JP2011115122A - Specimen pretreatment method and gene detection method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pretreatment method for easily detecting a gene without using particular device and apparatus. <P>SOLUTION: The specimen pretreatment for detecting a gene in a biospecimen comprises (A) a cell breaking step to break a cell in the specimen and (B) a protein removing step to remove proteins in the specimen. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a sample pretreatment method and a gene detection method for detecting a gene in a biological sample.

  Various methods are known for detecting and amplifying nucleic acids contained in biological samples, bacteria, viruses and the like. For example, a hybridization method for detecting a nucleic acid based on the presence or absence of hybridization between a probe and a nucleic acid to be detected, and a method for amplifying a nucleic acid to be detected by extending a primer are known.

  As a method for detecting a target gene by amplifying a nucleic acid, for example, a PCR method is generally used. However, since the PCR method requires a temperature cycle, a dedicated thermal cycler is required. Moreover, regarding the detection of gene mutations (for example, SNPs), the PCR method has a problem in detection sensitivity. As a gene amplification method capable of isothermal amplification and high detection accuracy, there is a method commonly called Smart Amplification Process 2 (SmartAmp2) developed by the present applicant (Patent Document 1). This method is an isothermal nucleic acid amplification method using an asymmetric primer set with a turnback primer (TP) and a folding primer (FP) as main primers. In this method, in principle, an increase in background is suppressed and the detection sensitivity of gene mutation is improved.

  Japanese Patent No. 3867926

  In general, in gene detection by nucleic acid amplification, removal of contaminants in a biological sample is performed as pretreatment of the sample before detection. On the other hand, in addition to seasonal influenza, recently a new type of influenza (new influenza) has occurred, and there is a sign of a pandemic. Identifying the type of influenza virus is extremely important in determining treatment strategy. Moreover, it is necessary to detect viral genes with high accuracy in clinical practice. For this reason, establishment of a simple virus gene detection technique that does not use special equipment and equipment is required. Such a demand is not limited to viral genes but is required for detection of all genes in biological samples such as detection of bacterial genes such as E. coli.

  Accordingly, an object of the present invention is to provide a simple biological sample pretreatment method and gene detection method that do not require special devices and equipment.

The pretreatment method of the present invention is a sample pretreatment method for detecting a gene in a biological sample, and is a sample pretreatment method including the following steps.
A) Cell destruction step for destroying cells in the sample B) Protein removal step for removing proteins in the sample

  The gene detection method of the present invention is a method of detecting a gene in a biological sample using a gene amplification method, wherein the sample is pretreated by the pretreatment method of the present invention, and then the gene is detected by the gene amplification method. It is characterized by doing.

  Thus, since the pretreatment method of the present invention has a cell destruction step and a protein removal step, it can be easily carried out without requiring special equipment and equipment. As a result, the gene detection method using the pretreatment method of the present invention can also carry out gene detection (including gene mutation) simply and with high accuracy without the need for special equipment and equipment.

  FIG. 1 is a diagram showing the results of Example 1 of the present invention.   FIG. 2 is a diagram showing the results of Example 2 of the present invention.

  In the pretreatment method of the present invention, the step A) is preferably a treatment using a surfactant. The surfactant is preferably an ionic surfactant. The ionic surfactant is preferably SDS.

  In the pretreatment method of the present invention, it is preferable that the step B) treats the sample after the step A) with a column. The column treatment is preferably a treatment of centrifuging the column filled with the sample.

  In the pretreatment method of the present invention, the gene is preferably a viral gene. The virus is preferably an influenza virus. The influenza virus is preferably a new influenza virus.

  In the gene detection method of the present invention, the gene amplification method is preferably an isothermal amplification method.

The isothermal amplification method is preferably an amplification method using the following TP.
TP: a sequence (Ac ′) that hybridizes to the sequence (A) at the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and 5 ′ from the sequence (A) in the target nucleic acid sequence A primer comprising a sequence (B ′) that hybridizes to a complementary sequence (Bc) of the sequence (B) present in 5 ′ of the sequence (Ac ′).

The isothermal amplification method is preferably an isothermal amplification method using a pair of TPs or an isothermal amplification method using TP and FP as a primer set.
FP: a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence at the 3 ′ end portion, and two nucleic acid sequences that hybridize to each other in the same strand A primer comprising the folded sequence (D-Dc ′) contained above on the 5 ′ side of the sequence (Cc ′).

