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.

The variable-region genes of monoclonal antibody againstBacillus cereus spores were cloned from mouse hybridoma cells by reverse transcription-PCR. The heavy- and light-chain variable-region genes were connected by a 45-base linker DNA to allow folding of the fusion protein into a functional tertiary structure. For detection of protein expression, a 10-amino-acid strep tag (biotin-like peptide) was attached to the C terminus of recombinant antibody as the reporter peptide. The single-chain antibody construct was inserted into the expression vector and expressed in Escherichia coliunder the control of the T7 RNA polymerase-T7 promoter expression system. The expressed single-chain antibody was detected on Western blots by using a streptavidin-conjugated enzyme system. This small recombinant antibody fragment (ca. 28,000 Da by calculation) hadB. cereus spore binding ability and antigen specificity similar to those of its parent native monoclonal antibody.

Figure 1. Detection of PCR products using 5 ′ nuclease activity and asymmetric fluorogenic probe set.

A series of simple and phenol-free silica-based protocols for isolating and cleaning RNA or DNA fragments from different sources were developed. Cytoplasmic RNA isolated from hybridoma cells by this method was used in RT-PCR. DNA fragments obtained using this protocol were suitable for further subcloning, gene transformation and DNA sequencing.

. 2000. A reverse transcription PCR (RT±PCR) method designed to reduce false-positive results due to the co-ampli®cation of contaminating genomic DNA is reported. Feasibility of the method was evaluated using 16S rRNA sequences speci®c to Bacillus cereus. A DNA oligonucleotide primer, consisting of 22-bases containing three consecutive mismatched bases near its 3 H terminus (primer B16RT), was used for reverse transcription and in subsequent cDNA ampli®cation. Speci®c rRNA was reverse transcribed at low temperature (40 C or 45 C) in the presence of primer B16RT. As subsequent PCR using primer B16RT at high (62 C) annealing temperatures is not nearly as ef®cient as ampli®cation using the speci®c primer, ampli®cation of genomic DNA was hindered relative to the ampli®cation of cDNA. The method was readily adapted to the selective ampli®cation of mRNA of the Listeria monocytogenes listeriolysin O (hly) gene.