Biotechnology & Biotechnological Equipment

ISSN: 1310-2818 (Print) 1314-3530 (Online) Journal homepage: https://www.tandfonline.com/loi/tbeq20

Detection and Typing of Human Papillomaviruses

by PCR

E. Shikova, I. Todorova, G. Ganchev & V. Kouseva-Dragneva

To cite this article: E. Shikova, I. Todorova, G. Ganchev & V. Kouseva-Dragneva (2009) Detection
and Typing of Human Papillomaviruses by PCR, Biotechnology & Biotechnological Equipment,
23:sup1, 877-880, DOI: 10.1080/13102818.2009.10818562

To link to this article: https://doi.org/10.1080/13102818.2009.10818562

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Published online: 15 Apr 2014.

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DETECTION AND TYPING OF HUMAN PAPILLOMAVIRUSES BY PCR

E. Shikova1, I. Todorova1, G. Ganchev2, V. Kouseva-Dragneva3

1
Bulgarian academy of sciences, Institute of Experimental Pathology and Parasitology, Sofia, Bulgaria
2
National Specialized Hospital for Active Treatment in Oncology, Sofia, Bulgaria
3
Sofia Dermatovenereological Dispensary, Sofia, Bulgaria
Correspondence to: Evelina Shikova

ABSTRACT
Human papillomavirus (HPV) infection is the leading risk factor for cervical intraepithelial neoplasia (CIN) and cervical
cancer. Detection of high-risk HPV infections might identify women who are at increased risk of development or progression
of a cervical lesion and may have prognostic significance. Therefore there is considerable interest in identifying and
accurately typing the viruses. PCR amplification of HPV genomes is the most sensitive method for the detection of
cervicovaginal HPV. The different PCR techniques used to detect HPV infection, however, affect the frequency with which
viral DNA is identified. Using different PCR primer sets, we tried to optimize the detection and typing of HPVs. We tested both
consensus and type-specific primers. PCR utilizing the two most commonly used consensus primer sets, MY09/MY11 and
GP5+/GP6+, located within the L1 region of HPV genome, amplified a broad spectrum of HPV genotypes in a single reaction.
To identify the HPV types, samples were also subjected to PCR using specific primers for HPV types 6/11, 16, 18, 31 and 33.
Our results suggest that testing with both PCR using L1 consensus primers MY09/MY11 and with PCR using type-specific
primers could provide the maximum accuracy of HPV identification.

