WO2017164354A1 - Method for detecting nucleic acid derived from hair - Google Patents

Method for detecting nucleic acid derived from hair Download PDF

Info

Publication number
WO2017164354A1
WO2017164354A1 PCT/JP2017/011935 JP2017011935W WO2017164354A1 WO 2017164354 A1 WO2017164354 A1 WO 2017164354A1 JP 2017011935 W JP2017011935 W JP 2017011935W WO 2017164354 A1 WO2017164354 A1 WO 2017164354A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
hair
target nucleic
detection
sample
Prior art date
Application number
PCT/JP2017/011935
Other languages
French (fr)
Japanese (ja)
Inventor
つかさ 前田
裕樹 山下
Original Assignee
倉敷紡績株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 倉敷紡績株式会社 filed Critical 倉敷紡績株式会社
Priority to JP2018507431A priority Critical patent/JPWO2017164354A1/en
Publication of WO2017164354A1 publication Critical patent/WO2017164354A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to a technique for detecting nucleic acids derived from human or animal hair.
  • Patent Documents 1 and 2 As a conventional technique for detecting nucleic acid from a small amount of sample such as a single hair, a technique has been adopted in which the nucleic acid is extracted from the sample, then the extracted nucleic acid is purified and the nucleic acid is amplified (for example, Patent Documents 1 and 2). Further techniques such as freezing the hair with liquid nitrogen (Patent Documents 3 and 4) and confirming the state of the hair with an image (Patent Document 5) are also employed.
  • Japanese Patent No. 5501570 JP-A-2015-73478 JP 2009-131253 A Japanese Unexamined Patent Publication No. 2007-20559 Japanese Patent No.4356783
  • nucleic acid detection technology it is common knowledge to acquire nucleic acid from a sample, and the nucleic acid is amplified by detecting, purifying, and collecting the nucleic acid from the sample, and the amplified nucleic acid is detected.
  • nucleic acid is collected from a sample such as extraction treatment such as ethanol precipitation or filtration that removes contaminants, the nucleic acid may be sheared or the nucleic acid may be lost.
  • a method of recovering nucleic acid from a sample by using chemical treatment or devising a crushing method has been taken, and the process itself has not been reviewed.
  • the present invention has been made in view of the above-mentioned present state of the art, and an object thereof is to provide a method for detecting a nucleic acid derived from hair more easily than conventional techniques.
  • the present inventors have intensively studied to solve the above problems, and have come up with the idea of simplifying the process itself without performing processing such as extraction, purification, and recovery of nucleic acids. Specifically, the present inventors crushed human or animal hair including hair by a physical technique in a liquid without performing a chemical treatment, and performed a nucleic acid amplification reaction using the crushed liquid. It has been found that amplified nucleic acids can be detected. The present inventors have further studied based on this discovery, and have completed the present invention.
  • the present invention provides the following.
  • [1] A method for detecting a target nucleic acid in mammalian hair, Crushing mammalian hair in solution by physical means, A step of amplifying a target nucleic acid by subjecting the disrupted solution to a nucleic acid amplification reaction; and Detecting the amplified target nucleic acid.
  • [2] The method according to [1] above, wherein the step of extracting nucleic acid is not performed.
  • [3] The method according to [1] or [2] above, wherein the hair is a hair fragment not containing a hair root.
  • [4] The method according to any one of [1] to [3] above, wherein the detection of the target nucleic acid is performed using a strip for nucleic acid chromatography.
  • [5] The method according to any one of [1] to [4] above, wherein the nucleic acid amplification reaction is a polymerase chain reaction.
  • nucleic acid derived from hair by eliminating a series of steps for extracting, purifying, and recovering nucleic acid from a sample, it is not necessary to perform complicated operations and shorten the time required for detection. Can do.
  • FIG. 3 is a diagram showing the results of nucleic acid detection using the nucleic acid detection strip in Example 1. It is a figure explaining the sample used in Example 2.
  • FIG. 4 is a diagram showing the results of nucleic acid detection using a nucleic acid detection strip in Example 2.
  • 6 is a diagram illustrating a sample used in Example 3.
  • FIG. It is a figure which shows the result of the detection of the nucleic acid using the strip for nucleic acid detection in Example 3. It is a figure which shows the result of the detection of the nucleic acid using the strip for nucleic acid detection in Example 4.
  • the type of mammal is not particularly limited. Examples include pets such as dogs and cats, primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees, and the like, preferably humans.
  • the hair collection site is not particularly limited, and any hair including hair, eyebrows, nasal hair, whiskers, pubic hair, and eyelashes can be used in the present invention.
  • the hair can be collected by drawing the hair from the root or excising the hair on the surface of the mammalian body. Or you may use the hair which fell off naturally.
  • the full length from the hair root to the tip may be sufficient as the hair, and the fragment
  • the position of the fragment is not limited.
  • the hair may or may not contain a hair root.
  • the hair to be subjected to the physical crushing process described later has a length of usually 0.1 to 10.0 cm, preferably 0.5 to 4.0 cm, more preferably 1.0 to 2.0 cm. .
  • a hair having a length of about 1 cm is easy to use for crushing with a crushing instrument as used in the examples.
  • the target nucleic acid is to be detected, and the type thereof is not particularly limited.
  • the nucleic acid may be DNA or RNA, but is preferably DNA.
  • Examples of the target nucleic acid include those whose expression is known to be related to the onset and risk of a specific disease or condition, therapeutic sensitivity, or a specific constitution.
  • Examples of the disease or condition include cancer, lifestyle-related diseases (for example, dyslipidemia, hypertension, diabetes, obesity), side effects of drugs, and the constitution includes alcohol tolerance.
  • a gene polymorphism (SNP) having a nucleotide sequence different from the wild type is related to the onset of a specific disease or condition, its risk, therapeutic sensitivity, or a specific constitution. Therefore, preferable genes include genes having such SNPs.
  • the gene include, but are not limited to, an alcohol dehydrogenase gene (ADH1B) and acetaldehyde dehydrogenase (ALDH2) involved in alcohol resistance.
  • ADH1B alcohol dehydrogenase gene
  • ADH2 acetaldehyde dehydrogenase
  • a DNA having a known sequence that has a different base sequence depending on the animal species may be used as the target nucleic acid.
  • DNA that can be used for species discrimination include mitochondrial DNA.
  • the target nucleic acid may be one, or two or more.
  • a plurality of target nucleic acids can be amplified by a multiplex nucleic acid amplification reaction, and the plurality of amplified target nucleic acids can be detected at once.
  • crushing by physical means of hair or physical crushing refers to a hair fragmentation process involving physical contact between a device used for crushing and hair.
  • the crushing can be performed by homogenization, shearing or grinding.
  • the means for physical crushing is not particularly limited, and a device such as a homogenizer can be used as appropriate, or the hair can be frozen with liquid nitrogen and crushed using a mortar, manual mill, or the like.
  • a disposable homogenizer such as a crushing device can be used conveniently and can be preferably used as the means.
  • the disposable homogenizer may be used manually, or a commercially available dedicated grinder may be used.
  • the present invention provides a method for detecting a target nucleic acid in mammalian hair (hereinafter also referred to as the method of the present invention).
  • the method of the present invention comprises: (Step 1) crushing mammalian hair in solution by physical means, (Step 2) Amplifying the target nucleic acid by subjecting the disrupted solution to a nucleic acid amplification reaction, and (Step 3) including a step of detecting the amplified target nucleic acid.
  • the method of the present invention preferably does not include the step of extracting or purifying the nucleic acid.
  • the method of the present invention preferably does not include a step of decomposing hair by chemical treatment.
  • Step 1 Step of crushing mammalian hair in solution by physical means Crushing can be carried out using the means for physical crushing as described above.
  • the crushing may be performed to such an extent that the hair cannot be visually recognized.
  • impurities attached to the hair sample may be removed using ethanol or the like.
  • the above solution may be any solution as long as it does not significantly inhibit the detection of the target nucleic acid by the method of the present invention, and is preferably a liquid having a buffering action. Since the solution after the crushing treatment is used as a sample for the nucleic acid amplification reaction in Step 2 described later, the solution is preferably the same as the buffer solution (buffer) used in the nucleic acid amplification reaction described later. It is preferable to put about 0.5 to 5 cm of hair in a solution of about 10 to 400 ⁇ L and perform crushing treatment. Further, after crushing, impurities may be precipitated and removed by centrifugation.
  • Step 2 Step of amplifying the target nucleic acid by subjecting the disrupted solution to a nucleic acid amplification reaction
  • the method of the nucleic acid amplification reaction is not limited as long as the target nucleic acid can be amplified.
  • amplification of a target nucleic acid does not necessarily amplify the entire target nucleic acid (for example, a target gene), as long as at least the detection target region of the nucleic acid can be amplified.
  • PCR polymerase chain reaction
  • TMA Transcription- Mediated Amplification
  • NASBA Nucleic Acid Sequence-Based Amplification
  • LCR Low-Cleukin-Reaction
  • CLR Cycling Ligation Reaction; technology described in JP2016-140287, JP2016-140288
  • PCR method is the most widely used nucleic acid amplification method, not only can various reagents and equipment optimized for the method be easily obtained, but there are various variations of the method as described above. Since it is extremely high, it is mentioned as a suitable technique for the present invention.
  • the nucleic acid amplification reaction solution is a solution containing reagents and the like necessary for performing the nucleic acid amplification reaction.
  • the composition of the reaction solution for nucleic acid amplification differs somewhat depending on the nucleic acid amplification method to be used, but usually four types of deoxynucleoside triphosphates (dATP, dTTP, dCTP, dGTP: hereinafter collectively referred to as dNTP) as substrates, Basically, a DNA polymerase as an enzyme, magnesium ions as a cofactor of the enzyme, and a primer set for nucleic acid amplification reaction are contained in a buffer.
  • dATP deoxynucleoside triphosphates
  • dNTP deoxynucleoside triphosphates
  • concentration of dNTP an optimum concentration may be examined in the range where each of the four final concentrations is in the range of 100 to 400 ⁇ M.
  • a DNA polymerase having a property suitable for the nucleic acid amplification method to be used is used.
