Type 1b hepatitis C virus 5 end of the 5223 bp half-genome amplification.docVIP

 PAGE \* MERGEFORMAT 15 Type 1b hepatitis C virus 5 ‘end of the 5223 bp half-genome amplification Authors: Xiao-Hong Wang, Xiong Yu-lin, Jun-Gang Li, Tan Zhaoxia, Hu Yajun [Keywords:] Hepatitis C virus Amplification of 5 ‘terminal 5223 bp fragment of hepatitis C virus genotype 1b [Abstract] AIM: To amplify 5 ‘terminal 5223 bp fragment of hepatitis C virus (HCV) genotype 1b. METHODS: Five human serum specimens infected with HCV 1b were obtained from HCV RNA positive repository. Total RNA was extracted by using 3 different methods including TRIzol LS Reagent, QIAamp viral RNA mini kit and Magnetic bead. The extracted RNA was reversely transcribed with 3 reverse transcriptases under different reaction conditions. Optimization for long PCR amplification was performed by comparing different Taq enzymes and cycle systems. The 5 ‘half of the HCV1b genome was amplified with the refined long RTPCR technique. RESULTS: The integrity of long RNA templates was achieved with TRIzol LS Reagent. Using ReverTra AceaTM and SuperScriptTM Ⅱ RNase H-reverse transcriptase, the complete cDNA molecules were made with incubation at 42 ℃ for 10 min followed by 30 cycles of 42 ℃ for 2 min and 48 ℃ for 2 min and then 75 ℃ for 10 min. Long PCR amplification of HCV fragment was established by using TaKaLa LA TaqTM or Platinum Taq DNA polymerase High Fidelity as well as the cycling condition of denaturation for 2 min at 94 ℃ followed by 30 cycles of 30 s at 94 ℃ , 30 s at 62 ℃ , and 4.5 min at 72 ℃ , or 5 cycles of 15 s at 94 ℃ , and 5 min at 68 ℃ followed by 20 cycles of 15 s at 94 ℃ , and 5 min with the increment of 10 s per cycle at 68 ℃ and finally 14.5 min at 72 ℃ . Both cycling conditions were feasible, but amplification efficiency of TaKaLa LA TaqTM is superior to Platinum Taq DNA polymerase High Fidelity under this reaction condition. 5223 bp HCV fragment was obtained from all 5 specimens by utilizing the optimized RTPCR protocol and their genotypes were confirmed by sequ