JP2004121229A - Method for identifying mixing ratio of animal hair fiber in animal hair fiber product - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for identifying a mixing ratio of animal hair fibers in an animal hair fiber product in which two or more animal hair fibers are mixed. <P>SOLUTION: The method for identifying the mixing ratio of animal hair fibers included in the animal hair fiber product includes following processes: (1) a process for extracting a DNA from the animal hair fiber product to be tested, (2) a process for obtaining a primer specific to each of animal species from which the animal hair fiber included in the animal hair fiber product to be tested originated from the extracted DNA in the process (1) as a mold and obtain a relative value ratio of a DNA quantity originated from various animal species by real time PCR (polymerase chain reaction) using a probe for specifically detecting a sequence amplified by the primer, (3) a process for obtaining the mixing ratio of the animal hair fibers to be tested by correcting the relative ratio of the DNA quantity originated from the various animal species obtained in (2) depending on a prepared correspondence table of the relative value ratio of the mixing ratio of animal hair fibers originated from the various animal species with the DNA amount originated from each of the animal species. <P>COPYRIGHT: (C)2004,JPO

Description

(4) The present invention relates to a method for assessing the mixing ratio of animal hair fibers in an animal hair fiber product in which a plurality of types of animal hair fibers are blended.

At present, JIS L 1030-1 (Japan Standards Association) is mainly used in Japan as a method for identifying the type of animal of origin used in animal hair fiber products and determining the mixing ratio. And a method of determining the fiber mixing ratio by counting the number of fibers using a microscope specified by JIS L 1030-2 (Japanese Standards Association). Other methods include, for example, a method for identifying animal hair fiber products by using an electron microscope or DNA.

Among the above, the discrimination methods of observing using an optical microscope or an electron microscope are all morphological discriminations that observe the structure of animal hair fibers such as animal hair fiber shape, scale shape, thickness, gloss and sliminess Is a discrimination method.

Animal hair fiber is a natural fiber derived from animal hair, so its fiber shape is not uniform, and its surface shape changes its external shape, which is a characteristic feature in judging animal species. Often disappears. Therefore, in order to distinguish the type of animal hair fiber by morphological observation with a microscope, specialized knowledge and experience are required.

For example, cashmere, which is one of the goat-derived animal hair fibers, is generally thin and soft, and has a good feel and texture. It is said that sheep-derived wool is generally thicker and harder than cashmere, and has a poor feel and texture. However, some cashmere are thick and hard, and some are wool thin and soft. In fact, it is said that the hair obtained from young sheep is often thin and soft. In recent years, processing to improve the feel and texture of wool by modifying the surface structure and shrink-proof processing to improve the phenomenon of animal hair fiber products shrinking after washing by removing specific surface structures have increased in recent years. ing. When such a treatment is performed, it is difficult for an expert to distinguish animal hair by observing with a microscope.

Yak is a bovine animal that lives in high altitudes of 4000 m or more, and has fine and soft hair unique to animals that inhabit high altitudes. Since the fiber obtained from this yak-derived animal hair is soft and has heat-retaining ability, it has a small amount of circulation and is very popular. However, the shape of yak-derived animal hair fibers is very similar to cashmere, and is one of the most difficult animal hairs to distinguish by microscopy. The transaction price is generally lower than cashmere and higher than wool.

However, there is a large difference in the transaction price between yak, cashmere and wool, so if the composition of animal hair fiber products is yak, cashmere, wool, or two or more, the mixing ratio will be different. There is a need for a discrimination method that can scientifically prove the degree.

In recent years, identification methods using DNA have been used for the purpose of identifying biological species in various fields. This utilizes a DNA sequence that is specific for each species, and it has been recognized that confirming this specific sequence has a sufficient scientific basis as a method for identifying a species.

At this time, as a method for simply confirming the target sequence in the trace sample, a desired sequence is amplified by a polymerase chain reaction (hereinafter, PCR), and it is determined whether or not the target sequence is contained. A method of confirming by electrophoresis is employed. Regarding the origin of animal hair species, a method for DNA identification of animal hair using this PCR method is known, and primers for each animal species for identification have been reported (Non-Patent Documents 1, 2 and the like). ). Also, for example, Patent Document 1 discloses a method for identifying animal hair fibers derived from cattle, camel, goat, and sheep species using DNA. According to such a method, discrimination with higher objectivity can be performed from discrimination relying on experience.

However, the currently known discrimination method is a qualitative test for determining whether or not DNA of a specific animal species is contained in animal hair fibers. It cannot be used to measure fiber mixing ratio.

As a method for quantifying a specific DNA contained in a sample by using the principle of the PCR method, a method for estimating the amount of DNA contained in a template solution by measuring the amount of an amplification product in a PCR step is a real-time PCR. Alternatively, a method called quantitative PCR (hereinafter referred to as real-time PCR) is known.

In real-time PCR, the amount of the template DNA contained in the sample before the PCR step is determined by detecting the amount of the PCR amplification product every cycle or several cycles using a fluorescent probe and measuring the change in the fluorescence intensity. It is an estimate. The principle and operation method of real-time PCR have already been established, and an apparatus for performing real-time PCR, such as ABI PRISM 7000 (Applied Biosystems Japan Co., Ltd.), is also known.

However, in addition to the fact that animal hair fibers are often treated with drugs, the activity of the protease used for DNA extraction is not constant, and the purification work is complicated, so that DNA that is originally susceptible to damage is There are many error factors related to DNA extraction, such as degradation or cleavage of DNA. In addition, since the amount of the inhibitor in the PCR step contained in the DNA extract from the sample is not constant, there is also an effect that underestimation of the measured value in real-time PCR occurs. Therefore, even if the absolute amount of DNA derived from each animal species contained in animal hair fibers is measured by real-time PCR, and these are compared, it is impossible to obtain an accurate mixing ratio.

JP 2000-325098 A Molecular speciation of animal fibers, Paul F Hamlyn et al., JSDC Volume 114 March 1998, P78-80

An object of the present invention is to provide a method capable of accurately and easily confirming the ratio of animal hair species used in animal fiber mixed fiber products by applying a real-time PCR method.

