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...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。展开更多
Objective:To classify 21 new isolates of Trypanosoma cruai(T.cruzi) according to the Discrete Typing Unit(DTU) which they belong to,as well as tune up a new pair of primers designed to detect the parasite in biologica...Objective:To classify 21 new isolates of Trypanosoma cruai(T.cruzi) according to the Discrete Typing Unit(DTU) which they belong to,as well as tune up a new pair of primers designed to detect the parasite in biological samples.Methods:Strains were isolated,DNA extracted,and classified by using three Polymerase Chain Reactions(PCR).Subsequently this DNA was used along with other isolates of various biological samples,for a new PCR using primers designed.Finally,the amplified fragments were sequenced.Results:It was observed the predominance of DTU i in Colombia,as well as the specificity of our primers for detection of T.cruzi,while no band was obtained when other species were used.Conclusions:This work reveals the genetic variability of 21 new isolates of T.cruzi in Colombia.Our primers confirmed their specificity for detecting the presence of T.cruzi.展开更多
基金This work is supported by the National Natural Science Foundation of China(30070958).
文摘目的: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。
基金funded by a FPU a grant from the Ministry of Education of Spain
文摘Objective:To classify 21 new isolates of Trypanosoma cruai(T.cruzi) according to the Discrete Typing Unit(DTU) which they belong to,as well as tune up a new pair of primers designed to detect the parasite in biological samples.Methods:Strains were isolated,DNA extracted,and classified by using three Polymerase Chain Reactions(PCR).Subsequently this DNA was used along with other isolates of various biological samples,for a new PCR using primers designed.Finally,the amplified fragments were sequenced.Results:It was observed the predominance of DTU i in Colombia,as well as the specificity of our primers for detection of T.cruzi,while no band was obtained when other species were used.Conclusions:This work reveals the genetic variability of 21 new isolates of T.cruzi in Colombia.Our primers confirmed their specificity for detecting the presence of T.cruzi.
