: *
!
"
#
"
$
%
&
.
() :*" + $ [email protected]
16 18 !" # $
Multiplex-PCR
1 2
3
* 4
1 -
! " #$ %
&
' ( )% *
&
+ .
2 - ./0 1!
+ 23 ! 1! " #$ )%
4
%4 + 43
&
+ .
3 - + 43 ' ( )% + 43 1! " #$ * 6& 7 8 " #$ % &
+ &
.
4 -
%
! " #$ %
&
' ( )% *
&
+ .
! / #
$%&
! / ' 4
/
$ ) 97
,$ $ / 78
: 1 / 8 / 97 :
12 / 9 / 97
:$ %&
' ' $( )* + ,
-
$(
. / 0 16 18 3 45 6%7 .% 7 93 $ 7 + : ;
<
=
$< ) 4 /
<
=
' ' , / 0 16 18 > 3 ( 93 $ 7 + : ( + 3 Multiplex-PCR
% ( .
> 3 : 7
3
= ( / $ ) 4
?@A B' 3 *
,
$ @ 7 , + 5 ( DNA
C
<
+ D <
E <(PCR
' 7 @ , F G 5 ( , / H
0 HPV-16 HPV-18
IJ GK .% L 3 M* NH 3 O) (PCR
1 P /٪
$ K/
Q .%
$ : 7
+
@/
60 % / $ ) 4 CK/ $ @ 19
C T ( 3
< .% ( )*HPV U
< $< + <V H
/
<
0 HPV-16
HPV-18 / / $(
; 8 ) 1 / 42٪ ( 11 ) 9 / 57 (٪ 3 ( .
$ \K(
, M :
= $ + V $ ) 4 ' (PCR
7 @ , F G 5 ( , HPV-16
HPV-18 ]
^ ;< > 3
<
_
.C U
= + V $ ) 4 ( ` /3 % 7
= 93 $ 7 + : HPV
% ( .
H :,% , 7
93 $ 7 + : -
' ' , Multiplex -PCR .
$ %&
B ?B* 7 + @ 6
C B DB#$
B BE
+ @ 6
+ ( )
1 .(
&
6 B + @ B B7 (
H + @
?
&
) B! I BE B
0B%3 .
+ : (
7 JK + 1
C
+ ( 4%
) 2 3 .(
?B* B 7 1 3 B!
L A M $ M N
DB B BO B
PQ 3 +
R # 7
!
#3 B
?B4 .
& B3 6
& 3 6 L M#$
N
&
O
&
6 + @ B B&
:
! ! BB # BBQ
) (HPV
BB R BB BB 1970
'
#BV MB $ + $ ?B* B 7 + @ B ( B
8 A
&
) 3
! ! .(
#
&
: 7 ) (PVs
W (
&
: 7
! +
Q7 DNA
. B&
6
&
: BB7 R #BB BB7
!
#3 BB R BB P)
* BB
BB XBB# 0 BB
) 4 ( .(
: BB& BBY BB
: & # ! !
&
: 7 7 ) B ( B ! K 8
BBQZ 20 DBB [BB#A BB4\
8 BB +
BB2 Y 3 2
2 BB + 1
2 L
BB BBQZ L
BB +
]3
) 5 .(
( + 8 + B2 Y 3 +
B7
E6
BB BB A& BB_ M& BB>3 : BB& & BB 3E7
&
` .0%
2
&
B_ " BK 6 6& .
a* . 0%3 B&
: & B ?B* B 7 B 2
2 7 D
( ( B \ 2 : B& DNA
) 6
* + 3 .(
80 (&
( B&
6 B&
:
20 B4 H B
&
BO B 7 + @ B
?*
) Q7 7 6& .(
&
: B7 ( ;B 3 B BY
b & 3
&
O B ? Qb3 + @ B
3
; B7
High-risk Low-risk
. BBBc
&
: BBB7 BBB # ! !
