains the standard nucleic acid are separately subjected to the nucleic acid extraction step without being mixed with each other. Subsequently, the obtained extract is subjected to an amplification step without being mixed separately, and the obtained amplification product is also subjected to a detection step without being mixed separately. That is, in each of the extraction, amplification and detection steps, the standard nucleic acid is not always mixed with the nucleic acid to be analyzed. Since the standard nucleic acid used as the standard substance in the extraction step is continuously used as the standard substance in the amplification step and the detection step, it is not necessary to use different types of standard nucleic acids each time in the extraction to detection steps.

  In addition, the sample that may contain the nucleic acid to be analyzed and the sample that contains the standard nucleic acid are subjected to the extraction step at substantially the same time under substantially the same conditions. Each extract is subjected to an amplification step at substantially the same time under substantially the same conditions, and each amplification product is subjected to a detection step at approximately the same time under substantially the same conditions. Therefore, since the nucleic acid to be analyzed and the standard nucleic acid go through almost the same process, it is confirmed whether or not the detection of the standard nucleic acid is normal even if the internal standard nucleic acid does not coexist with the nucleic acid to be analyzed as in the conventional method. By doing so, it can be confirmed whether the test | inspection of the nucleic acid sample was performed normally.

  In the present invention, normal detection means that a nucleic acid signal is detected with sufficient intensity for discrimination. In addition, when the size of the nucleic acid is known, it also means that the detected size matches this size.

  Moreover, substantially the same conditions indicate that the conditions are substantially the same. Specifically, when both the sample containing the nucleic acid to be analyzed and the sample containing the standard nucleic acid are normal, the nucleic acid to be analyzed and the standard nucleic acid are It shows that the error of the condition is small for each process. Here, the process conditions specifically refer to temperature, time, concentration, extraction operation, and the like.

  Also, “substantially the same time” indicates substantially the same time. Specifically, when both the sample containing the nucleic acid to be analyzed and the sample containing the standard nucleic acid are normal, the nucleic acid to be analyzed and the standard nucleic acid are It shows that the error of the timing of performing each process is small.

  In the present invention, the standard nucleic acid and the nucleic acid to be analyzed are not always mixed. Therefore, unlike the conventional method in which the internal standard nucleic acid and the nucleic acid to be analyzed coexist, the standard nucleic acid and the nucleic acid to be analyzed do not affect each other in each step of extraction to detection. Even if the nucleic acid to be analyzed is a trace amount, the nucleic acid sample can be inspected with high accuracy. For example, in the amplification step, if the concentration of either the coexisting internal standard nucleic acid or the nucleic acid to be analyzed becomes relatively high, the nucleic acid with the higher concentration is preferentially amplified, and the concentration with the lower concentration is reduced. Nucleic acids are difficult to amplify. In the present invention, there is no such inconvenience.

  In the present invention, the standard nucleic acid used in the extraction step as a standard substance is continuously used as the standard substance in the amplification step and the detection step, so that each step of extraction to detection can be greatly simplified. This will be described in detail with reference to FIGS. FIG. 1 is a flowchart illustrating the procedure of the inspection method of the present invention, and FIG. 2 is an explanatory diagram for explaining the procedure.

  Unlike the conventional methods shown in FIGS. 5 and 6, the test method of the present invention does not require a new standard nucleic acid different from that used in the extraction step in the amplification step and the detection step. That is, at least one standard nucleic acid may be used per test. Since at least one sample containing the standard nucleic acid is sufficient for each test, the amount of standard nucleic acid to be prepared can be small. This reduces the number of standard nucleic acids that need to be separated and detected. Therefore, the standard nucleic acid can be prepared easily and at low cost, and the time required for the extraction to detection process and the time required for determination of the detection result can be greatly shortened even in the implementation stage. That is, the inspection method of the present invention is high-throughput, and can be easily and inexpensively inspected. This effect becomes more prominent as the number of samples that may contain the nucleic acid to be analyzed for examination increases.

  In the present invention, it is preferable that the sample that may contain the nucleic acid to be analyzed and the sample that contains the standard nucleic acid have higher identity between these samples. Here, the sample identity means that, for example, the component composition error in the sample and the sample concentration and amount error are small between the nucleic acid to be analyzed and the standard nucleic acid.

