High-Resolution Melting Molecular Signatures for Rapid
Identification of Human Papillomavirus Genotypes
Ta-Hsien Lee1., Tzong-Shoon Wu1,2., Ching-Ping Tseng1,3,4*", Jiantai Timothy Qiu1,2,5*"
1Graduate Institute of Biomedical Sciences, College of Medicine, Taoyuan, Taiwan, 2Department of Biomedical Sciences, College of Medicine, Taoyuan, Taiwan,
3Department of Medical Biotechnology and Laboratory Science, College of Medicine, Taoyuan, Taiwan, 4Molecular Medicine Research Center, Chang Gung University,
Taoyuan, Taiwan, 5Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Taoyuan, Taiwan
Background: Genotyping of human papillomarvirus (HPV) is crucial for patient management in a clinical setting. This study
accesses the combined use of broad-range real-time PCR and high-resolution melting (HRM) analysis for rapid identification
of HPV genotypes.
Methods: Genomic DNA sequences of 8 high-risk genotypes (HPV16/18/39/45/52/56/58/68) were subject to bioinformatic
analysis to select for appropriate PCR amplicon. Asymmetric broad-range real-time PCR in the presence of HRM dye and two
unlabeled probes specific to HPV16 and 18 was employed to generate HRM molecular signatures for HPV genotyping. The
method was validated via assessment of 119 clinical HPV isolates.
Results: A DNA fragment within the L1 region was selected as the PCR amplicon ranging from 215–221 bp for different HPV
genotypes. Each genotype displayed a distinct HRM molecular signature with minimal inter-assay variability. According to
the HRM molecular signatures, HPV genotypes can be determined with one PCR within 3 h from the time of viral DNA
isolation. In the validation assay, a 91% accuracy rate was achieved when the genotypes were in the database.
Concomitantly, the HRM molecular signatures for additional 6 low-risk genotypes were established.
Conclusions: This assay provides a novel approach for HPV genotyping in a rapid and cost-effective manner.
Citation: Lee T-H, Wu T-S, Tseng C-P, Qiu JT (2012) High-Resolution Melting Molecular Signatures for Rapid Identification of Human Papillomavirus
Genotypes. PLoS ONE 7(8): e42051. doi:10.1371/journal.pone.0042051
Editor: Zhi-Ming Zheng, National Institute of Health - National Cancer Institute, United States of America
Received September 29, 2011; Accepted July 2, 2012; Published August 20, 2012
Copyright: ? 2012 Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Science Council [NMRPD180311 to J.T.Q.], Department of Health [DOH99-TD-C-111-006 to J.T.Q.] and Chang
Gung Molecular Medicine Research Center [EMRPD1B0151 to C.-P.T.]. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (JTQ); email@example.com (C)
. These authors contributed equally to this work.
" These authors also contributed equally to this work.