7 d
* B ?B*
H B ( R B
High-
>3 & BB 3 BB risk
&
M + BB BB 7 + @ BB BB d
?BB*
)
&
` ( ) 8 .(
D P$
7 R B
7 e 0 I A& _ 3 #& M ( " 8 d
?*
) 7 f (CIN
) 9 .(
6 I BA N g& B !
BBBB + BBBB%
BBBB BBBB BBBB4 3 15 ;BBBB 3
) 16 18 31 33 35 39 45 51 52 56 58 59 68 73 8 (
3
; 7 + @ B Q7 h A N ! (
B
) 10 .(
+ H !HPV
N + B $ MB $ i>B
B DB P$ ?B* + B + @ B 16
- HPV
18 - A2\ I V HPV
* 70
% B 7 + @ (
M ?*
b .
&
Q
&
) B3 + B4\ [@
90%
_ (
&
I A + @ k B H B ( + ( ?* +
16 - ) HPV
11 .(
4\ 7 16
-
HPV
b
&
Q
&
) H !HPV
N B2$ b
+ % D P$
B7 ) 12 .(
B&
6 B B\ R B2 *
# $ Z A3
7
&
8 D * 1
&
" B
&
! 7 " 3 ?4
&
:
! D8 B%
) ?* + + @
13 3 + b8 .(
&
DB%
! ! BB #
&
: BB7 BB BB BB i>BB
&
BB BB&
6
&
: 7
. M %
&
6 l 7 B B 4
&
6
1
&
+
&
6 l 7 l
& B7
! B B&
#Z 8 + M PCR
" O D 4
3 B !
BBB! BBBA N ( h BBB7 . BBBQ7 A3
BBB 6 BBB 8
! !
#
&
: 7 3
; 16 18 ' > + ( + @ B
7 l ?*
Multiplex-PCR
.
> 3 7
_ * A N
&
g V 3 A N P
- ANb ( D
R 4
1395 B4 3 B
R B 1396
. " O
&
6 A N 60 2 + m' B I BV
8 /3 ' > + ( (
?* 7 + @ 4\
+ 2
4 8 _ 0 ( >
& n . A
BBA N BB BB8 BB
BB7 BB DBB8
3 !)
&
g 3 (
&
( ?B* B 7 + @ \ B&
BY
( + ( ./0 g 1!
&
+ B2 o 0 B . B B DNA
( P D
O3 D
+ # `B! . B " O
8
! !
#
&
: 7 3
; 16 18 ' > + (
?* 7 + @ ...
>A
+ 27
(
BLAST
BB7 ( p q 0
BB BB
&
D
NCBI
Multiplex-PCR
4 ?O*
&
25 B B
M 5 / 5
PCR master mix 5X
) + #
BB&
+ BB* (
Taq DNA polymerase (0.05 U/µl)
MgCl2 3 mM
(
dNTPs (0.4mM
(
&
g B
(
7
&
g
! (
&
7 2 DBY#p 8 / 0 s B 1 B
( ) ) DNA
10 ("
5 / 10 q
Nb3 BBBB BBBB
BBBB
&
M ( P BBBB BBBB
&
DBBBB
3
&
# (+ 2 h ! ) 34
B M B I BV
(&
B t>3 q 0 uD8 " O MB&v I BV
E D R " : & M2$
&
94 \
50 E ( p R /3 " "
56 \ 30 E B
tQ " "
72 \ 2
K Bb
&
g 4 tQ
&
72 \ 10 K b B 8 BY
! .
&
+ B Is B/L
M-PCR
( B R
1%
* 3
&
"
&
)
µg/ml 0.5
( 8 (
&
.