In the Household Goods Quality Labeling Law, it is required that the labeling of the fiber composition show its mixing ratio (%). Therefore, the present inventors do not obtain the absolute amount of the DNA derived from each animal species contained in the animal hair fiber, but determine the mixing ratio of the DNA derived from each animal species contained in the animal hair fiber to a standard value of a known mixing ratio. By compensating for the value obtained from the mixed animal hair fiber, a method for obtaining the mixed rate of the animal hair fiber was completed.

That is, the present invention comprises the following steps:
1) a step of extracting DNA from a test animal hair fiber product;
2) Using the DNA extracted in 1) as a template, a primer specific to each animal species derived from animal hair contained in the test animal hair fiber product and a sequence amplified by the primer are specifically detected. Performing a real-time PCR using a probe to obtain a ratio of a relative value of a DNA amount derived from each animal species,
3) The mixing ratio of animal hair derived from each animal species obtained using a standard mixed animal hair fiber prepared by mixing a plurality of ratios of substantially 100% pure animal hair fibers prepared in advance and the origin of each animal species A step of correcting the ratio of the relative value of the DNA amount derived from various animal species obtained in 2) based on the correspondence table with the ratio of the relative value of the DNA amount to obtain the mixing ratio of the test animal hair fiber And a method for examining the mixing ratio of animal hair contained in animal hair fiber products.

に よ っ て The method of the present invention can quantitatively obtain the mixing ratio of animal hair in an animal hair fiber product containing two or more animal hair fibers.

The present invention further provides a primer pair specific to goat, yak and sheep species and a probe specifically binding to a gene fragment amplified by the primer pair, which is useful for examining the mixed ratio of the animal hair fiber of the present invention. I will provide a.

The present invention also provides a kit for detecting or quantifying each animal hair species gene, comprising the above-mentioned primer pair and probe.

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, "animal fiber" means an animal hair part. In the present specification, the term "animal fiber product" includes untreated raw wool, hair washed, scoured, bleached, dyed, processed, etc., which has been cut from an animal, and a yarn obtained by spinning these. Shall be included. In addition, the "animal fiber product" shall include any of products processed into yarn, woven fabric, knitted products, etc., and finished products such as clothing, and conventionally known processes, for example, Products that have been subjected to shrinkage prevention, wrinkle prevention, water repellency, insect repellency, deodorization, antifouling processing, and the like are also included. Further, animal hair provided as fur without cutting the hair is also included in the "animal hair fiber product" in this specification.

(4) In the method of the present invention, first, the kind of animal hair which can be mixed in the animal hair fiber product to be tested is confirmed. This step is unnecessary if the mixed animal hair species is known in advance. The animal hair species may be confirmed by any of the methods conventionally used, and for example, a method defined in JIS-1030, in particular, a microscopic method can be used.

(4) The animal hair species may be confirmed using a conventionally known DNA identification method. In the DNA discrimination method, a PCR reaction is performed using DNA extracted from the test target fiber as a template, and primers designed to be specific to animal hair that can be mixed are used, and PCR amplification products are analyzed and predicted. This is a qualitative one that determines whether or not the DNA sequence contains the selected DNA sequence. (Molecular speciation of animal fibers, Paul F Hamlyn et al., JSDC Volume 114 March 1998, P78-80). Various primers that can be used to identify animal hair species are known, and for example, those described in G. Nelson et al., Textile Research Journal 62 (10) 590-595 (1992) are suitably used. Alternatively, each primer pair specific to yak, sheep and goat provided in the present invention may be used.

Alternatively, whether or not animal hair fibers derived from bovine, camel, goat and sheep species are mixed may be confirmed by the method described in JP-A-2000-325098.

DNA is then extracted from the test animal hair fibers. If necessary, the animal hair fibers may be washed before the DNA extraction treatment. In particular, when fiber waste and oils and fats are attached to the animal hair fiber product to be inspected, it is preferable to wash. When a PCR reaction is used to confirm the type of animal hair, a part of the DNA extracted by the method described below is used to confirm the animal hair type, and a part is used to discriminate the mixing ratio of each animal hair type. It may be used.

ＤＮＡ Extraction of DNA from animal hair fiber products may be performed by any conventionally known method, and is not particularly limited. It is preferable that impurities in the DNA extract do not affect PCR, and that the amount of DNA is such that Ct is 30 or less in the subsequent measurement by real-time PCR.

Specifically, for example, there is a method in which animal hair fiber products are shredded and immersed in a DNA extraction solution. Examples of the DNA extract include those containing an appropriate protease and surfactants such as SDS and polyethylene glycol, and EDTA and DTT, and are not limited thereto, and any commercially available DNA extract is suitable. Used.

DNA extraction is typically performed by charging a sample into a protease-containing DNA extract and incubating at 50 ° C. for 12 hours or more with slow stirring. After the incubation is completed, the sample is stirred again, and insoluble substances are precipitated by centrifugation. The DNA contained in the obtained supernatant is isolated and purified by a conventional method, for example, a method using phenol and chloroform well known to those skilled in the art.

A primer pair specifically recognizing the DNA derived from each animal species identified above using the DNA isolated from the animal hair fiber product as a template, a label capable of emitting fluorescence according to the amount of amplification by the primer pair A PCR reaction is performed under conditions containing a probe and a DNA polymerase, and the fluorescence intensity indicating the amount of the obtained amplification product is monitored in real time, that is, at every fixed cycle, for example, at every cycle. This real-time PCR method is a method already established as described above, and may be performed using a commercially available device.

In order to make the fluorescence intensity obtained by the real-time PCR method correspond to the amount of the amplification product, a real-time PCR reaction using a standard DNA sample of various concentrations as a template is performed under the same conditions as the test sample, and the amount of the amplification product is reduced. What is necessary is just to monitor in real time and create a calibration curve. As a fluorescently labeled probe for obtaining an amplification product, for example, there is a TaqMan probe described below. In addition, SYBR Green, which is a dye that specifically binds to double-stranded DNA, is known, but a TaqMan probe is preferably used because of the accuracy of the measured value.

