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.

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 Misidentification of Bordetella bronchiseptica as Bordetella pertussis using a newly described real-time PCR targeting the pertactin gene

2007 ◽  
Vol 56 (12) ◽  
pp. 1608-1610 ◽  
Author(s):  
Karen B. Register ◽  
Tracy L. Nicholson
Keyword(s):  
Real Time ◽  
Bordetella Pertussis ◽  
Real Time Pcr ◽  
Gene Sequence ◽  
Significant Proportion ◽  
Bordetella Bronchiseptica ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Avian Origin ◽  
Pcr Targeting

Recently, a real-time PCR (RT-PCR) assay based on sequence from the gene for pertactin was proposed for identification of Bordetella pertussis. Here, it is reported that the B. pertussis pertactin gene sequence for the region that encompasses the RT-PCR probe and primers is nearly identical to that of many Bordetella bronchiseptica strains of human and avian origin. Additionally, it is demonstrated that such strains are erroneously identified as B. pertussis using the RT-PCR assay. These data suggest that the use of the assay without confirmatory testing may result in erroneous identification of a significant proportion of human isolates of B. bronchiseptica as B. pertussis.

scholarly journals Diagnostic Real-Time PCR Assays for the Detection of Emetic Bacillus cereus Strains in Foods and Recent Food-Borne Outbreaks

2007 ◽  
Vol 73 (6) ◽  
pp. 1892-1898 ◽  
Author(s):  
Martina Fricker ◽  
Ute Messelhäußer ◽  
Ulrich Busch ◽  
Siegfried Scherer ◽  
Monika Ehling-Schulz
Keyword(s):  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr ◽  
Care Center ◽  
Food Poisoning ◽  
Sybr Green ◽  
Sybr Green I ◽  
Taqman Assay ◽  
Pcr Assay ◽  
Food Borne

ABSTRACT Cereulide-producing Bacillus cereus can cause an emetic type of food-borne disease that mimics the symptoms provoked by Staphylococcus aureus. Based on the recently discovered genetic background for cereulide formation, a novel 5′ nuclease (TaqMan) real-time PCR assay was developed to provide a rapid and sensitive method for the specific detection of emetic B. cereus in food. The TaqMan assay includes an internal amplification control and primers and a probe designed to target a highly specific part of the cereulide synthetase genes. Additionally, a specific SYBR green I assay was developed and extended to create a duplex SYBR green I assay for the one-step identification and discrimination of the two emesis-causing food pathogens B. cereus and S. aureus. The inclusivity and exclusivity of the assay were assessed using a panel of 100 strains, including 23 emetic B. cereus and 14 S. aureus strains. Different methods for DNA isolation from artificially contaminated foods were evaluated, and established real-time assays were used to analyze two recent emetic food poisonings in southern Germany. One of the food-borne outbreaks included 17 children visiting a day care center who vomited after consuming a reheated rice dish, collapsed, and were hospitalized; the other case concerned a single food-poisoning incident occurring after consumption of cauliflower. Within 2 h, the etiological agent of these food poisonings was identified as emetic B. cereus by using the real-time PCR assay.

scholarly journals A misleading false-negative result of Pneumocystis real-time PCR assay due to a rare punctual mutation: A French multicenter study

2016 ◽  
Vol 55 (2) ◽  
pp. 180-184 ◽  
Author(s):  
Solène Le Gal ◽  
Florence Robert-Gangneux ◽  
Yann Pépino ◽  
Sorya Belaz ◽  
Céline Damiani ◽  
...  
Keyword(s):  
Real Time ◽  
Multicenter Study ◽  
Real Time Pcr ◽  
False Negative ◽  
False Negative Result ◽  
Pcr Assay

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

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