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5’-Nuclease PCR Assays for Fo
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2010-08-11
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Introduction
Foodborne disease is estimated to cause 76 million illnesses and 5,000 deaths
annually in the United States [1]. Of the known pathogens that cause foodborne
illness, , O157:H7, , and are
among the short list of micro-organisms that exert the largest disease burden.
Bacteriological culturing has traditionally been used to detect microbial patho-
gens in food, and this technique is still in widespread use today. However, the
time-consuming and low-throughput nature of culture-based detection methods
warrant their replacement by more rapid methods based on biomolecular
analysis, like the polymerase chain reaction (PCR). PCR, predicated on DNA
synthesis and amplification, is an established, core technique in molecular biol-
ogy, microbiology, diagnostics, genetics and environmental science applications.
A derivative of PCR, real-time quantitative PCR (Q-PCR) kinetically monitors the
reaction in ‘real time’. Q-PCR confers significant advantages over conventional
PCR, including increased reaction speed, sensitivity and specificity; it also
eliminates the need to open the reaction tubes for post-PCR analysis, thus
preventing cross-contamination. Q-PCR is a fluorogenic probe-based 5’-nuclease
assay that provides a higher level of specificity than conventional PCR, thus
assuring the correct DNA target is amplified, and it is the preferred method to
Foodborne disease is estimated to cause 76 million illnesses and 5,000 deaths
annually in the United States [1]. Of the known pathogens that cause foodborne
illness, , O157:H7, , and are
among the short list of micro-organisms that exert the largest disease burden.
Bacteriological culturing has traditionally been used to detect microbial patho-
gens in food, and this technique is still in widespread use today. However, the
time-consuming and low-throughput nature of culture-based detection methods
warrant their replacement by more rapid methods based on biomolecular
analysis, like the polymerase chain reaction (PCR). PCR, predicated on DNA
synthesis and amplification, is an established, core technique in molecular biol-
ogy, microbiology, diagnostics, genetics and environmental science applications.
A derivative of PCR, real-time quantitative PCR (Q-PCR) kinetically monitors the
reaction in ‘real time’. Q-PCR confers significant advantages over conventional
PCR, including increased reaction speed, sensitivity and specificity; it also
eliminates the need to open the reaction tubes for post-PCR analysis, thus
preventing cross-contamination. Q-PCR is a fluorogenic probe-based 5’-nuclease
assay that provides a higher level of specificity than conventional PCR, thus
assuring the correct DNA target is amplified, and it is the preferred method to
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