  • Taq DNA polymerase, Pfu DNA polymerase, and other heat-resistant DNA polymerases developed by other bioscience-related companies are preferably used.
  • magnesium ion concentration an optimum concentration may be examined within a final concentration range of 1 to 6 mM.
  • the amplification primer included in the primer set for nucleic acid amplification reaction is composed of nucleic acids such as DNA and RNA, modified nucleic acids, or combinations thereof. What is necessary is just to examine the density
  • the number of amplification primers is not particularly limited as long as the target nucleic acid can be amplified.
  • the number of bases of the amplification primer is preferably 15 to 60 bases, more preferably 25 to 40 bases. However, the sequence of the tag region not related to amplification of the amplification primer is not included as the number of bases.
  • the distance between the amplification primers, that is, the detection region is usually in the range of 50 to 5000 bases, preferably 100 to 2000 bases.
  • the sequence of the amplification primer is not particularly limited as long as the detection region can be amplified by the nucleic acid amplification method, and the Tm value is usually in the range of 50 ° C to 75 ° C, preferably in the range of 55 ° C to 68 ° C. .
  • the design of the amplification primer sequence may be performed manually, or an appropriate primer design software may be used. For example, Primer 3 software (http://frodo.wi.mit.edu) may be used.
  • the above buffer has an optimum pH and an optimum salt concentration at which the activity of the DNA polymerase can be obtained.
  • ribonuclease H RNase H
  • RT reverse transcriptase
  • various types of nucleic acid amplification reaction solutions are commercially available for reaction of each nucleic acid amplification method, and those attached to such commercially available kits may be used.
  • the solution after the disruption is mixed with the reaction solution for nucleic acid amplification.
  • the volume ratio is preferably about 5 to 50%, more preferably 10 to 30%, of the reaction solution for nucleic acid amplification.
  • the crushed solution may be subjected to centrifugation to precipitate impurities, and the supernatant may be used for mixing with the reaction solution.
  • the conditions, operations, etc. of the nucleic acid amplification reaction including PCR may be in accordance with conventional methods usually performed in this field.
  • the nucleic acid amplification reaction can be performed using commercially available equipment and reagents according to the instructions.
  • Step 3 Step of detecting the amplified target nucleic acid
  • the method for detecting the amplified target nucleic acid is not particularly limited as long as the amplification product can be detected.
  • a detection method using a strip for nucleic acid chromatography is preferred in that the reaction solution used in the PCR method or the like can be used as it is and the presence or absence of nucleic acid amplification can be confirmed quickly and easily.
  • nucleic acid chromatography is a method for detecting a target nucleic acid using chromatography, and a solid phase carrier in which a detection solution is immobilized on a specific region of a chromatography solution containing the target nucleic acid. And developing the hybridization product between the target nucleic acid and the detection probe, and detecting the hybridization product. Detection of the hybridized product is performed using a signal element imparted to the target nucleic acid prior to development (for example, during nucleic acid amplification) or at the time of development.
  • nucleic acid chromatography for example, described in JP2014-018150, JP2013-059319, JP2013-059320, JP2013-059321, JP2014-057565, JP2014-079260, and JP5503021 Can be used.
  • the embodiment of the nucleic acid chromatography is not particularly limited, and may be a so-called lateral flow type intended for development in a substantially horizontal state, or may be a so-called dipstick type intended for development in a substantially vertical direction. .
  • the solid phase carrier on which the detection probe is fixed is referred to as a strip.
  • a conventionally known strip can be used.
  • the strip may be plastic or glass, and the material is not particularly limited.
  • porous bodies such as a cellulose, a nitrocellulose, and nylon, may be sufficient. This type of porous body is particularly suitable for hybridization of the detection probe immobilized on the strip and the amplified fragment by affinity chromatography.
  • a typical strip is a product C-PAS manufactured by TBA. This strip is a test paper having a width of about 2 mm and a length of about 10 cm, and a nucleic acid is printed as a probe on a nitrocellulose film on the surface. Nucleic acid can be detected by using a primer having a tag that hybridizes complementary to this probe.
  • kits The present invention also provides a kit for use in the above-described method of the present invention (hereinafter also referred to as the kit of the present invention).
  • the kit of the present invention includes an instrument for physically crushing mammalian hair, and at least one of the above-described nucleic acid chromatography strip and primer set for nucleic acid amplification reaction.
  • the above crushing device the above-described devices can be used as appropriate.
  • the kit of the invention comprises the disruption device, the strip, and the primer set.
  • the kit of the present invention includes the strip
  • the kit preferably further includes a developing buffer.
  • this kit of this invention contains this primer set, it is preferable that this kit also contains the other components as mentioned above required in order to perform a nucleic acid amplification reaction.
  • Detection of hair-derived nucleic acids by the method of the present invention was carried out by the following procedure. Hereinafter, it demonstrates according to these order.
  • the detection in this example is visual confirmation using a nucleic acid chromatography strip, but the detection method is merely an example, and the detection can be performed by other methods.
  • Target nucleic acid detection reaction using nucleic acid chromatography
  • Sample Preparation Prepare human or animal hair samples. 1-1. Grinding the sample After washing the sample with ethanol to remove impurities attached to the sample, the sample is cut to an arbitrary length (at this time, the hair root is removed). The prepared sample is put in a 1.5 mL tube containing 20 ⁇ L of TE buffer, and is pulverized using BioMasher (registered trademark) II (manufactured by Nippi) to such an extent that the sample cannot be visually recognized. The sample preparation solution is used for the amplification reaction.
  • Amplification reaction of target nucleic acid 2-1. Preparation of reaction solution Using the sample preparation solution obtained in step 1, a nucleic acid amplification reaction is performed. The reaction solution is prepared in a 0.2 mL tube as shown in the following table. 1 ⁇ L of the supernatant is used for the amplification reaction. QIAGEN Multiplex PCR Mix (manufactured by QIAGEN) is used as a sample amplification reagent.
  • the primer sequence is not particularly limited as long as the target nucleic acid can be amplified.
  • PCR For PCR, a Veriti (registered trademark) thermal cycler (manufactured by Thermo Fisher Scientific) is used. A thermal cycle reaction is performed (9 minutes at 95 ° C., 30 cycles at 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, 72 ° C. for 30 seconds, then 72 ° C. for 5 minutes and then lowered to 4 ° C.).
  • Target Nucleic Acid Detection Reaction Detection is performed by a hybridization reaction by nucleic acid chromatography using a detection strip.
  • a strip manufactured by TBA was used (FIG. 1).
  • 3-1. Preparation of reaction solution The amplified nucleic acid obtained in step 2 is detected using nucleic acid chromatography.
  • the reaction solution was prepared as shown in the following table. TE buffer, developing solution (manufactured by Fujikura Kasei Co., Ltd.), and latex beads (manufactured by Fujikura Kasei Co., Ltd.) are used.
  • the composition of the reaction solution in one sample is as follows.
  • a nucleic acid chromatography strip is inserted into the above mixed reaction solution and allowed to stand for 40 minutes. If there is a target nucleic acid, it appears as a blue line on the strip, and therefore whether or not the target nucleic acid can be amplified is visually determined.
  • Example 1 In order to confirm the presence or absence of detection of target nucleic acid derived from human hair, three types of completely different hair were prepared and tested. Nucleic acid detection was performed according to the procedure of the experimental method. The sample was nucleic acid derived from human hair, one different human hair was prepared, the hair root was cut out, and the length was about 2 cm. N is a negative control. The detection results are shown in FIG. From this result, it was confirmed that the target nucleic acid could be detected from human-derived hair. Further, no line was detected from the N line, and it was confirmed that no reaction occurred in the absence of the sample.
  • Example 2 In order to confirm whether the detection success rate of nucleic acid derived from human hair changes depending on the collection site in the hair, two experiments were conducted. 47 cm of hair was prepared, and 9 types of 1 cm long hair including the hair root and 1 cm long hair at 5 cm intervals were prepared and tested (Sample 1). N is a negative control. 41 cm of hair was prepared, and eight types of 1 cm long hair were prepared at intervals of 5 cm excluding the roots (sample 2). N is a negative control (see FIG. 3). Nucleic acid detection was performed according to the procedure of the experimental method. The detection result is shown in FIG. From this result, it was confirmed that the target nucleic acid could be detected regardless of the presence or absence of the hair root or the hair site. Further, no line was detected from the N line, and it was confirmed that no reaction occurred in the absence of the sample.
  • Example 3 In order to confirm whether the detection success rate of nucleic acid derived from human hair is affected by the length of the hair, as shown in FIG. 5, the length of the hair is 0.5 to 4.0 cm from the tip of the hair in increments of 0.5 cm.
  • N is a negative control (see FIG. 5).
  • Nucleic acid detection was performed according to the procedure of the experimental method. The detection result is shown in FIG. From this result, it was confirmed that the target nucleic acid can be detected if the length of the hair is 0.5 cm or more. Further, no line was detected from the N line, and it was confirmed that no reaction occurred in the absence of the sample. The reason why the common primer line is thin when the hair length is 3.5 cm and 4.0 cm seems to be that the amount of hair is excessive and the amount of PCR-inhibiting substances increases accordingly.
  • Example 4 In order to confirm whether or not the target nucleic acid of animals other than humans can be detected, experiments were performed using human hair and animal hair of dogs and cats. Nucleic acid detection was performed according to the procedure of the experimental method. As positive controls, known DNAs of humans, dogs and cats were used. N is a negative control. The detection result is shown in FIG. The reaction was confirmed for the sample and the positive control. Further, no line was detected from the N line, and it was confirmed that no reaction occurred in the absence of the sample. From this result, it was confirmed that the species could be determined by detecting the target nucleic acid even when human hair and dog or cat animal hair were used. Furthermore, it was confirmed that human, dog and cat hair could be detected in multiplex.

