CAJ | 학술논문

目的建立一种同时检测HBV 3种核苷(酸)类似物耐药突变的方法.方法以基因库公布的981条HBV基因序列为基础,设计2对地高辛标记的通用引物,扩增HBV聚合酶的逆转录区.针对拉米夫定、阿德福韦和恩替卡韦10个耐药位点上的氨基酸替换形式,设计26条特异性寡核苷酸探针,包括I169T,V173L/G,L180M,A181T/V,T184G,S202I/G,M204V/I,Q215S,N236T,M250V/I/L,并固定于带正电荷的尼龙膜上.利用地高辛标记的靶DNA扩增产物与固定于膜上的特异性探针的反向杂交,检测HBV 3种核苷(酸)类似物的耐药突变.应用反向杂交法分别检测HBV野生和耐药标准株,以及40例患者血清标本,观察其检测特异性和准确性.结果反向杂交法检测HBV野生和耐药标准株,有效地区分了1169T,V173L,L180M,A1811T,T184G,S202I,M204V,Q215S,N236T,M250V 10个耐药位点上的特异性探针.40例患者血清标本中,37例临床病例的耐药检测结果与直接测序法一致.结论反向杂交法可以快速,准确的检测出HBV3种核苷类似物耐药突变,有助于这3种药物耐药的诊断和个体化治疗.
목적 건립일충동시검측HBV 3충핵감(산)유사물내약돌변적방법.방법 이기인고공포적981조HBV기인서렬위기출,설계2대지고신표기적통용인물,확증HBV취합매적역전록구.침대랍미부정、아덕복위화은체잡위10개내약위점상적안기산체환형식,설계26조특이성과핵감산탐침,포괄I169T,V173L/G,L180M,A181T/V,T184G,S202I/G,M204V/I,Q215S,N236T,M250V/I/L,병고정우대정전하적니룡막상.이용지고신표기적파DNA확증산물여고정우막상적특이성탐침적반향잡교,검측HBV 3충핵감(산)유사물적내약돌변.응용반향잡교법분별검측HBV야생화내약표준주,이급40례환자혈청표본,관찰기검측특이성화준학성.결과 반향잡교법검측HBV야생화내약표준주,유효지구분료1169T,V173L,L180M,A1811T,T184G,S202I,M204V,Q215S,N236T,M250V 10개내약위점상적특이성탐침.40례환자혈청표본중,37례림상병례적내약검측결과 여직접측서법일치.결론 반향잡교법가이쾌속,준학적검측출HBV3충핵감유사물내약돌변,유조우저3충약물내약적진단화개체화치료.
Objective To establish a method for simultaneous detection of HBV resistant mutations associated with three kinds of nucleoside analogues. Methods According to 981 HBV complete sequences in GenBank, two pairs of conserved primers labeled with digoxigenin were synthesized to amplify the region of HBV reverse transcriptase. To detect non-synonymous amino acid substitutions associated with lamivudine, adefovir and entecavir, 26 specific oligonucleotide probes covering ten different codon positions, I169T, V173L/G, L180M, A181T/V, T184G, S202I/G, M204V/I, Q215S, N236T and M250V/I/L were synthesized and immobilized on nylon membranes charged positively. The oligonucleotide probes immobilized on nylon membranes were then hybridized with PCR products labeled with digoxigenin to detect three drug-resistant mutations. In order to observe specificity and accuracy of probes, HBV wild-type, resistant reference strains and patients serums were assayed by reverse hybridization technique, respectively. Results The specific probes of 10 codon positions related to HBV wild-type and resistant reference strains, including I169T, V173L, L180M, A181T, T184G, S202I, M204V, Q215S, N236T, M250V, were distinguished effectively by reverse hybridization method. The results results of 37 samples applicated the method were in accordance with that Of DNA sequencing. Conclusion Reverse hybridization technique can be applied to detect HBV resistant mutations associated with Lamivudine, Adefovir and Entecavir rapidly and accurately.