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Detection of YMDD mutation using mutant-specific primers in chronic hepatitis B patients before and after lamivudine treatment 被引量:1

Detection of YMDD mutation using mutant-specific primers in chronic hepatitis B patients before and after lamivudine treatment
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摘要 瞄准:用异种特定的教材开发 PCR 试金检测 tyrosine-methionine-aspartate-aspartate (YMDD ) 的变化到 tyrosine-valine-aspartate-aspartate (YVDD ) 或 tyrosine-isoleucine-aspartate-aspartate (YIDD ) 的 HBV 的主题。方法:克隆野类型、变异的 HBV 序列被用作模板测试试金的敏感和特性。教材的许多教材构造,教材集中, dNTP 集中,和退火的温度系统地被检验。对 rtL180M 和 rtM204V 特定的对教材为 YVDD 察觉被选择。到有错配到异种和野类型的顺序的一个另外的 3'-penultimate 底的 rtM204I 的教材 specific 为 YIDD 察觉被选择。我们使用了这试金在 lamivudine 治疗前后在 28 个长期的肝炎 B 病人学习 YMDD 异种。结果:我们几乎不能象 -10(9) 用这试金野类型的 HBV 拷贝的在 10 共存的变异的病毒(8 ) 的 0.001%-0.00001% 一样检测。YMDD 异种在 lamivudine 治疗前在 12 个 HBeAg 积极的病人中的 8 个和 16 个 HBeAg 否定的病人中的 8 个被检测。术后疗法,在 HBeAg 积极的病人的二个更多的病人和在 HBeAg 否定的病人的七个更多的病人开发了 YMDD 变化。结论:我们为检测 YMDD 异种开发了高度敏感、特定的试金。这试金能被使用监视长期的肝炎 B 病人在前并且在 lamivudine 治疗期间。 AIM: To develop a PCR assay using mutant-specific primers to detect mutation of tyrosine-methionineaspartate-aspartate (YMDD) motif of HBV to tyrosinevaline-aspartate-aspartate (YVDD) or tyrosine-isoleucineaspartate-aspartate (YIDD). METHODS: Cloned wild-type and mutant HBV sequences were used as templates to test the sensitivity and specificity of the assay. A variety of primer construction, primer concentration, dNTP concentration, and annealing temperature of primers were systematically examined. Pair primers specific to rtL180M and rtM204V were selected for YVDD detection. Primer specific to rtM204I with an additional 3'-penultimate base mismatched to both the mutant and wild-type sequence was selected for YIDD detection. We applied this assay to study YMDD mutants in 28 chronic hepatitis B patients before and after lamivudine treatment. RESULTS: We could detect as little as 0.001%-0.00001% of mutant viruses coexisting in 108-109 copies of wildtype HBV using this assay. YMDD mutants were detected in 8 of 12 HBeAg-positive patients and 8 of 16 HBeAgnegative patients before lamivudine treatment. After treatment, two more patients in HBeAg-positive patients and seven more patients in HBeAg-negative patients developed YMDD mutations. CONCLUSION: We developed a highly sensitive and specific assay for detecting YMDD mutants. This assay can be applied to monitor chronic hepatitis B patients before and during lamivudine treatment.
作者 Cha-Ze Lee Hsuan-Shu Lee Guan-Tarn Huang Pei-Ming Yang Jin-Chuan Sheu Lee CZ;Lee HS;Huang GT;Yang PM;Sheu JC
出处 《世界胃肠病学杂志:英文版(电子版)》 SCIE CAS 2006年第33期5301-5305,共5页 World Journal of Gastroenterology
基金 ACKN0WLEDGMENTS We thank Ms. Hsiao-Mann Lin for her laboratory work.
关键词 基因突变 油漆 慢性乙型肝炎 天门冬氨酸盐 Hepatitis B virus Lamivudine Tyrosinemethionine-aspartate-aspartate, Mutant-specific primer
作者简介 Correspondence to: Cha-Ze Lee, MD, PhD, Departments of Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei 10016, TaJwan, China. czlee@ha.mc.ntu.edu.tw Telephone: +886-2-23123456-7243 Fax: +886-2-2381723
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