Abstract

The present invention provides a method for detecting a nucleic acid derived from hair more easily than by using conventional technology. This method for detecting a target nucleic acid contained in mammalian animal hair comprises a step for crushing mammalian animal hair in a solution using a physical means, a step for amplifying the target nucleic acid by subjecting the solution after crushing to a nucleic acid amplification reaction, and a step for detecting the amplified target nucleic acid.

Description

毛由来の核酸の検出方法Method for detecting nucleic acid derived from hair
　本発明は、ヒトや動物の毛由来の核酸の検出技術に関する。 The present invention relates to a technique for detecting nucleic acids derived from human or animal hair.
従来技術Conventional technology
　ヒトや動物の毛由来の遺伝子の検査により種判別を行う技術が産業上求められている。状況によっては、たった一本の毛から種判別を行うことが求められる。 There is a need in the industry for techniques for species discrimination by examining genes derived from human and animal hair. Depending on the situation, it is required to perform species discrimination from only one hair.
　従来からの、一本の毛髪のような少量のサンプルから核酸を検出する手法としては、サンプルから核酸を抽出した後、抽出した核酸を精製し、核酸を増幅させるという手法が採られてきた（例えば、特許文献１、２）。さらなる技術として、毛を液体窒素で凍結させたり（特許文献３、４）、毛の状態を画像で確認したり（特許文献５）といった手法も採られている。 As a conventional technique for detecting nucleic acid from a small amount of sample such as a single hair, a technique has been adopted in which the nucleic acid is extracted from the sample, then the extracted nucleic acid is purified and the nucleic acid is amplified ( For example, Patent Documents 1 and 2). Further techniques such as freezing the hair with liquid nitrogen (Patent Documents 3 and 4) and confirming the state of the hair with an image (Patent Document 5) are also employed.
　しかしながら、少量のサンプルにおいては、これらの作業は煩雑であり、サンプルから核酸を確実に取得することに不安があった。そのため、より簡易的に核酸検出を可能とする技術が求められている。 However, for a small amount of sample, these operations are complicated, and there was anxiety in reliably obtaining nucleic acid from the sample. Therefore, a technique that enables nucleic acid detection more simply is required.
特許第5501570号公報Japanese Patent No. 5501570 特開2015-73478号公報JP-A-2015-73478 特開2009-131253号公報JP 2009-131253 A 特開2007-20559号公報Japanese Unexamined Patent Publication No. 2007-20559 特許第4356783号公報Japanese Patent No.4356783
　核酸検出技術においては、サンプルから核酸を取得することが技術常識であり、サンプルから核酸を抽出、精製、回収するといった段階を経ることで、核酸を増幅し、増幅した核酸を検出している。
　しかしながら、一本の毛髪のような少量のサンプルから回収できる核酸はごく微量である。エタノール沈殿のような抽出処理や夾雑物を除くフィルトレーションなどのサンプルから核酸を回収する処理を行うと、核酸がせん断される、あるいは核酸が消失してしまうことがあり得る。
　従来技術としては、化学的処理を用いたり、破砕方法を工夫するなどして、サンプルから核酸を回収する手法がとられてきており、工程そのものが見直されることはなかった。 In the nucleic acid detection technology, it is common knowledge to acquire nucleic acid from a sample, and the nucleic acid is amplified by detecting, purifying, and collecting the nucleic acid from the sample, and the amplified nucleic acid is detected.
However, only a very small amount of nucleic acid can be recovered from a small sample such as a single hair. When nucleic acid is collected from a sample such as extraction treatment such as ethanol precipitation or filtration that removes contaminants, the nucleic acid may be sheared or the nucleic acid may be lost.
As a conventional technique, a method of recovering nucleic acid from a sample by using chemical treatment or devising a crushing method has been taken, and the process itself has not been reviewed.
　本発明は当該技術分野の上記現状に鑑み為されたものであり、従来技術よりも簡易に毛由来の核酸を検出するための方法を提供することをその目的とする。 The present invention has been made in view of the above-mentioned present state of the art, and an object thereof is to provide a method for detecting a nucleic acid derived from hair more easily than conventional techniques.
　本発明者らは、上記課題を解決するために鋭意検討を行い、核酸の抽出、精製、回収といった処理を行わず、工程そのものを簡略化することを着想した。具体的には、本発明者らは、毛髪を含むヒトや動物の毛を化学的処理を行うことなく液中で物理的手法により破砕し、その破砕液を用いて核酸の増幅反応を行い、増幅した核酸を検出することが可能であることを見出した。本発明者らは、かかる発見に基づいてさらに検討を進め、本発明を完成させるに至った。 The present inventors have intensively studied to solve the above problems, and have come up with the idea of simplifying the process itself without performing processing such as extraction, purification, and recovery of nucleic acids. Specifically, the present inventors crushed human or animal hair including hair by a physical technique in a liquid without performing a chemical treatment, and performed a nucleic acid amplification reaction using the crushed liquid. It has been found that amplified nucleic acids can be detected. The present inventors have further studied based on this discovery, and have completed the present invention.
　本発明は即ち、以下を提供する。
［１］哺乳動物の毛の中の標的核酸を検出する方法であって、
　哺乳動物の毛を溶液中で物理的手段により破砕する工程、
　破砕後の溶液を核酸増幅反応に供することにより標的核酸を増幅させる工程、および、
　増幅された該標的核酸を検出する工程
を含む、前記方法。
［２］核酸を抽出する工程が行われない、上記［１］に記載の方法。
［３］該毛が、毛根を含まない毛の断片である、上記［１］または［２］に記載の方法。
［４］該標的核酸の検出が、核酸クロマトグラフィー用ストリップを用いて行われる、上記［１］～［３］のいずれかに記載の方法。
［５］該核酸増幅反応がポリメラーゼ連鎖反応である、上記［１］～［４］のいずれかに記載の方法。
［６］哺乳動物の毛を物理的に破砕するための器具、ならびに、
　核酸クロマトグラフィー用ストリップおよび核酸増幅反応用プライマーセットの少なくとも１つ
を含む、上記［１］～［５］のいずれかに記載の方法に用いるためのキット。 The present invention provides the following.
[1] A method for detecting a target nucleic acid in mammalian hair,
Crushing mammalian hair in solution by physical means,
A step of amplifying a target nucleic acid by subjecting the disrupted solution to a nucleic acid amplification reaction; and
Detecting the amplified target nucleic acid.
[2] The method according to [1] above, wherein the step of extracting nucleic acid is not performed.
[3] The method according to [1] or [2] above, wherein the hair is a hair fragment not containing a hair root.
[4] The method according to any one of [1] to [3] above, wherein the detection of the target nucleic acid is performed using a strip for nucleic acid chromatography.
[5] The method according to any one of [1] to [4] above, wherein the nucleic acid amplification reaction is a polymerase chain reaction.
[6] An instrument for physically crushing mammalian hair, and
A kit for use in the method according to any one of [1] to [5] above, comprising at least one of a strip for nucleic acid chromatography and a primer set for nucleic acid amplification reaction.
　本発明によれば、毛由来の核酸の検出において、サンプルから核酸を抽出・精製・回収する一連の工程をなくすることにより、煩雑な作業を行う必要がなく、検出に要する時間も短縮することができる。 According to the present invention, in detecting nucleic acid derived from hair, by eliminating a series of steps for extracting, purifying, and recovering nucleic acid from a sample, it is not necessary to perform complicated operations and shorten the time required for detection. Can do.
実施例で用いた核酸検出用ストリップを説明する図である。It is a figure explaining the strip for nucleic acid detection used in the Example. 実施例１における核酸検出用ストリップを用いた核酸の検出の結果を示す図である。FIG. 3 is a diagram showing the results of nucleic acid detection using the nucleic acid detection strip in Example 1. 実施例２で用いたサンプルを説明する図である。It is a figure explaining the sample used in Example 2. FIG. 実施例２における核酸検出用ストリップを用いた核酸の検出の結果を示す図である。FIG. 4 is a diagram showing the results of nucleic acid detection using a nucleic acid detection strip in Example 2. 実施例３で用いたサンプルを説明する図である。6 is a diagram illustrating a sample used in Example 3. FIG. 実施例３における核酸検出用ストリップを用いた核酸の検出の結果を示す図である。It is a figure which shows the result of the detection of the nucleic acid using the strip for nucleic acid detection in Example 3. 実施例４における核酸検出用ストリップを用いた核酸の検出の結果を示す図である。It is a figure which shows the result of the detection of the nucleic acid using the strip for nucleic acid detection in Example 4.
（定義）
　本発明において、哺乳動物の種類は特に限定されず、例えば、マウス、ラット、ハムスター、モルモット等のげっ歯類やウサギ等の実験動物、ブタ、ウシ、ヤギ、ウマ、ヒツジ、ミンク等の家畜、イヌ、ネコ等のペット、ヒト、サル、アカゲザル、マーモセット、オランウータン、チンパンジーなどの霊長類等を挙げることができ、好ましくはヒトである。 (Definition)
In the present invention, the type of mammal is not particularly limited. Examples include pets such as dogs and cats, primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees, and the like, preferably humans.
　毛の採取部位は特に限定されず、毛髪、眉毛、鼻毛、ひげ、陰毛、腋毛を含むあらゆる部位の毛を本発明では使用することができる。毛は、哺乳動物の身体表面において、根元から毛を引き抜くか、あるいは毛を切除することにより採取することができる。あるいは、自然に脱落した毛を用いてもよい。
　毛は、毛根から先端までの全長であってもよいし、その断片であってもよい。断片の位置は限定されない。毛は毛根を含んでいてもよいし、含んでいなくてもよい。
　後述の物理的破砕処理に供する毛は、長さが、通常０．１～１０．０ｃｍであり、好ましくは０．５～４．０ｃｍであり、より好ましくは１．０～２．０ｃｍである。例えば、実施例で用いるような破砕器具による破砕のためには、１ｃｍ程度の長さの毛が使いやすい。 The hair collection site is not particularly limited, and any hair including hair, eyebrows, nasal hair, whiskers, pubic hair, and eyelashes can be used in the present invention. The hair can be collected by drawing the hair from the root or excising the hair on the surface of the mammalian body. Or you may use the hair which fell off naturally.
The full length from the hair root to the tip may be sufficient as the hair, and the fragment | piece may be sufficient as it. The position of the fragment is not limited. The hair may or may not contain a hair root.
The hair to be subjected to the physical crushing process described later has a length of usually 0.1 to 10.0 cm, preferably 0.5 to 4.0 cm, more preferably 1.0 to 2.0 cm. . For example, a hair having a length of about 1 cm is easy to use for crushing with a crushing instrument as used in the examples.