  The inventor has intensively studied a substance that reduces or suppresses the nucleic acid detection and amplification inhibitory action by impurities, destroys cells containing the target nucleic acid in the biological sample, and further detects the target nucleic acid in the biological sample and The present inventors have developed a method for removing proteins that inhibit amplification, and have found a method that can detect and / or amplify nucleic acids simply and quickly.

  The present inventors can use any of a cationic surfactant, an anionic surfactant, an amphoteric surfactant, and a nonionic surfactant for a biological sample. By adding to the sample at least one substance having a strong denaturing effect selected from the group consisting of any of those in the molecular system, cells containing the target nucleic acid in the biological sample are destroyed, and detection of the target nucleic acid and / or Or it turned out that amplification efficiency increases.

  Furthermore, the present invention detects and / or amplifies a target nucleic acid in a biological sample by adding a substance having a strong denaturing effect to the sample, and treating the sample in which the cells containing the target nucleic acid are destroyed. Has been developed, and a method has been found that can detect and / or amplify nucleic acids simply and quickly.

  Usually, in order to increase the efficiency of nucleic acid detection and / or amplification, nucleic acid extraction and purification treatment is performed in advance from a biological sample by a complicated means using an organic solvent such as phenol or chloroform. In the method of the present invention, Nucleic acids can be detected and / or amplified easily and quickly without using such an organic solvent.

  The step of complementary binding of the oligonucleotide can be a step of hybridizing the probe. After the probe is hybridized, Southern hybridization, Northern hybridization, Invader method, Pulsar method, Colony hybridization method, LCR The nucleic acid to be detected can be detected by a method selected from the group consisting of a method and a bDNA method.

  Complementary binding of the oligonucleotide may be a step of annealing the primer, and in this case, may further comprise the step of amplifying the nucleic acid by extending the nucleic acid strand from the 3 'end of the primer. The step of amplifying the nucleic acid may be performed by an isothermal amplification method. The step of amplifying the nucleic acid may be performed by a method selected from the group consisting of PCR method, TMA method, NASBA method, LAMP method, ICAN method, RCA method, TRC method, SDA method, HDA method and SmartAmp2 method. it can. In particular, the nucleic acid can be suitably amplified by the LAMP method or the SmartAmp2 method.

The biological sample and the solution in the biological sample processing step include a buffer and salts, and the buffer includes MES, acetate-sodium acetate buffer, Bis-tris, ADA, PIPES, ACES, BES, MOPS, A sample pretreatment method comprising at least one selected from the group consisting of phosphate buffer, TES, HEPES, Tris, Tricine, Bicine, TAPS, CHES and CAPS, wherein the salt contains at least one of NaCl and KCl. is there.

A “surfactant” can have a hydrophobic region or portion that can interact with a hydrophobic solvent and a hydrophobic portion of a cell membrane, and can have a positive or negative charge in solution, or a polar region that has no charge. Means a molecule or class of molecules having a hydrophilic region or portion.
“Nonionic surfactant” means a surfactant that contains at least one polar, uncharged group or ion in its hydrophilic region or portion.
"Ionic surfactant" means a surfactant that contains at least one positively or negatively charged group or ion in a hydrophilic region or portion.

    As the surfactant, any of a cationic surfactant, an anionic surfactant, an amphoteric surfactant and a nonionic surfactant can be used. Any of these can be used.

  Examples of column materials include various organic materials and inorganic materials.

  The size of the column must have a capacity for removing measurement-inhibiting substances, and can be appropriately selected according to the amount of the sample to be measured.

Examples of the carrier to be packed in the column include GE Healthcare's Sephacryl S-100HR, Sephacryl S-200HR, Sephacryl S-300HR, Sephacryl S-400HR, Sephacryl S-500HR, Sephacryl S-1000F, Sephacryl S-1000Sr. TOYOPEARLHW-40, TOYOPEARLHW-50, TOYOPEARLHW-55, TOYOPEARLHW-65, TOYOPEARLHW-75 from TOSOH, and CHROMA SPIN TE-10, CHROMA SPINTE-30 SP from Clontech (Takara Bio) , CHROMA SPIN TE-200, CHRO Although the like A SPIN TE-400, CHROMA SPIN TE-1000, is not limited only to these, either if separable carrier column, and further, it can also be used combinations thereof, and the like.

