Quantification of Campylobacter spp. in chicken carcass rinse by real‐time PCR

Aims: In this study, a real‐time quantitative polymerase chain reaction (PCR) method was examined for its ability to quantify Campylobacter spp. in chicken carcass rinses and compared with bacteriological culturing. Methods and Results: The linearity of the real‐time PCR quantification protocol was assessed on pure DNA. The amplification efficiency was 100% and the square regression coefficient (R²) was 0·998. Quantification was linear over at least 7 log units. Using a crude cell lysate gave the highest sensitivity and the detection limit of the method was 3·3 log CFU per carcass. The statistical correlation between the bacteriological enumeration and the real‐time quantitative (Q)‐PCR determined using chicken carcasses sampled at the end of the slaughter line was 0·733. The difference in detection levels was probably because of the detection of stressed, dead or viable but not culturable cells by Q‐PCR. Conclusion: The real‐time Q‐PCR method described in this study is a powerful tool for determining the number of Campylobacter cells on carcasses. Significance and Impact of the Study: The real‐time Q‐PCR method is available to quantify the Campylobacter contamination at the slaughterhouse level and could be used to evaluate primary production.     

A quantitative real-time PCR method for in planta monitoring of Phytophthora infestans growth

Aim: To develop a real‐time PCR‐based strategy for the detection of Paenibacillus larvae vegetative cells and spores to improve the diagnosis and the screening of American foulbrood (AFB), the most harmful pathology of honeybee brood. Methods and Results: A real‐time PCR that allowed selective identification and quantification of P. larvae 16S rRNA sequence was developed. Using standard samples quantified by flow cytometry, detection limits of 37·5 vegetative cells ml^?1 and 10 spores ml^?1 were determined. Compared to spread plate method, this real‐time PCR‐based strategy allowed, in only 2 h, the detection of P. larvae in contaminated honeys. No false‐positive results were obtained. Moreover, its detection limit was 100 times lower than that of the culture method (2 vs 200 spores g^?1 of honey). Conclusion: A rapid, selective, with low detection limit, sensitive and specific method to detect and quantify vegetative cells and spores of P. larvae is now available. Significance and Impact of Study: In addition to honey samples, this real‐time PCR‐based strategy may be also applied to confirm AFB diagnosis in honeybee brood and to screen other apiary supplies and products (bees, pollen, wax), thus broadening the control of AFB spreading.     

Identification and quantification of viable Bifidobacterium breve strain Yakult in human faeces by using strain-specific primers and propidium monoazide

Aims: To develop a quick and accurate PCR‐based method to evaluate viable Bifidobacterium breve strain Yakult (BbrY) in human faeces. Methods and Results: The number of BbrY in faeces was detected by using strain‐specific quantitative real‐time PCR (qPCR) derived from a randomly amplified polymorphic DNA analysis. And using propidium monoazide (PMA) treatment, which combined a DNA‐intercalating dye for covalently linking DNA in dead cells and photoactivation, only viable BbrY in the faeces highly and significantly correlated with the number of viable BbrY added to faecal samples within the range of 10⁵–10⁹ cells per g of faeces was enumerated. After 11 healthy subjects ingested 10·7 log CFU of BbrY daily for 10 days, 6·9 (±1·5) log CFU g^?1 [mean (±SD)] of BbrY was detected in faeces by using strain‐specific transgalactosylated oligosaccharide–carbenicillin (T‐CBPC) selective agar medium. Viable BbrY detected by qPCR with PMA treatment was 7·5 (±1·0) log cells per g and the total number (viable and dead) of BbrY detected by qPCR without PMA treatment was 8·1 (±0·8) log cells per g. Conclusions: Strain‐specific qPCR with PMA treatment evaluated viable BbrY in faeces quickly and accurately. Significance and Impact of the Study: Combination of strain‐specific qPCR and PMA treatment is useful for evaluating viable probiotics and its availability in humans.     

