Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR
Background 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. Results Amplification mixtures calibrated with a known number of pHV101 copies carry...
Ausführliche Beschreibung
Autor*in: |
Hernández-Arteaga, Socorro [verfasserIn] |
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E-Artikel |
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Sprache: |
Englisch |
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2010 |
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Anmerkung: |
© Hernández-Arteaga and López-Revilla; licensee BioMed Central Ltd. 2010 |
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Übergeordnetes Werk: |
Enthalten in: Infectious agents and cancer - London : Biomed Central, 2006, 5(2010), 1 vom: 14. Mai |
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Übergeordnetes Werk: |
volume:5 ; year:2010 ; number:1 ; day:14 ; month:05 |
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DOI / URN: |
10.1186/1750-9378-5-9 |
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Katalog-ID: |
SPR029518628 |
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520 | |a Background 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. Results 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^{2} $-2.5 × $ 10^{6} $ 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. Conclusions 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. | ||
650 | 4 | |a Invasive Cervical Cancer |7 (dpeaa)DE-He213 | |
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10.1186/1750-9378-5-9 doi (DE-627)SPR029518628 (SPR)1750-9378-5-9-e DE-627 ger DE-627 rakwb eng Hernández-Arteaga, Socorro verfasserin aut Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR 2010 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Hernández-Arteaga and López-Revilla; licensee BioMed Central Ltd. 2010 Background 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. Results 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^{2} $-2.5 × $ 10^{6} $ 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. Conclusions 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. Invasive Cervical Cancer (dpeaa)DE-He213 SiHa Cell (dpeaa)DE-He213 HPV16 Genome (dpeaa)DE-He213 SiHa Cell Line (dpeaa)DE-He213 Invasive Cervical Cancer Case (dpeaa)DE-He213 López-Revilla, Rubén aut Enthalten in Infectious agents and cancer London : Biomed Central, 2006 5(2010), 1 vom: 14. Mai (DE-627)51781403X (DE-600)2251117-9 1750-9378 nnns volume:5 year:2010 number:1 day:14 month:05 https://dx.doi.org/10.1186/1750-9378-5-9 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 5 2010 1 14 05 |
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10.1186/1750-9378-5-9 doi (DE-627)SPR029518628 (SPR)1750-9378-5-9-e DE-627 ger DE-627 rakwb eng Hernández-Arteaga, Socorro verfasserin aut Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR 2010 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Hernández-Arteaga and López-Revilla; licensee BioMed Central Ltd. 2010 Background 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. Results 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^{2} $-2.5 × $ 10^{6} $ 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. Conclusions 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. Invasive Cervical Cancer (dpeaa)DE-He213 SiHa Cell (dpeaa)DE-He213 HPV16 Genome (dpeaa)DE-He213 SiHa Cell Line (dpeaa)DE-He213 Invasive Cervical Cancer Case (dpeaa)DE-He213 López-Revilla, Rubén aut Enthalten in Infectious agents and cancer London : Biomed Central, 2006 5(2010), 1 vom: 14. Mai (DE-627)51781403X (DE-600)2251117-9 1750-9378 nnns volume:5 year:2010 number:1 day:14 month:05 https://dx.doi.org/10.1186/1750-9378-5-9 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 5 2010 1 14 05 |
allfields_unstemmed |
10.1186/1750-9378-5-9 doi (DE-627)SPR029518628 (SPR)1750-9378-5-9-e DE-627 ger DE-627 rakwb eng Hernández-Arteaga, Socorro verfasserin aut Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR 2010 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Hernández-Arteaga and López-Revilla; licensee BioMed Central Ltd. 2010 Background 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. Results 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^{2} $-2.5 × $ 10^{6} $ 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. Conclusions 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. Invasive Cervical Cancer (dpeaa)DE-He213 SiHa Cell (dpeaa)DE-He213 HPV16 Genome (dpeaa)DE-He213 SiHa Cell Line (dpeaa)DE-He213 Invasive Cervical Cancer Case (dpeaa)DE-He213 López-Revilla, Rubén aut Enthalten in Infectious agents and cancer London : Biomed Central, 2006 5(2010), 1 vom: 14. Mai (DE-627)51781403X (DE-600)2251117-9 1750-9378 nnns volume:5 year:2010 number:1 day:14 month:05 https://dx.doi.org/10.1186/1750-9378-5-9 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 5 2010 1 14 05 |
allfieldsGer |