  The method for passing the biological sample through the column is not particularly limited, such as liquid feeding by gravity, liquid feeding by centrifugal force, liquid feeding by pressurization, liquid feeding by suction, and the like.

  The present invention also relates to a method for detecting a gene by a gene amplification method after performing a cell destruction step for destroying cells in a sample and a protein removal step for removing proteins in the sample.

  The present invention can directly and easily detect and amplify a target nucleic acid even when a sample that has not been subjected to nucleic acid extraction and purification using an organic solvent is used. In addition, since the method of the present invention is simple, the target nucleic acid can be detected and / or amplified accurately without increasing the chance of contamination.

  In the present invention, a treatment process for destroying cells in a biological sample using a surfactant, and a protein removal treatment process through a simple column in order to remove proteins in the destroyed sample. The target nucleic acid can be detected and / or amplified effectively without being inhibited. In addition to these treatments, or in addition to these treatments, the target nucleic acid can be detected and / or amplified effectively without being inhibited by contaminants by heat-denaturing the sample. Can do.

Examples of genes to be detected in the present invention include viral genes, bacterial genes, human genome genes and the like, and in particular, viral genes such as influenza virus, new influenza virus, SARS virus, norovirus and cytomegalovirus are preferred. Applicable. In addition, the sample may contain a buffer and salts. Examples of the buffer include MES, acetate-sodium acetate buffer, Bis-tris, ADA, PIPES, ACES, BES, MOPS, phosphate buffer, TES, and HEPES. , Tris, Tricine, Bicine, TAPS, CHES, and CAPS, and examples of the salt include at least one of NaCl and KCl. Preferably, the salt is at least one of MES and an acetic acid-sodium acetate buffer, and the salts include at least one of NaCl and KCl.

  By performing the above treatment steps, it is considered that the complementary binding to the nucleic acid by the oligonucleotide having a sequence complementary to the target nucleic acid is not or is less susceptible to the inhibitory action by impurities. Therefore, hybridization to the target nucleic acid by the probe or annealing to the target nucleic acid by the primer is smoothly performed. As the efficiency of annealing of the primer to the target nucleic acid increases, the efficiency of nucleotide chain extension from the 3 'end of the primer also increases, and the efficiency of nucleic acid amplification improves.

  In addition to the above processing steps, heat treatment may be performed. The heat treatment can be performed before, during or after the above-described processing steps. The heating temperature can be 37 ° C. or higher, preferably 45 ° C. or higher, more preferably 55 ° C. or higher, and still more preferably 80 ° C. or higher. The heating time is not particularly limited, and an extremely short processing time is possible. For example, it can be performed for 0.01 seconds or more, or 3 seconds or more, preferably 1 minute or more, more preferably 3 minutes or more. For example, as one of the heating conditions, heating at 98 ° C. for 3 minutes can be exemplified, but those skilled in the art can set arbitrary time and temperature in addition to this condition. In addition, this heat treatment can be performed as one step of a reaction involving heating such as a PCR method.

  Further, in addition to the above treatment steps, an alkali treatment may be performed. Alkaline treatment can be performed by mixing an alkali solution with a sample before, during or after the above treatment step. Examples of the alkali solution include, but are not limited to, alkali metal hydroxide solutions such as sodium hydroxide solution and potassium hydroxide solution. The alkali concentration (concentration of OH-1) can be 0.1 mM or higher, or 1 mM or higher, preferably 10 mM or higher, more preferably 30 mM or higher. Alternatively, it can be adjusted to pH 7 or higher, preferably pH 8 or higher, more preferably pH 9 or higher. The alkali concentration and pH can be performed at any concentration and pH (for example, 10 M or less) as long as they do not inhibit the subsequent detection or amplification step.

  Furthermore, it is possible to perform both heat treatment and alkali treatment. For example, after adding the above substances to a sample and further mixing with an alkaline solution, a heat treatment can be performed at 98 ° C. for 3 minutes.

  The oligonucleotide can be a probe that hybridizes to a portion of the nucleic acid sequence to be detected or a primer that anneals to a portion of the nucleic acid sequence to be detected. The probe has a sequence complementary to the nucleic acid sequence to be detected or its complementary strand. As long as it can hybridize to the nucleic acid sequence to be detected or its complementary strand, all the bases of the probe need not be complementary to the nucleic acid sequence to be detected or its complementary strand. Moreover, you may have in a part of probe the sequence which is not complementary to the nucleic acid sequence which should be detected, or its complementary strand. Furthermore, the probe may be labeled for detection. For example, the probe may be labeled with a radioisotope, a fluorescent substance, an enzyme, another chemical substance, or the like. The probe can be in the form of being immobilized or adsorbed on the surface of the solid phase, or can be used in a free state. The immobilized or adsorbed probe can be in the form of, for example, a DNA chip or a DNA microarray.