Phylogenetic analysis of Bacteroidales 16S rRNA gene sequences from human and animal effluents and assessment of ruminant faecal pollution by real‐time PCR

Aims: The aims of this study were to evaluate the host‐specific distribution of Bacteroidales 16S rRNA gene sequences from human‐ and animal‐related effluents and faeces, and to define a ruminant‐specific marker. Methods and Results: Bacteroidales 16S rRNA gene clone libraries were constructed from samples of effluent (sewage, bovine manure and pig slurry) and faeces (human, bovine, pig and wild bird), using PCR primers targeting order Bacteroidales. The phylogenetic analysis revealed six main distinct human‐, bovine‐, pig‐ and wild bird‐specific clusters. From the bovine‐specific cluster II, we designed a ruminant‐specific marker, Rum‐2‐Bac, and this showed 97% sensitivity (n = 30) and 100% specificity (n = 40) when tested by TaqMan^® real‐time PCR. Average concentrations of this marker in bovine and sheep faeces and in bovine manure were 8·2 ± 0·5, 8·4 ± 1·3 and 7 ± 0·5 log₁₀ copies per gram, respectively. It was also quantified in samples of runoff water impacted by bovine manure, with average concentrations of 5·1 ± 0·3 log₁₀ copies per millilitre water. Conclusions: Our results confirmed that some members of Bacteroidales isolated from effluents and faeces had host‐specific distributions. Identification of a bovine‐specific cluster made it possible to design a reliable ruminant‐specific marker. Significance and Impact of the Study: The host‐specific distribution of Bacteroidales sequences from effluents mirrored the host‐specific distribution of sequences observed in individual faeces. This efficient new ruminant‐specific Bacteroidales 16S rRNA marker represents a useful addition to the microbial source tracking toolbox.     

Statistical correlation between enterovirus genome copy numbers and infectious viral particles in wastewater samples

Aims: Classic virological tests are time consuming and labour‐intensive; real‐time RT‐PCR has proven to be a fast method to detect and quantify enterovirus genomes in clinical and environmental samples. This method is unable to discriminate between infective and noninfective enterovirus particles; few clinical studies have compared real‐time RT‐PCR and viral culture. We wondered if the enterovirus genome quantification could be correlated to the infectivity. Methods and Results: We used the statistical approach to verify our hypotheses to correlate data, obtained by the standard method (most probable number of cytopathic units—MPNCU) and molecular test (real‐time RT‐PCR), on wastewater treatment plant samples. Chi‐squared test was used, considering several cut‐off values (‘50’‐‘100’‐‘200’ genome copy numbers), to determine statistical significance in comparison of the two methods. Chi‐square value was not significant when cut‐off of 50 (P = 0·103) and 100 (P = 0·178) was assumed but was significant with cut‐off of 200 (P = 0·044). Conclusion: This limit, 200 genome copy, could be used as cut‐off value to indicate enterovirus survival in environmental monitoring. Significant and Impact of the Study: To introduce a fast procedure that is able to compensate for disadvantages of cell culture method for viral environmental analyses.     

A real‐time PCR method to quantify spores carrying the Bacillus thuringiensis var. israelensis cry4Aa and cry4Ba genes in soil

Aim: To develop a rapid real‐time PCR method for the specific detection and quantification of Bacillus thuringiensis var. israelensis (Bti) spores present in the environment. Methods and Results: Seven soil samples as well as one sediment sample obtained from various regions of Switzerland and characterized by different granulometry, pH values, organic matter and carbonate content were artificially inoculated with known amounts of Bti spores. After DNA extraction, DNA templates were amplified using TaqMan real‐time PCR targeting the cry4Aa and cry4Ba plasmid genes encoding two insecticidal toxins (δ‐endotoxins), and quantitative standard curves were created for each sample. Physicochemical characteristics of the samples tested did not influence DNA extraction efficiency. Real‐time PCR inhibition because of the presence of co‐extracted humic substances from the soil was observed only for undiluted DNA extracts from samples with very high organic matter content (68%). The developed real‐time PCR system proved to be sensitive, detecting down to 1 × 10³ Bti spores per g soil. One‐way analysis of variance confirmed the accuracy of the method. Conclusions: Direct extraction of DNA from environmental samples without culturing, followed by a specific real‐time PCR allowed for a fast and reliable identification and quantification of Bti spores in soil and sediment. Significance and Impact of the Study: The developed real‐time PCR system can be used as a tool for ecological surveys of areas where treatments with Bti are carried out.     