解析 The method of analyzing the obtained fluorescence intensity and the method of creating a calibration curve can be easily performed by the attached computer program based on the instructions attached to the commercially available device.

Separately, a standard mixed animal hair fiber is prepared by mixing animal hair fibers containing substantially 100% of animal hair derived from each animal species mixed in the test animal hair fibers in a plurality of ratios. DNA is extracted from the standard mixed animal hair fiber in the same manner as described above. Using the extracted DNA as a template, real-time PCR was performed using a primer pair specific to each animal species and a probe that specifically detects the primer pair, and the relative value of the amount of DNA derived from each animal species in the extracted DNA Respectively. A comparison table of the mixing ratio of the fiber derived from each animal species in the standard mixed animal hair fiber and the ratio of the relative value of the amount of DNA derived from each animal is prepared in advance.

比 Based on the prepared comparison table, the ratio of the primary measurement value of each test animal fiber by each primer pair is corrected. By performing such a correction, an accurate mixing ratio, which could not be obtained conventionally, can be obtained.

The present invention further provides a primer pair specific to each animal species useful for the method of the present invention and a probe capable of specifically detecting the animal species. The primers and probes provided by the present invention are useful for detecting and quantifying yak, an animal from which yak is derived, sheep, which is an animal from which wool is derived, and goat, which is an animal from which cashmere is derived.

Preparation of primer pairs and probes:
Animal hair products are often already damaged in DNA due to various treatments before they become products. In addition, DNA sequences are partially similar between yak, goat and sheep species. For this reason, it is necessary to use primers and probes that target a DNA sequence portion that is particularly highly conserved and has high species specificity. Just because DNA sequences of yak species, goat species and sheep species are known, it is not easy to prepare practical primers and probes. Theoretically, in real-time PCR measurement, the more constant the amplification efficiency, the higher the reliability of the measured value, so not only the specificity is high, but also the ease of annealing of primers and probes to the template DNA must be considered. No. It is not easy to make a balanced primer and probe.

As described above, the TaqMan probe is obtained by binding a reporter fluorescent dye and a quencher fluorescent dye to an oligonucleotide probe. As the reporter fluorescent dye, any known various dyes can be used, and for example, a fluorescein-based fluorescent dye such as FAM (6-carboxy-fluorescein) is preferable. As the quencher fluorescent dye, any of various known quencher dyes can be used. For example, a rhodamine-based fluorescent dye such as TAMRA (6-carboxy-tetramethyl-rhodamine) is preferable. Also in this embodiment, FAM and TAMRA were used as reporters and quenchers.

The present inventors have made use of the known sequence of mitochondrial DNA, which is said to have a relatively good DNA preservation and a high mutation rate for yak, goat and sheep, and have a unique sequence for each species of organism. We designed our own primer pairs. In addition, a TaqMan probe for specifically detecting amplification by each primer was designed.

FAM (6-carboxy-fluorescein) was linked to the 5 'end as a reporter fluorescent dye of the probe, and TAMRA (6-carboxy-tetramethyl-rhodamine) was linked to the 3' end as a quencher fluorescent dye.

The newly designed primers and probes are as follows:
Sheep primer pair 1
woolF7404: 5'-AACCAGGAGAACTGCGTTTACT-3 '(SEQ ID NO: 1)
woolR7626: 5'-TTTGATCCGCAAATTTCTGA-3 '(SEQ ID NO: 2)
Sheep probe 1
woolT7562: 5'-FAM-ACAACCCTTATGTCAACTCGTCCAGGCC-TAMRA-3 '(SEQ ID NO: 3)

Sheep primer pair 2
Wool F 1788: 5'-AATCCCTTAGAAGTCCCACTACTCAA-3 '(SEQ ID NO: 10)
Wool R 1930: 5'-TACACGCCTAGTGCGATGGTAA-3 '(SEQ ID NO: 11)
Sheep probe 2
Wool T 1853: 5'-FAM-AGCTCACCATAGCCTCATAGAAGGGAACCG-TAMRA-3 '(SEQ ID NO: 12)

Goat primer pair
yagiF874: 5'-TTAGTTGAATTAGGCCATGAAGCA-3 '(SEQ ID NO: 4)
yagiR953: 5'-TTAATAGGCTTGAGTGCATTGTATTTACT-3 '(SEQ ID NO: 5)
Goat probe
yagiT901: 5'-FAM-ACACACCGCCCGTCACCCTCC-TAMRA-3 '(SEQ ID NO: 6)

Yak primer pair
Yak F 883: 5'-GCTTTCTCTATCCTAATCCTTGCTCTTA-3 '(SEQ ID NO: 13)
Yak R 964: 5'-GGCTGAGTGGTCGGAAGATTAT-3 '(SEQ ID NO: 14)
Yak probe
Yak T 913: 5'-FAM-CCCCTACTGCACACTTCCAAACAACGAA-TAMRA-3 '(SEQ ID NO: 15)

Yak, sheep, goat common primer pair
12SrRNAF531: 5'-TACAGAAACAAAATTATTCGCCAGAGT-3 '(SEQ ID NO: 7)
12SrRNAR607: 5'-GGGTATAAAGCACCGCCAAGT-3 '(SEQ ID NO: 8)
Yak, sheep, goat common probe
12SrRNAT560: 5'-FAM-TACCGGCAACAGCCCGAAACTCAAA-TAMRA-3 '(SEQ ID NO: 9)

キ ッ ト A kit for detecting or quantifying a gene derived from each animal hair species, including the above-mentioned primer pairs and probes, which can be used for real-time PCR, is also included in the scope of the present invention. In addition, the kit may include reagents and buffers that are known to be usually used for a PCR reaction, such as a DNA polymerase and a reaction solution.

In the examples of the present invention, TaqMan PCR was used as a means for detecting real-time PCR. The TaqMan PCR performs PCR in the presence of a probe to which a reporter dye and a quencher dye are bound, and measures the fluorescence intensity that increases with the amplification of the target nucleic acid, thereby detecting the target nucleic acid in the test sample. This is a detection and quantification technique [Christian et al., Genome Research 6: 986-994 (1996)]. This technique involves simultaneously performing an amplification reaction and measurement of fluorescence intensity, detecting in real time a fluorescent reporter dye released by the decomposition of a labeled probe hybridized to a template, and using a computer connected to a detector to amplify the amplified product. Analyze automatically.

