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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

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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...
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Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
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Multiplex Solid-Phase Real-Time Polymerase Chain Reaction without DNA Extraction: A Rapid Intraoperative Diagnosis

Satoko Nakano1, Yasuhiro Tomaru2, Toshiaki Kubota1

  • 1Department of Ophthalmology, Oita University, Yufu, Japan.

Ophthalmology
|September 28, 2020
PubMed
Summary
This summary is machine-generated.

A new Direct Strip PCR test offers rapid, easy diagnosis for infectious uveitis without DNA extraction. This method shows high accuracy and concordance with qPCR, improving patient prognosis.

Keywords:
DiagnosisInfectionMultiplex polymerase chain reactionReal-time polymerase chain reactionUveitis

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Area of Science:

  • Molecular diagnostics
  • Ophthalmology
  • Infectious disease detection

Background:

  • Current polymerase chain reaction (PCR) methods for diagnosing infections are often time-consuming and require large sample volumes and skilled technicians.
  • Uveitis, an inflammation of the eye, can be caused by various pathogens, necessitating accurate and rapid etiological diagnosis for effective treatment.

Purpose of the Study:

  • To develop and evaluate a novel, rapid, and user-friendly multiplex real-time PCR test for detecting nine common pathogens causing infectious uveitis.
  • To assess the performance of the Direct Strip PCR assay compared to quantitative PCR (qPCR) in terms of accuracy, speed, and ease of use.

Main Methods:

  • A multicenter prospective evaluation involving 511 participants (infectious uveitis patients and controls) across 18 international institutes.
  • Development of a novel Direct Strip PCR assay that bypasses the need for DNA extraction, enabling rapid detection of nine uveitis pathogens in small sample volumes (20 μl).
  • Validation and comparison of Direct Strip PCR results with established qPCR methods for etiological diagnosis, including intraoperative applications.

Main Results:

  • The Direct Strip PCR test demonstrated rapid detection (1-minute processing, 33-minute total time) with good repeatability, specificity, and long storage stability.
  • High concordance with qPCR was observed (positive concordance rate: 98.8%-100%; negative concordance rate: 99.8%-100%; κ coefficient: 0.969-1.000).
  • Results from Direct Strip PCR and qPCR were highly correlated (ρ = 0.748; P < 0.001), with low interinstitutional variability, even for PCR beginners.

Conclusions:

  • The Direct Strip PCR assay is a rapid, accurate, and user-friendly diagnostic tool for infectious uveitis, comparable in performance to qPCR.
  • This novel PCR method facilitates timely etiological evaluation at the initial patient visit and can be utilized for rapid intraoperative diagnosis.
  • The Direct Strip PCR test holds potential to improve patient prognosis for various infectious diseases by enabling faster diagnosis and treatment initiation.