| Backgrounds: Hepatitis C virus is the main pathogen of chronic hepatitis C. HCV antigen's variation is large because of its high frequency of gene mutation. At present, its diagnosisof hepatitis C depends on testing IgG antibody against HCV in mainly laboratory in China. But in acute period only 60% (37.2%-61.8%) patients with hepatitis C in acute period can be detected with positive antibodies, while others still may be negative. To say in other words, a great part of patients can be judged as antibody negative by ELISA. Measurement of HCV RNA is the important quota in the detection of HCV. It is significant for HCV diagnosis, therapy and prognosis. Since PCR appeared, it has been widely used in medical domain and increased the level of medical technology. TRFIA (time-resolved fluoro-immunoassay) is a new, high sensitive, quantitative immunoassay and has been widely used. We applied this method into the detection of HCV RNA. We prepared a europium labeled probe with BSA system and a new europium fluorescent chelate BHHCT[(4,4'-bis(l",l",l",2",2",3",3" -heptafluoro-4 " ,6" -hexanedion-6" -y l)-chlorosul-fo-o-terpheny 1) to link europium to the target DNA. Then the fluorescent of europium was measured. Objective:To establish a semi-quantitative RT-PCR method for measurement of HCV RNA using primers and probe which were designed by ourselves , and a new europium fluorescent chelate BHHCT. Optimize the reaction system and analyze its sensitivity specificity and precision. Materials and Methods:1. Patients samples: HCV RNA in sera of 44 patients with chronic HCV infection were detection by HCV fluorescence PCR diagnostic kit produced by Zhongshan University DAAN Gene Co Ltd. Normal sera come from the 20 healthy people who take physical examination in our hospital. Blood samples 5ml of blood were taken, then sera were isolated and stored at -70 without thawing until use.2. Primers and probes: According to the principle of primer design, we make the front and reverse primers by the GENE RUNNER: 5' B-GGC GAC ACT CCA CCA TGA ATC 3', 5' GCA AGC ACC CTA TCA GGC AG 3'.The length of the RT-PCR product is 291bp. We designed the capture probe by Prime Primer 5.0: 5' NH2-ACC CTA TCA GGC AGT ACC ACA AG Primers and probe were synthesized by Shanghai ShenYou Co, Ltd.3. RNA extraction: HCV RNA in serum sample was extracted by HCV fluorescence PCR diagnostic kit produced by Zhongshan University DAAN Gene Co Ltd.4. HCV RNA reverse transcription (RT): Reaction mix 20 1, which included 5-times RT buffer 4 u K 20mmol/L dNTP Mix 2 K l0umol/L reverse primer Ink Rnase inhibitor 201K DEPC H2O ll.5K AMV enzyme 10U was added into the RNA sediment, then incubate it at 42 for 55min.5. HCV PCR amplification: It carried out in a reaction mixture 30 u 1 , containing 10-times reaction buffer 3 1, 2mmol/L dNTP Mix 3 U 1, 25mmol/L MgCl2 3 u 1, 10 u mol/L front primer labeled with biotin and reverse primer 0.5 u 1 each, Taq enzyme 1U, DEPC H2O17.8ul, RT product cDNA 2 U 1. The final volume was 30 u 1. The PCR conditions for the amplification was 95 for 5 minutes (pre-degeneration), 35 cycles of 94 for 45 seconds(degeneration), 60 for 30 seconds (annealing), 72for 30 seconds(extension), followed by 7C for 7 minutes(final extension). After amplification, the product was diluted 10-fold with the hybridization solution(2 X SSC, l%casein, 0.02% SDS.0.1 % N-lauroylsarcosine) and boiled for 10 minutes, then was put into ice bath immediately.6. Labeling of SA with BHHCT: 0.2ml of dry ethanol solution containing O.8mg of BHHCT was added dropwise (25 1 each) into 6.6ml of 0.1 M carbonate buffer of pH 9.1 containing Img of SA with stirring for Ih at room temperature. Then the reaction mix was dialyzed twice against 1 liter of 0.1 M NaHCO3 containing 0.25g of NaN3, first for 24h and second for 7h. lOmg of BSA and 4mg of NaN3 was added to the dialyzed solution, and the pH was adjusted to 6.2 with IM HCI. The solution was stored at -20 . The concertration of SA-BHHCT was determined using the absorbance of the solution at 330nm divid...
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