Journal of Natural Fibers

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Identification of Cashmere and Wool by DNA

Barcode

Xiaying Zhang, Xiaofeng Wu, Hailiang Yang, Hailing Zheng & Yang Zhou

To cite this article: Xiaying Zhang, Xiaofeng Wu, Hailiang Yang, Hailing Zheng & Yang Zhou
(2023) Identification of Cashmere and Wool by DNA Barcode, Journal of Natural Fibers, 20:1,
2175100, DOI: 10.1080/15440478.2023.2175100

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JOURNAL OF NATURAL FIBERS
2023, VOL. 20, NO. 1, 2175100
https://doi.org/10.1080/15440478.2023.2175100

Identification of Cashmere and Wool by DNA Barcode

Xiaying Zhanga, Xiaofeng Wua, Hailiang Yangb, Hailing Zhengb, and Yang Zhoub
a
College of Animal Sciences, Zhejiang University, Hangzhou, China; bChinese Textiles Identification and Conservation
Center, China Silk Museum, Hangzhou, China

ABSTRACT KEYWORDS
In the textile market, it is common to use wool instead of precious cashmere, DNA barcode; COX I gene;
and it is difficult to identify those accurately by traditional methods such as cashmere; wool; species
appearance identification. To differentiate the cashmere and wool accurately identification; PCR
and efficiently, a polymerase chain reaction (PCR) method based on the 关键词
universal primers of DNA barcode was established according to the varia­ DNA条形码; COX I基因; 山
tions in the base sequence of mitochondrial COX I gene between goats and 羊绒; 绵羊毛; 种属鉴定;
sheep. The mitochondrial DNA of cashmere and wool was extracted and then PCR
amplified by a pair of universal primers designed based on the COX I gene of
goats and sheep. The amplified products were thus obtained with the length
of 380 bp as predicted and subsequently sequenced. The sequencing results
were verified with BOLD databases for homology comparison and the spe­
cies of cashmere and wool were identified successfully. In conclusion, the
PCR technology based on DNA barcode established in this study is quick and
accurate in differentiating the species of cashmere and wool.
摘要
纺织市场上, 用绵羊毛替代珍贵的山羊绒的情况普遍存在, 传统方法如外观
鉴别法难以准确地对二者进行鉴别。为了准确高效地对山羊绒和绵羊毛进
行区分, 本研究根据山羊与绵羊线粒体COX I基因的特定碱基序列的差异,
建立了一种基于山羊和绵羊DNA条形码通用引物的聚合酶链式反应
（PCR）方法。首先提取山羊绒和绵羊毛的DNA, 然后用一对基于山羊和绵
羊COX I基因设计的通用引物进行PCR扩增, 得到长度为380bp的扩增产
物。对扩增产物进行测序, 利用BOLD数据库对测序结果进行同源性比对,
成功对山羊绒和绵羊毛进行了种属鉴定。总之, 本研究中建立的基于DNA
条形码的PCR技术能够快速准确地对羊绒和羊毛进行种属鉴定。

Introduction
As a kind of special animal fiber, cashmere has many excellent characteristics, so it is often used in the
production of textiles. The products of cashmere are quite precious and expensive. For this reason,
there are cases of adulteration of cashmere products in the market, usually by using cheap wool instead
of cashmere. Therefore, the distinction between cashmere and wool is an important part of the
identification of animal sources of textiles. In addition, animal fiber is easy to obtain and not perish­
able in many cases, so the species identification of animal fiber can provide clues and a basis for wild
animal identification and protection (Jun et al. 2011), species evolution analysis (Clack, MacPhee, and
Poinar 2012), archeology (Li 2019), and forensic identification (Melton et al. 2005).
The structure and properties of cashmere and wool are similar, so it is difficult to identify them
accurately by burning and dissolving methods usually applied in textile identifications (Meiju et al.
2012). There are some traditional methods such as appearance identification usually uses an optical
microscope and electron microscope to observe the color, curvature, cross-section, scale, and medul­
lary cavity of the hair fibers. However, it is difficult to identify the hair fibers which were processed into

