JP2017051111A - Gene detection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique for improving the sensitivity of a detection without causing contamination to the surroundings of an amplification product by PCR or contamination into the next sample to solve a disadvantage of amplified products by general multiplex PCR in which contamination to the surroundings or contamination to the next sample causes a misjudgment.SOLUTION: The present invention provides a gene detection method for detecting a trace amount of mutant gene contained in a sample, the method including: a first PCR step of previously amplifying the sample 20 times or less by PCR; a second PCR step of further performing PCR on the amplified product obtained in the first PCR step; and a gene detection step of performing gene detection in a closed reaction field after the second PCR step. At least one of the first PCR step and the second PCR step is performed in the closed reaction field.SELECTED DRAWING: None

Description

  The present invention relates to a gene detection method.

In recent years, samples such as serum have been increasingly used for minimally invasive cancer diagnosis and cancer treatment monitoring. Collecting these samples is less burdensome for the patient, but nucleic acid amplification is necessary for accurate diagnosis because the amount of nucleic acid contained per unit amount is small. In addition, nucleic acids in samples collected at crime scenes and the like are in very small quantities and are often damaged, so nucleic acid amplification is also necessary for such forensic examination purposes.
Until now, when the amount of nucleic acid in a sample is small, a means for amplifying a plurality of regions of a target at once by a multiplex polymerase chain reaction (multiplex PCR) has been taken. In general, in multiplex PCR, in order to amplify a plurality of target regions at once, an upstream primer and a downstream primer corresponding to the number of regions are required. In making this selection, the temperature and time of denaturation, annealing, and elongation that determine the thermal cycle of PCR are common in all amplification regions, so the melting temperature of primers ( Tm) must be aligned.

However, depending on the amplification region, the selection range of primers is limited due to a high content of cystine (C) and guanine (G) that cause the dissolution temperature to increase or a high degree of homology with the non-amplification region. In some cases, it was difficult to adjust the dissolution temperature in all amplification regions.
Moreover, even if the dissolution temperatures of all the primers can be made close to each other, a difference may occur in the amplification efficiency, and it is difficult to amplify all the regions with the same amplification efficiency. As a result, a false positive or false negative determination result due to the heterogeneity of the amplification rate occurs at the detection stage, and therefore, the primer design becomes more difficult as the amplification region of the multiplex PCR increases.

Furthermore, in order to detect a specific mutation from an amplification product by multiplex PCR, it is necessary to dispense the amplification product into a reagent containing individual detection probes. In that case, the contamination of the surroundings of the amplification product due to aerosols from the amplification product and splashes of the remaining liquid when the pipette tip is exchanged has been a problem. On the other hand, a mechanism for continuously performing multiplex PCR and detection in a sealed space has been devised, but the mechanism is complicated and difficult to realize.
Patent Document 1 discloses a method of performing amplification and fluorescence detection in multiplex PCR, but does not disclose that nucleic acid is automatically dispensed in multiplex PCR.
Patent Document 2 discloses an apparatus for automatically performing from nucleic acid extraction to amplification and fluorescence detection.

Special table 2008-517632 gazette Japanese Patent No. 5003845

In multiplex PCR, since a plurality of nucleic acid sequence sites in a nucleic acid are simultaneously amplified, it is difficult to detect a mutant gene having a low concentration and a low mutation rate. This is because even if a low-mutation rate mutant portion is amplified by multiplex PCR, nucleic acid sequence sites other than the low mutation rate are also amplified. For this reason, it is difficult to efficiently detect the low-mutation-rate mutant portion because the low-mutation-rate mutation portion contained in the original sample and the other portions are amplified in the same way. It was.
Further, since 2 n of 3 billion base pairs (bp) is contained in one copy of the genome, 6 billion base pairs (6 × 10 ⁹ bp) of deoxyribonucleic acid (DNA) is contained per cell. When PCR of 150 bp of genome from this one cell is performed, it becomes 3 × 10 ¹¹ bp after 30 cycles, and contains 50 times as much DNA as the original genome. For this reason, general multiplex PCR amplification products have caused misjudgment due to contamination to the surroundings or contamination to the next sample.
The present invention is a technique capable of improving detection sensitivity and detecting a mutant gene having a low concentration and a low mutation rate with high sensitivity without causing contamination around the amplification product by PCR or contamination in the next sample. I will provide a.

  The gene detection method according to an aspect of the present invention is a method for detecting a trace amount of a mutant gene contained in a sample in a sealed reaction field, and first amplifying the sample by PCR 20 times or less in advance. A second PCR step for further performing PCR on the amplification product obtained in the first PCR step, and a gene detection step for performing gene detection in a closed reaction field after the second PCR step, At least one of the PCR step and the second PCR step is performed in a closed reaction field.

