JP6459438B2 - Method for determining position of probe in microarray and kit for labeling reaction - Google Patents

Description

  The present invention relates to a technique for identifying the type of organism based on a gene sequence, and more particularly, to a probe position determination method in a microarray and a labeling reaction kit.

In recent years, in food inspections and environmental inspections, probes that hybridize (complementarily bind) to the genes of organisms to be detected in order to identify the types of organisms such as microorganisms present in foods and the environment based on gene sequences. A microarray having a fixed spot is widely used. In this case, for example, by amplifying a DNA fragment in a target gene by PCR (polymerase chain reaction) method, labeling the amplified product with a probe on the microarray, and detecting the label of the amplified product, It is done to identify the type of organism.
Usually, microarrays have a large number of detection spots on which probes related to various organisms are immobilized. Therefore, when detecting a label, it is necessary to accurately identify the positions of detection spots on which probes are immobilized. There is.

  As a method for specifying the position of the detection spot on which the probe is immobilized, for example, the method described in Patent Document 1 below can be cited. In this method, a reference position spot for specifying the position of the probe is provided on the microarray, and a fluorescent substance is spotted on the reference position spot. Then, when detecting the label, the position of the reference position spot is detected by detecting the fluorescence at the reference position spot, whereby the position of the detection spot where the probe is immobilized can be specified from the position of the reference position spot. .

Further, as a method for specifying the position of the detection spot on which the probe is immobilized, for example, the method described in Patent Document 2 below can be cited. In this method, a reference position spot for specifying the position of the probe is provided on the microarray, and a reference position probe not labeled with the reference position spot is spotted. Also, a labeled marker polynucleotide that hybridizes to the reference position probe and a buffer used for hybridization are mixed to prepare a mixed solution. Then, after PCR amplification, the amplified product is added to the mixed solution and dropped onto the microarray. Upon detection of the amplified product, the marker polynucleotide label is detected and the position of the reference position spot is specified, thereby detecting the position of the reference position spot. Thus, the position of the detection spot where the probe is immobilized can be specified.
According to this method, in addition to being able to specify the position of the detection spot on which the probe is immobilized, it is possible to simultaneously confirm whether or not the hybridization step has been performed normally.

Japanese Patent No. 4261661 International Publication No. 2010/147167

However, in the method of spotting a reference position probe that is not labeled to such a reference position spot, it is necessary to prepare a marker polynucleotide for each test and mix it with a buffer used for hybridization. There is a problem that is complicated.
In addition, the optimal concentration of marker polynucleotide at the time of hybridization is very low, from several to several tens of nM, and it is necessary to adjust to this concentration. It is necessary to mix with the buffer used for soybean.
Specifically, for example, 1 μL of a marker polynucleotide stock solution (100 μM) is added to 999 μL of sterile water and diluted 1000 times to prepare a 100 nM solution. Then, 4 μL of a 100 nM solution of the marker polynucleotide is mixed with 96 μL of a buffer solution used for hybridization, and a 100 μL mixed solution is prepared and used.

However, the marker polynucleotide diluted to such a low concentration cannot be properly stored because it is easily adsorbed to the tube. For this reason, surplus marker polynucleotide dilution solution and mixed solution with buffer solution had to be discarded conventionally.
Specifically, in the above-described example, 996 μL of the 100 nM marker polynucleotide remaining unused and the remaining mixed solution are discarded, and there is a problem that the inspection cost increases.

  The present invention has been made in view of the above circumstances, and in a method of spotting a reference position probe that is not labeled with a reference position spot in order to identify the position of a detection spot on which the probe is immobilized, a work process It is an object of the present invention to provide a probe positioning method and a labeling reaction kit in a microarray capable of reducing the number and loss of reagents.

In order to achieve the above object, a method for determining the position of a probe in a microarray of the present invention includes a step of detecting hybridization between a probe and a target polynucleotide using the microarray after undergoing a reaction for labeling the target polynucleotide. A method of determining the position of the target polynucleotide, wherein the probe has a plurality of spots for detection and a microarray having a reference position spot on which a reference position probe is fixed; the labeled target polynucleotide; and comprising contacting the hybridizing labeled marker polynucleotide to the reference position probe, the reaction to label the target polynucleotides are PCR, the labeled marker polynucleotide, After modified so as not to the origin of synthesis of nucleic acid amplification by serial PCR, before the start of the reaction to label the target polynucleotides, is a method to be added pressure to the reaction solution to be used in the reaction.

  The labeling reaction kit of the present invention is a labeling reaction kit used for a labeling reaction in the probe position determination method in the microarray, and includes the marker polynucleotide.

  According to the present invention, it is possible to provide a probe position determination method and a labeling reaction kit in a microarray capable of reducing the number of work steps and reagent loss for determining the position of the probe in the microarray. .

