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Sensitive and Rapid Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA by Loop-Mediated Isothermal Amplification

Department of Microbiology,¹ Department of Ophthalmology, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima 960-1295,² Visual Sciences, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido 060-8638, Japan³

Received 4 October 2004/ Returned for modification 20 November 2004/ Accepted 10 March 2005

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react rapidly and efficiently, with a high specificity, under isothermal conditions. We used a LAMP assay for the detection of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV). The virus specificities of primers were confirmed by using 50 HSV-1, 50 HSV-2, and 8 VZV strains. The assay was performed for 45 min at 65°C. The LAMP assay had a 10-fold higher sensitivity than a PCR assay. An analysis of nucleotide sequence variations in the target and primer regions used for the LAMP assay indicated that 3 of 50 HSV-1 strains had single nucleotide polymorphisms. No HSV-2 or VZV strains had nucleotide polymorphisms. Regardless of the sequence variation, there were no differences in sensitivity with the HSV-1-specific LAMP assay. To evaluate the application of the LAMP assay for clinical diagnosis, we tested clinical samples from 40 genital herpes patients and 20 ocular herpes patients. With the LAMP assay, 41 samples with DNA extraction and 26 direct samples without DNA extraction were identified as positive for HSV-1 or HSV-2, although 37 samples with DNA extraction and just one without DNA extraction were positive by a PCR assay. Thus, the LAMP assay was less influenced than the PCR assay by the presence of inhibitory substances in clinical samples. These observations indicate that the LAMP assay is very useful for the diagnosis of HSV-1, HSV-2, and VZV infections.

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• Kaneko, H., Kawana, T., Ishioka, K., Fukushima, E., Suzutani, T. (2008). Discrimination of Herpes Simplex Virus Type 2 Strains by Nucleotide Sequence Variations. J. Clin. Microbiol. 46: 780-784 [Abstract] [Full Text]