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Ge Zhao, Qing Yuan; Rapid and sensitive diagnosis of Acanthamoeba keratitis by loop-mediated isothermal amplification. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2898.
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To develope a loop-mediated isothermal amplification (LAMP) assay for the detection of Acanthamoeba.
The sensitivity of the LAMP assay was tested using different copies of positive DNA. The specificity of the assay was tested using DNA extracted from Acanthamoeba, Pseudomonas aeruginosa, Candida albicans, herpes simplex virus-1 and human corneal epithelial cells. The effectiveness of the LAMP assay was evaluated and compared with culture, corneal smear examination and real-time PCR in the corneal samples of Acanthamoeba keratitis (AK) mice. We also tested 3 corneal samples from patients with suspected Acanthamoeba or fungal infection using LAMP.
LAMP was confirmed to be very sensitive, with the lowest detection limit being 10 copies/tube of Acanthamoeba DNA. The LAMP primers only amplified Acanthamoeba DNA. During the development of AK in mice, almost all of the positive rates of LAMP at each time point post-infection were higher than those of culture or corneal smear examination. The total positive rate of LAMP was significantly higher than those of culture and corneal smear examination (P<0.05), while the sensitivity of LAMP and real-time PCR was comparable. However, the trends of positive change in these different test methods were generally similar. Of the three clinical corneal specimens, two suspected AK were tested positive for Acanthamoeba using LAMP along with culture or corneal smear examination, while the other suspected fungal keratitis was tested negative.
The LAMP assay is a simple, rapid, highly specific and sensitive method for the diagnosis of keratitis caused by Acanthamoeba.
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