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Rapid diagnosis of adenoviral conjunctivitis by PCR and restriction fragment length polymorphism analysis
W Saitoh-Inagawa1, A Oshima, K Aoki
1Department of Ophthalmology, Yokohama City University School of Medicine, Kanagawa-ken, Japan.
Abstract:
To detect and identify adenovirus (Ad), we used a combination of PCR and restriction fragment length polymorphism (RFLP) analysis. Nested PCR with two primer sets that hybridize to the conserved region for hexon proteins of 14 prototypes of Ad, Ad serotype 1 (Ad1) to Ad8, -11, -14, -19, -37, -40, and -41, amplified a 956-bp DNA fragment. The amplified fragments from the 14 prototypes were completely differentiated with a combination of three restriction endonucleases, EcoT14I, HaeIII, and HintI. We applied this new method for 127 samples of conjunctival scrapings from patients with conjunctivitis and compared the results with those obtained with the combination of culture isolation and a neutralization test (NT). PCR gave a positive result in 69 of 127 cases (54.3%), while only 61 of the 127 samples (48.0%) tested positive by culture isolation. Compared with isolation, the PCR method had a sensitivity of 100% (61 of 61). Positive PCR samples were further classified as Ad37 (59.5%), -3(31.9%), -11 (4.3%), -8 (2.9%), and -4 (1.4%) by PCR-RFLP analysis. Of eight samples that were PCR positive and culture isolation negative, six were Ad37 and two were Ad8 by PCR-RFLP analysis. These differentiations of isolation-positive samples were identical to the results obtained by the NT. It took only 3 days to detect and identify Ad by PCR-RFLP analysis, whereas it took at least 3 weeks by culture isolation and NT. Our newly developed method of detecting and typing human Ad by PCR-RFLP analysis is more sensitive, accurate, and rapid than the conventional method of culture isolation and an NT.
Insights
A new PCR-RFLP method rapidly and accurately detects adenovirus (Ad) in conjunctivitis patients. This molecular technique offers higher sensitivity and speed than traditional culture isolation and neutralization tests.
Area of Science:
- Ophthalmology
- Virology
- Molecular Biology
Background:
- Adenovirus (Ad) is a common cause of conjunctivitis.
- Conventional detection methods, such as culture isolation and neutralization tests (NT), are time-consuming and may lack sensitivity.
Purpose of the Study:
- To develop and evaluate a novel, rapid, and sensitive method for detecting and identifying adenovirus in clinical samples.
- To compare the performance of the new method against established diagnostic techniques.
Main Methods:
- Developed a nested PCR assay targeting conserved hexon protein regions of 14 adenovirus prototypes.
- Utilized restriction fragment length polymorphism (RFLP) analysis with three restriction enzymes (EcoT14I, HaeIII, HintI) for serotype differentiation.
- Applied the PCR-RFLP method to 127 conjunctival scraping samples and compared results with culture isolation and NT.
Main Results:
- The PCR method detected Ad in 54.3% of samples, exceeding the 48.0% detection rate of culture isolation.
- PCR demonstrated 100% sensitivity compared to culture isolation.
- PCR-RFLP successfully identified adenovirus types, with Ad37 being the most prevalent (59.5%), followed by Ad3 (31.9%).
- The PCR-RFLP method provided results in 3 days, significantly faster than the 3 weeks required for culture isolation and NT.
Conclusions:
- The developed PCR-RFLP method is a highly sensitive, accurate, and rapid diagnostic tool for adenovirus detection and typing.
- This molecular approach offers a significant advantage over conventional methods for diagnosing adenovirus-related conjunctivitis.