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. 2005 Jul;43(7):3290-6.
doi: 10.1128/JCM.43.7.3290-3296.2005.

Sensitive and rapid detection of herpes simplex virus and varicella-zoster virus DNA by loop-mediated isothermal amplification

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Sensitive and rapid detection of herpes simplex virus and varicella-zoster virus DNA by loop-mediated isothermal amplification

Hisatoshi Kaneko et al. J Clin Microbiol. 2005 Jul.

Abstract

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 degrees 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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Figures

FIG. 1.
FIG. 1.
Location and sequence of LAMP target and primer regions for HSV-1 (A), HSV-2 (B), and VZV (C). The single nucleotide polymorphisms of three HSV-1 strains (the G1-4, conj10, and KH15 strains) are shaded. Arrows indicate the nucleotide bases of strains with polymorphism.
FIG. 1.
FIG. 1.
Location and sequence of LAMP target and primer regions for HSV-1 (A), HSV-2 (B), and VZV (C). The single nucleotide polymorphisms of three HSV-1 strains (the G1-4, conj10, and KH15 strains) are shaded. Arrows indicate the nucleotide bases of strains with polymorphism.
FIG. 1.
FIG. 1.
Location and sequence of LAMP target and primer regions for HSV-1 (A), HSV-2 (B), and VZV (C). The single nucleotide polymorphisms of three HSV-1 strains (the G1-4, conj10, and KH15 strains) are shaded. Arrows indicate the nucleotide bases of strains with polymorphism.
FIG. 2.
FIG. 2.
Electrophoretic analysis of LAMP-amplified products. Lanes 1, 2, and 3, LAMP carried out with HSV-1 primers and genomic DNAs from HSV-1, HSV-2, and VZV, respectively; lane 4, LAMP product from lane 1 after digestion with PstI; lanes 5, 10, and 15, LAMP carried out in the absence of template DNA with the HSV-1, HSV-2, and VZV primers, respectively; lanes 6, 7, and 8, LAMP carried out with HSV-2 primers in the presence of genomic DNAs from HSV-1, HSV-2, and VZV, respectively; lane 9, LAMP product from lane 7 after digestion with HaeIII; lanes 11, 12, and 13, LAMP carried out with VZV primers in the presence of genomic DNAs from HSV-1, HSV-2, and VZV, respectively; lane 14, LAMP product from lane 13 after digestion with AluI. The positions of size markers are shown on the left.
FIG. 3.
FIG. 3.
Comparative sensitivities of LAMP and PCR for the detection of HSV-1 (A) and HSV-2 (B). Amplification by LAMP shows a ladder-like pattern, whereas that by primer set 2 and primer set 3 shows single bands for the amplification products obtained by using the primer set shown in Table 2 and that from a commercial PCR kit, respectively. (C to E) Comparative sensitivities to single nucleotide polymorphisms of three HSV-1 strains. The electrophoretic profiles of the G1-4 strain (C), the conj10 strain (D), and the KH15 strain (E) are shown. Lanes: 1, 106 copies/tube; 2, 105 copies/tube; 3, 104 copies/tube; 4, 103 copies/tube; 5, 102 copies/tube; 6, 101 copies/tube; 7, 100 copies/tube; 8, negative control without target DNA.

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