Rapid diagnosis of human herpesvirus 6 infection by a novel DNA amplification method, loop-mediated isothermal amplification

Abstract

A novel nucleic acid amplification method, termed loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions, may be a valuable tool for the rapid detection of infectious agents. LAMP was developed for human herpesvirus 6 (HHV-6), and its reliability was evaluated in this study. Although LAMP products were detected in HHV-6 B and HHV-6 A DNA, they were not detected in HHV-7 and human cytomegalovirus DNA. The sensitivity of the original HHV-6 LAMP protocol was 50 copies/tube. In order to increase the method's sensitivity, HHV-6 LAMP was modified by increasing the primer concentration. As a result of the modification, sensitivity increased to 25 copies/tube. After these initial validation studies, 13 patients with fever were tested for HHV-6 by viral isolation, serological analysis, and HHV-6 LAMP. In three of the eight patients with primary HHV-6 infection, HHV-6 DNA was detected in whole blood by the original HHV-6 LAMP protocol in not only the acute phase but also the convalescent phase. HHV-6 DNA was detected by modified HHV-6 LAMP in all eight plasma samples collected in the acute phase; however, no HHV-6 DNA was detected in plasma samples collected in the convalescent phase. Although HHV-6 DNA was detected in both the acute and convalescent phases of whole-blood samples in patients with past HHV-6 infection, it was not detected in plasma samples that did not contain latent viral DNA. Thus, detection of HHV-6 DNA in plasma by using this modified HHV-6 LAMP protocol is appropriate for diagnosis of active HHV-6 infection.

FIG. 1.

Location and name of target sequences used as primers for HHV-6 LAMP on the HHV-6 U31 gene (A) and name and sequence of each primer for HHV-6 LAMP (B). B2c, sequence complementary to B2; F1c, sequence complementary to F1.

FIG. 2.

DNA extracted from Betaherpesvirinae-infected cells was amplified by using the original HHV-6 LAMP protocol to determine the specificity of the method. A fresh isolate of HHV-6 B was prepared from a patient with ES in our institute. Marker, 123-bp DNA ladder marker.

FIG. 3.

Serial dilutions of pGEMH6S12 plasmid DNA were amplified by the original (A) and modified HHV-6 LAMP (B) protocols to determine the respective sensitivities of each assay. Marker, 123-bp DNA ladder marker.