　標的核酸は、検出の対象とするものであって、その種類は特に限定されない。核酸はＤＮＡであってもＲＮＡであってもよいが、好ましくはＤＮＡである。標的核酸としては、その発現が、特定の疾患または状態の発症やそのリスク、治療感受性、あるいは特定の体質などに関連することが知られているものが挙げられる。上記の疾患または状態としては、癌、生活習慣病（例えば、脂質異常症、高血圧、糖尿病、肥満）、薬の副作用などが挙げられ、上記の体質としては、アルコール耐性などが挙げられる。例えば、野生型とは異なる塩基配列を有する遺伝子多型（ＳＮＰ）は、特定の疾患または状態の発症やそのリスク、治療感受性、あるいは特定の体質などに関連することが知られている。従って、好ましい遺伝子としては、そのようなＳＮＰを有する遺伝子が挙げられる。具体的な遺伝子としては、例えば、アルコール耐性に関与するアルコール脱水素酵素遺伝子（ＡＤＨ１Ｂ）やアセトアルデヒド脱水素酵素（ＡＬＤＨ２）などが挙げられるが、これらに限定されない。
　あるいは、遺伝子検査による動物種の判別のために、動物種によって塩基配列が異なる、配列が既知のＤＮＡを標的核酸としてもよい。種判別のために用い得るＤＮＡとしては、ミトコンドリアＤＮＡなどが挙げられる。 The target nucleic acid is to be detected, and the type thereof is not particularly limited. The nucleic acid may be DNA or RNA, but is preferably DNA. Examples of the target nucleic acid include those whose expression is known to be related to the onset and risk of a specific disease or condition, therapeutic sensitivity, or a specific constitution. Examples of the disease or condition include cancer, lifestyle-related diseases (for example, dyslipidemia, hypertension, diabetes, obesity), side effects of drugs, and the constitution includes alcohol tolerance. For example, it is known that a gene polymorphism (SNP) having a nucleotide sequence different from the wild type is related to the onset of a specific disease or condition, its risk, therapeutic sensitivity, or a specific constitution. Therefore, preferable genes include genes having such SNPs. Specific examples of the gene include, but are not limited to, an alcohol dehydrogenase gene (ADH1B) and acetaldehyde dehydrogenase (ALDH2) involved in alcohol resistance.
Alternatively, for discrimination of animal species by genetic testing, a DNA having a known sequence that has a different base sequence depending on the animal species may be used as the target nucleic acid. Examples of DNA that can be used for species discrimination include mitochondrial DNA.
　また、標的核酸は、１つであってもよいし、２つ以上であってもよい。本発明の一つの実施形態によれば、複数の標的核酸をマルチプレックスの核酸増幅反応により増幅させ、増幅された該複数の標的核酸を一括に検出することもできる。 Further, the target nucleic acid may be one, or two or more. According to one embodiment of the present invention, a plurality of target nucleic acids can be amplified by a multiplex nucleic acid amplification reaction, and the plurality of amplified target nucleic acids can be detected at once.
　本明細書において、毛の物理的手段による破砕または物理的破砕とは、破砕に用いる器具と毛との間の物理的な接触を伴う毛の断片化処理をいう。破砕は、均質化（ホモジナイズ）、せん断、またはすりつぶしなどにより行うことができる。物理的破砕のための手段は特に限定されず、ホモジナイザーなどの機器を適宜用いることや、液体窒素で毛を凍結し、乳鉢や手動式ミルなどを用いて破砕することができる。例えば破砕器具などの使い捨て型ホモジナイザーは簡便に使用できるため、該手段として好ましく使用できる。使い捨て型ホモジナイザーの使用は手動で行ってもよいし、市販されている専用のグラインダーを用いてもよい。 In this specification, crushing by physical means of hair or physical crushing refers to a hair fragmentation process involving physical contact between a device used for crushing and hair. The crushing can be performed by homogenization, shearing or grinding. The means for physical crushing is not particularly limited, and a device such as a homogenizer can be used as appropriate, or the hair can be frozen with liquid nitrogen and crushed using a mortar, manual mill, or the like. For example, a disposable homogenizer such as a crushing device can be used conveniently and can be preferably used as the means. The disposable homogenizer may be used manually, or a commercially available dedicated grinder may be used.
（核酸検出方法）
　本発明は、哺乳動物の毛の中の標的核酸を検出する方法（以下、本発明の方法ともいう。）を提供する。本発明の方法は、
（工程１）哺乳動物の毛を溶液中で物理的手段により破砕する工程、
（工程２）破砕後の溶液を核酸増幅反応に供することにより標的核酸を増幅させる工程、および、
（工程３）増幅された該標的核酸を検出する工程
を含む。本発明の方法は、好ましくは、核酸を抽出または精製する工程を含まない。また、本発明の方法は、好ましくは、化学的処理により毛を分解する工程を含まない。 (Nucleic acid detection method)
The present invention provides a method for detecting a target nucleic acid in mammalian hair (hereinafter also referred to as the method of the present invention). The method of the present invention comprises:
(Step 1) crushing mammalian hair in solution by physical means,
(Step 2) Amplifying the target nucleic acid by subjecting the disrupted solution to a nucleic acid amplification reaction, and
(Step 3) including a step of detecting the amplified target nucleic acid. The method of the present invention preferably does not include the step of extracting or purifying the nucleic acid. In addition, the method of the present invention preferably does not include a step of decomposing hair by chemical treatment.
（工程１）哺乳動物の毛を溶液中で物理的手段により破砕する工程
　破砕は、上述したような物理的破砕用の手段を用いて行うことができる。破砕は、例えば、目視で毛を視認できなくなる程度まで行えばよい。また、破砕の前に、エタノールなどを用いて毛のサンプルに付着した夾雑物を除去してもよい。 (Step 1) Step of crushing mammalian hair in solution by physical means Crushing can be carried out using the means for physical crushing as described above. For example, the crushing may be performed to such an extent that the hair cannot be visually recognized. Further, before crushing, impurities attached to the hair sample may be removed using ethanol or the like.
　上記の溶液は、本発明の方法による標的核酸の検出を有意に阻害しない限り任意の溶液であってよく、好ましくは、緩衝作用を有する液体である。後述の工程２における核酸増幅反応のサンプルとして、上記破砕処理後の溶液を用いるので、該溶液は後述の核酸増幅反応において用いる緩衝液（バッファー）と同じものであることが好ましい。１０～４００μＬ程度の溶液中に０．５～５ｃｍ程度の毛を入れ、破砕処理を行うことが好ましい。また、破砕後に、遠心分離により夾雑物を沈殿させ、除去してもよい。 The above solution may be any solution as long as it does not significantly inhibit the detection of the target nucleic acid by the method of the present invention, and is preferably a liquid having a buffering action. Since the solution after the crushing treatment is used as a sample for the nucleic acid amplification reaction in Step 2 described later, the solution is preferably the same as the buffer solution (buffer) used in the nucleic acid amplification reaction described later. It is preferable to put about 0.5 to 5 cm of hair in a solution of about 10 to 400 μL and perform crushing treatment. Further, after crushing, impurities may be precipitated and removed by centrifugation.
（工程２）破砕後の溶液を核酸増幅反応に供することにより標的核酸を増幅させる工程
　核酸増幅反応の方法としては、標的核酸を増幅できるものである限り制限されない。本明細書において、標的核酸の増幅とは、必ずしも標的核酸（例えば標的遺伝子）の全体を増幅するものでなくてもよく、少なくとも該核酸の検出対象領域を増幅できればよい。核酸増幅反応のための方法としては、例えば、ポリメラーゼ連鎖反応（ＰＣＲ）法（最も基本的な原理に基づくＰＣＲ法のみならず、それを基にした各種改良法を含む。）、ＴＭＡ（Transcription-Mediated Amplification）法、ＮＡＳＢＡ（Nucleic Acid Sequence-Based Amplification）法、ＬＣＲ（Ligase Chain Reaction）法、ＣＬＲ（Cycling Ligation Reaction；特開2016-140287、特開2016-140288に記載の技術）法などを用いることができる。ＰＣＲ法は最も広く用いられている核酸増幅法であることから当該方法に最適化された様々な試薬や機器が容易に入手できるだけでなく、上記の通り様々なバリエーションの方法があり、汎用性が極めて高いため、本発明のために好適な手法として挙げられる。 (Step 2) Step of amplifying the target nucleic acid by subjecting the disrupted solution to a nucleic acid amplification reaction The method of the nucleic acid amplification reaction is not limited as long as the target nucleic acid can be amplified. In this specification, amplification of a target nucleic acid does not necessarily amplify the entire target nucleic acid (for example, a target gene), as long as at least the detection target region of the nucleic acid can be amplified. As a method for nucleic acid amplification reaction, for example, polymerase chain reaction (PCR) method (including not only PCR method based on the most basic principle but also various improved methods based on it), TMA (Transcription- Mediated Amplification (NASBA) method, NASBA (Nucleic Acid Sequence-Based Amplification) method, LCR (Ligase Chain Reaction) method, CLR (Cycling Ligation Reaction; technology described in JP2016-140287, JP2016-140288), etc. are used. be able to. Since the PCR method is the most widely used nucleic acid amplification method, not only can various reagents and equipment optimized for the method be easily obtained, but there are various variations of the method as described above. Since it is extremely high, it is mentioned as a suitable technique for the present invention.
　核酸増幅用反応液とは、前記核酸増幅反応を行う上で必要な試薬等を含有する溶液である。核酸増幅用反応液の組成は、使用する核酸増幅法によって多少異なるが、通常は基質としての４種のデオキシヌクレオシド三リン酸（ｄＡＴＰ、ｄＴＴＰ、ｄＣＴＰ、ｄＧＴＰ：以下まとめてｄＮＴＰとする）と、酵素としてのＤＮＡポリメラーゼと、前記酵素の補因子としてのマグネシウムイオンと、核酸増幅反応用プライマーセットと、をバッファー中に含むことを基本とする。ｄＮＴＰの濃度は、４種の各終濃度が１００～４００μＭの範囲で最適な濃度を検討すればよい。ＤＮＡポリメラーゼは、用いる核酸増幅法に適する性質を有したものを使用する。例えば、ＰＣＲ法であれば、Ｔａｑ　ＤＮＡポリメラーゼ、Ｐｆｕ　ＤＮＡポリメラーゼ、その他バイオサイエンス関連の各社により開発されたもの等の耐熱性ＤＮＡポリメラーゼが好ましく使用される。マグネシウムイオン濃度は、終濃度が１～６ｍＭの範囲で最適な濃度を検討すればよい。 The nucleic acid amplification reaction solution is a solution containing reagents and the like necessary for performing the nucleic acid amplification reaction. The composition of the reaction solution for nucleic acid amplification differs somewhat depending on the nucleic acid amplification method to be used, but usually four types of deoxynucleoside triphosphates (dATP, dTTP, dCTP, dGTP: hereinafter collectively referred to as dNTP) as substrates, Basically, a DNA polymerase as an enzyme, magnesium ions as a cofactor of the enzyme, and a primer set for nucleic acid amplification reaction are contained in a buffer. As for the concentration of dNTP, an optimum concentration may be examined in the range where each of the four final concentrations is in the range of 100 to 400 μM. A DNA polymerase having a property suitable for the nucleic acid amplification method to be used is used. For example, in the case of the PCR method, Taq DNA polymerase, Pfu DNA polymerase, and other heat-resistant DNA polymerases developed by other bioscience-related companies are preferably used. As for the magnesium ion concentration, an optimum concentration may be examined within a final concentration range of 1 to 6 mM.