10.1186/1750-9378-5-9 doi (DE-627)SPR029518628 (SPR)1750-9378-5-9-e DE-627 ger DE-627 rakwb eng Hernández-Arteaga, Socorro verfasserin aut Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR 2010 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Hernández-Arteaga and López-Revilla; licensee BioMed Central Ltd. 2010 Background 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. Results 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^{2} $-2.5 × $ 10^{6} $ 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. Conclusions 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. Invasive Cervical Cancer (dpeaa)DE-He213 SiHa Cell (dpeaa)DE-He213 HPV16 Genome (dpeaa)DE-He213 SiHa Cell Line (dpeaa)DE-He213 Invasive Cervical Cancer Case (dpeaa)DE-He213 López-Revilla, Rubén aut Enthalten in Infectious agents and cancer London : Biomed Central, 2006 5(2010), 1 vom: 14. Mai (DE-627)51781403X (DE-600)2251117-9 1750-9378 nnns volume:5 year:2010 number:1 day:14 month:05 https://dx.doi.org/10.1186/1750-9378-5-9 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 5 2010 1 14 05 |
allfieldsSound |
10.1186/1750-9378-5-9 doi (DE-627)SPR029518628 (SPR)1750-9378-5-9-e DE-627 ger DE-627 rakwb eng Hernández-Arteaga, Socorro verfasserin aut Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR 2010 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Hernández-Arteaga and López-Revilla; licensee BioMed Central Ltd. 2010 Background 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. Results 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^{2} $-2.5 × $ 10^{6} $ 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. Conclusions 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. Invasive Cervical Cancer (dpeaa)DE-He213 SiHa Cell (dpeaa)DE-He213 HPV16 Genome (dpeaa)DE-He213 SiHa Cell Line (dpeaa)DE-He213 Invasive Cervical Cancer Case (dpeaa)DE-He213 López-Revilla, Rubén aut Enthalten in Infectious agents and cancer London : Biomed Central, 2006 5(2010), 1 vom: 14. Mai (DE-627)51781403X (DE-600)2251117-9 1750-9378 nnns volume:5 year:2010 number:1 day:14 month:05 https://dx.doi.org/10.1186/1750-9378-5-9 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 5 2010 1 14 05 |
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Hernández-Arteaga, Socorro |
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Hernández-Arteaga, Socorro misc Invasive Cervical Cancer misc SiHa Cell misc HPV16 Genome misc SiHa Cell Line misc Invasive Cervical Cancer Case Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR |
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Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR Invasive Cervical Cancer (dpeaa)DE-He213 SiHa Cell (dpeaa)DE-He213 HPV16 Genome (dpeaa)DE-He213 SiHa Cell Line (dpeaa)DE-He213 Invasive Cervical Cancer Case (dpeaa)DE-He213 |
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ultrasensitive quantitation of human papillomavirus type 16 e6 oncogene sequences by nested real time pcr |
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Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR |
abstract |
Background 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. Results 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^{2} $-2.5 × $ 10^{6} $ 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. Conclusions 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. © Hernández-Arteaga and López-Revilla; licensee BioMed Central Ltd. 2010 |
abstractGer |
Background 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. Results 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^{2} $-2.5 × $ 10^{6} $ 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. Conclusions 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. © Hernández-Arteaga and López-Revilla; licensee BioMed Central Ltd. 2010 |
abstract_unstemmed |
Background 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. Results 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^{2} $-2.5 × $ 10^{6} $ 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. Conclusions 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. © Hernández-Arteaga and López-Revilla; licensee BioMed Central Ltd. 2010 |
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Results 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^{2} $-2.5 × $ 10^{6} $ 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. Conclusions 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.</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Invasive Cervical Cancer</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">SiHa Cell</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">HPV16 Genome</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">SiHa Cell Line</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Invasive Cervical Cancer Case</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">López-Revilla, Rubén</subfield><subfield code="4">aut</subfield></datafield><datafield tag="773" ind1="0" ind2="8"><subfield code="i">Enthalten in</subfield><subfield code="t">Infectious agents and cancer</subfield><subfield code="d">London : Biomed Central, 2006</subfield><subfield code="g">5(2010), 1 vom: 14. 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