Human papillomavirus (HPV), a small and nonenveloped
double-stranded DNA virus, is established as the key etiological
factor in cervical neoplasms [1–3]. The recognition of the central
role of HPV infections in the etiology of virtually all cervical
cancers has dramatically changed the perspectives of diagnoses
and prevention of this neoplasia [4,5]. Currently, HPV DNA
testing plays a pivotal role for atypical squamous cells of
undetermined significance, primary screening in conjunction with
cytology for the detection of cervical cancer and cervical
intraepithelial neoplasia, and follow-up in a variety of clinical
settings [6–12]. Genotyping assays are also instrumental in
assessing the impact of HPV vaccination on the risk of acquisition
and on the distribution of individual HPV types in a population
HPV infection can be monitored by detection of thirteen high-
risk oncogenic HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52,
56, 58, 59 and 68) using a commercially available HPV testing
method such as the Hybrid Capture 2 assay (Digene Corporation,
Gaithersburg, MD), the only HPV assay approved by the US Food
and Drug Administration [11,15,16]. However, information on
HPV genotype is lacking in the cocktail detection method. Other
detection systems that determine HPV genotype include non-
amplification Southern and dot blot hybridization with type-
specific probes , type-specific PCR [18,19], and broad-range
PCR [5,20]. The disadvantage of type-specific PCR is that
multiple hybridization reactions are needed to access multiple
HPV genotypes in a single sample, while broad-range PCR such
as MY09/11 has the drawback of a large PCR fragment with less
sensitivity . Under these circumstances, there is a clinical
demand for developing a simple and accurate method for
identification of infecting HPV genotype with high specificity
Recently, a high-resolution melting (HRM) analysis method
which incorporates double-stranded DNA saturating dye and
specifically designed data collection and analyzing software has
been developed. HRM analysis was first developed as a closed-
tube technology for genotyping DNA variants and mutation
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screening with advantages over the other techniques such as high-
throughput, rapid and non-destructive nature [22–29]. Instead of
using a labeled primer to analyze the domain in which the
mutation resides, Wittwer and his coworkers developed HRM
analysis using a saturation dye LCGreen I to substitute the need of
labeled primer . The combined use of real-time PCR and
HRM for analysis of microbial DNA results in distinguishable
HRM profiles and generates unique molecular fingerprints that
facilitates its practical applications such as quantification of
pathogen load and microbial species identification [30–35]. If
required, heteroduplex formation or multiple PCR fragments can
be employed to distinguish microbial species with closely similar
HRM profile [30,31,36]. A modified HRM protocol incorporating
unlabeled probes has also been reported for genotyping of herpes
simplex virus that provides an alternative to detect and genotype
low copies of viral infection .
In this study, we reported a novel method based on the use of
HRM analysis and unlabeled probes to rapidly identify and
differentiate HPV genotypes in clinical specimens. Without
multiplexing, HPV genotypes can be completed with one PCR
within 3 h from the time of viral DNA isolation.
Materials and Methods
All cervical samples were collected from the Department of
Obstetrics and Gynecology, Chang Gung Memorial Hospital with
the approval by the Institutional Review Board (IRB 99-0112B)
and with informed consent from the patients. The genomic DNAs
of 140 consecutive HPV clinical specimens were obtained from the
sample bank for this study. The individuals who performed the
experiments do not know the genotype of these clinical specimens
until completion of HRM analysis.
The QIAamp DNA mini kit was purchased from Qiagen
(Hilden, Germany). The LightCycler 480 and the LightCycler 480
High Resolution Melting Master were purchased from Roche
Applied Science (Mannheim, Germany). The T&A cloning vector
was purchased from RBC Bioscience (Taipei, Taiwan). The
McTaq DNA polymerase was purchased from One-Star Biotech-
nology (Taipei, Taiwan). The UniPOL-Long Range PCR enzyme
mix containing Taq DNA polymerase and AccuPOL with 39R59
exonuclease activity was purchased from Ampliqon ApS (Skov-
lunde, Denmark). The EasyChip HPV genotyping system was
purchased from KingCar (Yilan, Taiwan).
Genomic DNA isolation
The QIAamp DNA mini kit was used to extract DNA from
cervical specimen and the genomic DNA was eluted in 50 ml of
elution buffer. The quality of the extracted DNA was checked by
conventional PCR to amplify a housekeeping gene glyceraldehyde
3-phosphate dehydrogenase (GAPDH) in a 50 ml of reaction
containing 5 ml of 106 PCR buffer (20 mM MgCl2), 2 ml of
10 mM dNTP, 0.5 ml of McTaq DNA polymerase (5 U/ml), 2 ml
of 5 mM forward primer GAPDH-F, 2 ml of 5 mM reverse primer
GAPDH-R (Table 1) and 2 ml of template DNA. The PCR
condition was 95uC for 5 min followed by 50 cycles of 95uC for
30 sec, 60uC for 40 sec, and 72uC for 60 sec. An additional 3 min
of extension at 72uC was performed after the last PCR cycle to
replenish PCR products followed by cooling at 4uC.