　核酸増幅反応用プライマーセットに含まれる増幅プライマーは、ＤＮＡ、ＲＮＡ等の核酸や修飾核酸、またはそれらの組み合わせによって構成される。増幅プライマーの濃度は、終濃度が１０ｎＭから２μＭの範囲で最適な濃度を検討すればよい。増幅プライマーの数は、標的核酸を増幅可能であれば特に問わない。増幅プライマーの塩基数は、好ましくは１５～６０塩基であり、より好ましくは２５～４０塩基である。ただし、増幅プライマーの増幅に関係しないタグ領域の配列は塩基数として含まない。増幅プライマー間の距離、即ち検出領域は通常５０～５０００塩基の範囲であり、好ましくは１００～２０００塩基である。増幅プライマーの配列は、検出領域を前記核酸増幅法によって増幅可能であり、Ｔｍ値が通常５０℃から７５℃の範囲内であり、好ましくは５５℃から６８℃の範囲内であれば特に問わない。増幅プライマーの配列のデザインは、マニュアルで行ってもよいし、適当なプライマーデザイン用のソフトウェアを用いてもよい。例えば、Ｐｒｉｍｅｒ３ソフトウェア（http://frodo.wi.mit.edu）等を用いてもよい。 The amplification primer included in the primer set for nucleic acid amplification reaction is composed of nucleic acids such as DNA and RNA, modified nucleic acids, or combinations thereof. What is necessary is just to examine the density | concentration of an amplification primer in the range whose final concentration is 10 nM to 2 micromol. The number of amplification primers is not particularly limited as long as the target nucleic acid can be amplified. The number of bases of the amplification primer is preferably 15 to 60 bases, more preferably 25 to 40 bases. However, the sequence of the tag region not related to amplification of the amplification primer is not included as the number of bases. The distance between the amplification primers, that is, the detection region is usually in the range of 50 to 5000 bases, preferably 100 to 2000 bases. The sequence of the amplification primer is not particularly limited as long as the detection region can be amplified by the nucleic acid amplification method, and the Tm value is usually in the range of 50 ° C to 75 ° C, preferably in the range of 55 ° C to 68 ° C. . The design of the amplification primer sequence may be performed manually, or an appropriate primer design software may be used. For example, Primer 3 software (http://frodo.wi.mit.edu) may be used.
　上記のバッファーは、前記ＤＮＡポリメラーゼの活性が得られる至適ｐＨ、および至適塩濃度を有する。使用する核酸増幅法によっては、更にリボヌクレアーゼＨ（ＲＮａｓｅＨ）や逆転写酵素（ＲＴ）等を含んでいてもよい。また、核酸増幅用反応液は、各核酸増幅法の反応用として様々なタイプが市販されており、そのような市販のキットに添付されたものを用いてもよい。 The above buffer has an optimum pH and an optimum salt concentration at which the activity of the DNA polymerase can be obtained. Depending on the nucleic acid amplification method to be used, ribonuclease H (RNase H), reverse transcriptase (RT), etc. may be further included. In addition, various types of nucleic acid amplification reaction solutions are commercially available for reaction of each nucleic acid amplification method, and those attached to such commercially available kits may be used.
　核酸増幅反応のテンプレートを含むサンプルとして、上記破砕後の溶液が核酸増幅用反応液と混合される。容量比として、核酸増幅用反応液に対して５～５０％程度、より好ましくは１０～３０％の上記破砕後の溶液を混合することが好ましい。また、破砕後の溶液を遠心処理に供して夾雑物を沈殿させた後、その上清を反応液との混合に用いてもよい。 As the sample containing the template for nucleic acid amplification reaction, the solution after the disruption is mixed with the reaction solution for nucleic acid amplification. The volume ratio is preferably about 5 to 50%, more preferably 10 to 30%, of the reaction solution for nucleic acid amplification. Alternatively, the crushed solution may be subjected to centrifugation to precipitate impurities, and the supernatant may be used for mixing with the reaction solution.
　ＰＣＲを含む核酸増幅反応の条件、操作等は、この分野で通常行われている常法に従えばよい。また、核酸増幅反応は、市販の機器や試薬を用い、その説明書に従って行うことができる。 The conditions, operations, etc. of the nucleic acid amplification reaction including PCR may be in accordance with conventional methods usually performed in this field. The nucleic acid amplification reaction can be performed using commercially available equipment and reagents according to the instructions.
（工程３）増幅された該標的核酸を検出する工程
　増幅された標的核酸の検出の方法は、該増幅産物を検出できるものであれば特に限定されない。ＰＣＲ法などに用いた反応液をそのまま利用し、核酸増幅の有無を迅速かつ簡便に確認できるという点で、核酸クロマトグラフィー用ストリップを用いた検出方法が好ましい。 (Step 3) Step of detecting the amplified target nucleic acid The method for detecting the amplified target nucleic acid is not particularly limited as long as the amplification product can be detected. A detection method using a strip for nucleic acid chromatography is preferred in that the reaction solution used in the PCR method or the like can be used as it is and the presence or absence of nucleic acid amplification can be confirmed quickly and easily.
　本明細書において、核酸クロマトグラフィーとは、クロマトグラフィーを用いて標的核酸を検出するための方法であって、標的核酸を含むクロマトグラフィー溶液を、特定の領域に検出用プローブを固定した固相担体の内部または表面上を展開させて、標的核酸と検出用プローブとのハイブリダイズ産物を形成させ、ハイブリダイズ産物を検出することを含む。ハイブリダイズ産物の検出は、展開に先立って（例えば、核酸増幅時に）または展開の際に、標的核酸に対して付与したシグナル要素を利用して行う。核酸クロマトグラフィーとして、例えば、特開2014-018150、特開2013-059319、特開2013-059320、特開2013-059321、特開2014-057565、特開2014-079260、特許第5503021号などに記載の技術を用いることができる。核酸クロマトグラフィーの実施形態は特に限定されず、ほぼ水平状態での展開を意図したいわゆるラテラルフロー型であってもよいし、ほぼ鉛直方向での展開を意図したいわゆるディップスティック型であってもよい。 In this specification, nucleic acid chromatography is a method for detecting a target nucleic acid using chromatography, and a solid phase carrier in which a detection solution is immobilized on a specific region of a chromatography solution containing the target nucleic acid. And developing the hybridization product between the target nucleic acid and the detection probe, and detecting the hybridization product. Detection of the hybridized product is performed using a signal element imparted to the target nucleic acid prior to development (for example, during nucleic acid amplification) or at the time of development. As nucleic acid chromatography, for example, described in JP2014-018150, JP2013-059319, JP2013-059320, JP2013-059321, JP2014-057565, JP2014-079260, and JP5503021 Can be used. The embodiment of the nucleic acid chromatography is not particularly limited, and may be a so-called lateral flow type intended for development in a substantially horizontal state, or may be a so-called dipstick type intended for development in a substantially vertical direction. .
　本明細書において、検出用プローブを固定した上記の固相担体をストリップと呼称する。ストリップは従来公知のものを用いることができる。例えば、ストリップはプラスチックであってもよいし、ガラスであってもよく、材質は特に限定されない。また、セルロース、ニトロセルロース、ナイロン等の多孔質体であってもよい。この種の多孔質体は、特に、アフィニティークロマトグラフィーにより、ストリップに固定化した検出用プローブと増幅断片とをハイブリダイゼーションさせるのに好適である。また、代表的なストリップとしては、ＴＢＡ社製の商品Ｃ-ＰＡＳがあげられる。このストリップは、幅２ｍｍ×長さ１０ｃｍ程度の検査試験紙であり、表面のニトロセルロース膜上に核酸をプローブとしてプリントしたものである。このプローブと相補的にハイブリダイゼーションするタグを持つプライマーをセットで用いることで核酸の検出が可能となる。 In the present specification, the solid phase carrier on which the detection probe is fixed is referred to as a strip. A conventionally known strip can be used. For example, the strip may be plastic or glass, and the material is not particularly limited. Moreover, porous bodies, such as a cellulose, a nitrocellulose, and nylon, may be sufficient. This type of porous body is particularly suitable for hybridization of the detection probe immobilized on the strip and the amplified fragment by affinity chromatography. A typical strip is a product C-PAS manufactured by TBA. This strip is a test paper having a width of about 2 mm and a length of about 10 cm, and a nucleic acid is printed as a probe on a nitrocellulose film on the surface. Nucleic acid can be detected by using a primer having a tag that hybridizes complementary to this probe.
（キット）
　本発明はまた、上述した本発明の方法に用いるためのキット（以下、本発明のキットともいう。）も提供する。本発明のキットは、哺乳動物の毛を物理的に破砕するための器具、ならびに、上述の核酸クロマトグラフィー用ストリップおよび核酸増幅反応用プライマーセットの少なくとも一つを含む。上記の破砕器具は、上述したような器具を適宜用いることができる。一実施形態において、本発明のキットは、該破砕器具、該ストリップ、および該プライマーセットを含む。本発明のキットが該ストリップを含む場合、該キットは展開用緩衝液をさらに含むことが好ましい。また、本発明のキットが該プライマーセットを含む場合、該キットは、核酸増幅反応を行うために必要な、上述したようなその他の構成要素も含むことが好ましい。 (kit)
The present invention also provides a kit for use in the above-described method of the present invention (hereinafter also referred to as the kit of the present invention). The kit of the present invention includes an instrument for physically crushing mammalian hair, and at least one of the above-described nucleic acid chromatography strip and primer set for nucleic acid amplification reaction. As the above crushing device, the above-described devices can be used as appropriate. In one embodiment, the kit of the invention comprises the disruption device, the strip, and the primer set. When the kit of the present invention includes the strip, the kit preferably further includes a developing buffer. Moreover, when the kit of this invention contains this primer set, it is preferable that this kit also contains the other components as mentioned above required in order to perform a nucleic acid amplification reaction.
　以下に実施例を挙げて本発明をより詳細に説明するが、本発明は以下の実施例により限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.
　本発明の方法による毛由来の核酸の検出を以下の手順で行った。以下、これらの順序に従って説明する。なお、本実施例での検出は、核酸クロマトグラフィーのストリップを用いた目視による確認であるが、検出方法はあくまでも一例であり、その他の方法でも検出は可能である。
手順：
　１．サンプルの調製
　２．標的核酸の増幅反応
　３．標的核酸の検出反応（核酸クロマトグラフィーを使用） Detection of hair-derived nucleic acids by the method of the present invention was carried out by the following procedure. Hereinafter, it demonstrates according to these order. The detection in this example is visual confirmation using a nucleic acid chromatography strip, but the detection method is merely an example, and the detection can be performed by other methods.
procedure:
1. Sample preparation 2. Amplification reaction of target nucleic acid Target nucleic acid detection reaction (using nucleic acid chromatography)
１．サンプル調製
　ヒトまたは動物の毛のサンプルを調製する。
１－１．サンプルの粉砕
　サンプルをエタノールで洗浄し、サンプルに付着した夾雑物を除去した後、サンプルを任意の長さに切り取る（この時、毛根部は除去）。ＴＥバッファー２０μＬを入れた１．５ｍＬチューブに調製したサンプルを入れ、バイオマッシャー（登録商標）ＩＩ（ニッピ社製）を用いて、サンプルが視認できない程度まで粉砕する。そのサンプル調製液を増幅反応に用いる。 1. Sample Preparation Prepare human or animal hair samples.
