Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR

<p>Abstract</p> <p>Background</p> <p>We have developed an ultrasensitive method based on conventional PCR preamplification followed by nested amplification through real time PCR (qPCR) in the presence of the DNA intercalating agent EvaGreen.</p> <p>Results&l...

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Main Authors: López-Revilla Rubén, Hernández-Arteaga Socorro
Format: Article
Language:English
Published: BMC 2010-05-01
Series:Infectious Agents and Cancer
Online Access:http://www.infectagentscancer.com/content/5/1/9
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spelling doaj-140fdc91f88244d48bc653b8797f6e422020-11-25T00:58:03ZengBMCInfectious Agents and Cancer1750-93782010-05-0151910.1186/1750-9378-5-9Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCRLópez-Revilla RubénHernández-Arteaga Socorro<p>Abstract</p> <p>Background</p> <p>We have developed an ultrasensitive method based on conventional PCR preamplification followed by nested amplification through real time PCR (qPCR) in the presence of the DNA intercalating agent EvaGreen.</p> <p>Results</p> <p>Amplification mixtures calibrated with a known number of pHV101 copies carrying a 645 base pair (bp)-long insert of the human papillomavirus type 16 (HPV16) E6 oncogene were used to generate the E6-1 amplicon of 645 bp by conventional PCR and then the E6-2 amplicon of 237 bp by nested qPCR. Direct and nested qPCR mixtures for E6-2 amplification corresponding to 2.5 × 10<sup>2</sup>-2.5 × 10<sup>6 </sup>initial pHV101 copies had threshold cycle (Ct) values in the ranges of 18.7-29.0 and 10.0-25.0, respectively. The Ct of qPCR mixtures prepared with 1/50 volumes of preamplified mixtures containing 50 ng of DNA of the SiHa cell line (derived from an invasive cervical cancer with one HPV16 genome per cell) was 19.9. Thermal fluorescence extinction profiles of E6-2 amplicons generated from pHV101 and SiHa DNA were identical, with a peak at 85.5°C.</p> <p>Conclusions</p> <p>Our method based on conventional preamplification for 15 cycles increased 10,750 times the sensitivity of nested qPCR for the quantitation of the E6 viral oncogene and confirmed that the SiHa cell line contains one E6-HPV16 copy per cell.</p> http://www.infectagentscancer.com/content/5/1/9
collection DOAJ
language English
format Article
sources DOAJ
author López-Revilla Rubén
Hernández-Arteaga Socorro
spellingShingle López-Revilla Rubén
Hernández-Arteaga Socorro
Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR
Infectious Agents and Cancer
author_facet López-Revilla Rubén
Hernández-Arteaga Socorro
author_sort López-Revilla Rubén
title Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR
title_short Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR
title_full Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR
title_fullStr Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR
title_full_unstemmed Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR
title_sort ultrasensitive quantitation of human papillomavirus type 16 e6 oncogene sequences by nested real time pcr
publisher BMC
series Infectious Agents and Cancer
issn 1750-9378
publishDate 2010-05-01
description <p>Abstract</p> <p>Background</p> <p>We have developed an ultrasensitive method based on conventional PCR preamplification followed by nested amplification through real time PCR (qPCR) in the presence of the DNA intercalating agent EvaGreen.</p> <p>Results</p> <p>Amplification mixtures calibrated with a known number of pHV101 copies carrying a 645 base pair (bp)-long insert of the human papillomavirus type 16 (HPV16) E6 oncogene were used to generate the E6-1 amplicon of 645 bp by conventional PCR and then the E6-2 amplicon of 237 bp by nested qPCR. Direct and nested qPCR mixtures for E6-2 amplification corresponding to 2.5 × 10<sup>2</sup>-2.5 × 10<sup>6 </sup>initial pHV101 copies had threshold cycle (Ct) values in the ranges of 18.7-29.0 and 10.0-25.0, respectively. The Ct of qPCR mixtures prepared with 1/50 volumes of preamplified mixtures containing 50 ng of DNA of the SiHa cell line (derived from an invasive cervical cancer with one HPV16 genome per cell) was 19.9. Thermal fluorescence extinction profiles of E6-2 amplicons generated from pHV101 and SiHa DNA were identical, with a peak at 85.5°C.</p> <p>Conclusions</p> <p>Our method based on conventional preamplification for 15 cycles increased 10,750 times the sensitivity of nested qPCR for the quantitation of the E6 viral oncogene and confirmed that the SiHa cell line contains one E6-HPV16 copy per cell.</p>
url http://www.infectagentscancer.com/content/5/1/9
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AT hernandezarteagasocorro ultrasensitivequantitationofhumanpapillomavirustype16e6oncogenesequencesbynestedrealtimepcr
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