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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
Summary
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.
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.