  The primer has a sequence complementary to the nucleic acid sequence to be detected or its complementary strand, and may be used as a primer set composed of two or more primers. As long as it can anneal to the nucleic acid sequence to be detected or its complementary strand, not all the bases of the primer may be complementary to the nucleic acid sequence to be detected or a sequence complementary to its complementary strand. Moreover, you may have in a part of primer the sequence which is not complementary to the nucleic acid sequence which should be detected, or the sequence complementary to the complementary strand. Examples of the sequence that is not complementary to the nucleic acid sequence to be detected contained in a part of the primer or a sequence complementary to its complementary strand include a promoter of RNA polymerase. Furthermore, the primer may be labeled for detection. For example, the primer may be labeled with a radioisotope, a fluorescent substance, an enzyme, another chemical substance, or the like.

  As long as the nucleic acid sequence to be detected is, for example, a gene composed of a double-stranded nucleic acid, either the sense-side base sequence or the antisense-side base sequence may be detected. For example, by detecting the nucleic acid sequence on the antisense side, the complementary nucleic acid sequence on the sense side (that is, the base sequence of the gene) can be detected. Nucleic acids to be detected are specific to organisms that contain nucleic acids such as, for example, genetic conditions such as bacteria, viruses, bacteria, protozoa, multicellular organisms, plasmids or genetic mutations associated with a predisposition or tendency to certain diseases Nucleic acid, particularly RNA or DNA.

  In the present invention, as the surfactant, any of a cationic surfactant, an anionic surfactant, an amphoteric surfactant, and a nonionic surfactant can be used. Any of low molecular weight materials can be used.

  Specific examples of surfactants as substances having a strong modifying effect include pure soap, alpha sulfo fatty acid ester salt, linear alkyl benzene sulfonate, alkyl sulfate ester, alkyl ether sulfate ester, alpha olefin sulfone. Acid salt, alkyl sulfonate, sucrose fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene fatty acid ester, fatty acid alkanolamide, alkyl glucoside, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, Including alkylamino fatty acid salt, alkylbetaine, alkylamine oxide, alkyltrimethylammonium salt, dialkyldimethylammonium salt, more specifically N, N-Bis 3-D-gluconamidopropyl) cholamide [BIGCHAP], N, N-Bis (3-D-gluconamidopropyl) deoxycholide [Deoxy-BIGCHAP], NIKKOL BL-9EX [Polyoxy9] [Polyoxy] -8], Nonanoyl-N-methylglucomide [MEGA-9], Decanoyl-N-methylglucamide [MEGA-10], Polyoxyethylene (8) Octylphenyl Ether [Triton X-114] ol, her [NP-40], Polyoxyethylene (10) Octylphenyl Ether [Triton X-100], Polyoxyethylene (20) Sorbitan Monolaurate [Tween20], Polyoxyethylene (20) Sorbitan Monopalmitate [Tween40], Polyoxyethylene (20) Sorbitan Monostearate [Tween60], Polyoxyethylene (20) Sorbitan Monooleate [Tween 80], Polyoxyethylene (20) Sorbitan Trioleate, Polyoxyethylene (23) Lauryl Ether [Brij3 ], Polyoxyethylene (20) CethylEther [Brij58], n-Dodecyl-β-D-maltopyranoside, n-Heptyl-β-D-thioglucopyranoidide, n-Octyl-β-Dy-pyl-β-D-O-yl-pyl-β-D-O-Gy-D-O-Gy-D-O-G-Dy-O-G-O-Dy-O-G-O-D-O-G-O-G-O-D , N-Nonyl-β-D-thiomaltoside, IGEPAL CA-630, Digitonin, Saponin, from Soybeans, 3-[(3-Cholamidopropyl) diathylammonio] -2-hydroxy3-] -Cholamidopropyl) d imethylammonio] -1-propanesulfonate [CHAPS], Sodium Dodecylsulfate [SDS], Sodiumdodecylbenzene sulphonate [SDBS], Lithium Dodecyl Sulfate [LDS], Lithium3,5-Diiodosalicylate, Tris (hydroxymethyl) aminomethane Dodecyl Sulfate [Tris DS], Sodium Cholate, N-Lauroylsarcosine, Sodium N-Dodecanoylsalcosinate, Cetyldimethylamylammonium Bromide, Cetyltrimethyla monium Bromide [CTAB], Cetyltrimethylammonium Chloride, may be either, or a mixture thereof, such as Guanidine Thiocyanate.

