Search

Objective: To establish a CRISPR/Cas13a based method for detecting TP53 R248W mutant molecules. Methods: TP53 R248W mutant plasmid was constructed by Over-lap PCR using TP53 wild-type plasmid as template. The CRISPR/Cas13a method for the detection of TP53 R248W variant was initially established by optimizing the amplification product size, amplification technology, the length and concentration of crRNA. The sensitivity of CRISPR/Cas13a method was evaluated by TP53 R248W variants with different mutation rates and simulated plasma ctDNA. Results: The size of the amplified product detected by CRISPR/Cas13a was about 368 bp. The concentration of crRNA had influence upon the detection intensity of CRISPR/Cas13a. In the range of 0.05~0.25μmol/L, the higher the concentration of crRNA, the higher the detection intensity of CRISPR/Cas13a was detected. The sensitivity of CRISPR/ Cas13a in detecting TP53 R248W variants was 104 copies/μL, and the minimum mutation rate was 0.01% . Conclusion: The established CRISPR/Cas13a method has the advantages of rapid, simple, sensitive and specific, and provides a new technology for the detection of TP53 R248W variant in tissues and plasma. [ABSTRACT FROM AUTHOR]