丙型肝炎病毒__(期刊论文)

丙型肝炎病毒1b型5′端5223 bp半基因组扩增

http://www.100md.com 《第四军医大学学报》 2006年第9期

丙型肝炎病毒,,丙型肝炎病毒；长链RTPCR；半基因组,丙型肝炎病毒1b型5′端5223bp半基因组扩增,0引言,1材料和方法,2结果,3讨论,【参考文献】

     Amplification of 5′ terminal 5223 bp fragment of hepatitis C virus genotype 1b

    WANG XiaoHong, XIONG YuLin, LI JunGang, TAN ZhaoXia, HU YaJun

    Chinese PLA Institute of Infectious Diseases，Southwest Hospital，Third Military Medical University，Chongqing 400038，China

    【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 HCV1b genome was amplified with the refined long RTPCR technique. RESULTS: The integrity of long RNA templates was achieved with TRIzol LS Reagent. Using ReverTra AceaTM and SuperScriptTM ⅡRNase 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 RTPCR protocol and their genotypes were confirmed by sequence analysis. CONCLUSION: The 5′ terminal 5223 bp fragment of HCV was successfully achieved, which should provide a basis for further construction of fulllength cDNA clone and replicon of HCV 1b. ......

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