Molecular and Cellular Probes xxx (2014) 1e4

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Molecular and Cellular Probes

journal homepage: www.elsevier.com/locate/ymcpr

Species-specific PCR for the identification of goat cashmere and sheep

wool
Rong-Qing Geng*
College of Life Science and Technology, Yancheng Teachers University, Yancheng, Jiangsu Province 224051, PR China

a r t i c l e i n f o a b s t r a c t

Article history: In order to establish rapid and species-specific method of goat cashmere and sheep wool identification, a
Received 30 August 2014 polymerase chain reaction using specific primer pairs targeting mitochondrial D-loop was developed.
Accepted 11 November 2014 The goat specific primers yielded a 294 bp PCR fragment and the sheep specific primers yielded three
Available online xxx
PCR fragments of which only the 404 bp fragment was found highly diagnostic. The specificity and
reliability of the developed species-specific PCR assay was validated by considering as many as 500
Keywords:
cashmere and wool samples. The developed species-specific PCR was found effective in detecting mixed
Species-specific PCR
samples of cashmere and wool precisely with the relative content over 9.09%. The species-specific PCR
Goat cashmere
Sheep wool
method proved to be low cost, fast, easy and reliable alternative to determine the addition of sheep wool
Identification in goat cashmere.
© 2014 Published by Elsevier Ltd.

1. Introduction mainly include macroscopical identification, microscopical exami-

Animal fibers from a range of domesticated and undomesticated
animal species have long provided valuable products for use by the
human population. Historically, goat hair fiber described as “cash-
mere” and sheep hair fiber described as “wool”, have been impor-
tant source of economic wealth for many countries with production
surpassing in recent times. To be classified as cashmere the mean
fiber diameter of the undercoat must generally be less than 18.5 mm
[1]. Cashmere, the fine undercoat produced from the secondary
hair follicles of some goat breeds, is one of the finest and softest
animal fibers used by the textile industry, which is next to the
world's most sought after animal fiber today. Hence, cashmere is
regarded as a specialty or luxury fiber due to its scarcity and high
economic value, and also due to its softness and luster [2,3].
For financial gains, various interested parties often adulterate
cashmere with cheap sheep wool. The qualitative and quantitative
identification of cashmere and wool is increasingly becoming ne-
cessity due to possible adulterations in raw and processed textiles.
Hence, identification of cashmere from wool is essential to
discourage its adulteration. Although a number of techniques have
been developed, accurate identification of cashmere and wool in
the textile industry is very difficult. These traditional methods
* Tel./fax: þ86 0515 88239395.
E-mail address: rqgeng@hotmail.com.
nation, physical and chemical experiment [4e7]. However, the
application of these methods is restricted due to different techno-
logical processes and is suited only to the identification of pure
cashmere or wool. It is difficult to quantitatively measure the
content of each kind in a mixture. As goat and sheep belong to
different genus, their genetic differences have been investigated for
their utility in distinguishing of cashmere and wool [8e10].
In this study, species-specific PCR assays were optimized and
evaluated for their suitability for species origin identification from
goat cashmere and sheep wool to assess the authenticity. This
approach can contribute to reinforce consumer's trust on tradi-
tional cashmere products and avoid unfair competition among
producers.
2. Materials and methods
2.1. Sample collection
Cashmere samples were collected from three standard special-
ized cashmere goat breeds (Inner Mongolia cashmere goat, n ¼ 52;
Liaoning cashmere goat, n ¼ 56; Shanbei white cashmere goat
n ¼ 53) and two non-descript cashmere goat breeds (Xinjiang
cashmere goat, n ¼ 54; Ziwuling black cashmere goat n ¼ 52). Wool
samples were collected from five standard fine wool sheep breeds
(Chinese Merino sheep, n ¼ 54; Xinjiang fine wool Sheep, n ¼ 52;

http://dx.doi.org/10.1016/j.mcp.2014.11.002
0890-8508/© 2014 Published by Elsevier Ltd.

