CAJ | 학술논문

目的建立一种HBV耐药突变的反向杂交检测方法,并对该方法进行优化和评估.方法针对拉米夫定、阿德福韦酯和恩替卡韦3种药物10个耐药位点的常见耐药突变形式,合成兼顾HBV 8种基因型的26条简并探针,并固定于同一张带正电荷的尼龙膜上,与地高辛标记的待测样本聚合酶链反应(PCR)产物进行杂交.为了提高检测灵敏度和特异性,从PCR产物标记地高辛的数目,紫外交联探针的能量强度,以及杂交和严格洗脱4个方面进行优化.为了证实方法的可行性,从检测特异性、灵敏度和临床标本的检测准确性3个方面进行评估.结果当检测体系为引物标记3个地高辛分子,紫外交联的能量强度选择1500× 0.1 mJ/cm2,杂交温度选择42℃,严格洗脱条件选择44℃,用0.5 ×SSC和0.1％十二烷基硫酸钠严格洗脱30 min时,可获得灵敏、特异的结果.在该检测体系的评估中,当PCR产量在10 mg/L以上,可以被检测到,且绝大多数探针特异性较好.临床标本的检测结果与直接测序法的检测结果符合率为93.9％ (31/33).结论该方法可以对HBV拉米夫定、阿德福韦和恩替卡韦耐药突变同时进行检测,是一种简便、快速、灵敏的分析方法,但部分探针的特异性有待进一步提高.对于180/181、202/204这4个位点的探针,由于两两距离较近,同一探针序列包含2个位点的密码子,杂交会相互干扰,通过组合距离较近的2个位点的密码子的各种形式来设计探针,可能有助于取得更好的结果.
목적 건립일충HBV내약돌변적반향잡교검측방법,병대해방법진행우화화평고.방법 침대랍미부정、아덕복위지화은체잡위3충약물10개내약위점적상견내약돌변형식,합성겸고HBV 8충기인형적26조간병탐침,병고정우동일장대정전하적니룡막상,여지고신표기적대측양본취합매련반응(PCR)산물진행잡교.위료제고검측령민도화특이성,종PCR산물표기지고신적수목,자외교련탐침적능량강도,이급잡교화엄격세탈4개방면진행우화.위료증실방법적가행성,종검측특이성、령민도화림상표본적검측준학성3개방면진행평고.결과 당검측체계위인물표기3개지고신분자,자외교련적능량강도선택1500× 0.1 mJ/cm2,잡교온도선택42℃,엄격세탈조건선택44℃,용0.5 ×SSC화0.1％십이완기류산납엄격세탈30 min시,가획득령민、특이적결과.재해검측체계적평고중,당PCR산량재10 mg/L이상,가이피검측도,차절대다수탐침특이성교호.림상표본적검측결과여직접측서법적검측결과부합솔위93.9％ (31/33).결론 해방법가이대HBV랍미부정、아덕복위화은체잡위내약돌변동시진행검측,시일충간편、쾌속、령민적분석방법,단부분탐침적특이성유대진일보제고.대우180/181、202/204저4개위점적탐침,유우량량거리교근,동일탐침서렬포함2개위점적밀마자,잡교회상호간우,통과조합거리교근적2개위점적밀마자적각충형식래설계탐침,가능유조우취득경호적결과.
Objective To establish a detection method for HBV drug-resistant mutations related to lamivudine,adefovir and entecavir by optimization and assessment of reverse hybridization system.Methods 26 degenerated probes covering 10 drug-resistant hotspots of 3 drugs were synthesized and immobilized on the same positively charged nylon membrane.PCR products labeled with digoxigenins were hybridized with corresponding probes.To improve the sensitivity and specificity,4 reaction steps of reverse hybridization were optimized including the number of labeled digoxigenin,the energy intensity of UV cross-linking,hybridization and stringency wash conditions.To prove the feasibility,the specificity,semitivity and accuracy of this system were assessed respectively.Results Sensitive and specific results are obtained by the optimization of the following 4 reaction steps: the primers labeled with 3 digoxigenins,energy intensity of UV cross-linking for 1 500 × 0.1 mJ/cm2,hybridization at 42℃ and stringency wash with 0.5 × SSC and 0.1％ SDS solution at 44℃ for 30min.In the assessment of system,the majority of probes have high specificity.The quantity of PCR product with a concentration of 10 ng/μl or above can be detected by this method.The concordant rate between reverse hybridization and direct sequencing is 93.9％ in the clinical sample test.Conclusion Though the specificity of several probes needs to be improved further,it is a simple,rapid and sensitive method which can detect HBV resistant mutations related to lamivudine,adefovir and entecavir simultaneously.Due to the short distance between 180 and 181,likewise 202 and 204,the sequence of the same probe covers two codon positions,and hybridization will be interfered by each other.To avoid such interference,the possible solution is that probes are designed by arranging and combining various forms of two near codons.