CONTACT Xiaofeng Wu wuxiaofeng@zju.edu.cn College of Animal Sciences, Zhejiang University, Hangzhou, 310058 China
© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
2 X. ZHANG ET AL.

textiles or came from closely related species by using these methods. Moreover, appearance identifica­
tion has high requirements for technicians, which is easily affected by subjective factors, and the
efficiency of identification and the reliability of the results are poor (Hongbo and Yaqin 2008).
Infrared spectroscopy is another traditional methods to identify hair fiber by analyzing the character­
istic atlas of animal hair measured by OMNI sampler of FT-IR Fourier transform infrared spectro­
meter. This method obtains the structural information of organic components on the surface of the
samples by collecting the reflection signal of the sample surface. However, this method is easily
affected by the state of the fiber itself. Usually, there is no significant differences in the characteristic
atlas of animals of the same family. Therefore, this method also has great limitations (Haitao,
Xiaoming, and Senlin 2011).
In contrast, the identification technique based on DNA has high specificity and sensitivity and is
much more reliable and efficient. The DNA in hair is mainly concentrated in hair follicle cells, and the
hair shaft contains only a small amount of mitochondrial DNA (Yalin 2008). The hair fibers used for
textiles are generally without hair follicles after a series of processing, so comparing the differences in
the base sequences of mitochondrial DNA is more suitable for the identification of animal hair fibers.
The difficulty of this method is extracting high-quality DNA from the animal hair shafts. Although the
content of DNA in the hair shaft is very low, a small amount of DNA can be obtained if the extraction
method is appropriate. The extracted DNA can meet the requirements of subsequent polymerase
chain reaction (PCR) amplification.
Some scholars have identified cashmere and wool according to the differences in the DNA base
sequences between goats and sheep. Selvi Subramanian identified cashmere fiber and wool fiber by
PCR-RFLP according to the mitochondrial Cyt b gene (Subramanian, Karthik, and Vijayaraaghavan
2005). Rajiv Kumar established a PCR technique for the detection of cashmere fiber in blended fabrics
using the specific primers of the mitochondrial 12s rRNA gene (Kumar et al. 2015). Wan Ji distin­
guished cashmere and wool by TaqMan PCR technology according to the mitochondrial 12s rRNA
gene (Ji et al. 2011). Qing-Rong Geng identified cashmere and wool by duplex PCR and PCR-RFLP
according to the D-loop gene and 12s rRNA gene (Geng 2016; Geng, Yuan, and Chen 2012).
Till now, no study has reported the use of the mitochondrial cytochrome c oxidase I (COX I) gene
to distinguish between cashmere and wool. The mitochondrial COX I gene of 650 bp has been
recognized as a barcode region for species identification in animals (Marshall 2005). It provides
more than 97% of species specificities to birds, mammals, fish, and various arthropods (Meusnier et al.
2008). In this study, we tried to extract the mitochondrial DNA from cashmere and wool respectively,
and a pair of universal primers of the mitochondrial COX I gene were designed for both goats and
sheep. Cashmere and wool were thus identified successfully, and a PCR technique for the identifica­
tion of cashmere and wool by mitochondrial COX I gene was well established.

Materials and methods

Animal fiber source
The cashmere from goats (Capra hircus) and wool from sheep (Ovis aries) used in this study are
provided by the China National Silk Museum. Both cashmere and wool were collected from Inner
Mongolia, China.