In TaqManPCR, perform PCR reaction while measuring fluorescence intensity. When the test sample contains DNA having the target sequence, DNA amplification occurs, and the fluorescently labeled probe hybridized to the amplification product is degraded by the action of DNA polymerase. After a certain number of cycles, the fluorescence intensity exceeds the detection limit and rapidly increases. Then, as the amount of DNA having the target sequence in the test sample increases, the fluorescence intensity increases rapidly with a small number of cycles. Therefore, by examining the number of cycles at which the rapid increase in the fluorescence intensity starts, the DNA having the target sequence in the test sample in the test sample can be quantified.

Specifically, if a calibration curve is prepared with the number of cycles on which the rapid increase in fluorescence intensity is detected on the abscissa and the amount of DNA on the abscissa, when DNA from the test sample is used as a template, By examining the number of cycles (Ct value) when the detection limit is exceeded, the amount of DNA in the test sample can be measured.

The present invention will be described in more detail with reference to Examples in which the mixture ratio of animal hair species derived from sheep, goats and yaks in animal hair fiber products was measured using the primers and probes of the present invention.

The mixing ratio of cashmere (goat) and wool (sheep) was measured from the animal hair fiber products (mixture of two types) derived from cashmere and wool by the method of the present invention.
The sample used was 100% cashmere wool and 100% wool wool, and was mixed at a weight ratio of 100: 0 to 0: 100 to obtain a standard value.
Reagents, instruments used, etc. were sterilized as much as possible, and those suitable for use in DNA manipulation were used.

DNA extraction from animal hair fiber products Samples in which cashmere wool and wool raw wool were mixed at various ratios were cut into small pieces, and 50 mg of each sample was taken and placed in a 2.0 ml Eppendorf test tube.
2. 1 ml of a DNA extraction buffer (DNA extraction buffer (DEB) (400 mM Tris-HCl pH 8.0, 60 mM EDTA, 1% SDS, 0.1% PEG-400, 5 mM DTT)) and 10 U of proteinase K are added, and slowly at 50 ° C. for 12 hours or more. And incubated with stirring.
3. The sample after the incubation was agitated and centrifuged at 9500G for 5 minutes. The supernatant was transferred to another tube, 300 μl of phenol was added and mixed for 15 minutes.
4. 300 μl of chloroform was added and mixed for 5 minutes.
5. Centrifuged at 270 G for 1 minute. The supernatant was transferred to another tube and kept at 0 ° C. for 15 minutes.
6. The mixture was centrifuged at 11000 G for 5 minutes, and the supernatant was purified using Microcon-100. The obtained DNA was suspended in 30 μl of 10 mM Tris-HCl (pH 8.0).

Using the thus obtained DNA from each sample as a template, measurement was performed by real-time PCR using the primers and probes of the present invention. (All are listed in order of primer (forward, reverse) and probe)
Goat species primers and probes yagiF874, yagiR953, yagiT901
Sheep species primers and probes woolF7404, woolR7626, woolT7562
Primers and probes for standard (preparation of calibration curve) 12SrRNAF531, 12SrRNAR607, 12SrRNAT560

PCR reaction solution Sterile water 20μl
TaqManUniversalPCRMasterMix 25μl
Primer (forward, reverse) 1μl each (50pmol)
TaqMan probe 1μl (12.5pmol)
DNA sample 2μl

TaTaqMan Universal PCR MasterMix from Applied Biosystems Japan was used for the PCR reaction solution. In addition, as a negative control, a sample into which 2 μl of 10 mM Tris-HCl (pH 8.0) had been introduced instead of the DNA sample was similarly subjected to the following PCR cycle.

PCR reaction conditions 50 ° C for 2 minutes 95 ° C for 10 minutes 95 ° C for 15 seconds, 60 ° C for 1 minute, 40 cycles

Setting of measurement conditions for real-time PCR ABI PRISM 7000 (Applied Biosystems Japan Co., Ltd.) was used as a real-time PCR device. As described above, since FAM was used as the reporter and TAMRA was used as the quencher for the probe, the detector settings were also adjusted to this. Each PCR reaction was performed in duplicate.

1) Setting of threshold line and preparation of calibration curve For DNA extract (standard) extracted from 100% wool wool, undiluted solution, 2-fold diluted, 4-fold diluted sample, use standard primers and probes 12SrRNAF531, 12SrRNAR607, 12SrRNAT560 The real-time PCR reaction was performed. The fluorescence intensity in each cycle was measured, and the fluorescence intensity at which the fluorescence intensity showed an index increase at each concentration was set as a threshold line. The concentration (Qty) of the DNA in the stock solution of the standard DNA sample, the 2-fold and 4-fold dilutions was set to 100, 50 and 25, respectively, and the number of cycles (Ct) and the logarithmic value of the concentration when the threshold line was reached were obtained. (Fig. 1).
In the following real-time PCR, this value was used for both the threshold line and the calibration curve.

2) Preparation of a comparison table of the ratio of the mixing ratio in the standard mixed animal hair fiber to the relative value of the DNA amount A sample containing 100% wool and 100% cashmere wool and containing cashmere in the amount shown in Table 1, That is, a standard mixed animal hair fiber in which the mixture ratio of goat in the sample was as shown in Table 1 was prepared. The DNA of each standard mixed animal hair fiber was extracted as described above. The DNA solution extracted from each sample was subjected to a real-time PCR reaction using primers and probes for goat species: yagiF874, yagiR953, yagiT901, primers and probes for sheep species woolF7404, woolR7626, and woolT7562.
The PCR reaction conditions were the same as above, and the relative value (Qty) of the amount of DNA was obtained from the number of cycles (Ct) at the time of reaching the threshold line obtained in 1) using a calibration curve. As the Qty value, an average value measured in duplicate was used.

