Food Control 18 (2007) 11491158

www.elsevier.com/locate/foodcont

A TaqMan real-time PCR system for the identication

and quantication of bovine DNA in meats,
milks and cheeses
Chun-Lai Zhang *, Mark R. Fowler, Nigel W. Scott, Graham Lawson, Adrian Slater

Forensics Research Unit/Systems Biology Research Laboratory, Faculty of Health and Life Sciences, De Montfort University,
Hawthorn Building, The Gateway, Leicester, LE1 9BH, UK
Received 22 January 2006; received in revised form 9 July 2006; accepted 16 July 2006

Abstract
Accurate quantitative assays are required for enforcing food labelling procedures and preventing food ingredient contamination,
misdescription and fraud. Simplex and duplex TaqMan real-time PCR systems have been tested for the identication and quantication
of DNA in meat, milk and cheese. DNA was isolated from meat and cheese using a standard CTAB protocol and from milk using a
Promega Wizard Magnetic kit and puried by Qiagen silicon spin columns. High quality DNA isolated from beef mince was used
for standard curve construction in the TaqMan real-time PCR assay using a bovine-specic primer pair for the mitochondrial cytb gene
and a FAM-labelled mammalian-specic cytb probe. The real-time PCR assay can quantitatively detect as little as 35 pg bovine DNA
and showed no cross-reaction with ovine, caprine or porcine DNA. The system has been successfully used to measure bovine DNA in
fresh and processed meat, milk and cheese, and will prove useful for bovine species identication and quantitative authentication of
animal-derived products.
 2006 Elsevier Ltd. All rights reserved.
Keywords: Real-time PCR; Bovine DNA; Quantitative detection; Meat; Milk; Cheese

1. Introduction
Food safety, quality and composition have become the
subjects of increasing public concern. Consumers have
been given more choices with regard to food composition
and dietary requirements via food labels. A number of people are allergic to specic molecules in meats, milks or
cheeses. Healthy diet followers tend to prefer chicken
instead of beef, pork or lamb, due to its low dietary fat content. Various religious groups avoid specic meats such as
beef or pork; whilst vegetarians choose not to consume any
meat. Each constituency has an interest in ensuring the
authenticity of the foods that they consume.
*

Corresponding authors.
E-mail addresses: clzhang@dmu.ac.uk (C.-L. Zhang), ads@dmu.ac.uk
(A. Slater).
0956-7135/$ - see front matter  2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2006.07.018

The fraudulent misdescription of food contents on product labels is a widespread problem, particularly with high
added-value products commanding a premium price
(Woolfe & Primrose, 2004). There can be intentional or
unintentional contamination in the production chain or
during processing. Proving conclusively that adulteration
or contamination has occurred requires the detection and
quantication of food constituents. This can be dicult
because the materials replaced are often biochemically very
similar and food matrices are extremely complex and
variable.
Lipid, protein and DNA based methods have been
established for food identication. Lipid analysis is only
applicable for gross measurement of animal-derived fats
(Lumley, 1996; Saeed, Ali, Abdul Rahman, & Sawaya,
1989). Protein-based methods such as high performance liquid chromatography (HPLC) (Espinoza, Kirms,

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C.-L. Zhang et al. / Food Control 18 (2007) 11491158

& Filipek, 1996), enzyme-linked immunosorbent assays

(ELISA) (Chen & Hsieh, 2000) or isoelectric focusing protein proles (Skarpeid, Kvaal, & Hildrum, 1998) are eective mainly for unprocessed food and are unable to
dierentiate species such as lamb and goat or chicken
and turkey. Both require complicated procedures and it
has proved dicult to accurately quantify the analytes in
a short time (Mayer, 2005). A variety of DNA-based methods including polymerase chain reaction (PCR) amplication, RFLP mapping and microarray gene chip assay
have now been successfully adapted for the detection of
food substitution (Peter, Brunen-Nieweler, Cammann, &
Borchers, 2004; Woolfe & Primrose, 2004). DNA based
methods have been well received because of the relative stability of the DNA molecule under extreme conditions and
its ecient amplication by PCR (Lanzilao, Burgalassi,
Fancelli, Settimelli, & Fani, 2005; Matsunaga et al., 1999;
Mayer, Hoein, Luthy, & Candrian, 1995; Sun & Lin,
2003; Zhang, Zheng, Zhou, Ouyang, & Li, 1999). The limitations of standard PCR assays include the insensitivity
and lack of quantitation of end-point analysis, and the
dependence on a low throughput technique (agarose gel
electrophoresis) for analysis of the products. Real-time
PCR assay has provided sensitive and safe solutions by
monitoring PCR products continuously using uorescent
markers (Heid, Stevens, Livak, & Williams, 1996; Holland,
Abramson, Watson, & Gelfand, 1991). Several real-time
PCR methodologies have been developed for meat species
identication and quantication using low copy nuclear
genes (Laube et al., 2003), high copy genomic short interspersed elements (Walker et al., 2003) or mitochondrial
genes (Dooley, Paine, Garrett, & Brown, 2004; Hird
et al., 2004; Lopez-Andreo, Lugo, Garrido-Pertierra, Prieto, & Puyet, 2005). The real-time system is applicable to
processed or mixed meats as well (Lopez-Andreo et al.,
2005).
Cows milk is more widely available and cheaper than
that of goat and water bualo. Cows milk is also processed
in large quantities to produce a range of dairy produce
including a wide variety of cheeses. On the other hand, specialist cheeses such as Greek Feta cheese made from goats
and sheeps milk and Italian mozzarella di bufala campana
cheese made from water bualo milk, both registered by
European law with the Protected Designation of Origin
(PDO), have become widely accepted throughout the EU
and command a premium price because of their production
from more costly milks than cows milk. It is also more difcult to stretch out mozzarella cheese prepared from buffalo milk and to spin it mechanically due to the dierent
rheologic characteristics of bualo milk casein compared
to cows milk casein. Adulteration of goat and water buffalo milk and their products by cows milk is therefore economically attractive. Their products are traditionally tested
for adulteration by immunological and/or electrophoretic
methods (Amigo, Ramos, Calhau, & Barbosa, 1992;
Cerquaglia & Avellini, 2004; Hurley, Ireland, Coleman,
& Williams, 2004; Levieux & Venien, 1994; Mayer, 2005;