Assessment of SCAR markers to design real‐time PCR primers for rhizosphere quantification of Azospirillum brasilense phytostimulatory inoculants of maize

Aims: To assess the applicability of sequence characterized amplified region (SCAR) markers obtained from BOX, ERIC and RAPD fragments to design primers for real‐time PCR quantification of the phytostimulatory maize inoculants Azospirillum brasilense UAP‐154 and CFN‐535 in the rhizosphere. Methods and Results: Primers were designed based on strain‐specific SCAR markers and were screened for successful amplification of target strain and absence of cross‐reaction with other Azospirillum strains. The specificity of primers thus selected was verified under real‐time PCR conditions using genomic DNA from strain collection and DNA from rhizosphere samples. The detection limit was 60 fg DNA with pure cultures and 4 × 10³ (for UAP‐154) and 4 × 10⁴ CFU g^?1 (for CFN‐535) in the maize rhizosphere. Inoculant quantification was effective from 10⁴ to 10⁸ CFU g^?1 soil. Conclusion: BOX‐based SCAR markers were useful to find primers for strain‐specific real‐time PCR quantification of each A. brasilense inoculant in the maize rhizosphere. Significance and Impact of the Study: Effective root colonization is a prerequisite for successful Azospirillum phytostimulation, but cultivation‐independent monitoring methods were lacking. The real‐time PCR methods developed here will help understand the effect of environmental conditions on root colonization and phytostimulation by A. brasilense UAP‐154 and CFN‐535.     

Usefulness of real-time PCR as a complementary tool to the monitoring of Legionella spp. and Legionella pneumophila by culture in industrial cooling systems

Aims: This study was designed to evaluate the usefulness of quantification by real‐time PCR as a management tool to monitor concentrations of Legionella spp. and Legionella pneumophila in industrial cooling systems and its ability to anticipate culture trends by the French standard method (AFNOR T90‐431). Methods and Results: Quantifications of Legionella bacteria were achieved by both methods on samples from nine cooling systems with different water qualities. Proportion of positive samples for L. pneumophila quantified by PCR was clearly lower in deionized or river waters submitted to a biocide treatment than in raw river waters, while positive samples for Legionella spp. were quantified for almost all the samples. For some samples containing PCR inhibitors, high quantification limits (up to 4·80 × 10⁵ GU l^?1) did not allow us to quantify L. pneumophila, when they were quantified by culture. Finally, the monitoring of concentrations of L. pneumophila by both methods showed similar trends for 57–100% of the samples. Conclusions: These results suggest that, if some methodological steps designed to reduce inhibitory problems and thus decrease the quantification limits, could be developed to quantify Legionella in complex waters, the real‐time PCR could be a valuable complementary tool to monitor the evolution of L. pneumophila concentrations. Significance and Impact of the Study: This study shows the possibility of using real‐time PCR to monitor L. pneumophila proliferations in cooling systems and the importance to adapt nucleic acid extraction and purification protocols to raw waters.     

