@article{koo_jaykus_2003, title={Detection of Listeria monocytogenes from a model food by fluorescence resonance energy transfer-based PCR with an asymmetric fluorogenic probe set}, volume={69}, ISSN={["0099-2240"]}, DOI={10.1128/AEM.69.2.1082-1088.2003}, abstractNote={ABSTRACT It has been shown that fluorescence resonance energy transfer (FRET)-based PCR, including the TaqMan assay and molecular beacons, has potential for rapid detection of pathogens. In these promising real-time detection assays a single internal oligonucleotide probe labeled on both the 5′ (reporter) and 3′ (quencher) ends is used for selective generation of fluorescence. In this paper, we describe the use of a previously reported novel probe design for FRET-based PCR detection of Listeria monocytogenes in pure culture and in a model food commodity. In the assay described here an asymmetric probe set is used; this probe set consists of a long 5′ fluorescein-labeled reporter probe and a short, complementary 3′ DABCYL-labeled quencher oligonucleotide, which are used in a 5′ nuclease amplification and detection assay. By using the listeriolysin O ( hly ) and p60 ( iap ) genes as amplification targets, the performance of two primer-probe sets in amplification and subsequent detection of target DNA was evaluated. In studies performed with pure cultures of L. monocytogenes , the PCR profiles indicated that the relative change in fluorescence intensity was correlated with both the initial number of cells and the accumulation of specific amplicons for both hly and iap gene fragments. Experiments were also done to determine the applicability of the method to the detection of L. monocytogenes by targeting hly DNA and its short-lived mRNA product in a model food commodity. Twenty-five-milliliter samples of reconstituted nonfat dry milk (NFDM) were seeded with L. monocytogenes and processed to concentrate the bacteria by centrifugation, and this was followed by nucleic acid extraction and amplification with hly -specific primers. Endpoint detection of PCR and reverse transcription-PCR amplicons could be achieved at inoculum levels of 10 3 and 10 4 CFU of L. monocytogenes /25 ml of NFDM, respectively. This study demonstrated that this asymmetric FRET-based amplification and detection protocol provides an alternative approach for endpoint detection of nucleic acid amplification products as applied to detection of pathogens in a model food.}, number={2}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Koo, K and Jaykus, LA}, year={2003}, month={Feb}, pages={1082–1088} }
 @article{koo_jaykus_2002, title={Detection of single nucleotide polymorphisms within the Listeria genus using an 'asymmetric' fluorogenic probe set and fluorescence resonance energy transfer based-PCR}, volume={35}, ISSN={["0266-8254"]}, DOI={10.1046/j.1472-765X.2002.01232.x}, abstractNote={We describe a novel and inexpensive fluorescence energy transfer (FRET)-based PCR protocol to distinguish single nucleotide polymorphisms (SNPs) within the genus Listeria.Sequence information for the 16S rRNA gene of representative Listeria species was used to design genus-specific primers and two species-specific probes that differed in sequence by one single nucleotide. The probes were 5' labelled with either fluorescein or Texas Red, quenched with a shorter yet complementary 3' dimethyl-amino-phenyloazo benzoic acid (DABCYL) labelled oligonucleotide, and then incorporated into a previously reported 'asymmetric' FRET-based PCR detection protocol.Listeria monocytogenes could be readily distinguished from other members of the Listeria genus after PCR amplification and measurement of endpoint fluorescence at two different wavelengths.The relatively low cost and high flexibility of this system will benefit laboratories in their efforts to develop rapid and specific methods to detect minor sequence differences between related microorganisms.}, number={6}, journal={LETTERS IN APPLIED MICROBIOLOGY}, author={Koo, K and Jaykus, LA}, year={2002}, pages={513–517} }
 @article{koo_jaykus_2000, title={Modified method to detect PCR products by 5 ' nuclease activity and an asymmetric fluorogenic probe set}, volume={29}, ISSN={["0736-6205"]}, DOI={10.2144/00294bm03}, number={4}, journal={BIOTECHNIQUES}, author={Koo, K and Jaykus, LA}, year={2000}, month={Oct}, pages={690-+} }
 @article{koo_jaykus_2000, title={Selective amplification of bacterial RNA: use of a DNA primer containing mismatched bases near its 3 ' terminus to reduce false-positive signals}, volume={31}, ISSN={["0266-8254"]}, DOI={10.1046/j.1365-2672.2000.00798.x}, abstractNote={A reverse transcription PCR (RT-PCR) method designed to reduce false-positive results due to the co-amplification of contaminating genomic DNA is reported. Feasibility of the method was evaluated using 16S rRNA sequences specific to Bacillus cereus. A DNA oligonucleotide primer, consisting of 22-bases containing three consecutive mismatched bases near its 3' terminus (primer B16RT), was used for reverse transcription and in subsequent cDNA amplification. Specific rRNA was reverse transcribed at low temperature (40 degrees C or 45 degrees C) in the presence of primer B16RT. As subsequent PCR using primer B16RT at high (62 degrees C) annealing temperatures is not nearly as efficient as amplification using the specific primer, amplification of genomic DNA was hindered relative to the amplification of cDNA. The method was readily adapted to the selective amplification of mRNA of the Listeria monocytogenes listeriolysin O (hly) gene.}, number={3}, journal={LETTERS IN APPLIED MICROBIOLOGY}, author={Koo, K and Jaykus, LA}, year={2000}, month={Sep}, pages={187–192} }