Mimmo & Pagani, 1998). Identication of milk by PCR

amplication of DNA is based on the presence of mammalian somatic cells in the milk (Herman, 2001). Several simplex PCR procedures have been developed for species
identication in milk, cheese and yogurt based on primers
designed to amplify a number of mitochondrial genes:
cytochrome b (cytb) (Bania, Ugorski, Polanowsk, &
Adamczyk, 2001; Di Pinto, Conversano, Forte, Novello,
& Tantillo, 2004; Herman, 2001), D-loop region (Maudet
& Taberlet, 2001), 12S ribosomal RNA gene (LopezCalleja et al., 2004; Lopez-Calleja et al., 2005), cytochrome
oxidase II (Mayer, 2005), cytochrome oxidase I (Feligini
et al., 2005), and nuclear encoded genes e.g. coat colour
MC1R (Maudet & Taberlet, 2002). Procedures have also
been developed for duplex PCR based on cow and bualospecic cytb primers (Bottero, Civera, Anastasio, Turi, &
Rosati, 2002; Rea et al., 2001), bovine and ovine 12S and
16S rRNA genes (Mafra, Ferreira, Faria, & Oliveira,
2004), and multiplex PCR based on bovine, ovine and
caprine 12S and 16S rRNA genes (Bottero et al., 2003).
Procedures based on polymerase chain reaction-restriction
fragment length polymorphism have also been developed
using cytb primers to dierentiate mozzarella cheese made
from water bualo milk and from less expensive bovine
milk and also Feta cheeses made from bovine, ovine,
and caprine milk (Branciari, Nijman, Plas, Di Antonio, &
Lenstra, 2000) and ovine yogurt (Stefos et al., 2004).
However, most of these procedures are not applicable for
accurate quantitative measurement and have the disadvantages of conventional PCR discussed above. This study
showed that TaqMan real-time PCR is applicable to the
authentication of milk and cheese.
2. Materials and methods
2.1. Sample preparation
Fresh and processed meats, milks and cheeses were purchased from several national food retailers and/or producers (randomly coded as AF) in Leicester, UK (Table 1).
Meats and cheeses were cut into small pieces with a
hand knife. DNA was isolated using a standard cetyltrimethylammonium bromide (CTAB) method (Murray &
Thompson, 1980; Zhang et al., 2001). Briey, about 1.8 g
of chopped tissues were mixed with 5 ml of extraction buffer and incubated at 65 C for 2 h. The above mixture was
extracted twice with an equal volume of chloroform and an
equal volume of isopropanol was added to the aqueous
fraction. After centrifugation the precipitate was washed
with ethanol. The dried pellet was dissolved in 0.5 ml sterilised Millipore MQ water.
DNA from milks was isolated by the Promega Wizard
Magnetic kit following the manufacturers instructions as
only a small amount of DNA was extracted from fresh
whole milk using the CTAB method. Firstly, 3 ml of milk
was centrifuged at 13,000 rpm for 10 min. The sediment
portions were collected and mixed with 0.4 ml lysis buer

C.-L. Zhang et al. / Food Control 18 (2007) 11491158

1151

Table 1
Commercial food products used in general and real-time PCR assays
Products

Retailers and/or producers

Descriptions

Beef mince
Lamb mince
Pork mince
Chicken portions
Turkey mince
Corned beef
Steak pie
Tinned oxtail soup
Fresh whole cows milk
Fresh whole cows milk
Fresh goats milk
Cheddar cheese
Cheese onion and chive quiche
Somerset goats cheese
Welsh goats cheese
Italian mozzarella cheese ball
Italian mozzarella cheese

E
B
A
A
B
B
B
C/F
B
C
B/D
B
A
B
A
B
A

Fresh minced beef

Fresh minced lamb
Fresh minced pork
Frozen chicken portion meat
Fresh minced turkey
Processed beef meat
Cooked beef in pie
Prepared soup with vegetables and oxtail
Standardized pasteurised homogenised cows milk
Standardized pasteurised homogenised cows milk
Fresh pasteurised homogenised goats milk
Contains milk
Processed quiche, containing milk, cheese and egg
Contains milk
Contains goats milk
Made in Italy, contains milk
Made in Italy, contains milk

A and 4 ll of RNaseA. This step avoided further processing of the cream and skimmed milk fractions which contain
lower concentrations of DNA than the sediment (Poms,
Glossl, & Foissy, 2001). Further DNA extraction procedures followed the manufactures instructions. The isolated
DNA was eluted from the magnetic beads with 50 ll of
MQ water.
All DNA preparations were puried through a Qiagen
silicon spin column and dissolved in MQ water. Initial
DNA analysis was carried out using agarose gel electrophoresis (1% SeaKem LE agarose gel containing ethidium
bromide). DNA aliquots (12 ll for meats, 15 ll for milks
and 20 ll for cheeses) were loaded into each well and electrophoresed at 75 V/cm for 1.5 h. Electrophoresis gels were
visualized by UV transillumination, photographed and
processed using GeneSnap software. DNA amount and
quality were determined with a Unicam Hekios spectrophotometer at 260 nm and 280 nm. DNA concentrations
of various samples were calculated and further DNA samples were obtained by dilution with MQ water or mixed
with lamb and pork, or chicken DNA (see Section 3 for
details). For serial dilution, 3 ll of DNA solution was
transferred into 27 ll MQ water and mixed thoroughly.
A water bualo milk DNA sample (40 ng/ll) and a goat
blood DNA sample (350 ng/ll) were provided by Dr. Isabel Gonzalez, Universidad Complutense de Madrid, Spain.
2.2. PCR primers and probes
PCR primers and probes were based on those described
by Dooley et al. (2004). Bovine-specic cytochrome b gene
(cytb) primers were Bovcytbf: CGG AGT AAT CCT TCT
GCT CAC AGT, Bovcytbr: GGA TTG CTG ATA AGA
GGT TGG TG to amplify a 116 bp fragment. Ovinespecic cytb gene primers were Ovicytbf: GAG TAA
TCC TCC TAT TTG CGA CA, Ovicytbr: AGG TTT