Keywords: consensus primers, genotyping, human
papillomavirus (HPV), PCR
Introduction
The association between cervical cancer and human
papillomavirus (HPV) is well established (7, 16). More than
100 HPV genotypes are identified, but several of them,
including HPV 16, 18, 31 and 33 are members of the high
risk (HR) HPV group as they promote carcinogenesis. HPV
type 16 is the most oncogenic type, followed by type 18.
Since persistence of oncogenic HPV types is important
risk factor and predictor of cervical intraepithelial neoplasia
(CIN) progression and cancer, identification of oncogenic
HPV types has prognostic significance. Therefore there is
considerable interest in detection and typing of these viruses.
Many molecular methods for HPV testing are currently
available but PCR amplification of HPV genomes is the most
sensitive technique and can detect between 5 and 100 DNA
molecules in a specimen. PCR utilizing consensus primers,
directed at relatively conserved regions of the HPV genome,
allows the amplification of a broad spectrum of HPV
genotypes in a single reaction. A number of different primer
combinations amplifying fragments from various regions of
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the HPV genome have been developed (14). The most
frequently used amplification systems for the detection of
HPV DNA in clinical samples are based on MY09/MY11
and GP5+/GP6+ consensus primer sets mediated PCR,
amplifying DNA fragments in the conserved L1 region of
approximately 450bp and 150bp respectively. Further, type-
specific PCR primer sets allow the identification of
individual genotypes.
Several studies indicate that any single method or
technique for the detection of HPV may underestimate the
true prevalence of HPV in cervical samples (4, 11). Since
detection of HR HPV infections is crucial for identifying
women who are at increased risk of development or
progression of a cervical lesion, optimization of methods for
HPV testing is an important task.
In order to optimize the PCR-based assays, used for HPV
identification, we compared the ability of different primer
sets to detect and type HPV DNA in clinical materials. We
evaluated several consensus and type-specific (TS) PCR
assays as tools to determine the HPV types in cervical
specimens.
Materials and methods
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Clinical materials and GP5+/GP6+ and type-specific primers for HPV6, 11, 16
In this study, 54 cervical scrapes with normal and abnormal (16L1 and 16E6/7), 18, 31 and 33 (1, 12, 13). All primers
cytomorphology were analysed. The samples with cytological used in this study are shown in Table 1.
abnormalities were from women with low- and high-grade The final 50μl PCR mixture contained 10-μl sample, 25
lesions of the cervix. The samples of cervical cells were taken μl PCR Master Mix (Promega), 3 mM MgCl2, 20 pmol of
using a cytobrush and placed in 1 ml 0,9% NaCl. each primer. Amplifications were performed with the
following cycling profile: incubation at 94°C for 5 min
Sample processing
followed by 40 cycles of 1-min denaturation at 95°C, 1-min
The samples were vigorously stirred and centrifuged at 4,000
annealing at 55°C, and 1-min elongation at 72°C. The last
x g for 10 min at room tempetrature. The cell pellet was
cycle was followed by a final extension of 10 min at 72°C.
resuspended in 500 μl of detergent buffer (10 mM Tris-HCl
The primer annealing step of GP5+/GP6+ primers-based
(pH 8,3), 50 mM KCl, 2,5 mM MgCl2, 0,45% Triton X100,
PCR was performed at 40°C for 2 min.
0,45% Tween 20) and digested with 100μg/ml proteinase K
(Promega) for 2 h at 56oC (12). The proteinase was To check the DNA quality the specimens were amplified
inactivated by incubation at 95oC for 10 minutes. The with beta-globin primers PC04 and GH20. During
processed samples were stored at -20°C antil analysis was amplification positive and negative control samples were
performed. 10μl of processed samples was used in each PCR included. As positive controls CaSki cell DNA (HPV16
assay. positive) and HeLa cell DNA (HPV18 positive) were used.
PCR products were analysed on a 2% agarose gel stained
PCR amplifications with ethidium bromide and visualized by UV-
Each sample was subjected to 9 paralell PCR reactions using transillumination.
the following primer sets: consensus primers MY09/MY11
TABLE 1
Primers used in this study
__________________________________________________________
Primers Sequence (5'-3') Amplicon (bp)
_____________________________________________________________________
Consensus primers
MY09 CGTCC(AC)A(AG)(AG)GGA(T)ACTGATC
MY11 GC(AC)CAGGG(AT)CATAA(CT)AATGG 450
GP5+ TTTGTTACTGTGGTAGATACTAC
GP6+ GAAAAATAAACTGTAAATCATATTC 150
Type-specific primers
6.1 TAGTGGGCCTATGGCTCGTC
6.2 TCCATTAGCCTCCACGGGTG 280
11.1 GGAATACATGCGCCATGTGG
11.2 CGAGCAGACGTCCGTCCTCG 360
16.L1-1 TGCTAGTGCTTATGCAGCAA
16.L1-2 ATTTACTGCAACATTGGTAC 152
16.E6/7-1 TTGCTTTTCGGGATTTATGC
16.E6/7-2 AGATCAGTTGTCTCTGGTTGCA 390
18.1 AAGGATGCTGCACCGGCTGA
18.2 CACGCACACGCTTGGCAGGT 216
31.1 ATGGTGATGTACACAACACC
31.2 GTAGTTGCAGGACAACTGAC 514
33.1 ATGATAGATGATGTAACGCC
33.2 GCACACTCCATGCGTATCAG 455
_____________________________________________________________________

by PCR using consensus and type-specific primers to

Results and Discussion determine if HPV detection varies when different primer sets
We examined 54 cervical specimens obtained from are used. First we have compared the two major consensus
cytologically normal women and from women with low- and primer sets used for PCR amplification of HPV DNA,
high-grade lesions of cervix for the presence of HPV DNA MY09/MY11 and GP5+/GP6+ primers. The MY09/M11

