(PCR)
!
"
!
"
#
$
$
%
&'()
*
+, - . /'
*
$ 0 1
/
#
$%
2&3'(&
4 56
0 .7 0 6
8 '
,0'
(
! /
0' 9
$
: . '
RFLP
; ,
,
( ( +<7 =' $7 <' > (: ? 7
PCR
5'(
@ A
. B 8
. ;
"
# ' (; . ; ?; 7
, # '
? 7 $
+A 8
.8
@ C <7 ='
8
359
! 7 B 8
2 C
5'(
-@ ; 8,6 .; 7
0
Tru91, RsaI, Hinf I, Hae III, Alu I, Taq I, Mob I
<7 ='
8
=3
DA ', @ 3E:
: =;F $
;
5'( ,7 <7 ='
8
9 0'
8,6
Hinf I
&- @ 3E 0
+A 8 .8
8
-198
117
44
; , ! 7 B 8
; : 8,G
Hae III
+A 8 .8
. ' 8
3E:
285
74
8,6
: ! 7 B 8
Alu I
.8 ; .;# . ' 8; = : 3EH
+A 8
190
169
(
+
5'(
4 56 @ 8,I
0
<7 ='
0
=F $
! 7 B 8
@ '7
JCF
@ 8,6 ><,
5'( ,7 4 56 @ ,
9 , 0
0 6
8 ' <
',
K
Identification of beef using restriction fragment length polymorphism–
polymerase chain reaction
R. A. Al-Sanjary
Department of Veterinary Public Health, College of Veterinary Medicine, University of Mosul, Mosul, Iraq
Abstract
To differentiate the beef from other types of meat consumed by human, DNA markers based on polymerase chain reaction
restriction fragment length polymorphism technique is performed by using universal primers designed on mitochondrial
cytochrome b gene to obtain amplified band 359 bp, then digested with some of restriction enzymes like
Tru
91,
Rsa
I,
Hinf
I,
Hae
III,
Alu
I
, Taq
I,
Mob
I. The result revealed that, the
Hinf
I enzyme produce three bands 198, 117, 44 bp and the
Hae
III
enzyme revealed two band 285, 74 bp, the
Alu
I enzyme also produced two band but the molecular weight are 190, 169 bp.
The other enzymes did not reveal any digestion of the amplified bands and this result is a characteristic unique to beef
compared with other types of meat when using same enzymes.
Available online at
http://www.vetmedmosul.org/ijvs
& &
.G
;
5 L CM
A 7
$ :
'
+ .
N
' +' @
' .
3
6 . O :
P $
1
+F @
' JCF . Q
? 7 A 5 @
9=
RM " (:
@ ,
3' ',
' '
Q 7 " (6 JCF
=
; E,: $ .
P 5
(6
;
: ,
:
' "
2
3
Q ;7
.S
,:
F . #' % (6 + . -
=F
T
RM
:
$ '()
+' Q # $# /
. JCF , - P5 :
,H
$
U
33
'
;
-
9 , ' +
5
0 6
RM' &-
, +<
,H
.
P;5 6
$ ,
-#
:
' #, + 2 C#
8 ,5
RM'
,7 3
5' $ 5
> 6
(
0 6
B ',G
-4
=G
? G
2 3'(
'F
J ; 5 . J #H' 3, 4 5: " (6 +A CM D',
F
.;
U 6
Q
5
JC;F %;
.;7 $0',' +'
# '/
+;' U ; 6 . F V
0 . , U
$-
CV6
@ ,
" '
;
%
.7 3' ( $ 0', + '
5
K
A 7
Q 7 Q -# %
@ 5'(
$ :
,:
F8 '
(6 @ C Q
,6
5
A
@ 7 , @ C 4 56
.7
.#
+;F 3 = :
W ' !C ,
$ ' > (: ? 7 ,
%
Q 7
N'
;=I
;
5 W E
$
J ' J
?; G
#
&5 9
+ J
+
.A
$3(
7
P; <
,
,
0'
<
6
@ 5'(
@ /
? 7 , Q 7
> (:
8; ' + 5'(' , $ '
;,:
P ;5'
,
,0' 3, 3' ',
@ ,
Species-specific PCR
7
8
2 C#
; 6 8; ' + @ /X
, . =
.