A real‐time PCR assay for detection and quantification of Lactococcus garvieae

Aims: To develop a rapid, sensitive, specific tool for the detection and quantification of Lactococcus garvieae in food and environmental samples. Methods and Results: A real‐time quantitative PCR (qPCR) assay with primers for CAU12F and CAU12R based on the 16S rRNA gene of L. garvieae was successfully established. The limit of detection for L. garvieae genomic DNA was 1 ng DNA in conventional PCR and 32 fg with a mean C_T value of 36·75 in qPCR. Quantification of L. garvieae vegetative cells was linear (R² = 0·99) over a 7‐log‐unit dynamic range down to ten L. garvieae cells. Conclusions: This method is highly specific, sensitive and reproducible for the detection of L. garvieae compared to gel‐based conventional PCR assays, thus providing precise quantification of L. garvieae in food and natural environments. Significance and Impact of the Study: This work provides efficient diagnostic and monitoring tools for the rapid identification of L. garvieae, an emerging pathogen in aquaculture and an occasional human pathogen from other members of the genus Lactobacillus.     

Detection of Mycobacterium avium ssp. paratuberculosis in cheese, milk powder and milk using IS900 and f57-based qPCR assays

Aims: To develop a quantitative PCR assay for sensitive and specific detection of Mycobacterium avium ssp. paratuberculosis (Map) in a range of dairy products. Methods and Results: TaqMan^® assays were designed to target the IS900 and f57 genetic elements of Map. Both real‐time PCR assays were integrated with the Adiapure^® Map DNA extraction kit and assessed separately for the detection/quantification of Map in spiked milk, Cheddar cheese and milk powder. Assays were validated against Cheddar cheese samples containing known concentrations of Map. The IS900 qPCR assay was significantly more sensitive than the assay based on the f57 primer/probe. At a threshold cycle value of 38, limits of detection (LOD) for the IS900 qPCR assay were 0·6 CFU ml^?1, 2·8 CFU g^?1 and 30 CFU g^?1 for artificially contaminated pasteurized milk, whole milk powder and Cheddar cheese, respectively. The respective LOD’s for the f57 assay were 6·2 CFU ml^?1, 26·7 CFU g^?1 and 316 CFU g^?1. Conclusion: The integrated Adiapure^® extraction – IS900 real time assay described is a sensitive, quantitative method for the detection of Map in dairy products. This is the first study to consider qPCR as a quantitative estimation of Map‐DNA in cheese and whole milk powder. Significance and Impact of the Study: The assay developed allows sensitive detection and quantification of Map DNA in a range of dairy products which is valuable for the screening and surveillance of this potential zoonotic organism.     

Quantification of Survival of Escherichia coli O157:H7 on Plants Affected by Contaminated Irrigation Water

Enterohemorrhagic E. coli O157: H7 (EHEC) is a major foodborne pathogen capable of causing diarrhea and vomiting, with further complications such as hemolytic‐uremic syndrome (HUS). The aim of this study was to use the real‐time PCR method to quantify the survival of Escherichia coli O157:H7/pGFP in phyllosphere (leaf surface), rhizosphere (volume of soil tightly held by plant roots), and non‐rhizosphere soils (sand and clay) irrigated with contaminated water and compare the results obtained between real‐time PCR method and conventional plate counts. The real‐time PCR probe was designed to hybridize with the (eae) gene of E. coli O157:H7. The probe was incorporated into real‐time PCR containing DNA extracted from the phyllosphere, rhizosphere, and non‐rhizosphere soils irrigated with water artificially contaminated with E. coli O157:H7. The detection limit for E. coli O157:H7 quantification by real‐time PCR was 2.3 × 10³ in the rhizosphere and phyllosphere samples. E. coli O157:H7 survived longer in rhizosphere soil than the non‐rhizosphere soil. The concentration of E. coli O157:H7/pGFP in rhizosphere soils was ≥ 10⁴ CFU/g in both soils at day 12 based on both plate count and real time PCR, with the clay soil significantly (P = 0.05) higher than the sandy soil. This data showed that E. coli O157H:7 can persist in the environment for more than 50 d, and this may pose some risk for both animal and human infection and provides a very significant pathway for pathogen recontamination in the environment.     

