A simple method for the detection of measles virus genome by loop-mediated isothermal amplification (LAMP)

Abstract

Approximately 20,000-30,000 measles patients were reported in a surveillance of infectious diseases because of low vaccine coverage of 80% in Japan. Among them, some were thought to be secondary vaccine failure (SVF) with generally mild or non-typical measles illness and sometimes became a source of further transmission. We have developed a new, sensitive, and rapid method to detect the measles virus genome by reverse transcription loop-mediated isothermal amplification (RT-LAMP). We examined 50 nasopharyngeal secretion (NPS) samples that were obtained during the 1999 outbreak and stored at -70 degrees C and fresh NPS, lymphocytes and sera from 11 patients in 2003. Total RNA was extracted from the samples and subjected to reverse transcription-polymerase chain reaction (RT-PCR) and RT-LAMP. We detected the genomic RNA corresponding to at least 0.01-0.04 TCID50, 30-100 copies in samples by RT-LAMP within 60 min after extraction of RNA, and all four genotypes isolated in Japan were equally amplified. Specific DNA amplification was monitored spectrophotometrically by real time turbidimeter and the quantity of RNA was calculated. Measles virus genome was detected in 44 of 50 stored NPS by RT-PCR and in 49 by RT-LAMP. The vaccine strain was discriminated from wild strains after sequencing the LAMP products. RT-LAMP is a useful rapid diagnostic method for the detection of measles virus without any special apparatus, showing higher sensitivity than RT-PCR, and expected to be applied for hospital-based infection control and for laboratory-based measles surveillance.