DNA extraction
The DNA extraction of cashmere and wool based on the protocol of QIAamp Fast DNA Stool Mini Kit
(cat. no. 51604) of QIAGEN Company, the method was improved and DNA was extracted. The
specific extraction steps are as follows: The samples of cashmere and wool were washed with deionized
water until there were no obvious impurities, then the samples were washed and soaked with
anhydrous ethanol. After natural drying, the samples were cut up with scissors and packed into 1.5
 JOURNAL OF NATURAL FIBERS 3

ml EP tubes for use. Each 1.5 ml EP tube contained 0.5–3.0 g samples. The samples were incubated and
digested for more than 24 hours with 600 μl inhibitEX Buffer, 20 μl DTT solution (2 M), 20 μl protease
K solution (20 mg/ml), and 600 μl Buffer AL. Subsequently, the supernatants were separated by
centrifugation (14,000rpm, 15–25°C, 1 min), and 600 μl anhydrous ethanol was added to the super­
natants. The mixed solutions were transferred into the collection columns and centrifuged
(14,000rpm, 15–25°C, 1 min), and then washed with 500 μl Buffer AW1 and 500 μl Buffer AW2.
Finally, DNA was eluted from the collection columns with 100 μl Buffer ATE heated to 56°C, and the
DNA samples were obtained.

Primer designing
The sequences of the mitochondrial COX I gene of goat (Capra hircus) and sheep (Ovis aries) were
searched from GeneBank (https://www.ncbi.nlm.nih.gov/). The sequences were compared by NCBI
Nucleotide BLAST and the universal primers for the mitochondrial COX I gene of goat and sheep
were designed using NCBI Primer-BLAST. Forward primer: 5′-ATCGGCACCCTCTACCTTCT-3′,
Reverse primer: 5′-GCTCCTGCATGGGCTAGATT-3′.

PCR amplification
PCR amplification was performed in a final volume of 20 μl containing 5 μl of DNA template
(Table 1), 0.5 μl of forward primer (10 μM), 0.5 μl of reverse primer (10 μM), 2 μl of TaKaRa 10× Ex
Taq Buffer, 2 μl of TaKaRa dNTP Mixture, 0.5 μl of TaKaRa Ex Taq Mix, and 9.5 μl of ddH2O. PCR
cycling parameters were as follows: 35 cycles each consisted of 94°C for 30 s, 55°C for 30 s, and 72°C
for 1 min, with an initial hot start at 94°C for 1 min and a final extension at 72°C for 5 min.

PCR products detection

The PCR products were electrophoresed in 2% agarose gel. Electrophoretic bands were observed by gel
imager, and PCR products were recovered from the target bands using V-ELUTE Gel Mini
Purification Kit (ZPV202). To ensure the accuracy of sequencing, PCR products related to the
T vector and then transformed into competent cells. After culture and identification, positive colonies
were selected for further identification, culture, and plasmid extraction. Sangon Biotech (Shanghai)
Co., Ltd. has been commissioned to sequence the plasmid containing the target gene fragment.

Results
DNA extraction results
DNA was extracted from cashmere and wool, and the concentration of DNA was determined by
a Qubit fluorescence quantitative analyzer. The results are shown in Table 1. Because the DNA
concentration will be affected by the sample quality and the volume of DNA eluent, DNA-specific
gravity is used to calculate and compare the contents of extracted DNA. The DNA-specific gravity is
the amount of DNA that can be extracted per gram of hair. The formula is as follows:
Qubit DNA concentration ðng=μlÞ � eluent volume ðμlÞ
DNA specific gravity ¼
hair weight ðgÞ
The DNA-specific gravity of the extracted DNA of cashmere and wool in this study is 35.42 ng/g and
22.49 ng/g respectively.
The DNA purity of the samples was analyzed by a NanoDrop nucleic acid protein analyzer. The
genomic DNA of the samples was electrophoresed by Agilent 2100 Bioanalyzer and the results are
shown in Figure 1.
4 X. ZHANG ET AL.

Table 1. DNA extraction results of cashmere and wool.

