乙型肝炎病毒基因分型的双通道荧光PCR检测及其在未成年乙型肝炎病毒感染者中的临床应用
|
摘要
目的评价双通道荧光PCR(d-qPCR)法检测乙型肝炎病毒(HBV)基因型的方法学性能,并在未成年HBV感染者中进行临床应用。方法对基于d-qPCR的HBV基因型检测方法进行性能验证,并将该方法用于未成年HBV感染者的基因型检测,分析其临床意义。结果用于HBV基因分型的dd-qPCR法能检测B型、C型和D型,准确度为100%;最低检测限为1×10~(3 )IU/ml,批内和批间精密度高,抗干扰能力强,特异性高。在本地区200例未成年慢性HBV型携带者基因型中,以B型和C型为主,B型、C型和D型分别占34.0%、65.5%和0.5%,未发现混合型和不能分型的标本。B型和C型HBV DNA定量、HBeAg阳性率差异无统计学意义(P>0.05),肝功能异常率(ALT>50 U/L)差异有统计学意义(P<0.05)。结论 d-qPCR法检测HBV基因型具有分型全面、性能可靠和操作简便等优点。本院就诊的HBV C型未成年HBV感染者存在较高比例的肝功能损害,临床应结合HBV基因分型结果,尽早进行治疗干预。
Objective To evaluate the method of HBV genotypes by dual-color qPCR(d-qPCR) and apply it for the juveniles infected with HBV. Methods The performance of HBV genotype detection by d-qPCR was validated. The d-qPCR was applied to detect the juveniles infected with HBV and its clinical significance was analyzed. Results d-qPCR for HBV genotyping can be used to detect type B, type C and type D, and the accuracy was 100%. The limit of detection reaches 1×10~(3 )IU/ml. The method also had high precision of within-run and between-run. Moreover, the assay had a strong anti-jamming ability and high specificity. The major HBV genotypes in this region included type B and type C in 200 cases of HBV infection, and the proportion of type B, type C and type D was 34.0%, 65.5% and 0.5%, respectively. However, no mixed and undivided cases were found. There was no statistical significance on the difference in the HBV DNA load and positive rate of HBeAg between type B and type C(P>0.05), while the difference in abnormal rate of liver function(ALT>50 U/L) was statistically significant(P<0.05). Conclusion d-qPCR for HBV genotyping has the advantages of more subtypes detection, reliable performance and easy operation. There is a certain proportion of liver function damage in juveniles infected with HBV type C. Therefore, combined with the results of HBV genotyping, the juveniles infected with HBV should be monitored and appropriate treatment measures should be applied as soon as possible.
Objective To evaluate the method of HBV genotypes by dual-color qPCR(d-qPCR) and apply it for the juveniles infected with HBV. Methods The performance of HBV genotype detection by d-qPCR was validated. The d-qPCR was applied to detect the juveniles infected with HBV and its clinical significance was analyzed. Results d-qPCR for HBV genotyping can be used to detect type B, type C and type D, and the accuracy was 100%. The limit of detection reaches 1×10~(3 )IU/ml. The method also had high precision of within-run and between-run. Moreover, the assay had a strong anti-jamming ability and high specificity. The major HBV genotypes in this region included type B and type C in 200 cases of HBV infection, and the proportion of type B, type C and type D was 34.0%, 65.5% and 0.5%, respectively. However, no mixed and undivided cases were found. There was no statistical significance on the difference in the HBV DNA load and positive rate of HBeAg between type B and type C(P>0.05), while the difference in abnormal rate of liver function(ALT>50 U/L) was statistically significant(P<0.05). Conclusion d-qPCR for HBV genotyping has the advantages of more subtypes detection, reliable performance and easy operation. There is a certain proportion of liver function damage in juveniles infected with HBV type C. Therefore, combined with the results of HBV genotyping, the juveniles infected with HBV should be monitored and appropriate treatment measures should be applied as soon as possible.
引文
[1] Yu R, Fan R, Hou J. Chronic hepatitis B virus infection: epidemiology, prevention, and treatment in China[J]. Front Med, 2014, 8(2): 135-144.
[2] Ott JJ, Stevens GA, Groeger J, et al. Global epidemiology of hepatitis B virus infection: new estimates of age-specific HBsAg seroprevalence and endemicity[J]. Vaccine, 2012, 30(12): 2212-2219.
[3] Sunbul M. Hepatitis B virus genotypes: global distribution and clinical importance[J]. World J Gastroenterol, 2014, 20(18): 5427-5434.
[4] 中华医学会肝病学分会, 中华医学会感染病学分会. 慢性乙型肝炎防治指南(2015更新版)[J]. 中华传染病杂志, 2015, 11(33): 641-662.
[5] Tong Y, Liu B, Liu H, et al. New universal primers for genotyping and resistance detection of low HBV DNA levels[J]. Medicine (Baltimore), 2016, 95(33): e4618.