N %A 1 . ) / 7 , '
@ , 3 %
=
$ ) 4
` 8 (bp) R @ 5`-3` 3 : & ;3
)
(10 2017
AAGGCCAACTAAATGTCAC F HPV-16 CTGCTTTTATACAACCGG R
100 ACCTTAATGAAAAACCACGA F HPV-18 CGTCGTTTAGAGTCGTTCCTG R
$ 7
BB
&
x BB&
6 BB\ BB + BB% BBA N 8
&
BB
Multiplex-PCR
&
B& DNA
:
HPV
2
.D i 7
) 6 + 2
A N DL3
3 /
±0 4 / 26 R L 18
3 56 BK R B
H 2O ( . 60
DL3 2 A3
19 2
) 6 /
%31 \ Y ( (
"
&
:
! !
# B . B D>c
8 2 ) 1 / 42٪ ( e
& 3
; 16 11 ) 2 8 / 57%
(
H ( 18 - 7 . HPV
y
"
B 2 ( 7 DBL3
3 7 A N
;
&
: 27 B@
B + B 1
&
. %
QO 1 . N GK 3 O) NH G/
M a* (PCR
N . F G 5 b
bC c N 4& d*
$ 7 ,
C c N P DNA
P 3 100 .bp
\K(
$ , M
" O + 3 7 I A N : 8
D P$
('! z A 3 #V M $ + $ HPV
! 2
#3 R # 3 *
&
D
&
{ 3 & ! D P$ (
; 7 6& N !
7 3( #zQ 6& 8 I V : &
) 12
& .(
6
&
: 7 ( ; 3 b & 3 Y
&
O Qb3 + @
? 3 :
; 7
High-risk
Low-risk
R 2A . 3
&
6 ; 3 H HPV
6 11 Q7
; 3 D P$
16 18 R 2A 1 )
13 .(
! A N (
60 + 2 D
(
' > + ( ?B* B 7 + @ B B
19 ) B 2 6 / 31%
(
\ Y ( 8 . D>c HPV
3
; 3 B
; 16
18 3 3 i 8 ) 1 / 42٪ ( 11 ) 9 / 57٪ ( B B .
7
+ 27
+ 7
B B 252
B2
?BB* BB 7 + @ BB BB ' BB>
BB >c BB 2 (
HPV
&
) 14
|27 .(
6 B ! + B 27 B
1 (
50 + @ 2
&
` + 2 Q 8 7 y
BB 2 ( DNA
) BB 7 BB% HPV
15 .(
BB
+ 27
s
! B
! ! B #
&
:
BBQ BB! H BB ) + BBN
16 - 18 HPV
- (HPV
@ BB DBB8 . BB ! + BBQ# + BB ?BB*
A N
&
6 bbL 6 H 2O ( 231
J2\ 2
BBA3 122 + BB ( BBB 2 109
BBB> ( BBB 2
JBB2\
BB7 . BB y
BB&
g BB 2 ( BB 7 (
B
! ! B B #
&
: &
DB8 B%
) 16 .(
g3( B
+ 27
B B&
B&
:
! ! B # BQ ) (HPV
)
&
: ) (CMV
B&
: B%!
6 ) B (EBV
&
: `! 7
` #Z2 H
1 2 )
HSV-1 HSV-2
(
`! 7
&
: Q 3
&
; 8 ) 8 - (HHV
69 2
' >
2 ?* + @ 7
p
@ B&
B
&
B7 3 8
3 } B B&
8
" B >
B B
) K
17 .(
&
6 BbbL 6 8 ) B 9 / 12٪ (
HSV-1
28 ) BBB 2 / 45٪ ( 47 EBV
BBB ) 8 / 75٪ ( 8 CMV
) BBBB 9 / 12٪ ( HPV
28 ) BBBB 2 / 45٪ (
HHV-8
&
+ B 27 ! B>\ . B
H
HPV-16
75 2 _
&
I A B + @ B
&
` 64
% l 1B
) 2
18 4\ .(
B + 27
&
J 3
&
6 H B
HPV
H B ( ?B* B 7 + @ B ' > + ( 16
B8 A
) 2
19 s ( .(
&
M B h' B
&
x B s B BA N
7 ! + 3
0B%3 B#* B .
