scholarly journals Quantitative Detection of Listeria monocytogenes and Listeria innocua by Real-Time PCR: Assessment of hly, iap, and lin02483 Targets and AmpliFluor Technology

2004 ◽  
Vol 70 (3) ◽  
pp. 1366-1377 ◽  
Author(s):  
David Rodr�guez-L�zaro ◽  
Marta Hern�ndez ◽  
Mariela Scortti ◽  
Teresa Esteve ◽  
Jos� A. V�zquez-Boland ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Target Genes ◽  
Dynamic Range ◽  
Significant Proportion ◽  
Quantitative Detection ◽  
Target Sequence ◽  
Food Borne ◽  
Highly Sensitive

ABSTRACT We developed and assessed real-time PCR (RTi-PCR) assays for the detection and quantification of the food-borne pathogen Listeria monocytogenes and the closely related nonpathogenic species L. innocua. The target genes were hly and iap for L. monocytogenes and lin02483 for L. innocua. The assays were 100% specific, as determined with 100 Listeria strains and 45 non-Listeria strains, and highly sensitive, with detection limits of one target molecule in 11 to 56% of the reactions with purified DNA and 3 CFU in 56 to 89% of the reactions with bacterial suspensions. Quantification was possible over a 5-log dynamic range, with a limit of 15 target molecules and R 2 values of >0.996. There was an excellent correspondence between the predicted and the actual numbers of CFU in the samples (deviations of <23%). The hly-based assay accurately quantified L. monocytogenes in all of the samples tested. The iap-based assay, in contrast, was unsuitable for quantification purposes, underestimating the bacterial counts by 3 to 4 log units in a significant proportion of the samples due to serovar-related target sequence variability. The combination of the two assays enabled us to classify L. monocytogenes isolates into one of the two major phylogenetic divisions of the species, I and II. We also assessed the new AmpliFluor technology for the quantitative detection of L. monocytogenes by RTi-PCR. The performance of this system was similar to that of the TaqMan system, although the former system was slightly less sensitive (detection limit of 15 molecules in 45% of the reactions) and had a higher quantification limit (60 molecules).

scholarly journals Use of Ethidium Monoazide and PCR in Combination for Quantification of Viable and Dead Cells in Complex Samples

2005 ◽  
Vol 71 (2) ◽  
pp. 1018-1024 ◽  
Author(s):  
Knut Rudi ◽  
Birgitte Moen ◽  
Signe Marie Drømtorp ◽  
Askild L. Holck
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dynamic Range ◽  
Covalent Binding ◽  
Biological Research ◽  
Complex Samples ◽  
Ethidium Monoazide ◽  
Food Borne ◽  
Pcr Method ◽  
Membrane Cell

ABSTRACT The distinction between viable and dead cells is a major issue in many aspects of biological research. The current technologies for determining viable versus dead cells cannot readily be used for quantitative differentiation of specific cells in mixed populations. This is a serious limitation. We have solved this problem by developing a new concept with the viable/dead stain ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR). A dynamic range of approximately 4 log10 was obtained for the EMA-PCR viable/dead assay. Viable/dead differentiation is obtained by covalent binding of EMA to DNA in dead cells by photoactivation. EMA penetrates only dead cells with compromised membrane/cell wall systems. DNA covalently bound to EMA cannot be PCR amplified. Thus, only DNA from viable cells can be detected. We evaluated EMA-PCR with the major food-borne bacterium Campylobacter jejuni as an example. Traditional diagnosis of this bacterium is very difficult due to its specific growth requirements and because it may enter a state where it is viable but not cultivable. The conditions analyzed included detection in mixed and natural samples, survival in food, and survival after disinfection or antibiotic treatment. We obtained reliable viable/dead quantifications for all conditions tested. Comparison with standard fluorescence-based viable/dead techniques showed that the EMA-PCR has a broader dynamic range and enables quantification in mixed and complex samples. In conclusion, EMA-PCR offers a novel real-time PCR method for quantitative distinction between viable and dead cells with potentially very wide application.

