scholarly journals Real-time TaqMan PCR for rapid detection and typing of genes encoding CTX-M extended-spectrum β-lactamases

2007 ◽  
Vol 56 (1) ◽  
pp. 52-55 ◽  
Author(s):  
Christopher I. Birkett ◽  
Hugo A. Ludlam ◽  
Neil Woodford ◽  
Derek F. J. Brown ◽  
Nicholas M. Brown ◽  
...  
Keyword(s):  
Real Time ◽  
Taqman Assay ◽  
Esbl Production ◽  
Pcr Assay ◽  
Extended Spectrum ◽  
Genes Encoding ◽  
Taqman Pcr ◽  
Assay Time ◽  
Phenotypic Methods ◽  
Single Reaction

The prevalence of CTX-M-producing members of the Enterobacteriaceae is increasing worldwide. A novel, multiplex, real-time TaqMan PCR assay to detect and type bla CTX-M genes is described which is an improvement on previously described techniques with respect to reduced assay time, elimination of the need for protracted post-PCR processing and the convenience of a single reaction vessel. Based on β-lactam antibiogram and MIC data, 478 of 1279 Enterobacteriaceae isolates from clinical blood and urine culture specimens were selected and tested for extended-spectrum β-lactamase (ESBL) production using phenotypic methods. The new TaqMan assay detected and typed bla CTX-M genes in 21 of 28 ESBL-producing isolates.

scholarly journals Automated 5′ Nuclease PCR Assay for Identification of Salmonella enterica

2000 ◽  
Vol 38 (9) ◽  
pp. 3429-3435 ◽  
Author(s):  
J. Hoorfar ◽  
P. Ahrens ◽  
P. Rådström
Keyword(s):  
Salmonella Enterica ◽  
Relative Sensitivity ◽  
Positive Control ◽  
Taqman Assay ◽  
Diagnostic Specificity ◽  
Pcr Assay ◽  
Sample Tube ◽  
Simple Alternative ◽  
Taqman Pcr ◽  
Internal Positive Control

A simple and ready-to-go test based on a 5′ nuclease (TaqMan) PCR technique was developed for identification of presumptiveSalmonella enterica isolates. The results were compared with those of conventional methods. The TaqMan assay was evaluated for its ability to accurately detect 210 S. enterica isolates, including 100 problematic “rough” isolates. An internal positive control was designed to use the same Salmonella primers for amplification of a spiked nonrelevant template (116 bp) in the sample tube. The PCR test correctly identified all the Salmonellastrains by resulting in positive end-point fluorescence (FAM) signals for the samples and positive control (TET) signals (relative sensitivity [ΔRn], >0.6). The diagnostic specificity of the method was assessed using 120 non-Salmonella strains, which all resulted in negative FAM signals (ΔRn, ≤0.5). All 100 roughSalmonella strains tested resulted in positive FAM and TET signals. In addition, it was found that the complete PCR mixture, predispensed in microwell plates, could be stored for up to 3 months at −20°C. Thus, the diagnostic TaqMan assay developed can be a useful and simple alternative method for identification ofSalmonella, particularly in large reference laboratories.

Differentiation of herpes simplex virus types 1 and 2 in clinical samples by a real-time taqman PCR assay

2005 ◽  
Vol 76 (3) ◽  
pp. 350-355 ◽  
Author(s):  
Lawrence Corey ◽  
Meei-Li Huang ◽  
Stacy Selke ◽  
Anna Wald
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Real Time ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Taqman Pcr ◽  
Simplex Virus

scholarly journals Performance of the Phoenix bacterial identification system compared with disc diffusion methods for identifying extended-spectrum β-lactamase, AmpC and KPC producers

2009 ◽  
Vol 58 (6) ◽  
pp. 774-778 ◽  
Author(s):  
Mark A. Fisher ◽  
Paul D. Stamper ◽  
Kristine M. Hujer ◽  
Zachary Love ◽  
Ann Croft ◽  
...  
Keyword(s):  
Boronic Acid ◽  
Identification System ◽  
Bacterial Identification ◽  
Esbl Production ◽  
Pcr Screening ◽  
Extended Spectrum ◽  
Manual Testing ◽  
Phenotypic Methods ◽  
Testing Algorithm ◽  
The Phoenix

