Objective: To develop a novel real-time PCR for sensitively quantitative detection of JAK2 V617F allele burden in peripheral blood.
Methods: Based on the real-time allele-specific PCR (AS-qPCR), the locked nucleic acid (LNA)-modified oligonucleotide probe was used for selectively blocking amplification of wild-type alleles in AS-qPCR, and then a novel AS-LNA-qPCR method was established. The percentages of sample JAK2 V617F alleles were directly calculated by its threshold cycle (Ct) values according to the standard curve which generated by JAK2 V617F alleles with its Ct values. We validated intra- and inter-assay variability for quantifying JAK2 V617F. We also assayed 623 apparent healthy donors by our method to validate its clinical application value.
Results: The quantitative lower limit of this method for JAK2 V617F was 0.01%, and the intra- and inter-assay average variability for quantifying percentage of JAK2 V617F in total DNA was 6.3% and 8.6%, respectively. Nineteen JAK2 V617F-positive individuals were identified using AS-LNA-qPCR in blood of 623 apparently healthy donors, and the range of percentages of JAK2 V617F alleles were 0.01%-5.49%.
Conclusion: The AS-LNA-qPCR with highly sensitive and reproducible quantification of JAK2 V617F mutant burden can be used clinically for diagnosis as well as evaluation of disease prognosis and efficacy of therapy in patients with myeloproliferative neoplasms.
目的 建立一种定量检测外周血细胞酪氨酸激酶2（JAK2）基因V617F突变率的等位基因特异性实时荧光定量PCR（AS-qPCR）方法。方法 在AS-qPCR基础上，引入1条锁核酸（LNA）修饰的寡核苷酸探针，用以选择性抑制AS-qPCR中突变引物对野生等位基因的非特异性扩增，定量检测JAK2 V617F突变率，称之为AS-LNA-qPCR法。通过AS-LNA-qPCR法测定样本的循环阈值（Ct值），根据AS-LNA-qPCR法检测不同JAK2 V617F突变率标准品的Ct值，绘制标准曲线，根据标准曲线直接获得检测样本中JAK2 V617F突变率。我们对该方法标准品的批内、批间检测结果进行了评价，并对623名表观健康人外周血细胞基因组DNA中JAK2 V617F突变进行检测，以探讨其临床应用价值。结果 AS-LNA-qPCR法定量检测JAK2 V617F突变率的下限可达到0.01%，批内、批间平均变异率分别是6.3%和8.6%。对623名表观健康人外周血细胞基因组DNA中JAK2 V617F突变检测显示，其中19名（3%）表现健康人JAK2 V617F突变阳性，突变率变化范围为0.01%~5.49%。结论 AS-LNA-qPCR方法直接定量检测外周血细胞JAK2 V617F突变率，具有检测灵敏度高和重复性好的特性，适合临床上用于对骨髓增殖性疾病诊断、疾病进程评估和疗效的监测。