GTG CCA ATA TAT GGA ATT to amplify a 133 bp

fragment. The universal mammalian-specic cytb gene
probe (mammalcytprobe) was TGA GGA CAA ATA
TCA TCA TTC TGA GGA GCW ARG TYA. A uorescent dye, 6-carboxyuorescein (FAM) was attached to the
5 0 end of the probe. The quencher moiety, 6-carboxytetramethylrhodamine (TAMRA), was added to the 3 0
end of the probe. The pair of chicken-specic cytb primers
was: Chicytbf: AGC AAT TCC CTA CAT TGG ACA
CA, Chicytbr: GAT GAT AGT AAT ACC TGC GAT
TGC A to amplify a 133 bp fragment. A universal poultry-specic cytb gene probe was ACA ACC CAA CCC
TTA CCC GAT TCT TC. TET, 6-carboxy-4,7,2 0 ,7 0 -tetrachlorouorescein, a uorescent dye, was attached to the
5 0 end of the probe. The quencher moiety TAMRA was
added to the 3 0 end of the probe. Turkey and pork-specic
cytb primer pairs were the same as those described by Dooley et al. (2004). The PCR primers were synthesized by
Invitrogen and the TaqMan probes were synthesized by
Applied Biosystems. The alignment of selected animal cytb
sequences was performed using the ClustalW programme
(Chenna et al., 2003) and shown in Fig. 1.
2.3. Conventional PCR protocol
MJ Research PTC-200 or Techne A thermal cyclers were
used in this study. Simple PCR reaction mixtures (50 ll)
comprised MQ water 38 ll, 10 buer 5 ll, 25 mM MgCl2
solution 2 ll, 10 mM each dNTPs 0.5 ll, 5 unit/ll Promega
Taq polymerase 0.5 ll, primers (25 lM) 1.5 ll each and
DNA (32.537.5 ng/ll) 1 ll. MQ water (1 ll) instead of
DNA was used as a negative control. PCR cycling parameters were 95 C 4 min, followed by 35 cycles of 94 C 30 s,
60 C 1 min, 72 C 1 min, and nal extension at 72 C
10 min. Duplex PCR reaction mixtures were performed
using the same PCR conditions, except that 0.52 ll of each
primer (25 lM) was used per 50 ll reaction (see Section 3.4).

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C.-L. Zhang et al. / Food Control 18 (2007) 11491158

Fig. 1. Alignment of selected animal cytb sequences using the ClustalW programme indicating the position of the mammalcytprobe, and bovine and
ovine-specic cytb primers. Bovcytbf and complementary sequence to Bovcytbr are boxed and shown in red colour. Ovicytbf and complementary sequence
to Ovicytbr are boxed and shown in green colour. The cytb sequences shown are: OvisX56284 for lamb (Ovis aries) X56284, CapraX56289 for goat (Capra
hircus) X56289, BubalusD82894 for water bualo (Bubalus bubalus) D82894, BosD34635 for beef (Bos taurus) D34635, SusX56295 for pork (Sus scrofa)
X56295, GallusL08376 for chicken (Gallus gallus) L08376 and MeleagL08381 for turkey (Meleagris gallopavo) L08381. * indicates identical nucleotide
base. (For interpretation of the references in color in this gure legend, the reader is referred to the web version of this article.)

2.4. Real-time PCR protocol

A MJ Research real-time PCR system, Chromo4 Continuous Fluorescence Detector, was used for these experiments. Real-time PCR cycling parameters were optimized
based on the Chromo4 Fluorescence Detector operation
manual as: 50 C 2 min, 95 C 10 min, followed by
55 cycles of 95 C 15 s, 60 C 45 s, reading products,
72 C 1 min, then 72 C 10 min and nally, melting point
analysis. This programme is controlled by Opticon Monitor 3 software. Experiments were repeated three to ve
times.
Real-time PCR reaction mixtures (25 ll) using the TaqMan master mix kit (Applied Biosystems) were: MQ water
10 ll, 2 TaqMan master mix 12.5 ll, primers (25 lM)
0.5 ll each, probe (10 lM) 0.5 ll, DNA (minute amount
up to 625 ng/ll) 1 ll. The 2 TaqMan master mix is optimized for 5 0 nuclease assay using TaqMan probes and
contains ROX as a passive reference dye, 2 units of AmpliTaq Gold DNA Polymerase, 0.4 units of AmpErase
uracil DNA glycosylase (UNG), 400 lM dATP, dCTP,
dGTP with 800 lM dUTP and 6 mM MgCl2. Duplex
PCR reaction mixtures were the same as above with the
inclusion of additional primers (0.5 ll each of bovine-specic cytb primers and 1 ll each of chicken-specic cytb
primers) and probes (0.5 ll each of mammalian and poultry-specic cytb probe).

General Promega Taq DNA polymerase and buersbased PCR mixture included MQ water 12.75 ll, 10 reaction buer 2.5 ll, 25 mM MgCl2 solution 5 ll, 10 mM each
dNTPs 1 ll, 5 u/ll Taq polymerase 0.25 ll, primers
(25 lM) 1 ll each, probe (10 lM) 0.5 ll, DNA solution
1 ll. MQ water (1 ll) instead of DNA was used as a negative control.
2.5. Gel electrophoresis of PCR products
Gel electrophoresis with SeaKem LE agarose (1.7% for
general PCR, 3% for duplex PCR) gel containing ethidium
bromide was used to separate the PCR products.
PCR (13 ll) products was loaded onto each well. Electrophoretic proles reecting dierent molecular sizes of
PCR products were visualized and analysed as described
in Section 2.1.
2.6. Data analysis
Primary real-time PCR data were analysed by the Opticon
Monitor 3 software and the threshold cycle (Ct) was
calculated. Replicate standard curves of Ct value (Y) vs
log10[DNA amount] (X) were analysed using Microsoft Excel software and a linear regression equation of the
Ct value plotted against the log10[DNA amount] was
calculated.

C.-L. Zhang et al. / Food Control 18 (2007) 11491158

3. Results and discussion

3.1. Extraction of DNA from fresh and processed foods
DNA was isolated from fresh meats, processed meats and
cheeses using the standard CTAB method, which was very
eective for solid products, and analysed by agarose gel electrophoresis (Fig. 2). DNA extracted from fresh beef mince
was of relatively good quality, as determined by an A260/
A280 ratio of 1.73 (Table 2) and lack of degradation (Fig
2), and high yield (357.8 lg/g fresh tissue), whilst DNA
obtained from cooked, processed cows meats was of lower
quality (A260/A280 ratio of 1.351.69), highly degraded (very
little high molecular weight DNA was observed in oxtail

Fig. 2. Gel electrophoresis of total DNA extracted from meats, milks and
cheeses. Lane 1, fresh beef mince DNA; lane 2, fresh lamb mince DNA;
lane 3, corned beef DNA; lane 4, steak pie DNA; lane 5, tinned oxtail soup
DNA; lane 6, C cows milk DNA; lane 7, B cows milk DNA; lane 8,
goats milk DNA; lane 9, Welsh goats cheese DNA; lane 10, B Mozzarella
cheese DNA; lane 11, cheese quiche DNA. Lane M1, HindIII digested k
DNA; lane M2, 1 kb DNA ladder.