It is a figure which shows the base sequence of the primer used in the Example of the position determination method of the probe in the microarray which concerns on embodiment of this invention. It is a figure which shows the result (1) of the confirmation test of the presence or absence of non-specific amplification by the probe position determination method in the microarray which concerns on embodiment of this invention. It is a figure which shows the base sequence of the marker polynucleotide used in the Example of the position determination method of the probe in the microarray which concerns on embodiment of this invention. It is a figure which shows the result (2) of the confirmation test of the presence or absence of non-specific amplification by the probe position determination method in the microarray which concerns on embodiment of this invention. It is a figure which shows the result (3) of the confirmation test of the presence or absence of non-specific amplification by the probe position determination method in the microarray which concerns on embodiment of this invention. It is a figure which shows the base sequence of the probe used in the Example of the position determination method of the probe in the microarray which concerns on embodiment of this invention. It is a figure which shows the result (fluorescence intensity value) of the detection test of the label | marker by the probe position determination method in the microarray which concerns on embodiment of this invention. It is a figure which shows the result (S / N ratio value) of the detection test of the label | marker by the probe position determination method in the microarray which concerns on embodiment of this invention. It is a figure which shows the result (microarray image) of the detection test of the label | marker by the probe position determination method in the microarray which concerns on embodiment of this invention.

Hereinafter, a probe position determination method and a label detection kit in a microarray according to an embodiment of the present invention will be described in detail.
The probe position determination method in the microarray according to the present embodiment is a probe position determination method in the microarray including a step of detecting hybridization between the probe and the target polynucleotide using the microarray after a reaction for labeling the target polynucleotide. The microarray having a plurality of detection spots on which the probes are immobilized and the reference position spot on which the reference position probe is immobilized is labeled with a labeled target polynucleotide and hybridized to the reference position probe. A marker polynucleotide is added to the reaction solution used for the reaction before the start of the labeling reaction.

As the “labeling reaction” in the present embodiment, for example, a nucleic acid amplification method can be suitably used. Examples of the nucleic acid amplification method include a PCR method, a LAMP method, an SDA method, and an LCA method. The “labeling reaction” includes a reaction that does not involve nucleic acid amplification, and includes, for example, a reaction in which labeled RNA is synthesized by a reverse transcriptase reaction using a sample RNA and a fluorescent dye.
In the following, the case where the PCR method is mainly used as the “labeling reaction” will be described as an example.

First, an outline of a scene where the probe position determination method in the microarray of the present embodiment is used will be described, and the features of the present invention will be described while being compared with the prior art.
The method for determining the position of the probe in the microarray of the present embodiment can be suitably used in a target gene detection method for specifying the type of organism based on the gene sequence using the microarray.
That is, in this target gene detection method, DNA is extracted from cells of a detection target organism, PCR is performed using a primer for amplifying a target amplification target region in this DNA, and a labeled amplification product ( Target polynucleotide) (labeling step).
In addition, when detecting microorganisms such as food poisoning bacteria and fungi, prior to the labeling step, the microorganisms are sampled from food and the environment, and a plurality of microorganisms are enriched and cultured at the same time. It is preferable to extract DNA from the same.

Next, the amplification product is brought into contact with a microarray having a detection spot on which a probe designed to hybridize with the amplification product is immobilized, so that the amplification product is hybridized to the probe (hybridization step).
Then, the presence of the detection target organism is confirmed by detecting the label in the amplification product (detection step).

In such a target gene detection method, the microarray is provided with a reference position spot for specifying the probe position in order to accurately specify the position of the spot on which the probe is immobilized. A reference position probe not labeled on the spot is immobilized.
Then, by using a labeled marker polynucleotide that hybridizes to the reference position probe (nucleic acid for reference position spot detection), by specifying the reference position spot, and by specifying the position of the detection spot from this reference position spot, The position of the detection spot where the label is detected can be accurately specified.

Here, conventionally, a marker polynucleotide is mixed with a buffer solution used for hybridization to prepare a mixed solution, and an amplification product is added to the mixed solution and dropped onto the microarray.
For this reason, each time a test is performed, it is necessary to prepare a mixed solution of a buffer solution and a marker polynucleotide, resulting in a problem that the operation becomes complicated. In addition, it is necessary to dilute the marker polynucleotide to about 1 / 1,000 before mixing, but the marker polynucleotide becomes unstable in a mixed solution with a low nucleic acid concentration and cannot be stored. The surplus mixed solution and diluted solution are discarded, and there is a problem that the inspection cost becomes high.
Examples of a method for preserving a polynucleotide diluted to a low concentration include a method of adding carrier DNA, a method of diluting with an adsorption inhibitor, and a method of using a low adsorption tube. However, in any case, there is a problem that the inspection cost becomes high.

Therefore, in the probe position determination method in the microarray of this embodiment, a marker polynucleotide is added to a reaction solution used for PCR before the start of PCR.
Thereby, it is not necessary to prepare a mixed solution of a buffer solution and a marker polynucleotide used for hybridization each time a test is performed, and it is possible to prevent forgetting to insert a marker polynucleotide. Furthermore, when adding the marker polynucleotide to the reaction solution, it is not necessary to substantially dilute, and it is possible to prevent the loss of reagents.

In addition, since the reaction solution contains nucleic acids such as nucleic acid synthesis substrates and primers, and the marker polynucleotide is contained undiluted, the marker polynucleotide can be stably stored by freezing the reaction solution. Is possible.
Furthermore, separately from the reaction solution, the marker polynucleotide is added to a primer mix consisting of a plurality of primer pairs used for PCR and frozen, so that it can be stored as a PCR amplification reagent for use as a component of the PCR reaction solution. It is also preferable. Such a PCR amplification reagent can be stably stored for a long time.

