Abstract
Background. The PIK3CA:H1047R mutation is one of the most common driver mutations in breast cancer that leads to aberrant activation of the PI3K/AKT/mTOR signaling pathway. Rapid and low-cost detection of this mutation can significantly contribute to a better outcome and personalized treatment protocols. This study aimed to develop and optimize an allele-specific LAMP assay for the visual detection of the H1047R mutation without the need for advanced laboratory equipment.
Methods. Allele-specific FIP and BIP primers were designed with a 5′ end complementary to the mutant nucleotide, along with a loop primer to enhance amplification speed and sensitivity. The assay was tested on DNA from MDA-MB-453 (mutant) and MCF-7 (wild-type) breast cancer cell lines. Sensitivity was evaluated using serial dilutions. Reaction outcomes were assessed visually using Neutral Red colorimetry and SYBR Green fluorescence. Additionally, RNA-sequencing data of H1047R-positive breast cancer samples from the TCGA database were analyzed to investigate gene expression changes within the PI3K/AKT/mTOR pathway.
Results. The developed assay accurately distinguished mutant from wild-type sequences with high specificity. The loop primer reduced reaction time from 25 to 20 minutes and improved sensitivity, achieving a detection limit of 0.033 ng/µL. Neutral Red enabled clear visual discrimination between positive and negative reactions, whereas SYBR Green showed only minor fluorescence differences. RNA-seq analysis revealed significant alterations in the expression of multiple PI3K/AKT/mTOR pathway genes in H1047R-positive samples.
Conclusion. The optimized PE-LAMP assay provides a rapid, sensitive, inexpensive, and naked-eye–readable method for detecting the PIK3CA:H1047R mutation. Given its simplicity and minimal equipment requirements, this approach holds strong potential for clinical application in resource-limited settings to support targeted therapy selection in breast cancer patients.
Practical Implications. The application of the developed method in this study can facilitate the detection of the PIK3CA:H1047R mutation, and potentially other mutations in the future, in clinical and laboratory settings with limited resources. Its high speed, low cost, and the ability to visualize results with the naked eye make this method suitable for initial screening of the patient and rapid selection of targeted therapies. Moreover, its use in point-of-care settings can improve patient access to accurate molecular diagnostic approaches.