  Examples of the column material include various organic materials and inorganic materials such as polymethyl methyl methacrylate (PMMA), polycarbonate, polypropylene, polyethylene, polymethylpentene, polystyrene, polytetrafluoroethylene, ABS resin, poly Resins such as dimethylsiloxane and silicon, and copolymers or composites containing such polymer compounds; Glasses such as quartz glass, Pyrex (registered trademark) glass, soda glass, borate glass, silicate glass, and borosilicate glass Ceramics and composites thereof are preferably used. The production method may be injection molding or cutting, but an injection molded product of polymethyl methyl methacrylate (PMMA) is particularly preferably used.

  The size of the column must have a capacity for removing measurement-inhibiting substances, and can be appropriately selected according to the amount of the sample to be measured. The shape of the column is not particularly limited, such as a circle or polygonal cross section, but the channel length is longer than the short diameter (diameter in the case of a circle, the shortest diameter passing through the center in the case of a polygon). Is preferred. For example, the carrier volume in the column is required to be about 20 times that of the biological sample, and the carrier volume in the column is required to be 1.0 mL or more with respect to 0.05 mL of the biological sample. This is not the case because it depends. For example, the minor axis of the normal section can be in the range of 0.1 mm to 100 mm, and the flow path length can be in the range of 1 mm to 100 cm.

Examples of the carrier to be packed in the column include GE Healthcare's Sephacryl S-100HR, Sephacryl S-200HR, Sephacryl S-300HR, Sephacryl S-400HR, Sephacryl S-500HR, Sephacryl S-1000F, Sephacryl S-1000Sr. TOYOPEARLHW-40, TOYOPEARLHW-50, TOYOPEARLHW-55, TOYOPEARLHW-65, TOYOPEARLHW-75 from TOSOH, and CHROMA SPIN TE-10, CHROMA SPINTE-30 SP from Clontech (Takara Bio) , CHROMA SPIN TE-200, CHRO Although the like A SPIN TE-400, CHROMA SPIN TE-1000, is not limited only to these, either if separable carrier column, and further, it can also be used combinations thereof, and the like.

  The method of passing the biological sample through the column is not particularly limited, such as liquid feeding by gravity, liquid feeding by centrifugal force, liquid feeding by pressurization, liquid feeding by suction, etc., but centrifugal liquid feeding with a high recovery rate of the biological sample is preferable. Used. By sending liquid through the column by centrifugal force, a simple system can be established without newly requiring a pump or the like.

  Examples of biological samples include, but are not limited to, samples derived from animals, plants, viruses, and bacteria. Animal-derived samples include blood (including whole blood and isolated blood cells), serum, plasma, cells (including cultured cell lines), tissues, body fluids (ear leakage, nasal discharge, pus, ascites, pleural effusion, bile, cerebrospinal fluid) , Sputum, etc.), mucosal cells (oral mucosal cells, gastric mucosal cells, airway mucosal cells, etc.), swabs collected from nasal mucosa and oral mucosa with cotton swabs, sweat, amniotic fluid, discharge (urine and feces etc.), internal Examples include, but are not limited to, brushing from various organs using a endoscope, collected liquid, biopsy sample, alveolar lavage fluid, and the like. Examples of plant-derived samples include, but are not limited to, whole plants, cultured cells, tissues, roots, stems, leaves, flowers, seeds, and other organs. Examples of the microorganism-derived sample include a microorganism culture. Moreover, it can apply not only to a biological sample but to arbitrary samples, such as seawater and soil.

  This method is useful for a method of examining the presence or absence of an infectious disease as a biological sample. Although this method is useful for detection of viruses and bacteria related to respiratory infections, especially influenza viruses, and new influenza viruses outbreaked in 2009 (new swine-derived H1N1 influenza virus (S-OIV)) However, the present invention is not limited to this detection.