Qubit DNA Eluent DNA Nano DNA Nano Nano
concentration (ng/ volume quantity DNA specific concentration(ng/ A260/ A260/
Sample Weight(g) μl) (μl) (ng) gravity(ng/g) μl) A280 A230
cashmere 1.31 0.4640 100 46.40 35.42 2.4 2.43 0.56
wool 2.17 0.4880 100 48.80 22.49 3.2 1.52 0.58

Figure 1. 2100 electrophoresis results of DNA from cashmere and wool. (A) the result of cashmere; (B) the result of wool.

PCR amplification results

Cashmere DNA and wool DNA were amplified by PCR using universal primers of mitochondrial
COX I gene of goat and sheep. The agarose gel electrophoresis of the PCR products is shown in
Figure 2. Cashmere and wool both have a clear band between 250-500bp. It can be preliminarily
concluded that the PCR products of both cashmere and wool DNA have the same length and the
length range is 250-500bp. The sizes of the two PCR products were determined to be 380bp by
sequencing.

Sequencing results of amplification products

The PCR products of cashmere and wool were sequenced, and the base sequences of the two PCR
products are shown in Figure 3 (OP740835 and OP740836). It can be observed that the universal
primers were used to amplify cashmere DNA and wool DNA respectively, and the amplification
products with a length of 380bp were obtained. The two sequences were compared by NCBI
Nucleotide BLAST, and 35 different bases sites between the two sequences have been found. The
specific differences between the two sequences are shown in Table 2. These different sites are the basis
for species identifications.

Homology comparison
The sequences of the amplified products of two samples were input into BOLD database (http://www.
boldsystems.org/index.php) for homology comparison. Judge whether the animals with the highest
 JOURNAL OF NATURAL FIBERS 5

Figure 2. Agarose gel electrophoresis result of PCR products of cashmere DNA and wool DNA.

Figure 3. The alignment data sheet between two cashmere and wool COI partial sequences using MEGA-X software.

Table 2. Characteristic variable sites of nucleotide sequences of PCR products of cashmere DNA and wool DNA.
Sample\Site 21 24 39 42 48 57 61 84 96 102 114 123
cashmere G C C A G G T T C T G T
wool A T T G A A C C T C A C
Sample\Site 135 141 147 171 180 183 186 189 196 207 213 228
cashmere T C C A G T A G G C A C
wool C T T G A C T A A T G T
Sample\Site 255 270 279 282 286 288 294 309 315 345 360
cashmere T C C T C A T A A T T
wool C T A C T G C G G C C
6 X. ZHANG ET AL.

Table 3. Homology comparison results.

Sample Best ID Search DB Top % Low %
cashmere Capra hircus COI FULL DATABASE (includes records without species designation) 99.73 99.73
wool Ovis aries COI FULL DATABASE (includes records without species designation) 99.04 98.64

Figure 4. Similarity scores of the top 100 matched results in homology comparison. (A) the result of cashmere; (B) the result of wool.

similarity are consistent with the animal sources of the two samples used in this study, and then judge
the genetic relationship between the samples and the most matching animals according to the highest
matching degree. The results are shown in Tables 3, Figure 4, Tables S1, and Tables S2. The species
with the highest matching degree of cashmere DNA was Capra hircus, and the highest matching
degree was 99.73%. The species with the highest matching degree of wool DNA was Ovis aries, and the
highest matching degree was 99.04%. According to “the homology of animals within the same species
is more than 96%, and the homology of species belonging to different genera of the same family is
70%-90%, and the homology of species belonging to different families is 65%-70%” (Fu-Zai et al.
2009), it can be determined that the identification results of the two fiber samples are consistent with
their actual animal sources. Therefore, the fiber sources of animal species can be successfully
determined by this experimental method.

