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Related Concept Videos

PCR01:32

PCR

Overview
Real Time RT-PCR02:57

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
DNA Isolation01:24

DNA Isolation

DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...
PCR - Polymerase Chain Reaction01:32

PCR - Polymerase Chain Reaction

Overview

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Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Antisense Protein (ASP) RNA Transcripts in Patients by Strand-Specific RT-PCR
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Aspergillus PCR: one step closer to standardization.

P Lewis White1, Stéphane Bretagne, Lena Klingspor

  • 1NPHS Microbiology Cardiff, University Hospital of Wales, Heath Park, Cardiff CF14 4XN, United Kingdom. lewis.white@nphs.wales.nhs.uk

Journal of Clinical Microbiology
|February 12, 2010
PubMed
Summary
This summary is machine-generated.

Standardizing the Polymerase Chain Reaction (PCR) for invasive aspergillosis diagnosis is crucial. Optimized DNA extraction and internal controls significantly improve PCR sensitivity, enabling reliable clinical use.

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Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
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Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

Published on: March 30, 2015

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Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Antisense Protein (ASP) RNA Transcripts in Patients by Strand-Specific RT-PCR
08:01

Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Antisense Protein (ASP) RNA Transcripts in Patients by Strand-Specific RT-PCR

Published on: November 27, 2019

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
08:37

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

Published on: March 30, 2015

Area of Science:

  • Medical Mycology
  • Molecular Diagnostics
  • Infectious Diseases

Background:

  • Polymerase Chain Reaction (PCR) has been utilized for invasive aspergillosis diagnosis for nearly two decades.
  • Lack of standardization has hindered its acceptance as a diagnostic tool and inclusion in disease criteria.
  • The European Aspergillus PCR Initiative was established in 2006 to address these limitations.

Purpose of the Study:

  • To develop and recommend standardized protocols for Aspergillus PCR to enable widespread clinical evaluation.
  • To determine the diagnostic role of Aspergillus PCR and facilitate its inclusion in disease diagnosis criteria.
  • To identify factors influencing the efficiency and sensitivity of Aspergillus PCR assays.

Main Methods:

  • Development and distribution of quality control panels to participating centers.
  • Classification of centers as 'compliant' or 'noncompliant' based on adherence to proposed recommendations.
  • Meta-regression analysis to evaluate performance parameters and identify correlations with methodology.

Main Results:

  • PCR amplification systems showed similar detection thresholds, but positivity depended on fungal burden.
  • 50% of centers failed to achieve consistent detection when DNA extraction was combined with PCR.
  • Sensitivity correlated positively with recommended extraction protocols, bead beating, white cell lysis buffer, and internal controls.
  • Elution volumes >100 microliters negatively correlated with sensitivity.
  • Overall efficiency was limited by DNA extraction, not PCR amplification.

Conclusions:

  • Standardized DNA extraction protocols, including efficient lysis of large blood volumes (>or=3 ml) and bead beating, are essential for Aspergillus PCR.
  • The use of an internal control PCR is critical to prevent false negatives.
  • Optimized extraction procedures and elution volumes (<100 microliters) are necessary for reliable Aspergillus PCR testing in whole blood.