  According to the present invention, cells containing a target nucleic acid are destroyed using a surfactant, and the sample is subjected to column treatment, thereby removing a protein that inhibits detection and / or amplification of the target nucleic acid in a biological sample, Nucleic acids can be detected and / or amplified simply and quickly.

  Examples of the method for detecting a nucleic acid with a probe include, but are not limited to, Southern hybridization for detecting DNA, Northern hybridization for detecting RNA, invader method, pulsar method, and bDNA method.

  Southern hybridization is a method for detecting DNA having a target sequence, and is generally performed by the following procedure. DNA is cleaved with restriction enzymes and separated by agarose electrophoresis. The DNA in the agarose gel is denatured to single strands by alkali treatment. Next, the DNA fragment is transferred from the gel after electrophoresis to a membrane such as a nitrocellulose membrane or nylon membrane, and DNA having a complementary sequence to the probe is obtained by hybridization with a probe labeled with an enzyme or a radioisotope. To detect.

  Northern hybridization is a method for detecting RNA having a target sequence, and is generally performed by the following procedure. RNA is separated by agarose electrophoresis under denaturing conditions. Subsequently, the RNA is transferred from the gel after electrophoresis to a membrane such as a nitrocellulose membrane or nylon membrane, and hybridization with a probe labeled with an enzyme or a radioisotope is used to detect RNA having a complementary sequence to the probe. To do.

  The invader method is a method for detecting a single nucleotide polymorphism (SNP) by detecting a fluorescent signal using an enzyme that specifically recognizes a structural change of a single nucleotide mismatch of a target DNA. It is done according to the procedure. Three types of probes called allele probe, invader probe, and FRET probe are prepared. The allele probe has a template-specific sequence on the 3 'side, and the side has an irrelevant sequence (flap) on the 5' side. The 5 'end of the 3' sequence specific to the template of the allele probe is a SNP site, which is designed to be complementary to the base of the allele to be detected. The invader probe is designed to bind complementarily from the SNP site to the 3 'side of the template, but the base corresponding to the SNP site may be any base. The FRET probe has a sequence complementary to the flap on the 3 'side and a palindromic sequence that forms a hairpin structure on the 5' side. A fluorescent dye is bound to the 5 'end of the FRET probe, and a quencher is bound to the vicinity thereof.

  These three probes are hybridized with the target DNA, and the enzyme cleavage is allowed to act. At this time, when the base of the allele probe corresponding to the SNP site binds complementarily to the base of the SNP site, the 3 'end of the invader probe enters. The enzyme cleavage recognizes this structure, and the allele probe is cleaved at the flap site to release the flap portion. The released flap binds to the FRET probe. When the flap binds to the FRET probe, this structure is further recognized by the enzyme cleavage, the 5 ′ end of the FREP probe is cleaved, the fluorescent dye quenched by the quencher is released, and fluorescence is detected. .

  In the pulsar method, two kinds of complementary DNA probes prepared in advance are labeled, and hybridization is repeated on them to bind and amplify them into a honeycomb-like shape. And make this a signal. Its presence can be confirmed by binding this signal to the gene to be detected. This method is simple because it does not require a nucleic acid synthesizing enzyme in the process of hybridization and can be carried out isothermally, the reaction can be carried out in a short time, and both DNA and RNA can be detected with high sensitivity.

  The bDNA method is a nucleic acid detection method also called a branched-chain probe method or bDNA probe method. A target nucleic acid is detected by hybridizing a probe and a nucleic acid, further hybridizing with an amplification molecule called branched DNA, and detecting it.

  Examples of techniques for amplifying nucleic acids using primers include, but are not limited to, the PCR method, the TMA method, the NASBA method, the LAMP method, the ICAN method, the RCA method, the SDA method, the HDA method, and the SmartAmp2 method. These amplification techniques may further include a step of detecting the target nucleic acid by detecting the amplified nucleic acid. Any of the amplification by cyclically changing the temperature such as PCR and the isothermal amplification method in which the reaction is carried out isothermally can be used. For example, as the isothermal amplification method, the TMA method, NASBA method, LAMP method, ICAN method, RCA method, SDA method, TRC method, HDA method and SmartAmp2 method can be exemplified, but the LAMP method or SmartAmp2 method using TP is particularly preferable. .