The HPV DNA testing was done routinely for patients who
attended our dysplasia unit. Clinical DNA samples were subjected
to EasyChip assay platform for HPV genotyping. The details of
HPV blot format and typing procedure were described previously
. Briefly, 20 ml of the denatured amplicon was hybridized to
the blot and the genotype was determined using streptavidin-
alkaline phosphatase conjugate and substrate. After the blot was
dried, the HPV genotypes displayed on the blot were determined
using a standard visual assessment protocol.
Plasmid construction of L1 fragment from various HPV
Partial HPV L1 region was amplified by PCR using the clinical
DNA samples of HPV16, 18, 39, 45, 52, 56, 58 and 68 and the
primer pair FRG5/FRG2 (Table 1). Briefly, the PCR reaction
(50 ml) was composed of 5 ml of 106 UniPOL buffer B (15 mM
MgCl2), 1 ml of 25 mM MgCl2, 2 ml of 10 mM dNTP, 0.5 ml of
the UniPOL-Long Range PCR Enzyme mix (5 U/ml), 2 ml of
forward primer FRG5 (5 mM), 2 ml of reverse primer FRG2
(5 mM) and 2 ml of template DNA. The PCR condition was 95uC
for 5 min followed by 50 cycles of 95uC for 30 sec, 46uC for
40 sec, and 72uC for 30 sec. An additional 3 min of extension at
72uC was performed after the last PCR cycle to replenish PCR
product followed by cooling at 4uC. The PCR product was then
cloned into the T&A cloning vector as described by the
manufacturer’s instruction (RBC Bioscience) and was confirmed
by DNA sequencing.
Symmetric and asymmetric broad-range real-time PCR
and HRM analysis
For symmetric amplification of the HPV genomic DNA, broad-
range real-time PCR was performed in a 384-well format using
LightCycler 480. Briefly, the PCR reaction (20 ml) was composed
of 10 ml of 26HRM master mix, 2 ml of 25 mM MgCl2, 0.8 ml of
5 mM forward primer FRG5, 0.8 ml of 5 mM reverse primer
FRG2, and 2 ml of template DNA. The amplification condition
was optimized for the use of FastStart Taq DNA polymerase by
incubating the reaction mixtures at 95uC for 15 min, followed by
50 cycles of 95uC for 15 sec, 46uC for 20 sec, and 72uC for 30 sec
with the ramp to 95uC at 4.8uC/s, to 46uC at 2.5uC/s, and to
72uC at 4.8uC/s. An additional 1 min at 72uC was added to
replenish the PCR product.
For asymmetric amplification of HPV genomic DNA, the PCR
reaction (20 ml) was composed of 10 ml of 26HRM master mix,
2 ml of 25 mM MgCl2, 0.4 ml of 2.5 mM forward primer FRG5,
2 ml of 5 mM reverse primer FRG2, 2 ml of 5 mM unlabeled probe
for HPV16 and HPV18 when indicated, and 2 ml of template
DNA. The unlabeled probe was modified by C6-amine or inverted
dT at the 39-end to prevent the probe from self-extension. The
amplification condition was optimized for the use of FastStart Taq
DNA polymerase by incubating the reaction mixtures at 95uC for
15 min, followed by 65 cycles of 95uC for 15 sec, 46uC for 20 sec,
and 72uC for 30 sec with the ramp to 95uC at 4.8uC/s, to 46uC at
2.5uC/s, and to 72uC at 4.8uC/s. An additional 1 min at 72uC was
added to replenish PCR product.
For HRM analysis, the PCR product was denatured by rising
temperature to 95uC at 4.8uC/s and was then cool down to 55uC
at 2.5uC/s for hybridization. The melting curve was acquired by
increasing the temperature from 55uC to 95uC at a ramp rate of
4.8uC/s with 25 acquisitions per degree of temperature.