smcL as a novel diagnostic marker for quantitative detection of Listeria ivanovii in biological samples

Aims: To develop a novel molecular tool for the quantitative detection of the ruminant pathogen Listeria ivanovii in different biological matrices. Methods and Results: A real‐time PCR (RTi‐PCR) for the quantitative and species‐specific identification of L. ivanovii was designed to target the region of the smcL gene. The assay includes an internal amplification control (IAC) to avoid false‐negative results. The smcL‐IAC RTi‐PCR assay was 100% selective and allowed the detection of as little as one genome equivalent in 45% of reactions. The quantification accuracy was excellent, as demonstrated by its high linearity (R² > 0·9989) and PCR efficiency (E > 0·984) over a 6‐log dynamic range, down to 10 genome equivalents. Finally, the applicability of this assay was evaluated with artificially contaminated biological matrices implicated in the transmission of this bacterium such as sheep raw milk, blood and amniotic fluid. The smcL‐IAC RTi‐PCR assay allowed the detection of as few as 50 colony forming unit numbers (CFUs) per 25 ml of raw milk, 43 CFUs per 1 ml of blood or 50 CFUs per 1 ml of amniotic fluid. Conclusions: This method can be an adequate alternative for the identification of L. ivanovii and for complete diagnosis of animal and human listeriosis. Significance and Impact of the Study: We present an alternative for the detection of another pathogenic member of Listeria genus, which can help to distinguish from Listeria monocytogenes and therefore facilitates the establishment of preventive and prophylactic measures in food and farm environments.     

Detection of Salmonella enterica in food using two-step enrichment and real-time polymerase chain reaction

Aims: A new real‐time polymerase chain reaction‐based method was developed for the detection of Salmonella enterica in food. Methods and Results: The method consisted of a novel two‐step enrichment involving overnight incubation in buffered peptone water and a 5‐h subculture in Rappaport–Vassiliadis medium, lysis of bacterial cells and a Salmonella‐specific 5′‐nuclease real‐time PCR with an exogenous internal amplification control. Because a two‐step enrichment was used, the detection limit for dead S. enterica cells in artificially contaminated ice cream and salami samples was high at 10⁷ CFU (25 g)^?1, eliminating potential false‐positive results. When the method was evaluated with a range of 100 naturally contaminated food samples, three positive samples were detected by both the real‐time PCR‐based method and by the standard microbiological method, according to EN ISO 6579. When the real‐time PCR‐based method was evaluated alongside the standard microbiological method according to EN ISO 6579 with 36 food samples artificially contaminated at a level of 10⁰ CFU (25 g)^?1, identical results were obtained from both methods. Conclusions: The real‐time PCR‐based method involving a two‐step enrichment produced equivalent results to EN ISO 6579 on the day after sample receipt. Significance and Impact of the Study: The developed method is suitable for rapid detection of S. enterica in food.     

Development and evaluation of a multiplex real-time PCR assay for the detection and differentiation of Moraxella bovis, Moraxella bovoculi and Moraxella ovis in pure culture isolates and lacrimal swabs collected from conventionally raised cattle

Aim: To develop a multiplex real‐time PCR assay for the detection and differentiation of Moraxella bovis (M. bovis), M. bovoculi and M. ovis. Methods and Results: The multiplex real‐time PCR assay was validated on three reference strains, 57 pure culture isolates and 45 lacrimal swab samples. All reference strains were identified correctly with no cross‐reactions between species. Sequencing of 53 of the 57 culture isolates confirmed the results obtained with the multiplex real‐time PCR, and the assay had 96·5% (55/57) concordance with a Moraxella spp. multiplex conventional PCR assay on the isolates. Among the lacrimal swab samples, the concordance between the multiplex real‐time PCR and culture was 86·7% (39/45) for M. bovoculi and 75·6% (34/45) for M. bovis. Conclusions: The multiplex real‐time PCR assay is specific and sensitive and can be used directly on lacrimal swab samples. Significance and Impact of Study: The lack of a rapid, specific and sensitive detection method is a barrier for determining the roles of M. bovis, M. bovoculi and M. ovis in infectious bovine keratoconjunctivitis cases, and the developed PCR assay will contribute to improved understanding of the epidemiology and pathogenesis of these three Moraxella species.     