  The PCR method is a technique in which a set of primers complementary to a template DNA is designed, and a region sandwiched between the primers is amplified in a reaction vessel by a DNA polymerase. There are various variations in the PCR method. RT-PCR method in which PCR is performed after synthesizing DNA from RNA using reverse transcriptase, specified by TaqMan probe and TaqDNA polymerase, which are fluorescent probes for typing SNPs Various improvements are known to those skilled in the art, such as the TaqMan PCR method, which amplifies only the alleles.

  The TMA method uses a forward primer having the same sequence as the target RNA, a reverse primer having a sequence complementary to the target RNA on the 3 ′ side and a promoter sequence recognized by T7 RNA polymerase on the 5 ′ side, and RNA having RNase activity This is a method of specifically amplifying a target RNA by combining a transcript obtained from a template RNA with a polymerase, and further synthesizing the transcript using the obtained transcript as a template.

  The NASBA method uses a DNA-dependent RNA polymerase using a forward primer having the same sequence as the target RNA, a reverse primer having a sequence complementary to the target RNA on the 3 ′ side and a promoter sequence recognized by T7 polymerase on the 5 ′ side. Is a method for specifically amplifying a target RNA by combining a transcription product obtained from the template RNA with the obtained transcription product as a template and further synthesizing the transcription product.

  The LAMP method is an amplification method comprising a total of four or more of two TPs (turnback primers) and two OPs (outer primers). Both ends of the amplified DNA form a loop structure, and the loop In this method, the primer is annealed inside and the target DNA is specifically amplified isothermally.

  The ICAM method uses a chimeric primer composed of RNA-DNA, a DNA polymerase having strand displacement activity, and RNase H to perform specific amplification of target DNA isothermally by strand displacement reaction, template exchange reaction, and nick introduction reaction. It is.

  In the SDA method, a DNA strand displaced by a strand-synthesized DNA polymerase lacking 5 ′ → 3 ′ exonuclease activity due to a single strand nick by a restriction enzyme serves as a template for the next replication. Is a method of performing amplification.

  The SmartAmp2 method is a method developed by the present inventor for continuously synthesizing a target nucleic acid under isothermal conditions using DNA or RNA as a template using a primer set comprising two kinds of primers. . The first primer included in the primer set comprises a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and in the target nucleic acid sequence, A sequence (B ′) that hybridizes to a complementary sequence (Bc) of the sequence (B) existing 5 ′ to the sequence (A) is included on the 5 ′ side of the sequence (Ac ′). The second primer comprises a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence at the 3 ′ end portion, and two hybridizing hybrids to each other. A folded sequence (D-Dc ′) containing a nucleic acid sequence on the same strand is included on the 5 ′ side of the sequence (Cc ′).

  These nucleic acid detection and nucleic acid amplification techniques are known to those skilled in the art, and the technique of the present invention can be applied to any reaction as long as it is based on detection involving probe hybridization and / or amplification reaction involving primer annealing. it can.

  In the following examples, nasal mucosal cells were used as a biological sample, an amplification reaction of a target nucleic acid sequence in a novel influenza virus gene contained therein was performed, and detection of the virus was attempted. However, the present invention is not limited or limited by the following examples.

Biological Sample Processing Method Nucleic acid preparation using the present invention was performed as follows. The tip of a swab collected from the nasal mucosa of one flu patient was cut out and collected in a 1.5 mL tube, 300 μL of 5% SDS solution was added thereto, and the mixture was thoroughly stirred using a vortex mixer to prepare a crude extract. . In this example, a column packed with Sephacryl (registered trademark) -400 swollen with TE buffer was used. 15 μL of the crude extract was applied to the column from which the swollen buffer had been removed beforehand by centrifugation, and centrifuged for 1 minute using a tabletop centrifuge to prepare a pretreated sample. A sample was collected from one healthy person and prepared in the same manner.

Nucleic acid amplification reaction A reaction by RT-LAMP was performed using the eluted nucleic acid as a template. That is, cDNA was generated by reverse transcriptase in solution, and at the same time, only the gene sequence specific to the new influenza virus was amplified. Detection by the LAMP method was performed as follows. The composition of the reaction system (25 μL) is as follows. 20 mM Tris-HCl (pH 8.0), 1.4 mM dNTPs, 30 mM CH ₃ COOK, 10 mM (NH ₄ ) ₂ SO ₄ , 8 mM MgSO ₄ , 0.1% Tween® 20, 12 U Aac polymerase, 0. Reactions were performed with 25 U AMV, 5 μL pretreated sample, 1.8 μM TP1, 1.8 μM TP2, 0.675 μM BP1, 0.9 μM BP2, 0.225 μM OP1, 0.225 μM OP2, 0.225 μM BP3. The reaction was performed at 60 ° C. for 40 minutes using the above reaction composition. The reaction was performed using MX-3000P (STRATAGENE) while measuring the fluorescence intensity over time. The target nucleic acid sequence in the new influenza virus gene and the sequence of the primer set for the LAMP method designed and used based on it are shown below.