Multiplex Quantitative Real-Time PCR for the Detection of t(14;18) Translocations with Breakpoints within 5 Different Regions of the BCL-2 Gene: MBR, 3′MBR, mcr, 5′mcr, icr.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2826-2826
Author(s):  
Frank Schüler ◽  
Sandra C. Dölken ◽  
Carsten Hirt ◽  
Gottfried Dolken
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Mononuclear Cells ◽  
Consensus Sequence ◽  
Degraded Dna ◽  
Quantitative Detection ◽  
Target Sequence ◽  
Quantitative Real Time Pcr ◽  
Consensus Primer ◽  
Target Sequences

Abstract Follicular lymphomas (FL) are associated with the chromosomal translocation t(14;18)(q32;q21). Most breakpoints of chromosome 18 (60%) occur in the major breakpoint region (MBR) of the BCL-2 gene. Further breakpoints have been detected in the minor cluster region (mcr), less frequent breakpoints are found in regions called 3′-MBR, 5′-mcr and icr. On chromosome 14 most breakpoints are located within one of the six JH-genes. Therefore, BCL-2 translocations with breakpoints within the MBR and mcr are generally detected by PCR using combinations of different BCL-2 primers with one JH-consensus primer. We have developed a multiplex quantitative real-time PCR strategy that that can be used to detect t(14;18) translocations with breakpoints located within all regions mentioned above. To minimize the costs for expensive probes we used the JH-consensus sequence as a target for one “consensus probe” (fluorescent labelled minor groove binder probe) for all assays in combination with 6 different JH intron primers. To reduce the size of amplified PCR fragments 12 BCL-2 primers were chosen in combination with 6 JH intron primers for the detection of all 5 breakpoint regions. It is very important to choose short DNA target sequences for amplification: (a) to establish a real-time PCR with a high amplification efficacy; (b) to be able to amplify target sequences also from partially degraded DNA isolated from formaldehyde-fixed paraffin-embedded tissue sections; (c) to achieve a high sensitivity to detect 1–3 copies per assay. Peripheral bood mononuclear cells (PBMNC) and formalin fixed, paraffin embedded lymph node tissue obtained from 139 FL patients at the time of diagnosis (LN and PBMNC, n = 54; LN only, n = 3; PBMNC only, n = 82) were tested by multiplex quantitative real-time PCR. 80 breakpoints were identified within the MBR (61%) region. For comparison, 78/80 breakpoints were also detected by our standard real-time PCR assay with one BCL-2-MBR- primer and one JH consensus primer in combination with a fluorescent probe located within the BCL-2 sequence [Doelken et al., BioTechniques, 1998]. Two additional translocations with breakpoints located 5′ of the target sequence of the standard PCR were found by using two additional MBR primers. In addition, five mcr breakpoints (5%), one breakpoint in the 3′MBR region and one breakpoint in the icr region were found. Based on these results the prevalence of breakpoints in various regions of the BCL-2 gene in FL patients is: MBR = 61% (80/139); mcr = 5% (5/139); 3′MBR = 1% (1/139); icr = 1% (1/139); 5′mcr = 0%). Furthermore, based on quantitative PCR results the t(14;18) translocations detected in this study were undoubtedly lymphoma associated and did not belong to t(14;18)-positive non-lymphoma B cell clones found in healthy persons. By applying this multiplex quantitative real-time PCR strategy t(14;18) translocations with breakpoints in five different breakpoint clusters can be detected in about 70% of patients with follicular lymphoma. The assays can be used for a fast and reliable quantitative detection of t(14;18) translocations on DNA isolated from fresh lymph nodes or pathological specimens as well as blood samples at the time of diagnosis. In almost all cases quantitative results will allow a distinction whether the translocation found is lymphoma associated or not, which will in turn allow a quantitative MRD analysis on follow-up samples during and after treatment.

scholarly journals Quantitative Detection of Listeria monocytogenes in Biofilms by Real-Time PCR

2005 ◽  
Vol 71 (4) ◽  
pp. 2190-2194 ◽  
Author(s):  
Morgan Guilbaud ◽  
Pierre de Coppet ◽  
Fabrice Bourion ◽  
Cinta Rachman ◽  
Hervé Prévost ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Method ◽  
Sybr Green ◽  
Sybr Green I ◽  
Quantitative Detection ◽  
Pcr Assay

