Detection of human papillomavirus in cervical cell specimens by hybrid capture and PCR with different primers.

Abstract

The purpose of this study was to compare hybrid capture assay with PCRs using different primers for the L1, E6-E7 regions for the detection of human papillomavirus (HPV) genome. One hundred twenty-five cervical smears with normal (n=42) and abnormal (n=83) cytology were investigated. Those at high-risk for HPV were studied by hybridization antibody capture assay and PCR with the pU-1M/pU-2R primers. Target DNA from the HPV L1 region was amplified by SPF10 primer set and home-PCR with MY09/MY11 primers. The presence of HPV DNA in cervical smears was detected by SPF10 (in 72% of cases), MY09/MY11 (58%), hybrid capture (55%) and pU-1M/pU-2R (39%). Results obtained with the SPF10 and MY09/MY11 consensus primer sets as well as hybrid capture and pU-1M/pU-2R specific for high-risk types differed significantly (chi2, P<0.0005). The correlation between assays with the use of SPF10 and MY09/MY11 was 86% and between hybrid capture and the pU-1M/pU2R technique--78%. In 49% of samples HPV DNA was detected by the four methods, whereas in 12% only by the SPF10 primers. The most sensitive technique was found to be PCR with the use of SPF10 primers, while the most specific--the MY09/11 PCR method. It seems that home-PCR with MY09/MY11 primers could be applied in screening tests.

Full text

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Communication
Detection of human papillomavirus in cervical cell specimens by
hybrid capture and PCR with different primers*
Slawa Szostek1½, Malgorzata Klimek2, Barbara Zawilinska1, Janusz Rys2,
Jolanta Kopec1 and Ewa Daszkiewicz1
1Jagiellonian University Medical College, Chair of Microbiology, Department of Virology, Kraków, Poland;
2Centre of Oncology, M. Sklodowska-Curie Memorial Institute, Kraków, Poland;
½e-mail: sszostek@cm-uj.krakow.pl
Received: 06 June, 2006; revised: 31 August, 2006; accepted: 18 September, 2006
available on-line: 01 October, 2006
The purpose of this study was to compare hybrid capture assay with PCRs using different prim-
ers for the L1, E6-E7 regions for the detection of human papillomavirus (HPV) genome. One
hundred twenty-five cervical smears with normal (n = 42) and abnormal (n = 83) cytology were
investigated. Those at high-risk for HPV were studied by hybridization antibody capture assay
and PCR with the pU-1M/pU-2R primers. Target DNA from the HPV L1 region was amplified by
SPF10 primer set and home-PCR with MY09/MY11 primers. The presence of HPV DNA in cervi-
cal smears was detected by SPF10 (in 72% of cases), MY09/MY11 (58%), hybrid capture (55%) and
pU-1M/pU-2R (39%). Results obtained with the SPF10 and MY09/MY11 consensus primer sets as
well as hybrid capture and pU-1M/pU-2R specific for high-risk types differed significantly (χ2,
P < 0.0005). The correlation between assays with the use of SPF10 and MY09/MY11 was 86% and
between hybrid capture and the pU-1M/pU2R technique — 78%. In 49% of samples HPV DNA
was detected by the four methods, whereas in 12% only by the SPF10 primers. The most sensi-
tive technique was found to be PCR with the use of SPF10 primers, while the most specific —
the MY09/11 PCR method. It seems that home-PCR with MY09/MY11 primers could be applied
in screening tests.
Keywords: papillomavirus, HPV detection, PCR, hybrid capture
INTRODUCTION
Human papillomavirus (HPV), the major cause
of cervical cancer and cervical dysplasia, is a mem-
ber of the Papillomaviridae family of DNA viruses.
So far, 118 types have been identified according to
their biological niche, oncogenic potential and phy-
logenetic position (de Villiers et al., 2004). There are
about 40 HPV viral types that are commonly found
in the genital tract. They are classified in the Alpha-
papillomavirus genus (Fauquet & Mayo, 2005).
HPV’s nonenveloped capsid has an icosahe-
dral symmetry, containing 22 capsomers with a di-
ameter of 52–55 nm. The HPV virion contains circu-
lar, double-stranded DNA. The HPV genome codes
for only eight early open reading frame proteins
(E1-E8, with E3 and E8 function unknown) and two
late open reading frame proteins (L1 and L2) on a
single strand of DNA (Lowy & Howley, 2001). The
late proteins L1 and L2 are the major and minor cap-
sid proteins of the virion. The DNA and amino-acid
sequences are highly conserved between HPV types,
especially of the L1 protein. The early genes of the
HPV genome code for proteins E1 and E2 which
are responsible for viral replication and transcrip-
tion, and E4 seems to aid virus release from infected
cells. The E6 and E7 genes are invariably expressed
in tumours and are sufficient to induce proliferation
and immortalize cells in culture. Expression of E6/E7
is required continuously to maintain the prolifera-
*The paper was presented at XXXIII Winter School of Biotechnology „Various Faces of Biotechnology” organized by the
Faculty of Biochemistry, Biophysics and Biotechnology Jagiellonian University, 25th February–2nd March 2006, Krynica,
Poland.
Abbreviations: HPV, human papillomavirus.
Vol. 53 No. 3/2006, 603–607
on-line at: www.actabp.pl

