基因分型
基因座(遗传学)
乙型肝炎病毒
分子生物学
杂交探针
生物
单核苷酸多态性
基因型
病毒学
DNA
病毒
遗传学
基因
作者
Xianbo Mou,Danni Sheng,Zhu Chen,Mei Liu,Yuan Liu,Yan Deng,Xu Kui,Ruixia Hou,Jingping Zhao,Yabin Zhu,Song Li
标识
DOI:10.1166/jbn.2019.2862
摘要
Hepatitis B virus (HBV) is closely related to occurrence and development of viral hepatitis. A mutation of 1896nt locus in its pre-C region can promote replication of HBV DNA and improve stability of pre-genome RNA structure, and can even help HBV evade immune clearance. In this study, magnetic beads-probe (MBs@probe) method, combined with single base extension (SBE) technology, was developed for in-situ mutation detection of HBV pre-C region 1896nt locus. Before successfully completing the genotyping of 165 HBV samples, the crucial reaction conditions were first optimized, such as SBE temperature, MBs size and amount, and probe concentration on the surface of MBs. Experimental results showed that these conditions had significant effects on MBs@probe in-situ mutation detection. Comprehensive considerations, such as 58 °C of SBE temperature, high fluorescence intensity and signal-to-noise ratios (SNRs) were obtained when MBs@probe complex was made by 100 μg of 300 nm-MBs and 3.0 μM of probes in the system. Finally, 1896nt locus mutation in pre-C region of 165 HBV samples was successfully genotyped, among which 71 HBV samples were wild types and the remaining 94 samples were mutant types. Meanwhile, 14 randomly chosen samples were taken to further analyze fluorescence intensity and SNRs respectively, and sequencing results for the first two samples were consistent with results from the MBs@probe in-situ mutation detection method. Compared with two-color fluorescence hybridization (TCFH) genotyping technology, this method generally improves the SNRs to more than 10 (which is more than 2-fold), has higher reliability and is more suitable to detect SNPs for known sites.
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