Specifically, the size of the standard nucleic acid is preferably approximately the same as the size of the nucleic acid to be analyzed, and more preferably 0.5 to 2.0 times the size of the nucleic acid to be analyzed.
The concentration of the standard nucleic acid in the sample is preferably substantially the same as the concentration of the nucleic acid to be analyzed in the sample, and is more preferably 0.5 to 2.0 times the concentration of the nucleic acid in the sample to be analyzed. preferable.
More preferably, the size of the standard nucleic acid is substantially the same as the size of the nucleic acid to be analyzed, and the concentration of the standard nucleic acid in the sample is substantially the same as the concentration of the nucleic acid to be analyzed in the sample. Is preferably 0.5 to 2.0 times the size of the nucleic acid to be analyzed, and the concentration of the standard nucleic acid in the sample is 0.5 to 2.0 times the concentration of the nucleic acid to be analyzed in the sample. .

The present invention is also suitable for a standard nucleic acid and / or nucleic acid to be analyzed whose concentration in a sample is extremely small, for example, about 1 pg / μl to 1 μg / μl. Conventionally, the concentration of a nucleic acid to be extracted for amplification and detection in a sample is at least about several tens of ng / μl to several μg / μl. However, it is difficult to use in a conventionally known method. A sample containing a small amount of nucleic acid can also be used in the inspection method of the present invention.
The present invention is also suitable for standard nucleic acids and / or nucleic acids to be analyzed, for example, those having a size of about 100 to 300 base pairs.
Examples of the sample containing a small amount of nucleic acid to be analyzed having a small size include plasma separated from blood and containing DNA derived from cancer cells.

When the analysis target nucleic acid contained in the sample is a trace amount, the content of the standard nucleic acid used in the sample is also preferably a trace amount. Therefore, when the concentration of the nucleic acid to be analyzed in the sample is, for example, 1 pg / μl to 1 μg / μl, the concentration of the standard nucleic acid in the sample is also preferably 1 pg / μl to 1 μg / μl.
When the size of the nucleic acid to be analyzed is small, it is preferable that the standard nucleic acid to be used is also small. Therefore, when the size of the nucleic acid to be analyzed is, for example, 100 to 300 base pairs, the size of the standard nucleic acid is preferably 100 to 300 base pairs.
That is, if a sample containing a standard nucleic acid having a size of 100 to 300 base pairs at a concentration of 1 pg / μl to 1 μg / μl is used, it is the most difficult to use with a conventionally known method. A sample containing a pair of nucleic acids to be analyzed at a concentration of 1 pg / μl to 1 μg / μl can also be suitably used in the present invention.

In the present invention, a conventionally known method may be applied as the extraction step, amplification step, and detection step, and is not particularly limited.
In the extraction step, for example, blood is centrifuged and plasma is collected, and this can be used as a sample that may contain the nucleic acid to be analyzed or a sample that contains a standard nucleic acid. Subsequently, the nucleic acid to be analyzed or the standard nucleic acid can be extracted from plasma using various commercially available DNA detection kits. Note that the sample to be used is not limited to plasma, and other samples can be used, and the extraction process is not limited to that shown here.

  The number of samples that may be subjected to the extraction step and may contain the nucleic acid to be analyzed is not particularly limited, and may be one or more. If there are a plurality of containers, for example, a 96-well plate such as a 96-well plate in which a plurality of containers for performing each step of extraction to detection are integrally provided (hereinafter abbreviated as a well-plate container) may be used. A plurality of tubular containers may be used separately. It goes without saying that such a container can be used even when there is only one sample that may contain the nucleic acid to be analyzed, which is subjected to the extraction step.

  On the other hand, the number of samples containing the standard nucleic acid used for the extraction process may be at least one per test. For example, when using a well plate-like container, a sample containing a standard nucleic acid may be added to at least one well to which a sample that may contain a nucleic acid to be analyzed is not added. When a plurality of well plate-like containers are used in one test, a sample containing a standard nucleic acid may be added to at least one well for each well plate-like container. Only one may contain a sample containing at least one standard nucleic acid.