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604????????????2006
S. Szostek and others
tive state of the cells (zur Hausen, 2000). The protein
products of these genes interfere with the normal
function of tumour suppressor genes. HPV E6 inter-
acts with p53, and E7 binds to retinoblastoma pro-
tein, leading to tissue proliferation. HPV types can
be classified according to various criteria, e.g. their
tissue tropism, oncogenic potential and phylogenetic
classification.
On the basis of epidemiological studies of the
frequencies of certain types of intraepithelial cervi-
cal lesions and cervical cancer, the anogenital HPV
types can be generally categorized, according to
their oncogenic potential, as being either high-risk
or low risk (Bosch et al., 2002; Muňoz et al., 2003).
Papillomaviruses cannot be grown in con-
ventional cell culture, and serological assays have
only limited accuracy. As infection with HPV is fol-
lowed by a humoral immune response against the
major capsid protein (Dillner, 1999), with antibod-
ies remaining detectable for many years, serology
is not suitable for distinguishing present and past
infections. Thus accurate diagnosis of HPV infection
is based on molecular methods, including hybridi-
sation (e.g. hybrid capture, Southern and dot blot
hybridisation) and PCR (Manos et al., 1989; Low et
al., 1990; Cox et al., 1995; Jacobs et al., 1997; Clavel
et al., 1999; Gravitt et al., 2000). Various PCR-based
methods have been described for the identification
of HPV genotypes. Individual genotypes can be de-
tected by type-specific PCR primer sets. However,
these require the performance of multiple parallel
assays for each sample, and type-specific PCR prim-
ers have not been reported for each HPV genotype.
Alternatively, general PCR primer sets can be used,
permitting simultaneous amplification of a broad
range of HPV genotypes. Recently a novel general
primer set, designated SPF10, amplifying a fragment
of only 65 bp of the L1 region of the HPV genome
was developed (Kleter et al., 1998). The aim of the
present study was a comparison of the hybridization
antibody capture assay versus PCRs using different
primer sets for the L1, E6/E7 regions for the detec-
tion of the HPV genome. The choice of these primer
sets was determined by the fact that they are most
commonly used in HPV detection.
MATERIALS AND METHODS
Cervical smears were obtained from 83 wom-
en (40 ± 15 years old) with low grade (LSIL, n = 44)
and high grade (HSIL, n = 12) squamous intraepi-
thelial lesions and cervical cancer (Ca, n = 27). All
women were diagnosed by cytological examination.
The diagnosis of patients with HSIL and Ca was
also confirmed by histopathological methods. Cervi-
cal cancer patients were hospitalised in the Centre of
Oncology, M. Sklodowska-Curie Memorial Institute
in Krakow (Poland) and none of them had radio-
therapy before material collection.
The control group consisted of 42 women
28 ± 5 years old undergoing preventive examina-
tions before planned pregnancy who did not display
atypical squamous cells of the cervix. All cervical
smears obtained before the cervical biopsy speci-
mens were taken with a cervical brush and collected
to specimen transport medium (Digene Diagnostic,
USA) and stored frozen at –70oC until tested. Sam-
ples were then thawed and divided into portions for
hybridisation and PCR methods.
Hybrid capture HPV assay (Digene Diag-
nostic, USA). This test is a signal-amplified solution
hybridisation antibody capture assay that utilizes
chemiluminescent detection (Lorincz, 1996). Speci-
mens containing the target DNA hybridize with
a specific HPV RNA probe cocktail. The resultant
RNA:DNA hybrids are captured onto the surface of
a tube coated with antibodies specific for hybrids.
Immobilized hybrids are then reacted with alkaline
phosphatase-conjugated antibodies specific for the
RNA:DNA hybrids, and detected with a chemilu-
minescent substrate. The substrate is cleaved by the
bound alkaline phosphatase. The intensity of the
light emitted denotes the amount of target DNA
in the specimen. In our study, the RNA probe mix
for the detection of high-risk HPV types (HPV 16,
18, 31, 33, 35, 45, 51, 52, 56) was used following the
supplied instructions.
PCR. DNA was isolated using Genomic DNA
Prep Plus kit (A&A Biotechnology, Gdynia, Poland).
Cell samples were lysed in a buffer with proteinase
K (1.25 mg/ml) and chaotropic salts. Addition of
ethanol caused DNA to bind when the lysate was
spun through a silica membrane in a microcentri-
fuge tube. Following washing to remove contami-
nants, DNA was eluted in 10 mM Tris/HCl, pH 8.5
(preheated to 75°C). PCR was performed in a final
reaction volume of 50 µl containing 10 µl of the iso-
lated DNA sample.
General PCR primers (MY09/MY11, SPF10)
to amplify a broad-spectrum of HPV genotypes and
pU-1M/pU-2R primers to amplify high-risk HPV
DNA were used separately. Table 1 presents the
primers used for the PCR technique and the reaction
conditions for each primer pair.
Each experiment was performed with separate
positive (CaSki cells) and negative (H2O) PCR con-
trols. Analysis of the PCR products was performed
by electrophoresis in 3% agarose gel. To ensure ad-
equate DNA preparation, PCR amplification of β-
globin gene was performed in a separate reaction,
resulting in a 110 bp product (Saiki et al., 1985).
The analytic sensitivity of the methods used
determined by analyses of serial dilutions of CaSki-

