Development of a new method for diagnosis of rubella virus infection by reverse transcription-loop-mediated isothermal amplification

Abstract

We developed a useful method for the detection of rubella virus genome RNA by reverse transcription loop-mediated isothermal amplification (RT-LAMP) and compared the sensitivity of RT-LAMP with that of other virological tests: reverse transcription-PCR (RT-PCR) and virus isolation. The rubella virus genome was amplified by RT-LAMP from clinical isolates obtained between 1987 and 2004 with similar sensitivities to the Takahashi vaccine strain. The detection limit of RT-LAMP was compared with that of RT-PCR using the Takahashi vaccine strain. We detected rubella virus genome material corresponding to 30 PFU/ml in a culture fluid sample by RT-LAMP within 60 min after the extraction of RNA with equal sensitivity to RT-nested PCR. The positive result rates of RT-LAMP, RT-PCR, and virus isolation were also compared using throat swabs obtained from patients who were clinically diagnosed with acute rubella virus infection in 2004 in Tochigi, Japan. Among nine patients with clinical rubella, the positive result rates were three/nine (33.3%) for virus isolation, six/nine (66.7%) for RT-PCR, and seven/nine (77.8%) for RT-LAMP. Consequently, RT-LAMP for rubella virus would be expected to be a reliable rapid diagnostic tool in the clinical setting.

FIG. 1.

Diagram of RT-LAMP primers for the detection of the rubella virus genome. The eight primer sites are shown in the upper panel. Sequence alignments of six LAMP primers are shown in the lower panel. Positive-sense F3 and complementary B3 were used as outer primers. FIP contains the sequence complementary to F1 linked with the F2 sequence. BIP contains the B1 sequence linked with the sequence complementary to B2. Two additional loop primers (F and B) are synthesized between F1 and F2 and between B1 and B2, respectively. The arrows show the direction of DNA synthesis.

FIG. 2.

Detection limit of RT-LAMP and RT-PCR. The Takahashi vaccine strain containing 10^5.5 PFU/0.1 ml was used. RNA was serially diluted by 1:10, and each dilution was subjected to RT-LAMP and RT-PCR. A result with a turbidity of ≥0.1 was considered to be LAMP positive. Numbers at left of the right panel are sizes in base pairs of the DNA marker.