Please cite this article in press as: Geng R-Q, Species-specific PCR for the identification of goat cashmere and sheep wool, Molecular and Cellular
Probes (2014), http://dx.doi.org/10.1016/j.mcp.2014.11.002
2 R.-Q. Geng / Molecular and Cellular Probes xxx (2014) 1e4
Northeast fine wool Sheep, n ¼ 55; Inner Mongolia fine wool Sheep,
n ¼ 53; Gansu fine wool Sheep, n ¼ 50).
2.2. Mitochondrial DNA extraction and preparation
Mitochondrial DNA was extracted using the method as
described by Geng et al., 2012 [11]. Briefly, all goat cashmere and
sheep wool samples were cleaned prior to mitochondrial DNA
extraction by successively shaked 10 s in 95% ethanol to wipe off
sundries. The air dried samples (about 15 mg) were then cut into
1 mm fragments and put into 2 ml microcentrifuge tube. The
mitochondrial DNA was extracted using Tissue and Hair Extraction
Kit (DNA IQ™, Promega, Madison, WI, USA) according to the
manufacturer's protocol. DNA concentration was assessed by
spectrophotometry (NanoDrop 1000 spectrophotometer, Thermo
Scientific, Wilmington, USA). DNA extracts of dissolved in TE buffer
and diluted so as to get 50 ng/ml DNA concentration.
2.3. Oligonucleotide primers and PCR procedure
The species-specific oligonucleotide primers used in this work
targeted mitochondrial D-loop sequences of goat and sheep, pre-
viously reported in the literature [12,13]. A pair of primers from
goat (F1: 50 -TTCTTCAGGGCCATCTCATC-30 and R1: 50 -GCGGATG-
CATGGTGAAAT-30 ) were used for PCR amplification so as to obtain
an expected fragment of 294 bp. Another pair of primers for sheep
(F2: 50 -CCACCCACGGACACGAG-30 and R2: 50 -AGTTCAATGCCCTA-
TATGCTTCAG-30 ) was to obtain an expected fragment of 404 bp. The
primers were synthesized by Nanjing GenScript Biotechnology Co.,
Ltd., Nanjing, China.
PCR amplification was performed in a final volume of 25 ml
containing 2.5 ml of 10  PCR buffer (100 mM Tris-HCl, pH 9.0,
15 mM MgCl2, 500 mM KCl and 0.1% gelatin), 1 ml of 10 mM dNTPs
mix, 1 ml each of primer (10 mM), 1 U of Taq DNA polymerase and
100 ng of DNA template. Amplification was performed in a Thermal
Cycler C1000 (Bio-Rad, USA). PCR cycling parameters were as fol-
lows: 35 cycles each consisted of 94  C for 30 s, 57  C for 30 s, and
72  C for 30 s, with an initial hot start at 94  C for 5 min and a final
extension at 72  C for 10 min. The PCR products were electro-
phoresed in 1% agarose gel and stained with ethidium bromide.
2.4. Reliability and sensitivity detection of the specific primer pairs
To test the reliability of the designed primers, the populations of
five cashmere goat breeds and five wool sheep breeds were
included as the target samples. All the 10 populations were used to
confirm the reliability of the species-specific primer pairs. In order
to evaluate the accuracy of the method developed, the PCR frag-
ments were purified and ligated into the pMD18-T Vector (Takara,
Dalian, China) for sequencing on an ABI 3700 sequencer (Applied
Biosystems) according to the manufacturer's protocol. The se-
quences of PCR products were used as the confirming analysis.
To determine the sensitivity of the species-specific PCR assay, a
serial mixture of goat cashmere and sheep wool were prepared
with the following weight/weight ratios: 14:1, 13:1, 12:1, 11:1, 10:1,
9:1, 8:1, 6:1,3:1, 1:1, 1:3, 1:6, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14. The
mitochondrial DNA extraction, PCR amplification and detection
were performed according to above-mentioned methods.
3. Results
Species-specific PCR primers for goat and sheep were selected as
molecular markers to amplify regions of the mitochondrial D-loop.
After optimizing different primer concentrations and annealing
temperatures, optimum conditions were found suitable for
Please cite this article in press as: Geng R-Q, Species-specific PCR for the identification of goat cashmere and sheep wool, Molecular and Cellular
Probes (2014), http://dx.doi.org/10.1016/j.mcp.2014.11.002
amplification standardized. A fragment of size 294 bp was suc-
cessfully amplified from goat cashmere using goat specific primer
pairs (Fig. 1). Although three PCR fragments were obtained from
sheep wool using sheep specific primer pairs, only one prominent
fragment of size 404 bp was found (Fig. 1). The nucleotide sequence
of the amplified fragments (294 bp and 404 bp), which were cloned
into the pMD18-T Vector for sequencing, was the same as that
expected from goat and sheep sequences reported previously.
The possibility of cross amplification was precluded by
analyzing specific primers with mitochondrial DNA extracted from
goat cashmere and sheep wool. The results clearly indicated that
the primers were only able to produce the expected fragments
when the corresponding DNA for which they were designed was
present, demonstrating their specificity. Repeatability of the PCR
assay was confirmed five times by testing species-specific primers
with mitochondrial DNA isolated from different samples. The
species-specific PCR method was then applied to detect the reli-
ability using different goat cashmere and sheep wool samples. After
PCR amplification using two sets of species-specific primers indi-
vidually, all samples were successfully amplified to obtain species-
specific fragments (267 cashmere samples and 264 wool samples).
The sensitivity of the developed PCR method was examined
with purified mitochondrial DNA from a set series of known
amount of goat cashmere and sheep wool mixtures. It was found
that the goat specific primers were found sensitive to detect mix-
tures when the ratio of goat cashmere and sheep wool was 1:10
(Fig. 2). The sheep specific primers successfully produced amplifi-
cation from mixtures when the ratio of goat cashmere and sheep
wool was 10:1 (Fig. 3). A similar degree of sensitivity in detection of
mixtures was observed by using goat specific primers and sheep
specific primers with species-specific PCR method.
4. Discussion
