PCR-RFLP联合AS-PCR检测bcr/abl融合基因T315I突变方法的建立

摘要

Objective To develop a appropriate assay for identifying T315I point mutation of BCR/ABL fusion gene by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed mutant enriched allele specific (AS)-PCR,and to improve screening sentivity of BCR/ABL gene T315I mutant.Methods Recommbinant plasmids harboring wild-type ABL gene and the T315I mutant were constructed respectively and used as the detection objects.A pair of universal primers was designed according to wild-type and T315I mutant gene sequence.The wild-type and T315I mutant plasmids were served as template,the screening of ABL gene T315I mutation was carried out by RFLP after PCR amplification.The AS-PCR for T315I mutation detection was established based on a pair of specific primer.The method was used to detect the status of T315I gene mutation in one patient with chronic myeloid leukemia (CML).Also,the specificity and sensitivity of this method were evaluated.Results The PCR amplicons of the target 273 bp were digested into 4 fragments,141 bp,68 bp,46 bp and 18 bp in the wildtype BCR/ABL fragment and 3 fragments,159 bp,68 bp and 46 bp in the T315I mutant ABL fragment.The sensitivity of PCR-RFLP for the mutation detection of T315I was 6％.The digested production was used as the template in the following AS-PCR reaction through optimizes the reaction condition.The lowest concentration the T315I mutant can by detected by AS-PCR was 0.18％.The result confirmed the presence of T315I gene mutation in the CML patient.Conclusions The PCR-RFLP followed AS-PCR method used in the mutation detection in this study has higher specificity and sensitivity,providing a new qualitative tool for ABL gene T315I mutation detection.%目的 建立聚合酶链反应限制性片段长度多态性(PCR-RFLP)联合等位基因特异性聚合酶链反应(AS-PCR)检测bcr/abl融合基因T315I点突变的方法,以提高bcr/abl基因T315I突变检出率.方法 方法学建立.构建abl基因野生型和T315I突变型重组质粒作为检测对象,设计一对野生型和突变型abl基因序列的通用引物,分别以野生型和T315I突变型质粒作为模板,PCR扩增出目的片段,经酶切初筛检测abl基因T315I突变情况,另设计一对仅针对T315I突变的特异性引物进行AS-PCR检测.并对具有该基因突变的1例慢性粒细胞白血病患者进行检测,同时对该方法的灵敏度和特异性进行检测.结果 PCR扩增的目的片段(273 bp)经限制性内切酶Dde Ⅰ酶切后,野生型abl基因片段产生141、68、46和18 bp4条片段,而T315I突变型abl基因片段酶切后产生159、68和46 bp3条片段.PCR-RFLP方法可以检测T315I突变,检测灵敏度为6％.再以酶切产物作为模板进行AS-PCR,优化反应条件,AS-PCR所能检测到的T315I最低突变浓度为0.18％.所测临床标本结果显示,该患者发生了bcr/abl融合基因T315I突变.结论 PCR-RFLP联合AS-PCR检测方法特异性好、灵敏度高,能够给临床检测abl基因T315I突变提供一种新型的检测方法.