Sentinel-base DNA genotyping using multiple sequencing primers for high-risk human papillomaviruses

Abstract

Despite the various technologies in place for genotyping human papillomaviruses (HPV), clinical use and clinical research demand a method that is fast, more reliable and cost-effective. The technology described here represents a breakthrough development in that direction. By combining the method of multiple sequencing primers with DNA sequencing, we have developed a rapid assay for genotyping HPV that relies on the identification of a single, type-specific 'sentinel' base. As described here, the prototype assay has been developed to recognize the 12 most high-risk HPV types (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59) and is capable of recognizing and simultaneously genotyping multiple HPV co-infections. By providing sequence information on multiple HPV infections, this method eliminates the need for labor- and cost-intensive PCR cloning. These proof-of-concept studies establish the assay to be accurate, reliable, rapid, flexible, and cost-effective, providing evidence of the feasibility this technique for use in clinical settings.

Does HPV-status 6–12 months after treatment of high grade dysplasia in the uterine cervix predict long term recurrence?

“…Among the women infected with HPV we found a high proportion of multiple infections (34%). This is in concordance with other results with the same basic techniques 19 and studies with other sensitive HPV-typing methods.20…”

“…18 Twelve typespecific sequencing primers for the high risk HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58 and -59 were designed. 19 The minimum detection limit was found to be 0.154 pmol of PCR product. Sequencing by pyrosequencing technology as described earlier 18 was performed on an automated plate-based bench-top PSQ™ HS96A System.…”

Pyrosequencing-An Alternative to Traditional Sanger Sequencing

“…Currently, pyrosequencing method is broadly being used in many applications such as Single Nucleotide Polymorphism (SNP) genotyping (Ahmadian et al, 2000a;Nordstrom et al, 2000;Milan et al, 2000), identification of bacteria (Gharizadeh, 2003;Grahn et al, 2003;Jonasson et al, 2002), fungal (Gharizadeh et al, 2005;Trama et al, 2005) and viral typing (Gharizadeh et al, 2001;. Moreover, the method has demonstrated the ability to determine difficult secondary structures (Ronaghi, 2001) and perform mutation detection (Ahmadian et al, 2000b;Garcia et al, 2000), DNA methylation analysis (Neve et al, 2002;Uhlmann et al, 2002), multiplex sequencing (Gharizadeh et al, 2003a;2006) (Gharizadeh et al, 2003b;Gharizadeh et al, 2006), tag sequencing of cDNA library (Nordstrom et al, 2001) and clone checking (Nourizad et al, 2003). Another highly significant application is whole genome sequencing (Margulies et al, 2005).…”

Pyrosequencing Technology for Short DNA Sequencing and Whole Genome Sequencing

“…Currently, the Pyrosequencing method is broadly being applied for many applications such as SNP genotyping Moreover, the method has shown the ability for determination of difficult secondary structures 24) , mutation detection 25), 26) , DNA methylation analysis 27)- 29) , multiplex sequencing 30), 31) , tag sequencing of cDNA library 7) and clone checking 32) .…”