HPV Genotyping by HRM
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Table 1. The primers and unlabeled probes sequences.
Primer/probe typePrimer/probe nameSequences
Unlabeled probe HPV-18-UP59-TGCTTCTACACAGTCTCCTGTACCTGGGCA-39
Figure 1. Sequence alignments of the L1 fragment PCR amplicons. Sequence alignments of the PCR amplicons corresponding to nt 6895 to
7115 of the L1 fragment (accession number NC_001526.1) for the indicated HPV genotypes. Only the sequences showing differences from the first
sequence are shown. Nucleotides identical to the nucleotide in the first sequence are indicated by dots. The underlined sequences were used for the
design of primers FRG5 and FRG2.
HPV Genotyping by HRM
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To assess the combined use of real-time PCR and HRM
analysis for rapid detection and differentiation of HPV genotypes,
genomic DNA sequences for 8 high-risk HPV genotypes (HPV16,
18, 39, 45, 52, 56, 58 and 68) were subject to bioinformatic
analysis. These genotypes represent 98% and 75% of the clinical
isolates in Southeast Asia and Europe/US, respectively [37,38].
After multiple-sequence alignment of HPV genomic DNA
sequences from different genotypes using the Vector NTI and
BioEdit software packages, the fragments that are highly
degenerated and flanked by conserved DNA sequences were
chosen as the candidate targets for design of broad-range PCR to
amplify HPV genomic DNA. Accordingly, a L1 fragment
corresponding to nt 6895 to nt 7115 of HPV16 (accession number
NC_001526.1) with the size ranging from 215 to 221 bp for
different HPV genotypes was found to fulfill our selection criteria
(Fig. 1). Due to the conserved nature of these nucleotides among
various HPV genotypes, the nt 6895 to 6923 and nt 7087 to 7115
were selected to design the forward primer FRG5 and reverse
primer FRG2, respectively (Table 1).
We determined whether symmetric PCR of the L1 fragment
followed by HRM analysis provides distinguishable melting
profiles for HPV genotyping. To facilitate the assay, the PCR
products corresponding to L1 fragment of various HPV genotypes
were subcloned into the T&A cloning vector. These plasmids were
used as the template for PCR amplification using the primers
FRG5 and FRG2. Despite that most of the HPV genotypes
displayed unique HRM profiles, the melting profiles for some
HPV genotypes were not distinguishable. For example, HPV18
and HPV45 exhibited almost identical derivative plots and the
genotypes were not likely to be determined accordingly (Fig. 2A).
To overcome this problem, we determined whether appropriate
use of type-specific unlabeled probe(s) generates HRM profiles
sufficient for differentiating HPV genotypes. To facilitate the
Figure 2. Asymmetric broad-range real-time PCR of L1 PCR amplicon. A and B. The L1 fragments of HPV18 and HPV45 were subject to
symmetric (panel A) or asymmetric (panel B) PCR amplification using the primers FRG5 and FRG2. An unlabeled probe complementary to HPV18
target sequence was included in the reaction during asymmetric PCR. HRM analysis was then performed and the derivative plots for the indicated
genotypes were shown. C–F. Serial dilution of the plasmid (from 1 pg to 10 ag) harboring the L1 fragment of HPV18 were used as the templates for
asymmetric broad-range real-time PCR with the primer pair FRG5 and FRG2. (C) A typical LightCycler 480 amplification plot. (D) The Ctplotted against
the plasmid DNA concentration. (E) A typical high-resolution melting plot. (F) A typical high-resolution derivative plots. NTC, no-template control.
HPV Genotyping by HRM
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binding of unlabeled probe to the PCR product, asymmetric PCR
was performed to selectively amplify the DNA strain complemen-
tary to the unlabeled probe. An unlabeled probe specific to
HPV18 was designed (Table 1) and was added into the
asymmetric PCR reaction. As illustrated in Fig. 2B, the HPV18-
specific unlabeled probe resulted in an additional melting peak for
HPV18 and thereby the genotypes of HPV18 and HPV45 can be
unambiguously differentiated (Fig. 2B).
