Title:
METHOD FOR QUANTITATIVE END-POINT PCR
Document Type and Number:
WIPO Patent Application WO2004035835
Kind Code:
A3
Abstract:
The present invention provides a sensitive and robust analytical approach to identifying and quantifying multiple pathogens within a single complex environmental sample. Numerous nucleic acid signatures may be screened and quantified within a single reaction tube using polymerase chain reaction (PCR) and chromatographically analyzing the amplification products with microchannel fluidic (e.g. Agilent 2100 Bioanalyzer, or Caliper AMS-90) or reverse-phase ion-pairing high-performance liquid chromatography RP IP HPLC (e.g. Transgenomic WAVE) instruments. The method may be employed in a multiplex fashion to allow identification and quantification of multiple combinations of up to five different nucleic acid signatures simultaneously within a single multiplex PCR reaction tube. This approach is quantitative across a dynamic range of up to five orders of magnitude. This method is suitable for target nucleic acid analysis in medium or high-throughput contexts such as routine clinical diagnotics or environmental monitoring. The method is also suitable for pathogen monitoring or surveillance in a biodefense context.
Inventors:
SWIGER ROY R
EMMERLING ERIC J
EMMERLING ERIC J
Application Number:
PCT/US2003/033183
Publication Date:
May 27, 2004
Filing Date:
October 17, 2003
Export Citation:
Assignee:
MIDWEST RES INST INC (US)
International Classes:
C12Q1/68; (IPC1-7): C12Q1/68
Foreign References:
| US5858658A | 1999-01-12 | |||
| US5219727A | 1993-06-15 |
Other References:
MARLOWE E M ET AL: "A METHOD FOR THE DETECTION AND QUANTITATION OF PCR TEMPLATE IN ENVIRONMENTAL SAMPLES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY", JOURNAL OF MICROBIOLOGICAL METHODS, ELSEVIER, AMSTERDAM,, NL, vol. 28, no. 1, 1997, pages 45 - 53, XP002918644, ISSN: 0167-7012
HAYWARD-LESTER A ET AL: "RAPID QUANTIFICATION OF GENE EXPRESSION BY COMPETITIVE RT-PCR AND ION-PAIR REVERSED PHASE HPLC", BIOTECHNIQUES, EATON PUBLISHING, NATICK, US, vol. 20, no. 2, February 1996 (1996-02-01), pages 250 - 257, XP001041581, ISSN: 0736-6205
DORIS P A ET AL: "Quantitative analysis of gene expression by ion-pair high-performance liquid chromatography", JOURNAL OF CHROMATOGRAPHY A, ELSEVIER SCIENCE, NL, vol. 806, no. 1, 8 May 1998 (1998-05-08), pages 47 - 60, XP004121167, ISSN: 0021-9673
REISCHL U ET AL: "QUANTITATIVE PCR A SURVEY OF THE PRESENT TECHNOLOGY", MOLECULAR BIOTECHNOLOGY, TOTOWA, NJ, US, vol. 3, 1995, pages 55 - 71, XP000600241, ISSN: 1073-6085
HENNINGER H-P ET AL: "PURIFICATION AND QUANTITATIVE ANALYSIS OF NUCLEIC ACIDS BY ANION-EXCHANGE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY", BIOLOGICAL CHEMISTRY HOPPE-SEYLER, WALTER DE GRUYTER, BERLIN, DE, vol. 374, no. 8, 1 August 1993 (1993-08-01), pages 625 - 634, XP000675214, ISSN: 0177-3593
WILLIAMS S J ET AL: "QUANTITATIVE COMPETITIVE POLYMERASE CHAIN REACTION: ANALYSIS OF AMPLIFIED PRODUCTS OF THE HIV-1 GAG GENE BY CAPILLARY ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION", ANALYTICAL BIOCHEMISTRY, ACADEMIC PRESS, SAN DIEGO, CA, US, vol. 236, no. 1, 5 April 1996 (1996-04-05), pages 146 - 152, XP000582427, ISSN: 0003-2697
DATABASE MEDLINE [online] US NATIONAL LIBRARY OF MEDICINE (NLM), BETHESDA, MD, US; 20 July 1990 (1990-07-20), KATZ E D ET AL: "Rapid separation, quantitation and purification of products of polymerase chain reaction by liquid chromatography.", XP002275638, Database accession no. NLM2229237
HAYWARD-LESTER A ET AL: "RAPID QUANTIFICATION OF GENE EXPRESSION BY COMPETITIVE RT-PCR AND ION-PAIR REVERSED PHASE HPLC", BIOTECHNIQUES, EATON PUBLISHING, NATICK, US, vol. 20, no. 2, February 1996 (1996-02-01), pages 250 - 257, XP001041581, ISSN: 0736-6205
DORIS P A ET AL: "Quantitative analysis of gene expression by ion-pair high-performance liquid chromatography", JOURNAL OF CHROMATOGRAPHY A, ELSEVIER SCIENCE, NL, vol. 806, no. 1, 8 May 1998 (1998-05-08), pages 47 - 60, XP004121167, ISSN: 0021-9673
REISCHL U ET AL: "QUANTITATIVE PCR A SURVEY OF THE PRESENT TECHNOLOGY", MOLECULAR BIOTECHNOLOGY, TOTOWA, NJ, US, vol. 3, 1995, pages 55 - 71, XP000600241, ISSN: 1073-6085
HENNINGER H-P ET AL: "PURIFICATION AND QUANTITATIVE ANALYSIS OF NUCLEIC ACIDS BY ANION-EXCHANGE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY", BIOLOGICAL CHEMISTRY HOPPE-SEYLER, WALTER DE GRUYTER, BERLIN, DE, vol. 374, no. 8, 1 August 1993 (1993-08-01), pages 625 - 634, XP000675214, ISSN: 0177-3593
WILLIAMS S J ET AL: "QUANTITATIVE COMPETITIVE POLYMERASE CHAIN REACTION: ANALYSIS OF AMPLIFIED PRODUCTS OF THE HIV-1 GAG GENE BY CAPILLARY ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION", ANALYTICAL BIOCHEMISTRY, ACADEMIC PRESS, SAN DIEGO, CA, US, vol. 236, no. 1, 5 April 1996 (1996-04-05), pages 146 - 152, XP000582427, ISSN: 0003-2697
DATABASE MEDLINE [online] US NATIONAL LIBRARY OF MEDICINE (NLM), BETHESDA, MD, US; 20 July 1990 (1990-07-20), KATZ E D ET AL: "Rapid separation, quantitation and purification of products of polymerase chain reaction by liquid chromatography.", XP002275638, Database accession no. NLM2229237
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