1153

soup) and low yield (33322 lg/g fresh weight). DNA isolated from cheeses and cooked dairy products (quiche) was
also degraded with a yield of 34349.6 lg/g fresh weight
(Table 3). Using the Wizard Magnetic kit (a mini-prep system) small amount (2.43 lg/ml) of DNA was extracted
from pasteurised cows milks. This was not degraded
(Fig. 2) and had A260/A280 ratios of 1.442.0. The DNA
yields of various products were calculated from the A260 values and are included in Tables 2 and 3 for reference though
these values obtained for highly degraded DNA or samples
with a low A260/A280 ratio may not be reliable.
3.2. Specicity of bovine-specic and ovine-specic cytb
primers
In conventional PCR analysis, the pair of bovine-specic cytb primers amplied bovine DNA isolated from beef
meat and produced a 116 bp fragment using 1 mM MgCl2
(lane 1 in Fig. 3a). No PCR product was obtained using the
bovine-specic primers with DNA extracted from lamb,
pork, goat, turkey and chicken tissue and water bualo
milk. The pork, turkey and chicken DNA were all PCR
positive with corresponding species-specic primers, indicating that the negative reaction was not the result of poor
quality DNA or PCR inhibitory contaminants (data not
shown). The ovine-specic cytb primers amplied the lamb
mince DNA and produced a 133 bp fragment (lane 10 in
Fig. 3a), but did not amplify DNA from beef, pork turkey
and water bualo samples. The ovine cytb primers did,
however, amplify a 133 bp fragment from goat DNA samples at a low eciency.
Bottero et al. (2003) reported that ovine-specic cytb
gene primer amplied caprine DNAs from a few breeds.

Table 2
Ct values and their derived target DNA amount for DNA extracted from fresh or frozen and processed meats and mixed DNA samples measured by realtime PCR with bovine-specic cytb primers and a FAM-labelled mammalian-specic cytb probe
DNA samples

DNA yield
(lg/g fresh tissue)

A260/A280

Input DNA
amount (ng)

Ct valuea

Target DNA
amount (ng) derived
from Ct value

Eciency
(target DNA/input
DNA 100)%

Beef DNA
Beef DNA
Beef DNA
Beef DNA
Beef DNA
Beef DNA
Beef DNA
Lamb DNA
Pork DNA
Chicken DNA
Turkey DNA
Beef DNA mixed with
lamb and pork DNA
Beef DNA mixed with
lamb and pork DNA
Corned beef
Steak pie
Tinned oxtail soup

357.8
357.8
357.8
357.8
357.8
357.8
357.8
498.2
673.0
1104.4
2157.2
N/A

1.73 0.17
1.73 0.17
1.73 0.17
1.73 0.17
1.73 0.17
1.73 0.17
1.73 0.17
1.81 0.14
2.12 0.05
1.90 0.25
2.11 0.01
N/A

18.73 1.90
21.12 1.07
24.71 2.42
27.77 0.95
32.1 0.83
37.03 3.35
55
54.87
44.23
50.4
51.99
25.81 1.11

350
35
3.5
0.35
0.035
0.0035
N/D
N/D
N/D
N/D
N/D
1.93

N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
275.71

N/A

N/A

350
35
3.5
0.35
0.035
0.0035
0.00035
37.5
32.5
70
80
0.7 + 37.5
(lamb) + 32.5 (pork)
0.07 + 37.5
(lamb) + 32.5 (pork)
500
200
60

30.67 1.08

0.088

125.71

17.94 2.46
19.30 1.17
35.21 7.01

287.45
121.12
0.0049

57.49
60.56
0.0082

32.7
189.2
322.0

1.69 0.08
1.56 0.22
1.35 0.15

Data (average SE) represent three repeats. N/D, not detectable. N/A, not applicable.

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C.-L. Zhang et al. / Food Control 18 (2007) 11491158

Table 3
Ct values and their derived target DNA amount for DNA isolated from milks and cheeses measured by real-time PCR with bovine-specic cytb primers
and a FAM-labelled mammalian-specic cytb probe
DNA samples

DNA yield
(lg/g tissue)

A260/A280

Input DNA
amount (ng)

Ct valuea

Target DNA
amount (ng) derived
from Ct value

Eciency
(target DNA/input
DNA 100)%

B cows milkb
C cows milkb
Cheddar cheese
Cheese quiche
B Italian mozzarella cheese ball
A Italian mozzarella cheese
Fresh goats milkb
Somerset goats cheese
Welsh goats cheese

3.0
2.4
180.0
258.8
34.0
35.0
0.75
349.6
162.7

1.44 0.23
2.0
1.66
1.27
1.48 0.18
1.63 0.37
2.00
1.86 0.09
1.73 0.06

25
20
5
50
15
150
15.6
200
625

25.50 0.49
26.05 0.70
28.13 1.76
29.68 1.02
31.86 2.44
24.12 2.39
55
55
55

2.36
1.66
0.443
0.17
0.041
5.66
N/D
N/D
N/D

9.44
8.30
8.86
0.34
0.27
3.77
N/A
N/A
N/A

a
b

Data (average SE) represent three repeats.

DNA amount indicted by lg/ml of milk. N/D, not detectable.

Fig. 3. PCR amplication of DNA isolated from meats, milks and cheeses. (a) PCR amplication of DNA with bovine and ovine-specic cytb primers.
Lanes 17, PCR amplication of bovine cytb gene with DNA from fresh beef mince, water bualo milk, lamb mince, goat blood, pork mince, chicken meat
and turkey mince respectively; lanes 811, PCR amplication using ovine cytb gene primers with DNA from fresh beef mince, water bualo milk, lamb
mince and goat blood, respectively. (b) PCR amplication of DNA using bovine-specic cytb primers. Lane 1, beef mince DNA; lane 2, B cows milk
DNA; lane 3, Cheddar cheese DNA; lane 4, B Italian mozzarella cheese DNA; lane 5, Somerset goats cheese DNA. Arrows indicate the molecular size of
expected PCR products: unlled arrow for the 116 bp bovine sequence and lled arrow for a 133 bp sequence.

