Promising Nucleic Acid Lateral Flow Assay Plus PCR for Shiga Toxin-Producing Escherichia coli.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a frequent cause of foodborne infections, and methods for rapid and reliable detection of STEC are needed. A nucleic acid lateral flow assay (NALFA) plus PCR was evaluated for detecting STEC after enrichment. When cell suspensions of 45 STEC strains, 14 non-STEC strains, and 13 non-E. coli strains were tested with the NALFA plus PCR, all of the STEC strains yielded positive results, and all of the non-STEC and non-E. coli strains yielded negative results. The lower detection limit for the STEC strains ranged from 0.1 to 1 pg of genomic DNA (about 20 to 200 CFU) per test, and the NALFA plus PCR was able to detect Stx1- and Stx2-producing E. coli strains with similar sensitivities. The ability of the NALFA plus PCR to detect STEC in enrichment cultures of radish sprouts, tomato, raw ground beef, and beef liver inoculated with 10-fold serially diluted STEC cultures was comparable to that of a real-time PCR assay (at a level of 100 to 100,000 CFU/ml in enrichment culture). The bacterial inoculation test in raw ground beef revealed that the lower detection limit of the NALFA plus PCR was also comparable to that obtained with a real-time PCR assay that followed the U.S. Department of Agriculture guidelines. Although further evaluation is required, these results suggest that the NALFA plus PCR is a specific and sensitive method for detecting STEC in a food manufacturing plant.

Potential rapid and simple lateral flow assay for Escherichia coli O111.

We developed and evaluated a lateral flow assay (LFA) as a simple and rapid method for direct detection of Escherichia coli O111 in food after enrichment. When cell suspensions of 8 E. coli O111 strains and 77 non-E. coli O111 strains were tested with the LFA, the former all yielded positive results and the latter all yielded negative results. The minimum detection limits for the E. coli O111 strains were 1.8 × 10(3) to 5.6 × 10(5) CFU/ml of cell suspension, and the LFA was able to detect live cultures or those killed by autoclaving at nearly the same level of sensitivity. To evaluate the ability of LFA to detect its target in food, enrichment cultures of meat samples inoculated with 10-fold serial dilutions of E. coli O111 were tested with the LFA and PCR. Even when there were very few E. coli O111 cells in the meat samples (1.6 × 10(0) to 1.6 × 10(1) CFU/25 g of food), when they were cultured in modified E. coli broth with novobiocin for 22 h at 42°C, the LFA yielded positive results that corresponded to the PCR results. Although the LFA requires further evaluation and field study, these results suggest that this assay has sufficient sensitivity and specificity. This procedure can be completed with a one-step incubation after the test strip has been inserted into the sample after 22 h of culture, whereas the standard culture method requires multiple cultures, skilled personnel, a well-equipped laboratory, and 4 or 5 days. The speed and simplicity of this LFA make it suitable for use as part of routine screening assays in the food industry.

Identification of antibody against porcine coronavirus in human milk.

A total of 239 human milk samples collected from mothers in Chiba City, Japan during 1994-1997 were tested for the presence of anti-coronavirus antibody. Interestingly, twelve human milk samples were positive for IgA against porcine transmissible gastroenteritis coronavirus, and this represented 5%. These findings provided evidence that the interspecies transmission of coronavirus between human and porcine might occur in nature. This report is noteworthy because it is the first, to the best of our knowledge, demonstrating the presence of antibody against porcine transmissible gastroenteritis coronavirus in human milk.