Validation of human papillomavirus genotyping by signature DNA sequence analysis

Abstract

Background: Screening with combined cytologic and HPV testing has led to the highest number of excessive colposcopic referrals due to high false positive rates of the current HPV testing in the USA. How best to capitalize on the enhanced sensitivity of HPV DNA testing while minimizing falsepositive results from its lower specificity is an important task for the clinical pathologists.

Detection of human papillomavirus (HPV) L1 gene DNA possibly bound to particulate aluminum adjuvant in the HPV vaccine Gardasil®

“…This HPV-11 DNA template does not exist in the environment and cannot come from any patient samples. In the author's laboratory serving a women population under the care of private gynecologists, HPV-18 is detected in about 6% of the routine HPV isolates [33], and about 80% of the HPV-18 isolates from the clinical samples belong to the European/Asian American subtype (author's unpublished data). All of the HPV DNA fragments detected in the vaccine samples other than those of the HPV-11 synthetic construct are of the African subtype of HPV-18.…”

“…A segment of 45-60 bases of the hypervariable region of the L1 gene sequence was excised from the computer-generated base-calling electropherogram for BLAST analyses for confirmation of the HPV DNA detected and to validate its genotype. The technical detail of this nested PCR/DNA sequencing methodology has been previously reported [30][31][32][33][34][35].…”

“…Short target sequence HPV genotyping was performed by direct automated cycle DNA sequencing [30][31][32][33][34][35]. Briefly, a trace of the positive nested PCR product was transferred directly with a micro-glass rod from the positive nested PCR tube into a 20 μL volume of a cycle sequencing reaction mixture consisting of 14.5 μL water, 3.5 μL of 5 X buffer, 1 μL of BigDye Terminator 1.1 (Applied Biosystems) and 1 μL of 10 μM GP6, or GP5 sequencing primer.…”

Detection of human papillomavirus L1 gene DNA fragments in postmortem blood and spleen after Gardasil<sup>®</sup> vaccination—A case report

“…As a result, a LoTemp ® PCR with a highly processive HiFi ® DNA polymerase system programmed at thermocycling steps not to exceed 85˚C was selected for this study. The general method used to detect HPV L1 gene DNA by heminested (nested) LoTemp ® PCR amplification with the GP/MY degenerate consensus primers and validation with direct automated DNA sequenceing for genotyping has been described in detail elsewhere for clinical samples [20][21][22][23][24][25] and for detecting residual HPV DNA fragments in the Gardasil ® vaccine [9]. Each primary PCR consisted of 1 μL of reconstituted DNA solution in molecular grade water containing about 0.5 μg human DNA, 2 μL of water, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, and 20 μL of ready-to-use LoTemp ® PCR master mix with HiFi ® DNA polymerase (www.hifidna.com) in a total volume of 25 μL.…”

Microfluidic vapor-diffusion barrier for pressure reduction in fully closed PCR modules

“…In addition, bubbles formed during heating of liquid are inherently separated by centrifugal buoyancy rendering the platform an ideal candidate for integrated PCR analysis 15,16 . We proofed the proper function of VDB's by applying them in a structure for PCR based pre-amplification, which is usually used to achieve lower detection limits in geometrically multiplexed PCR 31,32 or to enable the direct use of crude samples rendering nucleic acid purification redundant 33 . To demonstrate easy integration with further downstream processes, subsequently to the PCR amplification the reaction product is pumped radially inwards employing a centrifugo-dynamic pumping mechanism 34 .…”