Here we observed non-specic amplication of goat DNA

with ovine cytb primers at a low eciency (lane 11 in
Fig. 3a). Alignment of the ovine primers with the caprine
cytb sequence present in the Genbank database (Accession
number X56289) shows that there are four base dierences
at the 3 0 end of the primers (Fig. 1), indicating that they are
unlikely to amplify caprine DNA eciently. However, a
dierent caprine cytb sequence (AB110597), was identied
with greater similarity at 3 0 end to the ovine cytb primers
(data not shown). Thus, the ovine primers may be able to
amplify certain goat varieties, depending upon the cytb
DNA sequence. Lopez-Calleja et al. (2005) also reported
amplication of bualo DNA of a few breeds with a
bovine-specic 12S primer. For the development of a
robust quantitative animal products detection system it is
necessary to check those primers with DNA from many
breeds of cow, sheep and goat.
3.3. PCR amplication of DNA from milk and cheese
samples
The pair of bovine-specic cytb primers was able to
amplify DNA from cows milk and Cheddar cheese (lane

2&3 in Fig. 3b). Positive PCR results were also obtained

with the Italian mozzarella cheese samples indicating the
presence of bovine DNA in those cheeses (lane 4 in
Fig. 3b). Positive PCR results were obtained with goats
cheeses DNA using ovine cytb primers whilst negative
PCR results were obtained using the bovine-specic primers, indicating that the negative reactions were not the
results of poor quality DNA or PCR inhibitory contaminants (lane 5 in Fig. 3b and data not shown). This conrms
previous ndings that there are sucient somatic cells present in mammalian milk to enable the isolation of DNA of
suitable quantity and quality for subsequent PCR amplication (Herman, 2001).
3.4. Duplex PCR
Duplex PCR for bovine and caprine DNA detection was
developed using the bovine and ovine-specic cytb primers
(Fig. 4a). For equal amplication of bovine and ovine cytb
sequences, 1 ll each of forward and reverse bovine or
ovine-specic primers were optimal for the duplex PCR.
No preferential DNA amplication was observed. A further duplex PCR using bovine and chicken-specic cytb

C.-L. Zhang et al. / Food Control 18 (2007) 11491158

1155

optimal for equal amplication of beef and chicken DNAs.

These parameters were used in the real-time duplex PCR
for simultaneous detection of beef and chicken, or of complex foods containing eggs and milk.
3.5. Real-time PCR detection of DNA in meat products

Fig. 4. Duplex PCR amplication of mixed meats DNA with bovine and
ovine or chicken-specic cytb primers. (a) Duplex PCR amplication using
primers for bovine and ovine cytb genes with fresh beef/lamb mince DNA
(15 ng/15 ng). Lane 1, 1 ll ovine primers; lane 2, 0.5 ll bovine primers/1 ll
ovine primers; lane 3, 1 ll bovine primers/1 ll ovine primers; lane 4, 1 ll
bovine primers. The unlled and lled arrows indicate the expected 116 bp
bovine/133 bp ovine PCR products, respectively. (b) Duplex PCR
amplication using primers for bovine and chicken cytb genes with beef/
chicken mince DNA (30 ng/35 ng). Lane 1, 1 ll chicken primers; lane 2,
0.3 ll bovine primers/1 ll chicken primers; lane 3, 0.5 ll bovine primers/
1 ll chicken primers; lane 4, 1 ll each of bovine/chicken primers. The
unlled and lled arrows indicate the expected 116 bp bovine/133 bp
chicken PCR products, respectively. Lane M, 100 bp ladder molecular
marker.

primers was optimized for amplication of beef and

chicken meats DNA (Fig. 4b). 0.5 ll each of bovine cytb
primers and 1 ll each of chicken cytb primers were found

Serial dilutions of fresh beef mince DNA with sterilised

MQ water were tested in real-time PCR assays (Fig. 5a;
Table 2). The lowest Ct value (18.7) was obtained with
350 ng of beef DNA (Table 2). The lowest quantiable
level of bovine DNA was found to be 35 pg with Ct values
of about 32. The Ct values with high DNA concentrations
are in agreement with those reported (Dooley et al., 2004)
using the same primers. However, the sensitivity of this
assay is lower than 0.5 pg reported by Walker et al.
(2003) using primers and probe for a high copy genomic
short interspersed element. The mammalian-specic cytb
probe used here was aligned with various cytb genes
(Fig. 1). It was observed that the published sequence (Dooley et al., 2004) was not an exact match for the aligned
sequences though a reasonable signal was obtained in
this study. A modied universal mammalian-specic
cytb probe, TGA GGA CAA ATA TCA TTY TGA
GGR GCW ACR GTY A, is being tested for enhanced
sensitivity and wide applications, e.g. for the identication
of water bualo and goat DNA.

Fig. 5. Real-time PCR graph obtained with bovine DNA using bovine-specic cytb primers and a mammalian-specic cytb probe. (a) Quantitation graphs
of DNA isolated from fresh beef mince. The amount of DNA from 350 ng to 0.035 ng was used and was shown in red, green, blue, yellow and pink colour,
respectively (from left to right). Data from one typical experiment were shown here. (b) A generalised standard curve for real-time quantitation of bovine
DNA. Ct values of fresh beef mince DNA (3500.035 ng) in three repeated experiments were used for the construction of standard curve. (For
interpretation of the references in color in this gure legend, the reader is referred to the web version of this article.)