The detection limit of this method was evaluated by asymmetric
PCR amplification of 10-fold serially diluted plasmid DNA (from
1 pg to 10 ag) that contained 221-bp of HPV18 L1 fragment. The
amplification reaction was performed in the presence of 40 ng of
human genomic DNA to mimic the co-presence of HPV and
human genomic DNA in clinical specimens. As revealed by
amplification plots, as little as 10 ag plasmid DNA equivalent to 3
copies of HPV DNA was detectable in this assay condition
(Fig. 2C). The standard curve showed a dynamic linear range for
quantification across 6 logs of DNA concentrations and had a
correlation coefficient of 0.9986 (Fig. 2D). Notably, both the
melting and derivative plots were consistent when the amounts of
template DNA from 1 pg to 10 ag were subject to asymmetric
PCR amplification (Fig. 2E and 2F).
It is known that the last 6–7 nucleotides of PCR primers are
critical for amplification specificity and efficiency [39,40]. Due to
the degenerative nature of the FRG5 and FRG2 primers and the
HPV68 genomic DNA carrying 2 mismatches at the last 7
nucleotides of the FRG5 primer region, we determined whether
the efficiency for amplification of HPV68 DNA is affected. When
the amounts of template DNA ranging from 1 pg to 100 ag were
used, PCR amplification of HPV68 DNA was as efficient as for
HPV18 DNA with no potential skewing. The presence of human
genomic DNA also had no effect on amplification of HPV18 and
HPV68 DNA (Fig. S1). However, 10 ag of plasmid template DNA
for HPV18 but not HPV68 could be detected by this method,
indicating a decrease in amplification efficiency that either due to
skewing effect or a negative impact of human genomic DNA on
amplifying low amount of HPV68 DNA.
The HRM profiles for 8 high-risk HPV genotypes were then
generated by asymmetric broad-range real-time PCR in the
presence of the unlabeled probes specific for HPV16 and HPV18.
The melting temperatures and melting profiles provide molecular
signatures for the 8 high-risk genotypes that can be unambiguously
identified through high-resolution derivative plots (Table 2 and
Fig. 3A). When clinical samples with the indicated genotypes were
subject to the analyses, the melting patterns were in accord with
those obtained from plasmid DNA template (Fig. 3B). Agarose gel
electrophoresis further confirmed the generation of genotype-
specific PCR products (Fig. 3C). The distinct HRM molecular
signatures thereby provide a basis for genotyping of HPV
We further assessed our method in HPV genotyping of clinical
specimens retrospectively. A total of 140 clinical samples from
patients who were suspected of having precancerous lesion were
blind tested. Due to the nature of sample storage, only 119 of the
140 DNA samples were informative and generated PCR products
that were suited for HRM analysis (Table 3). As revealed by
EasyChip genotyping and DNA sequencing, 70 of the 119
informative cases were infected with high-risk HPV subtypes,
while 49 were infected with low-risk subtypes. In addition, 65 of
the 70 samples were infected with the HPV subtypes that were the
analytical subjects of this study. Of the 65 samples, 59 displayed
distinguishable HRM patterns that can be assigned to the correct
genotype with the typing rate equivalent to 91%. According to
EasyChip genotyping analysis, the remaining 6 samples were
infected with multiple HPV genotypes and can not be genotyped
accurately by HRM analysis (Table S1).
During our analysis of clinical specimens, several low-risk HPV
genotypes including HPV42, 62, 70, CP8304, CP8061 and MM8
were found to display their unique high-resolution derivative plots
(Fig. 4A and 4B). Of the 49 specimens that were infected with low-
risk genotypes, 32 of them belonged to HPV42, 62, 70, CP8304,
CP8061 and MM8 with 29 of them being genotyped accurately.