  On the other hand, adding a polynucleotide unrelated to the amplification of the target polynucleotide to the PCR reaction solution can cause non-specific amplification, and thus has been an operation never performed in the past. However, the present invention attempts to eliminate the occurrence of non-specific amplification by making a special contrivance not to cause non-specific amplification, as will be described later, while trying this. It is possible.

Next, each step in the probe position determination method in the microarray of this embodiment will be described in detail. As described above, the probe position determination method in the microarray of this embodiment can include a labeling step, a hybridization step, and a detection step.
In the labeling step, PCR is performed using a primer for amplifying the target amplification target region in the DNA of the cell of the detection target organism, and a labeled target polynucleotide is generated.

The reaction solution used for PCR in the present embodiment includes primers, DNA (or RNA) extracted from cells of the organism to be detected, marker polynucleotide, nucleic acid synthase, nucleic acid synthesis substrate, buffer solution, and water as the remaining components. What contains can be used suitably.
In the present embodiment, the primer contained in the reaction solution is composed of one or more kinds of primer pairs used for amplifying the target amplification target region.

In the present embodiment, the labeling method is not particularly limited, but a fluorescent label can be suitably used.
When fluorescent labeling is performed in PCR, a target polynucleotide labeled only at the end can be generated using a fluorescently labeled primer. In addition, a target polynucleotide containing a label therein can also be generated using a fluorescently labeled nucleic acid synthesis substrate. In any case, Cy5 or Cy3 can be suitably used as the fluorescent labeling component.
Furthermore, as a label, it is also possible to use a label other than fluorescence such as digoxigenin, biotin, and a radioisotope.

In the present embodiment, the target polynucleotide means a nucleic acid such as DNA or RNA to which a label is added. The nucleic acid may be a nucleic acid analog, and examples thereof include peptide nucleic acid (PNA), Bridged Nucleic Acid (BNA) / Locked Nucleic Acid (LNA, registered trademark), and the like.
When labeling a target polynucleotide by PCR, a target region selected from a gene in DNA (template) extracted from a cell of an organism to be detected can be amplified to obtain a labeled amplification product.

In the present embodiment, the marker polynucleotide is a nucleic acid that hybridizes to the reference position probe, and may be the nucleic acid analog described above.
As the marker polynucleotide, a labeled polynucleotide is used, and preferably a fluorescently labeled one is used. As the fluorescent label, Cy5, Cy3, etc. can be suitably used, and other labels described above may be used.
The marker polynucleotide is preferably composed of a base sequence that does not anneal to the target polynucleotide, and it is desirable that the marker polynucleotide does not function as a primer in error in PCR. However, according to the present invention, as described later, even when the marker polynucleotide is annealed to the target polynucleotide, it can be prevented from functioning as a primer.

  Further, in the hybridization step, the marker polynucleotide is composed of a base sequence that does not hybridize with the probe so that the marker polynucleotide does not hybridize with the probe to cause a false positive reaction. The target polynucleotide has a base sequence that does not hybridize with the reference position probe.

  Further, even when the marker polynucleotide is hybridized to the target polynucleotide, the marker polynucleotide is preferably chemically modified so that it does not serve as a PCR synthesis starting point so that it does not function as a primer. That is, when the marker polynucleotide becomes the starting point of PCR synthesis instead of the primer, a non-specific amplification product is generated. Therefore, it is preferable to process the marker polynucleotide so that it does not function as a primer.

  Here, if the marker polynucleotide is composed of a base sequence that does not hybridize to the target polynucleotide, basically, the marker polynucleotide can be made not to be a starting point for PCR synthesis. However, for example, in order to more reliably eliminate the possibility that the marker polynucleotide anneals to DNA derived from contaminant bacteria and functions as a primer, it is preferable to modify the marker polynucleotide so that it does not become the starting point of PCR synthesis. . Thus, if the marker polynucleotide is modified so that it does not become the starting point of PCR synthesis, it is possible to eliminate the generation of non-specific amplification even if the marker polynucleotide is annealed to the target polynucleotide.

  The method for modifying the marker polynucleotide so that it does not become the starting point for PCR synthesis is not particularly limited. For example, a method for phosphorylating and modifying the 3 'end of the marker polynucleotide can be preferably used. In addition, modification can also be made by using dideoxynucleotides, amination of the 3 'end, and the like.

  Such a marker polynucleotide is preferably used in the form of a reagent (labeling reaction kit) used in the labeling reaction. In particular, if it is used in the form of a PCR amplification reagent containing a marker polynucleotide and a primer and added to the reaction solution, it can be stored stably for a long period of time, and the marker polynucleotide can be added. Forgetting can be effectively prevented.

  Whether or not an amplification product is obtained by PCR can be confirmed by performing electrophoresis, for example. Examples of the electrophoresis include agarose gel electrophoresis, acrylamide electrophoresis, and microchip electrophoresis.

Next, in the hybridization step, a buffer solution is mixed with the reaction solution that has undergone the labeling reaction, and the obtained mixed solution is brought into contact with the microarray.
In the present embodiment, the microarray means a carrier on which a probe capable of hybridizing with a target polynucleotide is immobilized. Such a microarray is also called a DNA chip, and a plurality of detection spots are formed on the surface of the microarray and fixed by spotting probes composed of nucleic acid fragments.

