Abstract
BackgroundWe 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.ResultsAmplification 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 × 102-2.5 × 106 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.ConclusionsOur 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.
Highlights
We have developed an ultrasensitive method based on conventional PCR preamplification followed by nested amplification through real time PCR in the presence of the DNA intercalating agent EvaGreen
In this work we developed an ultrasensitive method to quantify E6-human papillomavirus type 16 (HPV16) oncogene sequences in two steps: 1) preamplification of the E6-1 (645 bp) sequence through 15 cycles of direct conventional PCR, and 2) amplification of the E6-2 (237 bp) sequence through nested qPCR in the presence of EvaGreen
The families of type curves required to calibrate the number of E6 copies and to quantify them in problem samples could be generated from nested qPCR mixtures derived from two types of preamplification mixtures: a) "complete" mixtures, prepared with serial logarithmic dilutions of the pHV101 template molecules before preamplification, and b) "minimum" mixtures, prepared with serial logarithmic dilutions from a single preamplified mixture containing the maximum number of template molecules used in complete mixtures
Summary
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. Invasive cervical cancer develops in women with persistent infection by high-risk human papillomavirus (HRHPV), among which HPV16 and HPV18 are the most frequent types [1], HPV16 being the most prevalent and associated with nearly half of the invasive cervical cancer cases in the world [2,3]
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