1156

C.-L. Zhang et al. / Food Control 18 (2007) 11491158

The real-time PCR data were less aected when beef

DNA samples were mixed with lamb and pork DNAs.
The cross-reactions with other species, i.e. pork, lamb,
goat, chicken and turkey were minimal (Tables 2 and 3).
Pork DNA (32.5 ng) had a Ct value of 44 which is well
below the Ct value of 37 produced by 3.5 pg bovine DNA.
A standard curve was constructed by plotting Ct values
against the log10[calculated DNA amount] (350 ng to
35 pg) (Fig. 5b). The linear regression equation, Y =
3.34X + 26.69, R2 = 0.924 (signicant at P = 0.05) was
used to calculate DNA amount from a range of DNA
samples isolated from processed meats and mixed foods
(Table 2). DNA isolated from a number of processed beef
products was tested. Corned beef DNA (500 ng) and steak
pie DNA (200 ng) produced Ct values of 17.9 and 19.3,
respectively, corresponding to equivalent amounts of fresh
meat DNA of 287 and 121 ng. This indicates that despite
the extensive degradation of DNA in these products
(Fig. 2), amplication occurred at around 60% eciency
compared to fresh meat DNA. On the other hand, the oxtail
soup sample (60 ng) had a low Ct value of 35, at the limits of
sensitivity of the calibration curve, corresponding to about
an equivalent of 0.005 ng fresh meat DNA. It is not yet clear
whether this reects the extensive degradation of DNA in
this sample, the presence of PCR contaminants, or the
low proportion of bovine DNA in the product.
The bovine cytb real-time PCR system has been developed as bovine products have been the subject of examination. This TaqMan system could be updated to detect dual
or multiple forensic subjects in a duplex or multiplex realtime PCR by using other compatible dyes (e.g. TET, VIC)
to label other species-specic probes, i.e. poultry, water
bualo or goat-specic DNA probes. Duplex real-time
PCR using bovine and chicken-specic cytb primer pairs
and FAM-labelled mammalian and TET-labelled poultryspecic cytb probes was carried out based on the optimized
duplex PCR described in Section 3.4. A DNA mixture
(fresh beef DNA 30 ng, and chicken DNA 35 ng) produced
Ct values of 22.22 0.66 and 25.15 1.90 which are comparable to data obtained using bovine and chicken single
species assays respectively developed in this laboratory.
For development of further duplex or multiplex real-time
PCR assays, primers and probes could be designed based
on those reported: e.g. bualo-specic mitochondrial cytb
genes (Bottero et al., 2002; Rea et al., 2001), ovine and caprine mitochondrial 12S and 16S rRNA genes (Bottero
et al., 2003; Mafra et al., 2004).
It has been demonstrated that the bovine cytb realtime PCR system is useful for the authentication of fresh
and processed meats. It may be valuable in the quality
assurance of meat products. The amount of DNA and
the size of DNA molecule in a food product are related
to the proportion of the meat and the method of preparation (Frezza et al., 2003; Matsunaga et al., 1999;
Mayer et al., 1995). The amount and quality of DNA
extracted from meat may also be aected by the species,
breed, tissue, feeding conditions and processing proce-

dures (Li et al., 2006; Zhao et al., 2005; Zhou et al.,

2001). One advantage of real-time PCR is that the amplicon is typically 100 bp, whereas conventional PCR
amplicons are several-fold longer. The application of
real-time PCR to the authentication of processed meat
products in which the DNA may be highly degraded is
therefore promising.
3.6. Real-time PCR detection of DNA in milk and cheese
products
The quantitative detection of bovine DNA in milks and
cheeses was possible using the real-time PCR assay developed for beef meat. The yield of DNA from milk was
low, but the DNA appeared to be high molecular weight.
The amplication pattern of DNA isolated from cows
milk monitored by real-time PCR was comparable to those
obtained with beef meat DNA (data not shown). However,
the target DNA amount derived from Ct values for DNA
samples of cows milk were only 89% of the amount of
milk DNA in the assay (Table 3).
The quantity and quality of DNA in cheese is thought to
be low because the processing of cheeses involves high temperature, microorganism and chemical treatments. It is
known that there are large numbers of microorganisms
involved in the cheese fermentation and ripening process
(for example, Rademaker, Hoolwerf, Wagendorp, & te
Giel, 2006). DNA from Cheddar cheese and Italian mozzarella cheese samples gave positive reactions, though with
Ct values equivalent to considerably lower amounts of
fresh meat DNA (Table 3). A positive identication of
bovine DNA in the cheese quiche (containing cows milk,
Cheddar cheese and hens eggs) was also made (Table 3).
Using the duplex real-time PCR assay, a further concentrated cheese quiche DNA sample produced Ct values of
28.11 1.11 (bovine results) and 34.87 2.38 (chicken
results). No reaction was recorded with goats milk and
goats cheese DNA, using the bovine cytb primers in realtime PCR.
The amount of target DNA estimated by Ct value in the
milks and cheeses used in this study was only a small fraction of the equivalent amount of fresh meat DNA. This
could indicate that the DNA was of low quality, either
due to degradation caused by food processing (in the case
of cheese) or the presence of PCR inhibitors which were
not suciently removed in the purication step. Another
possible reason is that the mtDNA amount may be lower
in somatic cells in milks than cells in meats, and that there
is a considerable amount of microbial DNA in cheese samples. Testing those possibilities and optimizing procedures
for DNA extraction and purication towards a higher
accuracy for DNA quantication in milks and cheeses
are in progress in this laboratory.
Rea et al. (2001) reported that the amount of DNA
recoverable from milk and cheese was directly related to
the somatic cell content of the raw milk, and also to the
strength of the technique used to process the product, as

C.-L. Zhang et al. / Food Control 18 (2007) 11491158

this can inuence the yield, integrity and extractability of

the DNA. Lopez-Calleja et al. (2005) analysed pure
(100%) bovine milk including raw, pasteurised and sterilised samples and found heat-treatment halved the number
of somatic cells though PCR amplication was less
aected. It was thought that the detection of low amounts
of cows milk adulteration in processed cheese will be dicult. We did not analyse samples with known percentages
of cow component in mixed goat or water bualo milks
or cheeses as nal DNA amount put into each PCR reaction determines the real-time PCR result.
Our results indicate that it is possible to detect bovine
sequences in meat mixtures by real-time PCR. They also
show that bovine DNA can be detected in cows milk
and cheeses. The negative results obtained with goats milk
and cheeses using bovine-specic primers, compared to the
positive results obtained in conventional PCR with ovinespecic primers suggest that it will be possible to use the
real-time procedure to detect adulteration of goat milk
products with cows milk.
It has been reported that more than half of Mozzarella
di bualo (POD) cheeses were contaminated or adulterated
with cows milk (Bottero et al., 2002; Di Pinto et al., 2004).
The procedures described here were able to isolate DNA
from mozzarella style cheese and obtain a positive reaction
with the bovine DNA real-time assay. The same primers
were negative against water bualo milk DNA. This realtime assay will be able to detect the adulteration of water
bualo mozzarella with cows milk.
3.7. Further consideration of real-time PCR system for
bovine DNA detection
The uptake of real-time PCR system by the food industry depends on its technical advantages and relatively
low cost. The most expensive chemicals in the real-time
PCR assay are TaqMan probe and the universal PCR
master mix. It was found that general PCR reagents were
compatible with TaqMan probes (data not shown) but
the sensitivity was reduced. This may indicate the importance of UNG in the universal PCR master mix (Longo,
Berninger, & Hartley, 1990).
Real-time PCR system based on SYBR Green chemistry
can be a cheap alternative to TaqMan system. SYBR
Green directly binds to double stranded DNA and facilitated detection of PCR products. However, this chemical
is thought not to be as sensitive as TaqMan chemistry
and does not permit the performance of multiple real-time
PCR assays.
4. Conclusions
The TaqMan bovine cytb real-time PCR system for the
identication of meats, milks and cheeses is sensitive, quick
and safe. Its capability to quantify low levels of bovine
DNA (35 pg) will meet the standard required by many
authentication measurements. A duplex real-time PCR sys-

