CAJ | 학술논문

目的建立一种单核苷酸多态性-聚合酶链反应(SNP-PCR)定量检测异基因造血干细胞移植后供受嵌合率的新方法 ,探讨其可行性、准确性及优越性.方法利用18个SNP位点筛选移植前每一对供、受者,采用实时定量PCR(RQ-PCR)对筛选出的差异位点行嵌合率定量分析,通过倍比稀释、模拟嵌合与微卫星重复序列-PCR(STR-PCR)、性染色体双色荧光原位杂交(XY-FISH)和融合基因的定量检测,比较、验证方法的准确性及敏感度.结果①利用内参质粒标准品扩增的17次标准曲线,平均斜率为-3.39,平均截距为39.97,相关系数均＞0.995,扩增效率接近理想水平;批内差及批间差分别为0.50%和1.10%,在可控范围;与模拟混合嵌合相关系数在0.99以上;可重复敏感度达0.01%.②40例移植患者中,95%以上可以筛选出供、受者差异SNP位点;SNP-PCR与STR-PCR结果吻合率达96.7%,与XY-FISH结果比较差异无统计学意义(P＞0.05);SNP-PCR与特定白血病融合基因检测结果比较,完全嵌合(CC)标本均未检出肿瘤相关的融合基因,部分嵌合(MC)标本融合基因均为阳性.结论 SNP-PCR检测嵌合率准确、敏感、易行,具有极高的临床应用前景,克服了STR-PCR竞争抑制及扩增平台期偏倚的缺点,可以替代其进行临床常规检测.
목적 건립일충단핵감산다태성-취합매련반응(SNP-PCR)정량검측이기인조혈간세포이식후공수감합솔적신방법 ,탐토기가행성、준학성급우월성.방법 이용18개SNP위점사선이식전매일대공、수자,채용실시정량PCR(RQ-PCR)대사선출적차이위점행감합솔정량분석,통과배비희석、모의감합여미위성중복서렬-PCR(STR-PCR)、성염색체쌍색형광원위잡교(XY-FISH)화융합기인적정량검측,비교、험증방법 적준학성급민감도.결과①이용내삼질립표준품확증적17차표준곡선,평균사솔위-3.39,평균절거위39.97,상관계수균＞0.995,확증효솔접근이상수평;비내차급비간차분별위0.50%화1.10%,재가공범위;여모의혼합감합상관계수재0.99이상;가중복민감도체0.01%.②40례이식환자중,95%이상가이사선출공、수자차이SNP위점;SNP-PCR여STR-PCR결과문합솔체96.7%,여XY-FISH결과비교차이무통계학의의(P＞0.05);SNP-PCR여특정백혈병융합기인검측결과비교,완전감합(CC)표본균미검출종류상관적융합기인,부분감합(MC)표본융합기인균위양성.결론 SNP-PCR검측감합솔준학、민감、역행,구유겁고적림상응용전경,극복료STR-PCR경쟁억제급확증평태기편의적결점,가이체대기진행림상상규검측.
Objective To develop a novel single nucleotide polymorphism (SNP)-PCR based method for quantitative detection of chimerism after allogeneic haemopoietic stem cell transplantation(allo-HSCT),and to explore its feasibility,accuracy and superiority.Methods 18 SNP loci were screened to identify informative markers for detecting chimerism in each donor/recipient pair before transplantation.Then the chimerism rate of each informative marker was analyzed by real-time quantitative PCR (RQ-PCR).The accuracy and sensitivity were verified by multiple proportion dilution and analogy chimerism compared with quantitative detection of short tandem repeat (STR)-PCR,fluorescence in situ hybridization (FISH) and fusion gene.Results ①The average slope of the 17 time amplications of the internal control plasmid was-3.39,the average intercept was 39.97,correlation coefficients were more than 0.995,which was close to the theoretical level.The intra-and interassay variability was 0.50% and 1.1%,respectively,which were both in the allowed ranges.A linear correlation with artificial mixed chimerism is above 0.99 and a sensitivity of 0.01% proved reproducible.②At least one informative marker could be found in over 95% of 40 donor/recipient pairs.The results of the chimerisms derived from SNP-PCR were consistent with that from STR-PCR (96.7%),FISH and fusion gene analasis (P＞0.05);the quantitative results of special fusion gene transcripts were negtive in complete chimerism samples,and positive in mixed chimerism samples.Conclusions This new assay which overcome the PCR competition and plateau biases of STR-PCR provides an accurate,reliable and rapid quantitative assessment of mixed chimerism after allo-transplantation.It is highly promising for of clinical application and may take the place of STR-PCR in the conventional chimerisim assessment.