ABSTRACT A quantitative method based on a real-time PCR assay to enumerate Listeria monocytogenes in biofilms was developed. The specificity for L. monocytogenes of primers targeting the listeriolysin gene was demonstrated using a SYBR Green I real-time PCR assay. The number of L. monocytogenes detected growing in biofilms was 6 × 102 CFU/cm2.

scholarly journals Quantitative Detection of Clostridium tyrobutyricum in Milk by Real-Time PCR

2007 ◽  
Vol 73 (11) ◽  
pp. 3747-3751 ◽  
Author(s):  
Lorena López-Enríquez ◽  
David Rodríguez-Lázaro ◽  
Marta Hernández
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dynamic Range ◽  
Quantitative Detection ◽  
Acid Extraction ◽  
Clostridium Tyrobutyricum ◽  
Analytical Sensitivity ◽  
Whole Milk ◽  
Detergent Treatment ◽  
Ultrahigh Temperature

ABSTRACT We developed a real-time PCR assay for the quantitative detection of Clostridium tyrobutyricum, which has been identified as the major causal agent of late blowing in cheese. The assay was 100% specific, with an analytical sensitivity of 1 genome equivalent in 40% of the reactions. The quantification was linear (R 2 > 0.9995) over a 5-log dynamic range, down to 10 genome equivalents, with a PCR efficiency of >0.946. With optimized detergent treatment and enzymatic pretreatment of the sample before centrifugation and nucleic acid extraction, the assay counted down to 300 C. tyrobutyricum spores, with a relative accuracy of 82.98 to 107.68, and detected as few as 25 spores in 25 ml of artificially contaminated raw or ultrahigh-temperature-treated whole milk.

scholarly journals A Novel Real-Time PCR for Listeria monocytogenes That Monitors Analytical Performance via an Internal Amplification Control

2005 ◽  
Vol 71 (12) ◽  
pp. 9008-9012 ◽  
Author(s):  
David Rodríguez-Lázaro ◽  
Maria Pla ◽  
Mariela Scortti ◽  
Héctor J. Monzó ◽  
José A. Vázquez-Boland
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Internal Amplification Control ◽  
Taqman Probe ◽  
Quantitative Detection ◽  
Pcr Assay ◽  
Double Stranded Dna ◽  
Specificity And Sensitivity ◽  
Food Borne ◽  
Smoked Salmon

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.

scholarly journals Real-Time PCR Assay To Differentiate Listeriolysin S-Positive and -Negative Strains ofListeria monocytogenes

2010 ◽  
Vol 77 (1) ◽  
pp. 163-171 ◽  
Author(s):  
Evelyn M. Clayton ◽  
Colin Hill ◽  
Paul D. Cotter ◽  
R. Paul Ross
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Food Industry ◽  
Significant Proportion ◽  
Pcr Assay ◽  
Deletion Mutagenesis ◽  
Positive Strain ◽  
Food Borne ◽  
Oflisteria Monocytogenes ◽  
Almost All

ABSTRACTDue to the severity of the food-borne infection listeriosis, strict legislation governs the detectable and permissible limits at whichListeria monocytogenesis permitted in foods. These requirements, coupled with the ubiquitous nature ofL. monocytogenesstrains and the potential for epidemic outbreaks, mean that the pathogen can devastate affected sectors of the food industry. Although almost allL. monocytogenesstrains have the potential to cause listeriosis, those implicated in the vast majority of epidemics belong to a subset of strains belonging to evolutionary lineage I. It has been established that a significant proportion of these strains, including those implicated in the majority of outbreaks, produce an additional hemolysin, designated listeriolysin S (LLS), which may be responsible for the enhanced virulence of these strains. In order to ultimately establish this definitively, it is important to first be able to rapidly discriminate between LLS-positive and -negative strains. Here, after essential genes within the LLS-encoding cluster,Listeriapathogenicity island 3, were identified by deletion mutagenesis, a real-time PCR assay which targets one such gene,llsX, was developed as a means of identifying LLS-positiveL. monocytogenes. The specificity of the assay was validated against a panel of 40L. monocytogenesstrains (20 of which were LLS positive) and 25 strains representative of otherListeriaspecies. Furthermore, 1 CFU of an LLS-positive strain per 25 g/ml of spiked foods was detected in less than 30 h when the assay was coupled with culture enrichment. The detection limit of this assay was 10 genome equivalents.

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