1157

tem based on bovine and chicken-specic cytb primers and

probes has also been used to measure DNA amounts in
meats, milk and egg products.
Acknowledgements
We are grateful to Dr. Isabel Gonzalez, Universidad
Complutense de Madrid, Spain for providing DNA samples extracted from authentic water bualo milk and goat
blood samples, to Drs. J. Hall, E. Taylor and R. Allsopp
for their help. This work was supported by the UK Higher
Education Innovation Fund (HEIF II).
References
Amigo, L., Ramos, M., Calhau, L., & Barbosa, M. (1992). Comparison of
electrophoresis, isoelectric focusing and immunodiusion in determinations of cows and goats milk in Serra da Estrella cheeses. Lait, 72,
95101.
Bania, J., Ugorski, M., Polanowsk, A., & Adamczyk, E. (2001).
Application of polymerase chain reaction for detection of goats
milk adulteration by milk of cow. Journal of Dairy Research, 68,
333336.
Bottero, M. T., Civera, T., Anastasio, A., Turi, R. M., & Rosati, S. (2002).
Identication of cows milk in bualo cheese by duplex polymerase
chain reaction. Journal of Food Protection, 65, 362366.
Bottero, M. T., Civera, T., Nucera, D., Rosati, S., Sacchi, P., & Turi, R.
M. (2003). A multiplex polymerase chain reaction for the identication
of cows, goats and sheeps milk in dairy products. International Dairy
Journal, 13, 277282.
Branciari, R., Nijman, I. J., Plas, M. E., Di Antonio, E., & Lenstra, J. A.
(2000). Species origin of milk in Italian mozzarella and Greek feta
cheese. Journal of Food Protection, 63, 408411.
Cerquaglia, O., & Avellini, P. (2004). A rapid c-casein isoelectrofocusing
method for detecting and quantifying bovine milk used in cheese
making: Application to sheep cheese. Italian Journal of Food Science,
16, 447455.
Chen, F. C., & Hsieh, Y. H. (2000). Detection of pork in heat-processed
meat products by monoclonal antibody-based ELISA. Journal of
AOAC International, 83, 7985.
Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins,
D. G., et al. (2003). Multiple sequence alignment with the Clustal
series of programs. Nucleic Acids Research, 31, 34973500.
Di Pinto, A., Conversano, M. C., Forte, V. T., Novello, L., & Tantillo, G.
M. (2004). Detection of cow milk in bualo mozzarella by polymerase chain reaction (PCR) assay. Journal of Food Quality, 27, 428435.
Dooley, J. J., Paine, K. E., Garrett, S. D., & Brown, H. M. (2004).
Detection of meat species using TaqMan real-time PCR assays. Meat
Science, 68, 431438.
Espinoza, E. O., Kirms, M. A., & Filipek, M. S. (1996). Identication and
quantitation of source from hemoglobin of blood and blood mixtures
by high performance liquid chromatography. Journal of Forensic
Science, 41, 804811.
Feligini, M., Bonizzi, I., Curik, V. C., Parma, P., Greppi, G. F., & Enne,
G. (2005). Detection of adulteration in Italian mozzarella cheese using
mitochondrial DNA templates as biomarkers. Food Technology and
Biotechnology, 43, 9195.
Frezza, D., Favaro, M., Vaccari, G., Von-Holst, C., Giambra, V.,
Anklam, E., et al. (2003). A competitive polymerase chain reactionbased approach for the identication and semiquantication of
mitochondrial DNA in dierently heat-treated bovine meat and bone
meal. Journal of Food Protection, 66, 103109.
Heid, C. A., Stevens, J., Livak, K. J., & Williams, P. M. (1996). Real time
quantitative PCR. Genome Research, 6, 986994.