  In the microarray used in the present embodiment, detection spots for detecting hybridization between the probe and the target polynucleotide are formed. That is, a probe that hybridizes with the target polynucleotide is immobilized on this detection spot. In each spot, the same probe may be immobilized, or different probes for identifying different genes and organisms may be immobilized.

The microarray is provided with one or more reference position spots, preferably two or more, for use in specifying the position of the probe. A reference position probe used for specifying the position of the probe is fixed to the reference position spot.
The reference position probe is composed of a nucleic acid such as DNA or RNA, and may be the nucleic acid analog described above.

Furthermore, in the detection step, the presence of the detection target organism is confirmed by detecting the label in the amplification product. At this time, the marker polynucleotide label in the reference position spot is detected, and the position of the detection spot to which the probe is immobilized is determined based on the position of the reference position spot.
The detection of the label can be performed using a general label detection apparatus such as a fluorescence scanning apparatus. For example, the label is detected by measuring the fluorescence intensity value of the amplification product using BIOSHOT (R) manufactured by Toyo Kohan Co., Ltd. be able to.

  In addition to the fluorescence intensity value, it is also preferable to calculate an S / N ratio value (Signal to Noise ratio, (median fluorescence intensity value−background value) ÷ background value) as a measurement result. This is because it is possible to accurately determine whether the measurement result is positive or negative based on the S / N ratio value. Generally, when the S / N ratio value is 3 or more, it is determined to be positive. Can do.

  A method for specifying the position of the detection spot from the position of the reference position spot is not particularly limited, and a known method can be used. For example, in the microarray, the detection spots can be arranged in a lattice pattern, and the reference position spots can be arranged at both ends of the lowermost stage, and the position of each detection spot can be specified based on the distance and angle from the reference position spot. is there.

  As described above, according to the probe position determination method and the labeling reaction kit in the microarray of the present embodiment, the marker polynucleotide can be mixed into the reaction solution before the labeling reaction. This makes it possible to dispense with preparation of a mixed solution that has to be performed for each test, and to prevent the marker polynucleotide from being forgotten. Such an effect is very useful because it can reduce the complexity of use and the occurrence of mistakes. This also makes it possible to reduce the loss of the reagent containing the marker polynucleotide and the buffer solution. Furthermore, although the marker polynucleotide cannot be stored in a solution having a low nucleic acid concentration, it can be stably stored depending on the form of the labeling reaction kit.

<Test 1: Confirmation of non-specific amplification>
Prior to the amplification reaction by PCR, a test was performed to confirm whether non-specific amplification occurred by adding a marker polynucleotide to the reaction solution.
Experiment 1 is an experiment when no marker polynucleotide is added, and Experiments 2 and 3 are experiments when a marker polynucleotide (unmodified 3 ′ end) is added. Experiments 4 and 5 are experiments in the case of adding a marker polynucleotide (3 ′ end phosphorylation).

(Experiment 1: No marker polynucleotide added)
A culture solution obtained by adding a medium for enrichment to a commercial scallion in which Staphylococcus aureus was detected and culturing overnight at 37 ° C. was used as a specimen. As this medium for enrichment, 10 g of peptone, 5 g of yeast extract, 0.5 g of magnesium sulfate, 15 g of sodium chloride, 3.5 g of disodium hydrogen phosphate, 1.5 g of potassium dihydrogen phosphate, and 964.5 g of sterilized water What was mixed and the pH was adjusted to 7.0 was used.

  Next, 1 mL of the culture solution was collected and centrifuged at 5000 × g for 10 minutes. The supernatant was discarded, and 20 mg / mL lysozyme solution (20 mM Tris-HCl, pH 8.0 / 2 mM EDTA, 1.2% Triton X-100) was added to the resulting precipitate, followed by lysis at 37 ° C. for 30 minutes. Processed. Furthermore, a DNA extract was obtained by performing column purification using DNeasy Blood & Tissue Kit (manufactured by Qiagen). This DNA extract was used as a template for PCR.

Next, a PCR reaction solution was prepared with the following composition. The primers were outsourced to Sigma Aldrich Japan GK, and the other reagents were from Takara Bio Inc.
・ Buffer (10 × Ex Taq buffer) (2.0μl)
・ Nucleic acid synthesis substrate (dNTP Mixture, 2.5mM each) (1.6μl)
・ Template (1.0μl)
・ Primer mix (8 types of primer pairs in Fig. 1) (1.0μl)
・ Nucleic acid synthase (TaKaRa Ex Taq Hot Start Version) (0.2μl)
・ Sterile water (remaining amount)
(Total 20 μl)

A thermal cycler ep gradient (Eppendorf Co., Ltd.) was used for gene amplification by PCR. The reaction conditions are as follows.
(1) 95 ° C for 2 minutes (2) 95 ° C for 10 seconds (DNA strand dissociation step)
(3) 52 ° C. for 30 seconds (annealing process)
(4) 72 ° C. for 30 seconds (DNA synthesis step)
(5) 72 ° C. 2 minutes (2) to (4) 40 cycles

Next, the PCR reaction solution containing the PCR amplification product thus obtained was subjected to a microchip electrophoresis apparatus MultiNA (Shimadzu Corporation), and size analysis of the PCR amplification product was performed. The result is shown in FIG.
As shown in the figure, as a result of electrophoresis, an amplification product (estimated size 238 bp) by the primer pair for amplifying the target gene (dnaJ) in the DNA of S. aureus was detected. On the other hand, a non-specific amplification product was not detected.