Evaluation of real‐time PCR methods for quantification of Acanthamoeba in anthropogenic water and biofilms

Aims: To assess two real‐time PCR methods (the Riviere and Qvarnstrom assays) for environmental Acanthamoeba. Methods and Results: DNA extracted from Acanthamoeba castellanii taken from water and biofilms of cooling towers was analysed by the Riviere and Qvarnstrom assays. To quantify environmental Acanthamoeba, the calibration curves (DNA quantity vs cell number) were constructed with samples spiked with A. castellanii. The calibration curves for both quantitative PCR assays showed low variation (coefficient of variation of C_t≤ 5·7%) and high linearity (R² ≥ 0·99) over six orders of magnitudes with detection limit of three cells per water sample. DNA quantity determined by Qvarnstrom assay was equivalent between trophozoites and cysts (P = 0·49), whereas a significant difference was observed with Riviere assay (P < 0·0001). Riviere assay failed to detect Acanthamoeba in 21% (15/71) of the environmental samples which were positively detected by Qvarnstrom assay, while one sample (1·4%) was shown positive by Riviere assay but negative by Qvarnstrom assay. Moreover, Acanthamoeba counts by Qvarnstrom assay were greater than those by Riviere assay (P < 0·0001). Conclusions: Qvarnstrom assay performs better than Riviere assay for detection and quantification of Acanthamoeba in anthropogenic water and biofilms. Significance and Impact of the Study: Qvarnstrom assay may significantly contribute to a better knowledge about the distribution and abundance of Acanthamoeba in environments.     

Rapid detection and differentiation of Erysipelothrix spp. by a novel multiplex real‐time PCR assay

Aim: The aim of this study was to develop a multiplex real‐time PCR assay for the identification and discrimination of Erysipelothrix rhusiopathiae, tonsillarum and Erysipelothrix sp. strain 2 for direct detection of Erysipelothrix spp. from animal specimens. Methods and Results: A primer set and three species‐specific probes with different end labelling were designed from the noncoding region downstream of the 5S rRNA coding region. The sensitivity, specificity and repeatability of the assay were validated by analysing 27 Erysipelothrix spp. reference serotype strains and ten septicemia‐associated non‐Erysipelothrix spp. bacterial isolates. Cross‐reactivity with Erysipelothrix sp. strain 1 was not observed with any of the primer probe combinations. The detection limit was determined to be <10 colony forming units and as low as one genome equivalent per PCR . Further evaluation of the Erysipelothrix spp. multiplex PCR was performed by comparing an enrichment isolation culture method and a conventional differential PCR on 15 samples from pigs experimentally inoculated with Erysipelothrix spp. and 22 samples from pigs with suspected natural infection. Conclusion: The multiplex real‐time PCR assay was found to be simple, rapid, reliable, specific and highly sensitive. Significance and Impact of the Study: The developed real‐time multiplex PCR assay does not require cumbersome and lengthy cultivation steps prior to DNA extraction, obtained comparable results to enrichment isolation, and will be useful in diagnostic laboratories for rapid detection of Erysipelothrix spp.     

