Quantitative Two-Step RT-PCR for the Detection of Human ABCA1 Transporter on LightCycler Using Hybridization Probes and External Standards

2002 ◽  
pp. 15-25
Author(s):  
Danuta Kielar ◽  
Wolfgang Dietmaier ◽  
Thomas Langmann ◽  
Charalampos Aslanidis ◽  
Mario Probst ◽  
...  
Keyword(s):  
Rt Pcr ◽  
Hybridization Probes

Performance Comparison of Multiplexed Fluorescent Resonance Emission Transfer Hybridization Probes Across Roche LightCycler® Real-Time PCR Systems for the Detection of Bartonella species

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S134-S135
Author(s):  
T Berent ◽  
T Rothstein ◽  
S Buckwalter ◽  
R Patel
Keyword(s):  
Real Time ◽  
Whole Blood ◽  
New Technologies ◽  
Limit Of Detection ◽  
Rt Pcr ◽  
Bartonella Species ◽  
Pcr Assay ◽  
Fixed Tissue ◽  
Hybridization Probes ◽  
Ffpe Samples

Abstract Introduction/Objective Molecular assays for Bartonella species are important in diagnosing infection and expediting patient treatment. Real time polymerase chain reaction (RT-PCR) using fluorescent resonance energy transfer (FRET) hybridization probes can be used to detect Bartonella species in blood and fresh/fixed tissue biopsies in RT-PCR instruments. Over time, new technologies and reagents are introduced and existing PCR primers and FRET probes must be re-validated on new platforms. This study aimed to compare the performance of a Bartonella RT-PCR assay using the sunsetting Roche LightCycler® 2.0 (Roche Diagnostics, Indianapolis, IN) and newer LightCycler® 480 RT- PCR instruments. Methods/Case Report DNA was extracted from 132 historically positive, whole organism spiked, and historically negative whole blood and formalin fixed paraffin embedded (FFPE) samples. Samples were run on the LightCycler® 2.0 using instrument specific LightCycler® FastStart DNA Master HybProbe enzyme and compared to results generated using the LightCycler® 480 and its instrument specific LightCycler® 480 Genotyping Master enzyme. During optimization, MgCl2 concentrations and thermocycling profiles were adjusted. Accuracy, specificity, inclusivity, and limit of detection studies were performed. Crossing point (Cp), melting temperature (Tm), fluorescent peak and fluorescent background values were compared between the two instruments. Results (if a Case Study enter NA) The agreement in accuracy between the LightCycler® 2.0 and the LightCycler® 480 was 100% for whole blood samples. For historically positive FFPE samples, LightCycler® 2.0 sensitivity and LightCycler® 480 sensitivity were 86% and 100%, respectively. Specificity and inclusivity of the assay were identical between the two instruments. The limit of detection in whole blood was 5-fold lower on the LightCycler® 480 (50 copies/µL) compared to the LightCycler® 2.0 (250 copies/µL). Mean Cp and fluorescent peak intensity values increased by 5.1% and 65-fold, respectively. Conclusion The study demonstrates similar performance and improved limit of detection for the Bartonella FRET hybridization probe RT-PCR assay on the LightCycler® 480 compared to the LightCycler® 2.0.

scholarly journals Comparative detection and enumeration strengths of quantitative real-time PCR & FISH for waterborne bacterial pathogens in municipal wastewater

2021 ◽  
Author(s):  
Merriam Haffar
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Municipal Wastewater ◽  
Bacterial Pathogens ◽  
Virulence Gene ◽  
Target Cells ◽  
Rt Pcr ◽  
Hybridization Probes ◽  
Gene Copies ◽  
Pure Cultures

This study comparatively evaluates the detection and enumeration strengths of Real-Time PCR (RT PCR) and FISH, for selected bacterial pathogens in municipal wastewater. Both assays were performed using three primer and probe sets complementary to the same chromosomal virulence gene sequences. Primer & probe specificity was confirmed with DNA & fixed cells from pure bacterial cultures as well as seeded wastewater samples. Detection limits calculated for the RT PCR assay were 25 to 3030 tir gene copies for Escherichia coli O157:H7 and 3 x 10⁴ to 293 x10⁷ invA gene copies for Salmonella enterica, using pure cultures and seeded wasewater samples, respectively. In spite of the confirmed specificity of the DNA hybridization probes with target nucleic acids, fluorescent signals from hybridized whole target cells were below the detection limit of the FISH assay, and consequently were not quantified. This research demonstrates both the utility of RT PCR in detecting bacterial pathogens and the need for further optimization with DNA-targeted FISH, using environmental samples.

scholarly journals A prolonged outbreak of Norwalk-like calicivirus (NLV) gastroenteritis in a rehabilitation centre due to environmental contamination

