Human papillomaviruses (HPV) are recognized as the causative agents of cervical cancer, its precursor lesions, and other anogenital cancers (1). Among more than 100 HPV types so far identified, nearly 40 types infecting the anogenital mucosa are classified as either low- or high-risk types on the basis of their oncogenic potentials (2). A previous large-scale case–control study revealed 15 high-risk types, HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82, which are closely linked to the development of cervical cancer, with HPV16 the predominant high-risk type worldwide (3). In contrast, low-risk HPV types, including HPV6 and 11, are associated almost exclusively with benign lesions. Due to the lack of a cell culture system to isolate HPV from clinical samples, detection of HPV-DNA is the only reliable means for diagnosis of HPV infection. HPV genotyping is of particular importance for understanding the natural history of HPV infection and management of cervical cancers. In addition, with the worldwide introduction of HPV vaccines that target the two prominent high-risk types, HPV16 and 18, there is a growing demand for reliable and practical HPV genotyping to monitor HPV prevalence and vaccine efficacy at both individual and population levels.
Various molecular techniques have been developed for detection of HPV-DNA, most of which rely on amplification of HPV-DNA by PCR. The PCR of HPV-DNA generally utilizes degenerate/consensus primer systems, such as MY09/11 (4), PGMY09/11 (5), GP5+/6+ (6), or SPF (7), all of which are designed to amplify the L1 region of the HPV genome. For HPV genotyping, PCR is followed by sequence analysis, restriction fragment length polymorphism analysis, or hybridization with type-specific oligonucleotide probes by a membrane-based RLB assay. Of the various HPV genotyping assays, the RLB assay has the advantage of being able to detect multiple HPV-type infections with greater sensitivity. Several RLB assays combined with different PCR schemes have been established and used for HPV research and diagnostic purposes (8–10). However, the RLB assays are relatively laborious, are limited to a maximum of about 40 samples per assay, and depend on visual read-out of the hybridization signal. To overcome these drawbacks, HPV genotyping using Luminex® suspension array technology has been developed (11–14). The Luminex®-based genotyping coupled with GP5+/6+ PCR allowed sensitive and specific genotyping of 27 mucosal HPV types in a 96-well plate format with a digital read-out (13). Moreover, a modified version of GP5+/6+ PCR was successfully introduced into the Luminex®-based assay, and showed improved sensitivity (15).
A VeraCode-ASPE method was first developed for the detection of SNP in the human genome (16) and has been applied to multiplex SNP genotyping on the Illumina BeadXpress® platform (17, 18). The ASPE primer is composed of two distinct regions: the 5′ region that contains the capture sequence, which is used in a subsequent hybridization reaction, and the 3′ region that contains the genomic target region with a SNP nucleotide at the extreme 3′ end. For SNP genotyping, the ASPE primer that matches the SNP nucleotide to the genome is extended by the primer extension reaction and is thus labeled with biotinylated nucleotides. After the primer extension, the products are mixed with VeraCode beads, so that the capture sequence on the primer hybridizes to its complementary sequence attached to the VeraCode beads. Labeling is then carried out with a streptavidin-fluorophore conjugate, followed by scanning and detection of the fluorescent signal using an Illumina BeadXpress® reader (Illumina Inc., San Diego, CA, USA).
In this work, the VeraCode-ASPE method on the Illumina BeadXpress® platform was evaluated for its suitability as a method to detect and genotype HPV-DNA (Fig. 1). The HPV-DNA amplified by PGMY-PCR was selected as a target for the VeraCode-ASPE genotyping, as PGMY-PCR has been validated as a sensitive and specific means for HPV-DNA amplification (19, 20). HPV-type-specific ASPE primers were designed to target the PCR amplicons of 16 HPV types (HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) in the 3′ region (Table 1), and with type-specific capture sequences in the 5′ region. The Tm values of the HPV-type-specific sequences, the lengths of which ranged from 19 to 28 bases, were adjusted to be between 54°C and 66°C using Primer3Plus software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) thus allowing similar annealing profiles.
