The technique of loop-mediated isothermal amplification (LAMP) utilizes four (or six) primers targeting six (or eight) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional PCR methods. The high concentrations of primers used leads to an increased likelihood of non-specific amplification induced by primer dimers. In this study, a set of LAMP primers were designed targeting the prfA gene sequence of Listeria monocytogenes, and dimethyl sulfoxide (DMSO) as well as Touchdown LAMP were employed to increase the sensitivity and specificity of the LAMP reactions. The results indicate that the detection limit of this novel LAMP assay with the newly designed primers and additives was 10 fg per reaction, which is ten-fold more sensitive than a commercial Isothermal Amplification Kit and hundred-fold more sensitive than previously reported LAMP assays. This highly sensitive LAMP assay has been shown to detect 11 strains of Listeria monocytogenes, and does not detect other Listeria species (including Listeria innocua and Listeria invanovii), providing some advantages in specificity over commercial Isothermal Amplification Kits and previously reported LAMP assay.

Centrifugation is widely used to isolate and concentrate bacteria from dairy products before assay. We found that more than 98% of common pathogenic bacteria added to pasteurized, homogenized, or pasteurized homogenized milk were recovered in the pellet after centrifugation, whereas less than 7% were recovered from raw milk. The remaining bacteria partitioned into the cream layer of raw milk within 5 min, and half-saturation of the cream layer required a bacterial load of approximately 5×10(8) cfu/mL. Known treatments (e.g., heat, enzymes or solvents) can disrupt cream layer binding and improve recovery from raw milk, but can also damage bacteria and compromise detection. We developed a simple, rapid agitation treatment that disrupted bacteria binding to the cream layer and provided more than 95% recovery without affecting bacteria viability. Combining this simple agitation treatment with a previously developed real-time quantitative PCR assay allowed the detection of Salmonella spp. in raw milk at 4 cfu/mL within 3 h. To our knowledge, this is the first report of an effective method for achieving high centrifugal recovery of bacteria from raw milk without impairing bacterial viability.

Shiga toxins 1 and 2 (Stx1 and Stx2) from Shiga toxin-producing E. coli (STEC) bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies) and pooled horseradish peroxidase (HRP)-conjugated monoclonal antibodies. Following the reaction of HRP with the precipitating chromogenic substrate (metal enhanced 3,3-diaminobenzidine tetrahydrochloride or DAB), the formation of a colored product was quantitatively measured with an inexpensive flatbed page scanner. The colorimetric ELISA microarray was demonstrated to detect Stx1 and Stx2 at levels as low as ~4.5 ng/mL within ~2 h of total assay time with a narrow linear dynamic range of ~1–2 orders of magnitude and saturation levels well above background. Stx1 and/or Stx2 produced by various strains of STEC were also detected following the treatment of cultured cells with mitomycin C (a toxin-inducing antibiotic) and/or B-PER (a cell-disrupting, protein extraction reagent). Semi-quantitative detection of Shiga toxins was demonstrated to be sporadic among various STEC strains following incubation with mitomycin C; however, further reaction with B-PER generally resulted in the detection of or increased detection of Stx1, relative to Stx2, produced by STECs inoculated into either axenic broth culture or culture broth containing ground beef.

Antibody microarray is a powerful analytical technique because of its inherent ability to simultaneously discriminate and measure numerous analytes, therefore making the technique conducive to both the multiplexed detection and identification of bacterial analytes (i.e., whole cells, as well as associated metabolites and/or toxins). We developed a sandwich fluorescent immunoassay combined with a high-throughput, multiwell plate microarray detection format. Inexpensive polystyrene plates were employed containing passively adsorbed, array-printed capture antibodies. During sample reaction, centrifugation was the only strategy found to significantly improve capture, and hence detection, of bacteria (pathogenic Escherichia coli O157:H7) to planar capture surfaces containing printed antibodies. Whereas several other sample incubation techniques (e.g., static vs. agitation) had minimal effect. Immobilized bacteria were labeled with a red-orange-fluorescent dye (Alexa Fluor 555) conjugated antibody to allow for quantitative detection of the captured bacteria with a laser scanner. Shiga toxin 1 (Stx1) could be simultaneously detected along with the cells, but none of the agitation techniques employed during incubation improved detection of the relatively small biomolecule. Under optimal conditions, the assay had demonstrated limits of detection of ~5.8 × 10⁵ cells/mL and 110 ng/mL for E. coli O157:H7 and Stx1, respectively, in a ~75 min total assay time.