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- W2393943886 abstract "Objective The aim of this study was to establish a new technique for the sensitive and noninvasive early diagnosis of Acanthamoeba keratitis(AK). Methods DNA was extracted from 5strains of Acanthamoebaisolated from soil and water samples.PCR products of the 18srDNA gene in Acanthamoeba were sequenced.These sequences were compared to 67sets of sequences of the 18srDNA gene of Acanthamoebain GenBank.Real-time PCR(qPCR)primers and TaqMan probes were designed in accordance with sequences of the 18srDNA gene of Acanthamoeba.A T cloning vector was used to produce a standard sample that was then sequenced.A standard curve was created by subjecting different diluted concentrations of the standard sample to qPCR.The minimum detectable sample concentration was determined.Conventional laboratory cultures and qPCR were used to test for other pathogenic microorganisms besides Acanthamoebaand to test for Acanthamoebain eye secretions from patients suspected of having AK.Data were subjected to an F-test in JMP5.0.1and diagnostic assessment software. Results In total,180samples were tested using cultures and qPCR.Acanthamoeba was detected by cultures at a rate of(5±1)%and by qPCR at a rate of(2.9±1.2)%.The difference in the rate of detection was statistically significantly(F=13in the F-test,P0.01).The CT value for qPCR of the standard sample served as the vertical axis(Y),and the log of the sample concentration served as the horizontal axis(X)to create a standard curve with Y=-3.24,X+40.74,R0.99.The detection limit for detection of Acanthamoeba18S rDNA by qPCR was 10copies/UL.The proposed qPCR technique did not detect other pathogenic microorganisms.Diagnostic assessment software was used to compare cultures and qPCR of eye secretions from patients suspected of having AK.qPCR had greater sensitivity(100%)than cultures(62.5%)in the 95% confidence intervals. Conclusion Realtime PCR with a TaqMan probe is a noninvasive,sensitive,specific,highly efficient,economical,and effective way totest for AK and screen patients with suspected AK." @default.
- W2393943886 created "2016-06-24" @default.
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- W2393943886 date "2014-01-01" @default.
- W2393943886 modified "2023-09-23" @default.
- W2393943886 title "Establishment and use of a real-time PCR technique to rapidly diagnosis early Acanthamoeba keratitis" @default.
- W2393943886 hasPublicationYear "2014" @default.
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