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Background <p>This study describes the development and analytical validation of a DNA-based quantitative PCR (qPCR) assay for detecting the NPM1 Type A mutation (<i>NPM1-A</i> <sup>mut</sup>) in acute myeloid leukemia (AML). The assay employs a modified wild-type blocker (WTB) to preferentially suppress amplification of wild-type (WT) alleles, thereby enriching mutant targets.</p> Methods and Results <p>The modified WTB was synthesized as a 3′-ddC–terminated LNA/DNA chimera and used together with a 6-FAM–MGB-Eclipse hydrolysis probe. The analytical performance of the WTB-qPCR assay was compared with that of an otherwise identical qPCR assay lacking WTB. Key parameters such as linearity, precision, limit of detection (LoD), and limit of quantification (LoQ) were evaluated following the guidelines outlined in CLSI EP17-A2. Concurrently, modified WTB was implemented in Sanger sequencing to improve the sensitivity of mutation detection. Incorporation of WTB yielded an LoD of 0.00302% and an LoQ of 0.00528%, corresponding to approximately 3.02 and 5.28 <i>NPM1-A</i><sup>mut</sup> copies, respectively, per 100,000 WT copies. This represented a clear improvement over the non-WTB assay. The WTB-qPCR showed excellent linearity (R<sup>2</sup> &gt; 0.999) and precision (CV &lt; 5%), with 100% analytical specificity and no detectable cross-reactivity. Application of modified WTB in Sanger sequencing increased analytical sensitivity to ~ 0.1% mutant allele frequency.</p> Conclusion <p>The modified WTB-qPCR assay offers a rapid, highly sensitive, and cost-effective DNA-based method for MRD monitoring in NPM1-A–mutated AML. Its strong analytical performance and straightforward workflow support its implementation as a routine diagnostic tool in clinical laboratories.</p>

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Analytical validation of a modified wild-type blocker qpcr assay for the sensitive DNA-based detection of NPM1 type A in acute myeloid leukemia

  • 期刊论文

  • 发表日期：

    2026年8月3日

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  • Pejman Hamedi-Asl,
  • Dariush Hamedi-Asl,
  • Shaharbano Rostami,
  • Mahmood Barati,
  • Ali Amini,
  • Davod Jafari,
  • Fatemeh Damerchiloo,
  • Rima Manafi,
  • Majid Safa

摘要

Background

This study describes the development and analytical validation of a DNA-based quantitative PCR (qPCR) assay for detecting the NPM1 Type A mutation (NPM1-A mut) in acute myeloid leukemia (AML). The assay employs a modified wild-type blocker (WTB) to preferentially suppress amplification of wild-type (WT) alleles, thereby enriching mutant targets.

Methods and Results

The modified WTB was synthesized as a 3′-ddC–terminated LNA/DNA chimera and used together with a 6-FAM–MGB-Eclipse hydrolysis probe. The analytical performance of the WTB-qPCR assay was compared with that of an otherwise identical qPCR assay lacking WTB. Key parameters such as linearity, precision, limit of detection (LoD), and limit of quantification (LoQ) were evaluated following the guidelines outlined in CLSI EP17-A2. Concurrently, modified WTB was implemented in Sanger sequencing to improve the sensitivity of mutation detection. Incorporation of WTB yielded an LoD of 0.00302% and an LoQ of 0.00528%, corresponding to approximately 3.02 and 5.28 NPM1-Amut copies, respectively, per 100,000 WT copies. This represented a clear improvement over the non-WTB assay. The WTB-qPCR showed excellent linearity (R2 > 0.999) and precision (CV < 5%), with 100% analytical specificity and no detectable cross-reactivity. Application of modified WTB in Sanger sequencing increased analytical sensitivity to ~ 0.1% mutant allele frequency.

Conclusion

The modified WTB-qPCR assay offers a rapid, highly sensitive, and cost-effective DNA-based method for MRD monitoring in NPM1-A–mutated AML. Its strong analytical performance and straightforward workflow support its implementation as a routine diagnostic tool in clinical laboratories.