We propose here a new enzyme immunoassay based on surface-enhanced Raman scattering (SERS). In the proposed system, antibody immobilized on a solid substrate reacts with antigen, which binds with another antibody labeled with peroxidase. If this immunocomplex is subjected to reaction with o-phenylenediamine and hydrogen peroxide at 37°C, azoaniline is generated. This azo compound is adsorbed on a silver colloid. In this system, only the azo compound gives a strong surface-enhanced resonance Raman (SERRS) spectrum. The spectrum shows intense bands at 1582 and 1442 cm -1 due to the CdC and NdN stretching modes, respectively. A linear relationship was observed between the peak intensity of the NdN stretching band and the concentration of antigen, revealing that one can determine the concentration of antigen by the SERRS measurement of the reaction product. The correlation coefficient between the peak intensity and the concentration was calculated to be 0.999 for the concentration range from 0.158 to 2.5 ng/mL. The detection limit of this SERS enzyme immunoassay method was found to be about 10 -15 mol/mL, which was lower by 1 order of magnitude than that found for a previously reported method employing SERRS.
Surface-enhanced Raman spectroscopy (SERS) was utilized for the quantitative analysis of double-stranded (ds) DNA amplified by a polymerase chain reaction (PCR). 4?, 6-Diamidino-2-phenylindole dihydrochloride (DAPI), which intercalates into ds-DNA but does not form a complex with single-stranded (ss) DNA, was added to a DNA solution after amplification by PCR. When the solution was mixed, including ds-DNA-DAPI complexes and free DAPI with silver colloid sol, only free DAPI was adsorbed on the colloid surface. The dye on the colloid gave very intense SERS signals with excitation at 514.5 nm, whereas DAPI engaging in the intercalation with ds-DNA did not show any SERS signal. The SERS spectrum of DAPI on the colloid showed a strong band at 1610 cm(-1) due to the C?N stretching mode, and a linear relationship was observed between the peak intensity of the C?N stretching band and the concentration of free DAPI. Therefore one can determine the concentration of free DAPI by the SERS measurement. The more ds-DNA there is in the solution, the less free DAPI there is. Thus it is possible to quantitatively analyze the ds-DNA amplified by PCR indirectly by using SERS. The correlation coefficient between the peak intensity of the C?N stretching band and the concentration of ds-DNA amplified by PCR was calculated to be 0.988 for a concentration range from 0.1 to 1.3 mg/ml.
We detected Legionella species in 111 bath water samples and 95 cooling tower water samples by using a combination of conventional plate culture, quantitative polymerase chain reaction qPCR and qPCR combined with ethidium monoazide treatment EMA-qPCR methods. In the case of bath water samples, Legionella spp. were detected in 30 samples by plate culture, in 85 samples by qPCR, and in 49 samples by EMA-qPCR. Of 81 samples determined to be Legionella-negative by plate culture, 56 and 23 samples were positive by qPCR and EMA-qPCR, respectively. Therefore, EMA treatment decreased the number of Legionellapositive bath water samples detected by qPCR. In contrast, EMA treatment had no effect on cooling tower water samples. We therefore expect that EMA-qPCR is a useful method for the rapid detection of viable Legionella spp. from bath water samples.
The accurate detection of Legionella from environmental water samples using conventional plate culture methods is often made difficult by the overgrowth of non-target microorganisms on the selective agar plates. Acid pretreatment is a very effective pretreatment to decrease the overgrowth of heterotrophic bacteria. However, acid pretreatment would not be expected to eliminate molds. We evaluated the effects of four kinds of antifungal agents, individually and in combination, on the growth of Leg/one/la strains and molds. Consequently, it was demonstrated that the combination of cycloheximide, amphotericin B and thiabendazole was very effective in eliminating molds on agar plates, and resulted in the improved detection of Legionella. Examination of 214 cooling tower water samples using the enhanced antifungal selective medium (CAT a) instead of GVPC a selective medium demonstrated a decrease in contamination by molds from 13.6 c/3 to 1.9 % without affecting the growth of Legionella.
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