$&5
@ A
5'(
;
?; 7
' ( .
"
#
.
+
. ' ,0'
(
, # '
0' 9
$
:
RFLP_PCR
9
K
' ( .
'
+
3
@ ,
. "
#
8 ' + 3, Q <'() .# +'
, # '
.
;,:
@ 0<
10
11
!C;
.
CF ? 7 @ A
' '
$7 <'
,0' $&5 . @
. & W7 =' $
O (
?; 7 +
.A #
,
' - .
,
( ( +<7 ='
> (:
9
CF
:
=F $&5 .
, 2 C P 5 9 ''
(
9 0 JCF
@ 8,I
;
0
;;
; ,0'
RFLP
12
13
' '
0
RFLP-PCR
(;
%
.
B ' ' ) +'
? G
F8 ,)
# @
B ' ' )
? G
N
5 @ A
' +
.A # ,
9 ''
=I
? G
-#
0 @ 8,6
;
' Q
;
+
;
;' 3 &5 .
;
8 '
,:
;
><,; 37 , . = 2 C# @ ,
;
>,
13
14
4 56
0 .7 0 6
8 ' U M
;
0 @ 8,) $ 7 $&5 .
JC;F @;
:
;(
5'(
. ; ?; 7
;
;
@ A
. B 8
8 ? 7 $
"
# ' (
7
+A 8 .8
359
! 7 B 8
3 =F
-U
@ 8,I
'
0
3', 0
@ A
><, 3 @ 5'( +'
0 ( @ (
DA ', 9
><,
@ 8,I
@ ,
? 7 .#
4 5:
K
&
&
'(
0 6
. @ , 7 @
+; Q 8;
@&
.
$
,
@ =
;
0
. , > #: +
-;
@
3, , P 5'(
'5
? G
'7
;
;0
? 7
15
2 C#
@&7 ;<'
;;7G ;'
W7 =;'
$;7 <'
; ,0'
,
( ( +<7 ='
+;
? 7 ; '7)
13
16
Y
$
5'( '
'
;
F
;
+
E, $
;
10X
X
V
0 L
;
; -&- ; ,
', ;7
0
.
. (# 6
0, @ <( <
, Q
8,:
;
.; B 8
@ A
' (; . ; ? 7
. ;
"
#
, # '
# / . Q83
Germany promega
. #
+
# F @ A
JCF 9 ''
Z
[
$ 6
CCA TCC AAC ATC TCA GCA TGA TGA AA
[
+,
-GCC CCT CAG AAT GAT ATT TGT CCT CA
'
9=
? 7
" ,6
Q #C
J&;7:
+;
!
;
;
;;
8 ;;3
Thermocycler
# ;;/ .;;
Germany Eppendorf
8 3
D
D ,
P ;5
$7 <' C3
95
\
;
Q
4
Q
Q
$&5 0
95
\
0
Q
55
\
0
Q
72
\
0
Q
. -&- $&5
Q
72
\
Q
7
Q
Q
$&5 0
13
14
;-8 3
(
D' , $
' '
$
'
+A ; 3#
;
]
&3
-K
@ C <7 ='
8
? 7 $
+A 8
.8
359
@ 8,6 U
5'( ' ! 7 B 8
0
; 8
=;3
Tru91, RsaI, Hinf I, Hae III,
Alu I,
Taq I,
Mob I
;,
Q
? 7
"(;
8,6
; 7 $&;5 @ 7 ( Y&- Q
+A
+
9
: D', ) : -#: : . '
D', W ( 8,6
=3
"(
,
5'(
9 0
8,6
K
)(
@ 3E:
(
5'( @
: +'
[
9
@ ,
Universal primer
; 8 +, -
$ 6 N 7 ,
@ , 7 9
Q
0 6
+;A 8 .8 ;
359
B 8
+;( 0 +;
$ ; 9;
8 $# ! 7
Ladder DNA 100 bp
+ #
$#/
1
8 '
@ ,
+ .7 0 6
4 56
0
'5 '
0 @ 8,6 .
