Ultrasensitive detection of gene-PIK3CA

Low abundance gene-PIK3CAH1047R mutation detection is crucial for the clinical diagnosis and treatment of breast cancer. Here, a fluorescent biosensor which combines cascaded strand displacement amplification (C-SDA) and trans-cleavage ability of CRISPR/Cas12a was established to ultra-sensitively detect gene-PIK3CAH1047R mutation. The mutated gene-PIK3CAH1047R can combine with complementary sequence to form an intact recognition site for endonuclease FspI. Mediated by FspI, it breaks at the mutation site to produce DNA fragment to trigger SDA or C-SDA. Then, the fluorescent biosensors based on SDA-CRISPR/Cas12a or C-SDA-CRISPR/Cas12a were constructed. Compared with biosensor based on SDA-CRISPR/Cas12a (5 pM), the minimum detection of the biosensor based on C-SDA-CRISPR/Cas12a is reduced two orders of magnitude (50 fM). In range of 0.001%-50%, we achieved the ultrasensitive detection of gene-PIK3CAH1047R mutation low to 0.001%. Besides, the proposed biosensor works well in human serum samples, showing its application potential in low-abundance gene-PIK3CAH1047R mutation detection.