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Published on: November 12, 2015
Identification of subgenus C adenoviruses by fiber-based multiplex PCR
Arun Kumar Adhikary1, Toshiki Inada, Urmila Banik
1Infectious Disease Surveillance Center, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan. arun@nih.go.jp
Abstract:
Subgenus C human adenoviruses, which include serotypes 1, 2, 5, and 6, are often associated with respiratory illness, ocular infections, gastroenteritis, and systemic infection among immunocompromised patients. To address the problems associated with the conventional typing methods, we developed a fiber-based multiplex PCR assay for simple and specific identification of adenovirus type 1, 2, 5, and 6 field isolates. To design type-specific primers, adenovirus type 1 and 6 fiber genes were sequenced. The assay correctly identified prototype strains of adenovirus serotypes 1, 2, 5, 6, as well as 21 previously typed adenovirus field isolates. Mixing two different prototype DNAs produced two amplicons of different lengths, thus clearly distinguishing the prototypes. The results correlated 100% with serological tests and 95% with the previously described PCR-restriction fragment length polymorphism method. The detection of dual infection is an added benefit of the assay. No nonspecific amplification was detected with other adenovirus serotypes or with nonadenoviral DNA. Our fiber-based multiplex PCR assay will provide a convenient tool for type-specific identification of subgenus C adenovirus isolates in various clinical situations and in epidemiological investigations and is a better alternative than the hexon-based assay.
Insights
A new fiber-based multiplex PCR assay accurately identifies human adenovirus types 1, 2, 5, and 6. This method offers a simple, specific, and convenient tool for clinical and epidemiological investigations of adenovirus infections.
Area of Science:
- Virology
- Molecular Biology
- Infectious Diseases
Background:
- Subgenus C human adenoviruses (serotypes 1, 2, 5, 6) are linked to various illnesses, including respiratory and ocular infections.
- Conventional typing methods for these adenoviruses can be problematic and time-consuming.
- Accurate identification is crucial for clinical management and epidemiological tracking.
Purpose of the Study:
- To develop a novel, simple, and specific method for identifying human adenovirus serotypes 1, 2, 5, and 6.
- To overcome limitations associated with existing adenovirus typing techniques.
- To provide a reliable tool for clinical and public health applications.
Main Methods:
- Development of a fiber-based multiplex PCR assay.
- Sequencing of adenovirus type 1 and 6 fiber genes for primer design.
- Testing the assay with prototype strains and previously typed adenovirus field isolates.
Main Results:
- The assay correctly identified prototype strains and 21 field isolates of adenovirus serotypes 1, 2, 5, and 6.
- Distinguishing between prototypes was achieved by observing amplicons of different lengths.
- The assay demonstrated 100% correlation with serological tests and 95% with PCR-restriction fragment length polymorphism.
- The assay also detected dual adenovirus infections and showed no cross-reactivity with other adenovirus serotypes or non-adenoviral DNA.
Conclusions:
- The developed fiber-based multiplex PCR assay is a convenient and accurate tool for type-specific identification of subgenus C adenovirus isolates.
- This assay is a superior alternative to hexon-based methods for clinical and epidemiological investigations.
- The assay facilitates rapid and specific diagnosis of adenovirus infections in diverse settings.