  When using a tube-like container, prepare at least one container containing only the sample containing the standard nucleic acid, in addition to the container containing only the sample that may contain the nucleic acid to be analyzed. Just do it. This will be described with reference to FIG. FIG. 3 is an explanatory view for explaining a method of using a tube-shaped container and supplying each sample-containing container to each step of extraction to detection one by one as an embodiment of the present invention. For example, as shown in FIG. 3A, there is a method in which one sample containing a standard nucleic acid is used and the sample is first subjected to each step. As a preferred method, for example, as shown in FIG. 3 (b), a plurality of samples containing standard nucleic acids are used, and the samples that may contain the nucleic acids to be analyzed are placed between a plurality of samples. The method of using for each process sequentially is mentioned so that it may pinch | interpose. By doing in this way, a nucleic acid sample can be examined with higher accuracy.

  In addition, regardless of which container is used, at least one standard nucleic acid may be used.

  In the amplification step, for example, a conventionally known amplification method such as a PCR method or a NASBA method can be applied, but the PCR method is preferable, and a multiplex PCR method or the like can also be used.

  In the detection step, for example, after separating amplification products by agarose gel electrophoresis, polyacrylamide gel electrophoresis or the like, a method of staining nucleic acid with ethidium bromide and detecting fluorescence upon UV irradiation can be applied. Alternatively, a method of detecting using an intercalator after separating the nucleic acid to be analyzed and the standard nucleic acid by capillary electrophoresis may be applied. Alternatively, methods other than the methods listed here may be applied. Among these, in particular, when the nucleic acid to be analyzed and the standard nucleic acid are very small, a method of separating and detecting by capillary electrophoresis is preferable.

  Thus, the nucleic acid sample can be inspected by confirming the presence or absence of amplification products of the standard nucleic acid and the nucleic acid to be analyzed. In detection, the presence or absence of amplification products of these nucleic acids may be confirmed based on the presence or absence of peaks of the standard nucleic acid and the nucleic acid to be analyzed, and if present, the amount of amplification product may be compared based on the intensity of the peaks. In addition, since the molecular size is determined from the migration distance in electrophoresis, for example, the number of base pairs can be confirmed. When both the standard nucleic acid and the nucleic acid to be analyzed are detected in the detection step, it can be determined that there is no abnormality in the used nucleic acid sample and each of the extraction to detection steps.

The inspection method of the present invention is suitable for analysis of a gene polymorphism, for example, if a nucleic acid having a base sequence containing a gene polymorphism is selected as the nucleic acid to be analyzed.
A gene polymorphism is a genetic polymorphism of a human chromosome and is derived from individual differences in the base sequence of a gene. It is said that such gene polymorphism exists in about one place every several hundred bases on average. By analyzing the gene polymorphism, the paternally derived chromosome (allyl) and the maternally derived chromosome (allyl) Can be distinguished. Therefore, a gene deletion can be detected by measuring loss of one allele (LOSS of Heterozygosity: LOH) in a heterozygote using a gene polymorphism. However, gene polymorphisms due to single nucleotide substitutions are at most 50% heterozygous, and in order to investigate the deletion of one gene, multiple genes with high heterozygous frequency It is desirable to combine polymorphisms. Moreover, since there are race differences in gene polymorphisms, it is necessary to select gene polymorphisms having a high frequency of occurrence in the race from the genes for which deletion is to be diagnosed.

(Method for judging nucleic acid sample test results)
In the present invention, the nucleic acid sample used and the presence or absence of abnormality in each step of extraction to detection can be determined in more detail from the test result obtained by the above-described nucleic acid sample test method.
The nucleic acid to be analyzed and the standard nucleic acid undergo substantially the same process in each step of extraction to detection. Therefore, if it is confirmed that there is no abnormality in the quality of the sample containing the standard nucleic acid, and if the standard nucleic acid is detected normally, the sample that may contain the nucleic acid to be analyzed and the standard nucleic acid are contained. For any of the samples to be performed, it is determined that each step of extraction to detection has been performed normally. Therefore, at this time, if the analysis target nucleic acid is also detected normally, it is determined that the analysis target nucleic acid was actually contained in the sample that may contain the analysis target nucleic acid.

