3’末端碱基游移混合引物提高多变区基因片段PCR检测的阳性率(英文)

目的:通过使用3’末端碱基游移混合引物,减少引物末端错配,提高多变区基因片段的PCR检测阳性率。方法:在HBV DNA P基因区设计一对检测阳性率相对较高的引物(原型引物),在原型引物序列基础上将两根引物的3’末端分别截去1个或2个碱基,设计出两对参数与原型引物近似的突变引物。分别单独用原型引物和原型引物与突变引物组成的混合引物,在相同条件下对27份HBV DNA阳性标本行PCR,比较二者阳性率。用混合引物扩增4例拉米呋丁治疗无效患者血清HBV DNA,将扩增产物测序并评价3’末端错配对PCR的影响。结果:原型引物和混合引物检测阳性率分别为70.4％(19/27)和85.2％(23/27),两者差异非常显著(P<0.05)。引物3’末端错配能导致PCR假阴性。结论:扩增保守性相对较差的基因片段,采用3’末端单个或多个碱基截短的引物,构成3’末端碱基游移混合引物,可以避免因3’末端错配产生的PCR假阴性,提高检测阳性率。

3'-terminus shifted bases degeneracy primers increasing sensitivity of polymerase chain reaction

Objective: To reduce the risk of 3'-terminal mismatch between primers and template and increase the sensitivity of polymerase chain reaction (PCR) in the detection of variable region of DNA. Methods: A pair of special primer(WU, WD) was designed to amplify a fragment of HBV DNA P gene by PCR, Other 2 similar pairs of primer (MU1, MD1, MU2, MD2) were obtained by knocking off 1 or 2 bases at the 3'-terminal of WU and WD. (1) Special primers (WU, WD) and degeneracy primers(WU, WD, MU1, MU2. MD1, MD2) were used to amplify 27 samples respectively by PCR under the same condition. The sensitivity of each PCR was compared. (2) Using degeneracy primers, serum HBV DNA was amplified from 4 patients who were resistant to lamivudine. The PCR products were sequenced to evaluate the effect of the 3'-terminal mismatch of primers upon PCR. Results: (1) The sensitivity of special primers and degeneracy primers were 70. 4%(19/27) and 85. 2% (23/27) respectively (P<0. 05). (2) The sequencing analysis of the PCR products suggested that the 3'-terminal mismatch of primers caused false negative in the PCR detection. Conclusion: When amplifying the variable region of DNA, the false negative result can be avoided by using 3'-terminus shifted degeneracy primers.