(Experiment 2: Marker polynucleotide (with 3 ′ end unmodified) added)
A pure culture solution obtained by culturing Staphylococcus aureus (Staphylococcus aureus, strain number: NCTC 10788 strain) overnight at 37 ° C. using BD Bacto ^TM tryptic soy broth (manufactured by Nippon Becton Dickinson Co., Ltd.) It was.
Next, 1 mL of this pure culture solution was recovered, and a DNA extract was obtained in the same manner as in Experiment 1. This DNA extract was used as a template for PCR.

Next, a PCR reaction solution was prepared with the following composition. Primer and marker polynucleotides were outsourced to Sigma Aldrich Japan GK, and other reagents were manufactured by Takara Bio Inc.
・ Buffer (10 × Ex Taq buffer) (2.0μl)
・ Nucleic acid synthesis substrate (dNTP Mixture, 2.5mM each) (1.6μl)
・ Template (1.0μl)
・ Primer mix (8 types of primer pairs in Fig. 1) (1.0μl)
・ Nucleic acid synthase (TaKaRa Ex Taq Hot Start Version) (0.2μl)
Marker polynucleotide (3 'end unmodified in Fig. 3) (160nM final conc.)
・ Sterile water (remaining amount)
(Total 20 μl)

Gene amplification by PCR was performed in the same manner as in Experiment 1. The obtained PCR reaction solution containing the PCR amplification product was applied to a microchip electrophoresis apparatus MultiNA (Shimadzu Corporation), and size analysis of the PCR amplification product was performed. The result is shown in FIG.
As shown in the figure, as a result of electrophoresis, an amplification product (estimated size 238 bp) by the primer pair for amplifying the target gene (dnaJ) in the DNA of S. aureus was detected. On the other hand, a non-specific amplification product was not detected.

(Experiment 3: marker polynucleotide (with 3 ′ end unmodified) added)
In the same manner as in Experiment 1, a culture solution obtained by adding a medium for enrichment to a commercial scallion in which Staphylococcus aureus was detected and culturing overnight at 37 ° C. was used as a specimen.
Next, 1 mL of the culture solution was collected, and a DNA extract was obtained in the same manner as in Experiment 1. This DNA extract was used as a template for PCR.

Next, a PCR reaction solution was prepared with the following composition. Primer and marker polynucleotides were outsourced to Sigma Aldrich Japan GK, and other reagents were manufactured by Takara Bio Inc.
・ Buffer (10 × Ex Taq buffer) (2.0μl)
・ Nucleic acid synthesis substrate (dNTP Mixture, 2.5mM each) (1.6μl)
・ Template (1.0μl)
・ Primer mix (8 types of primer pairs in Fig. 1) (1.0μl)
・ Nucleic acid synthase (TaKaRa Ex Taq Hot Start Version) (0.2μl)
Marker polynucleotide (3 'end unmodified in Fig. 3) (160nM final conc.)
・ Sterile water (remaining amount)
(Total 20 μl)

Gene amplification by PCR was performed in the same manner as in Experiment 1. The obtained PCR reaction solution containing the PCR amplification product was applied to a microchip electrophoresis apparatus MultiNA (Shimadzu Corporation), and size analysis of the PCR amplification product was performed. The result is shown in FIG.
As shown in the figure, as a result of electrophoresis, an amplification product (estimated size 238 bp) by the primer pair for amplifying the target gene (dnaJ) in the DNA of S. aureus was detected. In addition, non-specific amplification products were detected.

(Experiment 4: marker polynucleotide (with 3 ′ end phosphorylation) added)
In the same manner as in Experiment 1, a culture solution obtained by adding a medium for enrichment to a commercial scallion in which Staphylococcus aureus was detected and culturing overnight at 37 ° C. was used as a specimen.
Next, 1 mL of the culture solution was collected, and a DNA extract was obtained in the same manner as in Experiment 1. This DNA extract was used as a template for PCR.

Next, a PCR reaction solution was prepared with the following composition. Primer and marker polynucleotides were outsourced to Sigma Aldrich Japan GK, and other reagents were manufactured by Takara Bio Inc. In this experiment, the marker polynucleotide is Cy5 modified at the 5 ′ end and phosphorylated at the 3 ′ end.
・ Buffer (10 × Ex Taq buffer) (2.0μl)
・ Nucleic acid synthesis substrate (dNTP Mixture, 2.5mM each) (1.6μl)
・ Template (1.0μl)
・ Primer mix (8 types of primer pairs in Fig. 1) (1.0μl)
・ Nucleic acid synthase (TaKaRa Ex Taq Hot Start Version) (0.2μl)
Marker polynucleotide (3 'terminal phosphorylation in Fig. 3) (160nM final conc.)
・ Sterile water (remaining amount)
(Total 20 μl)

Gene amplification by PCR was performed in the same manner as in Experiment 1. The obtained PCR reaction solution containing the PCR amplification product was applied to a microchip electrophoresis apparatus MultiNA (Shimadzu Corporation), and size analysis of the PCR amplification product was performed. The result is shown in FIG.
As shown in the figure, as a result of electrophoresis, an amplification product (estimated size 238 bp) by the primer pair for amplifying the target gene (dnaJ) in the DNA of S. aureus was detected. In addition, a non-specific amplification product was not detected.

