In:
Applied and Environmental Microbiology, American Society for Microbiology, Vol. 71, No. 12 ( 2005-12), p. 9008-9012
Abstract:
We describe a novel quantitative real-time (Q)-PCR assay for Listeria monocytogenes based on the coamplification of a target hly gene fragment and an internal amplification control (IAC). The IAC is a chimeric double-stranded DNA containing a fragment of the rapeseed BnACCg8 gene flanked by the hly -specific target sequences. This IAC is detected using a second TaqMan probe labeled with a different fluorophore, enabling the simultaneous monitoring of the hly and IAC signals. The hly -IAC assay had a specificity and sensitivity of 100%, as assessed using 49 L. monocytogenes isolates of different serotypes and 96 strains of nontarget bacteria, including 51 Listeria isolates. The detection and quantification limits were 8 and 30 genome equivalents, and the coefficients for PCR linearity ( R 2 ) and efficiency ( E ) were 0.997 and 0.80, respectively. We tested the performance of the hly -IAC Q-PCR assay using various broth media and food matrices. Fraser and half-Fraser media, raw pork, and raw or cold-smoked salmon were strongly PCR-inhibitory. This Q-PCR assay for L. monocytogenes , the first incorporating an IAC to be described for quantitative detection of a food-borne pathogen, is a simple and robust tool facilitating the identification of false negatives or underestimations of contamination loads due to PCR failure.
Type of Medium:
Online Resource
ISSN:
0099-2240
,
1098-5336
DOI:
10.1128/AEM.71.12.9008-9012.2005
Language:
English
Publisher:
American Society for Microbiology
Publication Date:
2005
detail.hit.zdb_id:
223011-2
detail.hit.zdb_id:
1478346-0
SSG:
12
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