1-1. Grinding the sample After washing the sample with ethanol to remove impurities attached to the sample, the sample is cut to an arbitrary length (at this time, the hair root is removed). The prepared sample is put in a 1.5 mL tube containing 20 μL of TE buffer, and is pulverized using BioMasher (registered trademark) II (manufactured by Nippi) to such an extent that the sample cannot be visually recognized. The sample preparation solution is used for the amplification reaction.
２．標的核酸の増幅反応
２－１．反応液の調製
　１の工程で得たサンプル調製液を用いて核酸の増幅反応を行う。反応液は０．２ｍＬチューブに下記表の通りに調製する。調製液は上清１μＬを増幅反応に用いる。サンプル増幅試薬としてＱＩＡＧＥＮ　Ｍｕｌｔｉｐｌｅｘ　ＰＣＲ　Ｍｉｘ（ＱＩＡＧＥＮ社製）を用いる。 2. 2. Amplification reaction of target nucleic acid 2-1. Preparation of reaction solution Using the sample preparation solution obtained in step 1, a nucleic acid amplification reaction is performed. The reaction solution is prepared in a 0.2 mL tube as shown in the following table. 1 μL of the supernatant is used for the amplification reaction. QIAGEN Multiplex PCR Mix (manufactured by QIAGEN) is used as a sample amplification reagent.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　プライマー配列は、標的とする核酸を増幅できる限り、特に制限されない。
２－２．ＰＣＲ
　ＰＣＲにはＶｅｒｉｔｉ（登録商標）サーマルサイクラー（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製）を用いる。サーマルサイクル反応（９５℃で９分後、９４℃で３０秒、６０℃で３０秒、７２℃で３０秒を３０サイクル、その後７２℃で５分後、４℃に下げる）を行う。 The primer sequence is not particularly limited as long as the target nucleic acid can be amplified.
2-2. PCR
For PCR, a Veriti (registered trademark) thermal cycler (manufactured by Thermo Fisher Scientific) is used. A thermal cycle reaction is performed (9 minutes at 95 ° C., 30 cycles at 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, 72 ° C. for 30 seconds, then 72 ° C. for 5 minutes and then lowered to 4 ° C.).
３．標的核酸の検出反応
　検出用のストリップを用いて、核酸クロマトグラフィーによるハイブリダイゼーション反応によって検出を行う。ストリップはＴＢＡ社製のものを使用した（図１）。
３－１．反応液の調製
　２の工程で得られた増幅核酸を、核酸クロマトグラフィーを用いて検出する。反応液は下記表の通りに調製した。ＴＥバッファー、展開液（藤倉化成社製）、ラテックスビーズ（藤倉化成社製）を用いる。１サンプルにおける反応液の組成は下記の通りである。 3. Target Nucleic Acid Detection Reaction Detection is performed by a hybridization reaction by nucleic acid chromatography using a detection strip. A strip manufactured by TBA was used (FIG. 1).
3-1. Preparation of reaction solution The amplified nucleic acid obtained in step 2 is detected using nucleic acid chromatography. The reaction solution was prepared as shown in the following table. TE buffer, developing solution (manufactured by Fujikura Kasei Co., Ltd.), and latex beads (manufactured by Fujikura Kasei Co., Ltd.) are used. The composition of the reaction solution in one sample is as follows.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　反応液を１．５ｍＬチューブに３０．０μＬ加え、５０℃で５分間以上加温する。その後、ＰＣＲ反応液１０．０μＬをこの反応液に加えて混合し、２分間静置する。
３－２．標的核酸の確認
　上記の混合反応液に核酸クロマトグラフィーストリップを挿入し、４０分間静置する。標的の核酸があればストリップ上に青いラインとして現れるので、標的核酸の増幅可否を目視で判定する。 Add 30.0 μL of the reaction solution to a 1.5 mL tube and warm at 50 ° C. for 5 minutes or more. Thereafter, 10.0 μL of the PCR reaction solution is added to the reaction solution, mixed, and allowed to stand for 2 minutes.
3-2. Confirmation of target nucleic acid A nucleic acid chromatography strip is inserted into the above mixed reaction solution and allowed to stand for 40 minutes. If there is a target nucleic acid, it appears as a blue line on the strip, and therefore whether or not the target nucleic acid can be amplified is visually determined.
実施例１
　ヒトの毛髪由来の標的核酸の検出の有無を確認するため、全く別の由来の毛髪を３式用意して実験を行った。
　実験方法の手順の通りに核酸検出を行った。サンプルはヒトの毛髪由来の核酸とし、１本の異なるヒトの毛髪を用意し、毛根を切り取り、長さを２ｃｍ程度にした。また、Ｎはネガティブコントロールである。
　検出結果を図２に示す。この結果から、ヒト由来の毛髪から標的核酸が検出できることを確認できた。また、Ｎのラインからはラインが検出されず、サンプルがない状態では反応しないことが確認できた。 Example 1
In order to confirm the presence or absence of detection of target nucleic acid derived from human hair, three types of completely different hair were prepared and tested.
Nucleic acid detection was performed according to the procedure of the experimental method. The sample was nucleic acid derived from human hair, one different human hair was prepared, the hair root was cut out, and the length was about 2 cm. N is a negative control.
The detection results are shown in FIG. From this result, it was confirmed that the target nucleic acid could be detected from human-derived hair. Further, no line was detected from the N line, and it was confirmed that no reaction occurred in the absence of the sample.
実施例２
　ヒトの毛髪由来の核酸の検出成功率が、毛髪中の採取部位によって変化するかを確認するため、２式の実験を行った。４７ｃｍの毛髪を用意し、毛根を含む１ｃｍ長の毛髪と５ｃｍ間隔に１ｃｍ長の毛髪を９式用意して実験を行った（サンプル１）。また、Ｎはネガティブコントロールである。４１ｃｍの毛髪を用意し、毛根を除いた５ｃｍ間隔に１ｃｍ長の毛髪を８式用意して実験を行った（サンプル２）。また、Ｎはネガティブコントロールである（図３を参照）。
　実験方法の手順の通りに核酸検出を行った。検出結果を図４に示す。この結果から、毛根の有無や毛髪の部位に関係なく標的核酸が検出できることを確認できた。また、Ｎのラインからはラインが検出されず、サンプルがない状態では反応しないことが確認できた。 Example 2
In order to confirm whether the detection success rate of nucleic acid derived from human hair changes depending on the collection site in the hair, two experiments were conducted. 47 cm of hair was prepared, and 9 types of 1 cm long hair including the hair root and 1 cm long hair at 5 cm intervals were prepared and tested (Sample 1). N is a negative control. 41 cm of hair was prepared, and eight types of 1 cm long hair were prepared at intervals of 5 cm excluding the roots (sample 2). N is a negative control (see FIG. 3).
Nucleic acid detection was performed according to the procedure of the experimental method. The detection result is shown in FIG. From this result, it was confirmed that the target nucleic acid could be detected regardless of the presence or absence of the hair root or the hair site. Further, no line was detected from the N line, and it was confirmed that no reaction occurred in the absence of the sample.
実施例３
　ヒトの毛髪由来の核酸の検出成功率が毛髪の長さによって影響するか確認するため、図５のように、毛髪の毛先から長さを０．５ｃｍ刻みに０．５－４．０ｃｍの毛髪を調製して実験を行った。また、Ｎはネガティブコントロールである（図５を参照）。
　実験方法の手順の通りに核酸検出を行った。検出結果を図６に示す。この結果から、毛髪の長さが０．５ｃｍ以上であれば、標的核酸が検出できることを確認できた。また、Ｎのラインからはラインが検出されず、サンプルがない状態では反応しないことが確認できた。
　なお、毛髪の長さが３．５ｃｍ、４．０ｃｍにおいてコモンプライマーのラインが薄いのは、毛髪量が過剰でそれに伴いＰＣＲ阻害物質も増加したためだと思われる。 Example 3
In order to confirm whether the detection success rate of nucleic acid derived from human hair is affected by the length of the hair, as shown in FIG. 5, the length of the hair is 0.5 to 4.0 cm from the tip of the hair in increments of 0.5 cm. Were prepared for experiments. N is a negative control (see FIG. 5).
Nucleic acid detection was performed according to the procedure of the experimental method. The detection result is shown in FIG. From this result, it was confirmed that the target nucleic acid can be detected if the length of the hair is 0.5 cm or more. Further, no line was detected from the N line, and it was confirmed that no reaction occurred in the absence of the sample.
The reason why the common primer line is thin when the hair length is 3.5 cm and 4.0 cm seems to be that the amount of hair is excessive and the amount of PCR-inhibiting substances increases accordingly.
実施例４
　ヒト以外の動物の標的核酸を検出できるか否かを確認するため、ヒトの毛髪とイヌとネコの獣毛を用いて実験を行った。
　実験方法の手順の通りに核酸検出を行った。ポジティブコントロールとしてヒト、イヌ、ネコの既知ＤＮＡを用いた。また、Ｎはネガティブコントロールである。検出結果を図７に示す。サンプルとポジティブコントロールについて反応が確認できた。また、Ｎのラインからはラインが検出されず、サンプルがない状態では反応しないことが確認できた。この結果から、ヒトの毛髪とイヌやネコの獣毛を用いても標的核酸の検出により種判定ができることを確認できた。さらに、ヒトやイヌ、ネコの毛をマルチプレックスで検出できることが確認できた。 Example 4
In order to confirm whether or not the target nucleic acid of animals other than humans can be detected, experiments were performed using human hair and animal hair of dogs and cats.
Nucleic acid detection was performed according to the procedure of the experimental method. As positive controls, known DNAs of humans, dogs and cats were used. N is a negative control. The detection result is shown in FIG. The reaction was confirmed for the sample and the positive control. Further, no line was detected from the N line, and it was confirmed that no reaction occurred in the absence of the sample. From this result, it was confirmed that the species could be determined by detecting the target nucleic acid even when human hair and dog or cat animal hair were used. Furthermore, it was confirmed that human, dog and cat hair could be detected in multiplex.
結果：
　従来の技術では、毛根をすりつぶして核酸を抽出することが一般的であった。しかしながら、状況によっては毛根のない毛や長さが短い毛などがあり、単純に毛から核酸を抽出することができない。
　本発明により、毛根の有無や毛中の部位や長さに関係なく、酵素等による化学的な破砕処理を行わなくても、簡便、簡単、迅速に毛のサンプルを用いてマルチプレックスの遺伝子検査を行うことができる。さらには、核酸抽出を行わなくても、毛のサンプルを用いて、遺伝子検査を行えることが確認できた。 result:
In the prior art, it has been common to extract nucleic acids by grinding hair roots. However, depending on the situation, there are hairs without hair roots and short hairs, and it is not possible to simply extract nucleic acids from the hairs.
According to the present invention, regardless of the presence or absence of hair roots and the location and length of hair, it is simple, easy and quick to use a hair sample for multiplex genetic testing without performing chemical crushing treatment with enzymes, etc. It can be performed. Furthermore, it was confirmed that genetic testing could be performed using hair samples without nucleic acid extraction.
　本出願は、日本で出願された特願２０１６－０６０２０１（出願日：２０１６年３月２４日）を基礎としており、その内容は本明細書に全て包含されるものである。 This application is based on Japanese Patent Application No. 2016-060201 (filing date: March 24, 2016) filed in Japan, the contents of which are incorporated in full herein.