1158

C.-L. Zhang et al. / Food Control 18 (2007) 11491158

Herman, L. (2001). Determination of the animal origin of raw food by

species-specic PCR. Journal of Dairy Research, 68, 429436.
Hird, H., Goodier, R., Schneede, K., Boltz, C., Chisholm, J., Lloyd, J.,
et al. (2004). Truncation of oligonucleotide primers confers specicity
on real time-PCR assays for food authentication. Food Additives and
Contaminants, 21, 10351040.
Holland, P. M., Abramson, R. D., Watson, R., & Gelfand, D. H. (1991).
Detection of specic polymerase chain reaction product by utilization
the 5 0 to 3 0 exonuclease activity of Thermus aquaticus. Proceedings of
the National Academy of Sciences of United States of America, 88,
72767280.
Hurley, I. P., Ireland, H. E., Coleman, R. C., & Williams, J. H. H. (2004).
Application of immunological methods for the detection of species
adulteration in dairy products. International Journal of Food Science
and Technology, 39, 873878.
Lanzilao, I., Burgalassi, F., Fancelli, S., Settimelli, M., & Fani, R. (2005).
Polymerase chain reaction-restriction fragment length polymorphism
analysis of mitochondrial cytb gene from species of dairy interest.
Journal of AOAC International, 88, 128135.
Laube, I., Spiegelberg, A., Butschke, A., Zagon, J., Schauzu, M., Kroh,
L., et al. (2003). Methods for the detection of beef and pork in foods
using real-time polymerase chain reaction. International Journal of
Food Science and Technology, 38, 111118.
Levieux, D., & Venien, A. (1994). Rapid, sensitive two-site ELISA for
detection of cows milk in goats or ewes milk using monoclonal
antibodies. Journal of Dairy Research, 61, 9199.
Li, C. B., Chen, Y. J., Xu, X. L., Huang, M., Hu, T. J., & Zhou, G. H.
(2006). Eects of low-voltage electrical stimulation and rapid chilling
on meat quality characteristics of Chinese Yellow crossbred bulls.
Meat Science, 72, 917.
Longo, M. C., Berninger, M. S., & Hartley, J. L. (1990). Use of uracil
DNA glycosylase to control carry-over contamination in polymerase
chain reactions. Gene, 93, 125128.
Lopez-Andreo, M., Lugo, L., Garrido-Pertierra, A., Prieto, M. I., &
Puyet, A. (2005). Identication and quantitation of species in complex
DNA mixtures by real-time polymerase chain reaction. Analytical
Biochemistry, 339, 7382.
Lopez-Calleja, I., Gonzalez, I., Fajardo, V., Rodrguez, M. A., Hernandez, P. E., Garca, T., et al. (2004). Rapid detection of cows milk in
sheeps and goats milk by a species-specic PCR technique. Journal of
Dairy Science, 87, 28392845.
Lopez-Calleja, I., Gonzalez Alonso, I., Fajardo, V., Rodrguez, M. A.,
Hernandez, P. E., Garca, T., et al. (2005). PCR detection of cows
milk in water bualo milk and mozzarella cheese. International Dairy
Journal, 15, 11221129.
Lumley, I. D. (1996). Authenticity of meat ant meat products. In P. R.
Ashurst & M. J. Dennis (Eds.), Food authentication (pp. 108139).
Blackie Academic & Professional.
Mafra, I., Ferreira, I. M. P. L. V. O., Faria, M. A., & Oliveira, B. P. P.
(2004). A novel approach to the quantication of bovine milk in ovine
cheeses using a duplex polymerase chain reaction method. Journal of
Agricultural and Food Chemistry, 52, 49434947.
Matsunaga, T., Chikuni, K., Tanabe, R., Muroya, S., Shibata, K.,
Yamada, J., et al. (1999). A quick and simple method for the
identication of meat species and meat products by PCR assay. Meat
Science, 51, 143148.
Maudet, C., & Taberlet, P. (2001). Detection of cows milk in goats
cheeses inferred from mitochondrial DNA polymorphism. Journal of
Dairy Research, 68, 229235.
Maudet, C., & Taberlet, P. (2002). Holsteins milk detection in cheeses
inferred from melanocortin receptor 1 (MC1R) gene polymorphism.
Journal of Dairy Science, 85, 707715.

Mayer, H. K. (2005). Milk species identication in cheese varieties using

electrophoretic, chromatographic and PCR techniques. International
Dairy Journal, 15, 595604.
Mayer, R., Hoein, C., Luthy, J., & Candrian, U. (1995). Polymerase
chain reaction-restriction fragment length polymorphism analysis: A
simple method for species identication in food. Journal of AOAC
International, 78, 15421551.
Mimmo, P., & Pagani, S. (1998). Development of an ELISA for the
detection of caprine as1-casein in milk. Milchwissenschaft, 53, 363367.
Murray, M. G., & Thompson, W. F. (1980). Rapid isolation of high
molecular weight plant DNA. Nucleic Acids Research, 8, 43214325.
Peter, C., Brunen-Nieweler, C., Cammann, K., & Borchers, T. (2004).
Dierentiation of animal species in food by oligonucleotide microarray
hybridization. European Food Research and Technology, 219, 286293.
Poms, R. E., Glossl, J., & Foissy, H. (2001). Increased sensitivity for
detection of specic target DNA in milk by concentration in milk fat.
European Food Research and Technology, 213, 361365.
Rademaker, J. L. W., Hoolwerf, J. D., Wagendorp, A. A., & te Giel, M.
C. (2006). Assessment of microbial population dynamics during
yoghurt and hard cheese fermentation and ripening by DNA population ngerprinting. International Dairy Journal, 16, 457466.
Rea, S., Chikuni, K., Branciari, R., Sukasi Sangamayya, R., Ranucci, D.,
& Avellini, P. (2001). Use of duplex polymerase chain reaction (duplex
PCR) technique to identify bovine and water bualo milk used in
making mozzarella cheese. Journal of Dairy Research, 68, 689698.
Saeed, T., Ali, S. G., Abdul Rahman, H. A., & Sawaya, W. N. (1989).
Detection of pork lard as adulterants in processed meat: Liquid
chromatographic analysis of derivatized triglycerides. Journal of the
Association of Ocial Analytical Chemists, 72, 921925.
Skarpeid, H. J., Kvaal, K., & Hildrum, K. I. (1998). Identication of
animal species in ground meat mixtures by multivariate analysis of
isoelectric focusing protein proles. Electrophoresis, 19, 31033109.
Stefos, G., Argyrokastritis, A., Bizelis, I., Moatsou, G., Anifantakis, E., &
Rogdakis, E. (2004). Detection of bovine mitochondrial DNA specic
sequences in Feta cheese and ovine yogurt by PCR-RFLP. Milchwissenschaft, 59, 509511.
Sun, Y.-L., & Lin, C.-S. (2003). Establishment and application of a
uorescent polymerase chain reaction-restriction fragment length
polymorphism (PCR-RFLP) method for identifying porcine, caprine,
and bovine meats. Journal of Agricultural and Food Chemistry, 51,
17711776.
Walker, J. A., Hughes, D. A., Anders, B. A., Shewale, J., Sinha, S. K., &
Batzer, M. A. (2003). Quantitative intra-short interspersed element
PCR for species-specic DNA identication. Analytical Biochemistry,
316, 259269.
Woolfe, M., & Primrose, S. (2004). Food forensics: Using DNA
technology to combat misdescription and fraud. Trends in Biotechnology, 22, 222226.
Zhang, C. L., Chen, D. F., McCormac, A. C., Scott, N. W., Elliott, M. C.,
& Slater, A. (2001). Use of the GFP reporter as a vital marker for
Agrobacterium-mediated transformation of sugar beet (Beta vulgaris
L.). Molecular Biotechnology, 17, 109117.
Zhang, G., Zheng, M., Zhou, Z., Ouyang, H., & Li, Q. (1999).
Establishment and application of a polymerase chain reaction for the
identication of beef. Meat Science, 51, 233236.
Zhao, G. M., Zhou, G. H., Wang, Y. L., Xu, X. L., Huan, Y. J., & Wu, J.
Q. (2005). Time-related changes in cathepsin B and L activities during
processing of Jinhua ham as a function of pH, salt and temperature.
Meat Science, 70, 381388.
Zhou, G. H., Liu, L., Xiu, X. L., Jian, H. M., Wang, L. Z., Sun, B. Z.,
et al. (2001). Productivity and carcass characteristics of pure and
crossbred Chinese Yellow Cattle. Meat Science, 58, 359362.