Rapid and sensitive diagnosis of adenoviral keratoconjunctivitis by loop-mediated isothermal amplification (LAMP)
Taketoshi Wakabayashi1, Ryoko Yamashita, Tetsuhiko Kakita
1Department of Ophthalmology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan. wakatake@md.tsukuba.ac.jp
Purpose:
To develop a new method to detect and type adenoviruses directly from conjunctival scrapings using loop-mediated isothermal amplification (LAMP) with adenovirus (ad) type specific primer.
Methods:
Using primers specific for the gene of ad1, ad3, ad4, ad8, ad19 and ad37, heat denatured adenovirus DNA was amplified by the LAMP and polymerase chain reaction (PCR). Alkaline lysed adenovirus prototype and conjunctival scrapings were also used directly as templates.
Results:
Type specific primers amplified ad genes of the corresponding ad prototype specifically. The specific amplification was observed in both heat denatured and alkaline lysed samples. The amplified product was first detected within 45 min. Ad genotypes of clinical samples determined by the LAMP method were almost identical to those determined using the PCR-sequencing method.
Conclusions:
LAMP based isothermal amplification of adenovirus genome for detection and typing of adenoviruses is faster than PCR based methods. This new method will be useful for rapid diagnosis and typing of adenoviral conjunctivitis.
