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ABSTRACT: Sero-epidemiological studies are required to identify populations susceptible to measles. The hemagglutination inhibition (HI) test is no longer sensitive enough to confirm immunity to measles, and at present the particle agglutination (PA) test and enzyme-linked immunosorbent assay (EIA) are employed. The most reliable method is the neutralization test (NT), but it is time-consuming and requires experience. To simplify the NT, a recombinant measles AIK-C virus expressing green fluorescence protein (GFP-MVAIK) was constructed and used as a challenge virus. Plaques and cytopathic effects were visualized under ultraviolet light and detected easily, and measuring the intensity of the fluorescence enabled a reduction in the time-consuming steps. Neutralizing antibody titers of a complete inhibition neutralization test were equivalent to those of a 90% plaque reduction neutralization test. Comparison of four methods, HI, PA, EIA and the complete inhibition neutralization test, showed that only the results of EIA correlated well with those of the complete inhibition neutralization test, but sera with borderline levels by EIA were sometimes negative by the complete inhibition neutralization assay.
Journal of Virological Methods 07/2007; 142(1-2):15-20. · 2.01 Impact Factor
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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.
Journal of Clinical Microbiology 10/2006; 44(9):3268-73. · 4.15 Impact Factor
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ABSTRACT: Most mumps patients are clinically diagnosed without any virological examinations, but some diagnosed cases of mumps may be caused by other pathogens or secondary vaccine failure (SVF). To clarify these issues, a sensitive, specific, and rapid diagnostic method is required. We obtained 60 salivary swabs from 34 patients with natural infection during the course of the illness, 10 samples from patients with vaccine-associated parotitis, and 5 samples from patients with SVF. Total RNA was extracted and subjected to reverse transcription-PCR (RT-PCR) and loop-mediated isothermal amplification (LAMP) for genome amplification. We detected mumps virus RNA corresponding to 0.1 PFU by LAMP within 60 min after RNA extraction, with the same sensitivity as RT-nested PCR. Mumps virus was isolated in 30 of 33 samples within day 2, and mumps virus genome was amplified by LAMP in 32 of them. The quantity of virus titer was calculated by monitoring the time to reach the threshold of turbidity. The viral load decreased after day 3 and was lower in patients serologically diagnosed as having SVF with milder illness. Accuracy of LAMP for the detection of mumps virus genome was confirmed; furthermore, it is of benefit for calculating the viral load, which reflects disease pathogenesis.
Journal of Clinical Microbiology 05/2005; 43(4):1625-31. · 4.15 Impact Factor