  ADVANTAGE OF THE INVENTION According to this invention, the gene detection method which cannot detect the misjudgment by the contamination to the circumference | surroundings of the amplification product by PCR, or mixing into the following sample, and can detect the low concentration and the low mutation rate mutant gene with high sensitivity Can provide.

It is a figure which shows the measurement result in the Example of this invention. It is a figure which shows the measurement result in the Example of this invention.

In the present embodiment described below, genetic testing can be performed with a high sensitivity from a very small amount of sample without fear of contamination around the amplification product (amplicon) by PCR (for example, multiplex PCR) or contamination of the next sample. The present invention relates to an apparatus, a method, a reagent, and a composition.
The gene detection method of the present embodiment is a method for detecting a trace amount of mutated gene contained in a sample in a sealed reaction field, and the sample is limited times (for example, 20 times or less, more preferably 10 times or less). ), A second PCR step in which PCR is further performed on the amplification product obtained in the first PCR step, and a gene detection is performed in a closed reaction field after the second PCR step. A gene detection step, and at least one of the first PCR step and the second PCR step is performed in a closed reaction field.

However, the first PCR step is performed outside the sealed reaction field, the amplification product obtained in the first PCR process is dispensed into the sealed reaction field, the second PCR process is performed, and the sealed PCR is performed after the second PCR process. Gene detection may be performed in the reaction field. In addition, the first PCR step, the second PCR step, and the gene detection step may be performed in a closed reaction field. If gene amplification is performed in a closed reaction field, amplification products will not diffuse outside.
Furthermore, the first PCR step may be included in the second PCR step. Furthermore, amplification of specific sequences is adjusted using artificial nucleic acids such as peptide nucleic acids (PNA), LNA or BNA (2 ', 4'-Bridged Nucleic Acid) for PCR in the first PCR step or the second PCR step By (suppressing), the ratio of the gene other than the specific sequence in the sample may be increased.

Further, an invader reaction may be used for the reaction in the gene detection step. Further, the amount of the mutated gene may be 100 copies / μL or less.
The gene detection method of this embodiment will be described in further detail below.
In recent years, detection of a minute amount of mutated genes contained in specimens from cancer patients has become important for the selection of anticancer agents. In this case, “trace amount” refers to a ratio, and refers to 1% or less of a mutated gene contained in a normal gene. If a mutant gene present at a rate of 0.1% is considered, 1000 copies of the gene including the wild type are required to obtain one copy of the mutant gene. Considering this as a genome, when obtaining one copy of a specific gene variant, a genome set is about 3 pg, so a total of 3 ng of genome is required.

When multiple types of mutations are considered, such as mutations at codons 12 and 13 in the KRAS gene, detection wells (or tubes) as many as the number of mutants are required to identify the genotype of the mutation. Become. When codons 12 and 13 in the KRAS gene are considered, since seven types of mutations are generally considered, seven wells (tubes) are necessary for genotype identification. When wild-type detection is also performed, a total of eight wells (tubes) are required.
The probability that a small number of mutant genes are contained in the well follows a Poisson distribution. The Poisson distribution can be calculated by the following formula.

  Here, λ is an average value (average number) of molecules existing in a certain solution, k is a probability that exactly k molecules are included, and e is a base of natural logarithm. For example, since the 72 pg genome contains 24 (copy) mutated genes, when this genome solution is distributed to 24 wells, each well contains 1 (copy) gene on average. However, according to the Poisson distribution, the probability of not including one copy of a gene in the well can be calculated as in (Expression 1) to (Expression 2), which is 37%. That is, about 9 wells out of 24 wells may not contain the mutated gene.

For this reason, if the concentration is increased further, the probability of occurrence of empty wells that do not contain even one genome is reduced, but 4.98% at 3 times concentration, 0.67% at 7 times concentration, 7 times Since the concentration is 0.09%, even if a 7-fold concentration of the genome is prepared, an empty well is generated once every 1000 times. There is a possibility that. In the case of 10-fold concentration, this probability can be reduced to once in 10,000 times. Therefore, if the number of mutant genes can be increased 10 times in advance, the probability of empty wells decreases and the required detection sensitivity is low. I'll do it.
In PCR, the relationship between the number of cycles and the amplification factor is theoretically expressed by the following (formula 3).

  Since not all targets are replicated in one PCR, the value actually becomes n to a value smaller than 2 in Equation (3), but to increase 10 times, increase it 4 times or 100 times It can be seen that eight PCRs are sufficient (see Table 1).

As the amount of DNA increased at this time, if the PCR region is 150 bp, even if it is increased 100 times, it is only 15000 bp, which is insignificant compared to 6 × 10 ⁹ bp of the whole genome taken from one cell. Therefore, there is no danger of contamination due to PCR amplification products. On the other hand, the detection sensitivity can be reduced to 1/100 (the detection sensitivity of 10% is necessary for detection of 0.1% in the past), and there is a risk of erroneous determination due to the occurrence of an empty well. Less. Furthermore, at this amplification concentration, PCR amplification products can be pipetted into the gene analyzer, and thus the automation of the gene analyzer is easy.