The correct genotyping rate reached 91% (Table 3). Furthermore,
2 cases of HPV11 and 2 cases of HPV81 that were assigned as
‘‘others’’ (Table 3) in the low-risk group were, at the beginning,
mistakenly classified as HPV54 and CP8304, respectively. All the
rest of the samples (n=18) assigned in the ‘‘others’’ group for both
high-risk and low-risk genotypes was not falsely claimed as infected
with the HPV subtypes of interest. Together, an HRM database
for a total of 14 HPV genotypes was established that form the basis
for HPV genotyping.
Table 2. High-resolution melting profiles for high risk HPV genotypes disclosed by broad-range real-time PCR.
HPV genotype (n)a
Tm± SDb(6C) GC content (%) GenBank accession no.
HPV16 (5) 65.0960.49
HPV18 (5) 72.4560.26
HPV39 (5)78.2660.2036 M62849.1
HPV45 (5)79.8160.17 36 X74479.1
HPV56 (4) 77.0760.0333 EF177176.1
HPV58 (5) 73.8760.28
aNumber of test (strain).
bThe variance between 4–6 measurements with the indicated number of isolates.
HPV Genotyping by HRM
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HPV Genotyping by HRM
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HPV is well recognized as a major cause of cervical cancer. The
development of high grade pre-cancer and invasive cervical cancer
could be due to persistent high-risk HPV infection. A rapid,
accurate and sensitive method to detect and differentiate HPV
genotypes is essential to identify high risk patients who are
otherwise found to have normal cytological results and women
with cervical cancer potential from the screened population. HPV
genotyping provides a reference point for HPV vaccination and
HPV prevalence in natural history studies during clinical practice.
Unlike hepatitis B vaccine, there is no acceptable antibody test for
HPV vaccines. If it is affordable, HPV genotyping assay should be
the first choice for HPV testing. In this study, a novel approach for
HPV genotyping is developed. This method is based on the
combined use of asymmetric broad-range real-time PCR and
HRM analysis. Accordingly, 8 high-risk and 6 low-risk HPV
subtypes can be identified that offers a novel approach for HPV
A number of HPV DNA genotyping methods including real-
time multiplex PCR, Hybrid Capture II INNO-LiPA v2 HPV
genotyping PCR, Roche Amplicor MWP HPV test and Digene
HC2 assay have been reported in the literature [16,41–45].
Recent trends in the application of HRM analysis for microor-
ganism identification [30,31,35] lead us to explore a new avenue
to identify and differentiate HPV genotypes. Through bioinfor-
matic analysis, we revealed that the 153–163 bp interprimer
regions of FRG5 and FRG2 is relatively conserved within isolates
of the same genotype, whereas nucleotide divergence is present
among different HPV genotypes. Therefore, the PCR amplicon
likely contains information for at least partial phylogenetic
characterization for HPV genotyping. We demonstrated that 8
high-risk and 6 low-risk HPV genotypes can be identified through
the HRM molecular signatures that were generated by asymmetric
broad-range real-time PCR of the L1 DNA fragment in the
presence of two HPV16- and HPV18-specific unlabeled probes.
The relatively small PCR amplicon also results in an increase in
the detection limit of this method when compared with the
previously reported broad-range PCR such as MY09/11 that has
a drawback of a large PCR fragment with less sensitivity . Our
data indicate that as little as 10 ag of plasmid DNA carrying the
HPV18 PCR amplicon equivalent to 3 copies of HPV genome is
detectable by this method. Due to the 2 mismatches of the last 7
nucleotides in the FRG5 primer region, the detection limit for
HPV68 is slightly affected with 30 copies of genome being
detected. As judged by the Ct value obtained from the analysis of
clinical specimens, most of the samples contained more than 300
copies of HPV DNA. Hence, this assay should have sufficient
sensitivity for most of the clinical HPV genotyping analysis. When
it is combined with rapid-cycle PCR, HRM analysis requires
Table 3. The validation assay for 119 clinical HPV isolates.