(Experiment 5: Marker polynucleotide (3 'terminal phosphorylation added))
As in Example 2, Staphylococcus aureus (strain number: NCTC 10788 strain) was obtained by culturing overnight at 37 ° C. using BD Bacto ^™ Tryptic Soy Broth (manufactured by Becton Dickinson, Japan). The obtained pure culture solution was used as a specimen.
Next, 1 mL of this pure culture solution was recovered, and a DNA extract was obtained in the same manner as in Experiment 1. This DNA extract was used as a template for PCR.

Next, a PCR reaction solution was prepared with the following composition. Primer and marker polynucleotides were outsourced to Sigma Aldrich Japan GK, and other reagents were manufactured by Takara Bio Inc. In this experiment, the marker polynucleotide is Cy5 modified at the 5 ′ end and phosphorylated at the 3 ′ end.
・ Buffer (10 × Ex Taq buffer) (2.0μl)
・ Nucleic acid synthesis substrate (dNTP Mixture, 2.5mM each) (1.6μl)
・ Template (1.0μl)
・ Primer mix (8 types of primer pairs in Fig. 1) (1.0μl)
・ Nucleic acid synthase (TaKaRa Ex Taq Hot Start Version) (0.2μl)
Marker polynucleotide (3 'terminal phosphorylation in Fig. 3) (160nM final conc.)
・ Sterile water (remaining amount)
(Total 20 μl)

Gene amplification by PCR was performed in the same manner as in Experiment 1. The obtained PCR reaction solution containing the PCR amplification product was applied to a microchip electrophoresis apparatus MultiNA (Shimadzu Corporation), and size analysis of the PCR amplification product was performed. The result is shown in FIG.
As shown in the figure, as a result of electrophoresis, an amplification product (estimated size 238 bp) by the primer pair for amplifying the target gene (dnaJ) in the DNA of S. aureus was detected. In addition, a non-specific amplification product was not detected.

  In Experiment 1, a primer for detecting Staphylococcus aureus was added to the PCR reaction solution, but no marker polynucleotide was added, and amplification products by Staphylococcus aureus were detected. No amplification product was detected.

  In Experiment 2, a primer and a marker polynucleotide for detecting S. aureus were added to the PCR reaction solution, and amplification products by S. aureus were detected, but non-specific amplification products were detected. There wasn't. Thus, even when a marker polynucleotide is added to a PCR reaction solution, the marker polynucleotide usually does not anneal to the target polynucleotide and often does not cause non-specific amplification products.

  On the other hand, in Experiment 3, a primer and a marker polynucleotide for detecting Staphylococcus aureus were added to the PCR reaction solution, and amplification products by S. aureus were detected, and non-specific amplification products were also detected. Marker polynucleotides typically do not anneal to the target polynucleotide, but in rare cases this may cause the marker polynucleotide to anneal to the target polynucleotide, resulting in a non-specific amplification product.

  Therefore, in Experiment 4, instead of the marker polynucleotide of Experiment 3 (3 ′ end unmodified), a marker polynucleotide (3 ′ terminal phosphorylation) was added to the PCR reaction solution, and the other points were similar. Went. As a result, it can be seen that by using such a marker polynucleotide that does not become the starting point for nucleic acid amplification synthesis by PCR, the generation of non-specific amplification products based on the marker polynucleotide can be prevented.

<Test 2: Detection of label>
A test for confirming the detection of the label in the microarray was performed assuming various cases of the presence or absence of addition of the marker polynucleotide, the timing of addition of the marker polynucleotide, and the presence or absence of modification of the 3 ′ end of the marker polynucleotide. In Experiment 6, when a marker polynucleotide (unmodified 3 ′ end) was added to the mixture after the labeling reaction and before the hybridization step, Experiment 7 was performed when no marker polynucleotide was added. When nucleotide (3 ′ terminal unmodified) is added to the PCR reaction solution, Experiment 9 is an experiment when marker polynucleotide (3 ′ terminal phosphorylation) is added to the PCR reaction solution.

(Experiment 6: Marker polynucleotide (3 ′ end unmodified) is added to the mixture after the labeling reaction and before the hybridization step)
In the same manner as in Experiment 1, a culture solution obtained by adding a medium for enrichment to a commercial scallion in which Staphylococcus aureus was detected and culturing overnight at 37 ° C. was used as a specimen.
Next, 1 mL of the culture solution was collected, and a DNA extract was obtained in the same manner as in Experiment 1. This DNA extract was used as a template for PCR.

Next, a PCR reaction solution was prepared with the following composition, and gene amplification by PCR was performed as in Experiment 1. Primers and marker polynucleotides described below were outsourced to Sigma Aldrich Japan LLC, and the other reagents were from Takara Bio Inc.
・ Buffer (10 × Ex Taq buffer) (2.0μl)
・ Nucleic acid synthesis substrate (dNTP Mixture, 2.5mM each) (1.6μl)
・ Template (1.0μl)
・ Primer mix (8 types of primer pairs in Fig. 1) (1.0μl)
・ Nucleic acid synthase (TaKaRa Ex Taq Hot Start Version) (0.2μl)
・ Sterile water (remaining amount)
(Total 20 μl)

  In addition, a microarray having spots in which the probes shown in FIG. 6 were immobilized was prepared in advance. Specifically, in the microarray shown in FIG. 6, probes selected from the amplification target region targeted by Staphylococcus aureus are immobilized at spots 29 to 32, and other bacteria are identified at spots 25 to 28 and 37 to 40. Probes selected from species genes are immobilized. Spots 57 and 64 are reference position spots, and a reference position probe is fixed. The reference position probe consists of a base sequence selected from φ-X174 bacteriophage.