  For example, since the abundance ratio of the original mutant to the wild type is 0.1% and the DNA concentration is 10 copies / μL, in the case of the gene analysis chip of the fully automatic gene analysis system of Patent Document 2, the chip The reaction well volume of 12.5 μL should contain an average of 0.125 mutant genes, but the actual distribution follows the Poisson distribution. The probability that none of the wells with an average concentration of 0.125 is contained in the well is 0.8825, so 7.06 out of the 8 wells necessary for genotyping are empty. Well, i.e., may not contain mutated genes. On the other hand, when 10 cycles of PCR are applied, even if the amplification efficiency is 90%, the number of PCR increases on the order of 640 times, so on average 80 per well (= 0.125 × 640) Thus, the probability that an empty well is generated is almost zero.

Amplification (hereinafter sometimes referred to as “pre-amplification”) is performed in advance to detect a trace amount of mutated genes contained in a specimen (sample, sample). Pre-amplification is a reaction of a detection chip, tube, or the like. It can also be performed in a reaction vessel different from the vessel. At this time, a step of dispensing the pre-amplified sample solution into a detection reaction vessel is required. Alternatively, the pre-amplification can be performed in a detection reaction vessel and the detection reaction can be continued.
The “sealed reaction field” in the present embodiment means that at least a reaction field (for example, a well) is not open to the air with a high possibility of contamination. That is, a structure in which the upper surface (upper space) of the well is partitioned by a film, a lid, or the like and does not directly contact the external space that causes contamination is preferable. For example, the chip structure of Patent Document 2 is an example of a sealed reaction field because a sample passage and a well as a reaction field are blocked from the outside.
In this embodiment, the detection is performed by fluorescence, but by detecting the luminescence, turbidity, absorbance, and electrical measurement such as pH, electrical conductivity, etc., by detecting the detection reagent to be used. Is possible.

Examples are shown below. The following example is one embodiment of the present invention, and the present invention is not limited to the following description.
[Example 1]
(Preamplification of low copy number and low mutation rate template)
Using the synthesized artificial gene plasmid DNA as a template with a low copy number and a low mutation rate, a 0.1% G12A mutant (0.1% G12A, 99.9% Wt) sample was prepared. The final concentration of this sample is 10 copies / μL, and the primer, dNTPmix, Hawk Taq, MOPS buffer, magnesium chloride (MgCl ₂ ), and sodium chloride (NaCl) have the final concentrations shown in Table 1. 600 μL of the reaction solution mixed in the above was prepared. At this stage, 6 copies of G12A mutant and 5994 copies of wild type are present in 600 μL of the reaction solution.

This reaction solution was divided into 300 μL portions for 2 minutes to obtain reaction solution I and reaction solution II. At this stage, 300 μL of each reaction solution contains 3 copies of G12A mutant and 2997 copies of wild type.
Only the reaction solution I was subjected to polymerase chain reaction (PCR) for 10 cycles as template preamplification. For the polymerase chain reaction, a commercially available Light Cycler (registered trademark) 480 system (Roche) was used. The reaction conditions were 95 ° C. for 2 minutes, 95 ° C. for 15 seconds and 66 ° C. for 20 seconds for 10 cycles, and then 99 ° C. for 10 minutes.
Reaction solution II was not pre-amplified, and was heated only at 95 ° C. for 2 minutes and at 99 ° C. for 10 minutes in the same manner as described above.

(About preparation of gene analysis chip)
A chip having 23 wells (capacity: 12.5 μL) shown in Patent Document 2 was prepared by molding polypropylene resin. Then, the reagents necessary for performing PCR and invader reaction (see Table 2) are dried and solidified under conditions of 60 ° C. and 700 g for 15 minutes in the well of this chip, and then sealed with an aluminum sheet by thermocompression bonding. Thus, a plurality of gene analysis chips (KRAS gene mutation detection chips) for performing KRAS gene mutation detection were prepared. A peptide nucleic acid (PNA) complementary to the wild type sequence was added to this chip to give a function of inhibiting wild type amplification. Table 3 shows the sequence information of the primers.