2002 ◽  
Vol 129 (1) ◽  
pp. 133-138 ◽  
Author(s):  
M. KUUSI ◽  
J. P. NUORTI ◽  
L. MAUNULA ◽  
N. N. TRAN MINH ◽  
M. RATIA ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Environmental Contamination ◽  
Water Samples ◽  
Epidemiological Investigation ◽  
Rt Pcr ◽  
Rehabilitation Centre ◽  
Hybridization Probe ◽  
Staff Members ◽  
Hybridization Probes ◽  
Stool Samples

An outbreak of Norwalk-like calicivirus (NLV) gastroenteritis occurred in a rehabilitation centre in southern Finland between December 1999 and February 2000. An epidemiological investigation was conducted to determine the source and extent of the outbreak. More than 300 guests and staff members became ill during the outbreak. No food or activity in the centre could be linked epidemiologically to illness. NLV genogroup II was detected by RT–PCR in stool samples of symptomatic guests and employees. All strains reacted similarly with the microplate hybridization probe panel and showed the same nucleotide sequence, indicating that they represented the same NLV strain. Food and water samples were negative for NLV, whereas NLV was detected in three environmental specimens. The strains from patients and environment were identical based on microplate hybridization probes, suggesting that environmental contamination may have been important for the spread of calicivirus and the protracted course of the outbreak.

scholarly journals Real-time PCR assay for the diagnosis of pleural tuberculosis

2017 ◽  
pp. 47-52
Author(s):  
Martha Alejandra Casallas-Rivera ◽  
Ana María Cardenas Bernal ◽  
Luis Fernando Giraldo-Cadavid ◽  
Enrique Prieto Diago ◽  
Paola Santander
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reference Method ◽  
Operating Characteristics ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Cross Sectional ◽  
Pleural Tuberculosis ◽  
Hybridization Probes ◽  
Polymerase Chain

Introduction: The diagnosis of pleural tuberculosis requires an invasive and time-consuming reference method. Polymerase chain reaction (PCR) is rapid, but validation in pleural tuberculosis is still weak.Objective: To establish the operating characteristics of real-time polymerase chain reaction (RT-PCR) hybridization probes for the diagnosis of pleural tuberculosis.Method: The validity of the RT-PCR hybridization probes was evaluated compared to a composite reference method by a cross-sectional study at the Hospital Universitario de la Samaritana. 40 adults with lymphocytic pleural effusion were included. Pleural tuberculosis was confirmed (in 9 patients) if the patient had at least one of three tests using the positive reference method: Ziehl-Neelsen or Mycobacterium tuberculosis culture in fluid or pleural tissue, or pleural biopsy with granulomas. Pleural tuberculosis was ruled out (in 31 patients) if all three tests were negative. The operating characteristics of the RT-PCR, using the Mid-P Exact Test, were determined using the OpenEpi 2.3 Software (2009).Results: The RT-PCR hybridization probes showed a sensitivity of 66.7% (95% CI: 33.2%-90.7%) and a specificity of 93.5% (95% CI: 80.3%-98.9%). The PPV was 75.0% (95% CI: 38.8%-95.6%) and a NPV of 90.6% (95% CI: 76.6%-97.6%). Two false positives were found for the test, one with pleural mesothelioma and the other with chronic pleuritis with mesothelial hyperplasia.Conclusions: The RT-PCR hybridization probes had good specificity and acceptable sensitivity, but a negative value cannot rule out pleural tuberculosis.

scholarly journals Comparative detection and enumeration strengths of quantitative real-time PCR & FISH for waterborne bacterial pathogens in municipal wastewater

2021 ◽  
Author(s):  
Merriam Haffar
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Municipal Wastewater ◽  
Bacterial Pathogens ◽  
Virulence Gene ◽  
Target Cells ◽  
Rt Pcr ◽  
Hybridization Probes ◽  
Gene Copies ◽  
Pure Cultures

This study comparatively evaluates the detection and enumeration strengths of Real-Time PCR (RT PCR) and FISH, for selected bacterial pathogens in municipal wastewater. Both assays were performed using three primer and probe sets complementary to the same chromosomal virulence gene sequences. Primer & probe specificity was confirmed with DNA & fixed cells from pure bacterial cultures as well as seeded wastewater samples. Detection limits calculated for the RT PCR assay were 25 to 3030 tir gene copies for Escherichia coli O157:H7 and 3 x 10⁴ to 293 x10⁷ invA gene copies for Salmonella enterica, using pure cultures and seeded wasewater samples, respectively. In spite of the confirmed specificity of the DNA hybridization probes with target nucleic acids, fluorescent signals from hybridized whole target cells were below the detection limit of the FISH assay, and consequently were not quantified. This research demonstrates both the utility of RT PCR in detecting bacterial pathogens and the need for further optimization with DNA-targeted FISH, using environmental samples.

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