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Abstract：
The use of locked nucleic acid (LNA) probes and primers potentially improves sensitivity and specificity of quantitative PCR (qPCR) assays. One area of application is that of minimal residual cancer where PCR techniques have proved to be highly relevant tools in patient follow-up. We present here sensitive and specific consensus qPCR assays for quantification of the malignant lymphoma translocations, t(11;14) and t(14;18), by taking advantage of the thermodynamic properties of LNA. The assays were applied to genomic DNA from patients diagnosed with mantle cell lymphoma (MCL) and follicular lymphoma (FL), respectively. Two consensus forward primers targeting the BCL1 and BCL2 genes were designed together with a common consensus reverse primer and hydrolysis probe, the latter consisting exclusively of LNA, both targeting the J segments of the immunoglobulin heavy chain (IgH) gene. The quantitative range of both assays was 1×10(0) to 5×10(-5), and the sensitivity was 10(-5), without the need for patient-specific primers. Peripheral blood (PB) and bone marrow (BM) samples from 36 patients diagnosed with MCL and nine patients diagnosed with FL were analysed using this novel qPCR approach. The level of minimal residual disease (MRD) using t(11;14) and t(14;18) as genetic targets reflected the clinical status of the patients: low levels of MRD at clinical remission, and increasing levels at disease progression. The present assays could prove as useful tools in lymphoma therapy.
摘要：
锁核酸(LNA)探针和引物的使用潜在地提高了定量PCR(qPCR)测定的灵敏度和特异性。一个应用领域是最小残留癌，其中PCR技术已被证明是患者随访中高度相关的工具。我们在这里介绍了用于定量恶性淋巴瘤易位的灵敏和特异的共有qPCR测定法，t(11;14)和t(14;18)，利用LNA的热力学性质。该测定应用于诊断为套细胞淋巴瘤（MCL）和滤泡性淋巴瘤（FL）的患者的基因组DNA，分别。设计了两个针对BCL1和BCL2基因的共有正向引物，以及一个共有反向引物和水解探针，后者完全由LNA组成，均靶向免疫球蛋白重链(IgH)基因的J区段。两种测定的定量范围为1×10(0)至5×10(-5)，灵敏度为10（-5），无需患者特异性引物。使用这种新颖的qPCR方法分析了来自36例诊断为MCL的患者和9例诊断为FL的患者的外周血（PB）和骨髓（BM）样品。以t（11;14）和t（14;18）为遗传目标的微小残留病（MRD）水平反映了患者的临床状态：临床缓解时MRD水平低，并在疾病进展时增加水平。本测定法可证明是淋巴瘤治疗中的有用工具。