HPV typeGenotype No. of isolates tested No. of isolates assigned with correct genotypeCorrect genotyping rate (%)
HPV58 1412 86
Total65 59 91
Total 32 2991
aHigh-risk HPV genotypes that are not characterized in this study.
bLow-risk HPV genotypes that are not characterized in this study.
Figure 3. High-resolution derivative plots for 8 common and high-risk HPV genotypes. A. The plasmids carrying genotype-specific DNA
fragment were subject to asymmetric broad-range real-time PCR followed by HRM analysis. The derivative plots for all 8 genotypes were plotted
together to reveal the differences among different HPV genotypes. B. Two clinical samples (red and blue curves) for each indicated genotype were
subject to asymmetric broad-range real-time PCR and HRM analysis. Derivative plots for the indicated HPV genotypes were plotted together with the
derivative plot obtained from plasmid DNA (black curve) to reveal the consistent melting patterns between these two different sources of template
DNA. (C) Agarose gel electrophoresis was performed to demonstrate the generation of genotype-specific PCR products from asymmetric broad-
range real-time PCR.
HPV Genotyping by HRM
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minimal time, and the material cost is usually less than $3. The
time required for the differentiation of HPV genotypes is
considerably short when PCR is performed directly with clinical
specimens. Approximate 3 h is required for genotyping of the
clinical samples with the isolated DNA as the starting material.
Figure 4. Unique high-resolution derivative plots for 6 low-risk HPV genotypes. Clinical isolates that did not have derivative plots typical of
those in our HRM database were analyzed. The HPV42, 62, 70, CP8304, CP8061 and MM8 genotypes were found to display their unique high-
resolution derivative plots. The derivative plots were plotted together to reveal the differences among different HPV genotypes (panel A). The
derivative plots for two to three measurements of each HPV genotypes were plotted to reveal the minimal inter-assay variability (panel B). Note the
consistent derivative plot pattern for each virus subtype.
HPV Genotyping by HRM
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Multi-infection of HPV strains with various genotypes accounts
for approximate 8–22% of the HPV-infected patients [46–48].
The prevalence of multiple HPV infections varies in relation to the
method used to detect HPV DNA and the study population. In
addition, a potential skewing for amplification of HPV DNA in
specimens containing multiple HPV genotypes by PCR with
broad-range primers has been reported . A technical
limitation of the current method is the incapability to clearly
identify the genotypes in these scenarios. Hence, among the
clinical samples we analyzed in this study, the HPV genotypes for
7 HPV-positive samples that are infected with multiple HPV
genotypes can not be identified by HRM analysis. However, the
distinguished HRM patterns provide a hint that multiple infections
may occur that required further identification with other methods.
In conclusion, the molecular signatures from HRM analysis of
the broad-range real-time PCR products are useful for detection
and genotyping of HPV infection. This approach can be extended
further to cover all the 13 high-risk HPV genotypes. With an
appropriate HRM molecular signatures database, this method
should allow rapid and cost-effective differentiation of HPV
amplification of HPV DNA. A–C. Serial dilution of the
Effects of human genomic DNA on the
plasmid harboring the L1 fragment of HPV18 (panel A and C) or
HPV68 (panel B and C) corresponding to the indicated copy
number of template DNA were subject to amplification by
asymmetric broad-range real-time PCR in the presence or absence
of 20 ng of human genomic DNA. The copy numbers of the
template DNA were plotted against Ct values.
The genotypes for the samples with multiple
We would like to thank Drs. Chyong-Huey Lai and Chung-Ming Chang
for their support and helpful discussion in this study.
Conceived and designed the experiments: THL CPT JTQ. Performed the
experiments: THL. Analyzed the data: THL TSW CPT JTQ. Contributed
reagents/materials/analysis tools: TSW CPT JTQ. Wrote the paper: THL
TSW CPT JTQ.
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