B2
BB\
? BBQ 7 ) BB p + BB2
( BBY BB\
B7 B z3 ! B 6
B7 DBB 4 ~NB
8 •\ I 8'
&
. "
(
} O
&
6
&
: B% ?}'$
B B
\ 3 H
s +
&
+ 0%3 . + B JK
&
J + 3
>3 (
&
M
? ( B7 B!
@ B B
! + @ 8 B%
B#\
2 B&
l . B7 B
3 PCR
l
&
J 6z2N 0B%3
.
2
* M 2
.
3%^ OV/
&
+ Q
"(s b + 27 (
0 " L B
'B ( )B%
B*
B ! B B%7 {! 1
Bb3
&
2 %3
&
.
8
! !
#
&
: 7 3
; 16 18 ' > + (
?* 7 + @ ...
>A
+ 27
References
1. Baliga MS, Dsouza JJ. Amla (Emblica officinalis Gaertn), a wonder berry in the treatment and prevention of cancer.
European Journal of Cancer Prevention.
2011;20(3):225-239.
2. Howley PM. Human Papillomaviruses and Cancer. Annual Review of Pathology:
Mechanisms of Disease. 2018;13(1): 11-16.
3. Roberts CC, Tadesse AS, Sands J, Halvorsen T, Schofield TL, Dalen A, et al.
Detection of HPV in Norwegian cervical biopsy specimens with type-specific PCR and reverse line blot assays. Journal of clinical virology. 2006;36(4): 277-282.
4. Insinga RP, Dasbach EJ, Elbasha EH.
Epidemiologic natural history and clinical management of Human Papillomavirus (HPV) Disease: a critical and systematic review of the literature in the development of an HPV dynamic transmission model.
BMC Infectious diseases. 2009;9(1):2334- 2339
5. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012.
International journal of cancer. 2015:
1;136(5):E359-86.
6. Schiffman, Mark, and Nicolas Wentzensen.
Human papillomavirus infection and the multistage carcinogenesis of cervical cancer. 2013: 553-560.
7. Zutshi V, Dankher S, Malik A. Cervical Cancer Screening and Prevention: An Analysis of Beliefs and Predictors of
Knowledge, Attitude and Practice in Northern India. Indian Journal of Gynecologic Oncology. 2017 Dec 1;15(4):71-74
8. Singh A, Datta P, Jain SK, Bhatla N, Gupta SD, Dey B, et al. Human papilloma virus genotyping, variants and viral load in tumors, squamous intraepithelial lesions, and controls in a north Indian population subset. International Journal of Gynecological Cancer. 2009;19(9):1642- 1648.
9. Shukla S, Bharti AC, Mahata S, Hussain S, Kumar R, Hedau S, et al. Infection of human papillomaviruses in cancers of different human organ sites.
2009;130(2):222-233.
10. Arora R, Kumar A, Prusty BK, Kailash U, Batra S, Das BC. Prevalence of high-risk human papillomavirus (HR-HPV) types 16 and 18 in healthy women with cytologically negative Pap smear. European Journal of Obstetrics & Gynecology and Reproductive Biology. 2005;121(1):104-109.
11. Josefsson AM, Magnusson PK, Ylitalo N, Sørensen P, Qwarforth-Tubbin P, Andersen PK, et al. Viral load of human papilloma virus 16 as a determinant for development of cervical carcinoma in situ: a nested case- control study. The Lancet.
2000;355(9222):2189-2193.
12. Burd EM. Human papillomavirus and cervical cancer. Clinical microbiology reviews. 2003;16(1):1-17.
13. Kojic EM, Conley L, Bush T, Cu-Uvin S, Unger ER, Henry K, Hammer J, Escota G, Darragh TM, Palefsky JM, Brooks JT.
Prevalence and Incidence of Anal and Cervical High-Risk Human Papillomavirus (HPV) Types Covered by Current HPV Vaccines Among HIV-Infected Women in the SUN Study. The Journal of infectious diseases. 2018 Feb 14;217(10):1544-1552.