Species identification from natural animal fibers has been
greatly advanced by the application of DNA analysis. Particularly,
PCR-based DNA analytical methods have been widely used for
identifying animal fibers [9e11,14e16]. These methods highlight
the fact that molecular markers could be unequivocally linked to
species identification and are excellent for the qualitative identifi-
cation of animal fibers. Moreover, several methods are developed
and validated for quantitative analysis of animal fiber blends.
Mitochondrial DNA sequences are commonly used as genetic
markers for species identification. The advantage of mitochondrial-
based DNA analyses derives from the fact that these markers are
Fig. 1. Species-specific amplification results using sheep primers (lanes 1e4) and goat
primers (lanes 5e8). M: DL2000 DNA marker, fragments range from 2000 bp, 1000 bp,
750 bp, 500 bp, 250 bp to 100 bp vertically; C: negative control; lanes 1e2 and lanes
5e6: goat cashmere samples; lanes 3e4 and lanes 7e8: sheep wool samples.
 R.-Q. Geng / Molecular and Cellular Probes xxx (2014) 1e4
Fig. 2. Sensitivity of specific PCR using goat primer sets in identification of goat
cashmere and sheep wool mixtures. M: DL2000 DNA marker, fragments range from
2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp to 100 bp vertically; lanes 1e10: mixed
cashmere and wool samples with different ratio of 1:1, 1:3, 1:6, 1:8, 1:9, 1:10, 1:11, 1:12,
1:13, 1:14.
Fig. 3. Sensitivity of specific PCR using sheep primer sets in identification of goat
cashmere and sheep wool mixtures. M: DL2000 DNA marker, fragments range from
2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp to 100 bp vertically; lanes 1e10: mixed wool
and cashmere samples with different ratio of 1:1, 1:3, 1:6, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13,
1:14.
present in multiple copies per cell which increases the sensitivity of
the PCR assay [17]. Moreover, mitochondrial DNA is considered to
have evolved more rapidly than nuclear DNA and therefore con-
tains highly informative polymorphic sites. Among mitochondrial
genes, the D-loop region is a commonly targeted mitochondrial
sequence for species identification. The mitochondrial D-loop re-
gion is the most rapidly evolving region of the mitochondrial DNA
molecule and is one of the most widely used markers when
determining relationships among closely related species [18].
PCR using species-specific primers is one of the most popular
approaches since it offers simplicity, specificity and high sensitivity
for species identification [17]. In the particular case of mixed-
species identification, species-specific PCR offers the advantages
of cheaper, faster, and more useful for routine analysis of large
amount of samples. The purpose of this study was to establish a
simpler, more rapid and accurate PCR method to detect and identify
each species from goat cashmere and sheep wool. To accomplish
this aim, species-specific primers were used targeting the mito-
chondrial D-loop region due to its higher copy number per cell. The
goat specific primers which were designed against a conserved
region of goat mitochondrial D-loop yielded a 294 bp PCR product
confirming only the presence of goat DNA. Similar findings were
reported when targeted the same mitochondrial D-loop fragment
for differentiation of different meat species [12,19]. The sheep
Please cite this article in press as: Geng R-Q, Species-specific PCR for the identification of goat cashmere and sheep wool, Molecular and Cellular
Probes (2014), http://dx.doi.org/10.1016/j.mcp.2014.11.002
3
specific primers which were designed against a unique target re-
gion of sheep mitochondrial D-loop yielded three PCR products of
which only the 404 bp fragment was found highly diagnostic. Such
phenomenon might be due to the ‘heteroplasmy’ in occurrence of
two or more types (copy number) of mitochondria. It was observed
that as the template concentration (target copies) went on
reducing, the least common copies got exhausted first, leaving
behind the more abundant copies. This phenomenon was previ-
ously reported in animal species identification [13,20]. The speci-
ficity and reliability of the developed species-specific PCR assay was
validated by considering as many as more than five hundred of goat
cashmere and sheep wool samples (267 cashmere samples and 264
wool samples).
The PCR-RFLP technique was found effective in detecting mixed
samples precisely when mixed with the relative content over 11.1%
[11]. It could easily identify whether sheep wool was added to goat
cashmere when mixed with more than 11.1%. It was reported that
two sets of TaqMan PCR primers and probes were designed to
quantify mixed goat cashmere and sheep wool [14]. Using TaqMan
PCR, it can not only distinguish cashmere and wool but also
quantify their relative contents in a mixture. When cashmere and
wool were mixed together, it could estimate their relative content
within 10% (w/w). In this study, the species-specific PCR was found
to be effective in detecting mixed samples of cashmere and wool
precisely when mixed each other with the relative content over
9.09%. The method can be applied directly to examine the quality of
cashmere products and will help to avoid fraudulent substitution
and adulteration in cashmere market.
5. Conclusions
The method of species-specific PCR was proved to be a low cost,
fast, easy and reliable alternative to estimate the addition of sheep
wool into goat cashmere.
Acknowledgments
This research was supported by the National Natural Science
Foundation of China (31101684 and 31101747), the Natural Science
Foundation (BK20141259) of Jiangsu Province and sponsored by
Qing Lan Project of Jiangsu Province.
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Please cite this article in press as: Geng R-Q, Species-specific PCR for the identification of goat cashmere and sheep wool, Molecular and Cellular
Probes (2014), http://dx.doi.org/10.1016/j.mcp.2014.11.002