Quantitation of site-specific HPV 16 DNA methylation by pyrosequencing.

Human papillomavirus (HPV) is a necessary but insufficient cause of cervical cancer. Factors influencing transcription, such as epigenetic silencing through viral DNA methylation, may impact neoplastic progression. Pyrosequencing technology was applied to quantify methylation at 19 cytosine guanine dinucleotide (CpG) sites in the L1 3' and long control region (LCR) of HPV 16 DNA using cell lines, CaSki ( approximately 400 integrated copies of HPV 16) and SiHa (1-2 integrated copies of HPV 16) that differ in their transcriptional activity. Methylation levels ranged from 20 to 100% in CaSki and from 0 to 85% in SiHa over the entire 19 CpG sites, with a >40-fold difference in the methylation levels of their promoter and enhancer regions (SiHa<2% and CaSki 79%). The method was successful at a limiting dilution of 1-4 HPV 16 DNA copies/3000 cells, a level compatible with most clinical samples. The results were not affected by fixation in methanol-based liquid cytology collection fluid or method of extraction. Conditions optimized with cell lines were applicable to fixed exfoliated cervical cells. Pyrosequencing provides a quantitative site-specific assessment of methylation at multiple CpG sites without cloning, and is thus suited to large-scale molecular epidemiologic studies.

Human papillomavirus type 16 variant assignment by pyrosequencing.

Polymorphisms in human papillomavirus type 16 (HPV16) result in variants from the prototype sequence which can be designated according to geographic distribution and are broadly classified as European (E), African (Af), Asian (As), or Asian-American (AA). Detection of variants has been used to distinguish persistent HPV16 infection from re-infection in natural history studies, and variants have been associated with an increased risk of cervical disease in some populations. Variant determination usually relies on conventional Sanger sequencing of regions of the viral genome, with the major variant group assignments requiring the sequencing of only seven polymorphic sites spread over a 242-bp region of the E6 gene. We applied pyrosequencing to facilitate rapid sequencing and enable the simultaneous detection of multiple variants. A single-stranded template for pyrosequencing was prepared by amplifying a 314-bp fragment (nt 75-388) with a biotin at the 5'-end of the reverse primer to facilitate strand separation and purification. Polymorphisms at the nucleotide sites 109, 131, 132, 143, 145, 178 and 350 were determined in three separate sequencing reactions, one of which was a multiplex format. Pyrosequencing of 97 HPV16-positive exfoliated cervical samples confirmed the Sanger sequencing results; however pyrosequencing identified additional variants in several samples containing mixed variants.

Detection and differentiation of Cryptosporidium parasites that are pathogenic for humans by real-time PCR.

Cryptosporidiosis is a significant cause of food-borne and waterborne outbreaks of diarrheal diseases. To better understand the route of transmission of Cryptosporidium parasites, a number of genotyping techniques have been developed, based on PCR-restriction fragment length polymorphism or sequencing analysis of antigen, structural, and housekeeping genes. In this study, a real-time assay for the detection of Cryptosporidium oocysts is described. This technique had a detection limit of five oocysts. By melting curve analysis of PCR products with fluorescence-labeled hybridization probes, this technique was able to differentiate five common Cryptosporidium parasites that are pathogenic for humans in a single PCR. We evaluated and validated the test using samples from presently known Cryptosporidium parasites that are pathogenic for humans. This technique provides an alternative molecular tool in epidemiologic studies of human cryptosporidiosis.