JP5403463B2 - Sample nucleic acid preparation method - Google Patents

Description

  The present invention relates to a method for preparing sample nucleic acid for analyzing nucleic acid from natural leather products.

  Since natural leather products have different values depending on the type of leather, it is important to scientifically prove the type of leather. Conventionally, however, the surface and cross-section are mainly observed with an optical microscope or an electron microscope, and this means has not led to the determination of species.

  Recently, there has been reported a method for identifying a biological species by dissolving natural leather using collagenase, extracting DNA, amplifying a desired sequence by PCR reaction (Patent Document 1).

  However, even with this method, there are problems that DNA extraction efficiency is not sufficient, collagenase is a rare and expensive product, is expensive, and requires a cutting operation with a scissors or the like.

Japanese Patent No. 3896272

  The present invention relates to a method for efficiently preparing a sample nucleic acid used in a nucleic acid amplification reaction or the like from a natural leather product.

  In view of such circumstances, the present inventors have made various studies on methods for preparing nucleic acids from natural leather products. After degrading leather by allowing collagenase-producing microorganisms to act on leather pieces derived from natural leather products, nucleic acid extraction is performed. When the operation was performed, it was found that the amplification efficiency in the nucleic acid amplification reaction was superior to that when collagenase was used.

That is, the present invention relates to the following inventions 1) to 7).
1) A method for preparing a sample nucleic acid, which comprises extracting a nucleic acid after performing a degradation treatment that causes a collagenase-producing microorganism to act on a leather piece derived from a natural leather product in a method for preparing a sample nucleic acid from a natural leather product.
2) The method of 1) above, wherein the collagenase-producing microorganism is a Vibrio bacterium.
3) The method according to 2) above, wherein the Vibrio bacterium is Vibrio hollisae 1706B strain (NITE P-776).
4) The method according to any one of 1) to 3) above, wherein the degradation treatment by the collagenase-producing microorganism is cultured at 15 to 55 ° C. for 2 hours or more.
5) The method according to any one of 1) to 4) above, wherein the leather pieces are immersed in an organic acid, neutralized as desired, and then collagenase-producing microorganisms are allowed to act.
6) The method according to any one of 1) to 5) above, wherein the natural leather product is a chrome-tanned or tannin-tanned leather product.
7) A kit for performing the method according to any one of 1) to 6) above, which comprises an organic acid, a collagenase-producing microorganism, and a nucleic acid extraction solution.

  According to the method of the present invention, sample nucleic acids suitable for various nucleic acid amplification reactions such as PCR (Polymerase chain reaction) and LAMP (Loop-mediated isothermal amplification) can be prepared from natural leather products more easily and inexpensively. Thus, by using the sample nucleic acid of the present invention, nucleic acid analysis such as DNA identification can be performed with higher accuracy.

Electrophoretic pattern of PCR amplification product. A: Microbial treatment, B: Collagenase treatment Electrophoretic pattern of PCR amplification product. Electrophoretic pattern of PCR amplification product. An electrophoresis pattern of a PCR amplification product amplified using a known meat species discrimination primer.

  In this specification, “nucleic acid” is used as a concept including DNA, RNA, and derivatives thereof.

  In the present invention, “natural leather” means leather derived from various animal species such as cow, pig, sheep, goat, horse, deer, ostrich, snake, crocodile, lizard and the like. “Natural leather product” means various products manufactured using natural leather that has been subjected to a tanning treatment, regardless of whether there is a dyeing treatment or a surface coating treatment.

  In addition to products that are sewn in the form of the final product, the product is a semi-finished product that is supplied as a material for the production of the final product, as well as natural leather trimming scraps (generated when trimming the perimeter of leather and leather) Recycled leather products (recycled leather) that have been reconstructed by crimping and bonding natural leather fibers such as small pieces) and shaving waste (scrap generated when scraping the surface of the leather's flesh side with a rotating blade) Is included.

  In performing the treatment in the present invention, the natural leather product may be appropriately subjected to physical treatment such as peeling off the coating such as the surface film with a file or cutting it into an appropriate size with a scissor, a knife or the like. Although it is possible, it is not necessary to chop the leather pieces.

Further, natural leather products (leather pieces) are subjected to tanning treatment such as chrome tanning and tannin tanning. For example, when the chromium content is high (for example, 2% or more), decomposition treatment by microorganisms is hindered. Since there is a fear, it is preferable to remove such a tanning agent in advance.
Usually, sulfuric acid, oxalic acid, etc. are used for the dechromation treatment, but considering the influence on the nucleic acid amplification reaction, an organic acid such as succinic acid, maleic acid, citric acid, acetic acid, formic acid, etc. It is preferable to immerse in this for a fixed time (refer Example 2).

