Homogeneous Amplification of Genital Human Alpha Papillomaviruses by PCR Using Novel Broad-Spectrum GP5+ and GP6+ Primers

Abstract

Human papillomavirus (HPV) DNA detection and typing are important for diagnosis and management of HPV-associated diseases. One of the most commonly used PCR methods, GP5؉/6؉, shows weaknesses in amplifying certain types. To circumvent this limitation, we developed and validated broad-spectrum primers targeting the GP5؉/6؉ region. The addition of eight upstream and two downstream BSGP5؉/6؉ (BS) primers improved amplification of plasmids of 14 genital HPV types 10-to 1,000-fold versus GP5؉/6؉ PCR without altering sensitivity for the 10 others. For these 24 types, an analytic sensitivity of <1,000 plasmid copies in the presence of 100 ng cellular DNA was obtained. Additionally, we integrated an internal ␤-globin PCR into both HPV PCR systems, allowing simultaneous DNA quality control without affecting the sensitivity of HPV detection. Furthermore, we describe five additional low-risk HPV probes used in multiplex HPV genotyping (MPG) for simultaneous identification of all 15 high-risk, 3 putative high-risk, and 9 low-risk HPV genotypes. The performance of BSGP5؉/6؉ multiplexed with ␤-globin primers was compared to that of standard GP5؉/6؉ with DNA from 1,112 cervical scrapings. There was 79% overall agreement (kappa ‫؍‬ 0.816). BSGP5؉/6؉ was significantly more sensitive than GP5؉/6؉ for detection of HPV 30, 39, 42, 44, 51, 52, 53, 68, 73, and 82, detecting 212 additional HPV infections and increasing the proportion of multiple infections from 17.2 to 26.9% in cancer patients. In conclusion, BSGP5؉/6؉ multiplexed with ␤-globin PCR provides an improvement in type-specific amplification sensitivity and homogeneity compared to GP5؉/6؉ and offers simultaneous internal control of DNA quality. BSGP5؉/6؉-MPG, therefore, is suitable for epidemiologic and also diagnostic applications.High-risk human papillomavirus (HR HPV) types are causally associated with several malignant diseases, of which cervical cancer has particular significance, being the second most common cancer in women worldwide (11). Until now, more than 100 HPV genotypes are fully characterized based on the isolation of complete genomes, with evidence that a larger number exist (6). There are 48 known mucosal HPV types, which are further divided into three groups based on their epidemiological association with cancer: HR types, including types 16,18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82; putative HR (pHR) types, including types 26, 53, and 66; and low-risk (LR) types, including types 6, 11, 40, 42, 43, 44, and 70 (11). HPV genotyping is of particular importance for studying the natural history of HPV infections and the diseases associated therewith, for monitoring vaccine efficacy, and for identifying novel HPV candidates for vaccine development. Further, it is required to diagnose multiple HPV infections.At present, there are several PCR-based HPV detection methods; one of the most commonly applied uses the GP5ϩ/6ϩ (GP) primer set (18), which targets conserved sequences within the L1 region of the virus genome flanking highly variable type...

Biological activity of probable/possible high‐risk human papillomavirus types in cervical cancer

“…In our study, two highly sensitive PCR methods targeting two different regions in the viral genomes (L1 and E7) of all HR and seven pHR types, were used to identify HPV types; [32][33][34] to our knowledge, the most meticulous analysis published so far. Both BS-L1-MPG and TS-E7-MPG showed to be highly suitable and sensitive assays applicable to FFPE materials in our study.…”

“…[32][33][34] Analytic sensitivity of BS-L1-MPG reached 10-1,000 HPV copies per PCR for each HPV type. BS-L1-MPG was performed as described with some modifications.…”

“…BS-L1-MPG was performed as described with some modifications. 33 Briefly, DNA was amplified with the Multiplex PCR Kit (Qiagen, Hilden, Germany). Each 0.2-0.5 lM of the BSGP5þ and 5 0 -biotinylated BSGP6þ primers and 0.15 lM each of the b-globin primers MS3 and 5 0 -biotinylated MS10 were added to the PCR mixture.…”

“…34,35 The multiplex type-specific E7 PCR utilizes HPV type-specific primers targeting the E7 region for the detection of 19 HR-/pHR (HPV16, 18,26,31,33,35,39,45, 51, 52, 53, 56, 58, 59, 66, 68a, 68b, 70, 73, 82) and 2 LRtypes (HPV6, 11), with detection limits ranging from 10 to 1,000 copies of the viral genome. 34 Two primers for the amplification of b-globin were also included to provide a positive control for the quality of the template DNA.…”

“…1,2 Epidemiological studies have demonstrated that 20 mucosal HPV types from five phylogenetically related species of the genus alpha (a5, a6, a7, a9 and a11-termed as ''high-risk clade'') are consistently found as single HPV infections in CxCa worldwide. [3][4][5][6] Based on their frequency in CxCa and available biological data, 12 of these types, i.e., types 16,18,31,33,35,39, 45, 51, 52, 56, 58 and 59 have been defined as carcinogens (IARC Group 1A)-hereafter referred to as high-risk (HR)-HPV. For eight other types, in the high-risk clade, i.e., types 26, 53, 66, 67, 68, 70, 73 and 82, the combination of their low frequency, lack of data on their active transcription and their transforming potential in model systems, has led them to be classified as only probable/possible carcinogens (IARC Groups 2A and 2B)-hereafter referred to as possible highrisk (pHR)-HPV types.…”