The ratio Ps of the Qty value of the goat species to the Qty value obtained by using the primer and the probe of the goat species and the sheep species in the standard mixed animal hair fiber is represented by the following formula 1:
Ps = Y / (Y + W) × 100 (1)
Ps: ratio of the measured value of the goat species to the sum of the measured values of the goat and sheep Y: the measured value of the goat species W: calculated by the measured value of the sheep species. A table was prepared in which the primary amount of the obtained DNA was associated with the mixing ratio of goat in the standard mixed animal hair fiber (Table 1).

なお、ネガティブコントロールとしてＤＮＡ試料に代えてトリスバッファーを投入した場合には、蛍光が検出されず、Ｃｔ値は得られなかった。

When Tris buffer was added instead of the DNA sample as a negative control, no fluorescence was detected and no Ct value was obtained.

3) Measurement of mixing ratio A sample in which 100% cashmere 10% wool and 90% wool 90% wool was mixed was prepared, divided into five samples, DNA extraction was performed for each, and primers and probes for goats and sheep Was performed by using real-time PCR. Each real-time PCR was performed in duplicate. The obtained result was analyzed under the conditions set above, and an average Qty value was obtained. Table 2 shows the results.

　ヤギ種及びヒツジ種の測定結果である平均Qtyの値より式２：
Ｐ＝Ｙ／（Ｙ＋Ｗ）×１００　　　　（２）
　Ｐ：ヤギ種測定値の比率
　Ｙ：ヤギ種の測定値
　Ｗ：ヒツジ種の測定値
によりヤギ種測定値の比率（Ｐ値）を得た。次いで、得られたＰ値を式３に当てはめて混用率を測定した。

From the value of the average Qty which is the measurement result of the goat species and the sheep species, Equation 2:
P = Y / (Y + W) × 100 (2)
P: Ratio of goat species measured values Y: Goat species measured values W: Goat species measured values ratio (P value) was obtained from sheep species measured values. Next, the obtained P value was applied to Equation 3 to measure the mixing ratio.

Equation 3:
Q = (P-SM) × (BT-ST) ÷ (BM-SM) + ST (3)
In Equation 3, each symbol indicates the following value. Q: Goat mixing ratio SM: Among Ps values in Table 1, smaller than P value and maximum value BM: Ps value in Table 1 larger than P value And minimum value ST: Goat mix rate corresponding to Ps value applied in SM in Table 1 BT: Goat mix rate corresponding to Ps value applied in BM in Table 1

Table 3 shows the results obtained by calculating the results in Table 2 using the above Equation 3.

As is apparent from the calculated mixture ratio, the measurement result using the novel mixture ratio measurement method of the present invention was measured with an accuracy within ± 5%, which is the allowable range defined in the Household Goods Quality Labeling Law. . According to the present example, it was confirmed that the method, the primer, and the probe of the present invention are suitably used for measuring the mixture ratio of animal hair fiber products derived from goat species and sheep species.

DNA was extracted from a product containing three types of animal hair fibers derived from yak, cashmere and wool, and the mixing ratio of yak, cashmere (goat) and wool (sheep) was measured by the method of the present invention.
The sample used was 100% raw wool, 100% cashmere wool and 100% wool raw wool. In order to obtain a standard value, the weight ratio was 100: 0: 0 to 0: 100: 0 to 0: 0: 100. Was used at a ratio of 1.

1) Setting of detection limit and preparation of calibration curve For DNA extract (standard) extracted from 100% cashmere wool, undiluted solution, 2-fold diluted, 4-fold diluted sample, standard primer and probe 12SrRNAF531, 12SrRNAR607, 12SrRNAT560 were used. The real-time PCR reaction used was performed. The fluorescence intensity in each cycle was measured, and the fluorescence intensity at which the fluorescence intensity showed an index increase at each concentration was set as a detection limit (Threshold line). The concentration (Qty) of the DNA in the stock solution of the standard DNA sample, the 2-fold and 4-fold dilutions was set to 100, 50, and 25, respectively, and the number of cycles (Ct) and the logarithmic value of the concentration when the detection limit was reached were obtained. A calibration curve was created between
In the following real-time PCR, this value was used for both the detection limit and the calibration curve.

2) Setting of each animal hair fiber mixture ratio and standard value in the standard mixed animal hair sample The primers and probes used in the real-time PCR of Example 2 are as follows.
(In order, primer (forward, reverse), probe):
Goat species primers and probes yagiF874, yagiR953, yagiT901
Sheep primers and probes Wool F 1788, Wool R 1930, Wool T 1853
Yak F 883, Yak R 964, Yak T 913
Primers and probes for standard (preparation of calibration curve) 12SrRNAF531, 12SrRNAR607, 12SrRNAT560

A standard mixed animal hair sample was prepared by mixing raw wool of 100% yak, 100% wool and 100% cashmere in a weight ratio of 100: 0: 0 to 0: 100: 0 to 0: 0: 100. Specifically, the ratio of the content of one type to the total content of the two types of the standard mixed animal hair fibers created is as follows: yak and cashmere, yak and wool, and cashmere and wool. It is shown. The standard mixed animal hair fiber is a mixture of three types of animal hair fibers, and each table defines the mixing ratio between two of the types.

DNA of each standard mixed animal hair fiber was extracted in the same manner as in Example 1, and primers and probes for goat species: yagiF874, yagiR953, yagiT901, primers and probes for sheep species using the DNA extracted from each sample as a template. A real-time PCR reaction was carried out using F 1788, Wool R 1930, Wool T 1853, primers and probes for yak species Yak F 883, Yak R 964, and Yak T 913, respectively.

The PCR reaction conditions are the same as in Example 1. From the number of cycles (Ct) at the time of reaching the detection limit obtained in 1), a relative value of the DNA amount (relative value: Qty) was obtained from a calibration curve. As the Qty value, an average value measured in duplicate was used. From the measurements obtained using the goat, sheep and yak species primers and probes, respectively, the ratios between the yak and the goat, between the yak and the sheep, and between the sheep and the goat, respectively, were determined and the ratio between the two species was determined. It is associated with the mixing ratio of the hair fiber.

