Drop-off微滴式数字PCR检测急性髓系白血病NRAS基因突变及其临床应用

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摘要目的：建立检测急性髓系白血病（acute myeloid leukemia，AML）神经母细胞瘤RAS病毒癌基因同源物（NRAS）基因突变的drop-off微滴式数字PCR（droplet digital PCR，ddPCR）方法，并评价该方法的临床应用价值。方法: 针对NRAS G12和G13突变位点设计特异性drop-off ddPCR引物和探针，建立最佳反应体系，并对该方法进行性能验证。应用该方法对140例临床样本进行初筛，其检测结果与Sanger测序进行比较，并用二代测序（next generation sequencing，NGS）进行验证。结果:建立drop-off ddPCR检测NRAS基因G12/G13突变的方法，灵敏度达0.158%，重复性、线性良好，其空白限为5.40拷贝数/μL，最低检测下限为15.84拷贝数/μL。在140例初诊AML患者中，dropoff ddPCR检出NRAS突变28例（20%），其突变负荷为0.26%~52.85%（中位2.14%）；而Sanger测序仅检出7例（5%）阳性，为进一步验证，将21例Sanger测序结果阴性而dropoff ddPCR检测阳性的样本进行NRAS基因NGS，检出14例阳性，而另外7例NGS阴性的样本，其drop-off ddPCR检测突变频率为0.53%~1.50%（中位1.10%）。结论: 建立了drop-off ddPCR技术检测AML患者中NRAS基因突变的方法，该方法灵敏度高，可用于对NRAS基因G12/G13突变进行定量检测，有望可用于AML患者预后判断和疾病监测。

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关键词 ： drop-off微滴式数字PCRfont-family: ????, font-weight: 700, text-align: -webkit-right, ')" href="#">background-color: rgb(212, 226, 255);">, NRAS基因font-family: ????, font-weight: 700, text-align: -webkit-right, ')" href="#">background-color: rgb(212, 226, 255);">, 急性髓系白血病font-family: ????, font-weight: 700, text-align: -webkit-right, ')" href="#">background-color: rgb(212, 226, 255);">, 基因突变font-family: ????, font-weight: 700, text-align: -webkit-right, ')" href="#">background-color: rgb(212, 226, 255);">, 微小残留病灶font-family: ????, font-weight: 700, text-align: -webkit-right, ')" href="#">background-color: rgb(212, 226, 255);">, 预后

Abstract： Objective： To establish a new dropoff droplet digital PCR (ddPCR) method for detecting NRAS mutations in patients with acute myeloid leukemia (AML), and to evaluate its clinical value. Methods： Specialized primers and probes were designed for NRAS G12 and G13 mutation. This research established and optimized the reaction system of drop-off ddPCR and verified this method. A total of 140 clinical samples were screened by this method, and the results were compared with Sanger sequencing, and verified by next generation sequencing (NGS). Results： Dropoff droplet digital PCR method was established to detect different types of the G12 and G13 mutations of NRAS in AML patients. The method had high sensitivity (up to 0.158%), good repeatability and linearity. The limit of blank was 5.40 copies/μL and the limit of detection was 15.84 copies/μL. Among 140 newly diagnosed AML patients, 28 samples (20%) were detected by dropoff ddPCR, the mutation rate was from 0.26%-52.85% (median 2.14%); only 7 (5%) samples were detected positive by Sanger sequencing. For further verification, those samples which were detected negatively by Sanger but positively by drop-off ddPCR were sent for NGS. A total of 14 samples were detected positive by NGS, the mutant frequency of the residual 7 detected positive by dropoff ddPCR was 0.53%-1.50% (median 1.10%). Conclusion： Drop-off ddPCR can effectively detect the quantity of the G12 and G13 mutations of NRAS, which can be used for screening and judging the prognosis of AML patients.

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收稿日期: 2022-08-02

基金资助: 国家自然科学基金资助项目（81970118）；江苏省医学创新团队（CTXDB201702）；镇江市血液病临床医学研究中心项目（SS2018009）；镇江市社会发展项目（SH2019067，SH2022026）

通讯作者: 钱军（通讯作者），主任医师，教授，博士生导师，E-mail： qianjun0007@hotmail.com

作者简介: 向鹤麟（1997—），女，硕士研究生

引用本文:

向鹤麟，金晔，袁倩，等.. Drop-off微滴式数字PCR检测急性髓系白血病NRAS基因突变及其临床应用[J]. 江苏大学学报:医学版, 2023, 33(01): 49-53. XIANG Helin1, JIN Ye1, YUAN Qian2,3, YAO Dongming2,3, LI Ting1, YU Di1, LENG Jiayan1, LIN Jiang2,3, QIAN Jun1,2,3. Detection of NRAS mutation in acute myeloid leukemia by drop-off droplet digital PCR and its clinical value

. Journal of Jiangsu University(Medicine Edition), 2023, 33(01): 49-53.

链接本文:

http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2023/V33/I01/49

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