Diagnostic value of the LAMP method with real-time fluo-rescence detection on a model of herpesvirus infection

Abstract

Due to the high prevalence and clinical significance of herpesvirus diseases, improvement of methods of their diagnostic remains relevant.The aim of the work: improvement of the methodological approaches to the rapid differential diagnosis of herpesvirus infections based on the LAMP method with real-time fluorescence detection (LAMPRT). 201 urogenital swabs were examined using RT-PCR and LAMP-RT, 27of which contained DNAof Epstein-Barr virus (EBV), 34 — Cytomegalovirus (CMV), 14 — Herpes simplex virus type 1 (HSV-1), 36 — Herpes simplex virus type 2 (HSV-2). For LAMP-RT reaction we used Bst 2.0 WarmStart DNA polymerase (BioLabs, Great Britain), SYTO-82 dye (Invitrogen, USA), and sets of primers for herpesvirus DNA detection by LAMP. High efficiency of using the SYTO-82 dye for the detection of herpesvirus DNA in the LAMP-RT reactionwas shown. Under the optimal conditions, the LAMP-RT allows to reduce the reaction time to 25 minutes, that 3 times less then real-time PCR. The diagnostic sensitivity of the LAMP-RT reaction for HSV-1 was 100 %, HSV-2 — 94.50 %, EBV — 89 %, CMV — 94 %. The diagnostic specificity for all studied viruses was 100 %. The analytical sensitivity of EBV detection was 104 DNA copies/ ml, for HSV types 1 and 2 and CMV — 103 DNA copies/ml. Thus, the LAMP-RT reaction with SYTO-82 dye makes it possible to detect the DNA of various herpesviruses in clinical specimens with high sensitivity and specificity and can be considered as a promising method for point-of-care diagnostics. For the widespread implementation of the method into the practice of laboratory diagnostics, it is necessary to solve the problem of creating an internal positive control of the reaction and the development of portable specialized analyzers.