Next, 1 μL of a stock solution (100 μM) of a marker polynucleotide (3 ′ end unmodified in FIG. 3) was diluted 1000-fold by adding it to 999 μL of sterile water to prepare a 100 nM solution. Then, 4 μL of a 100 nM solution of the marker polynucleotide was mixed with 96 μL of a buffer solution (3 × SSC / 0.3% SDS citrate-saline-sodium dodecyl sulfate) used for hybridization to prepare a 100 μL mixture. And what mixed 2 microliters of obtained mixed liquids and PCR reaction liquid 4 microliters was dripped at the said microarray, and it was made to react at 45 degreeC for 1 hour. The remaining 996 μL of marker polynucleotide dilution and 98 μL of the mixture were discarded because they could not be stored.
After the reaction, the microarray is washed by immersing it in a cleaning solution (2 x SSC / 0.2% SDS solution, 2 x SSC solution in this order) at room temperature, and a cover glass is placed on the fluorescence detector Bioshot (Toyo Kohan Co., Ltd.) Was used to detect the fluorescence in the spot region of each probe.

  Specifically, the label component (Cy5) of the amplification product hybridized with the probe was excited by laser light to emit light, and the amount of light was detected by a CCD camera attached in the detector. In addition, the light intensity was replaced with an electric signal and digitized to obtain a fluorescence intensity value. This fluorescence intensity value is an intensity index in the apparatus and has no unit. In addition, the S / N ratio value was calculated. The results are shown in FIGS. Further, FIG. 9 shows an image of the microarray.

  As shown in these figures, strong fluorescence intensity is observed in the detection spot on which the probe for detecting S. aureus is immobilized, and an S / N ratio value determined to be positive is obtained. I understand that. In addition, strong fluorescence intensity is also observed at the reference position spot, and it can be seen that an S / N ratio value determined to be positive is obtained.

(Experiment 7: No marker polynucleotide added)
As in Experiment 1, Staphylococcus aureus was cultured to obtain a DNA extract. This DNA extract was used as a template for PCR. Then, the gene was amplified by PCR as in Experiment 1. This PCR reaction solution does not contain a marker polynucleotide.
A microarray similar to that in Experiment 6 was prepared in advance.

Next, 4 μL of the PCR reaction solution and 2 μL of hybridization buffer (3 × SSC / 0.3% SDS citrate-saline-sodium dodecyl sulfate) were added dropwise to the microarray at 45 ° C. The reaction was carried out for 1 hour. This mixed solution does not contain the marker polynucleotide.
After the reaction, the fluorescence in the spot region of each probe was detected in the same manner as in Experiment 6 to obtain the fluorescence intensity value and the S / N ratio value. The results are shown in FIGS. Further, FIG. 9 shows an image of the microarray.

  As shown in these figures, strong fluorescence intensity is observed in the detection spot on which the probe for detecting S. aureus is immobilized, and an S / N ratio value determined to be positive is obtained. I understand that. On the other hand, since no marker polynucleotide is added, it can be seen that no fluorescence is detected at the reference position spot (substantially the background value).

(Experiment 8: Marker polynucleotide (3 ′ terminal unmodified) is added to the PCR reaction solution)
In the same manner as in Experiment 1, a culture solution obtained by adding a medium for enrichment to a commercial scallion in which Staphylococcus aureus was detected and culturing overnight at 37 ° C. was used as a specimen.
Next, 1 mL of the culture solution was collected, and a DNA extract was obtained in the same manner as in Experiment 1. This DNA extract was used as a template for PCR.

Next, a PCR reaction solution was prepared with the following composition, and gene amplification by PCR was performed as in Experiment 1. Primer and marker polynucleotides were outsourced to Sigma Aldrich Japan GK, and other reagents were manufactured by Takara Bio Inc.
・ Buffer (10 × Ex Taq buffer) (2.0μl)
・ Nucleic acid synthesis substrate (dNTP Mixture, 2.5mM each) (1.6μl)
・ Template (1.0μl)
・ Primer mix (8 types of primer pairs in Fig. 1) (1.0μl)
・ Nucleic acid synthase (TaKaRa Ex Taq Hot Start Version) (0.2μl)
Marker polynucleotide (3 'end unmodified in Fig. 3) (160nM final conc.)
・ Sterile water (remaining amount)
(Total 20 μl)
A microarray similar to that in Experiment 6 was prepared in advance.

Next, 4 μL of the PCR reaction solution and 2 μL of hybridization buffer (3 × SSC / 0.3% SDS citrate-saline-sodium dodecyl sulfate) were added dropwise to the microarray at 45 ° C. The reaction was carried out for 1 hour.
After the reaction, the fluorescence in the spot region of each probe was detected in the same manner as in Experiment 6 to obtain the fluorescence intensity value and the S / N ratio value. The results are shown in FIGS. Further, FIG. 9 shows an image of the microarray.