(Detection of low copy number and low mutation rate template using gene analysis chip)
The reaction solution I is poured into one of the above gene analysis chips, the reaction solution II is poured into the other one, and PCR and invader reaction are performed using a fully automatic gene analyzer (for example, the one described in Patent Document 2). Carried out. The reaction conditions were 95 ° C. for 120 seconds, 35 ° C. for 15 seconds and 66 ° C. for 20 seconds, followed by 99 ° C. for 120 seconds and 63 ° C. for 10 minutes.
FIG. 1 shows the fluorescence signal of the KRAS gene mutation detection chip of the reaction solution II that was not pre-amplified by PCR, and FIG. 2 shows the fluorescence signal of the KRAS gene mutation detection chip of the reaction solution I that was pre-amplified by PCR. . The solid line in FIGS. 1 and 2 is a signal for detecting the wild type sequence of the KRAS gene, and the dotted line is a signal for detecting the G12A mutant type sequence of the KRAS gene.

In reaction solution II, since pre-amplification was not performed, only 3 copies of G12A mutant were present in 300 μL of reaction solution, and G12A mutant plasmid DNA was not present in all 23 wells. In addition, it was not detected even after the amplification step in each well (see FIG. 1). In reaction solution I, since pre-amplification was performed, theoretically, 3072 (3 × 2 ¹⁰ ) copies of the G12A mutant form exist in 300 μL of the reaction solution. Since the reaction solution was poured into the KRAS gene mutation detection chip in that state, the G12A mutant plasmid DNA was present in all 23 wells, and the mutation rate was improved by detecting inhibition of wild type amplification by PNA. It is thought that this was possible (see FIG. 2).
The above figure shows the reaction results of these two sheets. FIG. 1 shows the result of the sample not subjected to pre-amplification, and FIG. 2 shows the result of the sample subjected to pre-amplification (10 cycles). In these figures, the solid line represents the reaction of the wild-type KRAS gene, and the dotted line represents the reaction of the mutant KRAS gene. The sealed KRAS gene mutation detection chip used this time contains a substance that inhibits the amplification of the wild type KRAS gene in order to enhance the mutation detection sensitivity. From this result, it was confirmed that the mutation was clearly detected only when preamplification was performed.

[Example 2]
In Example 1, the gene was amplified by preamplification outside the chip, and the subsequent PCR and detection reaction were performed in the chip. In Example 2, all of preamplification, PCR, and detection reaction were performed in the chip. . More specifically, a liquid mixture of a PCR reagent for preamplification and a sample to be amplified was injected into the chip, sent to the main flow path part of the chip, and the liquid mixture was preamplified in the main flow path part of the chip. After completion of the pre-amplification, the chip was centrifuged, and the pre-amplified solution was sent to each reaction well.
Since each reaction well is preliminarily immobilized with a PCR reagent and a reagent for detection reaction, the mixture is sent to each reaction well and heated, so that each immobilized reagent is mixed with the mixture. And PCR and detection reaction are performed. PCR, the concentration of the detection reaction reagent, each reaction temperature, and the number of cycles were the same as in Example 1.
Thus, when the same reaction as Example 1 was implemented in the chip | tip, the variation | mutation was able to be detected only when preamplification was implemented like Example 1. FIG.

Example 3
In Example 3, the chip was improved to provide a portion for preamplification. Otherwise, as in Example 2, the sample was mixed with a PCR reagent for preamplification and injected into the chip. After completion of preamplification, the solution after preamplification was sent to each reaction well by centrifugation. A detection reaction was performed in each well. PCR, the concentration of the detection reaction reagent, each reaction temperature, and the number of cycles were the same as in Example 1.
Thus, when the same reaction as Example 1 was implemented in the chip | tip, the variation | mutation was able to be detected only when preamplification was implemented like Example 1. FIG.
In Examples 1 to 3, the PCR reagent for pre-amplification was injected into the chip together with the sample solution, but it can be immobilized at a desired location on the chip.

Claims (6)

A method for detecting a minute amount of a mutant gene contained in a sample in a sealed reaction field,
A first PCR step of pre-amplifying the sample by PCR 20 times or less;
A second PCR step of further performing PCR on the amplification product obtained in the first PCR step;
A gene detection step of performing gene detection in the sealed reaction field after the second PCR step;
Have
A gene detection method in which at least one of the first PCR step and the second PCR step is performed in the sealed reaction field.   The first PCR step is performed outside the sealed reaction field, the PCR product of the second PCR step is performed after dispensing the amplification product obtained in the first PCR step into the sealed reaction field, and the second PCR step. The gene detection method according to claim 1, wherein gene detection is performed in the sealed reaction field after.   The gene detection method according to claim 1, wherein the first PCR step, the second PCR step, and the gene detection step are performed in the sealed reaction field.   The gene detection method according to any one of claims 1 to 3, wherein an invader reaction is used for the reaction in the gene detection step.   The gene detection method according to any one of claims 1 to 4, wherein amplification of a specific sequence is adjusted by using an artificial nucleic acid for PCR in the second PCR step.   The gene detection method according to any one of claims 1 to 5, wherein the amount of the mutated gene is 100 copies / µL or less.

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