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

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°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.Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) and varicella-zoster virus (VZV) are alphaherpesviruses that infect, establish lifelong latency in, and subsequently reactivate from human sensory neuronal ganglia (1,20). The reactivation of a latent HSV or VZV infection can occur spontaneously or in association with physical or emotional stress and immune suppression. Following reactivation from latent ganglion reservoirs, each of these herpesviruses may cause significant clinical symptoms in the individual and may spread to uninfected persons. Symptomatic VZV reactivation is an infrequent, usually once-in-a-lifetime event in 10% of the population that results in zoster (shingles), while HSV-1 and HSV-2 reactivation occurs frequently and results in numerous symptomatic and asymptomatic recurrences.HSV-1 and HSV-2 infections and even some VZV infections cause similar clinical symptoms, for example, cutaneous vesicles, keratitis, and acute retinal necrosis (ARN), and the causative agent cannot be distinguished based on the clinical features. However, different clinical courses and prognoses are caused by each virus. HSV-2 genital herpes tends to recur, but HSV-1 genital herpes does not (7). VZV ARN is severer and has a worse prognosis than HSV ARN (4). The identification of the virus is important for the determination of treatment and for an understanding of the clinical progress and prognosis; therefore, an effective laboratory method is urgently needed for the diagnosis of HSV-1, HSV-2, and V...

Time Profile of Viral DNA in Aqueous Humor Samples of Patients Treated for Varicella-Zoster Virus Acute Retinal Necrosis by Use of Quantitative Real-Time PCR

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