【摘要】：目的利用Ion Torrent PGM深度測(cè)序技術(shù),探討乙型肝炎病毒e抗原(HBe Ag)陽(yáng)性慢性乙型肝炎(CHB)患者乙型肝炎病毒(HBV)基本核心啟動(dòng)子和前C區(qū)突變特征。方法收集HBe Ag陽(yáng)性CHB患者血清樣本25例,提取HBV DNA,采用巢式PCR方法擴(kuò)增HBV基本核心啟動(dòng)子和前C區(qū)片段,構(gòu)建深度測(cè)序文庫(kù),基于Ion Torrent PGM平臺(tái)深度測(cè)序,生物信息學(xué)分析突變位點(diǎn)及其突變率;構(gòu)建HBV基本核心啟動(dòng)子和前C區(qū)突變型和野生型參考質(zhì)粒,作為深度測(cè)序質(zhì)控品。結(jié)果在25例HBe Ag陽(yáng)性CHB患者HBV基本核心啟動(dòng)子和前C區(qū)檢出流行率達(dá)20%的突變位點(diǎn)10個(gè):G1746A、A1752G/T、T1753C/G、A1762T、G1764A、C1817G/A、T1825C/A、A1846T、G1896A、G1899A;G1746A和T1825C/A的流行率分別達(dá)92%和100%。突變位點(diǎn)在HBV B基因型和C基因型感染者的分布情況顯示,A1752G/T和G1896A主要流行于B基因型感染者(63.6%vs 0.0%,χ~2=12.374、P=0.0007;72.7%vs 28.6%,χ~2=4.812、P=0.0472),A1762T和G1764A主要流行于C基因型感染者(27.3%vs 78.6%,χ~2=6.579,P=0.0172);C基因型感染者A1762T/G1764A突變率顯著高于B基因型感染者,但差異無(wú)統(tǒng)計(jì)學(xué)意義(25.7%±28.4%vs 68.4%±42.7%,t=1.614、P=0.1326)。25例HBe Ag陽(yáng)性CHB患者中,32.0%患者僅有A1762T/G1764A突變,24.0%患者僅有G1896A突變,24.0%患者同時(shí)攜帶A1762T/G1764A和G1896A突變。結(jié)論深度測(cè)序分析可用于定量檢測(cè)HBV基本核心啟動(dòng)子和前C區(qū)突變,為HBV突變研究的臨床應(yīng)用提供了技術(shù)平臺(tái)。
[Abstract]:Objective to investigate the characteristics of hepatitis B virus (HBV) (HBV) core promoter and precore region mutation in patients with hepatitis B virus (HBe Ag) positive chronic hepatitis B virus (CHB) by Ion Torrent PGM deep sequencing. Methods Serum samples of 25 patients with HBe Ag positive CHB were collected and HBV DNA was extracted. The HBV core promoter and precore region fragments were amplified by nested PCR method, and a deep sequencing library was constructed, which was based on Ion Torrent PGM platform. The mutation site and mutation rate were analyzed by bioinformatics, and the reference plasmids of the basic core promoter and precore promoter and wild type of HBV were constructed, which were used as quality control materials for deep sequencing. Results in 25 patients with HBe Ag positive CHB, 10 mutation sites with 20% prevalence rate were found in the basic core promoter and preC region of HBV. The prevalence rates of G1746A / A1752G / T / T1753C / G1764A / A1764A / A181764A / A181764A / A18176A / A1825C / A1846A / A1846A / G1896AG1899A G1746A and T1825C/A were 92% and 100%, respectively. The prevalence of G1746A and G1899A G1746A in 25 patients with HBe Ag positive CHB was 92% and 100%, respectively. The distribution of mutation locus in HBV B genotype and genotype C infection showed that A1762T and G1896A were mainly prevalent in patients with genotype B (63.6%vs 0.00, 蠂 ~ 2 12.374% P 0.00072.7 vs 28.6T, 蠂 ~ 2 + 4.812P 0.0472). A1762T and G1764A were mainly prevalent in C genotype carriers (27.3%vs 78.6p, 蠂 ~ 26.579P ~ (0.0172). Higher than genotype B, But there was no significant difference (25.7% 鹵28.4%vs 68.4% 鹵42.7%). Among 25 HBe Ag positive CHB patients, 32.0% had only A1762T/G1764A mutation 24.0%, only G1896A mutation 24.0% carried both A1762T/G1764A and G1896A mutations. Conclusion Deep-sequencing analysis can be used for quantitative detection of HBV core promoter and precore mutation, which provides a technical platform for clinical application of HBV mutation research.
【作者單位】： 江蘇大學(xué)醫(yī)學(xué)院免疫學(xué)系;北京大學(xué)人民醫(yī)院 北京大學(xué)肝病研究所 丙型肝炎及肝病免疫治療北京市重點(diǎn)實(shí)驗(yàn)室;中國(guó)科學(xué)院微生物研究所生物信息中心;
【基金】：國(guó)家自然科學(xué)基金(No.81273202;No.31400773) 國(guó)家重大科技專(zhuān)項(xiàng)(No.2012ZX10002011-006) 江蘇省臨床醫(yī)學(xué)科技專(zhuān)項(xiàng)(No.BL2013024)
【分類(lèi)號(hào)】：R373.21;R392

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