Quantitative threefold allele-specific PCR (QuanTAS-PCR) for highly sensitive JAK2 V617F mutant allele detection

BMC Cancer

Giada V Zapparoli³  Robert N Jorissen²  Chelsee A Hewitt³  Michelle McBean³  David A Westerman⁴  Alexander Dobrovic¹ 

[1] Molecular Pathology Research & Development, Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria, 3002, Australia

[2] Department of Medical Biology, University of Melbourne, Parkville, Australia

[3] Department of Pathology, Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria, 3002, Australia

[4] Division of Cancer Medicine, Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria, 3002, Australia

关键词: EGFR T790M;    BRAF V600E;    JAK2 V617F;    Real time PCR;    qPCR;    Minimal residual disease;    Myeloproliferative neoplasms;    Mutation detection;   

Others  :  1079785
DOI  :  10.1186/1471-2407-13-206

 received in 2013-02-07, accepted in 2013-03-26,  发布年份 2013

【 摘 要 】

Background

The JAK2 V617F mutation is the most frequent somatic change in myeloproliferative neoplasms, making it an important tumour-specific marker for diagnostic purposes and for the detection of minimal residual disease. Sensitive quantitative assays are required for both applications, particularly for the monitoring of minimal residual disease, which requires not only high sensitivity but also very high specificity.

Methods

We developed a highly sensitive probe-free quantitative mutant-allele detection method, Quantitative Threefold Allele-Specific PCR (QuanTAS-PCR), that is performed in a closed-tube system, thus eliminating the manipulation of PCR products. QuantTAS-PCR uses a threefold approach to ensure allele-specific amplification of the mutant sequence: (i) a mutant allele-specific primer, (ii) a 3′dideoxy blocker to suppress false-positive amplification from the wild-type template and (iii) a PCR specificity enhancer, also to suppress false-positive amplification from the wild-type template. Mutant alleles were quantified relative to exon 9 of JAK2.

Results

We showed that the addition of the 3′dideoxy blocker suppressed but did not eliminate false-positive amplification from the wild-type template. However, the addition of the PCR specificity enhancer near eliminated false-positive amplification from the wild-type allele. Further discrimination between true and false positives was enabled by using the quantification cycle (Cq) value of a single mutant template as a cut-off point, thus enabling robust distinction between true and false positives. As 10,000 JAK2 templates were used per replicate, the assay had a sensitivity of 1/10^-4 per replicate. Greater sensitivity could be reached by increasing the number of replicates analysed. Variation in replicates when low mutant-allele templates were present necessitated the use of a statistics-based approach to estimate the load of mutant JAK2 copies. QuanTAS-PCR showed comparable quantitative results when validated against a commercial assay.

Conclusions

QuanTAS-PCR is a simple, cost-efficient, closed-tube method for JAK2 V617F mutation quantification that can detect very low levels of the mutant allele, thus enabling analysis of minimal residual disease. The approach can be extended to the detection of other recurrent single nucleotide somatic changes in cancer.

【 授权许可】

   
2013 Zapparoli et al.; licensee BioMed Central Ltd.

【 图 表 】

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