Discussion
In recent years, the application of DNA technology to species identification of biological samples
has developed rapidly. Mitochondrial deoxyribonucleic acid (mtDNA) is widely used in species
 JOURNAL OF NATURAL FIBERS 7

identification derived from its particular molecular properties, including its high evolutionary
rate, uniparental inheritance, and small sizes. COX I gene is a specific DNA segment of mtDNA.
The sequence of the COX I gene with a length of about 650 bp from the 5’ is highly conserved
and has the abundant mutation sites, so it is called DNA barcode and is widely used for species
classification and identifications (Hebert, Ratnasingham, and deWaard 2003; Rabie 2019). This
provides a theoretical and material bases for species identification of special samples such as
animal fiber.
In this study, the species of cashmere and wool have been successfully identified. Firstly, the DNA
of two kinds of animal fibers was extracted, and then analyzed, and the results showed that the amount
of DNA in the two samples was very scarce. The results of DNA-specific gravity show that the DNA
that can be extracted from per gram of hair is even far less than 1 μg. That is to say, the extraction of
DNA from hair is challenging because of the limited quantity. The A260/A280 values of the cashmere
are higher than 2.0. This ratio indicates the RNA contamination of the sample. In this study, RNA
contamination has little effect on the specific amplification of DNA. The A260/A230 values of the two
samples are lower than 2.0. This shows that the samples are easily affected by external pollution due to
the few extracted DNA, and the contaminants in the samples may come from guanidine salts in
nucleic acid extraction reagents.
In addition, according to the results of 2100 electrophoresis, there is no obvious DNA bands on
the electrophoresis bands of the two samples, and the background colors of the electrophoresis
bands are dark. Also, there is no obvious peak in the peak diagram, but there are weak peaks with
a span of 1,000–10,380 bp (the composition of DNA shorter than 10,380bp can be detected by
Agilent 2100 Bioanalyzer). Theoretically, the DNA in the hair shaft should be mitochondrial DNA
with a length of about 16.5 kbp. But, due to the high degree of keratosis of the hair shaft, the high
degradation of DNA in hair is very serious. This shows that the high degradation of DNA in the
hair shaft is very serious.
The results show that the DNA in cashmere and wool is very few and has degraded seriously, which
greatly limits the application of DNA in hair fiber identifications. As the sensitivity and specificity of
PCR technology are very high, and only a very small amount of sample DNA is needed to complete the
amplifications.
The DNA of the two samples was amplified by PCR using the designed universal primers of goat
and sheep, and the target bands could be observed by 2% agarose gel electrophoresis. The target bands
were recovered and sequenced, and the base sequences of PCR products were obtained. The sequences
were compared by the BOLD database, and the results showed that the two samples could be
accurately identified the animal origin.
Compared with traditional methods, using a DNA barcode to identify cashmere and wool avoids
the influence of subjective factors, and more importantly the detection accuracy and sensitivity of
this method are very high, so the results are more objective and reliable. Moreover, this method is
simple and easy to operate, so accurate results could be obtained in less time. Universal primers can
effectively identify cashmere and wool and improve the detection efficiency and sensitivity.
However, it is not entirely reliable to identify species only by the target bands from agarose gel
electrophoresis. Therefore, we obtained accurate sequence information of the target bands by the
Sanger sequencing after PCR amplification and using the BOLD database for homology compar­
isons, so that the species of cashmere and wool were identified successfully. Our study laid
a foundation for the promotion and application of gene technology in the field of identification of
cashmere and wool, and provided strong technical support for the qualitative detection of cashmere
and wool based on gene technology. It is also of great significances can be used to identify ancient
textiles and other animal samples.

Highlights
● Universal primers for the identification of cashmere and wool were designed using COX I gene for the first time.
8 X. ZHANG ET AL.

● DNA was successfully extracted from hair shaft samples with degraded seriously.
● Trace amounts of samples can be successfully identified.
● A PCR technique for the identification of cashmere and wool by DNA barcode was established.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The work was supported by the scientific project of Preservation of Cultural Relics of Zhejiang Province [2021010]; and
the Natural Science Foundation of Zhejiang Province [Z20C170008].

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