  In addition, if the target nucleic acid is not detected normally even though the standard nucleic acid is detected normally, the sample that may contain the target nucleic acid was not included in the sample that may contain the target nucleic acid. Determined. In this case, it can be seen that the sample that may contain the nucleic acid to be analyzed has its cause before the extraction, that is, the sample collected. Specifically, for example, (i) the sample subjected to the extraction step contains no or almost no nucleic acid to be analyzed, and (ii) the nucleic acid to be analyzed in the sample is decomposed before being subjected to the extraction step. (Iii) It is conceivable that impurities that inhibit any one of the steps of extraction to detection were mixed in the sample. In the case of (i), (ia) originally, when the sample contains no or almost no nucleic acid to be analyzed, (ib) there is an abnormality in the sample preparation process, The case where the nucleic acid to be analyzed to be analyzed is not contained in the sample is considered.

  As a method for distinguishing between (ia) and (ib), a plurality of samples are prepared for one corresponding level, and these are used to inspect by the method of the present invention. In the case of (ia), no nucleic acid to be analyzed is detected from any of these multiple samples. On the other hand, in the case of (ib), there is a high possibility that the nucleic acid to be analyzed is detected from some of the plurality of samples, and the nucleic acid to be analyzed is not detected from some. Therefore, when testing by the method of the present invention, a plurality of samples that may contain the nucleic acid to be analyzed are prepared in advance for one level, and if these are used for testing, This is preferable because it eliminates the trouble of confirmation.

  On the other hand, if neither the standard nucleic acid nor the nucleic acid to be analyzed is detected normally, it is determined that there is an abnormality in any of the extraction to detection steps. In this case, for example, if an extract and an amplification product are subjected to a detection process for a sample in which an abnormality is recognized, the process in which the abnormality has occurred can be identified.

  If the sample containing the standard nucleic acid is appropriately quality-controlled and it is confirmed that there is no abnormality in the quality before the inspection, the inspection method of the present invention becomes more reliable. For example, storage of a sample containing a standard nucleic acid is preferably performed at 4 ° C. or less, although it depends on the type of nucleic acid, and the presence or absence of a quality abnormality is confirmed by observing the presence or absence of a change in detection signal by electrophoresis or the like. Just do it.

(Nucleic acid sample inspection management method)
In the present invention, when an abnormality in any one of the extraction step, the amplification step and the detection step is recognized from the determination result obtained by the determination method of the nucleic acid sample test result, the method of the present invention is used. The inspection of the nucleic acid sample can be managed by performing the inspection of the nucleic acid sample in which the abnormality is recognized again and then determining the inspection result again by the method of the present invention.

The reexamination of the nucleic acid sample in which the process abnormality is recognized may be performed at least for the process after the process in which the abnormality has occurred as long as the process in which the abnormality has occurred is specified, but each process of extraction to detection is performed again. It is preferable.
On the other hand, if it is determined that there is no nucleic acid to be analyzed in a sample that may contain the nucleic acid to be analyzed, as described above, preferably, a plurality of samples are re-applied for each applicable level. It is only necessary to prepare and inspect and determine again by the method of the present invention. Thereby, it is possible to identify the cause of the failure to detect the nucleic acid to be analyzed.

  As described above, according to the present invention, it is possible to perform inspection of a nucleic acid sample, determination of the inspection result, and management of the inspection of the nucleic acid sample with high accuracy, simple and low cost and high throughput. The present invention is particularly suitable for a sample containing a very small amount of nucleic acid, which is difficult to use in the conventional method.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples.
[Experiment 1]
In order to examine the nucleic acid content in plasma, the amount of nucleic acid contained in a healthy human plasma sample was measured. Specifically, plasma was immediately centrifuged and collected from 96 samples of healthy human samples, and the amount of DNA was measured using Quant-iT PicoGreen dsDNA Reagent and Kits (Invitrogen, P7589). As a result, it was revealed that the total DNA content in 1 ml of plasma was 1.7 ng on average. It is known that the DNA content in plasma derived from cancer patients is about 10 to 100 times higher than that in healthy individuals, but the DNA content in either plasma is equal to the DNA content in general blood. It turned out to be very few compared.

