| Backgrounds: Hepatitis C virus is the main pathogens of chronic hepatitis C. It's detection by RT-PCR assay was established initially by weiner etc. in 1990. From then on, this method was widely used in the research field of hepatitis c virus. The detection of hepatitis c virus is largely dependent on polymerase chain reaction. It can be divided into two groups: One is real-time quantification fluorescence assay which detects the virus at the same time as amplification. The other is the evaluation of the amplification products which typically requires enzyme-labeled detective techniques. It can be cumbersome and time-consuming. TRFIA (time-resolved fluoroimmunoassay) is non-radiate labeled detective technique. It demonstrates superiorities in many aspects, such as: sensitivity, specificity, range, speed and stabilize, etc. Many researchers at home or abroad have systematic discussed its principle, traits, achievement and development prospect. We applied this method into the detection of hepatitis c virus. We prepared a labeled probe by taking advantages of BSA system and a new europium fluorescent chelate BHHCT [4, 4' - bis(l" , 1" , 1" , 2" , 2" , 3" , 3" -heptafluoro- 4" , 6" -hexanedion- 6" -yl)-chlorosulfo-o-terphenyl] to measure the amplification products. This method not only improved the sensitivity and specificity of the assay, but also simplified the operation, saved the time. The TRFIA technology is rapid has a user-friendly format with the potential for extend application and is adaptable for amplicon detection in wide range of PCR assays.Objective:1. To establish a method used a new europium fluorescent chelate BHHCT as a label for highly sensitive detection of hepatitis c virus. This method was compared with enzyme-labeled assay in sensitivity, specificity, reproducibility, etc.Materials:1. Patient samples: 17 Patients with chronic HCV infection and 13 healthy people were involved in our study. Blood samples 5 ml were obtained, then stored at -80C without thawing until use.2. Labeling of SA with BHHCT: To 33 ml of 0.1 M carbonate buffer of pH 9.1 containing 5 mg of SA, 0.2 ml of dry ethanol solution containing 4 mg of BHHCT was added dropwise (25 u 1 each) with stirring for 5 minutes. After the solution was stirred for 1 h at room temperature, it was dialyzed twice against 4 liter of 0.1 M NaHCO3 containing 0.25 g of NaN3, first for 24 h and second for 7 h. To the dialyzed solution 50 mg of BSA and 20 mg of NaN3 was added, and the pH was adjusted to 6.2 with 1 M HC1. The solution was stored at -20C. The labeling ratio of BHHCT to SA was determined by using the absorbance of the solution at 330 nm and the molar extinction coefficient of free BHHCT (3.41 X 104 M ?cm-1) to be ca. SA(BHHCT)21. The labeled SA solution can be stored for 1 year at -20C. The labeled SA solution was diluted 500-fold with 0.05 M Tris-HCl, pH 7.8, containing 1.0X 107 M EuCl3, 0.2% BSA, 0.1%NaN3, and 0.9% NaCl, heated at 56C for 2 h, and used for immunoassay.3. HCV RNA was isolated from 100 u 1 serum by chromatography extraction.4. The 5' untranslated region (UTR) was amplified by RT-PCR. It was carried out in a reaction mixture containing 10-times reaction buffer, Tth enzyme, dNTP, MgCl2, primers pre-labeled with biotin, RNaseinhibitor 10 u 1 and diethylpyro-carbonate (DEPC) treated water 10 u 1 and sera extracted products 5 u 1. The PCR condition for the amplification of 5' UTR was 50癈 for 2 minutes, 60C for 30 minutes (reverse transcription); 3 cycles of 94C for 15 seconds, 58C for 30 seconds; 45 cycles of 90癈 for 20 seconds (denaturation), 61C for 30 seconds (annealing and extension), followed by 72C for 15 minutes (final extension).5. Instrument and Measurement Conditions: For time-resolved fluorometric measurement, an Victor 1420 fluorometer from PE company was used. The measurement conditions are delay time 0.20 ms, window time 0.40 ms, and flash rate 1.00 ms. The 96-well microtiter plates were Fluoro Nunc module plates.6. Eu-labeled probe detection: Capture...
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