The ratio Ps1 of the Qty value of the yak species to the sum of the Qty values obtained using the primers and the probes of the yak species and the goat species in the standard mixed animal hair fiber is expressed by the following formula (4).
Ps1 = Y / (Y + C) (4)
Ps1: Ratio of measured values of yak species in total of measured values of goat and goat Y: Measured value of yak species C: Calculated by measured value of goat species. The obtained values were associated with the percentage of yak content in the total content of yak and goat in the standard mixed animal hair fiber (Table 4).

The ratio Ps2 of the Qty value of the yak species to the sum of the Qty values obtained using the primer and the probe of the yak species and the sheep species in the standard mixed animal hair fiber is represented by the following formula 5:
Ps2 = Y / (Y + W) (5)
Ps2: Ratio of measured values of yak species in total of measured values of yak and sheep Y: Measured value of yak species W: Calculated by measured value of sheep species. The obtained value was associated with the ratio of the yak content to the total content of yak and sheep in the standard mixed animal hair fiber (Table 5).

The ratio Ps3 of the Qty value of the goat species to the sum of the Qty values obtained using the primer and the probe of the goat species and the sheep species in the standard mixed animal hair fiber is represented by the following formula 6:
Ps3 = C / (C + W) (6)
Ps3: Ratio of measured values of goat species to total of measured values of goat and sheep C: Measured value of goat species W: Calculated by measured value of sheep species. The obtained values were associated with the ratio of the content of goat to the total content of goat and sheep in the standard mixed animal hair fiber (Table 6).

When Tris buffer was added instead of the DNA sample as a negative control, no fluorescence was detected and no Ct value was obtained.

3) Measurement of Mixing Ratio Samples were prepared by mixing cashmere, yak, and wool wool at the mixing ratios shown in Table 7, and DNA extraction operations were performed on the samples, and primers and probes for yak, goat, and sheep were respectively prepared. The real-time PCR used was performed. Each real-time PCR was performed in duplicate. The obtained result was analyzed under the conditions set above, and an average Qty value was obtained. Table 7 shows the results.

From the value of the average Qty, which is the measurement result of yak species and goat species, Equation 7:
Pyc = Y / (Y + C) (7)
Pyc: the ratio of the yak species measured value to the sum of the measured values of the yak species and the goat species. Y: the measured value of the yak species. C: the measured value of the yak species and the sum of the measured values of the yak species and the goat species. The ratio (Pyc value) was obtained. Next, the obtained Pyc value was applied to Equation 8, and the mixing ratio between the yak species and the goat species was measured.

Equation 8:
Qyc = (Pyc-SM) × (BT-ST) ÷ (BM-SM) + ST
Qcy = 100−Qyc
In Equation 8, each symbol indicates the following value. Qyc: ratio of the content of yak to the total content of yak and goat. Qcy: ratio of the content of goat in the ratio of yak to goat. SM: Ps1 value in Table 4. , Pyc value and maximum value BM: Ps1 value in Table 4 larger than Pyc value and minimum value ST: Yak mixing ratio corresponding to Ps1 value applied in SM in Table 4 BT: Yak mixing ratio corresponding to Ps1 value applied in BM in Table 4

From the value of the average Qty, which is the measurement result of the yak species and the sheep species, Equation 9:
Pyw = Y / (Y + W) (9)
Pyw: ratio of yak species measured value to total of measured values of yak species and sheep species Y: measured value of yak species W: measured value of yak species and sum of measured values of yak species and sheep species based on measured values of sheep species The ratio (Pyw value) was obtained. Next, the obtained Pyw value was applied to Equation 10 to measure the mixing ratio.

Equation 10:
Qyw = (Pyw-SM) × (BT-ST) ÷ (BM-SM) + ST
Qwy = 100−Qyw
In Equation 10, each symbol indicates the following value. Qyw: Ratio of yak content in total content of yak and sheep Qwy: Ratio of sheep content in total content of yak and sheep SM: Ps2 in Table 5 BM: Ps2 value in Table 5 larger than Pyw value and minimum value ST: Yak mixing ratio corresponding to Ps2 value applied in SM in Table 5 BT: Yak mixing ratio corresponding to Ps2 value applied in BM in Table 5

From the value of the average Qty, which is the measurement result of goat and sheep species, Equation 11:
Pcw = C / (C + W) (11)
Pcw: ratio of goat species measured value to total measured values of goat species and sheep species C: measured value of goat species W: measured value of goat species based on measured values of sheep species and sum of measured values of goat species and sheep species The ratio (Pcw value) was obtained. Next, the obtained Pcw value was applied to Expression 12, and the mixing ratio was measured.

Equation 12:
Qcw = (Pcw−SM) × (BT−ST) ÷ (BM−SM) + ST
Qwc = 100−Qcw
In Equation 12, each symbol indicates the following value. Qcw: ratio of goat content to total content of goat and sheep Qwc: ratio of sheep content to total content of goat and sheep SM: Ps3 in Table 6 Among the values, the maximum value smaller than the Pcw value and the maximum value BM: The maximum value of the Ps3 value in Table 6 larger than the Pcw value and the minimum value ST: The mixing ratio of the goat corresponding to the Ps3 value applied in the SM in Table 6 BT: Goat mix rate corresponding to Ps3 value applied in BM in Table 6

The above obtained Qyc, Qcy, Qyw, Qwy, Qcw, and Qwc were applied to the following Expression 13 to calculate each mixed ratio of yak, goat, and sheep.
Equation 13:
Qc1 = Qcy × Qyw
Qw1 = Qwy × Qyc
Qy1 = Qyc × Qyw
Rc1 = Qc1 / (Qc1 + Qw1 + Qy1) × 100
Rw1 = Qw1 / (Qc1 + Qw1 + Qy1) × 100
Qc2 = Qcw × Qcy
Qw2 = Qwc × Qcy
Qy2 = Qyc × Qcw
Rc2 = Qc2 / (Qc2 + Qw2 + Qy2) × 100
Rw2 = Qw2 / (Qc2 + Qw2 + Qy2) × 100
Rc = (Rc1 + Rc2) / 2
Rw = (Rw1 + Rw2) / 2
Ry = 100−Rc−Rw

In the above equation 13, each symbol indicates the following value.
Qyc: ratio of the content of yak to the total content of yak and goat Qcy: ratio of the content of goat to the total content of yak and goat Qyw: ratio of the content of yak to the total content of yak and sheep Qwy: Qcw: Ratio of goat content to total content of goat and sheep Qcw: Ratio of goat content to total content of goat and sheep Qwc: Ratio of sheep content to total content of goat and sheep Rw: Sheep mixing rate Ry: Yak mixing rate

Table 8 shows the results obtained by calculating the results in Table 7 by the above formulas.