Claims (6)

  1. 　哺乳動物の毛の中の標的核酸を検出する方法であって、
    　哺乳動物の毛を溶液中で物理的手段により破砕する工程、
    　破砕後の溶液を核酸増幅反応に供することにより標的核酸を増幅させる工程、および、
    　増幅された該標的核酸を検出する工程
    を含む、前記方法。     A method for detecting a target nucleic acid in mammalian hair comprising the steps of:
    Crushing mammalian hair in solution by physical means,
    A step of amplifying a target nucleic acid by subjecting the disrupted solution to a nucleic acid amplification reaction; and
    Detecting the amplified target nucleic acid.
  2. 　核酸を抽出する工程が行われない、請求項１に記載の方法。 The method according to claim 1, wherein the step of extracting nucleic acid is not performed.
  3. 　該毛が、毛根を含まない毛の断片である、請求項１または２に記載の方法。 The method according to claim 1 or 2, wherein the hair is a hair fragment not containing a hair root.
  4. 　該標的核酸の検出が、核酸クロマトグラフィー用ストリップを用いて行われる、請求項１～３のいずれか１項に記載の方法。 The method according to any one of claims 1 to 3, wherein the target nucleic acid is detected using a strip for nucleic acid chromatography.
  5. 　該核酸増幅反応がポリメラーゼ連鎖反応である、請求項１～４のいずれか１項に記載の方法。 The method according to any one of claims 1 to 4, wherein the nucleic acid amplification reaction is a polymerase chain reaction.
  6. 　哺乳動物の毛を物理的に破砕するための器具、ならびに、
    　核酸クロマトグラフィー用ストリップおよび核酸増幅反応用プライマーセットの少なくとも１つ
    を含む、請求項１～５のいずれか１項に記載の方法に用いるためのキット。     An instrument for physically breaking mammalian hair, and
    A kit for use in the method according to any one of claims 1 to 5, comprising at least one of a strip for nucleic acid chromatography and a primer set for nucleic acid amplification reaction.
PCT/JP2017/011935 2016-03-24 2017-03-24 Method for detecting nucleic acid derived from hair WO2017164354A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2018507431A JPWO2017164354A1 (en) 2016-03-24 2017-03-24 Method for detecting nucleic acid derived from hair