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Vol.?53???????605
Human papillomavirus detection
cell HPV-16 DNA (Meissner, 1999) revealed that hy-
brid capture had a sensitivity limit of 50 000 HPV-16
genomes/reaction, PCR using pU-1M/pU-2R – 300
genomes/reaction, MY09/MY11 primers — 250 ge-
nomes/reaction, SPF10 — 10 genomes/reaction (Ca-
vuslu et al., 1996; unpublished authors’ data). The
results were analysed by the Student’s t-test and χ2
test.
RESULTS
HPV DNA was detected in 90 of 125 cervi-
cal smears by at least one of the following methods:
hybrid capture and PCR with three different primers
(pU-1M/pU-2R, MY09/MY11, SPF10). Looking at the
results of all the methods, only SPF10/PCR showed all
HPV positive results. HPV infection was present in 9
(21%) women with normal cytology (control group)
and significantly more frequently in patients with
LSIL — 43 (98%), those with HSIL — 11 (92%) and
with cervical cancer — 27 (100%). Results of the com-
parison of the methods used are shown in Table 2.
Table 2. Comparison of positive results obtained by different methods (hybrid capture and PCRs) in women with dif-
ferent cytological diagnoses
Significant differences were found between
the results of the assays performed with the four
different methods (χ2, P<0.0005) except for the
techniques of hybrid capture and PCR with prim-
ers MY09/MY11. SPF10 PCR was found to be the
most sensitive and MY09/11 PCR the most spe-
cific method. The correlation between PCR with
the universal primers MY09/MY11 and SPF10, as
well as hybrid capture and PCR with pU-1M/pU-
2R primers specific for high-risk HPV types was
86% and 78%, respectively. It was also demon-
strated that PCR results with the use of universal
primers MY09/MY11 correlated in 86% with the
hybrid capture method which detected the high-
risk types.
A positive result of HPV infection was ob-
tained in 49% of women when all four test methods
were used. When only three test methods were used
the positive result was obtained in 26% of women
and for two test methods only — 13%. In 12% of ex-
amined women the infection could be detected only
with one technique — the SPF10 PCR (Table 3).
Table 1. HPV primers used in PCR
Primers
Localization
of sequence
HPV L1
Product sizePCR protocol PCR conditions References
MY 09
MY11
(consensus)
450 bp50 mM KCl, 6.5 mM MgCl2,
200 µM DTP, 2.5 U Taq polyme-
rase, 50 pmol of each primer
94oC/30 s
55oC/60 s
72oC/30 s
40 cycles
Manos et al., 1989
SPF10
(consensus)
HPV L1 65 bp 50 mM KCl, 10 mM Tris/HCl,
pH 9.0, 0.1% Triton X-100,
0.01% gelatin, 200 µM dNTP, 1.5
U Ampli Taq Gold DNA polyme-
rase, 100 pmol of each primer
94oC/9 min
94oC/30 s
52oC/45 s
72oC/45 s
72oC/5min
40 cycles
Kleter et al., 1998
pU-1M
pU-2R
(specific for high
risk HPV 16, 18,
31, 33, 35, 52, 58)
HPV E6/E7228–268 bp50 mM KCl, 10 mM Tris/HCl,
pH 8.3, 1.5 mM MgCl2, 200 µM
dNTP, 2.5 U Taq polymerase,
100 pmol of each primer
94oC/30 s
55oC/2min
72oC/2min
30 cycles
Fujinaga et al., 1991
Detection method*
No. of HPV-positive women with various
cytological diagnoses
No. of
HPV(+)
Sensitivity
%
Specificity
%
χ2 test
Normal LSILHSIL Ca
SPF 1094311279010066
P<0.0005
MY09/MY114349257280100
HC43710 1869 9268
P<0.0005
pU-1M/pU-2R021820 3965 93
*HC, hybrid capture assay; PCR with primers: SPF10, MY09/MY11, pU-1M/pU-2R