  For immersion in an organic acid, the leather piece is usually put into a solution prepared with an organic acid at a concentration of 1 to 10%, preferably 2 to 7%, for example, at a bath ratio of 1:10 to 1: 1000, It can be carried out by standing at 20 to 95 ° C., preferably 30 to 70 ° C., for 8 hours or longer, preferably about 12 to 72 hours.

After immersing in organic acid, the leather pieces are precipitated by centrifugation and separated from the organic acid, and can be subjected to microbial treatment described later, but after removing the organic acid, an appropriate neutralization treatment is performed. Also good.
Neutralization treatment is performed, for example, by adding a neutralization solution such as phosphate buffer (pH 7.0) or borate buffer (pH 7.0) to the leather piece, stirring with a vortex mixer, etc., and centrifuging the leather. A method of repeating the treatment of precipitating the pieces and removing the solution several times can be mentioned.

The decomposition treatment of the leather piece with microorganisms is performed using collagenase-producing microorganisms.
Here, collagenase-producing microorganisms, but may be any microorganism producing collagenase, for example, Clostridium (Clostridium) genus Vibrio (Vibrio) genus Bacillus (Bacillus) genus Streptomyces (Streptomyces) bacteria belonging to the genus Is mentioned.
More specifically, for example, Clostridium histolyticum , Clostridium perfringens , Vibrio alginolyticus , Vibrio hollisae , Bacillus licheniformis , Streptomyces sp., Etc. Vibrio hollisae 1706B, Vibrio alginolyticum, etc. deposited as NITE P-776 are listed in the Patent Microorganism Deposit Center (postal code 292-0818, Kazusa Kamafoot 2-5-8, Kisarazu City, Chiba Prefecture).

The decomposition process of the leather pieces by the collagenase-producing microorganism is performed by adding the above-mentioned microorganism culture solution to the leather pieces, and a temperature at which the collagenase-producing microorganism can grow, for example, 15 to 55 ° C, preferably 20 to 55 ° C, more preferably 25 to 25 The reaction can be carried out at 35 ° C. for 2 hours or longer, preferably 8 to 72 hours, more preferably 6 to 24 hours.
As a medium for preparing the culture solution, for example, gelatin medium (composition: gelatin 2.0 g, peptone 5.0 g, yeast extract 1.0 g, potassium dihydrogen phosphate 0.5 g, dipotassium hydrogen phosphate 0.2 g, Examples include magnesium sulfate heptahydrate 0.2 g, sodium chloride 5.0 g, distilled water 1000 mL, pH 7.0).
The microbial culture solution is preferably used so that the bath ratio is 1:10 to 1: 100, for example, for 10 to 50 mg leather pieces.

After completion of the microorganism treatment, the culture containing the decomposed leather pieces is pulverized with a homogenizer, and nucleic acids are extracted from the pulverized product. Nucleic acid extraction can be performed by a publicly known method. Examples of the method include a Marmur method, an enzyme method, a benzyl chloride method, a CTAB method, and the like, but a commercially available DNA extraction kit (for example, “QIAamp DNA Stool Mini Kit” (QIAGEN) or the like) may be used. good.
Moreover, isolation and purification of nucleic acid from the nucleic acid extract can be performed by a known phenol / chloroform method or a method using ethanol precipitation.

  The nucleic acid preparation solution thus obtained is superior in amplification rate in the nucleic acid amplification reaction as compared to the case where collagenase is used, as shown in Example 1 described later. Therefore, the sample nucleic acid preparation method of the present invention can be said to be a sample preparation method suitable for nucleic acid amplification reaction such as PCR method and LAMP method performed when analyzing nucleic acid such as DNA identification of natural leather products. . Then, using the genomic DNA in the obtained preparation as a template, a conventionally known species-specific primer (eg, meat species determination primer (Matsunaga Takamitsu et al., 1997 Livestock Product Development Research Project Report, p131) -137, Agricultural and Livestock Industry Promotion Corp.)), and by amplifying a specific site (for example, cytochrome b gene region of mitochondrial DNA) by PCR method etc., the animal species of the leather can be determined with higher accuracy can do.