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2016060201 2016-03-24
JP2016-060201 2016-03-24

Publications (1)

Publication Number Publication Date
WO2017164354A1 true WO2017164354A1 (en) 2017-09-28

Family

ID=59900564

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2017/011935 WO2017164354A1 (en) 2016-03-24 2017-03-24 Method for detecting nucleic acid derived from hair

Country Status (2)

Country Link
JP (1) JPWO2017164354A1 (en)
WO (1) WO2017164354A1 (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006506057A (en) * 2002-10-07 2006-02-23 マーリゲン、バイオサイエンシーズ、インコーポレーテッド DNA extraction from biological samples
JP2006201062A (en) * 2005-01-21 2006-08-03 Kainosu:Kk Nucleic acid detection or determination method
JP2008249700A (en) * 2007-03-07 2008-10-16 Nippon Menaade Keshohin Kk Method of evaluating hair condition and its application
JP2014018150A (en) * 2012-07-19 2014-02-03 Ngk Insulators Ltd Inspection tool for nucleic acid chromatography and producing method thereof
JP2015073478A (en) * 2013-10-09 2015-04-20 独立行政法人産業技術総合研究所 Determining method for animal hair product

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006506057A (en) * 2002-10-07 2006-02-23 マーリゲン、バイオサイエンシーズ、インコーポレーテッド DNA extraction from biological samples
JP2006201062A (en) * 2005-01-21 2006-08-03 Kainosu:Kk Nucleic acid detection or determination method
JP2008249700A (en) * 2007-03-07 2008-10-16 Nippon Menaade Keshohin Kk Method of evaluating hair condition and its application
JP2014018150A (en) * 2012-07-19 2014-02-03 Ngk Insulators Ltd Inspection tool for nucleic acid chromatography and producing method thereof
JP2015073478A (en) * 2013-10-09 2015-04-20 独立行政法人産業技術総合研究所 Determining method for animal hair product

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HANDT O. ET AL.: "The end of bad hair days", FORENSIC SCIENCE INTERNATIONAL: GENETICS, vol. 5, 2015, pages e146 - e148 *
HAYASHIDA M. ET AL.: "Single-Tube Genotyping from a Human Hair Root by Direct PCR", ANALYTICAL SCIENCES, vol. 25, 2009, pages 1487 - 1489 *
OTTENS R. ET AL.: "Successful direct amplification of nuclear markers from a single hair follicle", FORENSIC SCIENCE MEDICINE AND PATHOLOGY, vol. 9, 2013, pages 238 - 243 *

Also Published As

Publication number Publication date
JPWO2017164354A1 (en) 2019-02-07

Similar Documents

Publication Publication Date Title
CN107385040B (en) Amplicon rescue multiplex polymerase chain reaction for amplification of multiple targets
JP2007537746A (en) Amplicon contamination detection method during PCR showing two different annealing temperatures
EP1529841A1 (en) RNA extraction method, RNA extraction reagent, and method for analyzing biological materials
EP1069190B1 (en) Method for amplification of RNA
JP2020141707A (en) Methods to detect silent carrier genotype
JP2011520467A5 (en)
WO2016050123A1 (en) Pyrolysis composition as well as use and kit thereof, method for preparing nucleic acid by using pyrolysis composition, and method for nucleic acid analysis
JP6060567B2 (en) Nucleic acid extraction method from embedded tissue
JP3452717B2 (en) Nucleic acid synthesis method
JP4470275B2 (en) Nucleic acid synthesis method
KR20160106041A (en) Compositions and methods for multiplex analysis of nras and braf nucleic acids
Kasai et al. Detection of SYT–SSX fusion transcripts in both epithelial and spindle cell areas of biphasic synovial sarcoma using laser capture microdissection
US10160965B2 (en) Method and materials for nucleic acids extraction and purification
WO2017164354A1 (en) Method for detecting nucleic acid derived from hair
JPH1080280A (en) Synthesis of nucleic acid
Oh et al. Development of an ammonium sulfate DNA extraction method for obtaining amplifiable DNA in a small number of cells and its application to clinical specimens
EP0751226B1 (en) Process for amplifying nucleic acid sequences
JP4186270B2 (en) Nucleic acid synthesis method
KR101162422B1 (en) Composition for amplifying nucleic acid
JP2000093175A (en) Synthesis of nucleic acid
JPWO2019212023A1 (en) RNA virus processing method
Tripathi A Simplified and Cheapest Method for the Diagnosis of Sickle Cell using Whole Blood PCR and RFLP in Nepal
JPH1080279A (en) Synthesis of nucleic acid
JP4629167B2 (en) Nucleic acid synthesis method
JP2015096061A (en) Nucleic acid amplification method, method for producing nucleic acid sample for nucleic acid amplification and nucleic acid analyzing method

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 2018507431

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17770394

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 17770394

Country of ref document: EP

Kind code of ref document: A1