Polymerase chain reaction amplification length-dependent ethidium monoazide suppression power for heat-killed cells of Enterobacteriaceae.

The polymerase chain reaction (PCR) can confirm the presence of bacteria, but it is unable to differentiate between live and dead bacteria. Although ethidium monoazide (EMA)- and propidium monoazide (PMA)-based PCR have been evaluated, a quantity of ≥ 10(3)cells/ml dead cells produces a false-positive reading at 40 to 50 cycles (K. Rudi et al., Appl. Environ. Microbiol. 71 (2005) 1018-1024). After confirming the precision of real-time PCR of a long DNA target (16S or 23S ribosomal RNA [rRNA] gene, 1490 or 2840 bp), we evaluated the degree of suppression of an EMA treatment on the 16S/23S PCR using various amplification lengths (110-2840 bp) with heat-killed cells of Enterobacteriaceae (e.g., Salmonella enteritidis). We found that the inhibition rate was proportional to the PCR amplification length; short DNA (110 bp) amplification slightly delayed the threshold cycle (C(T)) of heat-killed cells of Enterobacteriaceae when compared with no EMA treatment. Regardless of the amplification length, the C(T) delay using live cells of Enterobacteriaceae with EMA was negligible. Thus, our real-time PCR of a long DNA (16S or 23S) template following EMA treatment is a rapid viable bacterial assay, which can potentially target all genera, for testing pasteurized milk that may have originally been contaminated with high levels of dead bacteria.

Rapid detection of viable bacteria by nested polymerase chain reaction via long DNA amplification after ethidium monoazide treatment.

In assays to determine whether viable cells of Enterobacteriaceae are present in pasteurized milk, the typical ethidium monoazide (EMA) polymerase chain reaction (PCR) targets a short stretch of DNA. This process often triggers false-positive results owing to the high level of dead cells of Enterobacteriaceae that had initially contaminated the sample. We have developed a novel, direct, real-time PCR that does not require DNA isolation (DQ-PCR) to detect low levels of cells of Enterobacteriaceae regardless of live and dead cells first. We confirmed that the DQ-PCR targeting a long DNA (the 16S ribosomal RNA [rRNA] gene, amplified length of 1514 bp) following EMA treatment is a promising tool to detect live bacteria of all genera owing to the complete suppression of background signal from high levels of dead bacteria in pasteurized milk. However, when identifying viable bacteria in pasteurized milk, commercial PCR primers designed for detecting long stretches of DNA are generally not available. Thus, we treated samples with EMA and then carried out an initial round of PCR of a long stretch of DNA (16S gene, 1514 bp). We then performed another round of PCR, a novel nested PCR to generate short products using commercial primers. This procedure resulted in the rapid detection of low levels of viable cells of Enterobacteriaceae.

Prevalence and amounts of Salmonella found on raw California almonds.

Data on the prevalence and populations of pathogens in individual foods are critical to the development of product-specific quantitative microbial risk assessments. An outbreak of salmonellosis associated with the consumption of raw almonds in 2000 to 2001 provided an opportunity to evaluate the levels of Salmonella in the recalled product. Duplicate 100-g samples from each of fifty 22.7-kg boxes of recalled almonds were enriched by one of two methods. Salmonella was isolated by at least one method from 42 boxes (84% positive). The levels of Salmonella determined by a three-tube most-probable-number (MPN) method were 8.5+/-1.3 MPN/100 g. In a subsequent study, raw almonds that arrived at almond processors were sampled from 2001 through 2005 to determine the overall prevalence and levels of Salmonella and to characterize the Salmonella isolates obtained. Aerobic plate counts, coliform counts, and MPN levels of Escherichia coli were also determined on positive samples. An isolation frequency for Salmonella of 81 (0.87%+/-0.2%) of 9,274 samples tested (100 g) was determined for raw almonds sampled from throughout California over the 5-year period. Salmonella was not isolated upon retesting in 59 of 65 positive samples. When detected, levels were 1.2 to 2.9 MPN/100 g. Of the 81 total isolates, 35 different serotypes of Salmonella were represented. Aerobic plate counts, coliform counts, and E. coli levels did not correlate with the presence of Salmonella.