7
0
DA ', @ 3E:
N,
5'(
. $#
8;,:
Tru 91, Rsa I, Taq I, Mob I
=3
W7 ='
,
0 6
=;F !: $ ;
;
+A 8
.8
@ C <7 ='
0
359
+; ! 7 B 8
8
9 0' : =F $
.
7 ='
5';( ,7 <
4 56
0 @ 8,6
8;,6 (,
Hinf I
@ ;3E
+A 8 .8
8
-&-198
117
44
8
T
;7
T
,
; : 8,G
Hae III
+A 8 .8
. ' 8
3E:
285
74
8,6
: ! 7 B 8
Alu I
.# . ' 8 = : 3EH
+A 8 .8
190
169
7 B 8
+ # !
$#/
2
K
$#/
1
@ C 8 $#/ ? 7 0 6
, W7 =' DA ',
+A 8 .8
359
[
5'( ! 7 B 8
+( 0 +
$
9
M
Ladder 100bp
&;F ? 7
8
#6
1.2
K^
* &
0' 9
$
: . ' ,0' .G
RFLP
4 G +F
+<7 =;' $;7 <'
; ,0' > (: ? 7 ,
,
@ /X
,
( (
PCR
; 8 ?; 7 ;3 &5 . $
, +'
<7 ='
.; . ;
.
? 7
[
,
5
8; ? 7 $
, 8
JCF =F
+
.A # @ ,
8 ' 3 &5 . 9 '(, < '5 $
: @ C
@ ,
13
K
? 7 @ ,
.
<7 ='
8
+A 8
.8
'
.
.
? 7
!C
[
2 ,F .H
Y
?; 7
; '7
, $#
5
0
+ W&'5
N 7
[
.
17
K
' 0 6 8 ' (
+<
@ A
5'(
.
? 7
12SrRNA
;
. ;#
8
223
8
T
7
T
18
8
. # ,
294
8
T
7
T
. ; ?; 7
;
[ ;
5'(
,7
28SrRNA
19
;, # '
.
. -
$M'(
3 .
.
-#:
@ A
' +
11
K
[
.G
. ; ?; 7
; [ ; ;F (
JCF + 5'(
8 N,7 D', !C
, # '
.
+ "
# ' (
+A 8 .8 @ C <7 ='
359
.;
! 7 B 8
$
13
9;
0 6
5 ,'(
DA ', @0<' C3
><, @ 5'( +'
4 5: @ (
DA ',
@0
.# [
@ C <7 =;'
8 ? 7 $
+ 4 5: @ ,
? 7
+;;A 8 .8
359
! ;;7 B 8
14
20
22
;;;,7
. ; +; N; ' 9;
N(;<,
[
@ 5'(
"
# ' (
W 3 ,
K
$#/
2
# '/;
8
=F DA ',
359
! ;7 B 8
6
;
0 @ 8,6
(
0
1
Tru 91
2
Hae III
3
RsaI
4
Alu I
5
Taq I
6
Hinf I
7
Mob I
+;( 0 +
$
9
Ladder 100 bp (M)
8
#6 &F ? 7
2
K^
.G
0 @ 8,6
(
!: +
5'(
"
.:
'
/ F '5
+# "( , $#
DA ', 3E'
O ('
3 &5 .
8 '
@ ,
.
< '5
,6
? 7 W ' 2 C#
: $; :
# 3= 9 3 5 : 3 8
+ ?'