Confirmation of virus detection The results are shown in FIG. One sample of an influenza patient was confirmed to be amplified within 40 minutes, and an influenza virus could be detected. The result was consistent with the result of a simple test using immunochromatography. Moreover, amplification was not confirmed in one sample of a healthy person within 40 minutes, and the virus could not be detected. By using a simple treatment method for biological samples, simple and rapid detection has become possible.

  Using the same pretreated sample as in Example 1, a reaction by RT-PCR was performed. That is, cDNA was generated by reverse transcriptase in solution, and at the same time, only the gene sequence specific to the new influenza virus was amplified. Detection in the PCR method was performed as follows. Using a commercially available new influenza detection kit (Roche: Influenza A / H1N1 Detection Set), detection was performed from the nucleic acid sample (pretreated sample) prepared by the nucleic acid preparation method. The composition of the reaction system (20 μL) is as follows. 1 × Enzyme blend, 1 × Reaction buffer, 0.5 μM forward primer, 0.5 μM reverse primer, 0.25 μM probe, and 5 μL of nucleic acid sample are added to these to make a 20 μL reaction solution for amplification reaction It was. With the above reaction composition, RT-PCR reaction was performed with the following temperature control according to the manual of the kit. The reaction was performed using MX-3000P (STRATAGENE) while measuring the fluorescence intensity over time.

<PCR cycle>
55 ° C 8 minutes 95 ° C 30 seconds 95 ° C 1 second, 60 ° C 20 seconds, 72 ° C 1 second (45 cycles)

Confirmation of virus detection The results are shown in FIG. One sample of an influenza patient was confirmed to be amplified by 45 cycles of PCR, and the influenza virus could be detected. The result was consistent with the result of a simple test using immunochromatography. Moreover, amplification was not confirmed in one sample of a healthy person, and the virus could not be detected. By using a simple treatment method for biological samples, simple and rapid detection has become possible.

Claims (13)

A sample pretreatment method for detecting a gene in a biological sample, the sample pretreatment method comprising the following steps: A) a cell destruction step for destroying cells in the sample B) removing proteins in the sample Protein removal process The sample pretreatment method according to claim 1, wherein the step A) is a treatment using a surfactant. The sample pretreatment method according to claim 2, wherein the surfactant is an ionic surfactant. The sample pretreatment method according to claim 3, wherein the ionic surfactant is SDS. The sample pretreatment method according to any one of claims 1 to 4, wherein the step B) comprises treating the sample after the step A) with a column. 6. The sample pretreatment method according to claim 5, wherein the column treatment is a treatment of centrifuging the column packed with the sample. The sample pretreatment method according to any one of claims 1 to 6, wherein the gene is a viral gene. The sample pretreatment method according to claim 7, wherein the virus is an influenza virus. The sample pretreatment method according to claim 8, wherein the influenza virus is a new influenza virus. A method for detecting a gene in a biological sample using a gene amplification method, wherein the sample is pretreated by the method according to any one of claims 1 to 9, and then a gene is detected by the gene amplification method. A method for detecting a characteristic gene. The gene detection method according to claim 10, wherein the gene amplification method is an isothermal amplification method. The method for detecting a gene according to claim 11, wherein the isothermal amplification method is an amplification method using the following TP.
TP: a sequence (Ac ′) that hybridizes to the sequence (A) at the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and 5 ′ from the sequence (A) in the target nucleic acid sequence A primer comprising a sequence (B ′) that hybridizes to a complementary sequence (Bc) of the sequence (B) present in 5 ′ of the sequence (Ac ′). The method for detecting a gene according to claim 12, wherein the isothermal amplification method is an isothermal amplification method using a pair of TPs or an isothermal amplification method using TP and FP.
FP: a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence at the 3 ′ end portion, and two nucleic acid sequences that hybridize to each other in the same strand A primer comprising the folded sequence (D-Dc ′) contained above on the 5 ′ side of the sequence (Cc ′).