|Type||DNA sequence (5′–3′)||Tm (°C)|
HPV-DNA, which was provided by the HPV laboratory network in the WHO as a quality-assured authentic panel for validation of HPV genotyping, was used to assess the sensitivity and specificity of the VeraCode-ASPE HPV genotyping. Fifty copies of HPV16- and 18-DNA and 500 copies of the other 14 HPV-type DNAs from the panel were subjected to PGMY-PCR with AmpliTaq Gold® polymerase (Applied Biosystems, Foster City, CA, USA) as described (21). One-third of the PCR products was treated with 2 U shrimp alkaline phosphatase and 5 U exonuclease I at 37°C for 45 min, followed by the ASPE reaction in a mixture containing 1× PCR buffer II (Roche, Indianapolis, IN, USA), 2.5 mM MgCl2, 5 μM of each dATP, dGTP and dTTP, 7.5 μM biotin-14-dCTP, 0.05 μM of each ASPE primer, 0.5 U AmpliTaq Gold® polymerase, with denaturation at 95°C for 10 min followed by 50 cycles of 94°C for 30 sec, 56°C for 30 sec, and 72°C for 45 sec. The reaction products were then incubated with the VeraCode bead mixture for 1 hr at 45°C in a VeraCode-bead plate, followed by staining with streptavidin-Alexa-647 in a buffer consisting of 3× standard saline citrate (SSC) and 0.1% Tween 20 for 15 min at room temperature. The VeraCode-bead plate was subjected to scanning by the BeadXpress® reader, and the read-out was expressed as the MFI obtained from each HPV type-assigned bead. As shown in Figure 2a, the 16 types of HPV-DNA were specifically detected with signals from their corresponding VeraCode beads. Signal values from non-target HPV-DNAs were as low as those from DNA-negative samples, and were classified as background noises. Furthermore, when the panel DNA containing a mixture of HPV-DNA was analyzed, corresponding signals from included HPV types were correctly detected (Fig. 2b), which indicates that VeraCode-ASPE typing is applicable to the simultaneous detection of multiple HPV-type DNAs.
To test the suitability of this assay for diagnostic purposes, DNA samples prepared from clinical specimens were analyzed by VeraCode-ASPE HPV genotyping. DNA was purified using the QIAamp® DNA blood kit (QIAGEN, Hilden, Germany) from cervical exfoliated cells that had been collected from outpatients with their informed consent for HPV genotyping. The study design was approved by the institutional review board of the NTT Medical Center, Tokyo. DNA samples were previously genotyped by PGMY-reverse blot hybridization (PGMY-RBH) assay, which had been validated as to be sensitive and specific for genotyping of the 16 HPV types in the studies of the WHO HPV-DNA proficiency panel (20). The same PGMY-PCR products derived from these DNA samples were subjected to VeraCode-ASPE HPV genotyping as carried out for the WHO HPV-DNA panel. A positive result was defined as a signal value more than three-fold the average background value for each HPV-type-specific VeraCode bead. Of 50 clinical samples analyzed by the VeraCode-ASPE assay, 20 samples gave HPV-positive results, whereas the remaining 30 samples were judged to be negative. Table 2 shows raw MFI data and typing results of the VeraCode-ASPE assay with 20 positive samples and one negative sample. Overall, the typing results were identical to those obtained by the PGMY-RBH assay, which strongly suggests that the VeraCode-ASPE assay can substitute for the reverse blot hybridization on the same platform of PGMY-PCR.
|DNA no.||HPV6 HPV45 Typing result||HPV11 HPV51||HPV16 HPV52||HPV18 HPV56||HPV31 HPV58||HPV33 HPV59||HPV35 HPV66||HPV39 HPV68|
|HPV16, 58, 68|
The principle of the allele-specific primer extension was previously used in tag-array-based HPV genotyping (22, 23); however, the array format of this assay hampers its application to high-throughput HPV genotyping. In contrast, the 96-well plate format of the VeraCode-ASPE method enables HPV genotyping for large amounts of clinical samples. Furthermore, there are a total of 144 different sets of VeraCode beads, and thus it is possible to include more HPV types in the VeraCode-ASPE genotyping format. In conclusion, the VeraCode-ASPE genotyping is a powerful new tool for the high-throughput HPV genotyping that will be required for large-scale surveillance of HPV-type distribution at the population level in the near future.