Phenotypic identification of AmpC, KPC and extended-spectrum β-lactamases (ESBLs) among members of the Enterobacteriaceae remains challenging. This study compared the Phoenix Automated Microbiology System (BD Diagnostics) with the Clinical and Laboratory Standards Institute confirmatory method to identify ESBL production among 200 Escherichia coli and Klebsiella pneumoniae clinical isolates. The Phoenix system misclassified nearly half of the isolates as ESBL-positive, requiring manual testing for confirmation. Inclusion of aztreonam±clavulanic acid (CA) and cefpodoxime±CA in the testing algorithm increased the ESBL detection rate by 6 %. Boronic acid-based screening identified 24 isolates as AmpC+, but in a subset of genotypically characterized isolates, appeared to have a high false-positivity rate. PCR screening revealed eight KPC+ isolates, all of which tested as ESBL+ or ESBL+ AmpC+ by phenotypic methods, but half were reported as carbapenem-susceptible by the Phoenix system. Overall, these results indicate that laboratories should use the Phoenix ESBL results only as an initial screen followed by confirmation with an alternative method.

scholarly journals An Internal Reaction Control for Routine Detection of Clavibacter michiganensis subsp. sepedonicus Using a Real-Time TaqMan PCR-Based Assay

Plant Disease ◽  
2008 ◽  
Vol 92 (5) ◽  
pp. 684-693 ◽  
Author(s):  
Donna S. Smith ◽  
Solke H. De Boer ◽  
Jane Gourley
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Taqman Assay ◽  
Clavibacter Michiganensis ◽  
Causal Organism ◽  
Melt Temperature ◽  
Clavibacter Michiganensis Subsp ◽  
Internal Reaction ◽  
Control Plasmid ◽  
Taqman Pcr

An internal reaction control was integrated into a TaqMan polymerase chain reaction (PCR) assay for the detection of Clavibacter michiganensis subsp. sepedonicus, the causal organism of bacterial ring rot of potato. The reaction control, cloned into plasmid pCmsC4, consisted of a sequence unrelated to C. michiganensis subsp. sepedonicus flanked by the primer sequences used in the TaqMan PCR, thus eliminating the need for multiplexing. Inclusion of the reaction control plasmid in the TaqMan assay had no effect on either the limit of detection or the specificity of the method. Addition of SYBR Green permitted melt analysis of PCR products. The 242-bp reaction control amplicon, with a melt temperature of approximately 94.5°C, could easily be distinguished from the 152-bp primary diagnostic target amplicon, which had a melt temperature of about 85.5°C. Electrophoretic analysis showed that appearance of either melt peak correlated well with the presence of the appropriate amplicon. Two different substances, guanidine-HCl and humic acid, inhibited the amplification of the reaction control at concentrations lower than those that inhibited the primary diagnostic target, demonstrating the reaction control's effectiveness in detecting inhibition or reaction failure. Using the reaction control plasmid, a quantitative threshold for inhibitor detection was established. This permitted the validation of negative results, and thus facilitated the use of TaqMan real-time PCR in the routine testing of diagnostic samples for C. michiganensis subsp. sepedonicus.

scholarly journals Quantitative Assessment of the Tetracycline Resistance Gene Pool in Cheese Samples by Real-Time TaqMan PCR

2007 ◽  
Vol 73 (5) ◽  
pp. 1676-1677 ◽  
Author(s):  
Michele Y. Manuzon ◽  
Scott E. Hanna ◽  
Hongliang Luo ◽  
Zhongtang Yu ◽  
W. James Harper ◽  
...  
Keyword(s):  
Real Time ◽  
Gene Pool ◽  
Antibiotic Resistance Genes ◽  
Tetracycline Resistance ◽  
Food Samples ◽  
Independent Method ◽  
Pcr Assay ◽  
Tetracycline Resistance Gene ◽  
Culture Independent ◽  
Taqman Pcr

ABSTRACT A TaqMan real-time PCR assay was developed to quantify the tetS gene pool present in retail cheeses. This protocol offers a rapid, specific, sensitive, and culture-independent method for assessing antibiotic resistance genes in food samples rich in fats and proteins.

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