A real-time qPCR assay to quantify Fusarium graminearum biomass in wheat kernels

Aims: To develop a real‐time PCR assay to quantify Fusarium graminearum biomass in blighted wheat kernels. Methods and Results: Primers designed to amplify a gene in the trichothecene biosynthetic cluster (TRI6) were evaluated for sensitivity and specificity. Primer pair Tri6_10F/Tri6_4R specifically and consistently amplified a 245‐bp DNA fragment from F. graminearum. A workflow was developed and validated to extract DNA from infested grain. The assay detected as little as 10 μg of F. graminearum mycelia in 1 g of ground wheat grain with a high correlation between fungal biomass and cycle threshold values (R² = 0·9912; P = 0·004). In field‐inoculated grain, qPCR measurements of biomass correlated closely with deoxynivalenol levels (R = 0·82, P < 0·0001) and two visual techniques to assess grain quality (R = 0·88, P < 0·0001 and R = 0·81, P < 0·0001). Conclusions: The qPCR assay provided accurate and precise assessments of the amount of F. graminearum biomass in blighted wheat kernels. This method represents a technical advance over other approaches to quantify kernel colonization and real‐time PCR detection methodologies for F. graminearum that do not correlate quantification of fungal genomic DNA to biomass. Significance and Impact of the Study: Quantifying F. graminearum biomass, especially low levels of growth associated with kernels that are visually asymptomatic, represents a new approach to screen for resistance to kernel infection, an understudied yet potentially important avenue to reduce the impact of head blight.     

A Validated Normal Phase LC Method for Enantiomeric Separation of Rasagiline Mesylate and Its (S)‐Enantiomer on Cellulose Derivative‐Based Chiral Stationary Phase

A simple, sensitive, and robust normal‐phase isocratic HPLC‐UV method was developed and validated for the enantiomeric separation of rasagiline mesylate and its (S)‐enantiomer. The rasagiline and its (S)‐enantiomer were resolved on a Chiralcel‐OJ‐H (4‐methylbenzoate cellulose coated on silica) column using a mobile phase consisting of n‐hexane:isopropyl alcohol:ethanol:diethyl amine (96:2:2:0.01) at a flow rate of 1.0 ml/min. The column temperature was maintained at 27 °C and elution was monitored at 215 nm. The resolution (R_s) between the enantiomers was found to be more than 2.0. The limit of detection and the limit of quantification of the (S)‐enantiomer were found to be 0.35 and 1.05 µg/ml, respectively. The developed method was validated as per ICH guidelines with respect to linearity, limit of detection and quantification, accuracy, precision, and robustness—and satisfactory results were obtained. The sample solution and mobile phase were found to be stable up to 48 h. The method is useful for routine evaluation of the quality of rasagiline mesylate in bulk drug‐manufacturing units. Chirality 25:324–327, 2013. © 2013 Wiley Periodicals, Inc.     

Limitation in the detection of Listeria monocytogenes in food in the presence of competing Listeria innocua

Aims: Detectability of Listeria monocytogenes at 10⁰ CFU per food sample in the presence of Listeria innocua using standard microbiological detection was evaluated and compared with the real‐time PCR‐based method. Methods and Results: Enrichment in half‐Fraser broth followed by subculture in Fraser broth according to EN ISO 11290‐1 was used. False‐negative detection of 10⁰ CFU L. monocytogenes was obtained in the presence of 10¹ CFU L. innocua per sample using the standard detection method in contrast to more than 10⁵ CFU L. innocua per sample using real‐time PCR. Identification of L. monocytogenes on the chromogenic medium by the standard procedure was impossible if L. innocua was able to overgrow L. monocytogenes by more than three orders of magnitude after the enrichment in model samples. These results were confirmed using naturally contaminated food samples. Conclusions: Standard microbiological method was insufficient for the reliable detection of 10⁰ CFU L. monocytogenes in the presence of more than 10⁰ CFU of L. innocua per sample. On the other hand, if the growth of L. monocytogenes was sufficient to reach the concentration equal to the detection limit of PCR, the amount of the other microflora present in the food sample including L. innocua was not relevant for success of the PCR detection of L. monocytogenes. Significance and Impact of the Study: After the enrichment, the PCR detection is more convenient than the standard one as PCR detection is not compromised by other present microflora.