As shown in these figures, strong fluorescence intensity is observed in the detection spot on which the probe for detecting S. aureus is immobilized, and an S / N ratio value determined to be positive is obtained. I understand that. In addition, strong fluorescence intensity is also observed at the reference position spot, and it can be seen that an S / N ratio value determined to be positive is obtained.
On the other hand, an S / N ratio value determined to be positive is also obtained in a detection spot where a probe for detecting Vibrio parahaemolyticus is immobilized, indicating that a false positive reaction has occurred.

(Experiment 9: Addition of marker polynucleotide (3 ′ end phosphorylation) to PCR reaction solution)
In the same manner as in Experiment 1, a culture solution obtained by adding a medium for enrichment to a commercial scallion in which Staphylococcus aureus was detected and culturing overnight at 37 ° C. was used as a specimen.
Next, 1 mL of the culture solution was collected, and a DNA extract was obtained in the same manner as in Experiment 1. This DNA extract was used as a template for PCR.

Next, a PCR reaction solution was prepared with the following composition, and gene amplification by PCR was performed as in Experiment 1. Primer and marker polynucleotides were outsourced to Sigma Aldrich Japan GK, and other reagents were manufactured by Takara Bio Inc.
・ Buffer (10 × Ex Taq buffer) (2.0μl)
・ Nucleic acid synthesis substrate (dNTP Mixture, 2.5mM each) (1.6μl)
・ Template (1.0μl)
・ Primer mix (8 types of primer pairs in Fig. 1) (1.0μl)
・ Nucleic acid synthase (TaKaRa Ex Taq Hot Start Version) (0.2μl)
Marker polynucleotide (3 'terminal phosphorylation in Fig. 3) (160nM final conc.)
・ Sterile water (remaining amount)
(Total 20 μl)
A microarray similar to that in Experiment 6 was prepared in advance.

Next, 4 μL of the PCR reaction solution and 2 μL of hybridization buffer (3 × SSC / 0.3% SDS citrate-saline-sodium dodecyl sulfate) were added dropwise to the microarray at 45 ° C. The reaction was carried out for 1 hour.
After the reaction, the fluorescence in the spot region of each probe was detected in the same manner as in Experiment 6 to obtain the fluorescence intensity value and the S / N ratio value. The results are shown in FIGS. Further, FIG. 9 shows an image of the microarray.

  As shown in these figures, strong fluorescence intensity is observed in the detection spot on which the probe for detecting S. aureus is immobilized, and an S / N ratio value determined to be positive is obtained. I understand that. In addition, strong fluorescence intensity is also observed at the reference position spot, and it can be seen that an S / N ratio value determined to be positive is obtained. And it turns out that the false positive reaction has not arisen about other microbial species.

  As described above, according to Experiment 9 in which the marker polynucleotide was added to the PCR reaction solution, a result equivalent to the result of Experiment 6 in which the marker polynucleotide was added to the mixture before the hybridization step can be obtained, and It is possible to reduce the number of processes and reagent loss. Furthermore, according to Experiment 9, it can be seen that the occurrence of a false positive reaction in Experiment 8 in which a 3'-end unmodified marker polynucleotide was added to the PCR reaction solution could be eliminated.

  The present invention is not limited to the above-described embodiments and examples, and various modifications can be made within the scope of the present invention. For example, in the probe position determination method and the labeling reaction kit in the microarray according to the present embodiment, the base sequences of the target polynucleotide, probe, marker polynucleotide, reference position probe, and primer are not essential and arbitrary. Can be made. In the examples, experiments are performed on microorganisms, but the present invention can be applied without limiting the types of target organisms.

  The present invention can be suitably used for detecting various targets using a microarray in food inspection, epidemiological environmental inspection, environmental inspection, clinical test, livestock hygiene, and the like.

Claims (6)

A method for determining the position of a probe in a microarray comprising a step of detecting hybridization between a probe and a target polynucleotide by a microarray after undergoing a reaction for labeling the target polynucleotide,
A label that hybridizes to the labeled target polynucleotide and the reference position probe on a microarray having a plurality of detection spots to which the probe is immobilized and a reference position spot to which a reference position probe is immobilized Contacting the labeled marker polynucleotide,
The reaction for labeling the target polynucleotide is PCR,
The labeled marker polynucleotide, after modified so as not to the origin of synthesis of nucleic acid amplification by the PCR, before the start of the reaction to label the target polynucleotides is added pressure to the reaction solution to be used in the reaction A method for determining a position of a probe in a microarray. It said target polynucleotide as a mixture of buffer solution to the reaction solution which has passed through the indicator react to the resulting mixture is contacted with the microarray, and detecting the label of the labeled marker polynucleotide in said reference position spot, The method of determining a position of a probe in a microarray according to claim 1, wherein the position of the probe is determined based on the position of the reference position spot.   3. The method for determining a position of a probe in a microarray according to claim 1, wherein the reference position spot is two or more. The method for determining the position of a probe in a microarray according to any one of claims 1 to 3, wherein the modification of the labeled marker polynucleotide is a phosphorylation modification at the 3 'end. A labeling reaction kit for use in a labeling reaction in the probe position determination method in the microarray according to any one of claims 1 to 4 , comprising the labeled marker polynucleotide. For kit. 6. The labeling reaction kit according to claim 5 , further comprising one or more kinds of primer pairs for amplifying the target polynucleotide.