[Example 1]
(Preparation of sample containing standard nucleic acid)
5 ml of blood was collected from 51 healthy people and 45 cancer patients and added to a test tube containing an anticoagulant. Then, (1) Centrifugation was performed at 3000 rpm for 10 minutes at 4 ° C., and the supernatant (plasma) was collected in another test tube. Furthermore, the centrifugation in (1) was repeated again to purify the plasma. DNA was extracted from the purified plasma using Qiamp DNA Blood Midi Kit (51183, manufactured by QIAGEN). DNA extracted from plasma was concentrated using Speedvac (AES1010, manufactured by ThermoSavant), and the DNA concentration was measured with a plate reader using Quant-iT Picogreen dsDNA Reagent and Kits (P7589, manufactured by Molecular Probes). And the extracted DNA after a measurement was preserve | saved at -80 degreeC.
When this extracted DNA having a known concentration was electrophoresed to confirm the migration position, a band appeared in the vicinity of 200 base pairs, and it was found that the size was about 200 base pairs. Then, this extracted DNA was prepared as a standard nucleic acid at about 100 pg / μl and used as a sample containing a standard nucleic acid of known size at a constant concentration.

(Preparation of sample containing nucleic acid to be analyzed)
About 5 ml of blood is collected from 51 healthy subjects and 45 cancer patients, and plasma and buffy coat are separated in the same way as when preparing standard nucleic acids, and contains nucleic acids to be analyzed A sample was prepared.

(Nucleic acid extraction)
The obtained sample containing the nucleic acid to be analyzed and the sample containing the above-mentioned standard nucleic acid were collected in separate containers, and the DNA was extracted simultaneously under the same conditions as when preparing the standard nucleic acid. One sample containing standard nucleic acid was used for each extraction unit.

(Amplification of nucleic acid)
The microsatellite marker D13S153 was selected from a database (UniSTS. Www.ncbi.nlm.nih.gov/genome/sts/). First, conditions for PCR amplification were set. That is, as PCR reaction reagents, 0.25 pM each of primers consisting of the nucleotide sequences shown in SEQ ID NOs: 1 and 2, 4 nM each nucleotide triphosphate (dNTP), and 0. Taq DNA Polymerase (RR001A, manufactured by TAKARA). A 5U mixture was prepared. 1 μl of the sample containing the above-mentioned standard nucleic acid was added thereto to make a total volume of 20 μl in a 96-well plate, and PCR amplification was performed by changing the temperature as shown in (A) below. Then, agarose gel electrophoresis was performed using the obtained amplification product, and amplification proceeded best under the temperature condition (B) below, and a 121 base pair amplification product consisting of the base sequence shown in SEQ ID NO: 3 It was confirmed that
Therefore, 1 μl of an extract from a sample containing a standard nucleic acid, 1 μl of an extract from a sample containing a nucleic acid to be analyzed, and 1 μl of water as a negative control were separately collected in each well of a 96-well plate, The total amount was 20 μl by mixing with the PCR reaction reagent. And PCR amplification operation was performed simultaneously on the temperature conditions of the following (B). After the operation, each amplification product obtained was stored at 4 ° C.
(A) 95 ° C./1 minute → (95 ° C./30 seconds → 50 ° C. to 70 ° C./30 seconds → 72 ° C./30 seconds) × 30 cycles (B) 95 ° C./1 minute → (95 ° C./30 seconds → 60 ° C / 30 seconds → 72 ° C / 30 seconds) x 30 cycles

(Detection of genetic polymorphism)
Take 1 μl of each amplification product in a 96-well plate, dilute by adding 9.5 μl of Hi-Di Formatide (ABI, 431320) and 0.5 μl of GeneScan-500ROX Standard (ABI, 401734E). Nucleic acid was simultaneously detected using an electrophoresis apparatus (ABI PRISM3100 Genetic Analyzer, Applied Biosystems).

(Determination of genetic polymorphism analysis results)
A part of the detection result is shown in FIG. When the detection result of the standard nucleic acid was analyzed, the peak of the standard nucleic acid was observed as shown in FIG. 4A, and a signal intensity sufficient for peak discrimination was obtained. On the other hand, as shown in FIG. 4C, no peak was observed in the case of the negative control. On the other hand, when the analysis result of the nucleic acid to be analyzed was analyzed, as shown in FIG. 4B, the peak of the nucleic acid to be analyzed consisting of the base sequence shown in SEQ ID NO: 4 is also a signal sufficient for peak discrimination as in the case of the standard nucleic acid. Obtained with strength. Both the standard nucleic acid and the nucleic acid to be analyzed could be detected with sufficient signal intensity, and the results shown in Table 1 were obtained. That is, it was determined that the sample contained a small amount of the nucleic acid to be analyzed, that there was no abnormality in the quality of the sample, and that there was no abnormality in the inspection process. When this series of inspections and determinations were repeated 25 times, the result was 1 in 23 out of 25 times.