  The kit of the present invention is a kit for performing the above-described method for preparing a sample nucleic acid of the present invention, and contains at least an organic acid, a collagenase-producing microorganism, a medium and a nucleic acid extraction solution, and ethanol. In addition, a solution for neutralization treatment, a reaction vessel, a homogenizer, and the like may be included.

Example 1
(1) Chromium-tanned natural cowhide products are cut into 7 pieces with a blade, further chopped, soaked in a 5% strength citric acid solution, allowed to stand at 60 ° C for 12 hours or more, centrifuged, and leather pieces And citric acid solution.
The supernatant citrate solution was removed and replaced with a phosphate buffer solution. After stirring with a vortex mixer, the leather pieces were precipitated by centrifugation. The solution replacement with the phosphate buffer was performed twice or more to neutralize the leather pieces.

(2) Preparation of culture solution of Vibrio hollisae 1706B (NITE P-776)
Vibrio hollisae 1706B (NITE P-776) strain and gelatin liquid medium (composition: gelatin 2.0 g, peptone 5.0 g, yeast extract 1.0 g, potassium dihydrogen phosphate 0.5 g, dipotassium hydrogen phosphate in an L-shaped test tube 0.2 g, 0.2 g of magnesium sulfate heptahydrate, 5.0 g of sodium chloride, 1000 mL of distilled water, pH 7.0) were added. The test tube was shaken at 30 ° C. for 6 hours. When the cloudiness was confirmed visually, it was used for leather decomposition.

(3) 50 mg of each of the seven neutralized leather pieces prepared in (1) is put into a 2.0 mL Eppendorf tube, and 1 mL of the Vibrio hollisae 1706B strain culture medium prepared in (2) is added to this, and 30 ° C is added. For 24 hours. After completion of the incubation, the sample was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was removed.

(4) The precipitated sample was pulverized with a homogenizer, and DNA was extracted from the pulverized sample with a DNA extraction kit (QIAGEN, QIAamp DNA Stool Mini Kit).
200 μL of the obtained DNA extract was concentrated to 10 μL by ethanol precipitation, and this DNA solution was used as a template and subjected to PCR reaction.
Primers were designed as follows based on the cDNA sequence information of bovine mitochondrial DNA and cytochrome b region.
Forward side: 5'-TCT CCT CTG TTA CCC ATA TCT GCC GAG ACG-3 '(SEQ ID NO: 1)
Reverse side: 5'-TTT GTG CCG ATG TAT GGG ATT GCT GAT AAG-3 '(SEQ ID NO: 2)
In addition, the well-known primer for meat type discrimination ("Journal of Japan Society for Food Science and Technology 46 (3)" (1999), Takamitsu Matsunaga, Kiyohiro Shibata, Junichi Yamada, Hiroshi Shinmura, Japan Food Science and Technology Society, 187-194) could also be used (see FIG. 4).

The PCR reaction was 98 ° C for 2 minutes, 1 cycle, 98 ° C for 15 seconds, 66 ° C for 15 seconds, 72 ° C for 45 seconds, 35 cycles, 72 ° C for 2 minutes, and the DNA polymerase was TaKaRa manufactured by Takara Bio Inc.
Ex Taq was performed under the above conditions using an Applied Biosystems 2720 thermal cycler as the thermal cycler.
However, if DNA amplification could not be confirmed in the PCR reaction, the PCR product was used as a template, 98 ° C for 2 minutes 1 cycle, 98 ° C 15 seconds, 68 ° C 15 seconds, 72 ° C 45 seconds 35 cycles, 72 ° C 2 Secondary PCR was performed under the condition of 1 cycle per minute.
After completion of the PCR reaction, the obtained PCR product (289 bp) was analyzed by electrophoresis using 8% acrylamide gel or 2% agarose gel. The electrophoresis conditions were 20 mA for 60 minutes when using acrylamide gel and 100 V for 40 minutes when using agarose gel. A pGEM DNA marker manufactured by Promega was simultaneously subjected to electrophoresis as a molecular weight marker. After electrophoresis, the position of the band was observed by a conventional method. The results are shown in FIG. 1A.
Samples were prepared in the same manner as in (1) to (3), and PCR reaction (98 ° C for 2 minutes for 1 cycle, 98 ° C for 10 seconds for 35 cycles, 62 ° C for 15 minutes) was performed using the above-described meat type identification primer. FIG. 4 shows the electrophoresis pattern of the PCR product when a second cycle of 72 ° C. for 1 minute and 72 ° C. for 3 minutes is performed.

