Rapid and specific detection of RNA base sequence using fluorescence polarization

Rapid and specific determination of the RNA gene of hepatitis C virus (HCV), which had been multiplied by NASBA, was performed using a fluorescence polarization assay. The polarization of the probe DNA in the presence of HCV positive sample, amplified by NASBA, was obviously different from those in the presence of negative control samples. The total time for the gene amplification and detection was about 90 min, while the polarization detection was completed within 10 min. The slight increase of polarization was also confirmed with the hybridization between probe oligo-DNA 25-mers and the synthesized complementary oligo-RNA 25-mers. The polarization of positive and negative samples showed excellent agreement with the results obtained from electrophoresis and dotblot hybridization. INTRODUCTION The fluorescence polarization method seems interesting because it could be used for physico-chemical analysis of nucleic acid hybridization") and has potential for the identification of a specific microorganism-> with applications to the rapid measurement of gene base sequences. In basic research, the conditions for the rapid DNA hybridization was investigated by observing the time course of fluorescence polarization.) In DNA detection, we showed both the utility of asymmetric PCR and the lack of polarization change or reproducibility for the standard PCR in conjunction with the probing method using fluorescence polarization assay (FPA), and performed rapid and specific determination of the Shiga toxin (ST2) gene of enterohaemorrhagic Escherichia co//0157:H7.) To detect RNA of microorganism or virus, the combination of FPA with RNA amplification technique is suggested,) though recently, a direct probing method without dissociation of target DNA using hairpin-like oligonucleotide has been proposed.' The reversetranscriptase PCR is known for amplifying RNA sequence, but also in that case, the amplified product is usually double stranded and some more helpful means would be needed for direct hybridization with the probe in FPA. Isothermal nucleic acid sequence based amplification (NASBA) for the rapid amplification of single stranded RNA with template RNA is known and could be a good candidate for the combination with FPA, because a single stranded probe oligo-DNA would relatively easily hybridize with the amplified ssEXPERIMENTAL The processes and conditions for reagent preparation, gene amplification with NASBA method,> hybridization and instrumentation for fluorescence polarization measurement) used in these experiments were practically the same as those reported previously. Single stranded RNA having 230 bases from the gene of hepatitis C virus (HCV) > was amplified by the NASBA method and the amplicon was described as AP. Negative and positive control samples for FPA were prepared. The negative were salmon sperm DNA (64 ng/assay, Funakoshi, Japan), oligo-RNA noncomplementary to the probe DNA and the NASBA product using purified water instead of the template, which were described as N1, N2 and N3, respectively. The positive control (PC) was the synthesized oligo-RNA complementary to the probe. Ten minutes after mixing the probe DNA with the amplicon (AP) or control samples (N1, N2, N3 or PC), the fluorescence polarization value was measured for 1, 8, 8, 1 or 9 times, respectively, and each mean value was plotted. The RNA amplification of HCV using NASBA was confirmed with dot-blot hybridization and polyacrylamide gel electrophoresis. The probe sequence for dotblotting was the same with that for FPA, but alkaline phosphatase was labelled to the probe in the former. The detection in dot-blotting was done by staining with the dye product catalyzed by the enzyme.'The oligonucleotide base sequences of primers (a), (b), probe DNA (c) for detecting the non-coding region of the HCV genome,' and positive control RNA (d) are shown below. The sequence (a) includes 25 bases for T7 RNA polymerase promoter. The oligo-RNA (d) is complementary to the probe (c). F represents a fluorescein label. (a) 5' AAT.TCT.AAT.ACG.ACT.CAC.TAT.AGG.GCA. AGC.ACC.CTA.TCA.GGC.AGT.A 3 (b) 5' GTC.TAG.CCA.TGG.CGT.TAG.TA 3' (c) 5 FGTG.GTC.TGC.GGA.ACC.GGT.GAG.TAC.A 3 (d) 5 UGU.ACU.CAC.CGG.UUC.CGC.AGA.CCA.C 3 RESULTS AND DISCUSSION The result of measuring the fluorescence polarization for the amplified ss-RNA of HCV, the negative and positive control samples is shown in Figure 1. For the amplicon (AP), obviously high polarization was observed, while that was low for all the negative samples (N1, N2 and N3). Polarization for the positive control (PC) was slightly high, because the complementary oligo-