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606????????????2006
S. Szostek and others
DISCUSSION
HPV infection is closely linked to the devel-
opment of squamous cell benign and malignant neo-
plasms of the lower genital tract, in both men and
women. This relationship can be estimated at many
levels, as shown by epidemiological, clinical and mo-
lecular data. Thus, the development of methods for
simple, rapid and accurate detection HPV has a cen-
tral role in many strategies designed to reduce the
risk of cervical cancer. In this study we applied the
hybrid capture test (approved by FDA for diagnostic
use), the PCR technique with the use of commercial-
ly available primers (pU-1M/pU-2R and SPF10), and
home-PCR with the MY09/MY11 primers. Hybrid
capture is a simple technique that could be readily
automated for large-scale use if required. There is
only a slight risk of contamination and false-positive
results, since no DNA amplification step is neces-
sary. In the hybrid capture method, a probe detect-
ing the high oncogenic potential types was applied
so the results could be compared with the results of
a more sensitive method, PCR with primers detect-
ing high-risk types, pU-1M/pU-2R. This assay did
not result in more cases of HPV infections being
detected. In 20 women, the result obtained with the
use of the hybrid capture technique was not con-
firmed by PCR amplification with the use of pU-1M/
pU-2R primers. A possible explanation for this could
be that the probe used in the hybrid capture assay
had a broader spectrum of types it could identify
(HPV 45, 51, 56 additionally). So, the next step of
this research was the application of universal prim-
ers for the region L1 which could detect both high
and low oncogenic potential HPV types. The results
of the PCR assay with the use of MY09/MY11 prim-
ers compared to the results of hybrid capture tech-
nique were found to be similar (69 and 72 patients
HPV-positive, respectively). Other universal primers
which detected a 65 bp fragment localized inside
the sequence amplified by the MY09/MY11 primers,
were also applied. The best results were obtained by
means of this method. HPV infection was detected
in 90 (72%) of examined women. Our previous, un-
published data suggest that SPF10 PCR is the most
sensitive technique for HPV 16 (which occurs latent-
ly in the CaSki cell-line) detection. Therefore it may
be suggested that this method is the most sensitive
one also for other HPV types. The differences be-
tween the results of PCR with either MY09/MY11 or
SPF10 primers could also be explained by the differ-
ences in the ranges of HPV types detected. Accord-
ing to other authors, the SPF10 primer can detect
57 different types of HPV whereas the MY09/MY11
primers distinguish only 25 of them (Gravitt et al.,
2000; Perrons et al., 2005).
In the Perrons et al. (2002) study, HPV DNA
was amplified from 80% of samples by SPF10 and
from 42% only by MY09/MY11. They obtained con-
cordant positive results in 42% of samples while in
38/100 samples only the SPF10 primer detected HPV
DNA. In our study the results were compatible in as
many as 80% of the samples tested (72/90).
Comparison of the hybrid capture method
and PCR with various primer demonstrated that in
most cases (75%) positive results could be achieved
by means of three or four methods. This suggests
that the high-risk types dominated in the examined
tissue samples. Only in 12% of samples the posi-
tive result was obtained with the use of only one
method, the most sensitive one detecting the broad-
est of spectrum HPV types. However, the high cost
of HPV detection with the SPF10 PCR method lim-
its the clinical applicability of this assay in screen-
ing studies. Therefore it seems that home-PCR with
MY09/MY11 primers, the sensitivity of which is
comparable to the hybrid capture method, is a good
solution for screening examinations.
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