Comparative Example 1
(1) 50 mg of each neutralized leather piece prepared in Example 1 (1) was placed in a 2.0 mL Eppendorf tube, and a DNA extract (400 mM Tris-HCl pH 8.0, 60 mM EDTA, 1% SDS, 1 mL of 0.1% PEG-400, 5 mM DTT) was added. In order to remove the eluted dye, the mixture was stirred upside down several times at room temperature, then centrifuged at 12000 rpm for 5 minutes, and 500 μl of the supernatant was discarded. This operation was performed until there was no elution of the dye.
Proteinase K was added and incubated at 50 ° C. for 12 hours. Subsequently, 10 units of collagenase (from Clostridium histolyticum , used by Seikagaku Corporation) were added and incubated at 37 ° C. for 12 hours with gentle agitation.
Collagenase 10 units were added again and incubated for 12 hours, and then the sample was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was removed.
(2) The precipitated sample was pulverized with a homogenizer, and DNA was extracted from the pulverized sample in the same manner as in Example 1 and subjected to PCR reaction to confirm the PCR product. The results are shown in FIG. 1B.

  From FIG. 1A and FIG. 1B, it was shown that the sample prepared by carrying out the degradation treatment with the microorganism of the present invention has better DNA amplification efficiency than the sample subjected to the collagenase treatment.

Example 2
(1) Cut chrome-tanned natural cowhide products with a knife, immerse them in a 5% concentration organic acid solution (succinic acid, maleic acid, citric acid, formic acid) without chopping them, and at 60 ° C for 12 hours or longer After leaving still, it centrifuged and isolate | separated into the leather piece and the organic acid solution.
The organic acid solution in the supernatant was removed and replaced with a phosphate buffer solution. After stirring with a vortex mixer, the leather pieces were precipitated by centrifugation. The solution replacement with the phosphate buffer was performed twice or more to neutralize the leather pieces.

(2) 50 mg of each neutralized leather piece prepared in (1) is put into a 2.0 mL Eppendorf tube, and 1 mL of the culture solution of Vibrio hollisae 1706B prepared in Example 1 (2) is added thereto, 30 Incubated for 24 hours at ° C. After completion of the incubation, the sample was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was removed.

(3) The precipitated sample was pulverized with a homogenizer, and DNA was extracted from the pulverized sample in the same manner as in Example 1 and subjected to PCR reaction to confirm the PCR product. The results are shown in FIG.

  FIG. 2 shows that DNA can be amplified efficiently in any organic acid treatment.

Example 3
(1) Recycled cowhide products composed of two layers having different properties were divided into each layer (upper layer and lower layer) and cut from each layer to obtain two leather pieces. The leather pieces were immersed in a 5% strength citric acid solution as they were without being chopped, and allowed to stand at 60 ° C. for 12 hours or more, and then centrifuged to separate the leather pieces into a citric acid solution.
The supernatant citrate solution was removed and replaced with a phosphate buffer solution. After stirring with a vortex mixer, the leather pieces were precipitated by centrifugation. The solution replacement with the phosphate buffer was performed twice or more to neutralize the leather pieces.

(2) 50 mg of each neutralized leather piece prepared in (1) is put into a 2.0 mL Eppendorf tube, and 1 mL of the culture solution of Vibrio hollisae 1706B prepared in Example 1 (2) is added thereto, 30 Incubated for 24 hours at ° C. After completion of the incubation, the sample was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was removed.

(3) The precipitated sample was pulverized with a homogenizer, and DNA was extracted from the pulverized sample in the same manner as in Example 1 and subjected to PCR reaction to confirm the PCR product. The results are shown in FIG.

  From FIG. 3, it was shown that DNA can be efficiently amplified from the leather pieces of any layer.

Claims (5)

In the method for preparing a sample nucleic acid from a natural leather product, the sample is characterized in that the nucleic acid is extracted after subjecting a leather piece derived from a natural leather product to a Vibrio hollisae1706B strain (NITE P-776). Nucleic acid preparation method. Vibrio degradation treatment with hollisae1706B strain (NITE P-776) The method of claim 1 wherein the culture 2 hours or more at 15 to 55 ° C.. The method according to claim 1 or 2 , wherein Vibrio hollisae 1706B strain (NITE P-776) is allowed to act after the leather piece is immersed in an organic acid and neutralized if desired. The method according to any one of claims 1 to 3 , wherein the natural leather product is a chrome-tanned or tannin-tanned leather product. The kit for performing the method of any one of Claims 1-4 containing an organic acid, Vibrio hollisae1706B strain (NITE P-776), and a nucleic acid extraction solution.