3' ',
A 7
-
9 , '
+
12
=G
? G
2; C
;
.
0
RFLP
0 >
5'('
+
'
;,6 . ;
8
@ ,
.
< '5
0
>
,V:
K
K
F V
,G
MW
400 300
200
100 1000
1500
500
9 '('
2 C#
@ ,
8 '
=
, .
13
K
+<
(
JCF
DA ', @ 3E:
8
9 0' : =F $
N,
5';( ,7 <7 ='
: # '/
;
0 @ ; 8,6
4 56
8 .8
8
-&- @ 3E
+A
198
117
44
8
T
7
T
8,6 (,
Hinf I
,
; : 8,G
Hae III
+A 8 .8
. ' 8
3E:
285
74
; : ! ;7 B 8
8,6
Alu I
+;A 8 .8 .# . ' 8 = : 3EH
190
169
8
T
7
T
F
;
A ', JC
;
.; $# DA ', 9 %<'' D
14
(
+
+ 3'
8 '
,
-,:
@ ;,
.
3, = .
0 6
@ 5'(
3
:
@ 8,G
= F
'
8
<7 =
DA ', 9 2 C#
13
C
.
5'(
'(
@ 8,G
Q /7 8 '
,:
@ ,
.
3, ;= .
0 6
'(
,7
>;<,
[ ;
;
. ; ?; 7
# ' (
"
+A 8 .8
359
! 7 B 8
. ; +
(
DA ', @< '5
,'
; 7 .
' , 9 0
Y
.
(
DA ', 9
0 @ 8,6
=3
4 5:
12
. C
5'(
[
"
# ' ( .
? 7
B ',I
; 8
.8
<7 ='
+A 8
464
! 7 B 8
8 ' '
23
7 ,
T
5'(
@ ,
.
11
8,G
T
T
JC;F +; 2 C#
N,: DA ', @ 3E: (
; 0
=;F !: $ ;
;
+A 8
.8
@ C <7 ='
359
8
T
;7
T
;,7
;
5'(
; $#
;
.
8;,:
Tru 91, Rsa I, Taq I, Mob I
9 , 0
0 6
8 ' <
' ' = : JCF
;
@ 8,6 ><,
5'( ,7 4 56 @ ,
JCF 5'('
@ C
() @ ,
U 8 ' + 9( $#/
,0'
) +'
3= 9
0
0
F8 ' .#
)G
5';(
@ 8,I
0
23
24
K
+*
:
!
"
# Q 7 #/
;
$;
Q83 6
' ? 7
?; 7 .(;
:
7
'#
! 8'
Y
CF +
5'(
0 @ 8,6
K
$&
1. Skrokki A, Hormi O. Composition of minced meat. Part A: Methods. Meat Science 1994; 38: 497-501.
2. Buntjer JB, Lenstra JA, Haagsma N. Rapid species identification in meat by using satellite DNA probes.Z lebensm Unters Forsch 1995; 201 : 577-582.
3. Lenstra JA, Bradley DG. Systematics and phylogeny of cattle.CAB Intl. Ed. by R. Fries and A. Ruvenski 1999;1: 1-14.
4. King NL. Species identification of cooked meats by enzyme staining of isoelectric focussing gels. Meat Science 1984 ;11: 59-64.
5. Brodmann P. Development and Validation of species identification, Ph.D. Thesis. University of Basel ; 2002.
6. Cano RJ, Poinar HN, Pieniazek NJ, Acra A, Poinar GO. Amplification and sequencing of DNA from a 120 - 135 million–year–old weevil. Nature 1993; 363:536-538.
7. Al-Sanjary RA, Jubrael JMS. Differentiation between different animal meats using a species-specific Polymerase Chain Reaction.12th
Scientific Congress 10-12 December,Faculty of Veterinary Medicine, Assiut University ; 2006.
