Ultrasensitive detection of gene-PIK3CAH1047R mutation based on cascaded strand displacement amplification and trans-cleavage ability of CRISPR/Cas12a

Low abundance gene-PIK3CA(H1047R) 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-PIK3CA(H1047R) mutation. The mutated gene-PIK3CA(H1047R) 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-PIK3CA(H1047R) mutation low to 0.001%. Besides, the proposed biosensor works well in human serum samples, showing its application potential in low-abundance gene-PIK3CA(H1047R) mutation detection.

Automated summary

A fluorescent biosensor combining cascaded strand displacement amplification and CRISPR/Cas12a trans-cleavage ability was established for ultra-sensitive detection of low abundance gene-PIK3CA(H1047R) mutation in breast cancer. The biosensor showed improved sensitivity compared to previous methods, achieving detection of gene-PIK3CA(H1047R) mutation as low as 0.001% in human serum samples. This proposed biosensor has potential applications in low-abundance gene-PIK3CA(H1047R) mutation detection.