CAJ | 학술논문

利用无标记的分子信标及核酸染料Hoechst 33258建立了一种高灵敏、高选择性的特定序列核酸检测方法,并以野生型乙型肝炎病毒的一段寡核苷酸序列为目标DNA,对这种方法进行了验证。在此体系中,分子信标的茎完全设计成C/G碱基对。在没有目标DNA时,Hoechst 33258与分子信标作用较弱,其荧光信号很弱；当有目标DNA存在时,分子信标与目标DNA杂交形成双链,Hoechst 33258与双链DNA作用后荧光信号显著增强。在优化条件下,目标DNA浓度在2×10-10~2×10-8 mol/L 范围内时,Hoechst 33258的荧光强度(△I)与目标DNA的浓度(C)之间具有良好的线性关系,回归方程为△I=3.3439C﹢18.6949(R2=0.9982),方法检出限(3σ)为9×10-11 mol/L。此方法操作简单、检测速度快、灵敏度高、重现性好、检出限低。
이용무표기적분자신표급핵산염료Hoechst 33258건립료일충고령민、고선택성적특정서렬핵산검측방법,병이야생형을형간염병독적일단과핵감산서렬위목표DNA,대저충방법진행료험증。재차체계중,분자신표적경완전설계성C/G감기대。재몰유목표DNA시,Hoechst 33258여분자신표작용교약,기형광신호흔약；당유목표DNA존재시,분자신표여목표DNA잡교형성쌍련,Hoechst 33258여쌍련DNA작용후형광신호현저증강。재우화조건하,목표DNA농도재2×10-10~2×10-8 mol/L 범위내시,Hoechst 33258적형광강도(△I)여목표DNA적농도(C)지간구유량호적선성관계,회귀방정위△I=3.3439C﹢18.6949(R2=0.9982),방법검출한(3σ)위9×10-11 mol/L。차방법조작간단、검측속도쾌、령민도고、중현성호、검출한저。
A highly sensitive and selective method for specific DNA sequence detection is developed using a non-labeled molecular beacon (MB) and a nucleic acid dye Hoechst 33258. It is demonstrated by a specific DNA sequence of wild-type HBV as a model system. In this strategy, the stem of MB is completely designed as C/G base pairs. In the absence of target DNA, the interaction between Hoechst 33258 and the MBs is very weak,and the fluorescence signals of Hoechst 33258 is very low. In the presence of target DNA, the MBs hybridize with the target DNA and form double-stranded structure. Hoechst 33258 binds to dsDNA, and the fluorescence intensity is significantly enhanced. Under the optimum conditions, the fluorescence intensity of Hoechst 33258 exhibits good linear dependence on target DNA concentration in the range of 2 × 10-10-2 × 10-8 mol/L. The fitted regression equation is △I=3. 3439C(10-10 mol/L) ﹢18. 6949(R2=0. 9982) with a correlation coefficient of 0. 9982 (R2), and the detection limit is 9 × 10-11 mol/L (3σ). The proposed method has good precision, simple operation, fast detection speed, low detection limit, high accuracy and high sensitivity.