WO2017164354A1 - Procédé de détection d'un acide nucléique dérivé des cheveux - Google Patents

Procédé de détection d'un acide nucléique dérivé des cheveux Download PDF

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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
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nucleic acid
hair
target nucleic
detection
sample
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PCT/JP2017/011935
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English (en)
Japanese (ja)
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つかさ 前田
裕樹 山下
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倉敷紡績株式会社
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Publication of WO2017164354A1 publication Critical patent/WO2017164354A1/fr

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    • 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

La présente invention décrit un procédé permettant de détecter un acide nucléique dérivé des cheveux plus facilement qu'en utilisant une technologie classique. Ledit procédé de détection d'un acide nucléique cible contenu dans un cheveu d'animal mammifère comprend une étape de broyage du cheveu d'animal mammifère dans une solution en utilisant un moyen physique, une étape d'amplification de l'acide nucléique cible en soumettant la solution après broyage à une réaction d'amplification d'acide nucléique, et une étape de détection de l'acide nucléique cible amplifié.
PCT/JP2017/011935 2016-03-24 2017-03-24 Procédé de détection d'un acide nucléique dérivé des cheveux WO2017164354A1 (fr)

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JP2006201062A (ja) * 2005-01-21 2006-08-03 Kainosu:Kk 核酸の検出あるいは定量方法
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