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primers are synthesized with several degenerate nucleotides
in each primer and are thus a mixture of 25 primers, capable
of amplifying a wide range of HPV types (6). The
GP5+/GP6+ primers consist of fixed nucleotide sequence for
each primer and detect a wide range of HPV types by using a
lowered annealing temperature during PCR (1). The
MY09/MY11 primer set has been used predominantly in
studies in America and Asia, whereas the GP5+/GP6+ primer
system has been used primarily in Europe (9). Fragments of
450 bp and 150 bp are amplified by PCR using MY09/MY11
and GP5+/GP6+ primers, respectively (Fig. 1). In our study
both, MY09/MY11 and GP5+/GP6+ consensus primer sets,
exhibited approximately equal sensitivity, as defined by the
ability to detect HPV DNA. 15 (27,8%) and 14 (25,9%) of
samples were HPV positive in PCR using MY09/MY11
primers and GP5+/GP6+ primers, respectively. GP5+/GP6+
mediated PCR failed to detect HPV DNA in 1 sample found
HPV positive by MY09/11 PCR. All HPV DNA negative
samples showed positivity in beta-globin gene PCR analysis.
Fig.1. HPV detection by consensus primers PCR: 1. PCR with MY09/MY11
primers; 2. PCR with GP5+/GP6+ primers; 3. Molecular weight marker (100
bp)
Fig. 2. HPV16 detection by type-specific PCR: 1. Molecular weight marker
(100 bp ); 2. PCR with E6/E7 primers; 3. PCR with L1 primers
HPV typing is of great importance for cervical cancer
risk assessment, as well as planning individual treatment and
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vaccination strategies (3, 10).We tested cervical specimens
by PCR using type-specific primers for the most common
HPV types - HPV6, 11, 16, 18, 31 and 33 (6, 12, 13). The
most prevalent was HPV16, detected with an L1 gene-
specific primer pair in 5 (9.2%) samples, followed by HPV31
– in 3(5.6%) samples, HPV6 – in 2 (3.7%) samples and
HPV33 and HPV11 – in 1 sample each (1.85% each). We
were unable to detect HPV18 DNA in analyzed samples.
Three of samples, found HPV positive by consensus primers,
were untiped with type-specific primers. The relatively high
rate of untyped samples in this study indicates that PCR using
primer sets for more HPV types should be included in the
future analysis of cervical specimens.
Using TS PCR with primers directed to the L1gene we
found that HPV16 is the most common type. In addition,
HPV16 is the main risk factor for cervical cancer. At the
same time HPV type 16 is often found integrated into the
chromosomes of cervical cells that disruptеs the integrity of
the E1 and E2 genes, while the E6 and E7 genes are retained.
Therefore, we tried to optimize the TS PCR-based detection
of HPV16 by using primers that amplify 390bp fragment in
E6/7 region of the HPV genome (Fig. 2). With this primer set
we detected HPV16DNA in 2 more samples in addition to the
samples found HPV16 positive with L1 primer set. They
were both negative by PCR with MY09/MY11 and
GP5+/GP6+ consensus primers and by HPV16 PCR using L1
type-specific primers. These results showed that the E6/7
gene directed TS PCR increased the positivity rate for
HPV16 DNA detection. Similar results are reported by other
authors (2, 4, 5). The lower HPV16 positivity rate found with
TS PCR based on L1-gene specific primers could be
explained with partial deletion of the L1 region during the
integration of HPV 16 into the host DNA. The HPV
integration followed by deletion of virus genome could occur
not only in the late phases of the neoplasia but is also found
in some of low-grade lesions indicating that it may be an
early event in cancer progression (8). In addition, a recent
study showed that the frequency of integrated HR HPV
genomes is with marked differences for individual HR HPV
types (15) with HPV16, 18, and 45 found substantially more
often in the integrated state compared with HPV types 31 and
33.
In conclusion, primer configurations used in HPV DNA
amplification are important factor that determine sensitivity
and specificity of PCR-based assays. Our results indicate that
complementary PCR techniques based on both consensus and
type-specific primers should be used to avoid missing
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potential HR HPV infections. An appropriate HPV diagnostic
system could include preselection of samples with the
MY09/MY11 primers, followed by HPV typing by TS PCR
with primers from E6-E7 regions of HPV genome.
Acknowledgment
This work was supported by Bulgarian National Science
Fund, Grand No 1513-05.
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