As is apparent from the calculated mixture ratio, the measurement result using the novel mixture ratio measurement method of the present invention was measured with an accuracy within ± 5%, which is the allowable range defined in the Household Goods Quality Labeling Law. . According to this example, it was confirmed that the method, the primer, and the probe of the present application are suitably used for measuring the mixing ratio of animal hair fiber products derived from yak species, goat species and sheep species.

The mixing ratio of each animal hair fiber in the dyed animal hair fiber product was measured.
As a sample, a wool of cashmere and wool mixed with a cashmere: wool ratio of 90:10 and 50:50 and a wool of 100% each of cashmere and wool were used. Each sample was dyed yellow or black using an acid dye. The dyes used are as follows:
Yellow: Lanyl Yellow G extra conc.
Black: Lanyl Black BG extra conc.

原 The raw wool sample was immersed in a dye solution (pH 4.0) containing 3% dye, 1% acetic acid and 0.3% emulgen 910 in water, and dyed at 90 ° C. for 15 minutes. After dyeing, it was poured into water at 60 ° C., slowly cooled, and then washed with water at normal temperature.

For each stained sample, the mixture ratio of cashmere and wool was measured in the same manner as in Example 1, and compared with a known mixture ratio. The results are shown in the table.

通 り As is clear from the results shown in Table 9, the mixing ratio can be accurately measured even for animal hair fibers modified by dyeing.

3 shows a calibration curve of real-time PCR. The horizontal axis shows the logarithmic value of the relative DNA amount, and the vertical axis shows the number of cycles that reached the threshold line.

Claims (25)

