Loop-mediated isothermal amplification assay for the diagnosis of retinitis caused by herpes simplex virus-1

Abstract

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of herpes simplex virus 1 (HSV-1). The specificity of the assay was tested using DNA extracted from HSV-1-infected rabbit corneal epithelium cultures, HSV-2 grown on Vero cell line, cytomegalovirus (CMV) (AD-169), varicella zoster virus (VZV) (Oka-vaccine), adenovirus, Aspergillus flavus and Staphylococcus aureus. The specificity of LAMP was confirmed by bidirectional sequencing of the amplicons. The sensitivity of the LAMP assay was tested using different concentrations of HSV-1 DNA. To evaluate the application of the LAMP assay in clinical diagnosis, we tested vitreous samples from 20 patients with suspected viral retinitis using LAMP and real-time PCR for HSV-1. The LAMP primers amplified only HSV-1 DNA; no LAMP products were detected with the DNAs of HSV-2, CMV, VZV, adenovirus A. flavus and S. aureus. The sequences of the positive HSV-1 LAMP products perfectly (99–100%) matched the HSV-1 sequences deposited in the GenBank database. LAMP is as sensitive as real-time PCR, with the lowest detection limit being 10 copies/μL of HSV-1 DNA. Of the 20 patients with suspected viral retinitis, four tested positive for HSV-1 using real- time PCR and LAMP. A 100% concordance was observed across the two methods. The LAMP assay is a rapid, highly specific and sensitive method for the diagnosis of retinitis caused by HSV-1.