Abstract
High-resolution melt analysis (HRMA) is a cheap and reliable post-polymerase chain reaction (PCR) cancer mutation screening technique, which is fast gaining clinical relevance. The HRMA capabilities of the LightScanner (Idaho Technology) have been severally studied. However, the ABI 7500 HRM has not been tested against the purpose-built HRM instrument such as the LightScanner.DNA from formalin-fixed, paraffin-embedded gastric cancer, colorectal cancer, and normal tissue as well as from colorectal cancer cell lines were amplified at exons 2, 3, and 4 of KRAS, and at exons 11 and 15 of BRAF in the ABI 7500 fast real-time PCR machine and subjected to melting both on the ABI and on the LightScanner. HRMA data were analysed with the ABI HRM software v2.0.1 and the LightScanner Call-IT 2.5. We tested the ABI 7500 HRM for internal precision, accuracy, sensitivity, and specificity at mutation screening relative to the LightScanner, using crude percentage concordance, kappa statistics, and the area under the receiver operator characteristics (AUROC) curve on SPSS version 19.The results show that the ABI 7500 HRMA has a high internal precision, and excellent concordance, sensitivity, and specificity at mutation screening compared with the LightScanner. However, in contrast to the LightScanner HRM software analysis, the ABI HRM software v.2.0.1, cannot distinguish real from certain pseudovariations in PCR amplicons that are sometimes brought about by the artefacts of the melting process.In conclusion, the ABI HRM has a comparable performance level with the LightScanner, although in certain respects mentioned previously, the LightScanner has an edge over the ABI.
Highlights
High-resolution melt analysis is a highly reliable polymerase chain reaction (PCR)-based technique which has found use in genotyping and mutation scanning and is fast gaining clinical relevance [1, 2]
There are two classes of instruments utilized for High-resolution melt analysis (HRMA), namely real-time PCR instruments with HRM modules and HRM instruments without PCR capabilities [1, 3]
In the HRM instruments such as the LightScanner, PCR is carried out in a thermocycler/qPCR instruments, and amplicon melting is done in the HRM machines [4]
Summary
High-resolution melt analysis is a highly reliable PCR-based technique which has found use in genotyping and mutation scanning and is fast gaining clinical relevance [1, 2]. The aims of this study are to determine the degree of concordance between the ABI 7500 real-time PCR and the LightScanner in mutation screening and to compare the sensitivity and specificity of the ABI 7500 fast real-time PCR HRMA relative to that of the LightScanner. Our hypotheses are that there is a high rate of concordance between the HRMA results of the ABI 7500 real-time PCR and the LightScanner, and the ABI fast real-time instrument has an excellent sensitivity and specificity relative to the LightScanner. To test these hypotheses, the HRM results obtained with these instruments were analysed with the appropriate statistical tests of agreement.
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