1) a step of extracting DNA from a test animal hair fiber product;
2) Using the DNA extracted in 1) as a template, a primer specific to each animal species derived from animal hair contained in the test animal hair fiber product, and a probe for specifically detecting a sequence amplified by the primer Performing real-time PCR using to obtain the ratio of the relative value of the amount of DNA from each animal species,
3) The mixing ratio of animal hair derived from each animal species obtained using a standard mixed animal hair fiber prepared by mixing a plurality of ratios of substantially 100% pure animal hair fibers prepared in advance and the origin of each animal species The step of correcting the ratio of the relative value of the DNA amount derived from various animal species obtained in 2) based on the correspondence table with the ratio of the relative value of the DNA amount to obtain the mixing ratio of the test animal hair fiber product A method for determining the mixing ratio of animal hair contained in animal hair fiber products. The method according to claim 1, further comprising a step of determining an animal species from which animal hair contained in the test animal hair fiber product is derived. The method according to claim 1, wherein the step of determining the animal species from which the animal hair contained in the test animal hair fiber product is derived includes the step of determining the animal species contained in the animal hair fiber by a qualitative polymerase chain reaction. The method according to any of the preceding claims, wherein the animal hair fibers comprise fibers from two or more animal species selected from the group consisting of yak species, sheep species and goat species. The method according to any one of claims 1 to 4, wherein a TaqMan probe is used as a probe. A sheep species-specific primer pair having the following nucleotide sequence:
5'-AACCAGGAGAACTGCGTTTACT-3 '(SEQ ID NO: 1)
5'-TTTGATCCGCAAATTTCTGA-3 '(SEQ ID NO: 2). A probe having the following nucleotide sequence and capable of specifically detecting a sheep species gene:
5'-ACAACCCTTATGTCAACTCGTCCAGGCC-3 '(SEQ ID NO: 3). A sheep species-specific primer pair having the following nucleotide sequence:
5'-AATCCCTTAGAAGTCCCACTACTCAA-3 '(SEQ ID NO: 10)
5'-TACACGCCTAGTGCGATGGTAA-3 '(SEQ ID NO: 11). A probe having the following nucleotide sequence and capable of specifically detecting a sheep species gene:
5'-AGCTCACCATAGCCTCATAGAAGGGAACCG-3 '(SEQ ID NO: 12). A kit for detecting or quantifying a sheep species gene, comprising (a) the primer pair according to claim 6 and the probe according to claim 7, or (b) the primer pair according to claim 8 and the probe according to claim 9. Goat species-specific primer pairs having the following sequences:
5'-TTAGTTGAATTAGGCCATGAAGCA-3 '(SEQ ID NO: 4), and
5'-TTAATAGGCTTGAGTGCATTGTATTTACT-3 '(SEQ ID NO: 5). A probe having the following nucleotide sequence and specifically detecting a goat species gene:
5'-ACACACCGCCCGTCACCCTCC-3 '(SEQ ID NO: 6). [14] A kit for detecting or quantifying a goat species gene, comprising the primer pair according to claim 11 and the probe according to claim 12. Yak species-specific primer pairs having the following sequences:
5'-GCTTTCTCTATCCTAATCCTTGCTCTTA-3 '(SEQ ID NO: 13), and
5'-GGCTGAGTGGTCGGAAGATTAT-3 '(SEQ ID NO: 14). A probe having the following nucleotide sequence and specifically detecting a Yak species gene:
5'-CCCCTACTGCACACTTCCAAACAACGAA-3 '(SEQ ID NO: 15). A kit for detecting or quantifying a Yak species gene, comprising the primer pair according to claim 11 and the probe according to claim 12. Primer pairs having the following base sequences and specific to sequences common to yak, sheep and goat species:
5'-TACAGAAACAAAATTATTCGCCAGAGT-3 '(SEQ ID NO: 7) and
5'-GGGTATAAAGCACCGCCAAGT-3 '(SEQ ID NO: 8). A probe having the following nucleotide sequence and capable of specifically detecting an amplification product by the primer pair according to claim 17:
5'-TACCGGCAACAGCCCGAAACTCAAA-3 '(SEQ ID NO: 9). A kit for detecting or quantifying a gene of a yak species, a sheep species and a goat species, comprising the primer pair according to claim 17 and the probe according to claim 18. The probe according to any one of claims 7, 9, 12, 15 and 18, wherein the probe is a TaqMan probe having a reporter fluorescent dye bound to the 5 'end and a quencher fluorescent dye bound to the 3' end. The kit according to any one of claims 10, 13, 16, and 19, wherein the probe is a TaqMan probe having a reporter fluorescent dye bound to the 5 'end and a quencher fluorescent dye bound to the 3' end. A method for examining the mixing ratio of each fiber in animal hair fibers in which sheep species-derived fibers and goat species-derived fibers are mixed,
1) a step of extracting DNA from animal hair fibers,
2) Using the extracted DNA as a template, a primer pair consisting of 5'-AACCAGGAGAACTGCGTTTACT-3 '(SEQ ID NO: 1) and 5'-TTTGATCCGCAAATTTCTGA-3' (SEQ ID NO: 2) and 5'-ACAACCCTTATGTCAACTCGTCCAGGCC-3 '(SEQ ID NO: 3) ) Or a primer pair consisting of 5'-AATCCCTTAGAAGTCCCACTACTCAA-3 '(SEQ ID NO: 10) and 5'-TACACGCCTAGTGCGATGGTAA-3' (SEQ ID NO: 11) and 5'-AGCTCACCATAGCCTCATAGAAGGGAACCG-3 '(SEQ ID NO: 12) Performing a real-time PCR using a probe to obtain a relative value of the amount of sheep-derived DNA in the extracted DNA sample,
3) Using the extracted DNA as a template, a primer pair consisting of 5'-TTAGTTGAATTAGGCCATGAAGCA-3 '(SEQ ID NO: 4) and 5'-TTAATAGGCTTGAGTGCATTGTATTTACT-3' (SEQ ID NO: 5) and 5'-ACACACCGCCCGTCACCCTCC-3 '(SEQ ID NO: 6) A) performing a real-time PCR using the probe containing the probe to obtain a relative value of the amount of DNA derived from goat species in the extracted DNA sample;
4) DNA is extracted from previously prepared sheep 100% raw wool, goat 100% raw wool, and standard mixed animal hair fibers in which both are mixed at a plurality of ratios, and the extracted DNA is used as a template to obtain 2) and 3). Under the same conditions, the relative value of the amount of DNA derived from sheep species and the relative value of the amount of DNA derived from goat species were obtained, and the ratio of the relative values was corrected based on a table in which the ratio of both was associated with the mixing ratio of the raw wool. A method for assessing the mixing ratio of animal hair fiber products, comprising the step of obtaining the mixing ratio of goat- and sheep-derived fibers. Yak species, sheep species and goat species in animal hair fibers are mixed, a method for examining the mixing ratio of each fiber,
1) a step of extracting DNA from animal hair fibers,
2) Using the extracted DNA as a template, a primer pair consisting of 5'-AACCAGGAGAACTGCGTTTACT-3 '(SEQ ID NO: 1) and 5'-TTTGATCCGCAAATTTCTGA-3' (SEQ ID NO: 2) and 5'-ACAACCCTTATGTCAACTCGTCCAGGCC-3 '(SEQ ID NO: 3) ) Or a primer pair consisting of 5'-AATCCCTTAGAAGTCCCACTACTCAA-3 '(SEQ ID NO: 10) and 5'-TACACGCCTAGTGCGATGGTAA-3' (SEQ ID NO: 11) and 5'-AGCTCACCATAGCCTCATAGAAGGGAACCG-3 '(SEQ ID NO: 12) Performing a real-time PCR using a probe to obtain a relative value of the amount of sheep-derived DNA in the extracted DNA sample,
3) Using the extracted DNA as a template, a primer pair consisting of 5'-TTAGTTGAATTAGGCCATGAAGCA-3 '(SEQ ID NO: 4) and 5'-TTAATAGGCTTGAGTGCATTGTATTTACT-3' (SEQ ID NO: 5) and 5'-ACACACCGCCCGTCACCCTCC-3 '(SEQ ID NO: 6) A) performing a real-time PCR using the probe containing the probe to obtain a relative value of the amount of DNA derived from goat species in the extracted DNA sample;
4) Using the extracted DNA as a template, a primer pair consisting of 5′-GCTTTCTCTATCCTAATCCTTGCTCTTA-3 ′ (SEQ ID NO: 13) and 5′-GGCTGAGTGGTCGGAAGATTAT-3 ′ (SEQ ID NO: 14) and 5′-CCCCTACTGCACACTTCCAAACAACGAA-3 ′ (SEQ ID NO: 15) A) performing a real-time PCR using the probe containing the probe to obtain a relative value of the amount of DNA derived from Yak species in the extracted DNA sample;
5) DNA is extracted from previously prepared 100% raw wool, 100% raw sheep and 100% goat raw wool, and a standard mixed animal hair fiber obtained by mixing these at a plurality of ratios, and using the extracted DNA as a template. Under the same conditions as in 2) to 4), the relative values of the amounts of DNA from the respective animal species were obtained, and the two types of the yak species and the sheep species, the sheep species and the goat species, and the combinations of the yak species and the goat species were obtained. Animal hair, comprising the step of correcting the ratio of relative values based on a table associating the ratio of relative values and the mixing ratio of two types of raw wool to obtain the mixing ratio of fibers derived from yak, goat and sheep species Appraisal method of mixed ratio of textile products. In the real-time PCR, a calibration curve for obtaining a relative value of the DNA amount was obtained by using 5′-TACAGAAACAAAATTATTCGCCAGAGT-3 ′ (SEQ ID NO: 7) and 5′-GGGTATAAAGCACCGCCAAGT-3 ′ (SEQ ID NO: 8) as primers, and 5′-TACCGGCAACAGCCCGAAACTCAAA. 24. The method according to claim 22 or 23, wherein the method is prepared using -3 '(SEQ ID NO: 9) as a probe. The method according to any one of claims 22 to 24, wherein the probe is a TaqMan probe.

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