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Anal Biochem. 2011 Nov 1;418(1):37-43. doi: 10.1016/j.ab.2011.06.027. Epub 2011 Jun 29.

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

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Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan.


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.

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