14. Katiyar S, Hedau S, Jain N, Kar P, Khuroo MS, Mohanta J, Kumar S, Gopalkrishna V, Kumar N, Das BC. p53 gene mutation and human papillomavirus (HPV) infection in esophageal carcinoma from three different endemic geographic regions of India.
Cancer Lett. 2005 Jan 31; 218(1):69-79.
15. Peter M, Stransky N, Couturier J, Hupé P, Barillot E, de Cremoux P, Cottu P, Radvanyi F, Sastre‐Garau X. Frequent genomic structural alterations at HPV insertion sites in cervical carcinoma. The Journal of pathology. 2010: 1; 221(3):320- 330.
16. Moradi A, Mobasheri E, Tabarraei A, Bakhshandeh Nosrat S, Azarhosh R, Alizadeh SH, Bazori M. Molecular epidemiology of Human Papillomaviruses
in breast cancer, Golestan province of Iran.
Medical Laboratory Journal. 2009:
15;3(1):0-0 (In Persian).
17. Szostek S, Zawilinska B, Kopec J, Kosz- Vnenchak M. Herpes viruses as possible cofactors in HPV-16-related oncogenesis.
Acta Biochim Pol. 2009; 56(2):337-342.
18. Jabbarpour Bonyadi M, Esmaeili M, Dasranj A. Determine the types of human papillomavirus oncogene multiplex PCR in cervical cancer lesions in North West of Iran. Article in Persian] J Infect Dis. 2008;
13(41):29-34(In Persian).
19. Jahdi F, Khademi K, Merghati Khoei E, Haghani H, Yazdanpanahi Z. Evaluation of the Individual and Medical Factors Associated With Genital Human Papillomavirus in Iranian Women. Scimetr.
2014; 2(4): e17495 (In Persian).
8
! !
#
&
: 7 3
; 16 18 ' > + (
?* 7 + @ ...
>A
+ 27
The Prevalence of Papillomavirus-16 and -18 isolated from women with cervical cancer using Multiplex PCR
Shabani M 1, Sadeh M 2, Falsafi S3, Amini K *4
1.Department of Microbiology, School of Basic Sciences, Saveh Branch, Islamic Azad University, Saveh, Iran.
2. Microbiology Group, Shahid Beheshti University of Medical sciences, Tehran, Iran.
3.Assistant Professor, Department of Microbiology, Faculty of Advance Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran.
4. Associate Professor, Department of Microbiology, School of Basic Sciences, Saveh Branch, Islamic Azad University, Saveh, Iran, [email protected].
Received: 23 Oct 2018 Accepted: 3 Dec 2018
Abstract
Background: Women infected with human papillomavirus, especially types 16 and 18, are at risk of cervical cancer. The purpose of this study was to determine the frequency of papillomavirus-16 and -18 in women with cervical cancer using multiplex-PCR.
Materials and Methods: In this experimental study, after collecting blood samples, viral DNA was extracted using a Cinaclone kit, and PCR, with specific primers, was performed to detect HPV- 16 and HPV-18. PCR products were analyzed by 1% agarose gel electrophoresis.
Results: Of 60 samples, 19 were infected with HPV. The results showed that the frequency of genotype HPV-16 and HPV-18 was 8 (42.1%) and 11 (57.9%), respectively.
Conclusion: The study showed that using PCR with specific primers for the detection of HPV-16 and HPV-18 is a convenient and accurate method. The results of this study indicate the relationship between HPV and cervical cancer.
Keywords: Cervix cancer, Human Papilloma Virus, Multiplex-PCR.
*Citation: Shabani M, Sadeh M.Falsafi S, Amini K. The Prevalence of papillomavirus-16 and 18 isolated from women with cervical cancer by Multiplex-PCR. Yafte.2018; 20(1):1-7.