[Example 2]
In one out of 25 tests and determinations shown in Example 1, neither the standard nucleic acid nor the nucleic acid to be analyzed could be observed with a sufficient signal intensity, and the result shown in Result 3 of Table 1 was obtained. It was. Since the standard nucleic acid could not be detected normally, it was determined that there was an abnormality in any of the steps (extraction, amplification, detection) after the nucleic acid extraction step in the flowchart shown in FIG.

[Example 3]
In one of the 25 tests and determinations shown in Example 1, the standard nucleic acid could be detected with sufficient signal intensity, but the analysis target nucleic acid could not observe a peak with sufficient signal intensity. Table 1 The result shown in 2 was obtained. Since the standard nucleic acid can be detected normally, there is no abnormality in the steps after the extraction step (extraction, amplification, detection) in the flowchart shown in FIG. 1, and the stage before the sample is subjected to the extraction step, that is, the analysis target It was determined that there was an abnormality in the quality of the sample containing the nucleic acid.

  The present invention is useful in the medical-related field for analyzing a large number of nucleic acid samples, particularly samples containing a small amount of nucleic acid, and is suitable for simplification of clinical tests and cost reduction.

It is a flowchart which illustrates the procedure of the inspection method of this invention. It is explanatory drawing for demonstrating an example of the procedure of the test | inspection method of this invention. It is explanatory drawing explaining the example of embodiment of the test | inspection method of this invention at the time of using a tubular container. 2 is a graph showing detection results of standard nucleic acids and analysis target nucleic acids in Example 1. It is a flowchart which illustrates the procedure of the conventional inspection method. It is explanatory drawing for demonstrating an example of the procedure of the conventional inspection method.

Claims (7)

  The sample that may contain the nucleic acid to be analyzed and the sample that contains the standard nucleic acid are subjected to the nucleic acid extraction step at approximately the same time under substantially the same conditions without mixing, and the resulting extract is mixed. Without being mixed, subjected to the amplification step at approximately the same time, and the obtained amplification product is subjected to the detection step at approximately the same condition under approximately the same condition without mixing, and the amplification product of the nucleic acid to be analyzed and the standard nucleic acid A method for inspecting a nucleic acid sample, wherein the presence or absence of the nucleic acid sample is confirmed.   The nucleic acid sample according to claim 1, wherein the size of the standard nucleic acid is substantially the same as the size of the nucleic acid to be analyzed, and the concentration of the standard nucleic acid in the sample is substantially the same as the concentration of the nucleic acid to be analyzed in the sample. Inspection method.   The size of the standard nucleic acid is 0.5 to 2.0 times the size of the target nucleic acid, and the concentration of the standard nucleic acid in the sample is 0.5 to 2.0 times the concentration of the target nucleic acid in the sample. The method for examining a nucleic acid sample according to claim 2.   The size of the standard nucleic acid is 100 to 300 base pairs, and the concentration of the standard nucleic acid in the sample is 1 pg / μl to 1 μg / μl. Nucleic acid sample inspection method.   There are a plurality of samples possibly containing the nucleic acid to be analyzed to be subjected to the nucleic acid extraction step, at least one standard nucleic acid to be subjected to the nucleic acid extraction step, and at least one sample containing the standard nucleic acid to be subjected to the nucleic acid extraction step. The method for examining a nucleic acid sample according to any one of claims 1 to 4, wherein   After examining the nucleic acid sample by the method according to any one of claims 1 to 5, the presence or absence of abnormality in any of the extraction step, the amplification step, and the detection step is determined from the detection result of the standard nucleic acid. In addition, when it is determined that there is no abnormality, the presence or absence of the nucleic acid to be analyzed in the sample that may contain the nucleic acid to be analyzed is determined from the detection result of the nucleic acid to be analyzed. Method for determining the inspection results. After determining the test result of the nucleic acid sample by the method according to claim 6, if an abnormality in any of the extraction process, the amplification process and the detection process is recognized from the determination result, the test method A method for managing inspection of a nucleic acid sample, wherein the inspection of the nucleic acid sample in which an abnormality is recognized is performed again, and then the determination of the inspection result is performed again by the determination method.

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