8. Matsunaga T, Chikuni K, Tanabe R, Muroya S, Shibata K, Yamada J, Shinmura Y. A quick and simple method for the identification of meat products by PCR assay. Meat Science 1999 ; 51(2): 143-148. 9. Verkaar ELC, Nijman I J, Boutaga K, Lenstra JA. Differentiation of
cattle species in beef by PCR-RFLP of mitochondrial and satellite DNA. Meat Science 2002 ; 60: 365-369.
10.Chikuni K, Tabata T, Kosugiyama M, Monma M, Saito M. Polymerase Chain Reaction assay for detection of Sheep and Goat meats. Meat Science 1994 ; 37: 337-345.
11.Kocher TD, Thomas WK, Meyer A, Edwards SV, Paabo S, Villablanca FX, Wilson AC. Dynamics of mitochondrial DNA evolution in animals : Amplification and sequencing with conserved primers. Proc. Natl. Acad. Sci. USA.1989 ; 86: 6196-6200.
12.Wolf C, Rentsch J, Hubner P. PCR-RFLP Analysis of mitochondrial DNA : A reliable methods for species identification. J. Agric. Food Chem. 1999 ; 47: 1350-1355.
13.Meyer R, Hofelein C, Luthy J, Candrian U. Polymerase chain reaction-restriction fragment length polymorphism analysis: a simple method for species identification in food. J. AOAC Int. 1995 ; 78: 1542-1551. 14.Abdulmawjood A, Bulte M. Identification of Ostrich Meat by
Restriction Fragment Length Polymorphism’s (RFLP) Analysis of cytochrome b Gene. Journal of Food Science 2002 ; 67(5): 1688-1691. 15.Maniatis T, Fritsh EF, Sambrook J. Molecular cloning: A Laboratory
Manual, Cold spring Harbor laboratory, New York. 1982.
16.Bottero MT, Civera T, Anastasio A, Turi RM, Rosati S. Identification of cow ’s milk in "buffalo" cheese by duplex polymerase chain reaction. J. Food Prot. 2002 ;65(2): 362-366.
17.Dalmasso A, Fontanella E, Piatti P, Civera T, Rosati S, Bottero MT. A multiplex PCR assay for identification of animal species in feedstuffs. Molecular and Cellular Probes. 2004 ;18: 81-87.
18.Lopez-Calleja I, Gonzalez I, Fajardo V, Rodriguez MA, Hernandez PE, Garcia T, Martin R. Rapid Detection of Cows milk in Sheep and Goats milk by a species-specific polymerase chain reaction technique. J. Dairy Sci. 2004 ;87: 2839-2845.
19.Bellis C, Ashton KJ, Freney L, Blair B, Griffiths LR. A molecular genetic approach for forensic animal species identification. Forensic science international 2003; 134: 99-108.
20.Bellagamba F, Moretti VM, Comincini S, Valfre F. Identification of species in animal Feedstuffs by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Analysis of Mitochondrial DNA. J. Agric. Food Chem. 2001 ; 49: 3775-3781.
21.Wang H, Lee S, Chong T, Wong M. Examination of meat Components in commercial Dog and Cat feed by using Polymearase Chain Reaction–Restriction Fragment Length Polymorphism’s (PCR-RFLPs) Technique. J. Vet. Med. Sci. 2004 ; 66(7) : 855-859.
22.Partis I, Croan D, Guo Z, Clark R, Coldham T, Murby J. Evaluation of DNA fingerprinting method for determining the species origin of meats. Meat Science 2000 ; 54: 369-376.
23.Matsunaga T, Chikuni K, Tanabe R, Muroya S, Nakai H, Shibata K, Yamada J, Shinmura Y. Determination of Mitochondrial Cytochrome B Gene sequence for Red Deer (Cervus elaphus) and the Differentiation of closely related Deer meats. Meat Science 1998 ; 49(4): 379-385.