[6] 吴斌, 李彩东, 段正军, 等. PCR荧光法对乙型肝炎病毒基因分型的检测及临床意义[J]. 中华医院感染学杂志, 2012, 22(6): 1315-1317.
[7] Trepo C, Chan HL, Lok A. Hepatitis B virus infection[J]. Lancet, 2014, 384(9959): 2053-2063.
[8] Nannini P, Sokal EM. Hepatitis B: changing epidemiology and interventions[J]. Arch Dis Child, 2017, 102(7): 676-680.
[9] Takano T, Tajiri H, Hosono S, et al. Natural history of chronic hepatitis B virus infection in children in Japan: a comparison of mother-to-child transmission with horizontal transmission[J]. J Gastroenterol, 2017, 52(9): 1041-1050.
[10] 郑卫波, 许文芳, 李烨佳. HBeAg阳性慢性乙型肝炎患者 HBV基因型分布及其与抗病毒治疗疗效的关系[J]. 检验医学, 2015, 30(4): 349-352.
[11] 张文艳, 李明, 汤仁树, 等. 合肥地区慢性无症状乙肝病毒携带者HBV s基因变异研究[J]. 中国卫生检验杂志, 2015, 25(15): 2553-2555.
[12] 申静, 刘薇拉, 王菲. 太原地区乙肝病毒基因分型与临床指标的相关性分析[J]. 中国卫生检验杂志, 2015, 25(14): 2383-2385.
[13] 阳帆, 路滟, 张仁利, 等. 深圳市居民乙型肝炎病毒感染现状及基因型分析[J]. 中国卫生检验杂志, 2015, 25(15): 2605-2607.
[14] 马幼丽, 王黎芳, 谢服役, 等. 乙型肝炎病毒基因分型检测及临床意义分析[J]. 中华实验和临床病毒学杂志, 2015, 29(3): 248-250.
[15] 董绍斌, 王富珍, 张爽, 等. 原发性肝癌合并HBV感染者的HBV基因特征分析[J].中华实验和临床病毒学杂志, 2017, 31(2): 92-97.
[2] Ott JJ, Stevens GA, Groeger J, et al. Global epidemiology of hepatitis B virus infection: new estimates of age-specific HBsAg seroprevalence and endemicity[J]. Vaccine, 2012, 30(12): 2212-2219.
[3] Sunbul M. Hepatitis B virus genotypes: global distribution and clinical importance[J]. World J Gastroenterol, 2014, 20(18): 5427-5434.
[4] 中华医学会肝病学分会, 中华医学会感染病学分会. 慢性乙型肝炎防治指南(2015更新版)[J]. 中华传染病杂志, 2015, 11(33): 641-662.
[5] Tong Y, Liu B, Liu H, et al. New universal primers for genotyping and resistance detection of low HBV DNA levels[J]. Medicine (Baltimore), 2016, 95(33): e4618.
[6] 吴斌, 李彩东, 段正军, 等. PCR荧光法对乙型肝炎病毒基因分型的检测及临床意义[J]. 中华医院感染学杂志, 2012, 22(6): 1315-1317.
[7] Trepo C, Chan HL, Lok A. Hepatitis B virus infection[J]. Lancet, 2014, 384(9959): 2053-2063.
[8] Nannini P, Sokal EM. Hepatitis B: changing epidemiology and interventions[J]. Arch Dis Child, 2017, 102(7): 676-680.
[9] Takano T, Tajiri H, Hosono S, et al. Natural history of chronic hepatitis B virus infection in children in Japan: a comparison of mother-to-child transmission with horizontal transmission[J]. J Gastroenterol, 2017, 52(9): 1041-1050.
[10] 郑卫波, 许文芳, 李烨佳. HBeAg阳性慢性乙型肝炎患者 HBV基因型分布及其与抗病毒治疗疗效的关系[J]. 检验医学, 2015, 30(4): 349-352.
[11] 张文艳, 李明, 汤仁树, 等. 合肥地区慢性无症状乙肝病毒携带者HBV s基因变异研究[J]. 中国卫生检验杂志, 2015, 25(15): 2553-2555.
[12] 申静, 刘薇拉, 王菲. 太原地区乙肝病毒基因分型与临床指标的相关性分析[J]. 中国卫生检验杂志, 2015, 25(14): 2383-2385.
[13] 阳帆, 路滟, 张仁利, 等. 深圳市居民乙型肝炎病毒感染现状及基因型分析[J]. 中国卫生检验杂志, 2015, 25(15): 2605-2607.
[14] 马幼丽, 王黎芳, 谢服役, 等. 乙型肝炎病毒基因分型检测及临床意义分析[J]. 中华实验和临床病毒学杂志, 2015, 29(3): 248-250.
[15] 董绍斌, 王富珍, 张爽, 等. 原发性肝癌合并HBV感染者的HBV基因特征分析[J].中华实验和临床病毒学杂志, 2017, 31(2): 92-97.