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目的优化乙型肝炎病毒(HBV)不同长度基因片段PCR扩增条件,为后续机制研究提供实验基础。方法运用PCR或巢式PCR(Nested PCR)方法扩增6例临床乙肝患者HBV DNA,针对特异性目的片段,通过优化Mg2+浓度、引物浓度、Taq酶用量、DMSO浓度等,获得特异性扩增条带,PCR产物直接测序并使用CHROMAS、MEGA等生物学软件进行结果分析。结果通过综合分析各种实验条件对PCR扩增结果的影响,最终确认在50μL扩增体系中,2.5 mmol/L Mg2+、200 nmol/L特异性引物、1.5 U Taq酶、56℃退火在本实验室可获得良好的扩增效果,适量二甲基亚砜(DMSO)可减少非特异性干扰,同时建议对短片段可酌量减少Taq酶使用量。生物信息学分析示6例HBV感染标本中1例为B基因型,5例为C基因型,共发现S基因21个核酸变异及9个氨基酸可改变。结论建立了适合本实验室的HBV DNA扩增体系以及后续生物信息学分析方法,明确了不同的扩增体系条件对PCR扩增效果具有重要影响。
Objective To optimize the PCR amplification conditions of different lengths of Hepatitis B virus (HBV) gene fragments and provide the experimental basis for the follow-up mechanism study. Methods HBV DNA of 6 patients with clinical hepatitis B were amplified by PCR or nested PCR (Nested PCR), and specific target fragments were obtained. Specific primers were obtained by optimizing Mg2 + concentration, primer concentration, Taq enzyme dosage and DMSO concentration. The PCR products were directly sequenced and analyzed using biological softwares such as CHROMAS and MEGA. Results The effects of various experimental conditions on PCR amplification were analyzed. In this experiment, 2.5 mmol / L Mg2 +, 200 nmol / L specific primer, 1.5 U Taq enzyme and 56 ℃ annealing were confirmed in 50 μL amplification system. Room can get a good amplification effect, the amount of dimethyl sulfoxide (DMSO) can reduce the non-specific interference, and suggested that short segments can be reduced Taq enzyme dosage. Bioinformatics analysis showed that in 6 cases of HBV infection, 1 case was genotype B and 5 cases genotype C, 21 mutations of S gene and 9 amino acids were found. Conclusion The HBV DNA amplification system suitable for our laboratory and the subsequent bioinformatics analysis methods were established. It was clarified that different amplification system conditions had an important influence on PCR amplification.