摘要
目的 建立急性早幼粒细胞白血病(APL)患者治疗监测相关lnc-LOC100506453的微滴式数字PCR(ddPCR)定量检测方法,完成方法学评价,并用于APL患者的外周血检测。方法 设计和合成lnc-LOC100506453 ddPCR的引物和探针,优化ddPCR的反应体系和条件。评价所建立ddPCR的方法学性能,包括重复性、特异性、检测下限与定量检测范围。最后用于APL患者的外周血标本的检测。结果 lnc-LOC100506453 ddPCR方法的最佳退火温度为59℃,最佳引物浓度为900 nmol/L。该ddPCR方法的特异性为100%,批间和批内重复性的CV值均<10%。ddPCR方法的检测下限达到0.5 copies/μl,定量检测值为0.5 copies/μl~5 000 copies/μl。在对临床标本的ddPCR检测中,APL患者外周血lnc-LOC100506453表达量明显高于非APL患者和健康对照者(P<0.001)。APL患者诱导分化后的外周血lncLOC100506453表达量低于APL治疗前(P<0.001),APL患者完全缓解后的表达量进一步下降(P<0.001)。结论 本研究所建立的lnc-LOC100506453 ddPCR方法具有较好的方法学检测性能,可用于APL初诊患者的诊断以及APL随访患者的微创治疗监测。
Objective To establish a droplet digital PCR(ddPCR) method for quantitative detection of lnc-LOC100506453associated with treatment surveillance for acute promyelocytic leukemia(APL) patients,complete the methodology evaluation,and use it for peripheral blood( PB) detection in APL patients.Method The special primers and probe for lnc-LOC100506453 ddPCR were designed and synthesized,and the ddPCR reaction system and reaction conditions were optimized.The methodological performance of the established ddPCR was evaluated,including repeatability,specificity,detection limit and quantitative detection range.Finally,the established ddPCR was used for detecting PB samples from APL patients.Results The optimum annealing temperature of the lnc-LOC100506453 ddPCR is 59 ℃ and the optimum concentration of each primer is 900 nmol/L.The specificity of this ddPCR is 100%,and theCVvalue of repeatability between or within batches is less than 10%.The detection limit of the ddPCR is 0.5 copies/μl,and the quantitative detection range is from 0.5 copies/μl to 5 000 copies/μl.The PB lnc-LOC100506453 expression in APL patients was significantly higher than that in non-APL patients or healthy controls(P< 0.001).The PB lnc-LOC100506453 expression in APL patients after induction was lower than that in APL at diagnosis(P< 0.001) and further decreased in APL patients after complete remission(P< 0.001).Conclusion The established lnc-LOC100506453 ddPCR method has good methodological performance,and can be used for the diagnosis of APL and micro-invasive monitoring of APL follow-up.
作者
白园园
邵麒晖
赵京炜
王归然
陈占国
BAI Yuan-yuan;SHAO Qi-hui;ZHAO Jing-wei;WANG Gui-ran;CHEN Zhan-guo(Department of Clinical Laboratory,The Second Affiliated Hospital of Wenzhou Medical University,Wenzhou,Zhejiang 325000,China;不详)
出处
《中国卫生检验杂志》
CAS
2022年第15期1796-1800,共5页
Chinese Journal of Health Laboratory Technology
基金
浙江省自然科学基金项目(LGF20H200005)
浙江省卫生健康科技计划项目(2021KY216)
温州市基础性科研计划项目(Y20190090)
贺林院士新医学临床转化工作站科研基金项目(18331203)。
