Development and Evaluation of Loop-Mediated Isothermal Amplification Assay for Rapid and Inexpensive Detection of Cytomegalovirus DNA in Vitreous Specimens from Suspected Cases of Viral Retinitis

A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of cytomegalovirus (CMV) was developed and evaluated. The LAMP assay specifically amplified only CMV DNA, and no cross-reactivity with the DNA of herpes simplex virus type 1, varicella-zoster virus, adenovirus, Aspergillus flavus, or Staphylococcus aureus was observed. The sequences of the LAMP assay-positive CMV products were perfectly (100%) matched with the CMV sequence deposited in the GenBank database. The sensitivity of the LAMP assay was found to be 10 copies/μl of CMV DNA. Vitreous samples from 40 patients with suspected retinitis were subjected to LAMP and real-time PCR for the detection of CMV. Of 40 patients with suspected viral retinitis, 10 tested positive for CMV by the real-time PCR and LAMP assays. A 100% concordance was observed between the results of the two methods. The LAMP assay is a rapid, highly specific, and sensitive method for the diagnosis of retinitis caused by CMV.Viral retinitis is commonly caused by herpes simplex virus type 1 (HSV-1), HSV-2, varicella-zoster virus (VZV), cytomegalovirus (CMV), and occasionally, Epstein-Barr virus (EBV) (7). In patients with atypical features and in the early stages of ocular manifestations, clinical differentiation between cases of retinitis associated with CMV and other herpesvirus infections is often difficult (6). The differentiation of CMV retinitis from HSV and VZV retinitis is very important early in the course of the disease, as the therapeutic agent to be used for treatment differs from virus to virus (7). Conventional methods for the diagnosis of viral retinitis include the detection of viral antigen and virus isolation from intraocular specimens (2). These tests have been shown to have low sensitivities for the detection of viruses and are not currently recommended for use for the diagnosis of viral retinitis (2).PCR has proved to be of great utility for the diagnosis of viral retinitis (3, 4, 5, 6, 8). However, owing to the expensive systems required, PCR is still not a very common diagnostic test. Notomi et al. have reported on a novel nucleic acid amplification assay termed the loop-mediated isothermal amplification (LAMP) assay (10). The assay amplifies the DNA under isothermal conditions (63 to 65°C) with high degrees of specificity, efficiency, and speed. The assay can be conducted in the laboratory in a water bath or heating block (10). Thus, the thermal cycling needs of a PCR are avoided. The assay can be used for the rapid detection of pathogens in peripheral health care settings in developing countries. The present study describes the development and evaluation of a simple and cost-effective LAMP assay for the rapid detection of CMV DNA in patients with viral retinitis.