A faster method to detect norovirus in oysters using probe hybridization to isolate target RNA before RT-PCR.

Human Noroviruses (HuNoVs) are the most frequent cause of outbreaks of acute gastroenteritis following the ingestion of raw or improperly cooked oysters. Although highly sensitive methods to detect HuNoV in oysters using reverse transcriptase-polymerase chain reaction (RT-PCR) are available, rapid methods to process samples for RT-PCR are still needed. The conventional approach is to concentrate the virus first before RNA purification to maximize assay sensitivity, but the procedures used are cumbersome. We developed a new hybridization method that is much faster and more effective compared to existing technology. The procedure includes an initial extraction of total RNA from the digestive diverticula of oysters using TRI Reagent, followed by HuNoV RNA purification using a capture probe and then HuNoV detection by real-time RT-PCR. The detection limit is approximately 100 PCR detection units of HuNoV per sample. Compared to published methods that require an initial virus concentration step before RNA extraction, the new method is much faster to complete. Approximately 3 h are needed to purify HuNoV RNA using the new method compared to at least 8 h using conventional methods. Coupled with real-time RT-PCR, the new method can detect HuNoV in contaminated oysters within 8 h. The effectiveness of the method was demonstrated using live artificially contaminated oysters and wild oysters.

Methods to increase fidelity of repetitive extragenic palindromic PCR fingerprint-based bacterial source tracking efforts.

The goal of the study was to determine which similarity coefficient and statistical method to use to produce the highest rate of correct assignment (RCA) in repetitive extragenic palindromic PCR-based bacterial source tracking. In addition, the use of standards for deciding whether to accept or reject source assignments was investigated. The use of curve-based coefficients Cosine Coefficient and Pearson's Product Moment Correlation yielded higher RCAs than the use of band-based coefficients Jaccard, Dice, Jeffrey's x, and Ochiai. When enterococcal and Escherichia coli isolates from known sources were used in a blind test, the use of maximum similarity produced consistently higher RCAs than the use of average similarity. We also found that the use of a similarity value threshold and/or a quality factor threshold (the ratio of the average fingerprint similarity within a source to the average similarity of this source's isolates to an unknown) to decide whether to accept source assignments of unknowns increases the reliability of source assignments. Applying a similarity value threshold improved the overall RCA (ORCA) by 15 to 27% when enterococcal fingerprints were used and 8 to 29% when E. coli fingerprints were used. Applying the quality factor threshold resulted in a 22 to 32% improvement in the ORCA, depending on the fingerprinting technique used. This increase in reliability was, however, achieved at the expense of decreased numbers of isolates that were assigned a source.

Isolation, Enumeration, and Identification of Gram-Positive Cocci from Frozen Crabmeat.

Bacterial levels in frozen crabmeat samples were determined by plate counts using four staphylococcal isolation media incubated for 24, 48, and 72 h at 26 and 35°C. Staphylococcal counts determined by the spread-plate Food and Drug Administration Baird-Parker protocol incubated at 35°C for 48 h (FDABP48-35) served as the standard for comparison. When FDABP48-35 counts were compared to counts from 29 combinations of media, time of incubation, and incubation temperature, only FDABP and Borrego, Florido, Mrocek, and Romero (BFMR) counts, representing 11 combinations, were statistically comparable to FDABP48-35 counts. Cocci (91.5%) were the dominant bacterial type; gram-positive rods (8.3%) and gram-negative isolates (0.2%) were also detected. Isolates tested by the coagulase reaction were predominantly coagulase negative (CN) (97.7%). Of 100 isolates analyzed by the BIOLOG identification procedure, 62% were classified as Staphylococcus lentus , S. hominis , and S. epidermidis . Three isolates were identified as Staphylococcus aureus . These data indicate that species identification of staphylococci from crabmeat can assist in determining the source of contamination, and that staphylococcal isolates from crabmeat are more likely to be coagulase negative.

Rapid Methods to Evaluate the Bacteriological Quality of Frozen Crabmeat.

The standard methods plate count (SMPC) of frozen crabmeat samples was compared with counts of two alternative aerobic plate count methods (Redigel, Petrifilm). The differences in counts were compared after incubation at two temperatures (35°C and room temperature; RT) and three intervals of time (24, 48, and 72 h). No statistical differences were found when the time of analysis or the method of analysis was compared. However, differences were observed within SMPC values and within Petrifilm plate count values when RT was compared to 35°C, Redigel plate counts at RT and 35°C were not significantly different. The results suggest that seafood plants could use the Redigel media, incubate samples at room temperature for 48 h, and furnish data comparable to SMPC.